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Archive for the ‘Neutropenia’ Category


Neutrophil Serine Proteases in Disease and Therapeutic Considerations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

SERPINB1 Regulates the activity of the neutrophil proteases elastase, cathepsin G, proteinase-3, chymase,
chymotrypsin, and kallikrein-3. Belongs to the serpin family. Ov-serpin subfamily. Note: This description may
include information from UniProtKB.
Chromosomal Location of Human Ortholog: 6p25
Cellular Component: extracellular space; membrane; cytoplasm
Molecular Function: serine-type endopeptidase inhibitor activity
Reference #:  P30740 (UniProtKB)
Alt. Names/Synonyms: anti-elastase; EI; ELANH2; ILEU; LEI; Leukocyte elastase inhibitor; M/NEI; MNEI; Monocyte/neutrophil elastase inhibitor; Peptidase inhibitor 2; PI-2; PI2; protease inhibitor 2 (anti-elastase), monocyte/neutrophil derived; serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1; Serpin B1; serpin peptidase inhibitor, clade B (ovalbumin), member 1; SERPINB1
Gene Symbols: SERPINB1
Molecular weight: 42,742 Da
 

SERPIN PEPTIDASE INHIBITOR, CLADE B (OVALBUMIN), MEMBER 1; SERPINB1

Alternative titles; symbols
PROTEASE INHIBITOR 2, MONOCYTE/NEUTROPHIL DERIVED; ELANH2
ELASTASE INHIBITOR, MONOCYTE/NEUTROPHIL; EI
HGNC Approved Gene Symbol: SERPINB1
Cloning and Expression
Monocyte/neutrophil elastase inhibitor (EI) is a protein of approximately 42,000 Mr with serpin-like functional properties.
Remold-O’Donnell et al. (1992) cloned EI cDNA and identified 3 EI mRNA species of 1.5, 1.9, and 2.6 kb in monocyte-like cells
and no hybridizing mRNA in lymphoblastoid cells lacking detectable EI enzymatic activity. The cDNA open reading frame encoded
a 379-amino acid protein. Its sequence established EI as a member of the serpin superfamily. Sequence alignment indicated that
the reactive center P1 residue is cys-344, consistent with abrogation of elastase inhibitory activity by iodoacetamide and making
EI a naturally occurring cys-serpin.
 

 

Mapping

In the course of studying 4 closely linked genes encoding members of the ovalbumin family of serine proteinase inhibitors
(Ov-serpins) located on 18q21.3, Schneider et al. (1995) investigated the mapping of elastase inhibitor. They prepared PCR
primer sets of the gene, and by using the NIGMS monochromosomal somatic cell hybrid panel, showed that the EI gene maps
to chromosome 6.

By amplifying DNA of a somatic cell hybrid panel, Evans et al. (1995) unambiguously localized ELANH2 to chromosome 6.
With the use of a panel of radiation and somatic cell hybrids specific for chromosome 6, they refined the localization to
the short arm telomeric of D6S89, F13A (134570), and D6S202 at 6pter-p24.

http://www.phosphosite.org/getImageAction.do?id=27292293

 

 

REFERENCES
Evans, E., Cooley, J., Remold-O’Donnell, E. Characterization and chromosomal localization of ELANH2, the gene encoding human
monocyte/neutrophil elastase inhibitor. Genomics 28: 235-240, 1995. [PubMed: 8530031related citations] [Full Text]
Remold-O’Donnell, E., Chin, J., Alberts, M. Sequence and molecular characterization of human monocyte/neutrophil elastase inhibitor.
Proc. Nat. Acad. Sci. 89: 5635-5639, 1992. [PubMed: 1376927related citations][Full Text]
Schneider, S. S., Schick, C., Fish, K. E., Miller, E., Pena, J. C., Treter, S. D., Hui, S. M., Silverman, G. A. A serine proteinase inhibitor locus at
18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc. Nat. Acad. Sci. 92: 3147-3151, 1995.
[PubMed: 7724531,related citations] [Full Text]

 

Leukocyte elastase inhibitor (serpin B1) (IPR015557)

Short name: Serpin_B1

Family relationships

  • Serpin family (IPR000215)
    • Leukocyte elastase inhibitor (serpin B1) (IPR015557)

Description

Leukocyte elastase inhibitor is also known as serpin B1. Serpins (SERine Proteinase INhibitors) belong to MEROPS inhibitor family I4 (clan ID)
[PMID: 14705960].

Serpin B1 regulates the activity of neutrophil serine proteases such as elastase, cathepsin G and proteinase-3 and may play a regulatory role to
limit inflammatory damage due to proteases of cellular origin [PMID: 11747453]. It also functions as a potent intracellular inhibitor of granzyme
H [PMID: 23269243]. In mouse, four different homologues of human serpin B1 have been described [PMID: 12189154].

 

The neutrophil serine protease inhibitor SerpinB1 protects against inflammatory lung injury and morbidity in influenza virus infection

Dapeng Gong1,2, Charaf Benarafa1,2, Kevan L Hartshorn3 and Eileen Remold-O’Donnell1,2
J Immunol April 2009; 182(Meeting Abstract Supplement) 43.10
http://www.jimmunol.org/cgi/content/meeting_abstract/182/1_MeetingAbstracts/43.10

SerpinB1 is an efficient inhibitor of neutrophil serine proteases. SerpinB1-/- mice fail to clear bacterial lung infection with increased inflammation and neutrophil death. Here, we investigated the role of serpinB1 in influenza virus infection, where infiltrating neutrophils and monocytes facilitate virus clearance but can also cause tissue injury. Influenza virus (H3N2 A/Phil/82) infection caused greater and more protracted body weight loss in serpinB1-/- vs. WT mice (20% vs. 15%; nadir on day 4 vs. day 3). Increased morbidity was not associated with defective virus clearance. Cytokines (IFN, TNF, IL-17, IFN, G-CSF) and chemokines (MIP-1, KC, MIP-2) were increased in serpinB1-/- mice vs. WT on days 2-7 post-infection but not on day 1. In WT mice, histology indicated large infiltration of neutrophils peaking on day 1 and maximal airway injury on day 2 that resolved on day 3 coincident with the influx of monocytes/macrophages. In serpinB1-/- mice, neutrophils also peaked on day 1; epithelial injury was severe and sustained with accumulation of dead cells on day 2 and 3. Immunophenotyping of lung digests on day 2 and 3 showed delayed recruitment of monocytes, macrophages and DC in serpinB1-/- mice, but increase of activated CD4 (day 2-3) and CD8 (day 3) T cells. Our findings demonstrate that serpinB1 protects against morbidity and inflammatory lung injury associated with influenza infection.

 

The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosainfection

Charaf Benarafa 1 , 2 Gregory P. Priebe 3 , 4 , and Eileen Remold-O’Donnell 1 , 2
JEM July 30, 2007; 204(8): 1901-1909   http://dx.doi.org:/10.1084/jem.20070494

Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1−/− mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein–D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance inserpinb1−/− mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection.

 

Neutrophils are the first and most abundant phagocytes mobilized to clear pathogenic bacteria during acute lung infection. Prominent among their antimicrobial weapons, neutrophils carry high concentrations of a unique set of serine proteases in their granules, including neu trophil elastase (NE), cathepsin G (CG), and proteinase-3. These neutrophil serine proteases (NSPs) are required to kill phagocytosed bacteria and fungi (12). Indeed, neutrophils lacking NE fail to kill phagocytosed pathogens, and mice deficient for NE and/or CG have increased mortality after infection with pulmonary pathogens (34). However, NSPs in the lung airspace can have a detrimental effect in severe inflammatory lung disease through degradation of host defense and matrix proteins (57). Thus, understanding of the mechanisms that regulate NSP actions during lung infections associated with neutrophilia will help identify strategies to balance host defense and prevent infection-induced tissue injury.

 

SERPINB1, also known as monocyte NE inhibitor (8), is an ancestral serpin super-family protein and one of the most efficient inhibitors of NE, CG, and proteinase-3 (910). SERPINB1 is broadly expressed and is at particularly high levels in the cytoplasm of neutrophils (1112). SERPINB1 has been found complexed to neutro phil proteases in lung fluids of cystic fibrosis patients and in a baboon model of bronchopulmonary dysplasia (1314). Although these studies suggest a role for SERPINB1 in regulating NSP activity, it is unclear whether these complexes reflect an important physiological role for SERPINB1 in the lung air space.

RESULTS

To define the physiological importance of SERPINB1 in shaping the outcome of bacterial lung infection, we generated mice deficient for serpinb1 (serpinb1−/−) by targeted mutagenesis in embryonic stem (ES) cells (Fig. 1, A–C). Crossings of heterozygous mice produced WT (+/+), heterozygous (+/−), and KO (−/−) mice for serpinb1 at expected Mendelian ratios (25% +/+, 51% +/−, and 24% −/−; n = 225; Fig. 1 D), indicating no embryonic lethality. Bone marrow neutrophils of serpinb1−/− mice lacked expression of the protein, whereas heterozygous serpinb1+/− mice had reduced levels compared with WT mice (Fig. 1 E). Importantly, levels of the cognate neutrophil proteases NE and CG, measured as antigenic units, were not altered by deletion of serpinb1 (Fig. 1 F). When maintained in a specific pathogen-free environment, serpinb1−/− mice did not differ from WT littermates in growth, litter size, or life span (followed up to 12 mo), and no gross or histopathological defects were observed at necropsy in 8-wk-old mice.

6–8-wk-old animals were intranasally inoculated with the nonmucoid Pseudomonas aeruginosa strain PAO1. Using two infection doses (3 × 106 and 7 × 106 CFU/mouse),serpinb1−/− mice had a significantly lower survival probability and a shorter median survival time compared with WT mice (Fig. 2 A). Further groups of infected mice were used to evaluate bacterial clearance. At 6 h after infection, the bacteria were similarly restricted in mice of the two genotypes, suggesting that the serpinb1−/− mice have a normal initial response to infection. At 24 h, the median bacterial count in the lungs of serpinb1−/− mice was five logs higher than that of the WT mice (P < 0.001), and the infection had spread systemically in serpinb1−/− mice but not in WT mice, as shown by high median CFU counts in the spleen (Fig. 2 B). Histological examination at 24 h after infection revealed abundant neutrophil infiltration in the lungs of both WT and serpinb1−/− mice, and consistent with the bacteriological findings, numerous foci of bacterial colonies and large areas of alveolar exudates were found in serpinb1−/− mice only (Fig. 2 C). When challenged with the mucoid P. aeruginosa clinical strain PA M57-15 isolated from a cystic fibrosis patient, WT mice cleared >99.9% of the inoculum within 24 h, whereas serpinb1-deficient mice failed to clear the infection (Fig. 2 D). Thus, the NSP inhibitor serpinb1 is essential for maximal protection against pneumonia induced by mucoid and nonmucoid strains of P. aeruginosa.

Figure 2.

Serpinb1−/− mice fail to clear P. aeruginosalung infection. (A) Kaplan-Meier survival curves of WT (+/+) and serpinb1-deficient (−/−) mice intranasally inoculated with nonmucoid P. aeruginosa strain PAO1. Increased mortality of serpinb1−/− mice was statistically significant (P = 0.03 at 3 × 106CFU/mouse; P < 0.0001 at 7 × 106CFU/mouse). (B) CFUs per milligram of lung (left) and splenic (right) tissue determined 6 and 24 h after inoculation with 3 × 106 CFUP. aeruginosa PAO1 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Each symbol represents a value for an individual mouse. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test. Data below the limit of detection (dotted line) are plotted as 0.5 CFU × dilution factor. (C) Lung sections stained with hematoxylin and eosin show bacterial colonies (arrowheads) and alveolar exudate in lungs of serpinb1−/− mice 24 h after infection with P. aeruginosa PAO1. Bars, 50 μm. (D) Total CFUs in the lung and spleen 24 h after inoculation with 2 × 108 CFU of the mucoid P. aeruginosa strain PA M57-15 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test.

To verify specificity of the gene deletion, we tested whether delivering rSERPINB1 would correct the defective phenotype. Indeed, intranasal instillation of rSERPINB1 to serpinb1−/− mice at the time of inoculation significantly improved clearance of P. aeruginosa PAO1 from the lungs assessed at 24 h and reduced bacteremia compared with infectedserpinb1−/− mice that received PBS instead of the recombinant protein (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20070494/DC1). We have previously demonstrated that rSERPINB1 has no effect on the growth of P. aeruginosa in vitro (15) and does not induce bacterial aggrega tion (16). Also, rSERPINB1 mixed with PAO1 had no effect on adherence of the bacteria to human bronchial epithelial and corneal epithelial cell lines (unpublished data). Therefore, the improved bacterial clearance in treated serpinb1−/− mice is not related to a direct antibacterial role for rSERPINB1 but rather to reducing injury induced by excess neutrophil proteases. In addition, previous in vivo studies in WT rats showed that rSERPINB1 can protect against elastase-induced lung injury (17) and accelerate bacterial clearance two- to threefold in the Pseudomonas agar bead model (15).

Evidence of excess NSP action was examined in the lungs of infected serpinb1−/− mice by measuring surfactant protein–D (SP-D). SP-D, a multimeric collagenous C-type lectin produced by alveolar epithelial cells, is highly relevant as a host defense molecule, because it functions as an opsonin in microbial clearance (18) and acts on alveolar macrophages to regulate pro- and antiinflammatory cytokine production (19). SP-D is also relevant as an NSP target because it is degraded in vitro by trace levels of each of the NSPs (1620). SP-D levels in lung homogenates of WT and serpinb1−/− mice were similar 6 h after P. aeruginosa infection. At 24 h, SP-D levels were reduced in the lungs ofserpinb1−/− mice compared with WT mice, as indicated by immunoblots. A lower molecular mass band indicative of proteolytic degradation is also apparent (Fig. 3 A). Densitometry analysis of the 43-kD SP-D band relative to β-actin indicated that the reduction of SP-D level was statistically significant (+/+, 45 ± 6 [n = 8]; −/−, 10 ± 2 [n = 8]; P < 0.0001 according to the Student’s t test). Furthermore, rSERPINB1 treatment ofP. aeruginosa–infected serpinb1−/− mice partly prevented the degradation of SP-D in lung homogenates compared with nontreated mice (Fig. S1 B). As a further test of the impact of serpinb1 deletion on NSP activity, isolated neutrophils of serpinb1−/− mice were treated with LPS and FMLP and tested for their ability to cleave recombinant rat SP-D (rrSP-D) in vitro. The extent of rrSP-D cleavage by serpinb1−/− neutrophils was fourfold greater than by WT neutrophils, as determined by densitometry. The cleavage was specific for NSPs because it was abrogated by rSERPINB1 and diisopropyl fluorophosphate (Fig. 3 B). Collectively, these findings indicate a direct role for serpinb1 in regulating NSP activity released by neutrophils and in preserving SP-D, an important-host defense molecule.

Efficient clearance of P. aeruginosa infection requires an early cytokine and chemokine response coordinated by both resident alveolar macrophages and lung parenchymal cells (2122). The IL-8 homologue keratinocyte-derived chemokine (KC) and the cytokines TNF-α, IL-1β, and G-CSF were measured in cell-free bronchoalveolar (BAL) samples. Although the tested cytokines were undetectable in sham-infected mice of both genotypes (unpublished data), comparable induc tion of these cytokines was observed in BAL of WT and serpinb1−/− mice at 6 h after infection, demonstrating that there is no early defect in cytokine production in serpinb1−/− mice. At 24 h, levels of TNF-α, KC, and IL-1β were sustained or increased in serpinb1−/− mice and significantly higher than cytokine levels in WT mice. G-CSF levels at 24 h were elevated to a similar extent in BAL of WT and KO mice (Fig. 3 C). However, G-CSF levels were significantly higher in the serum of serpinb1−/− mice (WT, 336 ± 80 ng/ml; KO, 601 ± 13 ng/ml; n = 6 of each genotype; P < 0.01). In addition, serpinb1−/− mice that were treated at the time of infection with rSERPINB1 had cytokine levels in 24-h lung homogenates that were indistinguishable from those of infected WT mice (Fig. S1 C). The increased cytokine production in the lungs of infected serpinb1−/− mice may be caused by failed bacterial clearance but also by excess NSPs, which directly induce cytokine and neutrophil chemokine production in pulmonary parenchymal cells and alveolar macrophages (2324).

Neutrophil recruitment to the lungs was next examined as a pivotal event of the response to P. aeruginosa infection (25). Lung homogenates were assayed for the neutrophil-specific enzyme myeloperoxidase (MPO) to quantify marginating, interstitial, and alveolar neutrophils. Neutrophils in BAL fluid were directly counted as a measure of neutrophil accumulation in the alveolar and airway lumen. MPO in lung homo genates was undetectable in uninfected mice and was comparably increased in mice of both genotypes at 6 h, suggesting normal early serpinb1−/− neutrophil margination and migration into the interstitium. However, by 24 h after infection, MPO levels in lung homogenates remained high in WT mice but were significantly decreased in serpinb1−/− mice (Fig. 4 A). Importantly, the content of MPO per cell was the same for isolated neutrophils of WT andserpinb1−/− mice (+/+, 369 ± 33 mU/106 cells; −/−, 396 ± 27 mU/106 cells). The numbers of neutrophils in BAL were negligible in uninfected mice and were similarly increased in WT and serpinb1−/− mice at 6 h after infection. Neutrophil counts in BAL further increased at 24 h, but the mean BAL neutrophil numbers were significantly lower in serpinb1−/− mice compared with WT mice (Fig. 4 B). The evidence from the 6-h quantitation of MPO in homogenates and neutrophils in BAL strongly suggests that neutrophil recruitment is not defective in infected serpinb1−/− mice. Moreover, the high levels of cytokines and neutrophil chemoattractant KC in serpinb1−/− mice at 24 h (Fig. 3 C) also suggest that, potentially, more neutrophils should be recruited. Therefore, to examine neutrophil recruitment in serpinb1−/− mice, we used a noninfectious model in which neutrophils are mobilized to migrate to the lung after intranasal delivery of P. aeruginosa LPS. MPO levels in lung homogenate and neutrophil numbers in BAL were not statistically different in WT and serpinb1−/− mice 24 h after LPS instillation (Fig. 4, C and D). Furthermore, the number of circulating blood neutrophils and recruited peritoneal neutrophils after injection of sterile irritants glycogen and thioglycollate did not differ in WT and serpinb1−/− mice (unpublished data). Alveolar macrophage numbers were similar in uninfected mice of both genotypes (∼5 × 105 cells/mouse) and did not substantially change upon infection. Collectively, these findings show that neutrophil recruitment to the lungs in response to P. aeruginosa infection is not defective in serpinb1−/− mice, and therefore, the recovery of lower numbers of serpinb1−/− neutrophils at 24 h after infection suggests their decreased survival.

To examine the putative increased death of serpinb1−/− neutrophils in the lungs after P. aeruginosa infection, lung sections were analyzed by immunohistochemistry. Caspase-3–positive leukocytes were more relevant in the alveolar space of serpinb1−/− mice compared with WT mice at 24 h after infection, suggesting increased neutrophil apoptosis (Fig. 5 A). The positive cells were counted in 50 high power fields (hpf’s), and mean numbers of caspase-3–stained cells were increased in the lungs of serpinb1/− mice (1.8 ± 0.2 cells/hpf) compared with WT mice (0.4 ± 0.1 cells/hpf; P < 0.0001). To characterize neutrophils in the alveoli and airways, neutrophils in BAL were identified in flow cytometry by forward scatter (FSC) and side scatter and were stained with annexin V (AnV) and propidium iodide (PI). At 24 h after infection, the proportion of late apoptotic/necrotic neutrophils (AnV+PI+) was increased at the expense of viable neutrophils (AnVPI) in the BAL of serpinb1−/− mice compared with WT mice (Fig. 5 B). Neutrophil fragments in BAL were also identified in flow cytometry by low FSC (FSClow) within the neutrophil population defined by the neutrophil marker Gr-1. The number of neutrophil fragments (FSClow, Gr-1+) relative to intact neutrophils was increased two- to threefold at 24 h after infection for serpinb1−/− compared with WT mice (Fig. 5 C). Moreover, free MPO in BAL supernatants was increased in serpinb1−/− mice compared with WT mice at 24 h after infection, indicating increased PMN lysis or degranulation (Fig. 5 D).

Finally, we questioned whether the enhanced death of serpinb1−/− pulmonary neutrophils was a primary effect of gene deletion or a secondary effect caused by, for example, bacteria or components of inflammation. To address this, neutrophils were collected using the noninfectious LPS recruitment model and were cultured in vitro to allow for spontaneous cell death. After 24 h, the percentages of apoptotic and necrotic neutrophils evaluated by microscopy were increased in serpinb1−/− neutrophils compared with WT neutrophils (Fig. 6, A–C). A similar increase in apoptotic cells was observed using AnV/PI staining and measurements of hypodiploid DNA (unpublished data). Moreover, live cell numbers from serpinb1−/− mice remaining in culture after 24 h were significantly decreased compared with WT mice (Fig. 6 D). The in vitro findings indicate that enhanced death of pulmonary neutrophils of infected serpinb1−/− mice is at least in part a cell-autonomous defect likely mediated by unchecked NSP actions.

 

In this paper, we have demonstrated that serpinb1, an intracellular serpin family member, regulates the innate immune response and protects the host during lung bacterial infection. Serpinb1 is among the most potent inhibitors of NSPs and is carried at high levels within neutrophils. Serpinb1-deficient mice fail to clear P. aeruginosa PAO1 lung infection and succumb from systemic bacterial spreading. The defective immune function in serpinb1−/− mice stems at least in part from an increased rate of neutrophil necrosis, reducing the number of phagocytes and leading to increased NSP activity in the lungs with proteolysis of SP-D. In addition, serpinb1-deficient mice also have impaired clearance of the mucoid clinical strain PA M57-15. Interestingly, mucoid strains of P. aeruginosa are cleared with a very high efficiency from the lungs of WT and cystic fibrosis transmembrane conductance regulator–deficient mice (26). The phenotype of serpinb1−/− mice reproduces major pathologic features of human pulmonary diseases characterized by excessive inflammation, massive neutrophil recruitment to the air space, and destruction of cellular and molecular protective mechanisms. Importantly, serpinb1 deficiency may be helpful as an alternative or additional model of the inflammatory lung pathology of cystic fibrosis.

The present study documents a key protective role for serpinb1 in regulating NSP actions in the lung. This role has previously been attributed to the NSP inhibitors α1-antitrypsin and secretory leukocyte protease inhibitor, which are found in the airway and alveolar lining fluid (2728). However, patients with α1-antitrypsin deficiency do not present with pulmonary infection secondary to innate immune defects despite increased NSP activity that leads to reduced lung elasticity and emphysema. Moreover, there is so far no evidence that deficiency in secretory leukocyte protease inhibitor results in failure to clear pulmonary infection. Because synthesis and storage of NSPs in granules is an event that exclusively takes place in bone marrow promyelocytes (29), the regulation of NSPs in the lung relies entirely on NSP inhibitors. Thus, the extent of the innate immune defect inserpinb1−/− mice and the normalization of bacterial clearance with topical rSERPINB1 treatment indicate that serpinb1 is required to regulate NSP activity in the airway fluids and that, during acute lung infection associated with high neutrophilic recruitment, there is insufficient compensation by other NSP inhibitors. The devastating effects of NSPs when released in the lungs by degranulating and necrotic neutrophils are well documented in human pulmonary diseases (5630). Therefore, our findings clearly establish a physiological and nonredundant role for serpinb1 in regulating NSPs during pulmonary infection.

NSPs also cleave molecules involved in apoptotic cell clearance, including the surfactant protein SP-D and the phosphatidylserine receptor on macrophages (3132), thereby tipping the balance further toward a detrimental outcome. The increased numbers of leukocytes with active caspase-3 in the alveolar space of P. aeruginosa–infectedserpinb1−/− mice suggest that the removal of apoptotic cells may be inadequate during infection. SP-D has been shown to stimulate phagocytosis of P. aeruginosa by alveolar macrophages in vitro (33), and SP-D–deficient mice were found to have defective early (6-h) clearance of P. aeruginosa from the lung (34). Although the destruction of SP-D alone may not entirely account for the defective phenotype of serpinb1−/− mice, loss of SP-D likely diminishes bacterial clearance and removal of apop totic neutrophils.

Given that NSPs also mediate bacterial killing, why would NSP excess lead to a failed bacterial clearance? In the NE KO mice, the decreased killing activity of neutrophils is a direct consequence of the loss of the bactericidal activity of NE. The absence of an early bacterial clearance defect at 6 h after infection in serpinb1−/− mice suggests that there is initially normal bacterial killing. The current understanding is that the compartmentalization of the NSPs is crucial to the outcome of their actions: on the one hand, NSPs are protective when killing microbes within phagosomes, and on the other hand, extracellular NSPs destroy innate immune defense molecules such as lung collectins, immunoglobulins, and complement receptors. We have shown that the regulation of NSP activity is essential and that cytoplasmic serpinb1 provides this crucial shield. Neutrophils undergoing cell death gradually transition from apoptosis, characterized by a nonpermeable plasma membrane, to necrosis and lysis, where cellular and granule contents, including NSPs, are released. The increased pace of serpinb1−/− neutrophil cell death strongly suggests that unopposed NSPs may precipitate neutrophil demise and, therefore, reduce the neutrophil numbers leading to a late-onset innate immune defect. High levels of G-CSF, a prosurvival cytokine for neutrophils, also indicate that increased cell death is likely independent or downstream of G-CSF.

In conclusion, serpinb1 deficiency unleashes unbridled proteolytic activity during inflammation and thereby disables two critical components of the host response to bacterial infection, the neutrophil and the collectin SP-D. The phenotype of the infectedserpinb1-deficient mouse, characterized by a normal early antibacterial response that degenerates over time, highlights the delicate balance of protease–antiprotease systems that protect the host against its own defenses as well as invading microbes during infection-induced inflammation.

 

 

Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin

K Kessenbrock,1 LFröhlich,2 M Sixt,3 …., A Belaaouaj,5 J Ring,6,7 M Ollert,6 R Fässler,3 and DE. Jenne1
J Clin Invest. 2008 Jul 1; 118(7): 2438–2447.   http://dx.doi.org:/10.1172/JCI34694

Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.

 

Neutrophils belong to the body’s first line of cellular defense and respond quickly to tissue injury and invading microorganisms (1). In a variety of human diseases, like autoimmune disorders, infections, or hypersensitivity reactions, the underlying pathogenic mechanism is the formation of antigen-antibody complexes, so-called immune complexes (ICs), which trigger an inflammatory response by inducing the infiltration of neutrophils (2). The subsequent stimulation of neutrophils by C3b-opsonized ICs results in the generation of ROS and the release of intracellularly stored proteases leading to tissue damage and inflammation (3). It is therefore important to identify the mechanisms that control the activation of infiltrating neutrophils.

Neutrophils abundantly express a unique set of neutrophil serine proteases (NSPs), namely cathepsin G (CG), proteinase 3 (PR3; encoded by Prtn3), and neutrophil elastase (NE; encoded by Ela2), which are stored in the cytoplasmic, azurophilic granules. PR3 and NE are closely related enzymes, with overlapping and potentially redundant substrate specificities different from those of CG. All 3 NSPs are implicated in antimicrobial defense by degrading engulfed microorganisms inside the phagolysosomes of neutrophils (48). Among many other functions ascribed to these enzymes, PR3 and NE were also suggested to play a fundamental role in granulocyte development in the bone marrow (911).

While the vast majority of the enzymes is stored intracellularly, minor quantities of PR3 and NE are externalized early during neutrophil activation and remain bound to the cell surface, where they are protected against protease inhibitors (1213). These membrane presented proteases were suggested to act as path clearers for neutrophil migration by degrading components of the extracellular matrix (14). This notion has been addressed in a number of studies, which yielded conflicting results (1517). Thus, the role of PR3 and NE in leukocyte extravasation and interstitial migration still remains controversial.

Emerging data suggest that externalized NSPs can contribute to inflammatory processes in a more complex way than by simple proteolytic tissue degradation (18). For instance, recent observations using mice double-deficient for CG and NE indicate that pericellular CG enhances IC-mediated neutrophil activation and inflammation by modulating integrin clustering on the neutrophil cell surface (1920). Because to our knowledge no Prtn3–/– mice have previously been generated, the role of this NSP in inflammatory processes has not been deciphered. Moreover, NE-dependent functions that can be compensated by PR3 in Ela2–/–animals are still elusive.

One mechanism by which NSPs could upregulate the inflammatory response has recently been proposed. The ubiquitously expressed progranulin (PGRN) is a growth factor implicated in tissue regeneration, tumorigenesis, and inflammation (2123). PGRN was previously shown to directly inhibit adhesion-dependent neutrophil activation by suppressing the production of ROS and the release of neutrophil proteases in vitro (23). This antiinflammatory activity was degraded by NE-mediated proteolysis of PGRN to granulin (GRN) peptides (23). In contrast, GRN peptides may enhance inflammation (23) and have been detected in neutrophil-rich peritoneal exudates (24). In short, recent studies proposed PGRN as a regulator of the innate immune response, but the factors that control PGRN function are still poorly defined and its relevance to inflammation needs to be elucidated in vivo.

In the present study, we generated double-deficient Prtn3–/–Ela2–/– mice to investigate the role of these highly similar serine proteases in noninfectious neutrophilic inflammation. We established that PR3 and NE are required for acute inflammation in response to subcutaneous IC formation. The proteases were found to be directly involved in early neutrophil activation events, because isolated Prtn3–/–Ela2–/– neutrophils were poorly activated by ICs in vitro. These defects in Prtn3–/–Ela2–/– mice were accompanied by accumulation of PGRN. We demonstrated that PGRN represents a potent inflammation-suppressing factor that is cleaved by both PR3 and NE. Our data delineate what we believe to be a previously unknown proinflammatory role for PR3 and NE, which is accomplished via the local inactivation of antiinflammatory PGRN.

 

Generation of Prtn3–/–Ela2–/– mice.

To analyze the role of PR3 and NE in neutrophilic inflammation, we generated a Prtn3–/–Ela2–/– mouse line by targeted gene disruption in embryonic stem cells (see Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI34694DS1). Positive recombination of the Prtn3/Ela2locus was proven by Southern blotting of embryonic stem cell clones (Figure ​(Figure1A).1A). Prtn3–/–Ela2–/– mice showed no expression of mRNA for PR3 and NE in bone marrow cells, as assessed by RT-PCR (Figure ​(Figure1B).1B). The successful elimination of PR3 and NE was confirmed at the level of proteolytic activity in neutrophil lysates using a PR3/NE-specific chromogenic substrate (Supplemental Figure 3) as well as by casein zymography (Figure ​(Figure1C).1C). The substantially reduced casein degradation by heterozygous neutrophils indicates gene-dosage dependence of PR3/NE activities. Furthermore, PR3 and NE deficiency was proven by Western blotting using cell lysates from bone marrow–derived neutrophils, while other enzymes stored in azurophilic granula, such as CG and myeloperoxidase (MPO), were normally detected (Figure ​(Figure1D).1D). Crossing of heterozygous Prtn3+/–Ela2+/– mice resulted in regular offspring of WT, heterozygous, and homozygous genotype according to the Mendelian ratio. Despite the absence of 2 abundant serine proteases, and in contrast to expectations based on previous reports (911), we found unchanged neutrophil morphology (Figure ​(Figure1E)1E) and regular neutrophil populations in the peripheral blood of the mutant mice, the latter as assessed via flow cytometry to determine the differentiation markers CD11b and Gr-1 (Figure ​(Figure1F)1F) (2526). Moreover, Prtn3–/–Ela2–/– mice demonstrated normal percentages of the leukocyte subpopulations in the peripheral blood, as determined by the Diff-Quick staining protocol and by hemocytometric counting (Supplemental Figure 2, A and B). Hence, the proteases are not crucially involved in granulopoiesis, and ablating PR3 and NE in the germ line represents a valid approach to assess their biological significance in vivo.

 

Figure 1

Generation and characterization of Prtn3–/–Ela2–/– mice.

PR3 and NE are dispensable for neutrophil extravasation and interstitial migration.

To examine neutrophil infiltration into the perivascular tissue, we applied phorbol esters (croton oil) to the mouse ears. At 4 h after stimulation, we assessed the neutrophil distribution in relation to the extravascular basement membrane (EBM) by immunofluorescence microscopy of fixed whole-mount specimens (Figure ​(Figure2A).2A). We found that Prtn3–/–Ela2–/– neutrophils transmigrated into the interstitium without retention at the EBM (Figure ​(Figure2B),2B), resulting in quantitatively normal and widespread neutrophil influx compared with WT mice (Figure ​(Figure2C).2C). Moreover, we analyzed chemotactic migration of isolated neutrophils through a 3-dimensional collagen meshwork in vitro (Supplemental Video 1) and found unhampered chemotaxis toward a C5a gradient, based on the directionality (Figure ​(Figure2D)2D) and velocity (Figure ​(Figure2E)2E) of Prtn3–/–Ela2–/–neutrophils. These findings led us to conclude that PR3 and NE are not principally required for neutrophil extravasation or interstitial migration.

 

Figure 2

PR3 and NE are not principally required for neutrophil extravasation and interstitial migration.

Reduced inflammatory response to ICs in Prtn3–/–Ela2–/– mice.

The formation of ICs represents an important trigger of neutrophil-dependent inflammation in many human diseases (2). To determine the role of PR3 and NE in this context, we induced a classic model of subcutaneous IC-mediated inflammation, namely the reverse passive Arthus reaction (RPA) (27). At 4 h after RPA induction, we assessed the cellular inflammatory infiltrates by histology using H&E-stained skin sections (Figure ​(Figure3A).3A). Neutrophils, which were additionally identified by Gr-1 immunohistochemistry, made up the vast majority of all cellular infiltrates (Figure ​(Figure3A).3A). We found that neutrophil infiltration to the sites of IC formation was severely diminished in Prtn3–/–Ela2–/– mice. Indeed, histological quantification revealed significantly reduced neutrophil influx in Prtn3–/–Ela2–/– mice compared with WT mice, while Ela2–/– mice showed marginally reduced neutrophil counts (Figure ​(Figure3B).3B). These results indicate that PR3 and NE fulfill an important proinflammatory function during IC-mediated inflammation.

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Figure 3

Impaired inflammatory response to locally formed ICs inPrtn3–/–Ela2–/– mice.

(A) Representative photomicrographs of inflamed skin sections 4 h after IC formation. Neutrophils were identified morphologically (polymorphic nucleus) in H&E stainings and by Gr-1 staining (red). The cellular infiltrates were located to the adipose tissue next to the panniculus carnosus muscle (asterisks) and were primarily composed of neutrophil granulocytes. Scale bars: 200 μm. (B) Neutrophil infiltrates in lesions from Prtn3–/–Ela2–/– mice were significantly diminished compared with Ela2–/– mice and WT mice. Neutrophil influx in Ela2–/–mice was slightly, but not significantly, diminished compared with WT mice. Results are mean ± SEM infiltrated neutrophils per HPF. *P < 0.05.

PR3 and NE enhance neutrophil activation by ICs in vitro.

PR3 and NE enhance neutrophil activation by ICs in vitro.

Because PR3 and NE were required for the inflammatory response to IC (Figure ​(Figure3),3), but not to phorbol esters (Figure ​(Figure2),2), we considered the enzymes as enhancers of the neutrophil response to IC. We therefore assessed the oxidative burst using dihydrorhodamine as a readout for cellular activation of isolated, TNF-α–primed neutrophils in the presence of ICs in vitro. While both WT and Prtn3–/–Ela2–/– neutrophils showed a similar, approximately 20-min lag phase before the oxidative burst commenced, the ROS production over time was markedly reduced, by 30%–40%, in the absence of PR3 and NE (Figure ​(Figure4A).4A). In contrast, oxidative burst triggered by 25 nM PMA was not hindered in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure4B),4B), which indicated no general defect in producing ROS. We also performed a titration series ranging from 0.1 to 50 nM PMA and found no reduction in oxidative burst activity in Prtn3–/–Ela2–/– neutrophils at any PMA concentration used (Supplemental Figure 4). These data are consistent with our in vivo experiments showing that neutrophil influx to ICs was impaired (Figure ​(Figure3),3), whereas the inflammatory response to phorbol esters was normal (Figure ​(Figure2,2, A–C), in Prtn3–/–Ela2–/– mice. To compare neutrophil priming in WT and Prtn3–/–Ela2–/–neutrophils, we analyzed cell surface expression of CD11b after 30 min of incubation at various concentrations of TNF-α and found no difference (Supplemental Figure 5). Moreover, we observed normal neutrophil adhesion to IC-coated surfaces (Supplemental Figure 6A) and unaltered phagocytosis of opsonized, fluorescently labeled E. coli bacteria (Supplemental Figure 6, B and C) in the absence of both proteases. We therefore hypothesized that PR3 and NE enhance early events of adhesion-dependent neutrophil activation after TNF-α priming and binding of ICs. It is important to note that Ela2–/– neutrophils were previously shown to react normally in the same setup (20). Regarding the highly similar cleavage specificities of both proteases, we suggested that PR3 and NE complemented each other during the process of neutrophil activation and inflammation.

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Figure 4

Impaired oxidative burst and PGRN degradation by IC-activatedPrtn3–/–Ela2–/– neutrophils.

Oxidative burst as the readout for neutrophil activation by ICs was measured over time. (A) While no difference was observed during the initial 20-min lag phase of the oxidative burst, Prtn3–/–Ela2–/– neutrophils exhibited diminished ROS production over time compared with WT neutrophils. (B) Bypassing receptor-mediated activation using 25 nM PMA restored the diminished oxidative burst of Prtn3–/–Ela2–/–neutrophils. Results are presented as normalized fluorescence in AU (relative to maximum fluorescence produced by WT cells). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate. (C) Isolated mouse neutrophils were activated by ICs in vitro and tested for PGRN degradation by IB. In the cellular fraction, the PGRN (~80 kDa) signal was markedly increased in Prtn3–/–Ela2–/–cells compared with WT and Ela2–/– neutrophils. Intact PGRN was present in the supernatant (SN) of IC-activated Prtn3–/–Ela2–/–neutrophils only, not of WT or Ela2–/– cells. (D and E) Exogenous administration of 100 nM PGRN significantly reduced ROS production of neutrophils activated by ICs (D), but not when activated by PMA (E). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate.

Antiinflammatory PGRN is degraded by PR3 and NE during IC-mediated neutrophil activation.

PGRN inhibits neutrophil activation by ICs in vitro.

Both PR3 and NE process PGRN in vitro.

Figure 5

PR3 and NE are major PGRN processing enzymes of neutrophils.

PGRN inhibits IC-mediated inflammation in vivo.

Figure 6

PGRN is a potent inhibitor of IC-stimulated inflammation in vivo.

PR3 and NE cleave PGRN during inflammation in vivo.

Finally, we aimed to demonstrate defective PGRN degradation in Prtn3–/–Ela2–/– mice during neutrophilic inflammation in vivo. For practical reasons, we harvested infiltrated neutrophils from the inflamed peritoneum 4 h after casein injection and subjected the lysates of these cells to anti-PGRN Western blot. Intact, inhibitory PGRN was detected in Prtn3–/–Ela2–/– neutrophils, but not in WT cells (Figure ​(Figure6D).6D). These data prove that neutrophilic inflammation is accompanied by proteolytic removal of antiinflammatory PGRN and that the process of PGRN degradation is essentially impaired in vivo in the absence of PR3 and NE.

 

Chronic inflammatory and autoimmune diseases are often perpetuated by continuous neutrophil infiltration and activation. According to the current view, the role of NSPs in these diseases is mainly associated with proteolytic tissue degradation after their release from activated or dying neutrophils. However, recent observations suggest that NSPs such as CG may contribute to noninfectious diseases in a more complex manner, namely as specific regulators of inflammation (18). Here, we demonstrate that PR3 and NE cooperatively fulfilled an important proinflammatory role during neutrophilic inflammation. PR3 and NE directly enhanced neutrophil activation by degrading oxidative burst–suppressing PGRN. These findings support the use of specific serine protease inhibitors as antiinflammatory agents.

Much attention has been paid to the degradation of extracellular matrix components by NSPs. We therefore expected that ablation of both PR3 and NE would cause impaired neutrophil extravasation and interstitial migration. Surprisingly, we found that the proteases were principally dispensable for these processes:Prtn3–/–Ela2–/– neutrophils migrated normally through a dense, 3-dimensional collagen matrix in vitro and demonstrated regular extravasation in vivo when phorbol esters were applied (Figure ​(Figure2).2). This finding is in agreement with recent reports that neutrophils preferentially and readily cross the EBM through regions of low matrix density in the absence of NE (28).

Conversely, we observed that PR3 and NE were required for the inflammatory response to locally formed ICs (Figure ​(Figure3).3). Even isolated Prtn3–/–Ela2–/– neutrophils were challenged in performing oxidative burst after IC stimulation in vitro (Figure ​(Figure4A),4A), showing that the proteases directly enhanced the activation of neutrophils also in the absence of extracellular matrix. However, when receptor-mediated signal transduction was bypassed by means of PMA, neutrophils from Prtn3–/–Ela2–/– mice performed normal oxidative burst (Figure ​(Figure4B),4B), indicating that the function of the phagocyte oxidase (phox) complex was not altered in the absence of PR3 and NE. These findings substantiate what we believe to be a novel paradigm: that all 3 serine proteases of azurophilic granules (CG, PR3, and NE), after their release in response to IC encounter, potentiate a positive autocrine feedback on neutrophil activation.

In contrast to CG, the highly related proteases PR3 and NE cooperate in the effacement of antiinflammatory PGRN, leading to enhanced neutrophil activation. Previous studies already demonstrated that PGRN is a potent inhibitor of the adhesion-dependent oxidative burst of neutrophils in vitro, which can be degraded by NE (23). Here, we showed that PR3 and NE play an equally important role in the regulation of PGRN function. Ela2–/– neutrophils were sufficiently able to degrade PGRN. Only in the absence of both PR3 and NE was PGRN degradation substantially impaired, resulting in the accumulation of antiinflammatory PGRN during neutrophil activation in vitro (Figure ​(Figure4C)4C) and neutrophilic inflammation in vivo (Figure ​(Figure6D).6D). Moreover, we provided in vivo evidence for the crucial role of PGRN as an inflammation-suppressing mediator, because administration of recombinant PGRN potently inhibited the neutrophil influx to sites of IC formation (Figure ​(Figure6,6, A–C). Hence, the cooperative degradation of PGRN by PR3 and NE is a decisive step for the establishment of neutrophilic inflammation.

The molecular mechanism of PGRN function is not yet completely understood, but it seems to interfere with integrin (CD11b/CD18) outside-in signaling by blocking the function of pyk2 and thus dampens adhesion-related oxidative burst even when added after the initial lag phase of oxidase activation (23). PGRN is produced by neutrophils and stored in highly mobile secretory granules (29). It was recently shown that PGRN can bind to heparan-sulfated proteoglycans (30), which are abundant components of the EBM and various cell surfaces, including those of neutrophils. Also, PR3 and NE are known to interact with heparan sulfates on the outer membrane of neutrophils, where the enzymes appear to be protected against protease inhibitors (121331). These circumstantial observations support the notion that PGRN cleavage by PR3 and NE takes place at the pericellular microenvironment of the neutrophil cell surface.

Impaired outside-in signaling most likely reduced the oxidative burst in Prtn3–/–Ela2–/– neutrophils adhering to ICs. In support of this hypothesis, we excluded an altered response to TNF-α priming (Supplemental Figure 5) as well as reduced adhesion to immobilized ICs and defective endocytosis of serum-opsonized E. coli in Prtn3–/–Ela2–/– neutrophils (Supplemental Figure 6). MPO content and processing was also unchanged in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure1D);1D); hence, the previously discussed inhibitory effect of MPO on phox activity (3233) does not appear to be stronger in neutrophils lacking PR3 and NE. Because there was no difference in the lag phase of the oxidative burst, initial IC-triggered receptor activation was probably not affected by either PRGN or PR3/NE. Our concept is consistent with all these observations and takes into account that PGRN unfolds its suppressing effects in the second phase, when additional membrane receptors, endogenous PGRN, and some PR3/NE from highly mobile intracellular pools are translocated to the cell surface. The decline and cessation of ROS production suggested to us that outside-in signaling was not sustained and that active oxidase complexes were no longer replenished in the absence of PR3 and NE. Our present findings, however, do not allow us to exclude other potential mechanisms, such as accelerated disassembly of the active oxidase complex.

 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430496/bin/JCI0834694.f7.jpg

Proposed function of PR3 and NE in IC-mediated inflammation.

TNF-α–primed neutrophils extravasate from blood vessels, translocate PR3/NE to the cellular surface, and discharge PGRN to the pericellular environment (i). During transmigration of interstitial tissues (ii), neutrophil activation is initially suppressed by relatively high pericellular levels of antiinflammatory PGRN (green shading), which is also produced locally by keratinocytes and epithelial cells of the skin. Until IC depots are reached, neutrophil activation is inhibited by PGRN. Surface receptors (e.g., Mac-1) recognize ICs, which results in signal transduction (black dotted arrow) and activation of the phox. The molecular pathway of PGRN-mediated inhibition is not completely understood, but it may interfere with integrin signaling after IC encounter (green dotted line inside the cell). Adherence of neutrophils to ICs (iii) further increases pericellular PR3 and NE activity. PR3 and NE cooperatively degrade PGRN in the early stage of neutrophilic activation to facilitate optimal neutrophil activation (red shading), resulting in sustained integrin signaling (red arrow) and robust production of ROS by the phox system. Subsequently, neutrophils release ROS together with other proinflammatory mediators and chemotactic agents, thereby enhancing the recruitment of further neutrophils and establishing inflammation (iv). In the absence of PR3/NE, the switch from inflammation-suppressing (ii) to inflammation-enhancing (iii) conditions is substantially delayed, resulting in diminished inflammation in response to ICs (iv).

 

NSPs are strongly implicated as effector molecules in a large number of destructive diseases, such as emphysema or the autoimmune blistering skin disease bullous pemphigoid (143537). Normally, PR3/NE activity is tightly controlled by high plasma levels of α1-antitrypsin. This balance between proteases and protease inhibitors is disrupted in patients with genetic α1-antitrypsin deficiency, which represents a high risk factor for the development of emphysema and certain autoimmune disorders (38). The pathogenic effects of NSPs in these diseases have so far been associated with tissue destruction by the proteases after their release from dying neutrophils. Our findings showed that PR3 and NE were already involved in much earlier events of the inflammatory process, because the enzymes directly regulated cellular activation of infiltrating neutrophils by degrading inflammation-suppressing PGRN. This concept is further supported by previous studies showing increased inflammation in mice lacking serine protease inhibitors such as SERPINB1 or SLPI (3940). Blocking PR3/NE activity using specific inhibitors therefore represents a promising therapeutic strategy to treat chronic, noninfectious inflammation. Serine protease inhibitors as antiinflammatory agents can interfere with the disease process at 2 different stages, because they attenuate both early events of neutrophil activation and proteolytic tissue injury caused by released NSPs.

 

 

 

 

Editorial: Serine proteases, serpins, and neutropenia

David C. Dale

J Leuko Biol July 2011;  90(1): 3-4   http://dx.doi.org:/10.1189/jlb.1010592

Cyclic neutropenia and severe congenital neutropenia are autosomal-dominant diseases usually attributable to mutations in the gene for neutrophil elastase orELANE. Patients with these diseases are predisposed to recurrent and life-threatening infections [1]. Neutrophil elastase, the product of the ELANE gene, is a serine protease that is synthesized and packaged in the primary granules of neutrophils. These granules are formed at the promyelocytes stage of neutrophil development. Synthesis of mutant neutrophil elastase in promyelocytes triggers the unfolded protein response and a cascade of intracellular events, which culminates in death of neutrophil precursors through apoptosis [2]. This loss of cells causes the marrow abnormality often referred to as “maturation arrest” [34].

Neutrophil elastase is one of the serine proteases normally inhibited by serpinB1. In this issue of JLB, Benarafa and coauthors [5] present their intriguing studies of serpinB1 expression in human myeloid cells and their extensive investigations ofSERPINB1−/− mice. They observed that serpinB1 expression parallels protease expression. The peak of serpinB1 expression occurs in promyelocytes. Benarafa et al. [5] found that SERPINB1−/− mice have a deficiency of postmitotic neutrophils in the bone marrow. This change was accompanied by an increase in the plasma levels of G-CSF. The decreased supply of marrow neutrophils reduced the number of neutrophils that could be mobilized to an inflammatory site. Using colony-forming cell assays, they determined that the early myeloid progenitor pool was intact. Separate assays showed that maturing myeloid cells were being lost through accelerated apoptosis of maturing neutrophils in the marrow. The authors concluded that serpinB1 is required for maintenance of a healthy reserve of marrow neutrophils and a normal acute immune response [5].

This paper provides new and fascinating insights for understanding the mechanism for neutropenia. It also suggests opportunities to investigate potential therapies for patients with neutropenia and prompts several questions. As inhibition of the activity of intracellular serine proteases is the only known function of serpinB1, the findings reported by Benarafa et al. [5] suggest that uninhibited serine proteases perturbed neutrophil production severely. The SERPINB1−/− mice used in their work have accelerated apoptosis of myeloid cells, a finding suggesting that uninhibited serine proteases or mutant neutrophil elastase perturb myelopoiesis by similar mechanisms. It is now important to determine whether the defect in the SERPINB1−/− mice is, indeed, attributable to uninhibited activity of normal neutrophil elastase, other neutrophil proteases, or another mechanism. ″Double-knockout″ studies in mice deficient in neutrophil elastase and serpinB1 might provide an answer.

This report provides evidence regarding the intracellular mechanisms for the apoptosis of myeloid cells and indicates that other studies are ongoing. The key antiapoptotic proteins, Mcl-1, Bcl-XL, and A1/Bfl-I, are apparently not involved. A more precise understanding of the mechanisms of cell death is important for development of targeted therapies for neutropenia. It is also important to discover whether only cells of the neutrophil lineage are involved or whether monocytes are also affected. In cyclic and congenital neutropenia, patients failed to produce neutrophils, but they can produce monocytes; in fact, they overproduce monocytes and have significantly elevated blood monocyte counts. Neutropenia with monocytosis is probably attributable to differences in the expression of ELANE in the two lineages. Benarafa et al. [5] reported that human bone marrow monocytes contain substantially less serpinB1 than marrow neutrophils, suggesting that the expression of serpinB1 and the serine proteases are closely coordinated.

This report shows the importance of the marrow neutrophil reserves in the normal response to infections. Compared with humans, healthy mice are always neutropenic, but they have a bigger marrow neutrophil reserve, and their mature neutrophils in the marrow and blood look like human band neutrophils. These differences are well known, but they are critical for considering the clinical inferences that can be made from this report. For example, although theSERPINB1−/− mice were not neutropenic, human SERPINB1−/− might cause neutropenia because of physiological differences between the species. If some but not all mutations in SERPINB1 cause neutropenia, we might gain a better understanding about how serpinB1 normally inhibits the neutrophil’s serine proteases.

We do not know if some or all of the mutant neutrophil elastases can be inhibited by serpinB1. We do not know whether cyclic or congenital neutropenia are attributable to defects in this interaction. However, we do know that there are chemical inhibitors of neutrophil elastase that can abrogate apoptosis of myeloid cells in a cellular model for congenital neutropenia [6]. It would be interesting to see if these chemical inhibitors can replace the natural inhibitor and normalize neutrophil production in the SERPINB1−/− mice. This would provide evidence to support use of chemical protease inhibitors as a treatment for cyclic and congenital neutropenia.

Concerns with the use of G-CSF for the treatment of cyclic and congenital neutropenia are how and why some of these patients are at risk of developing leukemia. Are the SERPINB1−/− mice with a hyperproliferative marrow and high G-CSF levels also at risk of developing myeloid leukemia?

This is a very provocative paper, and much will be learned from further studies of the SERPINB1−/− mice.

 

SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G

Mathias Baumann1,2, Christine T. N. Pham3, and Charaf Benarafa1

Blood May 9, 2013; 121(19)   http://www.bloodjournal.org/content/121/19/3900

Key Points

  • Serine protease inhibitor serpinB1 protects neutrophils by inhibition of their own azurophil granule protease cathepsin G.
  • Granule permeabilization in neutrophils leads to cathepsin G–mediated death upstream and independent of apoptotic caspases.

Abstract

Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1−/−) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia ofsB1−/− mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1−/− mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1−/− PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent L-leucyl-L-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity.

Introduction

Polymorphonuclear neutrophil (PMN) granulocytes are essential components of the innate immune response to infection. PMNs are relatively short-lived leukocytes that originate from hematopoietic stem cells in the bone marrow (BM) in a process called granulopoiesis. Granulopoiesis proceeds through a proliferative phase followed by a maturation phase. After maturation, the BM retains a large reserve of mature PMNs, which includes over 90% of the mature PMNs in the body while only a small proportion (1%-5%) is in the blood.1,2 Even in noninflammatory conditions, granulopoiesis is remarkable as >1011 PMNs are produced daily in an adult human, only to be disposed of, largely unused, a few hours later.3 There is evidence that the majority of PMNs produced never reach circulation and die within the BM.4 Congenital or acquired forms of neutropenia are associated with the highest risks of bacterial and fungal infection,5 indicating a strong evolutionary pressure to maintain granulopoiesis at high levels and sustain a large mobilizable pool of PMNs in the BM.

In steady state, PMNs die by apoptosis, a form of programmed cell death that allows for the safe disposal of aging PMNs and their potentially toxic cargo. Like in other cells, caspases participate in the initiation, amplification, and execution steps of apoptosis in PMNs.6,7 Interestingly, noncaspase cysteine proteases calpain and cathepsin D were reported to induce PMN apoptosis through activation of caspases.811 In addition, PMNs carry a unique set of serine proteases (neutrophil serine proteases [NSPs]) including elastase (NE), cathepsin G (CG), and proteinase-3 (PR3) stored active in primary granules. There is strong evidence for a role of NSPs in killing pathogens and inducing tissue injury when released extracellularly.1214 In contrast, the function of NSPs in PMN homeostasis and cell death remains elusive. In particular, no defects in granulopoiesis or PMN homeostasis have been reported in mice deficient in cathepsin G (CG−/−),15 neutrophil elastase (NE−/−),16,17 or dipeptidylpeptidase I (DPPI−/−), which lack active NSPs.18 We have recently shown that mice lacking the serine protease inhibitor serpinB1 (sB1−/−) have reduced PMN survival in the lungs following Pseudomonas infection and that these mice have a profound reduction in mature PMN numbers in the BM.19,20SerpinB1, also known as monocyte NE inhibitor, is expressed at high levels in the cytoplasm of PMNs and is one of the most potent inhibitors of NE, CG, and PR3.21,22 In this study, we tested the hypothesis that serpinB1 promotes PMN survival by inhibiting 1 or several NSPs, and we discovered a novel regulatory pathway in PMN homeostasis in vivo.

 

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Figure 1

Defective PMN reserve in BM chimera depends on serpinB1 deficiency in the hematopoietic compartment. Flow cytometry analysis of major BM leukocyte subsets of lethally irradiated mice was performed 8 to 10 weeks after BM transfer. (A) Irradiated WT (CD45.1) mice were transferred with WT (●) or sB1−/− (○) BM cells. (B) Irradiated WT (●) andsB1−/− (○) mice both CD45.2 were transferred with WT (CD45.1) BM cells. Each circle represents leukocyte numbers for 1 mouse and horizontal line indicates the median. Median subsets numbers were compared by the Mann-Whitney test (*P < .05; ***P < .001).

CG regulates neutrophil numbers in the BM

Because serpinB1 is an efficient inhibitor of NE, CG, and PR3, we then examined PMN numbers in mice deficient in 1 or several NSPs in combination with serpinB1 deletion. As expected, sB1−/− mice had significantly reduced numbers and percentage of mature PMNs in the BM compared with WT and heterozygous sB1+/− mice. In addition, PMN numbers were normal in mice deficient in either DPPI, NE, or CG (Figure 2A). DPPI is not inhibited by serpinB1 but is required for the activation of all NSPs, and no NSP activity is detectable in DPPI−/− mice.18,23 PMN counts in DPPI−/−.sB1−/− BM were significantly higher than in sB1−/− BM, suggesting that 1 or several NSPs contribute to the PMN survival defect. To examine the role of NSPs in this process, we crossed several NSP-deficient strains with sB1−/− mice. We found that NE.CG.sB1−/− mice had normal PMN numbers indicating that these NSPs play a key role in the defective phenotype of sB1−/− PMNs (Figure 2A). Furthermore, CG.sB1−/− mice showed normal PMN numbers whereasNE.sB1−/− mice retained the BM neutropenia phenotype indicating that CG, but not NE, plays a significant role in the death of sB1−/− PMNs (Figure 2A). In addition, the double-deficient NE.sB1−/− mice had significantly lower BM myelocyte numbers than sB1−/− mice while the myelocyte numbers in singly deficient NE−/− and sB1−/− BM were normal (Figure 2B). These results suggest that NE may promote myeloid cell proliferation, an activity that is revealed only when serpinB1 is absent. This complex interaction between sB1 and NE requires further investigation. On the other hand, B-cell and monocyte numbers and relative percentage in the BM were largely similar in all genotypes (supplemental Figure 2). Total numbers of blood leukocytes, erythrocytes, and platelets were normal in mice deficient in NSPs and/or serpinB1 (supplemental Figure 3). PMN numbers in blood were normal insB1−/− mice in steady state and combined deficiency of NSPs did not significantly alter these numbers (Figure 2C). Taken together, our results indicate that serpinB1 likely sustains the survival of postmitotic PMNs through its interaction with CG.

Figure 2

PMN and myelocyte numbers in BM and blood of mice deficient in NSPs and serpinB1.

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CG-mediated PMN death proceeds independent of caspase activity

Figure 4

sB1−/− PMN death mediated by CG does not require caspase activity

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Granule membrane permeabilization induces CG-mediated death in PMNs

To test whether granule disruption contributes to the serpinB1-regulated CG-dependent cell death, BM cells were treated with the lysosomotropic agent LLME. LLME accumulates in lysosomes where the acyl transferase activity of DPPI generates hydrophobic (Leu-Leu)n-OMe polymers that induce lysosomal membrane permeabilization (LMP) and cytotoxicity in granule-bearing cells such as cytotoxic T lymphocytes, NK cells, and myeloid cells.29,30

Figure 5

LMP induces CG-mediated death in PMNs

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G-CSF therapy increases sB1−/− PMN numbers via enhanced granulopoiesis

G-CSF therapy is an effective long-term treatment in many cases of severe congenital neutropenia and it is also used to prevent chemotherapy-induced febrile neutropenia by enhancing PMN production. In addition, G-CSF delays neutrophil apoptosis by differentially regulating proapoptotic and antiapoptotic factors.10 To test whether G-CSF could rescue sB1−/− PMN survival defect, WT and sB1−/− mice were treated with therapeutic doses of G-CSF or saline for 5 days and BM and blood PMNs were analyzed 24 hours after the last injection. Total counts of myelocytes and PMNs were significantly increased in the BM of treated mice compared with their respective untreated genotype controls (Figure 6A-B). The increase in myelocyte numbers was identical in G-CSF–treated WT and sB1−/− mice, indicating that G-CSF–induced granulopoiesis proceeds normally in sB1−/−myeloid progenitors (Figure 6B).

Figure 6

In vivo G-CSF therapy increases PMN numbers in BM of sB1−/− mice.

 

SerpinB1 is a member of the clade B serpins, a subfamily composed of leaderless proteins with nucleocytoplasmic localization. Clade B serpins are often expressed in cells that also carry target proteases, which led to the hypothesis that intracellular serpins protect against misdirected granule proteases and/or protect bystander cells from released proteases.31 We previously reported that deficiency in serpinB1 is associated with reduced PMN survival in the BM and at inflammatory sites.19,20 The evidence presented here demonstrates that the cytoprotective function of serpinB1 in PMNs is based on the inhibition of granule protease CG. Deficiency in CG was sufficient to rescue the defect of sB1−/− mice as illustrated by normal PMN counts in the BM of double knockout CG.sB1−/− mice. We also showed that the protease-serpin interaction occurred within PMNs. Indeed, WT PMNs had a greater survival over sB1−/− PMNs in mixed BM chimera, whereas the survival of CG.sB1−/− PMNs was similar to WT PMNs after BM transfer. SerpinB1 is an ancestral clade B serpin with a conserved specificity determining reactive center loop in all vertebrates.32 Furthermore, human and mouse serpinB1 have the same specificity for chymotrypsin-like and elastase-like serine proteases.21,22 Likewise, human and mouse CG have identical substrate specificities and the phenotype of CG−/− murine PMN can be rescued by human CG.33 Therefore, it is highly likely that the antagonistic functions of CG and serpinB1 in cellular homeostasis observed in mice can be extended to other species.

Extracellular CG was previously reported to promote detachment-induced apoptosis (anoikis) in human and mouse cardiomyocytes.34 This activity is mediated through the shedding and transactivation of epidermal growth factor receptor and downregulation of focal adhesion signaling.35,36 In our study, exogenous human CG also induced PMN death in vitro but these effects were not enhanced in sB1−/− PMNs and the neutropenia associated with serpinB1 deficiency was principally cell intrinsic. How intracellular CG induces PMN death remains to be fully investigated. However, our studies provide some indications on the potential pathways. Like other NSPs, the expression of CG is transcriptionally restricted to the promyelocyte stage during PMN development and NSPs are then stored in active form in primary azurophil granules.37 Because serpinB1 is equally efficient at inhibiting NE, CG, and PR3, it was surprising that deletion of CG alone was sufficient to achieve a complete reversal of the PMN survival defect in CG.sB1−/− mice. A possible explanation would be that CG gains access to targets more readily than other granule proteases. There is evidence that binding to serglycin proteoglycans differs between NE and CG resulting in altered sorting of NE but not CG into granules of serglycin-deficient PMNs.38 Different interactions with granule matrix may thus contribute to differential release of CG from the granules compared with other NSPs. However, because sB1−/− PMNs have similar levels of CG and NE as WT PMNs20 and because LLME-induced granule permeabilization likely releases all granule contents equally, we favor an alternative interpretation where CG specifically targets essential cellular components that are not cleaved by the other serpinB1-inhibitable granule proteases. Upon granule permeabilization, we found that CG can induce cell death upstream of caspases as well as independent of caspases. CG was previously shown to activate caspase-7 in vitro and it functions at neutral pH, which is consistent with a physiological role in the nucleocytoplasmic environment.39 Cell death induced by lysosomal/granule membrane permeabilization has previously been linked to cysteine cathepsins in other cell types. However, these proteases appear to depend on caspase activation to trigger apoptosis and they function poorly at neutral pH, questioning their potential role as regulators of cell death.40 In contrast, CG-mediated cell death is not completely blocked by caspase inhibition, which is a property reminiscent of granzymes in cytotoxic T cells.41 In fact, CG is phylogenetically most closely related to serine proteases granzyme B and H.42 Granzymes have numerous nuclear, mitochondrial, and cytoplasmic target proteins leading to cell death41 and we anticipate that this may also be the case for CG.

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G-CSF therapy is successfully used to treat most congenital and acquired neutropenia through increased granulopoiesis, mobilization from the BM, and increased survival of PMNs. Prosurvival effects of G-CSF include the upregulation of antiapoptotic Bcl-2 family members, which act upstream of the mitochondria and the activation of effector caspases. In sB1−/− mice, G-CSF levels in serum are fourfold higher than in WT mice in steady state and this is accompanied by an upregulation of the antiapoptotic Bcl-2 family member Mcl-1 in sB1−/− PMNs.19 Here, G-CSF therapy significantly increased granulopoiesis in both WT and sB1−/− mice. However, the PMN numbers in treated sB1−/− BM and blood were significantly lower than those of treated WT mice, indicating only a partial rescue of the survival defect. This is consistent with our findings that CG-mediated death can proceed independent of caspases and can thus bypass antiapoptotic effects mediated by G-CSF.

CG has largely been studied in association with antimicrobial and inflammatory functions due to its presence in PMNs.1214,49 In this context, we have previously shown that serpinB1 contributes to prevent increased mortality and morbidity associated with production of inflammatory cytokines upon infection with Pseudomonas aeruginosa and influenza A virus.20,50 In this study, we demonstrate that serpinB1 inhibition of the primary granule protease CG in PMNs is essential for PMN survival and this ultimately regulates PMN numbers in vivo. Our findings also extend the roles of CG from antimicrobial and immunoregulatory functions to a novel role in inducing cell death.

 

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

Brice KorkmazMarshall S. HorwitzDieter E. Jenne and Francis Gauthier
Pharma Rev Dec 2010; 62(4):726-759  http://dx.doi.org:/10.1124/pr.110.002733

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.

 

Human polymorphonuclear neutrophils represent 35 to 75% of the population of circulating leukocytes and are the most abundant type of white blood cell in mammals (Borregaard et al., 2005). They are classified as granulocytes because of their intracytoplasmic granule content and are characterized by a multilobular nucleus. Neutrophils develop from pluripotent stem cells in the bone marrow and are released into the bloodstream where they reach a concentration of 1.5 to 5 × 109 cells/liter. Their half-life in the circulation is only on the order of a few hours. They play an essential role in innate immune defense against invading pathogens and are among the primary mediators of inflammatory response. During the acute phase of inflammation, neutrophils are the first inflammatory cells to leave the vasculature, where they migrate toward sites of inflammation, following a gradient of inflammatory stimuli. They are responsible for short-term phagocytosis during the initial stages of infection (Borregaard and Cowland, 1997Hampton et al., 1998Segal, 2005). Neutrophils use complementary oxidative and nonoxidative pathways to defend the host against invading pathogens (Kobayashi et al., 2005).

The three serine proteases neutrophil elastase (NE1), proteinase 3 (PR3), and cathepsin G (CG) are major components of neutrophil azurophilic granules and participate in the nonoxidative pathway of intracellular and extracellular pathogen destruction. These neutrophil serine proteases (NSPs) act intracellularly within phagolysosomes to digest phagocytized microorganisms in combination with microbicidal peptides and the membrane-associated NADPH oxidase system, which produces reactive oxygen metabolites (Segal, 2005). An additional extracellular antimicrobial mechanism, neutrophil extracellular traps (NET), has been described that is made of a web-like structure of DNA secreted by activated neutrophils (Papayannopoulos and Zychlinsky, 2009) (Fig. 1). NETs are composed of chromatin bound to positively charged molecules, such as histones and NSPs, and serve as physical barriers that kill pathogens extracellularly, thus preventing further spreading. NET-associated NSPs participate in pathogen killing by degrading bacterial virulence factors extracellularly (Brinkmann et al., 2004;Papayannopoulos and Zychlinsky, 2009).

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Fig. 1.

Polymorphonuclear neutrophil. Quiescent (A) and chemically activated (B) neutrophils purified from peripheral blood. C, PMA-activated neutrophils embedded within NET and neutrophil spreading on insoluble elastin.

In addition to their involvement in pathogen destruction and the regulation of proinflammatory processes, NSPs are also involved in a variety of inflammatory human conditions, including chronic lung diseases (chronic obstructive pulmonary disease, cystic fibrosis, acute lung injury, and acute respiratory distress syndrome) (Lee and Downey, 2001Shapiro, 2002Moraes et al., 2003Owen, 2008b). In these disorders, accumulation and activation of neutrophils in the airways result in excessive secretion of active NSPs, thus causing lung matrix destruction and inflammation. NSPs are also involved in other human disorders as a consequence of gene mutations, altered cellular trafficking, or, for PR3, autoimmune disease. Mutations in the ELA2/ELANE gene encoding HNE are the cause of human cyclic neutropenia and severe congenital neutropenia (Horwitz et al., 19992007). Neutrophil membrane-bound proteinase 3 (mPR3) is the major target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA), which are associated with Wegener granulomatosis (Jenne et al., 1990). All three proteases are affected by mutation of the gene (CTSC) encoding dipeptidyl peptidase I (DPPI), which activates several granular hematopoietic serine proteases (Pham and Ley, 1999Adkison et al., 2002). Mutations of CTSC cause Papillon-Lefèvre syndrome and palmoplantar keratosis (Hart et al., 1999Toomes et al., 1999).

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Fully processed mature HNE, PR3, and CG isolated from azurophilic granules contain, respectively, 218 (Bode et al., 1986Sinha et al., 1987), 222 (Campanelli et al., 1990b), and 235 (Salvesen et al., 1987Hof et al., 1996) residues. They are present in several isoforms depending on their carbohydrate content, with apparent mass of 29 to 33 kDa upon SDS-polyacrylamide gel electrophoresis (Twumasi and Liener, 1977Watorek et al., 1993). HNE and PR3 display two sites of N-glycosylation, whereas CG possesses only one. NSPs are stored mainly in neutrophil azurophilic granules, but HNE is also localized in the nuclear envelope, as revealed by immunostaining and electron microscopy (Clark et al., 1980;Benson et al., 2003), whereas PR3 is also found in secretory vesicles (Witko-Sarsat et al., 1999a). Upon neutrophil activation, granular HNE, PR3, and CG are secreted extracellularly, although some molecules nevertheless remain at the cell surface (Owen and Campbell, 1999Owen, 2008a). The mechanism through which NSPs are sorted from the trans-Golgi network to the granules has not been completely defined, even though an intracellular proteoglycan, serglycin, has been identified as playing a role in elastase sorting and packaging into azurophilic granules (Niemann et al., 2007). Unlike HNE and CG, PR3 is constitutively expressed on the membranes of freshly isolated neutrophils (Csernok et al., 1990Halbwachs-Mecarelli et al., 1995). Stimulation of neutrophils at inflammatory sites triggers intracytoplasmic granules to translocate to the phagosomes and plasma membrane, thereby liberating their contents. The first step of the translocation to the target membrane depends on cytoskeleton remodeling and microtubule assembly (Burgoyne and Morgan, 2003). This is followed by a second step of granule tethering and docking, which are dependent on the sequential intervention of SNARE proteins (Jog et al., 2007).

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Exposure of neutrophils to cytokines (TNF-α), chemoattractants (platelet-activating factor, formyl-Met-Leu-Phe, or IL-8), or bacterial lipopolysaccharide leads to rapid granule translocation to the cell surface with secretion of HNE, PR3, and CG into the extracellular medium (Owen and Campbell, 1999). A fraction of secreted HNE, PR3, and CG is detected at the surface of activated neutrophils (Owen et al., 1995a1997Campbell et al., 2000). Resting purified neutrophils from peripheral blood express variable amounts of PR3 on their surface. A bimodal, apparently genetically determined, distribution has been observed with two populations of quiescent neutrophils that express or do not express the protease at their surface (Halbwachs-Mecarelli et al., 1995Schreiber et al., 2003). The percentage of mPR3-positive neutrophils ranges from 0 to 100% of the total neutrophil population within individuals. Furthermore, the percentage of mPR3-positive neutrophils remains stable over time and is not affected by neutrophil activation (Halbwachs-Mecarelli et al., 1995).

The mechanism through which HNE and CG are associated with the outer surface of the plasma membrane of neutrophils mainly involves electrostatic interactions with the sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans (Campbell and Owen, 2007). These two proteases are released from neutrophil cell surfaces by high concentrations of salt (Owen et al., 1995b1997;Korkmaz et al., 2005a) and after treatment with chondroitinase ABC and heparinase (Campbell and Owen, 2007). Membrane PR3 is not solubilized by high salt concentrations, which means that its membrane association is not charge dependant (Witko-Sarsat et al., 1999aKorkmaz et al., 2009). Unlike HNE and CG, PR3 bears at its surface a hydrophobic patch formed by residues Phe166, Ile217, Trp218, Leu223, and Phe224 that is involved in membrane binding (Goldmann et al., 1999Hajjar et al., 2008) (Fig. 3B). Several membrane partners of PR3 have been identified, including CD16/FcγRIIIb (David et al., 2005Fridlich et al., 2006), phospholipid scramblase-1, a myristoylated membrane protein with translocase activity present in lipid rafts (Kantari et al., 2007), CD11b/CD18 (David et al., 2003), and human neutrophil antigen NB1/CD177 (von Vietinghoff et al., 2007Hu et al., 2009), a 58- to 64-kDa glycosyl-phosphatidylinositol anchored surface receptor belonging to the urokinase plasminogen activator receptor superfamily (Stroncek, 2007). NB1 shows a bimodal distribution that superimposes with that of PR3 on purified blood neutrophils (Bauer et al., 2007). Active, mature forms of PR3 but not pro-PR3 can bind to the surface of NB1-transfected human embryonic kidney 293 cells (von Vietinghoff et al., 2008) and Chinese hamster ovary cells (Korkmaz et al., 2008b). Interaction involves the hydrophobic patch of PR3 because specific amino acid substitutions disrupting this patch in the closely related gibbon PR3 prevent binding to NB1-transfected cells (Korkmaz et al., 2008b). Decreased interaction of pro-PR3 with NB1-transfected cells is explained by the topological changes affecting the activation domain containing the hydrophobic patch residues. Together, these results support the hydrophobic nature of PR3-membrane interaction.

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Roles in Inflammatory Process Regulation

NSPs are abundantly secreted into the extracellular environment upon neutrophil activation at inflammatory sites. A fraction of the released proteases remain bound in an active form on the external surface of the plasma membrane so that both soluble and membrane-bound NSPs are able to proteolytically regulate the activities of a variety of chemokines, cytokines, growth factors, and cell surface receptors. Secreted proteases also activate lymphocytes and cleave apoptotic and adhesion molecules (Bank and Ansorge, 2001Pham, 2006Meyer-Hoffert, 2009). Thus, they retain pro- and anti-inflammatory activities, resulting in a modulation of the immune response at sites of inflammation.

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Processing of Cytokines, Chemokines, and Growth Factors.

Processing and Activation of Cellular Receptors.

Induction of Apoptosis by Proteinase 3.

Physiological Inhibitors of Elastase, Proteinase 3, and Cathepsin G

During phagocytosis and neutrophil turnover, HNE, PR3, and CG are released into the extracellular space as active proteases. The proteolytic activity of HNE, PR3, and CG seems to be tightly regulated in the extracellular and pericellular space to avoid degradation of connective tissue proteins including elastin, collagen, and proteoglycans (Janoff, 1985). Protein inhibitors that belong to three main families, the serpins, the chelonianins, and the macroglobulins, ultimately control proteolytic activity of HNE, PR3, and CG activities. The individual contributions of these families depend on their tissue localization and that of their target proteases. The main characteristics of HNE, PR3, and CG physiological inhibitors are presented in Table 2.

 

Serine Protease Inhibitors

Serpins are the largest and most diverse family of protease inhibitors; more than 1000 members have been identified in human, plant, fungi, bacteria, archaea, and certain viruses (Silverman et al., 2001Mangan et al., 2008). They share a similar highly conserved tertiary structure and similar molecular weight of approximately 50 kDa. Human serpins belong to the first nine clades (A–I) of the 16 that have been described based on phylogenic relationships (Irving et al., 2000Silverman et al., 2001Mangan et al., 2008). For historical reasons, α1-protease inhibitor (α1-PI) was assigned to the first clade. Clade B, also known as the ov-serpin clan because of the similarity of its members to ovalbumin (a protein that belongs to the serpin family but lacks inhibitory activity), is the second largest clan in humans, with 15 members identified so far. Ov-serpin clan members are generally located in the cytoplasm and, to a lesser extent, on the cell surface and nucleus (Remold-O’Donnell, 1993).

Serpins play important regulatory functions in intracellular and extracellular proteolytic events, including blood coagulation, complement activation, fibrinolysis, cell migration, angiogenesis, and apoptosis (Potempa et al., 1994). Serpin dysfunction is known to contribute to diseases such as emphysema, thrombosis, angioedema, and cancer (Carrell and Lomas, 1997Lomas and Carrell, 2002). Most inhibitory serpins target trypsin-/chymotrypsin-like serine proteases, but some, termed “cross-class inhibitors,” have been shown to target cysteine proteases (Annand et al., 1999). The crystal structure of the prototype plasma inhibitor α1-PI revealed the archetype native serpin fold (Loebermann et al., 1984). All serpins typically have three β-sheets (termed A, B, and C) and eight or nine α-helices (hA–hI) arranged in a stressed configuration. The so-called reactive center loop (RCL) of inhibitory molecules determines specificity and forms the initial encounter complex with the target protease (Potempa et al., 1994Silverman et al., 2001). Serpins inhibit proteases by a suicide substrate inhibition mechanism. The protease initially recognizes the serpin as a potential substrate using residues of the reactive center loop and cleaves it between P1 and P1′ This cleavage allows insertion of the cleaved RCL into the β-sheet A of the serpin, dragging the protease with it and moving it over 71 Å to the distal end of the serpin to form a 1:1 stoichiometric covalent inhibitory complex (Huntington et al., 2000). Such cleavage generates a ∼4-kDa C-terminal fragment that remains noncovalently bound to the cleaved serpin. Displacement of the covalently attached active site serine residue from its catalytic partner histidine explains the loss of catalytic function in the covalent complex. The distortion of the catalytic site structure prevents the release of the protease from the complex, and the structural disorder induces its proteolytic inactivation (Huntington et al., 2000). Covalent complex formation between serpin and serine proteases triggers a number of conformational changes, particularly in the activation domain loops of the bound protease (Dementiev et al., 2006).

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Pathophysiology of Elastase, Proteinase 3 and Cathepsin G in Human Diseases

In many instances, the initiation and propagation of lung damage is a consequence of an exaggerated inappropriate inflammatory response, which includes the release of proteases and leukocyte-derived cytotoxic products (Owen, 2008b;Roghanian and Sallenave, 2008). Inflammation is a physiological protective response to injury or infection consisting of endothelial activation, leukocyte recruitment and activation, vasodilation, and increased vascular permeability. Although designed to curtail tissue injury and facilitate repair, the inflammatory response sometimes results in further injury and organ dysfunction. Inflammatory chronic lung diseases, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, and cystic fibrosis are syndromes of severe pulmonary dysfunction resulting from a massive inflammatory response and affecting millions of people worldwide. The histological hallmark of these chronic inflammatory lung diseases is the accumulation of neutrophils in the microvasculature of the lung. Neutrophils are crucial to the innate immune response, and their activation leads to the release of multiple cytotoxic products, including reactive oxygen species and proteases (serine, cysteine, and metalloproteases). The physiological balance between proteases and antiproteases is required for the maintenance of the lung’s connective tissue, and an imbalance in favor of proteases results in lung injury (Umeki et al., 1988Tetley, 1993). A number of studies in animal and cell culture models have demonstrated a contribution of HNE and related NSPs to the development of chronic inflammatory lung diseases. Available preclinical and clinical data suggest that inhibition of NSP in lung diseases suppresses or attenuates the contribution of NSP to pathogenesis (Chughtai and O’Riordan, 2004Voynow et al., 2008Quinn et al., 2010). HNE could also participate in fibrotic lung remodeling by playing a focused role in the conversion of latent transforming growth factor-β into its biologically active form (Chua and Laurent, 2006Lungarella et al., 2008).

Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides

ANCA-associated vasculitides encompasses a variety of diseases characterized by inflammation of blood vessels and by the presence of autoantibodies directed against neutrophil constituents. These autoantibodies are known as ANCAs (Kallenberg et al., 2006). In Wegener granulomatosis (WG), antibodies are mostly directed against PR3. WG is a relatively uncommon chronic inflammatory disorder first described in 1931 by Heinz Karl Ernst Klinger as a variant of polyarteritis nodosa (Klinger, 1931). In 1936, the German pathologist Friedrich Wegener described the disease as a distinct pathological entity (Wegener, 19361939). WG is characterized by necrotizing granulomatous inflammation and vasculitis of small vessels and can affect any organ (Fauci and Wolff, 1973Sarraf and Sneller, 2005). The most common sites of involvement are the upper and lower respiratory tract and the kidneys. WG affects approximately 1 in 20,000 people; it can occur in persons of any age but most often affects those aged 40 to 60 years (Walton, 1958Cotch et al., 1996). Approximately 90% of patients have cold or sinusitis symptoms that fail to respond to the usual therapeutic measures and that last considerably longer than the usual upper respiratory tract infection. Lung involvement occurs in approximately 85% of the patients. Other symptoms include nasal membrane ulcerations and crusting, saddle-nose deformity, inflammation of the ear with hearing problems, inflammation of the eye with sight problems, and cough (with or without hemoptysis).

Hereditary Neutropenias

Neutropenia is a hematological disorder characterized by an abnormally low number of neutrophils (Horwitz et al., 2007). The normal neutrophil count fluctuates across human populations and within individual patients in response to infection but typically lies in the range of 1.5 to 5 × 109 cells/liter. Neutropenia is categorized as severe when the cell count falls below 0.5 × 109 cells/liter. Hence, patients with neutropenia are more susceptible to bacterial infections and, without prompt medical attention, the condition may become life-threatening. Common causes of neutropenia include cancer chemotherapy, drug reactions, autoimmune diseases, and hereditary disorders (Berliner et al., 2004Schwartzberg, 2006).

Papillon-Lefèvre Syndrome

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New Strategies for Fighting Neutrophil Serine Protease-Related Human Diseases

Administration of therapeutic inhibitors to control unwanted proteolysis at inflammation sites has been tested as a therapy for a variety of inflammatory and infectious lung diseases (Chughtai and O’Riordan, 2004). Depending on the size and chemical nature of the inhibitors, they may be administered orally, intravenously, or by an aerosol route. Whatever the mode of administration, the access of therapeutic inhibitors to active proteases is often hampered by physicochemical constraints in the extravascular space and/or by the partitioning of proteases between soluble and solid phases.

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Concluding Remarks

NSPs were first recognized as protein-degrading enzymes but have now proven to be multifunctional components participating in a variety of pathophysiological processes. Thus, they appear as potential therapeutic targets for drugs that inhibit their active site or impair activation from their precursor. Overall, the available preclinical and clinical data suggest that inhibition of NSPs using therapeutic inhibitors would suppress or attenuate deleterious effects of inflammatory diseases, including lung diseases. Depending on the size and chemical nature of inhibitors, those may be administered orally, intravenously, or by aerosolization. But the results obtained until now have not been fully convincing because of the poor knowledge of the biological function of each protease, their spatiotemporal regulation during the course of the disease, the physicochemical constraints associated with inhibitor administration, or the use of animal models in which NSP regulation and specificity differ from those in human. Two different and complementary approaches may help bypass these putative problems. One is to target active proteases by inhibitors at the inflammatory site in animal models in which lung anatomy and physiology are close to those in human to allow in vitro and in vivo assays of human-directed drugs/inhibitors. The other is to prevent neutrophil accumulation at inflammatory sites by impairing production of proteolytically active NSPs using an inhibitor of their maturation protease, DPPI. Preventing neutrophil accumulation at the inflammatory sites by therapeutic inhibition of DPPI represents an original and novel approach, the exploration of which has just started (Méthot et al., 2008). Thus pharmacological inactivation of DPPI in human neutrophils could well reduce membrane binding of PR3 and, as a consequence, neutrophil priming by pathogenic auto-antibodies in WG. In addition, it has been recognized that the intracellular level of NSPs depends on their correct intracellular trafficking. In the future, pharmacological targeting of molecules specifically involved in the correct intracellular trafficking of each NSP could possibly regulate their production and activity, a feature that could be exploited as a therapeutic strategy for inflammatory diseases.

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Granulocyte colony stimulating factor (G-CSF)

Larry H. Bernstein, MD, FCAP, Curator

LPBI

G-CSF (granulocyte colony stimulating factor)

http://www.cancerresearchuk.org/about-cancer/cancers-in-general/treatment/cancer-drugs/gcsf

G-CSF is a type of growth factor. Growth factors are proteins made in the body and some of them make the bone marrow produce blood cells. G-CSF makes the body produce white blood cells to reduce the risk of infection after some types of cancer treatment. It also makes some stem cells move from the bone marrow into the blood. Stem cells are the cells in the bone marrow from which red blood cells, white cells and platelets develop.

The full name for G-CSF is granulocyte colony stimulating factor.

There are different types, including

  • Lenograstim (Granocyte)
  • Filgrastim (Neupogen, Zarzio, Nivestim, Ratiograstim)
  • Long acting (pegylated) filgrastim (pegfilgrastim, Neulasta) and lipegfilgrastim (Longquex)

Pegylated G-CSF stays in the body for longer so you have treatment less often than with the other types of G-CSF.

G-CSF after chemotherapy

A common side effect of chemotherapy is a drop in the number of white blood cells, which leads to an increased risk of getting an infection. Most people’s white blood cells recover quickly after chemotherapy and they don’t need treatment with G-CSF. But with some types of chemotherapy, such as high dose treatment, it can take a long time for the number of white blood cells to rise again.

Having G-CSF treatment can make white blood cell levels go up faster. So if your white blood cell count doesn’t go up as it should you may have G-CSF treatment. With some types of chemotherapy you have G-CSF as a standard part of your treatment plan.

G-CSF before and after a stem cell transplant

You may have G-CSF as part of a stem cell transplant. Before the treatment you have G-CSF to stimulate the bone marrow to produce stem cells and release them into the blood. The stem cells are collected and then you have high dose chemotherapy.

The chemotherapy stops your bone marrow producing blood cells. So you have the stem cells back into your bloodstream. They go into the bone marrow and start making the different types of blood cells again.

You can have G-CSF either

You may have G-CSF as a drip into your bloodstream (intravenously). You can have it through a thin, short tube (a cannula) put into a vein in your arm each time you have treatment. Or you may have it through a central line, a portacath or aPICC line. These are long, plastic tubes that give the drugs directly into a large vein in your chest. You have the tube put in just before your course of treatment starts and it stays in place as long as you need it.

If you have G-CSF as an injection under the skin you have it daily for up to 14 days. You have regular blood tests to check your white blood cell count. If you are having it as part of a planned treatment you start at least a day after your chemotherapy and continue until the number of white blood cells called neutrophils is within the normal range.

If you are having pegylated G-CSF you only need to have one injection at least 24 hours after the end of the chemotherapy.

Common side effects

More than 10 in every 100 people have one or more of the side effects listed below.

  • Headaches
  • Loss of appetite
  • Redness and irritation at the injection site
  • Feeling or being sick, but this is usually well controlled with anti sickness medicines
  • Bone pain happens in up to 4 out of 10 people (40%). It is caused by the bone marrow making blood cells. Paracetamol can help to control any pain
  • Diarrhoea – drink plenty of fluids. Tell your doctor or nurse if diarrhoea becomes severe, or continues for more than 3 days
  • Constipation – your doctor or nurse may give you laxatives to help prevent this but do tell them if you are constipated for more than 3 days
  • Liver changes that are very mild and unlikely to cause symptoms – the liver will almost certainly go back to normal when the treatment ends. You will have regular blood tests to check how well your liver is working

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Blood Transfusions

Larry H Bernstein, MD, FCAP, Curator

LPBI

What Is a Blood Transfusion?  

A blood transfusion is a safe, common procedure in which blood is given to you through an intravenous (IV) line in one of your blood vessels.

Blood transfusions are done to replace blood lost during surgery or due to a serious injury. A transfusion also may be done if your body can’t make blood properly because of an illness.

During a blood transfusion, a small needle is used to insert an IV line into one of your blood vessels. Through this line, you receive healthy blood. The procedure usually takes 1 to 4 hours, depending on how much blood you need.

Blood transfusions are very common. Each year, almost 5 million Americans need a blood transfusion. Most blood transfusions go well. Mild complications can occur. Very rarely, serious problems develop.

Blood is made up of various parts, including red blood cells, white blood cells, platelets (PLATE-lets), and plasma. Blood is transfused either as whole blood (with all its parts) or, more often, as individual parts.

Blood Types

Every person has one of the following blood types: A, B, AB, or O. Also, every person’s blood is either Rh-positive or Rh-negative. So, if you have type A blood, it’s either A positive or A negative.

The blood used in a transfusion must work with your blood type. If it doesn’t, antibodies (proteins) in your blood attack the new blood and make you sick.

Type O blood is safe for almost everyone. About 40 percent of the population has type O blood. People who have this blood type are called universal donors. Type O blood is used for emergencies when there’s no time to test a person’s blood type.

People who have type AB blood are called universal recipients. This means they can get any type of blood.

If you have Rh-positive blood, you can get Rh-positive or Rh-negative blood. But if you have Rh-negative blood, you should only get Rh-negative blood. Rh-negative blood is used for emergencies when there’s no time to test a person’s Rh type.

Blood Banks

Blood banks collect, test, and store blood. They carefully screen all donated blood for possible infectious agents, such as viruses, that could make you sick. (For more information, see“What Are the Risks of a Blood Transfusion?”)

Blood bank staff also screen each blood donation to find out whether it’s type A, B, AB, or O and whether it’s Rh-positive or Rh-negative. Getting a blood type that doesn’t work with your own blood type will make you very sick. That’s why blood banks are very careful when they test the blood.

To prepare blood for a transfusion, some blood banks remove white blood cells. This process is called white cell or leukocyte (LU-ko-site) reduction. Although rare, some people are allergic to white blood cells in donated blood. Removing these cells makes allergic reactions less likely.

Not all transfusions use blood donated from a stranger. If you’re going to have surgery, you may need a blood transfusion because of blood loss during the operation. If it’s surgery that you’re able to schedule months in advance, your doctor may ask whether you would like to use your own blood, rather than donated blood.

Alternatives to Blood Transfusions 

Researchers are trying to find ways to make blood. There’s currently no man-made alternative to human blood. However, researchers have developed medicines that may help do the job of some blood parts.

For example, some people who have kidney problems can now take a medicine called erythropoietin that helps their bodies make more red blood cells. This means they may need fewer blood transfusions.

Surgeons try to reduce the amount of blood lost during surgery so that fewer patients need blood transfusions. Sometimes they can collect and reuse the blood for the patient.

https://www.nhlbi.nih.gov/health/health-topics/topics/bt

Your options may be limited by time and health factors, so it is important to begin carrying out your decision as soon as possible. For example, if friends or family members are donating blood for a patient (directed donors), their blood should be drawn several days prior to the anticipated need to allow adequate time for testing and labeling. The exact protocols are hospital and donor site specific.

The safest blood product is your own, so if a transfusion is likely, this is your lowest risk choice. Unfortunately this option is usually only practical when preparing for elective surgery. In most other instances the patient cannot donate their own blood due to the acute nature of the need for blood. Although you have the right to refuse a blood transfusion, this decision may have life-threatening consequences. If you are a parent deciding for your child, you as the parent or guardian must understand that in a life-threatening situation your doctors will act in your child’s best interest to insure your child’s health and wellbeing in accordance with standards of medical care regardless of religious beliefs. Please carefully review this material and decide with your doctor which option(s) you prefer, understanding that your doctor will always act in the best interest of his or her patient.

To assure a safe transfusion make sure your healthcare provider who starts the transfusion verifies your name and matches it to the blood that is going to be transfused. Besides your name, a second personal identifier usually used is your birthday. This assures the blood is given to the correct patient.

If during the transfusion you have symptoms of shortness of breath, itching,fever or chills or just not feeling well, alert the person transfusing the blood immediately.

Blood can be provided from two sources: autologous blood (using your own blood) or donor blood (using someone else’s blood).

Autologous blood (using your own blood)

Pre-operative donation: donating your own blood before surgery. The blood bank draws your blood and stores it until you need it during or after surgery. This option is only for non-emergency (elective) surgery. It has the advantage of eliminating or minimizing the need for someone else’s blood during and after surgery. The disadvantage is that it requires advanced planning which may delay surgery. Some medical conditions may prevent the pre-operative donation of blood products.

Intra-operative autologous transfusion: recycling your blood during surgery. Blood lost during surgery is filtered, and put back into your body during surgery. This can be done in emergency and elective surgeries. It has the advantage of eliminating or minimizing the need for someone else’s blood during surgery. Large amounts of blood can be recycled. This process cannot be used if cancer or infection is present.

Post-operative autologous transfusion: recycling your blood after surgery. Blood lost after surgery is collected, filtered and returned to your body. This can be done in emergency and elective surgeries. It has the advantage of eliminating or minimizing the need for someone else’s blood during surgery. This process can’t be used in patients where cancer or infection is present.

Hemodilution: donating your own blood during surgery. Immediately before surgery, some of your blood is taken and replaced with IV fluids. After surgery, your blood is filtered and returned to you. This is done only for elective surgeries. This process dilutes your own blood so you lose less concentrated blood during surgery. It has the advantage of eliminating or minimizing the need for someone else’s blood during surgery. The disadvantage of this process is that only a limited amount of blood can be removed, and certain medical conditions may prevent the use of this technique.

Apheresis: donating your own platelets and plasma. Before surgery, your platelets and plasma, which help stop bleeding, are withdrawn, filtered and returned to you when you need it later. This can be done only for elective surgeries. This process may eliminate the need for donor platelets and plasma, especially in high blood-loss procedures. The disadvantage of this process is that some medical conditions may prevent apheresis, and in actual practice it has limited applications. 

http://www.medicinenet.com/blood_transfusion/article.htm

Diseases Requiring Blood Transfusion

Cancer

Some illnesses cause your body to make too few platelets or clotting factors. You may need transfusions of just those blood components to make up for low levels.

Cancer may decrease your body’s production of red blood cells, white blood cells and platelets by impacting the organs that influence blood count, such as the kidneys, bone marrow and the spleen. Radiation and chemotherapy drugs also can decrease components of the blood. Blood transfusions may be used to counter such effects.

Other illness

Some illnesses cause your body to make too few platelets or clotting factors. You may need transfusions of just those blood components to make up for low levels.

Infection, liver failure or severe burns

If you experience an infection, liver failure or severe burns, you may need a transfusion of plasma. Plasma is the liquid part of blood.

Blood disorders

People with blood diseases may receive transfusions of red blood cells, platelets or clotting factors.

Severe liver malfunction

If you have severe liver problems, you may receive a transfusion of albumin, a blood protein.

Risks

By Mayo Clinic Staff

Blood transfusions are generally considered to be safe. But they do carry some risk of complications. Complications may happen during the transfusion or not for weeks, months or even years afterward. They include the following:

Allergic reaction and hives

If you have an allergic reaction to the transfusion, you may experience hives and itching during the procedure or very soon after. This type of reaction is usually treated with antihistamines. Rarely, a more serious allergic reaction causes difficulty breathing, low blood pressure and nausea.

Fever

If you quickly develop a fever during the transfusion, you may be having a febrile transfusion reaction. Your doctor will stop the transfusion to do further tests before deciding whether to continue. A febrile reaction can also occur shortly after the transfusion. Fever may be accompanied by chills and shaking.

Acute immune hemolytic reaction

This is a very rare but serious transfusion reaction in which your body attacks the transfused red blood cells because the donor blood type is not a good match. In response, your immune system attacks the transfused red blood cells, which are viewed as foreign. These destroyed cells release a substance into your blood that harms your kidneys. This usually occurs during or right after a transfusion. Signs and symptoms include fever, nausea, chills, lower back or chest pain, and dark urine.

Lung injury

Transfusion-related acute lung injury (TRALI) is thought to occur due to antibodies or other biologic substances in the blood components. With TRALI, the lungs become damaged, making it difficult to breathe. Usually, TRALI occurs within one to six hours of the transfusion. People usually recover, especially when treated quickly. Most people who die after TRALI were very sick before the transfusion.

Bloodborne infections

Blood banks screen donors for risk factors and test donated blood to reduce the risk of transfusion-related infections. Infections related to blood transfusion still rarely may occur. It can take weeks or months after a blood transfusion to determine that you’ve been infected with a virus, bacterium or parasite.

The National Institutes of Health offers the following estimates for the risk of a blood donation carrying an infectious disease:

  • HIV — 1 in 2 million donations, which is lower than the risk of being killed by lightning
  • Hepatitis B — 1 in 205,000 donations
  • Hepatitis C — 1 in 2 million donations

Delayed hemolytic reaction

This type of reaction is similar to an acute immune hemolytic reaction, but it occurs much more slowly. Your body gradually attacks the donor red blood cells. It could take one to four weeks to notice a decrease in red blood cell levels.

Iron overload  

If you receive multiple blood transfusions, you may end up with too much iron in your blood. Iron overload (hemochromatosis) can damage parts of your body, including the liver and the heart. You may receive iron chelation therapy, which uses medication to remove excess iron.

Graft-versus-host disease

Transfusion-associated graft-versus-host disease is a very rare condition in which transfused white blood cells attack the recipient’s bone marrow. This disease is usually fatal. It is more likely to affect people with severely weakened immune systems, such as those being treated for leukemia or lymphoma. Signs and symptoms include fever, rash, diarrhea and abnormal liver function test results. Irradiating the blood before transfusing it reduces the risk.

Most of the donated blood collected by the American Red Cross is used for direct blood transfusions. Common types of blood transfusions including platelet, plasma and red blood cell transfusions.

A patient suffering from an iron deficiency or anemia, a condition where the body does not have enough red blood cells, may receive a Red Blood Cell Transfusion. This type of transfusion increases a patient’s hemoglobin and iron levels, while improving the amount of oxygen in the body.

Platelets are a component of blood that stops the body from bleeding. Often patients suffering from leukemia, or other types of cancer, have lower platelet counts as a side effect of their chemotherapy treatments. Patients who have illnesses that prevent the body from making enough platelets have to get regular transfusions to stay healthy.

Plasma is the liquid part of the body’s blood. It contains important proteins and other substances crucial to one’s overall health. Plasma transfusions are used for patients with liver failure, severe infections, and serious burns.

If you experience an infection, liver failure or severe burns, you may need a transfusion of plasma. Plasma is the liquid part of blood.

Blood disorders

People with blood diseases may receive transfusions of red blood cells, platelets or clotting factors.

Severe liver malfunction

If you have severe liver problems, you may receive a transfusion of albumin, a blood protein.

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Hematological Malignancy Diagnostics

Author and Curator: Larry H. Bernstein, MD, FCAP

 

2.4.3 Diagnostics

2.4.3.1 Computer-aided diagnostics

Back-to-Front Design

Robert Didner
Bell Laboratories

Decision-making in the clinical setting
Didner, R  Mar 1999  Amer Clin Lab

Mr. Didner is an Independent Consultant in Systems Analysis, Information Architecture (Informatics) Operations Research, and Human Factors Engineering (Cognitive Psychology),  Decision Information Designs, 29 Skyline Dr., Morristown, NJ07960, U.S.A.; tel.: 973-455-0489; fax/e-mail: bdidner@hotmail.com

A common problem in the medical profession is the level of effort dedicated to administration and paperwork necessitated by various agencies, which contributes to the high cost of medical care. Costs would be reduced and accuracy improved if the clinical data could be captured directly at the point they are generated in a form suitable for transmission to insurers or machine transformable into other formats. Such a capability could also be used to improve the form and the structure of information presented to physicians and support a more comprehensive database linking clinical protocols to outcomes, with the prospect of improving clinical outcomes. Although the problem centers on the physician’s process of determining the diagnosis and treatment of patients and the timely and accurate recording of that process in the medical system, it substantially involves the pathologist and laboratorian, who interact significantly throughout the in-formation-gathering process. Each of the currently predominant ways of collecting information from diagnostic protocols has drawbacks. Using blank paper to collect free-form notes from the physician is not amenable to computerization; such free-form data are also poorly formulated, formatted, and organized for the clinical decision-making they support. The alternative of preprinted forms listing the possible tests, results, and other in-formation gathered during the diagnostic process facilitates the desired computerization, but the fixed sequence of tests and questions they present impede the physician from using an optimal decision-making sequence. This follows because:

  • People tend to make decisions and consider information in a step-by-step manner in which intermediate decisions are intermixed with data acquisition steps.
  • The sequence in which components of decisions are made may alter the decision outcome.
  • People tend to consider information in the sequence it is requested or displayed.
  • Since there is a separate optimum sequence of tests and questions for each cluster of history and presenting symptoms, there is no one sequence of tests and questions that can be optimal for all presenting clusters.
  • As additional data and test results are acquired, the optimal sequence of further testing and data acquisition changes, depending on the already acquired information.

Therefore, promoting an arbitrary sequence of information requests with preprinted forms may detract from outcomes by contributing to a non-optimal decision-making sequence. Unlike the decisions resulting from theoretical or normative processes, decisions made by humans are path dependent; that is, the out-come of a decision process may be different if the same components are considered in a different sequence.

Proposed solution

This paper proposes a general approach to gathering data at their source in computer-based form so as to improve the expected outcomes. Such a means must be interactive and dynamic, so that at any point in the clinical process the patient’s presenting symptoms, history, and the data already collected are used to determine the next data or tests requested. That de-termination must derive from a decision-making strategy designed to produce outcomes with the greatest value and supported by appropriate data collection and display techniques. The strategy must be based on the knowledge of the possible outcomes at any given stage of testing and information gathering, coupled with a metric, or hierarchy of values for assessing the relative desirability of the possible outcomes.

A value hierarchy

  • The numbered list below illustrates a value hierarchy. In any particular instance, the higher-numbered values should only be considered once the lower- numbered values have been satisfied. Thus, a diagnostic sequence that is very time or cost efficient should only be considered if it does not increase the likelihood (relative to some other diagnostic sequence) that a life-threatening disorder may be missed, or that one of the diagnostic procedures may cause discomfort.
  • Minimize the likelihood that a treatable, life-threatening disorder is not treated.
  • Minimize the likelihood that a treatable, discomfort-causing disorder is not treated.
  • Minimize the likelihood that a risky procedure(treatment or diagnostic procedure) is inappropriately administered.
  • Minimize the likelihood that a discomfort-causing procedure is inappropriately administered.
  • Minimize the likelihood that a costly procedure is inappropriately administered.
  • Minimize the time of diagnosing and treating thepatient.8.Minimize the cost of diagnosing and treating the patient.

The above hierarchy is relative, not absolute; for many patients, a little bit of testing discomfort may be worth a lot of time. There are also some factors and graduations intentionally left out for expository simplicity (e.g., acute versus chronic disorders).This value hierarchy is based on a hypothetical patient. Clearly, the hierarchy of a health insurance carrier might be different, as might that of another patient (e.g., a geriatric patient). If the approach outlined herein were to be followed, a value hierarchy agreed to by a majority of stakeholders should be adopted.

Efficiency

Once the higher values are satisfied, the time and cost of diagnosis and treatment should be minimized. One way to do so would be to optimize the sequence in which tests are performed, so as to minimize the number, cost, and time of tests that need to be per-formed to reach a definitive decision regarding treatment. Such an optimum sequence could be constructed using Claude Shannon’s information theory.

According to this theory, the best next question to ask under any given situation (assuming the question has two possible outcomes) is that question that divides the possible outcomes into two equally likely sets. In the real world, all tests or questions are not equally valuable, costly, or time consuming; therefore, value(risk factors), cost, and time should be used as weighting factors to optimize the test sequence, but this is a complicating detail at this point.

A value scale

For dynamic computation of outcome values, the hierarchy could be converted into a weighted value scale so differing outcomes at more than one level of the hierarchy could be readily compared. An example of such a weighted value scale is Quality Adjusted Life Years (QALY).

Although QALY does not incorporate all of the factors in this example, it is a good conceptual starting place.

The display, request, decision-making relationship

For each clinical determination, the pertinent information should be gathered, organized, formatted, and formulated in a way that facilitates the accuracy, reliability, and efficiency with which that determination is made. A physician treating a patient with high cholesterol and blood pressure (BP), for example, may need to know whether or not the patient’s cholesterol and BP respond to weight changes to determine an appropriate treatment (e.g., weight control versus medication). This requires searching records for BP, certain blood chemicals (e.g., HDLs, LDLs, triglycerides, etc.), and weight from several

sources, then attempting to track them against each other over time. Manually reorganizing this clinical information each time it is used is extremely inefficient. More important, the current organization and formatting defies principles of human factors for optimally displaying information to enhance human information-processing characteristics, particularly for decision support.

While a discussion of human factors and cognitive psychology principles is beyond the scope of this paper, following are a few of the system design principles of concern:

  • Minimize the load on short-term memory.
  • Provide information pertinent to a given decision or component of a decision in a compact, contiguous space.
  • Take advantage of basic human perceptual and pat-tern recognition facilities.
  • Design the form of an information display to com-plement the decision-making task it supports.

F i g u re 1 shows fictitious, quasi-random data from a hypothetical patient with moderately elevated cholesterol. This one-page display pulls together all the pertinent data from six years of blood tests and related clinical measurements. At a glance, the physician’s innate pattern recognition, color, and shape perception facilities recognize the patient’s steadily increasing weight, cholesterol, BP, and triglycerides as well as the declining high-density lipoproteins. It would have taken considerably more time and effort to grasp this information from the raw data collection and blood test reports as they are currently presented in independent, tabular time slices.

Design the formulation of an information display to complement the decision-making task.

The physician may wish to know only the relationship between weight and cardiac risk factors rather than whether these measures are increasing or decreasing, or are within acceptable or marginal ranges. If so, Table 1 shows the correlations between weight and the other factors in a much more direct and simple way using the same data as in Figure 1. One can readily see the same conclusions about relations that were drawn from Figure 1.This type of abstract, symbolic display of derived information also makes it easier to spot relationships when the individual variables are bouncing up and down, unlike the more or less steady rise of most values in Figure 1. This increase in precision of relationship information is gained at the expense of other types of information (e.g., trends). To display information in an optimum form then, the system designer must know what the information demands of the task are at the point in the task when the display is to be used.

Present the sequence of information display clusters to complement an optimum decision-making strategy.

Just as a fixed sequence of gathering clinical, diagnostic information may lead to a far from optimum outcome, there exists an optimum sequence of testing, considering information, and gathering data that will lead to an optimum outcome (as defined by the value hierarchy) with a minimum of time and expense. The task of the information system designer, then, is to provide or request the right information, in the best form, at each stage of the procedure. For ex-ample, Figure 1 is suitable for the diagnostic phase since it shows the current state of the risk factors and their trends. Table 1, on the other hand, might be more appropriate in determining treatment, where there may be a choice of first trying a strict dietary treatment, or going straight to a combination of diet plus medication. The fact that Figure 1 and Table 1 have somewhat redundant information is not a problem, since they are intended to optimally provide information for different decision-making tasks. The critical need, at this point, is for a model of how to determine what information should be requested, what tests to order, what information to request and display, and in what form at each step of the decision-making process. Commitment to a collaborative relationship between physicians and laboratorians and other information providers would be an essential requirement for such an undertaking. The ideal diagnostic data-collection instrument is a flexible, computer-based device, such as a notebook computer or Personal Digital Assistant (PDA) sized device.

Barriers to interactive, computer-driven data collection at the source

As with any major change, it may be difficult to induce many physicians to change their behavior by interacting directly with a computer instead of with paper and pen. Unlike office workers, who have had to make this transition over the past three decades, most physicians’ livelihoods will not depend on converting to computer interaction. Therefore, the transition must be made attractive and the changes less onerous. Some suggestions follow:

  1. Make the data collection a natural part of the clinical process.
  2. Ensure that the user interface is extremely friendly, easy to learn, and easy to use.
  3. Use a small, portable device.
  4. Use the same device for collection and display of existing information (e.g., test results and his-tory).
  5. Minimize the need for free-form written data entry (use check boxes, forms, etc.).
  6. Allow the entry of notes in pen-based free-form (with the option of automated conversion of numeric data to machine-manipulable form).
  7. Give the physicians a more direct benefit for collecting data, not just a means of helping a clerk at an HMO second-guess the physician’s judgment.
  8. Improve administrative efficiency in the office.
  9. Make the data collection complement the clinical decision-making process.
  10. Improve information displays, leading to better outcomes.
  11. Make better use of the physician’s time and mental effort.

Conclusion

The medical profession is facing a crisis of information. Gathering information is costing a typical practice more and more while fees are being restricted by third parties, and the process of gathering this in-formation may be detrimental to current outcomes. Gathered properly, in machine-manipulable form, these data could be reformatted so as to greatly improve their value immediately in the clinical setting by leading to decisions with better outcomes and, in the long run, by contributing to a clinical data warehouse that could greatly improve medical knowledge. The challenge is to create a mechanism for data collection that facilitates, hastens, and improves the outcomes of clinical activity while minimizing the inconvenience and resistance to change on the part of clinical practitioners. This paper is intended to provide a high-level overview of how this may be accomplished, and start a dialogue along these lines.

References

  1. Tversky A. Elimination by aspects: a theory of choice. Psych Rev 1972; 79:281–99.
  2. Didner RS. Back-to-front design: a guns and butter approach. Ergonomics 1982; 25(6):2564–5.
  3. Shannon CE. A mathematical theory of communication. Bell System Technical J 1948; 27:379–423 (July), 623–56 (Oct).
  4. Feeny DH, Torrance GW. Incorporating utility-based quality-of-life assessment measures in clinical trials: two examples. Med Care 1989; 27:S190–204.
  5. Smith S, Mosier J. Guidelines for designing user interface soft-ware. ESD-TR-86-278, Aug 1986.
  6. Miller GA. The magical number seven plus or minus two. Psych Rev 1956; 65(2):81–97.
  7. Sternberg S. High-speed scanning in human memory. Science 1966; 153: 652–4.

Table 1

Correlation of weight with other cardiac risk factors

Cholesterol 0.759384
HDL 0.53908
LDL 0.177297
BP-syst. 0.424728
BP-dia. 0.516167
Triglycerides 0.637817

Figure 1  Hypothetical patient data.

(not shown)

Realtime Clinical Expert Support

https://pharmaceuticalintelligence.com/2015/05/10/realtime-clinical-expert-support/

Regression: A richly textured method for comparison and classification of predictor variables

https://pharmaceuticalintelligence.com/2012/08/14/regression-a-richly-textured-method-for-comparison-and-classification-of-predictor-variables/

Converting Hematology Based Data into an Inferential Interpretation

Larry H. Bernstein, Gil David, James Rucinski and Ronald R. Coifman
In Hematology – Science and Practice
Lawrie CH, Ch 22. Pp541-552.
InTech Feb 2012, ISBN 978-953-51-0174-1
https://www.researchgate.net/profile/Larry_Bernstein/publication/221927033_Converting_Hematology_Based_Data_into_an_Inferential_Interpretation/links/0fcfd507f28c14c8a2000000.pdf

A model for Thalassemia Screening using Hematology Measurements

https://www.researchgate.net/profile/Larry_Bernstein/publication/258848064_A_model_for_Thalassemia_Screening_using_Hematology_Measurements/links/0c9605293c3048060b000000.pdf

2.4.3.2 A model for automated screening of thalassemia in hematology (math study).

Kneifati-Hayek J, Fleischman W, Bernstein LH, Riccioli A, Bellevue R.
Lab Hematol. 2007; 13(4):119-23. http://dx.doi.org:/10.1532/LH96.07003.

The results of 398 patient screens were collected. Data from the set were divided into training and validation subsets. The Mentzer ratio was determined through a receiver operating characteristic (ROC) curve on the first subset, and screened for thalassemia using the second subset. HgbA2 levels were used to confirm beta-thalassemia.

RESULTS: We determined the correct decision point of the Mentzer index to be a ratio of 20. Physicians can screen patients using this index before further evaluation for beta-thalassemia (P < .05).

CONCLUSION: The proposed method can be implemented by hospitals and laboratories to flag positive matches for further definitive evaluation, and will enable beta-thalassemia screening of a much larger population at little to no additional cost.

Measurement of granulocyte maturation may improve the early diagnosis of the septic state.

2.4.3.3 Bernstein LH, Rucinski J. Clin Chem Lab Med. 2011 Sep 21;49(12):2089-95.
http://dx.doi.org:/10.1515/CCLM.2011.688.

2.4.3.4 The automated malnutrition assessment.

David G, Bernstein LH, Coifman RR. Nutrition. 2013 Jan; 29(1):113-21.
http://dx.doi.org:/10.1016/j.nut.2012.04.017

2.4.3.5 Molecular Diagnostics

Genomic Analysis of Hematological Malignancies

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy that occurs in children. Although more than 90% of children with ALL now survive to adulthood, those with the rarest and high-risk forms of the disease continue to have poor prognoses. Through the Pediatric Cancer Genome Project (PCGP), investigators in the Hematological Malignancies Program are identifying the genetic aberrations that cause these aggressive forms of leukemias. Here we present two studies on the genetic bases of early T-cell precursor ALL and acute megakaryoblastic leukemia.

  • Early T-Cell Precursor ALL Is Characterized by Activating Mutations
  • The CBFA2T3-GLIS2Fusion Gene Defines an Aggressive Subtype of Acute Megakaryoblastic Leukemia in Children

Early T-cell precursor ALL (ETP-ALL), which comprises 15% of all pediatric T-cell leukemias, is an aggressive disease that is typically resistant to contemporary therapies. Children with ETP-ALL have a high rate of relapse and an extremely poor prognosis (i.e., 5-year survival is approximately 20%). The genetic basis of ETP-ALL has remained elusive. Although ETP-ALL is associated with a high burden of DNA copy number aberrations, none are consistently found or suggest a unifying genetic alteration that drives this disease.

Through the efforts of the PCGP, Jinghui Zhang, PhD (Computational Biology), James R. Downing, MD (Pathology), Charles G. Mullighan, MBBS(Hons), MSc, MD (Pathology), and colleagues analyzed the whole-genome sequences of leukemic cells and matched normal DNA from 12 pediatric patients with ETP-ALL. The identified genetic mutations were confirmed in a validation cohort of 52 ETP-ALL specimens and 42 non-ETP T-lineage ALLs (T-ALL).

In the journal Nature, the investigators reported that each ETP-ALL sample carried an average of 1140 sequence mutations and 12 structural variations. Of the structural variations, 51% were breakpoints in genes with well-established roles in hematopoiesis or leukemogenesis (e.g., MLH2,SUZ12, and RUNX1). Eighty-four percent of the structural variations either caused loss of function of the gene in question or resulted in the formation of a fusion gene such as ETV6-INO80D. The ETV6 gene, which encodes a protein that is essential for hematopoiesis, is frequently mutated in leukemia. Among the DNA samples sequenced in this study, ETV6 was altered in 33% of ETP-ALL but only 10% of T-ALL cases.

Next-generation sequencing in hematologic malignancies: what will be the dividends?

Jason D. MerkerAnton Valouev, and Jason Gotlib
Ther Adv Hematol. 2012 Dec; 3(6): 333–339.
http://dx.doi.org:/10.1177/2040620712458948

The application of high-throughput, massively parallel sequencing technologies to hematologic malignancies over the past several years has provided novel insights into disease initiation, progression, and response to therapy. Here, we describe how these new DNA sequencing technologies have been applied to hematolymphoid malignancies. With further improvements in the sequencing and analysis methods as well as integration of the resulting data with clinical information, we expect these technologies will facilitate more precise and tailored treatment for patients with hematologic neoplasms.

Leveraging cancer genome information in hematologic malignancies.

Rampal R1Levine RL.
J Clin Oncol. 2013 May 20; 31(15):1885-92.
http://dx.doi.org:/10.1200/JCO.2013.48.7447

The use of candidate gene and genome-wide discovery studies in the last several years has led to an expansion of our knowledge of the spectrum of recurrent, somatic disease alleles, which contribute to the pathogenesis of hematologic malignancies. Notably, these studies have also begun to fundamentally change our ability to develop informative prognostic schema that inform outcome and therapeutic response, yielding substantive insights into mechanisms of hematopoietic transformation in different tissue compartments. Although these studies have already had important biologic and translational impact, significant challenges remain in systematically applying these findings to clinical decision making and in implementing new technologies for genetic analysis into clinical practice to inform real-time decision making. Here, we review recent major genetic advances in myeloid and lymphoid malignancies, the impact of these findings on prognostic models, our understanding of disease initiation and evolution, and the implication of genomic discoveries on clinical decision making. Finally, we discuss general concepts in genetic modeling and the current state-of-the-art technology used in genetic investigation.

p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies

E Wattel, C Preudhomme, B Hecquet, M Vanrumbeke, et AL.
Blood, (Nov 1), 1994; 84(9): pp 3148-3157
http://www.bloodjournal.org/content/bloodjournal/84/9/3148.full.pdf

We analyzed the prognostic value of p53 mutations for response to chemotherapy and survival in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic lymphocytic leukemia (CLL). Mutations were detected by single-stranded conformation polymorphism (SSCP) analysis of exons 4 to 10 of the P53 gene, and confirmed by direct sequencing. A p53 mutation was found in 16 of 107 (15%) AML, 20 of 182 (11%) MDS, and 9 of 81 (11%) CLL tested. In AML, three of nine (33%) mutated cases and 66 of 81 (81%) nonmutated cases treated with intensive chemotherapy achieved complete remission (CR) (P = .005) and none of five mutated cases and three of six nonmutated cases treated by low-dose Ara C achieved CR or partial remission (PR) (P = .06). Median actuarial survival was 2.5 months in mutated cases, and 15 months in nonmutated cases (P < lo-‘). In the MDS patients who received chemotherapy (intensive chemotherapy or low-dose Ara C), 1 of 13 (8%) mutated cases and 23 of 38 (60%) nonmutated cases achieved CR or PR (P = .004), and median actuarial survival was 2.5 and 13.5 months, respectively (P C lo-’). In all MDS cases (treated and untreated), the survival difference between mutated cases and nonmutated cases was also highly significant. In CLL, 1 of 8 (12.5%) mutated cases treated by chemotherapy (chlorambucil andlor CHOP andlor fludarabine) responded, as compared with 29 of 36 (80%) nonmutated cases (P = .02). In all CLL cases, survival from p53 analysis was significantly shorter in mutated cases (median 7 months) than in nonmutated cases (median not reached) (P < IO-’). In 35 of the 45 mutated cases of AML, MDS, and CLL, cytogenetic analysis or SSCP and sequence findings showed loss of the nonmutated P53 allele. Our findings show that p53 mutations are a strong prognostic indicator of response to chemotherapy and survival in AML, MDS, and CLL. The usual association of p53 mutations to loss of the nonmutated P53 allele, in those disorders, ie, to absence of normal p53 in tumor cells, suggests that p53 mutations could induce drug resistance, at least in part, by interfering with normal apoptotic pathways in tumor cells.

Genomic approaches to hematologic malignancies

Benjamin L. Ebert and Todd R. Golub
Blood. 2004; 104:923-932
https://www.broadinstitute.org/mpr/publications/projects/genomics/Review%20Genomics%20of%20Heme%20Malig,%20Blood%202004.pdf

In the past several years, experiments using DNA microarrays have contributed to an increasingly refined molecular taxonomy of hematologic malignancies. In addition to the characterization of molecular profiles for known diagnostic classifications, studies have defined patterns of gene expression corresponding to specific molecular abnormalities, oncologic phenotypes, and clinical outcomes. Furthermore, novel subclasses with distinct molecular profiles and clinical behaviors have been identified. In some cases, specific cellular pathways have been highlighted that can be therapeutically targeted. The findings of microarray studies are beginning to enter clinical practice as novel diagnostic tests, and clinical trials are ongoing in which therapeutic agents are being used to target pathways that were identified by gene expression profiling. While the technology of DNA microarrays is becoming well established, genome-wide surveys of gene expression generate large data sets that can easily lead to spurious conclusions. Many challenges remain in the statistical interpretation of gene expression data and the biologic validation of findings. As data accumulate and analyses become more sophisticated, genomic technologies offer the potential to generate increasingly sophisticated insights into the complex molecular circuitry of hematologic malignancies. This review summarizes the current state of discovery and addresses key areas for future research.

2.4.3.6 Flow cytometry

Introduction to Flow Cytometry: Blood Cell Identification

Dana L. Van Laeys
https://www.labce.com/flow_cytometry.aspx

No other laboratory method provides as rapid and detailed analysis of cellular populations as flow cytometry, making it a valuable tool for diagnosis and management of several hematologic and immunologic diseases. Understanding this relevant methodology is important for any medical laboratory scientist.

Whether you have no previous experience with flow cytometry or just need a refresher, this course will help you to understand the basic principles, with the help of video tutorials and interactive case studies.

Basic principles include:

  1. Immunophenotypic features of various types of hematologic cells
  2. Labeling cellular elements with fluorochromes
  3. Blood cell identification, specifically B and T lymphocyte identification and analysis
  4. Cell sorting to isolate select cell population for further analysis
  5. Analyzing and interpreting result reports and printouts

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Hematologic Malignancies , Table of Contents

Writer and Curator:  Larry H. Bernstein, MD, FCAP

Hematologic Malignancies 

Not excluding lymphomas [solid tumors]

The following series of articles are discussions of current identifications, classification, and treatments of leukemias, myelodysplastic syndromes and myelomas.

2.4 Hematological Malignancies

2.4.1 Ontogenesis of blood elements

Erythropoiesis

White blood cell series: myelopoiesis

Thrombocytogenesis

2.4.2 Classification of hematopoietic cancers

Primary Classification

Acute leukemias

Myelodysplastic syndromes

Acute myeloid leukemia

Acute lymphoblastic leukemia

Myeloproliferative Disorders

Chronic myeloproliferative disorders

Chronic myelogenous leukemia and related disorders

Myelofibrosis, including chronic idiopathic

Polycythemia, including polycythemia rubra vera

Thrombocytosis, including essential thrombocythemia

Chronic lymphoid leukemia and other lymphoid leukemias

Lymphomas

Non-Hodgkin Lymphoma

Hodgkin lymphoma

Lymphoproliferative disorders associated with immunodeficiency

Plasma Cell dyscrasias

Mast cell disease and Histiocytic neoplasms

Secondary Classification

Nuance – PathologyOutlines

2.4.3 Diagnostics

Computer-aided diagnostics

Back-to-Front Design

Realtime Clinical Expert Support

Regression: A richly textured method for comparison and classification of predictor variables

Converting Hematology Based Data into an Inferential Interpretation

A model for Thalassemia Screening using Hematology Measurements

Measurement of granulocyte maturation may improve the early diagnosis of the septic state.

The automated malnutrition assessment.

Molecular Diagnostics

Genomic Analysis of Hematological Malignancies

Next-generation sequencing in hematologic malignancies: what will be the dividends?

Leveraging cancer genome information in hematologic malignancies.

p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies

Genomic approaches to hematologic malignancies

2.4.4 Treatment of hematopoietic cancers

2.4.4.1 Treatments for leukemia by type

2.4.4..2 Acute lymphocytic leukemias

2.4..4.3 Treatment of Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia

Gene-Expression Patterns in Drug-Resistant Acute Lymphoblastic Leukemia Cells and Response to Treatment

Leukemias Treatment & Management

Treatments and drugs

2.4.5 Acute Myeloid Leukemia

New treatment approaches in acute myeloid leukemia: review of recent clinical studies

Novel approaches to the treatment of acute myeloid leukemia.

Current treatment of acute myeloid leukemia

Adult Acute Myeloid Leukemia Treatment (PDQ®)

2.4.6 Treatment for CML

Chronic Myelogenous Leukemia Treatment (PDQ®)

What`s new in chronic myeloid leukemia research and treatment?

4.2.7 Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia Treatment (PDQ®)

Results from the Phase 3 Resonate™ Trial

Typical treatment of chronic lymphocytic leukemia

4.2.8 Lymphoma treatment

4.2.8.1 Overview

4.2.8.2 Chemotherapy

………………………………..

Chapter 6

Total body irradiation (TBI)

Bone marrow (BM) transplantation

Autologous stem cell transplantation

Hematopoietic stem cell transplantation

Supportive Therapies

Blood transfusions

Erythropoietin

G-CSF (granulocyte-colony stimulating factor)

Plasma exchange (plasmapheresis)

Platelet transfusions

Steroids

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Neonatal Pathophysiology


Neonatal Pathophysiology

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

Introduction

This curation deals with a large and specialized branch of medicine that grew since the mid 20th century in concert with the developments in genetics and as a result of a growing population, with large urban populations, increasing problems of premature deliveries.  The problems of prematurity grew very preterm to very low birth weight babies with special problems.  While there were nurseries, the need for intensive care nurseries became evident in the 1960s, and the need for perinatal care of pregnant mothers also grew as a result of metabolic problems of the mother, intrauterine positioning of the fetus, and increasing numbers of teen age pregnancies as well as nutritional problems of the mother.  There was also a period when the manufacturers of nutritional products displaced the customary use of breast feeding, which was consequential.  This discussion is quite comprehensive, as it involves a consideration of the heart, the lungs, the brain, and the liver, to a large extent, and also the kidneys and skeletal development.

It is possible to outline, with a proportionate emphasis based on frequency and severity, this as follows:

  1. Genetic and metabolic diseases
  2. Nervous system
  3. Cardiovascular
  4. Pulmonary
  5. Skeletal – bone and muscle
  6. Hematological
  7. Liver
  8. Esophagus, stomach, and intestines
  9. Kidneys
  10. Immune system

Fetal Development

Gestation is the period of time between conception and birth when a baby grows and develops inside the mother’s womb. Because it’s impossible to know exactly when conception occurs, gestational age is measured from the first day of the mother’s last menstrual cycle to the current date. It is measured in weeks. A normal gestation lasts anywhere from 37 to 41 weeks.

Week 5 is the start of the “embryonic period.” This is when all the baby’s major systems and structures develop. The embryo’s cells multiply and start to take on specific functions. This is called differentiation. Blood cells, kidney cells, and nerve cells all develop. The embryo grows rapidly, and the baby’s external features begin to form.

Week 6-9:   Brain forms into five different areas. Some cranial nerves are visible. Eyes and ears begin to form. Tissue grows that will the baby’s spine and other bones. Baby’s heart continues to grow and now beats at a regular rhythm. Blood pumps through the main vessels. Your baby’s brain continues to grow. The lungs start to form. Limbs look like paddles. Essential organs begin to grow.

Weeks 11-18: Limbs extended. Baby makes sucking motion. Movement of limbs. Liver and pancreas produce secretions. Muscle and bones developing.

Week 19-21: Baby can hear. Mom feels baby – and quickening.

http://www.nlm.nih.gov/medlineplus/ency/article/002398.htm

fetal-development

fetal-development

https://polination.files.wordpress.com/2014/02/abortion-new-research-into-fetal-development.jpg

Inherited Metabolic Disorders

The original cause of most genetic metabolic disorders is a gene mutation that occurred many, many generations ago. The gene mutation is passed along through the generations, ensuring its preservation.

Each inherited metabolic disorder is quite rare in the general population. Considered all together, inherited metabolic disorders may affect about 1 in 1,000 to 2,500 newborns. In certain ethnic populations, such as Ashkenazi Jews (Jews of central and eastern European ancestry), the rate of inherited metabolic disorders is higher.

Hundreds of inherited metabolic disorders have been identified, and new ones continue to be discovered. Some of the more common and important genetic metabolic disorders include:

Lysosomal storage disorders : Lysosomes are spaces inside cells that break down waste products of metabolism. Various enzyme deficiencies inside lysosomes can result in buildup of toxic substances, causing metabolic disorders including:

  • Hurler syndrome (abnormal bone structure and developmental delay)
  • Niemann-Pick disease (babies develop liver enlargement, difficulty feeding, and nerve damage)
  • Tay-Sachs disease (progressive weakness in a months-old child, progressing to severe nerve damage; the child usually lives only until age 4 or 5)
  • Gauchers disease and others

Galactosemia: Impaired breakdown of the sugar galactose leads to jaundice, vomiting, and liver enlargement after breast or formula feeding by a newborn.

Maple syrup urine disease: Deficiency of an enzyme called BCKD causes buildup of amino acids in the body. Nerve damage results, and the urine smells like syrup.

Phenylketonuria (PKU): Deficiency of the enzyme PAH results in high levels of phenylalanine in the blood. Mental retardation results if the condition is not recognized.

Glycogen storage diseases: Problems with sugar storage lead to low blood sugar levels, muscle pain, and weakness.

Metal metabolism disorders: Levels of trace metals in the blood are controlled by special proteins. Inherited metabolic disorders can result in protein malfunction and toxic accumulation of metal in the body:

Wilson disease (toxic copper levels accumulate in the liver, brain, and other organs)

Hemochromatosis (the intestines absorb excessive iron, which builds up in the liver, pancreas, joints, and heart, causing damage)

Organic acidemias: methylmalonic acidemia and propionic acidemia.

Urea cycle disorders: ornithine transcarbamylase deficiency and citrullinemia

Hemoglobinopathies – thalassemias, sickle cell disease

Red cell enzyme disorders – glucose-6-phosphate dehydrogenase, pyruvate kinase

This list is by no means complete.

http://www.webmd.com/a-to-z-guides/inherited-metabolic-disorder-types-and-treatments

New variations in the galactose-1-phosphate uridyltransferase (GALT) gene

Clinical and molecular spectra in galactosemic patients from neonatal screening in northeastern Italy: Structural and functional characterization of new variations in the galactose-1-phosphate uridyltransferase (GALT) gene

E Viggiano, A Marabotti, AP Burlina, C Cazzorla, MR D’Apice, et al.
Gene 559 (2015) 112–118
http://dx.doi.org/10.1016/j.gene.2015.01.013
Galactosemia (OMIM 230400) is a rare autosomal recessive inherited disorder caused by deficiency of galactose-1-phosphate uridyltransferase (GALT; OMIM 606999) activity. The incidence of galactosemia is 1 in 30,000–60,000, with a prevalence of 1 in 47,000 in the white population. Neonates with galactosemia can present acute symptoms, such as severe hepatic and renal failure, cataract and sepsis after milk introduction. Dietary restriction of galactose determines the clinical improvement in these patients. However, despite early diagnosis by neonatal screening and dietary treatment, a high percentage of patients develop long-term complications such as cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction.

With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30 years.

No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications.

A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of < 1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype–phenotype correlation in classical galactosemia and underlines the importance of molecular diagnostic testing prior to making any treatment.

Diagnosis and Management of Hereditary Hemochromatosis

Reena J. Salgia, Kimberly Brown
Clin Liver Dis 19 (2015) 187–198
http://dx.doi.org/10.1016/j.cld.2014.09.011

Hereditary hemochromatosis (HH) is a diagnosis most commonly made in patients with elevated iron indices (transferrin saturation and ferritin), and HFE genetic mutation testing showing C282Y homozygosity.

The HFE mutation is believed to result in clinical iron overload through altering hepcidin levels resulting in increased iron absorption.

The most common clinical complications of HH include cirrhosis, diabetes, nonischemic cardiomyopathy, and hepatocellular carcinoma.

Liver biopsy should be performed in patients with HH if the liver enzymes are elevated or serum ferritin is greater than 1000 mg/L. This is useful to determine the degree of iron overload and stage the fibrosis.

Treatment of HH with clinical iron overload involves a combination of phlebotomy and/or chelation therapy. Liver transplantation should be considered for patients with HH-related decompensated cirrhosis.

Health economic evaluation of plasma oxysterol screening in the diagnosis of Niemann–Pick Type C disease among intellectually disabled using discrete event simulation

CDM van Karnebeek, Tima Mohammadi, Nicole Tsaod, Graham Sinclair, et al.
Molecular Genetics and Metabolism 114 (2015) 226–232
http://dx.doi.org/10.1016/j.ymgme.2014.07.004

Background: Recently a less invasive method of screening and diagnosing Niemann–Pick C (NP-C) disease has emerged. This approach involves the use of a metabolic screening test (oxysterol assay) instead of the current practice of clinical assessment of patients suspected of NP-C (review of medical history, family history and clinical examination for the signs and symptoms). Our objective is to compare costs and outcomes of plasma oxysterol screening versus current practice in diagnosis of NP-C disease among intellectually disabled (ID) patients using decision-analytic methods.
Methods: A discrete event simulation model was conducted to follow ID patients through the diagnosis and treatment of NP-C, forecast the costs and effectiveness for a cohort of ID patients and compare the outcomes and costs in two different arms of the model: plasma oxysterol screening and routine diagnosis procedure (anno 2013) over 5 years of follow up. Data from published sources and clinical trials were used in simulation model. Unit costs and quality-adjusted life-years (QALYs) were discounted at a 3% annual rate in the base case analysis. Deterministic and probabilistic sensitivity analyses were conducted.
Results: The outcomes of the base case model showed that using plasma oxysterol screening for diagnosis of NP-C disease among ID patients is a dominant strategy. It would result in lower total cost and would slightly improve patients’ quality of life. The average amount of cost saving was $3642 CAD and the incremental QALYs per each individual ID patient in oxysterol screening arm versus current practice of diagnosis NP-C was 0.0022 QALYs. Results of sensitivity analysis demonstrated robustness of the outcomes over the wide range of changes in model inputs.
Conclusion: Whilst acknowledging the limitations of this study, we conclude that screening ID children and adolescents with oxysterol tests compared to current practice for the diagnosis of NP-C is a dominant strategy with clinical and economic benefits. The less costly, more sensitive and specific oxysterol test has potential to save costs to the healthcare system while improving patients’ quality of life and may be considered as a routine tool in the NP-C diagnosis armamentarium for ID. Further research is needed to elucidate its effectiveness in patients presenting characteristics other than ID in childhood and adolescence.

Neurological and Behavioral Disorders

Estrogen receptor signaling during vertebrate development

Maria Bondesson, Ruixin Hao, Chin-Yo Lin, Cecilia Williams, Jan-Åke Gustafsson
Biochimica et Biophysica Acta 1849 (2015) 142–151
http://dx.doi.org/10.1016/j.bbagrm.2014.06.005

Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affectingboth the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

 

Memory function and hippocampal volumes in preterm born very-low-birth-weight (VLBW) young adults

Synne Aanes, Knut Jørgen Bjuland, Jon Skranes, Gro C.C. Løhaugen
NeuroImage 105 (2015) 76–83
http://dx.doi.org/10.1016/j.neuroimage.2014.10.023

The hippocampi are regarded as core structures for learning and memory functions, which is important for daily functioning and educational achievements. Previous studies have linked reduction in hippocampal volume to working memory problems in very low birth weight (VLBW; ≤1500 g) children and reduced general cognitive ability in VLBW adolescents. However, the relationship between memory function and hippocampal volume has not been described in VLBW subjects reaching adulthood. The aim of the study was to investigate memory function and hippocampal volume in VLBW young adults, both in relation to perinatal risk factors and compared to term born controls, and to look for structure–function relationships. Using Wechsler Memory Scale-III and MRI, we included 42 non-disabled VLBW and 61 control individuals at age 19–20 years, and related our findings to perinatal risk factors in the VLBW-group. The VLBW young adults achieved lower scores on several subtests of the Wechsler Memory Scale-III, resulting in lower results in the immediate memory indices (visual and auditory), the working memory index, and in the visual delayed and general memory delayed indices, but not in the auditory delayed and auditory recognition delayed indices. The VLBW group had smaller absolute and relative hippocampal volumes than the controls. In the VLBW group inferior memory function, especially for the working memory index, was related to smaller hippocampal volume, and both correlated with lower birth weight and more days in the neonatal intensive care unit (NICU). Our results may indicate a structural–functional relationship in the VLBW group due to aberrant hippocampal development and functioning after preterm birth.

The relation of infant attachment to attachment and cognitive and behavioural outcomes in early childhood

Yan-hua Ding, Xiu Xua, Zheng-yan Wang, Hui-rong Li, Wei-ping Wang
Early Human Development 90 (2014) 459–464
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.004

Background: In China, research on the relation of mother–infant attachment to children’s development is scarce.
Aims: This study sought to investigate the relation of mother–infant attachment to attachment, cognitive and behavioral development in young children.                                                                                                                            Study design: This study used a longitudinal study design.
Subjects: The subjects included healthy infants (n=160) aged 12 to 18 months.
Outcome measures: Ainsworth’s “Strange Situation Procedure” was used to evaluate mother–infant attachment types. The attachment Q-set (AQS) was used to evaluate the attachment between young children and their mothers. The Bayley scale of infant development-second edition (BSID-II) was used to evaluate cognitive developmental level in early childhood. Achenbach’s child behavior checklist (CBCL) for 2- to 3-year-oldswas used to investigate behavioral problems.
Results: In total, 118 young children (73.8%) completed the follow-up; 89.7% of infants with secure attachment and 85.0% of infants with insecure attachment still demonstrated this type of attachment in early childhood (κ = 0.738, p b 0.05). Infants with insecure attachment collectively exhibited a significantly lower mental development index (MDI) in early childhood than did infants with secure attachment, especially the resistant type. In addition, resistant infants were reported to have greater social withdrawal, sleep problems and aggressive behavior in early childhood.
Conclusion: There is a high consistency in attachment development from infancy to early childhood. Secure mother–infant attachment predicts a better cognitive and behavioral outcome; whereas insecure attachment, especially the resistant attachment, may lead to a lower cognitive level and greater behavioral problems in early childhood.

representations of the HPA axis

representations of the HPA axis

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

Fetal programming of schizophrenia: Select mechanisms

Monojit Debnatha, Ganesan Venkatasubramanian, Michael Berk
Neuroscience and Biobehavioral Reviews 49 (2015) 90–104
http://dx.doi.org/10.1016/j.neubiorev.2014.12.003

Mounting evidence indicates that schizophrenia is associated with adverse intrauterine experiences. An adverse or suboptimal fetal environment can cause irreversible changes in brain that can subsequently exert long-lasting effects through resetting a diverse array of biological systems including endocrine, immune and nervous. It is evident from animal and imaging studies that subtle variations in the intrauterine environment can cause recognizable differences in brain structure and cognitive functions in the offspring. A wide variety of environmental factors may play a role in precipitating the emergent developmental dysregulation and the consequent evolution of psychiatric traits in early adulthood by inducing inflammatory, oxidative and nitrosative stress (IO&NS) pathways, mitochondrial dysfunction, apoptosis, and epigenetic dysregulation. However, the precise mechanisms behind such relationships and the specificity of the risk factors for schizophrenia remain exploratory. Considering the paucity of knowledge on fetal programming of schizophrenia, it is timely to consolidate the recent advances in the field and put forward an integrated overview of the mechanisms associated with fetal origin of schizophrenia.

NMDA receptor dysfunction in autism spectrum disorders

Eun-Jae Lee, Su Yeon Choi and Eunjoon Kim
Current Opinion in Pharmacology 2015, 20:8–13
http://dx.doi.org/10.1016/j.coph.2014.10.007

Autism spectrum disorders (ASDs) represent neurodevelopmental disorders characterized by two core symptoms;

(1)  impaired social interaction and communication, and
(2)  restricted and repetitive behaviors, interests, and activities.

ASDs affect ~ 1% of the population, and are considered to be highly genetic in nature. A large number (~600) of ASD-related genetic variations have been identified (sfari.org), and target gene functions are apparently quite diverse. However, some fall onto common pathways, including synaptic function and chromosome remodeling, suggesting that core mechanisms may exist.

Abnormalities and imbalances in neuronal excitatory and inhibitory synapses have been implicated in diverse neuropsychiatric disorders including autism spectrum disorders (ASDs). Increasing evidence indicates that dysfunction of NMDA receptors (NMDARs) at excitatory synapses is associated with ASDs. In support of this, human ASD-associated genetic variations are found in genes encoding NMDAR subunits. Pharmacological enhancement or suppression of NMDAR function ameliorates ASD symptoms in humans. Animal models of ASD display bidirectional NMDAR dysfunction, and correcting this deficit rescues ASD-like behaviors. These findings suggest that deviation of NMDAR function in either direction contributes to the development of ASDs, and that correcting NMDAR dysfunction has therapeutic potential for ASDs.

Among known synaptic proteins implicated in ASD are metabotropic glutamate receptors (mGluRs). Functional enhancement and suppression of mGluR5 are associated with fragile X syndrome and tuberous sclerosis, respectively, which share autism as a common phenotype. More recently, ionotropic glutamate receptors, namely NMDA receptors (NMDARs) and AMPA receptors (AMPARs), have also been implicated in ASDs. In this review, we will focus on NMDA receptors and summarize evidence supporting the hypothesis that NMDAR dysfunction contributes to ASDs, and, by extension, that correcting NMDAR dysfunction has therapeutic potential for ASDs. ASD-related human NMDAR genetic variants.

Chemokines roles within the hippocampus

Chemokines roles within the hippocampus

IL-1 mediates stress-induced activation of the HPA axis

IL-1 mediates stress-induced activation of the HPA axis

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

Three Classes of Glutamate Receptors

Three Classes of Glutamate Receptors

Clinical studies on ASDs have identified genetic variants of NMDAR subunit genes. Specifically, de novo mutations have been identified in the GRIN2B gene, encoding the GluN2B subunit. In addition, SNP analyses have linked both GRIN2A (GluN2A subunit) and GRIN2B with ASDs. Because assembled NMDARs contain four subunits, each with distinct properties, ASD-related GRIN2A/ GRIN2B variants likely alter the functional properties of NMDARs and/or NMDAR-dependent plasticity.

Pharmacological modulation of NMDAR function can improve ASD symptoms. D-cycloserine (DCS), an NMDAR agonist, significantly ameliorates social withdrawal and repetitive behavior in individuals with ASD. These results suggest that reduced NMDAR function may contribute to the development of ASDs in humans.

We can divide animal studies into two groups. The first group consists of animals in which NMDAR modulators were shown to normalize both NMDAR dysfunction and ASD-like behaviors, establishing strong association between NMDARs and ASD phenotypes (Fig.). In the second group, NMDAR modulators were shown to rescue ASD-like behaviors, but NMDAR dysfunction and its correction have not been demonstrated.

ASD models with data showing rescue of both NMDAR dysfunction and ASD like behaviors Mice lacking neuroligin-1, an excitatory postsynaptic adhesion molecule, show reduced NMDAR function in the hippocampus and striatum, as evidenced by a decrease in NMDA/AMPA ratio and long-term potentiation (LTP). Neuroligin-1 is thought to enhance synaptic NMDAR function, by directly interacting with and promoting synaptic localization of NMDARs.

Fig not shown.

Bidirectional NMDAR dysfunction in animal models of ASD. Animal models of ASD with bidirectional NMDAR dysfunction can be positioned on either side of an NMDAR function curve. Model animals were divided into two groups.

Group 1: NMDAR modulators normalize both NMDAR dysfunction and ASD-like behaviors (green).

Group 2: NMDAR modulators rescue ASD-like behaviors, but NMDAR dysfunction and its rescue have not been demonstrated (orange). Note that Group 2 animals are tentatively placed on the left-hand side of the slope based on the observed DCS rescue of their ASD-like phenotypes, but the directions of their NMDAR dysfunctions remain to be experimentally determined.

ASD models with data showing rescue of ASD-like behaviors but no demonstrated NMDAR dysfunction

Tbr1 is a transcriptional regulator, one of whose targets is the gene encoding the GluN2B subunit of NMDARs. Mice haploinsufficient for Tbr1 (Tbr1+/-) show structural abnormalities in the amygdala and limited GluN2B induction upon behavioral stimulation. Both systemic injection and local amygdalar infusion of DCS rescue social deficits and impaired associative memory in Tbr1+/- mice. However, reduced NMDAR function and its DCS-dependent correction have not been demonstrated.

Spatial working memory and attention skills are predicted by maternal stress during pregnancy

André Plamondon, Emis Akbari, Leslie Atkinson, Meir Steiner
Early Human Development 91 (2015) 23–29
http://dx.doi.org/10.1016/j.earlhumdev.2014.11.004

Introduction: Experimental evidence in rodents shows that maternal stress during pregnancy (MSDP) negatively impacts spatial learning and memory in the offspring. We aim to investigate the association between MSDP (i.e., life events) and spatial working memory, as well as attention skills (attention shifting and attention focusing), in humans. The moderating roles of child sex, maternal anxiety during pregnancy and postnatal care are also investigated.  Methods: Participants were 236mother–child dyads that were followed from the second trimester of pregnancy until 4 years postpartum. Measurements included questionnaires and independent observations.
Results: MSDP was negatively associated with attention shifting at 18monthswhen concurrent maternal anxiety was low. MSDP was associated with poorer spatial working memory at 4 years of age, but only for boys who experienced poorer postnatal care.
Conclusion: Consistent with results observed in rodents, MSDP was found to be associated with spatial working memory and attention skills. These results point to postnatal care and maternal anxiety during pregnancy as potential targets for interventions that aim to buffer children from the detrimental effects of MSDP.

Acute and massive bleeding from placenta previa and infants’ brain damage

Ken Furuta, Shuichi Tokunaga, Seishi Furukawa, Hiroshi Sameshima
Early Human Development 90 (2014) 455–458
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.002

Background: Among the causes of third trimester bleeding, the impact of placenta previa on cerebral palsy is not well known.
Aims: To clarify the effect ofmaternal bleeding fromplacenta previa on cerebral palsy, and in particular when and how it occurs.
Study design: A descriptive study.
Subjects: Sixty infants born to mothers with placenta previa in our regional population-based study of 160,000 deliveries from 1998 to 2012. Premature deliveries occurring atb26 weeks of gestation and placenta accrete were excluded.
Outcome measures: Prevalence of cystic periventricular leukomalacia (PVL) and cerebral palsy (CP).
Results: Five infants had PVL and 4 of these infants developed CP (1/40,000 deliveries). Acute and massive bleeding (>500 g) within 8 h) occurred at around 30–31 weeks of gestation, and was severe enough to deliver the fetus. None of the 5 infants with PVL underwent antenatal corticosteroid treatment, and 1 infant had mild neonatal hypocapnia with a PaCO2 < 25 mm Hg. However, none of the 5 PVL infants showed umbilical arterial academia with pH < 7.2, an abnormal fetal heart rate monitoring pattern, or neonatal hypotension.
Conclusions: Our descriptive study showed that acute and massive bleeding from placenta previa at around 30 weeks of gestation may be a risk factor for CP, and requires careful neonatal follow-up. The underlying process connecting massive placental bleeding and PVL requires further investigation.

Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes

Courtney J. Wusthoff, Irene M. Loe
Seminars in Fetal & Neonatal Medicine 20 (2015) 52e57
http://dx.doi.org/10.1016/j.siny.2014.12.003

Extreme neonatal hyperbilirubinemia has long been known to cause the clinical syndrome of kernicterus, or chronic bilirubin encephalopathy (CBE). Kernicterus most usually is characterized by choreoathetoid cerebral palsy (CP), impaired upward gaze, and sensorineural hearing loss, whereas cognition is relatively spared. The chronic condition of kernicterus may be, but is not always, preceded in the acute stage by acute bilirubin encephalopathy (ABE). This acute neonatal condition is also due to hyperbilirubinemia, and is characterized by lethargy and abnormal behavior, evolving to frank neonatal encephalopathy, opisthotonus, and seizures. Less completely defined is the syndrome of bilirubin-induced neurologic dysfunction (BIND).

Bilirubin-induced neurologic dysfunction (BIND) is the constellation of neurologic sequelae following milder degrees of neonatal hyperbilirubinemia than are associated with kernicterus. Clinically, BIND may manifest after the neonatal period as developmental delay, cognitive impairment, disordered executive function, and behavioral and psychiatric disorders. However, there is controversy regarding the relative contribution of neonatal hyperbilirubinemia versus other risk factors to the development of later neurodevelopmental disorders in children with BIND. In this review, we focus on the empiric data from the past 25 years regarding neurodevelopmental outcomes and BIND, including specific effects on developmental delay, cognition, speech and language development, executive function, and the neurobehavioral disorders, such as attention deficit/hyperactivity disorder and autism.

As noted in a technical report by the American Academy of Pediatrics Subcommittee on Hyperbilirubinemia, “it is apparent that the use of a single total serum bilirubin level to predict long-term outcomes is inadequate and will lead to conflicting results”. As described above, this has certainly been the case in research to date. To clarify how hyperbilirubinemia influences neurodevelopmental outcome, more sophisticated consideration is needed both of how to assess bilirubin exposure leading to neurotoxicity, and of those comorbid conditions which may lower the threshold for brain injury.

For example, premature infants are known to be especially susceptible to bilirubin neurotoxicity, with kernicterus reported following TB levels far lower than the threshold expected in term neonates. Similarly, among extremely preterm neonates, BBC is proportional to gestational age, meaning that the most premature infants have the highest UB, even for similar TB levels. Thus, future studies must be adequately powered to examine preterm infants separately from term infants, and should consider not just peak TB, but also BBC, as independent variables in neonates with hyperbilirubinemia. Similarly, an analysis by the NICHD NRN found that, among ELBW infants, higher UB levels were associated with a higher risk of death or NDI. However, increased TB levels were only associated with death or NDI in unstable infants. Again, UB or BBC appeared to be more useful than TB.

Are the neuromotor disabilities of bilirubin-induced neurologic dysfunction disorders related to the cerebellum and its connections?

Jon F. Watchko, Michael J. Painter, Ashok Panigrahy
Seminars in Fetal & Neonatal Medicine 20 (2015) 47e51
http://dx.doi.org/10.1016/j.siny.2014.12.004

Investigators have hypothesized a range of subcortical neuropathology in the genesis of bilirubin induced neurologic dysfunction (BIND). The current review builds on this speculation with a specific focus on the cerebellum and its connections in the development of the subtle neuromotor disabilities of BIND. The focus on the cerebellum derives from the following observations:
(i) the cerebellum is vulnerable to bilirubin-induced injury; perhaps the most vulnerable region within the central nervous system;
(ii) infants with cerebellar injury exhibit a neuromotor phenotype similar to BIND; and                                                       (iii) the cerebellum has extensive bidirectional circuitry projections to motor and non-motor regions of the brain-stem and cerebral cortex that impact a variety of neurobehaviors.
Future study using advanced magnetic resonance neuroimaging techniques have the potential to shed new insights into bilirubin’s effect on neural network topology via both structural and functional brain connectivity measurements.

Bilirubin-induced neurologic damage is most often thought of in terms of severe adverse neuromotor (dystonia with or without athetosis) and auditory (hearing impairment or deafness) sequelae. Observed together, they comprise the classic neurodevelopmental phenotype of chronic bilirubin encephalopathy or kernicterus, and may also be seen individually as motor or auditory predominant subtypes. These injuries reflect both a predilection of bilirubin toxicity for neurons (relative to glial cells) and the regional topography of bilirubin-induced neuronal damage characterized by prominent involvement of the globus pallidus, subthalamic nucleus, VIII cranial nerve, and cochlear nucleus.

It is also asserted that bilirubin neurotoxicity may be associated with other less severe neurodevelopmental disabilities, a condition termed “subtle kernicterus” or “bilirubin-induced neurologic dysfunction” (BIND). BIND is defined by a constellation of “subtle neurodevelopmental disabilities without the classical findings of kernicterus that, after careful evaluation and exclusion of other possible etiologies, appear to be due to bilirubin neurotoxicity”. These purportedly include:

(i) mild-to-moderate disorders of movement (e.g., incoordination, clumsiness, gait abnormalities, disturbances in static and dynamic balance, impaired fine motor skills, and ataxia);                                                                                             (ii) disturbances in muscle tone; and
(iii) altered sensorimotor integration. Isolated disturbances of central auditory processing are also included in the spectrum of BIND.

  • Cerebellar vulnerability to bilirubin-induced injury
  • Cerebellar injury phenotypes and BIND
  • Cerebellar projections
Transverse section of cerebellum and brainstem

Transverse section of cerebellum and brainstem

Transverse section of cerebellum and brain-stem from a 34 gestational-week premature kernicteric infant formalin-fixed for two weeks. Yellow staining is evident in the cerebellar dentate nuclei (upper arrow) and vestibular nuclei at the pontomedullary junction (lower arrowhead). Photo is courtesy of Mahmdouha Ahdab-Barmada and reprinted with permission from Taylor-Francis Group (Ahdab Barmada M. The neuropathology of kernicterus: definitions and debate. In: Maisel MJ, Watchko JF editors. Neonatal jaundice. Amsterdam: Harwood Academic Publishers; 2000. p. 75e88

Whether cerebellar injury is primal or an integral part of disturbed neural circuitry in bilirubin-induced CNS damage is unclear. Movement disorders, however, are increasingly recognized to arise from abnormalities of neuronal circuitry rather than localized, circumscribed lesions. The cerebellum has extensive bidirectional circuitry projections to an array of brainstem nuclei and the cerebral cortex that modulate and refine motor activities. In this regard, the cerebellum is characteristically subdivided into three lobes based on neuroanatomic and phylogenetic criteria as well as by their primary afferent and efferent connections. They include:
(i) flocculonodular lobe (archicerebellum);
(ii) anterior lobe (paleocerebellum); and
(iii) posterior lobe (neocerebellum).

The archicerebellum, the oldest division phylogenically, receives extensive input from the vestibular system and is therefore also known as the vestibulocerebellum and is important for equilibrium control. The paleocerebellum, also a primitive region, receives extensive somatosensory input from the spinal cord, including the anterior and posterior spinocerebellar pathways that convey unconscious proprioception, and is therefore also known as the spinocerebellum. The neocerebellum is the most recently evolved region, receives most of the input from the cerebral cortex, and is thus termed the cerebrocerebellum. This area has greatly expanded in association with the extensive development of the cerebral cortex in mammals and especially primates. To cause serious longstanding dysfunction, cerebellar injury must typically involve the deep cerebellar nuclei and their projections.

Schematic of the bidirectional connectivity between the cerebellum and other

Schematic of the bidirectional connectivity between the cerebellum and other

Schematic of the bidirectional connectivity between the cerebellum and other brain regions including the cerebral cortex. Most cerebro-cerebellar afferent projections pass through the basal (anterior or ventral) pontine nuclei and intermediate cerebellar peduncle, whereas most cerebello-cerebral efferent projections pass through the dentate and ventrolateral thalamic nuclei. DCN, deep cerebellar nuclei; RN, red nucleus; ATN, anterior thalamic nucleus; PFC, prefrontal cortex; MC, motor cortex; PC, parietal cortex; TC, temporal cortex; STN, subthalamic nucleus; APN, anterior pontine nuclei. Reprinted under the terms of the Creative Commons Attribution License from D’Angelo E, Casali S. Seeking a unified framework for cerebellar function and dysfunction: from circuit to cognition. Front Neural Circuits 2013; 6:116.

Given the vulnerability of the cerebellum to bilirubin-induced injury, cerebellar involvement should also be evident in classic kernicterus, contributing to neuromotor deficits observed therein. It is of interest, therefore, that cerebellar damage may play a role in the genesis of bilirubin-induced dystonia, a prominent neuromotor feature of chronic bilirubin encephalopathy in preterm and term neonates alike. This complex movement disorder is characterized by involuntary sustained muscle contractions that result in abnormal position and posture. Moreover, dystonia that is brief in duration results in chorea, and, if brief and repetitive, leads to athetosis ‒ conditions also classically observed in kernicterus. Recent evidence suggests that dystonic movements may depend on disruption of both basal ganglia and cerebellar neuronal networks, rather than isolated dysfunction of only one motor system.

Dystonia is also a prominent feature in Gunn rat pups and neonatal Ugt1‒/‒-deficient mice both robust models of kernicterus. The former is used as an experimental model of dystonia. Although these models show basal ganglia injury, the sine qua non of bilirubin-induced murine neuropathology is cerebellar damage and resultant cerebellar hypoplasia.

Studies are needed to define more precisely the motor network abnormalities in kernicterus and BIND. Magnetic resonance imaging (MRI) has been widely used in evaluating infants at risk for bilirubin-induced brain injury using conventional structural T1-and T2-weighted imaging. Infants with chronic bilirubin encephalopathy often demonstrate abnormal bilateral, symmetric, high-signal intensity on T2-weighted MRI of the globus pallidus and subthalamic nucleus, consistent with the neuropathology of kernicterus. Early postnatal MRI of at-risk infants, although frequently showing increased T1-signal in these regions, may give false-positive findings due to the presence of myelin in these structures.

Diffusion tensor imaging and tractography could be used to delineate long-term changes involving specific white matter pathways, further elucidating the neural basis of long-term disability in infants and children with chronic bilirubin encephalopathy and BIND. It will be equally valuable to use blood oxygen level-dependent (BOLD) “resting state” functional MRI to study intrinsic connectivity in order to identify vulnerable brain networks in neonates with kernicterus and BIND. Structural networks of the CNS (connectome) and functional network topology can be characterized in infants with kernicterus and BIND to determine disease-related pattern(s) with respect to both long- and short-range connectivity. These findings have the potential to shed novel insights into the pathogenesis of these disorders and their impact on complex anatomical connections and resultant functional deficits.

Audiologic impairment associated with bilirubin-induced neurologic damage

Cristen Olds, John S. Oghalai
Seminars in Fetal & Neonatal Medicine 20 (2015) 42e46
http://dx.doi.org/10.1016/j.siny.2014.12.006

Hyperbilirubinemia affects up to 84% of term and late preterm infants in the first week of life. The elevation of total serum/plasma bilirubin (TB) levels is generally mild, transitory, and, for most children, inconsequential. However, a subset of infants experiences lifelong neurological sequelae. Although the prevalence of classic kernicterus has fallen steadily in the USA in recent years, the incidence of jaundice in term and premature infants has increased, and kernicterus remains a significant problem in the global arena. Bilirubin-induced neurologic dysfunction (BIND) is a spectrum of neurological injury due to acute or sustained exposure of the central nervous system(CNS) to bilirubin. The BIND spectrum includes kernicterus, acute bilirubin encephalopathy, and isolated neural pathway dysfunction.

Animal studies have shown that unconjugated bilirubin passively diffuses across cell membranes and the blood‒brain barrier (BBB), and bilirubin not removed by organic anion efflux pumps accumulates within the cytoplasm and becomes toxic. Exposure of neurons to bilirubin results in increased oxidative stress and decreased neuronal proliferation and presynaptic neuro-degeneration at central glutaminergic synapses. Furthermore, bilirubin administration results in smaller spiral ganglion cell bodies, with decreased cellular density and selective loss of large cranial nerve VIII myelinated fibers. When exposed to bilirubin, neuronal supporting cells have been found to secrete inflammatory markers, which contribute to increased BBB permeability and bilirubin loading.

The jaundiced Gunn rat is the classic animal model of bilirubin toxicity. It is homozygous for a premature stop codon within the gene for UDP-glucuronosyltransferase family 1 (UGT1). The resultant gene product has reduced bilirubin-conjugating activity, leading to a state of hyperbilirubinemia. Studies with this rat model have led to the concept that impaired calcium homeostasis is an important mechanism of neuronal toxicity, with reduced expression of calcium-binding proteins in affected cells being a sensitive index of bilirubin-induced neurotoxicity. Similarly, application of bilirubin to cultured auditory neurons from brainstem cochlear nuclei results in hyperexcitability and excitotoxicity.

The auditory pathway and normal auditory brainstem response (ABR).

The auditory pathway and normal auditory brainstem response (ABR).

The auditory pathway and normal auditory brain-stem response (ABR). The ipsilateral (green) and contralateral (blue) auditory pathways are shown, with structures that are known to be affected by hyperbilirubinemia highlighted in red. Roman numerals in parentheses indicate corresponding waves in the normal human ABR (inset). Illustration adapted from the “Ear Anatomy” series by Robert Jackler and Christine Gralapp, with permission.

Bilirubin-induced neurologic dysfunction (BIND)

Vinod K. Bhutani, Ronald Wong
Seminars in Fetal & Neonatal Medicine 20 (2015) 1
http://dx.doi.org/10.1016/j.siny.2014.12.010

Beyond the traditional recognized areas of fulminant injury to the globus pallidus as seen in infants with kernicterus, other vulnerable areas include the cerebellum, hippocampus, and subthalamic nuclear bodies as well as certain cranial nerves. The hippocampus is a brain region that is particularly affected by age related morphological changes. It is generally assumed that a loss in hippocampal volume results in functional deficits that contribute to age-related cognitive deficits. Lower grey matter volumes within the limbic-striato-thalamic circuitry are common to other etiological mechanisms of subtle neurologic injury. Lower grey matter volumes in the amygdala, caudate, frontal and medial gyrus are found in schizophrenia and in the putamen in autism. Thus, in terms of brain volumetrics, schizophrenia and autism spectrum disorders have a clear degree of overlap that may reflect shared etiological mechanisms. Overlap with injuries observed in infants with BIND raises the question about how these lesions are arrived at in the context of the impact of common etiologies.

Stress-induced perinatal and transgenerational epigenetic programming of brain development and mental health

Olena Babenko, Igor Kovalchuk, Gerlinde A.S. Metz
Neuroscience and Biobehavioral Reviews 48 (2015) 70–91
http://dx.doi.org/10.1016/j.neubiorev.2014.11.013

Research efforts during the past decades have provided intriguing evidence suggesting that stressful experiences during pregnancy exert long-term consequences on the future mental wellbeing of both the mother and her baby. Recent human epidemiological and animal studies indicate that stressful experiences in utero or during early life may increase the risk of neurological and psychiatric disorders, arguably via altered epigenetic regulation. Epigenetic mechanisms, such as miRNA expression, DNA methylation, and histone modifications are prone to changes in response to stressful experiences and hostile environmental factors. Altered epigenetic regulation may potentially influence fetal endocrine programming and brain development across several generations. Only recently, however, more attention has been paid to possible transgenerational effects of stress. In this review we discuss the evidence of transgenerational epigenetic inheritance of stress exposure in human studies and animal models. We highlight the complex interplay between prenatal stress exposure, associated changes in miRNA expression and DNA methylation in placenta and brain and possible links to greater risks of schizophrenia, attention deficit hyperactivity disorder, autism, anxiety- or depression-related disorders later in life. Based on existing evidence, we propose that prenatal stress, through the generation of epigenetic alterations, becomes one of the most powerful influences on mental health in later life. The consideration of ancestral and prenatal stress effects on lifetime health trajectories is critical for improving strategies that support healthy development and successful aging.

Sensitive time-windows for susceptibility in neurodevelopmental disorders

Rhiannon M. Meredith, Julia Dawitz and Ioannis Kramvis
Trends in Neurosciences, June 2012; 35(6): 335-344
http://dx.doi.org:/10.1016/j.tins.2012.03.005

Many neurodevelopmental disorders (NDDs) are characterized by age-dependent symptom onset and regression, particularly during early postnatal periods of life. The neurobiological mechanisms preceding and underlying these developmental cognitive and behavioral impairments are, however, not clearly understood. Recent evidence using animal models for monogenic NDDs demonstrates the existence of time-regulated windows of neuronal and synaptic impairments. We propose that these developmentally-dependent impairments can be unified into a key concept: namely, time-restricted windows for impaired synaptic phenotypes exist in NDDs, akin to critical periods during normal sensory development in the brain. Existence of sensitive time-windows has significant implications for our understanding of early brain development underlying NDDs and may indicate vulnerable periods when the brain is more susceptible to current therapeutic treatments.

Fig (not shown)

Misregulated mechanisms underlying spine morphology in NDDs. Several proteins implicated in monogenic NDDs (highlighted in red) are linked to the regulation of the synaptic cytoskeleton via F-actin through different Rho-mediated signaling pathways (highlighted in green). Mutations in OPHN1, TSC1/2, FMRP, p21-activated kinase (PAK) are directly linked to human NDDs of intellectual disability. For instance, point mutations in OPHN1 and a PAK isoform are linked to non-syndromic mental retardation, whereas mutations or altered expression of TSC1/2 and FMRP are linked to TSC and FXS, respectively. Cytoplasmic interacting protein (CYFIP) and LIM-domain kinase 1 (LIMK1) are known to interact with FMRP and PAK, respectively [105]. LIMK1 is one of many dysregulated proteins contributing to the NDD Williams syndrome. Mouse models are available for all highlighted (red) proteins and reveal specific synaptic and behavioral deficits. Local protein synthesis in synapses, dendrites and glia is also regulated by proteins such as TSC1/2 and the FMRP/CYFIP complex. Abbreviations: 4EBP, 4E binding protein; eIF4E, eukaryotic translation initiation factor 4E.

Fig (not shown)

Sensitive time-windows, synaptic phenotypes and NDD gene targets. Sensitive time-windows exist in neural circuits, during which gene targets implicated in NDDs are normally expressed. Misregulation of these genes can affect multiple synaptic phenotypes during a restricted developmental period. The effect upon synaptic phenotypes is dependent upon the temporal expression of these NDD genes and their targets. (a) Expression outside a critical period of development will have no effect upon synaptic phenotypes. (b,c) A temporal expression pattern that overlaps with the onset (b) or closure (c) of a known critical period can alter the synaptic phenotype during that developmental time-window.

Outstanding questions

(1) Can treatment at early presymptomatic stages in animal models for NDDs prevent or ease the later synaptic, neuronal, and behavioral impairments?

(2) Are all sensory critical periods equally misregulated in mouse models for a specific NDD? Are there different susceptibilities for auditory, visual and somatosensory neurocircuits that reflect the degree of impairments observed in patients?

(3) If one critical period is missed or delayed during formation of a layer-specific connection in a network, does the network overcome this misregulated connectivity or plasticity window?

(4) In monogenic NDDs, does the severity of misregulating one particular time-window for synaptic establishment during development correlate with the importance of that gene for that synaptic circuit?

(5) Why do critical periods close in brain development?

(6) What underlies the regression of some altered synaptic phenotypes in Fmr1-KO mice?

(7) Can the concept of susceptible time-windows be applied to other NDDs, including schizophrenia and Tourette’s syndrome?

Cardiovascular

Cardiac output monitoring in newborns

Willem-Pieter de Boode
Early Human Development 86 (2010) 143–148
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.032

There is an increased interest in methods of objective cardiac output measurement in critically ill patients. Several techniques are available for measurement of cardiac output in children, although this remains very complex in newborns. Cardiac output monitoring could provide essential information to guide hemodynamic management. An overview is given of various methods of cardiac output monitoring with advantages and major limitations of each technology together with a short explanation of the basic principles.

Fick principle

According to the Fick principle the volume of blood flow in a given period equals the amount of substance entering the blood stream in the same period divided by the difference in concentrations of the substrate upstream respectively downstream to the point of entry in the circulation. This substance can be oxygen (O2-Fick) or carbon dioxide (CO2-FICK), so cardiac output can be calculated by dividing measured pulmonary oxygen uptake by the arteriovenous oxygen concentration difference. The direct O2-Fick method is regarded as gold standard in cardiac output monitoring in a research setting, despite its limitations. When the Fick principle is applied for carbon dioxide (CO2 Fick), the pulmonary carbon dioxide exchange is divided by the venoarterial CO2 concentration difference to calculate cardiac output.

In the modified CO2 Fick method pulmonary CO2 exchange is measured at the endotracheal tube. Measurement of total CO2 concentration in blood is more complex and simultaneous sampling of arterial and central venous blood is required. However, frequent blood sampling will result in an unacceptable blood loss in the neonatal population.

Blood flow can be calculated if the change in concentration of a known quantity of injected indicator is measured in time distal to the point of injection, so an indicator dilution curve can be obtained. Cardiac output can then be calculated with the use of the Stewart–Hamilton equation. Several indicators are used, such as indocyanine green, Evans blue and brilliant red in dye dilution, cold solutions in thermodilution, lithium in lithium dilution, and isotonic saline in ultrasound dilution.

Cardiovascular adaptation to extra uterine life

Alice Lawford, Robert MR Tulloh
Paediatrics And Child Health 2014; 25(1): 1-6.

The adaptation to extra uterine life is of interest because of its complexity and the ability to cause significant health concerns. In this article we describe the normal changes that occur and the commoner abnormalities that are due to failure of normal development and the effect of congenital cardiac disease. Abnormal development may occur as a result of problems with the mother, or with the fetus before birth. After birth it is essential to determine whether there is an underlying abnormality of the fetal pulmonary or cardiac development and to determine the best course of management of pulmonary hypertension or congenital cardiac disease. Causes of underdevelopment, maldevelopment and maladaptation are described as are the causes of critical congenital heart disease. The methods of diagnosis and management are described to allow the neonatologist to successfully manage such newborns.

Fetal vascular structures that exist to direct blood flow

Fetal structure Function
Arterial duct Connects pulmonary artery to the aorta and shunts blood right to left; diverting flow away from fetal lungs
Foramen ovale Opening between the two atria thatdirects blood flow returning to right

atrium through the septal wall into the left atrium bypassing lungs

Ductus venosus Receives oxygenated blood fromumbilical vein and directs it to the

inferior vena cava and right atrium

Umbilical arteries Carrying deoxygenated blood fromthe fetus to the placenta
Umbilical vein Carrying oxygenated blood from theplacenta to the fetus

Maternal causes of congenital heart disease

Maternal disorders rubella, SLE, diabetes mellitus
Maternal drug use Warfarin, alcohol
Chromosomal abnormality Down, Edward, Patau, Turner, William, Noonan

 

Fetal and Neonatal Circulation  The fetal circulation is specifically adapted to efficiently exchange gases, nutrients, and wastes through placental circulation. Upon birth, the shunts (foramen ovale, ductus arteriosus, and ductus venosus) close and the placental circulation is disrupted, producing the series circulation of blood through the lungs, left atrium, left ventricle, systemic circulation, right heart, and back to the lungs.

Clinical monitoring of systemic hemodynamics in critically ill newborns

Willem-Pieter de Boode
Early Human Development 86 (2010) 137–141
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.031

Circulatory failure is a major cause of mortality and morbidity in critically ill newborn infants. Since objective measurement of systemic blood flow remains very challenging, neonatal hemodynamics is usually assessed by the interpretation of various clinical and biochemical parameters. An overview is given about the predictive value of the most used indicators of circulatory failure, which are blood pressure, heart rate, urine output, capillary refill time, serum lactate concentration, central–peripheral temperature difference, pH, standard base excess, central venous oxygen saturation and color.

Key guidelines

➢ The clinical assessment of cardiac output by the interpretation of indirect parameters of systemic blood flow is inaccurate, irrespective of the level of experience of the clinician

➢ Using blood pressure to diagnose low systemic blood flow will consequently mean that too many patients will potentially be undertreated or overtreated, both with substantial risk of adverse effects and iatrogenic damage.

➢ Combining different clinical hemodynamic parameters enhances the predictive value in the detection of circulatory failure, although accuracy is still limited.

➢ Variation in time (trend monitoring) might possibly be more informative than individual, static values of clinical and biochemical parameters to evaluate the adequacy of neonatal circulation.

Monitoring oxygen saturation and heart rate in the early neonatal period

J.A. Dawson, C.J. Morley
Seminars in Fetal & Neonatal Medicine 15 (2010) 203e207
http://dx.doi.org:/10.1016/j.siny.2010.03.004

Pulse oximetry is commonly used to assist clinicians in assessment and management of newly born infants in the delivery room (DR). In many DRs, pulse oximetry is now the standard of care for managing high risk infants, enabling immediate and dynamic assessment of oxygenation and heart rate. However, there is little evidence that using pulse oximetry in the DR improves short and long term outcomes. We review the current literature on using pulse oximetry to measure oxygen saturation and heart rate and how to apply current evidence to management in the DR.

Practice points

  • Understand how SpO2 changes in the first minutes after birth.
  • Apply a sensor to an infant’s right wrist as soon as possible after birth.
  • Attach sensor to infant then to oximeter cable.
  • Use two second averaging and maximum sensitivity.

Using pulse oximetry assists clinicians:

  1. Assess changes in HR in real time during transition.
  2. Assess oxygenation and titrate the administration of oxygen to maintain oxygenation within the appropriate range for SpO2 during the first minutes after birth.

Research directions

  • What are the appropriate centiles to target during the minutes after birth to prevent hypoxia and hyperoxia: 25th to 75th, or 10th to 90th, or just the 50th (median)?
  • Can the inspired oxygen be titrated against the SpO2 to keep the SpO2 in the ‘normal range’?
  • Does the use of centile charts in the DR for HR and oxygen saturation reduce the rate of hyperoxia when infants are treated with oxygen.
  • Does the use of pulse oximetry immediately after birth improve short term outcomes, e.g. efficacy of immediate respiratory support, intubation rates in the DR, percentage of inspired oxygen, rate of use of adrenalin or chest compressions, duration of hypoxia/hyperoxia and bradycardia.
  • Does the use of pulse oximetry in the DR improve short term respiratory and long term neurodevelopmental outcomes for preterm infants, e.g. rate of intubation, use of surfactant, and duration of ventilation, continuous positive airway pressure, or supplemental oxygen?
  • Can all modern pulse oximeters be used effectively in the DR or do some have a longer delay before giving an accurate signal and more movement artefact?
  • Would a longer averaging time result in more stable data?

Peripheral haemodynamics in newborns: Best practice guidelines

Michael Weindling, Fauzia Paize
Early Human Development 86 (2010) 159–165
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.033

Peripheral hemodynamics refers to blood flow, which determines oxygen and nutrient delivery to the tissues. Peripheral blood flow is affected by vascular resistance and blood pressure, which in turn varies with cardiac function. Arterial oxygen content depends on the blood hemoglobin concentration (Hb) and arterial pO2; tissue oxygen delivery depends on the position of the oxygen-dissociation curve, which is determined by temperature and the amount of adult or fetal hemoglobin. Methods available to study tissue perfusion include near-infrared spectroscopy, Doppler flowmetry, orthogonal polarization spectral imaging and the peripheral perfusion index. Cardiac function, blood gases, Hb, and peripheral temperature all affect blood flow and oxygen extraction. Blood pressure appears to be less important. Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing the intrathoracic pressure.

graphic

NIRS with partial venous occlusion to measure venous oxygen saturation

NIRS with partial venous occlusion to measure venous oxygen saturation

NIRS with partial venous occlusion to measure venous oxygen saturation. Taken from Yoxall and Weindling

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue

graphic

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue.  (a) oxygen delivery (DO2). (b) As DO2 decreases, VO2 is dependent on DO2. The slope of the line indicates the FOE, which in this case is about 0.50. (c) The slope of the line indicates the FOE in the normal situation where oxygenation is DO2 independent, usually < 0.35

The oxygen-dissociation curve

The oxygen-dissociation curve

graphic

The oxygen-dissociation curve

Considerable information about the response of the peripheral circulation has been obtained using NIRS with venous occlusion. Although these measurements were validated against blood co-oximetry in human adults and infants, they can only be made intermittently by a trained operator and are thus not appropriate for general clinical use. Further research is needed to find other better measures of peripheral perfusion and oxygenation which may be easily and continuously monitored, and which could be useful in a clinical setting.

Peripheral oxygenation and management in the perinatal period

Michael Weindling
Seminars in Fetal & Neonatal Medicine 15 (2010) 208e215
http://dx.doi.org:/10.1016/j.siny.2010.03.005

The mechanisms for the adequate provision of oxygen to the peripheral tissues are complex. They involve control of the microcirculation and peripheral blood flow, the position of the oxygen dissociation curve including the proportion of fetal and adult hemoglobin, blood gases and viscosity. Systemic blood pressure appears to have little effect, at least in the non-shocked state. The adequate delivery of oxygen (DO2) depends on consumption (VO2), which is variable. The balance between VO2 and DO2 is given by fractional oxygen extraction (FOE ¼ VO2/DO2). FOE varies from organ to organ and with levels of activity. Measurements of FOE for the whole body produce a range of about 0.15-0.33, i.e. the body consumes 15-33% of oxygen transported.

Fig (not shown)

Biphasic relationship between oxygen delivery (DO2) and oxygen consumption (VO2) in tissue. Dotted lines show fractional oxygen extraction (FOE). ‘A’ indicates the normal situation when VO2 is independent ofDO2 and FOE is about 0.30. AsDO2 decreases in the direction of the arrow, VO2 remains independent of DO2 until the critical point is reached at ‘B’; in this illustration, FOE is about 0.50. The slope of the dotted line indicates the FOE (¼ VO2/DO2), which increases progressively as DO2 decreases.

Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction

Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction

Graphic
(A)Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction in anaemic and control infants. (From Wardle et al.)  (B) HbF synthesis and concentration. (From Bard and Widness.) (C) Oxygen dissociation curve.

Peripheral fractional oxygen extraction in babies

Peripheral fractional oxygen extraction in babies

graphic

Peripheral fractional oxygen extraction in babies with asymptomatic or symptomatic anemia compared to controls. Bars represent the median for each group. (From Wardle et al.)

Practice points

  • Peripheral tissue DO2 is complex: cardiac function, blood gases, Hb concentration and the proportion of HbF, and peripheral temperature all play a part in determining blood flow and oxygen extraction in the sick, preterm infant. Blood pressure appears to be less important.
  • Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing intrathoracic pressure.
  • Central blood pressure is a poor surrogate measurement for the adequacy of DO2 to the periphery. Direct measurement, using NIRS, laser Doppler flowmetry or other means, may give more useful information.
  • Reasons for total hemoglobin concentration (Hb) being a relatively poor indicator of the adequacy of the provision of oxygen to the tissues:
  1. Hb is only indirectly related to red blood cell volume, which may be a better indicator of the body’s oxygen delivering capacity.
  2. Hb-dependent oxygen availability depends on the position of the oxygen-hemoglobin dissociation curve.
  3. An individual’s oxygen requirements vary with time and from organ to organ. This means that DO2 also needs to vary.
  4. It is possible to compensate for a low Hb by increasing cardiac output and ventilation, and so the ability to compensate for anemia depends on an individual’s cardio-respiratory reserve as well as Hb.
  5. The normal decrease of Hb during the first few weeks of life in both full-term and preterm babies usually occurs without symptoms or signs of anemia or clinical consequences.

The relationship between VO2 and DO2 is complex and various factors need to be taken into account, including the position of the oxygen dissociation curve, determined by the proportion of HbA and HbF, temperature and pH. Furthermore, diffusion of oxygen from capillaries to the cell depends on the oxygen tension gradient between erythrocytes and the mitochondria, which depends on microcirculatory conditions, e.g. capillary PO2, distance of the cell from the capillary (characterized by intercapillary distances) and the surface area of open capillaries. The latter can change rapidly, for example, in septic shock where arteriovenous shunting occurs associated with tissue hypoxia in spite of high DO2 and a low FOE.

Changes in local temperature deserve particular consideration. When the blood pressure is low, there may be peripheral vasoconstriction with decreased local perfusion and DO2. However, the fall in local tissue temperature would also be expected to be associated with a decreased metabolic rate and a consequent decrease in VO2. Thus a decreased DO2 may still be appropriate for tissue needs.

Pulmonary

Accurate Measurements of Oxygen Saturation in Neonates: Paired Arterial and Venous Blood Analyses

Shyang-Yun Pamela K. Shiao
Newborn and Infant Nurs Rev,  2005; 5(4): 170–178
http://dx.doi.org:/10.1053/j.nainr.2005.09.001

Oxygen saturation (So2) measurements (functional measurement, So2; and fractional measurement, oxyhemoglobin [Hbo2]) and monitoring are commonly investigated as a method of assessing oxygenation in neonates. Differences exist between the So2 and Hbo2 when blood tests are performed, and clinical monitors indicate So2 values. Oxyhemoglobin will decrease with the increased levels of carbon monoxide hemoglobin (Hbco) and methemo-globin (MetHb), and it is the most accurate measurements of oxygen (O2) association of hemoglobin (Hb). Pulse oximeter (for pulse oximetry saturation [Spo2] measurement) is commonly used in neonates. However, it will not detect the changes of Hb variations in the blood for accurate So2 measurements. Thus, the measurements from clinical oximeters should be used with caution. In neonates, fetal hemoglobin (HbF) accounts for most of the circulating Hb in their blood. Fetal hemoglobin has a high O2 affinity, thus releases less O2 to the body tissues, presenting a left-shifted Hbo2 dissociation curve.5,6 To date, however, limited data are available with HbF correction, for accurate arterial and venous (AV) So2 measurements (arterial oxygen saturation [Sao2] and venous oxygen saturation [Svo2]) in neonates, using paired AV blood samples.

In a study of critically ill adult patients, increased pulmonary CO production and elevation in arterial Hbco but not venous Hbco were documented by inflammatory stimuli inducing pulmonary heme oxygenase–1. In normal adults, venous Hbco level might be slightly higher than or equal to arterial Hbco because of production of CO by enzyme heme oxygenase–2, which is predominantly produced in the liver and spleen. However, hypoxia or pulmonary inflammation could induce heme oxygenase–1 to increase endogenous CO, thus elevating pulmonary arterial and systemic arterial Hbco levels in adults. Both endogenous and exogenous CO can suppress proliferation of pulmonary smooth muscles, a significant consideration for the prevention of chronic lung diseases in newborns. Despite these considerations, a later study in healthy adults indicated that the AV differences in Hbco were from technical artifacts and perhaps from inadequate control of different instruments. Thus, further studies are needed to provide more definitive answers for the AV differences of Hbco for adults and neonates with acute and chronic lung diseases.

Methemoglobin is an indicator of Hb oxidation and is essential for accurate measurement of Hbo2, So2, and oxygenation status. No evidence exists to show the AV MetHb difference, although this difference was elucidated with the potential changes of MetHb with different O2 levels.  Methemoglobin can be increased with nitric oxide (NO) therapy, used in respiratory distress syndrome (RDS) to reduce pulmonary hypertension and during heart surgery. Nitric oxide, in vitro, is an oxidant of Hb, with increased O2 during ischemia reperfusion. In hypoxemic conditions in vivo, nitrohemoglobin is a product generated by vessel responsiveness to nitrovasodilators. Nitro-hemoglobin can be spontaneously reversible in vivo, requiring no chemical agents or reductase. However, when O2 levels were increased experimentally in vitro following acidic conditions (pH 6.5) to simulate reperfusion conditions, MetHb levels were increased for the hemolysates (broken red cells). Nitrite-induced oxidation of Hb was associated with an increase in red blood cell membrane rigidity, thus contributing to Hb breakdown. A newer in vitro study of whole blood cells, however, concluded that MetHb formation is not dependent on increased O2 levels. Additional studies are needed to examine in vivo reperfusion of O2 and MetHb effects.

Purpose: The aim of this study was to examine the accuracy of arterial oxygen saturation (Sao2) and venous oxygen saturation (Svo2) with paired arterial and venous (AV) blood in relation to pulse oximetry saturation (Spo2) and oxyhemoglobin (Hbo2) with fetal hemoglobin determination, and their Hbo2 dissociation curves. Method: Twelve preterm neonates with gestational ages ranging from 27 to 34 weeks at birth, who had umbilical AV lines inserted, were investigated. Analyses were performed with 37 pairs of AV blood samples by using a blood volume safety protocol. Results: The mean differences between Sao2 and Svo2, and AV Hbo2 were both 6 percent (F6.9 and F6.7 percent, respectively), with higher Svo2 than those reported for adults. Biases were 2.1 – 0.49 for Sao2, 2.0 – 0.44 for Svo2, and 3.1 – 0.45 for Spo2, compared against Hbo2. With left-shifted Hbo2 dissociation curves in neonates, for the critical values of oxygen tension values between 50 and 75 millimeters of mercury, Hbo2 ranged from 92 to 93.4 percent; Sao2 ranged from 94.5 to 95.7 percent; and Spo2 ranged from 93.7 to 96.3 percent (compared to 85–94 percent in healthy adults). Conclusions: In neonates, both left-shifted Hbo2 dissociation curve and lower AV differences of oxygen saturation measurements indicated low flow of oxygen to the body tissues. These findings demonstrate the importance of accurate assessment of oxygenation statues in neonates.

In these neonates, the mean AV blood differences for both So2 and Hbo2 were about 6 percent, which was much lower than those reported for healthy adults (23 percent) for O2 supply and demand. In addition, with very high levels of HbF releasing less O2 to the body tissue, the results of blood analyses are worrisome for these critically ill neonates for low systemic oxygen states.  O’Connor and Hall determined AV So2 in neonates without HbF determination. Much of the AV So2 difference is dependent on Svo2 measurement. The ranges of Svo2 spanned for 35 percent, and the ranges of Sao2 spanned 6 percent in these neonates. The greater intervals for Svo2 measurements contribute to greater sensitivity for the measurements (than Sao2 measurements) in responding to nursing care and changes of O2 demand. Thus, Svo2 measurement is essential for better assessment of oxygenation status in neonates.

The findings of this study on AV differences of So2 were limited with very small number of paired AV blood samples. However, critically ill neonates need accurate assessment of oxygenation status because of HbF, which releases less O2 to the tissues. Decreased differences of AV So2 measurements added further possibilities of lower flow of O2 to the body tissues and demonstrated the greater need to accurately assess the proper oxygenation in the neonates. The findings of this study continued to clarify the accuracy of So2 measurements for neonates. Additional studies are needed to examine So2 levels in neonates to further validate these findings by using larger sample sizes.

Neonatal ventilation strategies and long-term respiratory outcomes

Sandeep Shetty, Anne Greenough
Early Human Development 90 (2014) 735–739
http://dx.doi.org/10.1016/j.earlhumdev.2014.08.020

Long-term respiratory morbidity is common, particularly in those born very prematurely and who have developed bronchopulmonary dysplasia (BPD), but it does occur in those without BPD and in infants born at term. A variety of neonatal strategies have been developed, all with short-term advantages, but meta-analyses of randomized controlled trials (RCTs) have demonstrated that only volume-targeted ventilation and prophylactic high-frequency oscillatory ventilation (HFOV) may reduce BPD. Few RCTs have incorporated long-term follow-up, but one has demonstrated that prophylactic HFOV improves respiratory and functional outcomes at school age, despite not reducing BPD. Results from other neonatal interventions have demonstrated that any impact on BPD may not translate into changes in long-term outcomes. All future neonatal  ventilation RCTs should have long-term outcomes rather than BPD as their primary outcome if they are to impact on clinical practice.

A Model Analysis of Arterial Oxygen Desaturation during Apnea in Preterm Infants

Scott A. Sands, BA Edwards, VJ Kelly, MR Davidson, MH Wilkinson, PJ Berger
PLoS Comput Biol 5(12): e1000588
http://dx.doi.org:/10.1371/journal.pcbi.1000588

Rapid arterial O2 desaturation during apnea in the preterm infant has obvious clinical implications but to date no adequate explanation for why it exists. Understanding the factors influencing the rate of arterial O2 desaturation during apnea (_SSaO2 ) is complicated by the non-linear O2 dissociation curve, falling pulmonary O2 uptake, and by the fact that O2 desaturation is biphasic, exhibiting a rapid phase (stage 1) followed by a slower phase when severe desaturation develops (stage 2). Using a mathematical model incorporating pulmonary uptake dynamics, we found that elevated metabolic O2 consumption accelerates _SSaO2 throughout the entire desaturation process. By contrast, the remaining factors have a restricted temporal influence: low pre-apneic alveolar PO2 causes an early onset of desaturation, but thereafter has little impact; reduced lung volume, hemoglobin content or cardiac output, accelerates _SSaO2 during stage 1, and finally, total blood O2 capacity (blood volume and hemoglobin content) alone determines _SSaO2 during stage 2. Preterm infants with elevated metabolic rate, respiratory depression, low lung volume, impaired cardiac reserve, anemia, or hypovolemia, are at risk for rapid and profound apneic hypoxemia. Our insights provide a basic physiological framework that may guide clinical interpretation and design of interventions for preventing sudden apneic hypoxemia.

A novel approach to study oxidative stress in neonatal respiratory distress syndrome

Reena Negi, D Pande, K Karki, A Kumar, RS Khanna, HD Khanna
BBA Clinical 3 (2015) 65–69
http://dx.doi.org/10.1016/j.bbacli.2014.12.001

Oxidative stress is an imbalance between the systemic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage. It is a physiological event in the fetal-to-neonatal transition, which is actually a great stress to the fetus. These physiological changes and processes greatly increase the production of free radicals, which must be controlled by the antioxidant defense system, the maturation of which follows the course of the gestation. This could lead to several functional alterations with important repercussions for the infants. Adequately mature and healthy infants are able to tolerate this drastic change in the oxygen concentration. A problem occurs when the intrauterine development is incomplete or abnormal. Preterm or intrauterine growth retarded (IUGR) and low birth weight neonates are typically of this kind. An oxidant/antioxidant imbalance in infants is implicated in the pathogenesis of the major complications of prematurity including respiratory distress syndrome (RDS), necrotizing enterocolitis (NEC), chronic lung disease, retinopathy of prematurity and intraventricular hemorrhage (IVH).

Background: Respiratory distress syndrome of the neonate (neonatal RDS) is still an important problem in treatment of preterm infants. It is accompanied by inflammatory processes with free radical generation and oxidative stress. The aim of study was to determine the role of oxidative stress in the development of neonatal RDS. Methods: Markers of oxidative stress and antioxidant activity in umbilical cord blood were studied in infants with neonatal respiratory distress syndrome with reference to healthy newborns. Results: Status of markers of oxidative stress (malondialdehyde, protein carbonyl and 8-hydroxy-2-deoxy guanosine) showed a significant increase with depleted levels of total antioxidant capacity in neonatal RDS when compared to healthy newborns. Conclusion: The study provides convincing evidence of oxidative damage and diminished antioxidant defenses in newborns with RDS. Neonatal RDS is characterized by damage of lipid, protein and DNA, which indicates the augmentation of oxidative stress. General significance: The identification of the potential biomarker of oxidative stress consists of a promising strategy to study the pathophysiology of neonatal RDS.

Neonatal respiratory distress syndrome represents the major lung complications of newborn babies. Preterm neonates suffer from respiratory distress syndrome (RDS) due to immature lungs and require assisted ventilation with high concentrations of oxygen. The pathogenesis of this disorder is based on the rapid formation of the oxygen reactive species, which surpasses the detoxification capacity of antioxidative defense system. The high chemical reactivity of free radical leads to damage to a variety of cellular macro molecules including proteins, lipids and nucleic acid. This results in cell injury and may induce respiratory cell death.

Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation. The present study showed increased concentration of MDA in neonates with respiratory disorders than that of control in consonance with the reported study.

Anemia, Apnea of Prematurity, and Blood Transfusions

Kelley Zagol, Douglas E. Lake, Brooke Vergales, Marion E. Moorman, et al
J Pediatr 2012;161:417-21
http://dx.doi.org:/10.1016/j.jpeds.2012.02.044

The etiology of apnea of prematurity is multifactorial; however, decreased oxygen carrying capacity may play a role. The respiratory neuronal network in neonates is immature, particularly in those born preterm, as demonstrated by their paradoxical response to hypoxemia. Although adults increase the minute ventilation in response to hypoxemia, newborns have a brief increase in ventilation followed by periodic breathing, respiratory depression, and occasionally cessation of respiratory effort. This phenomenon may be exacerbated by anemia in preterm newborns, where a decreased oxygen carrying capacity may result in decreased oxygen delivery to the central nervous system, a decreased efferent output of the respiratory neuronal network, and an increase in apnea.

Objective Compare the frequency and severity of apneic events in very low birth weight (VLBW) infants before and after blood transfusions using continuous electronic waveform analysis. Study design We continuously collected waveform, heart rate, and oxygen saturation data from patients in all 45 neonatal intensive care unit beds at the University of Virginia for 120 weeks. Central apneas were detected using continuous computer processing of chest impedance, electrocardiographic, and oximetry signals. Apnea was defined as respiratory pauses of >10, >20, and >30 seconds when accompanied by bradycardia (<100 beats per minute) and hypoxemia (<80% oxyhemoglobin saturation as detected by pulse oximetry). Times of packed red blood cell transfusions were determined from bedside charts. Two cohorts were analyzed. In the transfusion cohort, waveforms were analyzed for 3 days before and after the transfusion for all VLBW infants who received a blood transfusion while also breathing spontaneously. Mean apnea rates for the previous 12 hours were quantified and differences for 12 hours before and after transfusion were compared. In the hematocrit cohort, 1453 hematocrit values from all VLBW infants admitted and breathing spontaneously during the time period were retrieved, and the association of hematocrit and apnea in the next 12 hours was tested using logistic regression. Results Sixty-seven infants had 110 blood transfusions during times when complete monitoring data were available. Transfusion was associated with fewer computer-detected apneic events (P < .01). Probability of future apnea occurring within 12 hours increased with decreasing hematocrit values (P < .001). Conclusions Blood transfusions are associated with decreased apnea in VLBW infants, and apneas are less frequent at higher hematocrits.

Bronchopulmonary dysplasia: The earliest and perhaps the longest lasting obstructive lung disease in humans

Silvia Carraro, M Filippone, L Da Dalt, V Ferraro, M Maretti, S Bressan, et al.
Early Human Development 89 (2013) S3–S5
http://dx.doi.org/10.1016/j.earlhumdev.2013.07.015

Bronchopulmonary dysplasia (BPD) is one of the most important sequelae of premature birth and the most common form of chronic lung disease of infancy, an umbrella term for a number of different diseases that evolve as a consequence of a neonatal respiratory disorder. BPD is defined as the need for supplemental oxygen for at least 28 days after birth, and its severity is graded according to the respiratory support required at 36 post-menstrual weeks.

BPD was initially described as a chronic respiratory disease occurring in premature infants exposed to mechanical ventilation and oxygen supplementation. This respiratory disease (later named “old BPD”) occurred in relatively large premature newborn and, from a pathological standpoint, it was characterized by intense airway inflammation, disruption of normal pulmonary structures and lung fibrosis.

Bronchopulmonary dysplasia (BPD) is one of the most important sequelae of premature birth and the most common form of chronic lung disease of infancy. From a clinical standpoint BPD subjects are characterized by recurrent respiratory symptoms, which are very frequent during the first years of life and, although becoming less severe as children grow up, they remain more common than in term-born controls throughout childhood, adolescence and into adulthood. From a functional point of view BPD subjects show a significant airflow limitation that persists during adolescence and adulthood and they may experience an earlier and steeper decline in lung function during adulthood. Interestingly, patients born prematurely but not developing BPD usually fare better, but they too have airflow limitations during childhood and later on, suggesting that also prematurity per se has life-long detrimental effects on pulmonary function. For the time being, little is known about the presence and nature of pathological mechanisms underlying the clinical and functional picture presented by BPD survivors. Nonetheless, recent data suggest the presence of persistent neutrophilic airway inflammation and oxidative stress and it has been suggested that BPD may be sustained in the long term by inflammatory pathogenic mechanisms similar to those underlying COPD. This hypothesis is intriguing but more pathological data are needed.  A better understanding of these pathogenetic mechanisms, in fact, may be able to orient the development of novel targeted therapies or prevention strategies to improve the overall respiratory health of BPD patients.

We have a limited understanding of the presence and nature of pathological mechanisms in the lung of BPD survivors. The possible role of asthma-like inflammation has been investigated because BPD subjects often present with recurrent wheezing and other symptoms resembling asthma during their childhood and adolescence. But BPD subjects have normal or lower than normal exhaled nitric oxide levels and exhaled air temperatures, whereas they are higher than normal in asthmatic patients.

Of all obstructive lung diseases in humans, BPD has the earliest onset and is possibly the longest lasting. Given its frequent association with other conditions related to preterm birth (e.g. growth retardation, pulmonary hypertension, neurodevelopmental delay, hearing defects, and retinopathy of prematurity), it often warrants a multidisciplinary management.

Effects of Sustained Lung Inflation, a lung recruitment maneuver in primary acute respiratory distress syndrome, in respiratory and cerebral outcomes in preterm infants

Chiara Grasso, Pietro Sciacca, Valentina Giacchi, Caterina Carpinato, et al.
Early Human Development 91 (2015) 71–75
http://dx.doi.org/10.1016/j.earlhumdev.2014.12.002

Background: Sustained Lung Inflation (SLI) is a maneuver of lung recruitment in preterm newborns at birth that can facilitate the achieving of larger inflation volumes, leading to the clearance of lung fluid and formation of functional residual capacity (FRC). Aim: To investigate if Sustained Lung Inflation (SLI) reduces the need of invasive procedures and iatrogenic risks. Study design: 78 newborns (gestational age ≤ 34 weeks, weighing ≤ 2000 g) who didn’t breathe adequately at birth and needed to receive SLI in addition to other resuscitation maneuvers (2010 guidelines). Subjects: 78 preterm infants born one after the other in our department of Neonatology of Catania University from 2010 to 2012. Outcome measures: The need of intubation and surfactant, the ventilation required, radiological signs, the incidence of intraventricular hemorrhage (IVH), periventricular leukomalacia, retinopathy in prematurity from III to IV plus grades, bronchopulmonary dysplasia, patent ductus arteriosus, pneumothorax and necrotizing enterocolitis. Results: In the SLI group infants needed less intubation in the delivery room (6% vs 21%; p b 0.01), less invasive mechanical ventilation (14% vs 55%; p ≤ 0.001) and shorter duration of ventilation (9.1 days vs 13.8 days; p ≤ 0.001). There wasn’t any difference for nasal continuous positive airway pressure (82% vs 77%; p = 0.43); but there was less surfactant administration (54% vs 85%; p ≤ 0.001) and more infants received INSURE (40% vs 29%; p=0.17). We didn’t found any differences in the outcomes, except for more mild intraventricular hemorrhage in the SLI group (23% vs 14%; p = 0.15; OR= 1.83). Conclusion: SLI is easier to perform even with a single operator, it reduces the necessity of more complicated maneuvers and surfactant without statistically evident adverse effects.

Long-term respiratory consequences of premature birth at less than 32 weeks of gestation

Anne Greenough
Early Human Development 89 (2013) S25–S27
http://dx.doi.org/10.1016/j.earlhumdev.2013.07.004

Chronic respiratory morbidity is a common adverse outcome of very premature birth, particularly in infants who had developed bronchopulmonary dysplasia (BPD). Prematurely born infants who had BPD may require supplementary oxygen at home for many months and affected infants have increased healthcare utilization until school age. Chest radiograph abnormalities are common; computed tomography of the chest gives predictive information in children with ongoing respiratory problems. Readmission to hospital is common, particularly for those who have BPD and suffer respiratory syncytial virus lower respiratory infections (RSV LRTIs). Recurrent respiratory symptoms requiring treatment are common and are associated with evidence of airways obstruction and gas trapping. Pulmonary function improves with increasing age, but children with BPD may have ongoing airflow limitation. Lung function abnormalities may be more severe in those who had RSV LRTIs, although this may partly be explained by worse premorbid lung function. Worryingly, lung function may deteriorate during the first year. Longitudinal studies are required to determine if there is catch up growth.

Long-term pulmonary outcomes of patients with bronchopulmonary dysplasia

Anita Bhandari and Sharon McGrath-Morrow
Seminars in Perinatology 37 (2013)132–137
http://dx.doi.org/10.1053/j.semperi.2013.01.010

Bronchopulmonary dysplasia (BPD) is the commonest cause of chronic lung disease in infancy. The incidence of BPD has remained unchanged despite many advances in neonatal care. BPD starts in the neonatal period but its effects can persist long term. Premature infants with BPD have a greater incidence of hospitalization, and continue to have a greater respiratory morbidity and need for respiratory medications, compared to those without BPD. Lung function abnormalities, especially small airway abnormalities, often persist. Even in the absence of clinical symptoms, BPD survivors have persistent radiological abnormalities and presence of emphysema has been reported on chest computed tomography scans. Concern regarding their exercise tolerance remains. Long-term effects of BPD are still unknown, but given reports of a more rapid decline in lung function and their susceptibility to develop chronic obstructive pulmonary disease phenotype with aging, it is imperative that lung function of survivors of BPD be closely monitored.

Neonatal ventilation strategies and long-term respiratory outcomes

Sandeep Shetty, Anne Greenough
Early Human Development 90 (2014) 735–739
http://dx.doi.org/10.1016/j.earlhumdev.2014.08.020

Long-term respiratory morbidity is common, particularly in those born very prematurely and who have developed bronchopulmonary dysplasia (BPD), but it does occur in those without BPD and in infants born at term. A variety of neonatal strategies have been developed, all with short-term advantages, but meta-analyses of randomized controlled trials (RCTs) have demonstrated that only volume-targeted ventilation and prophylactic high-frequency oscillatory ventilation (HFOV) may reduce BPD. Few RCTs have incorporated long-term follow-up, but one has demonstrated that prophylactic HFOV improves respiratory and functional outcomes at school age, despite not reducing BPD. Results from other neonatal interventions have demonstrated that any impact on BPD may not translate into changes in long-term outcomes. All future neonatal ventilation RCTs should have long-term outcomes rather than BPD as their primary outcome if they are to impact on clinical practice.

Prediction of neonatal respiratory distress syndrome in term pregnancies by assessment of fetal lung volume and pulmonary artery resistance index

Mohamed Laban, GM Mansour, MSE Elsafty, AS Hassanin, SS EzzElarab
International Journal of Gynecology and Obstetrics 128 (2015) 246–250
http://dx.doi.org/10.1016/j.ijgo.2014.09.018

Objective: To develop reference cutoff values for mean fetal lung volume (FLV) and pulmonary artery resistance index (PA-RI) for prediction of neonatal respiratory distress syndrome (RDS) in low-risk term pregnancies. Methods: As part of a cross-sectional study, women aged 20–35 years were enrolled and admitted to a tertiary hospital in Cairo, Egypt, for elective repeat cesarean at 37–40 weeks of pregnancy between January 1, 2012, and July 31, 2013. FLV was calculated by virtual organ computer-aided analysis, and PA-RI was measured by Doppler ultrasonography before delivery. Results: A total of 80 women were enrolled. Neonatal RDS developed in 11 (13.8%) of the 80 newborns. Compared with neonates with RDS, healthy neonates had significantly higher FLVs (P b 0.001) and lower PA-RIs (P b 0.001). Neonatal RDS is less likely with FLV of at least 32 cm3 or PA-RI less than or equal to 0.74. Combining these two measures improved the accuracy of prediction. Conclusion: The use of either FLV or PA-RI predicted neonatal RDS. The predictive value increased when these two measures were combined

Pulmonary surfactant - a front line of lung host defense, 2003 JCI0318650.f2

Pulmonary surfactant – a front line of lung host defense, 2003 JCI0318650.f2

Pulmonary hypertension in bronchopulmonary dysplasia

Sara K.Berkelhamer, Karen K.Mestan, and Robin H. Steinhorn
Seminars In  Perinatology 37 (2013)124–131
http://dx.doi.org/10.1053/j.semperi.2013.01.009

Pulmonary hypertension (PH) is a common complication of neonatal respiratory diseases, including bronchopulmonary dysplasia (BPD), and recent studies have increased aware- ness that PH worsens the clinical course, morbidity and mortality of BPD. Recent evidence indicates that up to 18% of all extremely low-birth-weight infants will develop some degree of PH during their hospitalization, and the incidence rises to 25–40% of the infants with established BPD. Risk factors are not yet well understood, but new evidence shows that fetal growth restriction is a significant predictor of PH. Echocardiography remains the primary method for evaluation of BPD-associated PH, and the development of standardized screening timelines and techniques for identification of infants with BPD-associated PH remains an important ongoing topic of investigation. The use of pulmonary vasodilator medications, such as nitric oxide, sildenafil, and others, in the BPD population is steadily growing, but additional studies are needed regarding their long-term safety and efficacy.
An update on pharmacologic approaches to bronchopulmonary dysplasia

Sailaja Ghanta, Kristen Tropea Leeman, and Helen Christou
Seminars In Perinatology 37 (2013)115–123
http://dx.doi.org/10.1053/j.semperi.2013.01.008

Bronchopulmonary dysplasia (BPD) is the most prevalent long-term morbidity in surviving extremely preterm infants and is linked to increased risk of reactive airways disease, pulmonary hypertension, post-neonatal mortality, and adverse neurodevelopmental outcomes. BPD affects approximately 20% of premature newborns, and up to 60% of premature infants born before completing 26 weeks of gestation. It is characterized by the need for assisted ventilation and/or supplemental oxygen at 36 weeks postmenstrual age. Approaches to prevention and treatment of BPD have evolved with improved understanding of its pathogenesis. This review will focus on recent advancements and detail current research in pharmacotherapy for BPD. The evidence for both current and potential future experimental therapies will be reviewed in detail. As our understanding of the complex and multifactorial pathophysiology of BPD changes, research into these current and future approaches must continue to evolve.

Methylxanthines
Diuretics and bronchodilators
Corticosteroids
Macrolide antibiotics
Recombinant human Clara cell 10-kilodalton protein(rhCC10)
Vitamin A
Surfactant
Leukotriene receptor antagonist
Pulmonary vasodilators

Skeletal and Muscle

Skeletal Stem Cells in Space and Time

Moustapha Kassem and Paolo Bianco
Cell  Jan 15, 2015; 160: 17-19
http://dx.doi.org/10.1016/j.cell.2014.12.034

The nature, biological characteristics, and contribution to organ physiology of skeletal stem cells are not completely determined. Chan et al. and Worthley et al. demonstrate that a stem cell for skeletal tissues, and a system of more restricted, downstream progenitors, can be identified in mice and demonstrate its role in skeletal tissue maintenance and regeneration.

The groundbreaking concept that bone, cartilage, marrow adipocytes, and hematopoiesis-supporting stroma could originate from a common progenitor and putative stem cell was surprising at the time when it was formulated (Owen and Friedenstein, 1988). The putative stem cell, nonhematopoietic in nature, would be found in the postnatal bone marrow stroma, generate tissues previously thought of as foreign to each other, and support the turnover of tissues and organs that self-renew at a much slower rate compared to other tissues associated with stem cells (blood, epithelia). This concept also connected bone and bone marrow as parts of a single-organ system, implying their functional interplay. For many years, the evidence underpinning the concept has been incomplete.

While multipotency of stromal progenitors has been demonstrated by in vivo transplantation experiments, self-renewal, the defining property of a stem cell, has not been easily demonstrated until recently in humans (Sacchetti et al., 2007) and mice (Mendez-Ferrer et al., 2010). Meanwhile, a confusing and plethoric terminology has been introduced into the literature, which diverted and confounded the search for a skeletal stem cell and its physiological significance (Bianco et al., 2013).

Two studies in this issue of Cell (Chan et al., 2015; Worthley et al., 2015), using a combination of rigorous single-cell analyses and lineage tracing technologies, mark significant steps toward rectifying the course of skeletal stem cell discovery by making several important points, within and beyond skeletal physiology.

First, a stem cell for skeletal tissues, and a system of more restricted, downstream progenitors can in fact be identified and linked to defined phenotype(s) in the mouse. The system is framed conceptually, and approached experimentally, similar to the hematopoietic system.

Second, based on its assayable functions and potential, the stem cell at the top of the hierarchy is defined as a skeletal stem cell (SSC). As noted earlier (Sacchetti et al., 2007) (Bianco et al., 2013), this term clarifies, well beyond semantics, that the range of tissues that the self-renewing stromal progenitor (originally referred to as an ‘‘osteogenic’’ or ‘‘stromal’’ stem cell) (Owen and Friedenstein, 1988) can actually generate in vivo, overlaps with the range of tissues that make up the skeleton.

Third, these cells are spatially restricted, local residents of the bone/bone marrow organ. The systemic circulation is not a sizable contributor to their recruitment to locally deployed functions.

Fourth, a native skeletogenic potential is inherent to the system of progenitor/ stem cells found in the skeleton, and internally regulated by bone morphogenetic protein (BMP) signaling. This is reflected in the expression of regulators and antagonists of BMP signaling within the system, highlighting potential feedback mechanisms modulating expansion or quiescence of specific cell compartments.

Fifth, in cells isolated from other tissues, an assayable skeletogenic potential is not inherent: it can only be induced de novo by BMP reprogramming. These two studies (Chan et al., 2015, Worthley et al., 2015) corroborate the classical concept of ‘‘determined’’ and ‘‘inducible’’ skeletal progenitors (Owen and Friedenstein, 1988): the former residing in the skeleton, the latter found in nonskeletal tissues; the former capable of generating skeletal tissues, in vivo and spontaneously, the latter requiring reprogramming signals in order to acquire a skeletogenic capacity; the former operating in physiological bone formation, the latter in unwanted, ectopic bone formation in diseases such as fibrodysplasia ossificans progressiva.

To optimize our ability to obtain specific skeletal tissues for medical application, the study by Chan et al. offers a glimpse of another facet of the biology of SSC lineages and progenitors. Chan et al. show that a homogeneous cell population inherently committed to chondrogenesis can alter its output to generate bone if cotransplanted with multipotent progenitors. Conversely, osteogenic cells can be shifted to a chondrogenic fate by blockade of vascular endothelial growth factor receptor, consistent with the avascular and hypoxic milieu of cartilage. This has two important implications:

  • commitment is flexible in the system;
  • the choir is as important as the soloist and can modulate the solo tune.

Reversibility and population behavior thus emerge as two features that may be characteristic, albeit not unique, of the stromal system, resonating with conceptually comparable evidence in the human system.

The two studies by Chan et al. and Worthely et al. emphasize the relevance not only of their new data, but also of a proper concept of a skeletal stem cell per se, for proper clinical use. Confusion arising from improper conceptualization of skeletal stem cells has markedly limited clinical development of skeletal stem cell biology.

Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

Daniel L. Worthley, Michael Churchill, Jocelyn T. Compton, Yagnesh Tailor, et al.
Cell, Jan 15, 2015; 160: 269–284
http://dx.doi.org/10.1016/j.cell.2014.11.042

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Identification and Specification of the Mouse Skeletal Stem Cell

Charles K.F. Chan, Eun Young Seo, James Y. Chen, David Lo, A McArdle, et al.
Cell, Jan 15, 2015; 160: 285–298
http://dx.doi.org/10.1016/j.cell.2014.12.002

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Bone mesenchymal development

Bone mesenchymal development

Bone mesenchymal development

The bone-remodeling cycle

The bone-remodeling cycle

Nuclear receptor modulation – Role of coregulators in selective estrogen receptor modulator (SERM) actions

Qin Feng, Bert W. O’Malley
Steroids 90 (2014) 39–43
http://dx.doi.org/10.1016/j.steroids.2014.06.008

Selective estrogen receptor modulators (SERMs) are a class of small-molecule chemical compounds that bind to estrogen receptor (ER) ligand binding domain (LBD) with high affinity and selectively modulate ER transcriptional activity in a cell- and tissue-dependent manner. The prototype of SERMs is tamoxifen, which has agonist activity in bone, but has antagonist activity in breast. Tamoxifen can reduce the risk of breast cancer and, at same time, prevent osteoporosis in postmenopausal women. Tamoxifen is widely prescribed for treatment and prevention of breast cancer. Mechanistically the activity of SERMs is determined by the selective recruitment of coactivators and corepressors in different cell types and tissues. Therefore, understanding the coregulator function is the key to understanding the tissue selective activity of SERMs.

Hematopoietic

Hematopoietic Stem Cell Arrival Triggers Dynamic Remodeling of the Perivascular Niche

Owen J. Tamplin, Ellen M. Durand, Logan A. Carr, Sarah J. Childs, et al.
Cell, Jan 15, 2015; 160: 241–252
http://dx.doi.org/10.1016/j.cell.2014.12.032

Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization.

Neonatal anemia

Sanjay Aher, Kedar Malwatkar, Sandeep Kadam
Seminars in Fetal & Neonatal Medicine (2008) 13, 239e247
http://dx.doi.org:/10.1016/j.siny.2008.02.009

Neonatal anemia and the need for red blood cell (RBC) transfusions are very common in neonatal intensive care units. Neonatal anemia can be due to blood loss, decreased RBC production, or increased destruction of erythrocytes. Physiologic anemia of the newborn and anemia of prematurity are the two most common causes of anemia in neonates. Phlebotomy losses result in much of the anemia seen in extremely low birthweight infants (ELBW). Accepting a lower threshold level for transfusion in ELBW infants can prevent these infants being exposed to multiple donors.

Management of anemia in the newborn

Naomi L.C. Luban
Early Human Development (2008) 84, 493–498
http://dx.doi.org:/10.1016/j.earlhumdev.2008.06.007

Red blood cell (RBC) transfusions are administered to neonates and premature infants using poorly defined indications that may result in unintentional adverse consequences. Blood products are often manipulated to limit potential adverse events, and meet the unique needs of neonates with specific diagnoses. Selection of RBCs for small volume (5–20 mL/kg) transfusions and for massive transfusion, defined as extracorporeal bypass and exchange transfusions, are of particular concern to neonatologists. Mechanisms and therapeutic treatments to avoid transfusion are another area of significant investigation. RBCs collected in anticoagulant additive solutions and administered in small aliquots to neonates over the shelf life of the product can decrease donor exposure and has supplanted the use of fresh RBCs where each transfusion resulted in a donor exposure. The safety of this practice has been documented and procedures established to aid transfusion services in ensuring that these products are available. Less well established are the indications for transfusion in this population; hemoglobin or hematocrit alone are insufficient indications unless clinical criteria (e.g. oxygen desaturation, apnea and bradycardia, poor weight gain) also augment the justification to transfuse. Comorbidities increase oxygen consumption demands in these infants and include bronchopulmonary dysplasia, rapid growth and cardiac dysfunction. Noninvasive methods or assays have been developed to measure tissue oxygenation; however, a true measure of peripheral oxygen offloading is needed to improve transfusion practice and determine the value of recombinant products that stimulate erythropoiesis. The development of such noninvasive methods is especially important since randomized, controlled clinical trials to support specific practices are often lacking, due at least in part, to the difficulty of performing such studies in tiny infants.
The Effect of Blood Transfusion on the Hemoglobin Oxygen Dissociation Curve of Very Early Preterm Infants During the First Week of Life

Virginie De HaUeux, Anita Truttmann, Carmen Gagnon, and Harry Bard
Seminars in Perinatology, 2002; 26(6): 411-415
http://dx.doi.org:/10.1053/sper.2002.37313

This study was conducted during the first week of life to determine the changes in Ps0 (PO2 required to achieve a saturation of 50% at pH 7.4 and 37~ and the proportions of fetal hemoglobin (I-IbF) and adult hemoglobin (HbA) prior to and after transfusion in very early preterm infants. Eleven infants with a gestational age <–27 weeks have been included in study. The hemoglobin dissociation curve and the Ps0 was determined by Hemox-analyser. Liquid chromatography was also performed to determine the proportions of HbF and HbA. The mean gestational age of the 11 infants was 25.1 weeks (-+1 weeks) and their mean birth weight was 736 g (-+125 g). They received 26.9 mL/kg of packed red cells. The mean Ps0 prior and after transfusion was 18.5 +- 0.8 and 21.0 + 1 mm Hg (P = .0003) while the mean percentage of HbF was 92.9 -+ 1.1 and 42.6 -+ 5.7%, respectively. The data of this study show a decrease of hemoglobin oxygen affinity as a result of blood transfusion in very early preterm infants prone to O 2 toxicity. The shift in HbO 2 curve after transfusion should be taken into consideration when oxygen therapy is being regulated for these infants.

Effect of neonatal hemoglobin concentration on long-term outcome of infants affected by fetomaternal hemorrhage

Mizuho Kadooka, H Katob, A Kato, S Ibara, H Minakami, Yuko Maruyama
Early Human Development 90 (2014) 431–434
http://dx.doi.org/10.1016/j.earlhumdev.2014.05.010

Background: Fetomaternal hemorrhage (FMH) can cause severe morbidity. However, perinatal risk factors for long-term poor outcome due to FMH have not been extensively studied.                                                                                 Aims: To determine which FMH infants are likely to have neurological sequelae.
Study design: A single-center retrospective observational study. Perinatal factors, including demographic characteristics, Kleihauer–Betke test, blood gas analysis, and neonatal blood hemoglobin concentration ([Hb]), were analyzed in association with long-term outcomes.
Subjects: All 18 neonates referred to a Neonatal Intensive Care Unit of Kagoshima City Hospital and diagnosed with FMH during a 15-year study period. All had a neonatal [Hb] b7.5 g/dL and 15 of 17 neonates tested had Kleihauer–Betke test result N4.0%.
Outcome measures: Poor long-term outcome was defined as any of the following determined at 12 month old or more: cerebral palsy, mental retardation, attention deficit/hyperactivity disorder, and epilepsy.
Results: Nine of the 18 neonates exhibited poor outcomes. Among demographic characteristics and blood variables compared between two groups with poor and favorable outcomes, significant differences were observed in [Hb] (3.6 ± 1.4 vs. 5.4 ± 1.1 g/dL, P = 0.01), pH (7.09 ± 0.11 vs. 7.25 ± 0.13, P = 0.02) and base deficits (17.5 ± 5.4 vs. 10.4 ± 6.0 mmol/L, P = 0.02) in neonatal blood, and a number of infants with [Hb] ≤ 4.5 g/dL (78%[7/9] vs. 22%[2/9], P= 0.03), respectively. The base deficit in neonatal arterial blood increased significantly with decreasing neonatal [Hb].
Conclusions: Severe anemia causing severe base deficit is associated with neurological sequelae in FMH infants

Clinical and hematological presentation among Indian patients with common hemoglobin variants

Khushnooma Italia, Dipti Upadhye, Pooja Dabke, Harshada Kangane, et al.
Clinica Chimica Acta 431 (2014) 46–51
http://dx.doi.org/10.1016/j.cca.2014.01.028

Background: Co-inheritance of structural hemoglobin variants like HbS, HbD Punjab and HbE can lead to a variable clinical presentation and only few cases have been described so far in the Indian population.
Methods: We present the varied clinical and hematological presentation of 22 cases (HbSD Punjab disease-15, HbSE disease-4, HbD Punjab E disease-3) referred to us for diagnosis.
Results: Two of the 15 HbSDPunjab disease patients had moderate crisis, one presented with mild hemolytic anemia; however, the other 12 patients had a severe clinical presentation with frequent blood transfusion requirements, vaso occlusive crisis, avascular necrosis of the femur and febrile illness. The 4 HbSE disease patients had a mild to moderate presentation. Two of the 3 HbD Punjab E patients were asymptomatic with one patient’s sibling having a mild presentation. The hemoglobin levels of the HbSD Punjab disease patients ranged from 2.3 to 8.5 g/dl and MCV from 76.3 to 111.6 fl. The hemoglobin levels of the HbD Punjab E and HbSE patients ranged from 10.8 to 11.9 and 9.8 to 10.0 g/dl whereas MCV ranged from 67.1 to 78.2 and 74.5 to 76.0 fl respectively.
Conclusions: HbSD Punjab disease patients should be identified during newborn screening programs and managed in a way similar to sickle cell disease. Couple at risk of having HbSD Punjab disease children may be given the option of prenatal diagnosis in subsequent pregnancies.

Sickle cell anemia is the most common hemoglobinopathy seen across the world. It is caused by a point mutation in the 6th codon of the beta (β) globin gene leading to the substitution of the amino acid glutamic acid to valine. The sickle gene is frequently seen in Africa, some Mediterranean countries, India, Middle East—Saudi Arabia and North America. In India the prevalence of hemoglobin S (HbS) carriers varies from 2 to 40% among different population groups and HbS is mainly seen among the scheduled tribe, scheduled caste and other backward class populations in the western, central and parts of eastern and southern India. Sickle cell anemia has a variable clinical presentation in India with the most severe clinical presentation seen in central India whereas patients in the western region show a mild to moderate clinical presentation.

Hemoglobin D Punjab (HbD Punjab) (also known as HbD Los-Angeles, HbD Portugal, HbD North Carolina, D Oak Ridge and D Chicago) is another hemoglobin variant due to a point mutation in codon 121 of the β globin gene resulting in the substitution of the amino acid glutamic acid to glycine. It is a widely distributed hemoglobin with a relatively low prevalence of 0.86% in the Indo-Pak subcontinent, 1–3% in north-western India, 1–3% in the Black population in the Caribbean and North America and has also been reported among the English. It accounts for 55.6% of all the Hb variants seen in the Xenjiang province of China.

Hemoglobin E (HbE) is the most common abnormal hemoglobin in Southeast Asia. In India, the frequency ranges from 4% to 51% in the north eastern region and 3% to 4% in West Bengal in the east. The HbE mutation (β26 GAG→AAG) creates an alternative splice site and the βE chain is insufficiently synthesized, hence the phenotype of this disorder is that of a mild form of β thalassemia.

Though these 3 structural variants are prevalent in different regions of India, their interaction is increasingly seen in all states of the country due to migration of people to different regions for a better livelihood. There are very few reports on interaction of these commonly seen Hb variants and the phenotypic–genotypic presentation of these cases is important for genetic counseling and management.

HbF of patients with HbSD Punjab disease with variable clinical severity. The HbF values of 4 patients are not included as they were post blood transfusion

The genotypes of the patients were confirmed by restriction enzyme digestion and ARMS (Fig). Patients 1 to 15 were characterized as compound heterozygous for HbS and HbD Punjab whereas patients 16 to 19 were characterized as compound heterozygous for HbS and HbE. Patient nos. 20 to 22 were characterized as compound heterozygous for HbE and HbD Punjab.

Molecular characterization of HbS and HbDPunjab by restriction enzyme digestion and of HbE by ARMS.

Molecular characterization of HbS and HbDPunjab by restriction enzyme digestion and of HbE by ARMS.

Molecular characterization of HbS and HbDPunjab by restriction enzyme digestion and of HbE by ARMS.

The 3 common β globin gene variants of hemoglobin, HbS, HbE and HbD Punjab are commonly seen in India, with HbS having a high prevalence in the central belt and some parts of western, eastern and southern India, HbE in the eastern and north eastern region whereas HbD is mostly seen in the north western part of India. These hemoglobin variants have been reported in different population groups. However, with migration and intermixing of the different populations from different geographic regions, occasional cases of HbSD Punjab and HbSE are being reported. There are several HbD variants like HbD Punjab, HbD Iran, HbD Ibadan. However, of these only HbD Punjab interacts with HbS to form a clinically significant condition as the glutamine residue facilitates polymerization of HbS. HbD Iran and HbD Ibadan are non-interacting and produce benign conditions like the sickle cell trait. The first case of HbSD Punjab disease was a brother and sister considered to have atypical sickle cell disease in 1934. This family was further reinvestigated and reported as the first case of HbD Los Angeles which has the same mutation as the HbD Punjab. Serjeant et al. reported HbD Punjab in an English parent in 6 out of 11 HbSD-Punjab disease cases. This has been suggested to be due to the stationing of nearly 50,000 British troops on the Indian continent for a period of 200 y and the introduction into Britain of their Anglo-Indian children.

HbSD Punjab disease shows a similar pattern to HbS homozygous on alkaline hemoglobin electrophoresis but can be differentiated on acid agar gel electrophoresis and on HPLC. In HbSD Punjab disease cases, the peripheral blood films show anisocytosis, poikilocytosis, target cells and irreversibly sickled cells. Values of HbF and HbA2 are similar to those in sickle homozygous cases. HbSD Punjab disease is characterized by a moderately severe hemolytic anemia.

Twenty-one cases of HbSDPunjab were reported by Serjeant of which 16 were reported by different workers among patients originating from Caucasian, Spanish, Australian, Irish, English, Portuguese, Black, American, Venezuelan, Caribbean, Mexican, Turkish and Jamaican backgrounds. Yavarian et al. 2009 reported a multi centric origin of HbD Punjab which in combination with HbS results in sickle cell disease. Patel et al. 2010 have also reported 12 cases of HbSD Punjab from the Orissa state of eastern India. Majority of these cases were symptomatic, presenting with chronic hemolytic anemia and frequent painful crises.

HbF levels >20% were seen in 4 out of our 11 clinically severe patients of HbSD-Punjab disease with the mean HbF levels of 16.8% in 8 clinically severe patients, while 3 clinically severe patients were post transfused. However, the 3 patients with a mild to moderate clinical presentation showed a mean HbF level of 8.6%. This is in contrast to the relatively milder clinical presentation associated with high HbF seen in patients with sickle cell anemia. This was also reported by Adekile et al. 2010 in 5 cases of HbS-DLos Angeles where high HbF did not ameliorate the severe clinical presentation seen in these patients.

These 15 cases of HbSDPunjab disease give us an overall idea of the severe clinical presentation of the disease in different regions of India. However the HbDPunjabE cases were milder or asymptomatic and the HbSE cases were moderately symptomatic. Since most of the cases of HbSDPunjab disease were clinically severe, it is important to pick up these cases during newborn screening and enroll them into a comprehensive care program with the other sickle cell disease patients with introduction of therapeutic interventions such as penicillin prophylaxis if required and pneumococcal immunization. In fact, 2 of our cases (No. 6 and 7) were identified during newborn screening for sickle cell disorders. The parents can be given information on home care and educated to detect symptoms that may lead to serious medical emergencies. The parents of these patients as well as the couples who are at risk of having a child with HbSDPunjab disease could also be counseled about the option of prenatal diagnosis in subsequent pregnancies. It is thus important to document the clinical and hematological presentation of compound heterozygotes with these common β globin chain variants.

Common Hematologic Problems in the Newborn Nursery

Jon F. Watchko
Pediatr Clin N Am – (2015) xxx-xxx
http://dx.doi.org/10.1016/j.pcl.2014.11.011

Common RBC disorders include hemolytic disease of the newborn, anemia, and polycythemia. Another clinically relevant hematologic issue in neonates to be covered herein is thrombocytopenia. Disorders of white blood cells will not be reviewed.

KEY POINTS

(1)               Early clinical jaundice or rapidly developing hyperbilirubinemia are often signs of hemolysis, the differential diagnosis of which commonly includes immune-mediated disorders, red-cell enzyme deficiencies, and red-cell membrane defects.

(2)             Knowledge of the maternal blood type and antibody screen is critical in identifying non-ABO alloantibodies in the maternal serum that may pose a risk for severe hemolytic disease in the newborn.

(3)             Moderate to severe thrombocytopenia in an otherwise well-appearing newborn strongly suggests immune-mediated (alloimmune or autoimmune) thrombocytopenia.

Hemolytic conditions in the neonate

1. Immune-mediated (positive direct Coombs test)  a. Rhesus blood group: Anti-D, -c, -C, -e, -E, CW, and several others

  b. Non-Rhesus blood groups: Kell, Duffy, Kidd, Xg, Lewis, MNS, and others

  c. ABO blood group: Anti-A, -B

2. Red blood cell (RBC) enzyme defects

  a. Glucose-6-phosphate dehydrogenase (G6PD) deficiency

  b. Pyruvate kinase deficiency

  c. Others

3. RBC membrane defects

  a. Hereditary spherocytosis

  b. Elliptocytosis

  c. Stomatocytosis

  d. Pyknocytosis

  e. Others

4. Hemoglobinopathies

  a. alpha-thalassemia

  b. gamma-thalassemia

Standard maternal antibody screeningAlloantibody                                 Blood Group

D, C, c, E, e, f, CW, V                     Rhesus

K, k, Kpa, Jsa                                  Kell

Fya, Fyb                                          Duffy

Jka, Jkb                                           Kidd

Xga                                                  Xg

Lea, Leb                                          Lewis

S, s, M, N                                        MNS

P1                                                    P

Lub                                                  Lutheran

Non-ABO alloantibodies reported to cause moderate to severe hemolytic disease of the newbornWithin Rh system: Anti-D, -c, -C, -Cw, -Cx, -e, -E, -Ew, -ce, -Ces, -Rh29, -Rh32, -Rh42, -f, -G, -Goa, -Bea, -Evans, -Rh17, -Hro, -Hr, -Tar, -Sec, -JAL, -STEM

Outside Rh system:  Anti-LW, -K, -k, -Kpa, -Kpb, -Jka, -Jsa, -Jsb, -Ku, -K11, -K22, -Fya, -M, -N, -S, -s, -U, -PP1 pk, -Dib, -Far, -MUT, -En3, -Hut, -Hil, -Vel, -MAM, -JONES, -HJK, -REIT

 

Red Blood Cell Enzymopathies

G6PD9 and pyruvate kinase (PK) deficiency are the 2 most common red-cell enzyme disorders associated with marked neonatal hyperbilirubinemia. Of these, G6PD deficiency is the more frequently encountered and it remains an important cause of kernicterus worldwide, including the United States, Canada, and the United Kingdom, the prevalence in Western countries a reflection in part of immigration patterns and intermarriage. The risk of kernicterus in G6PD deficiency also relates to the potential for unexpected rapidly developing extreme hyperbilirubinemia in this disorder associated with acute severe hemolysis.

Red Blood Cell Membrane Defects

Establishing a diagnosis of RBC membrane defects is classically based on the development of Coombs-negative hyperbilirubinemia, a positive family history, and abnormal RBC smear, albeit it is often difficult because newborns normally exhibit a marked variation in red-cell membrane size and shape. Spherocytes, however, are not often seen on RBC smears of hematologically normal newborns and this morphologic abnormality, when prominent, may yield a diagnosis of hereditary spherocytosis (HS) in the immediate neonatal period. Given that approximately 75% of families affected with hereditary spherocytosis manifest an autosomal dominant phenotype, a positive family history can often be elicited and provide further support for this diagnosis. More recently, Christensen and Henry highlighted the use of an elevated mean corpuscular hemoglobin concentration (MCHC) (>36.0 g/dL) and/or elevated ratio of MCHC to mean corpuscular volume, the latter they term the “neonatal HS index” (>0.36, likely >0.40) as screening tools for HS. An index of greater than 0.36 had 97% sensitivity, greater than 99% specificity, and greater than 99% negative predictive value for identifying HS in neonates. Christensen and colleagues also provided a concise update of morphologic RBC features that may be helpful in diagnosing this and other underlying hemolytic conditions in newborns.

The diagnosis of HS can be confirmed using the incubated osmotic fragility test when coupled with fetal red-cell controls or eosin-5-maleimide flow cytometry. One must rule out symptomatic ABO hemolytic disease by performing a direct Coombs test, as infants so affected also may manifest prominent micro-spherocytosis. Moreover, HS and symptomatic ABO hemolytic disease can occur in the same infant and result in severe hyperbilirubinemia and anemia.  Of other red-cell membrane defects, only hereditary elliptocytosis,  stomato-cytosis, and infantile pyknocytosis have been reported to exhibit significant hemolysis in the newborn period. Hereditary elliptocytosis and stomatocytosis are both rare. Infantile pyknocytosis, a transient red-cell membrane abnormality manifesting itself during the first few months of life, is more common.

Risk factors for bilirubin neurotoxicityIsoimmune hemolytic disease

G6PD deficiency

Asphyxia

Sepsis

Acidosis

Albumin less than 3.0 g/dL
Data from Maisels MJ, Bhutani VK, Bogen D, et al. Hyperbilirubinemia in the newborn infant > or 535 weeks’ gestation: an update with clarifications. Pediatrics 2009; 124:1193–8.

Polycythemia

Polycythemia (venous hematocrit 65%) in seen in infants across a range of conditions associated with active erythropoiesis or passive transfusion.76,77 They include, among others, placental insufficiency, the infant of a diabetic mother, recipient in twin-twin transfusion syndrome, and several aneuploidies, including trisomy. The clinical concern related to polycythemia is the risk for microcirculatory complications of hyperviscosity. However, determining which polycythemic infants are hyperviscous and when to intervene is a challenge.

 

 

Liver

Metabolic disorders presenting as liver disease

Germaine Pierre, Efstathia Chronopoulou
Paediatrics and Child Health 2013; 23(12): 509-514
The liver is a highly metabolically active organ and many inherited metabolic disorders have hepatic manifestations. The clinical presentation in these patients cannot usually be distinguished from liver disease due to acquired causes like infection, drugs or hematological disorders. Manifestations include acute and chronic liver failure, cholestasis and hepatomegaly. Metabolic causes of acute liver failure in childhood can be as high as 35%. Certain disorders like citrin deficiency and Niemann-Pick C disease may present in infancy with self-limiting cholestasis before presenting in later childhood or adulthood with irreversible disease. This article reviews important details from the history and clinical examination when evaluating the pediatric patient with suspected metabolic disease, the specialist and genetic tests when investigating, and also discusses specific disorders, their clinical course and treatment. The role of liver transplantation is also briefly discussed. Increased awareness of this group of disorders is important as in many cases, early diagnosis leads to early intervention with improved outcome. Diagnosis also allows genetic counselling and future family planning.

Adult liver disorders caused by inborn errors of metabolism: Review and update

Sirisak Chanprasert, Fernando Scaglia
Molecular Genetics and Metabolism 114 (2015) 1–10
http://dx.doi.org/10.1016/j.ymgme.2014.10.011

Inborn errors of metabolism (IEMs) are a group of genetic diseases that have protean clinical manifestations and can involve several organ systems. The age of onset is highly variable but IEMs afflict mostly the pediatric population. However, in the past decades, the advancement in management and new therapeutic approaches have led to the improvement in IEM patient care. As a result, many patients with IEMs are surviving into adulthood and developing their own set of complications. In addition, some IEMs will present in adulthood. It is important for internists to have the knowledge and be familiar with these conditions because it is predicted that more and more adult patients with IEMs will need continuity of care in the near future. The review will focus on Wilson disease, alpha-1 antitrypsin deficiency, citrin deficiency, and HFE-associated hemochromatosis which are typically found in the adult population. Clinical manifestations and pathophysiology, particularly those that relate to hepatic disease as well as diagnosis and management will be discussed in detail.

Inborn errors of metabolism (IEMs) are a group of genetic diseases characterized by abnormal processing of biochemical reactions, resulting in accumulation of toxic substances that could interfere with normal organ functions, and failure to synthesize essential compounds. IEMs are individually rare, but collectively numerous. The clinical presentations cover a broad spectrum and can involve almost any organ system. The age of onset is highly variable but IEMs afflict mostly the pediatric population.

Wilson disease is an autosomal recessive genetic disorder of copper metabolism. It is characterized by an abnormal accumulation of inorganic copper in various tissues, most notably in the liver and the brain, especially in the basal ganglia. The disease was first described in 1912 by Kinnier Wilson, and affects between 1 in 30,000 and 1 in 100,000 individuals. Clinical features are variable and depend on the extent  and the severity of copper deposition. Typically, patients tend to develop hepatic disease at a younger age than the neuropsychiatric manifestations. Individuals withWilson disease eventually succumb to complications of end stage liver disease or become debilitated from neurological problems, if they are left untreated.

The clinical presentations of Wilson disease are varied affecting many organ systems. However, the overwhelming majority of cases display hepatic and neurologic symptoms. In general, patients with hepatic disease present between the first and second decades of life although patients as young as 3 years old or over 50 years old have also been reported. The most common modes of presentations are acute self-limited hepatitis and chronic active hepatitis that are indistinguishable from other hepatic disorders although liver aminotransferases are generally much lower than in autoimmune or viral hepatitis. Acute fulminant hepatic failure is less common but is observed in approximately 3% of all cases of acute liver failure. Symptoms of acute liver failure include jaundice, coagulopathy, and hepatic encephalopathy. Cirrhosis can develop over time and may be clinically silent. Hepatocellular carcinoma (HCC) is rarely associated with Wilson disease, but may occur in the setting of cirrhosis and chronic inflammation.

Copper is an essential element, and is required for the proper functioning of various proteins and enzymes. The total body content of copper in a healthy adult individual is approximately 70–100 mg, while the daily requirements are estimated to be between 1 and 5 mg. Absorption occurs in the small intestine. Copper is taken up to the hepatocytes via the copper transporter hTR1. Once inside the cell, copper is bound to various proteins including metallothionein and glutathione, however, it is the metal chaperone, ATOX1 that helps direct copper to the ATP7B protein for intracellular transport and excretion. At the steady state, copper will be bound to ATP7B and is then incorporated to ceruloplasmin and secreted into the systemic circulation. When the cellular copper concentration arises, ATP7B protein will be redistributed from the trans-Golgi network to the prelysosomal vesicles facilitating copper excretion into the bile. The molecular defects in ATP7B lead to a reduction of copper excretion. Excess copper is accumulated in the liver causing tissue injury. The rate of accumulation of copper varies among individuals, and it may depend on other factors such as alcohol consumption, or viral hepatitis infections. If the liver damage is not severe, patients will accumulate copper in various tissues including the brain, the kidney, the eyes, and the musculoskeletal system leading to clinical disease. A failure of copper to incorporate into ceruloplasmin leads to secretion of the unsteady protein that has a shorter half-life, resulting in the reduced concentrations of ceruloplasmin seen in most patients with Wilson disease.

Wilson disease used to be a progressive fatal condition during the first half of the 20th century because there was no effective treatment available at that time. Penicillamine was the first pharmacologic agent introduced in 1956 for treating this condition. Penicillamine is a sulfhydryl-bearing amino acid cysteine doubly substituted with methyl groups. This drug acts as a chelating agent that promotes the urinary excretion of copper. It is rapidly absorbed in the gastrointestinal track, and over 80% of circulating penicillamine is excreted via the kidneys. Although it is very effective, approximately 10%–50% of Wilson disease patients with neuropsychiatric presentations may experience worsening of their symptoms, and often times the worsening symptoms may not be reversible.

Alpha1-antitrypsin deficiency

Alpha1-antitrypsin deficiency (AATD) is one of the most common genetic liver diseases in children and adults, affecting 1 in 2000 to 1 in 3000 live births worldwide. It is transmitted in an autosomal co-dominant fashion with variable expressivity. Alpha1 antitrypsin (A1AT) is a member of the serine protease inhibitor (SERPIN) family. Its function is to counteract the proteolytic effect of neutrophil elastase and other neutrophil proteases. Mutations in the SERPINA1, the gene encoding A1AT, result in changes in the protein structure with the PiZZ phenotype being the most common cause of liver and lung disease-associated AATDs. Although, it classically causes early onset chronic obstructive pulmonary disease (COPD) in adults, liver disease characterized by chronic inflammation, hepatic fibrosis, and cirrhosis is not uncommon in the adult population. Decreased plasma concentration of A1AT predisposes lung tissue to be more susceptible to injury from protease enzymes. However, the underlying mechanism of liver injury is different, and is believed to be caused by accumulation of polymerized mutant A1AT in the hepatocyte endoplasmic reticulum (ER). Currently, there is no specific treatment for liver disease-associated AATD, but A1AT augmentation therapy is available for patients affected with pulmonary involvement.

A1AT is a single-chain, 52-kDa polypeptide of approximately 394 amino acids [56]. It is synthesized in the liver, circulates in the plasma, and functions as an inhibitor of neutrophil elastase and other proteases such as cathepsin G, and proteinase 3. A1AT has a globular shape composed of two central β sheets surrounded by a small β sheet and nine α helices. The pathophysiology underlying liver disease is thought to be a toxic gain-of-function mutation associated with the PiZZ phenotypes. This hypothesis has been supported by the fact that null alleles which produce no detectable plasma A1AT, are not associated with liver disease. In addition, the transgenic mouse model of AATD PiZZ developed periodic acid-Schiff-positive diastase-resistant intrahepatic globule early in life similar to AATD patients. The PiZZ phenotype results in the blockade of the final processing of A1AT in the liver, as only 15% of the A1AT reaches the circulation whereas 85% of non-secreted protein is accumulated in the hepatocytes.

Citrin deficiency

Citrin deficiency is a relatively newly-defined autosomal recessive disease. It encompasses two different sub-groups of patients, neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and adult onset citrullinemia type 2 (CTLN 2).

AGC2 exports aspartate out of the mitochondrial matrix in exchange for glutamate and a proton. Thus, this protein has an important role in ureagenesis and gluconeogenesis. In CTLN2, a defect in this protein is believed to limit the supply of aspartate for the formation of argininosuccinate in the cytosol resulting in impairment of ureagenesis. Interestingly, the mouse model of citrin deficiency (Ctrn−/−) fails to develop symptoms of CTLN2 suggesting that the mitochondrial aspartate is not the only source of ureagenesis. However, it should be noted that the rodent liver expresses higher glycerol-phosphate shuttle activity than the human counterpart. With the intact glycerol-phosphate dehydrogenase, it can compensate for the deficiency of AGC2, as demonstrated by the AGC2 and glycerol-phosphate dehydrogenase double knock-out mice that exhibit similar features to those observed in human CTLN2.

HFE-associated hemochromatosis

HFE-associated hemochromatosis is an inborn error of iron metabolism characterized by excessive iron storage resulting in tissue and organ damage. It is the most common autosomal recessive disorder in the Caucasian population, affecting 0.3%–0.5% of individuals of Northern European descent. The term “hemochromatosis” was coined in 1889 by the German pathologist Friedrich Daniel Von Recklinghausen, who described it as bronze stain of organs caused by a blood borne pigment.

The classic clinical triad of cirrhosis, diabetes, and bronze skin pigmentation is rarely observed nowadays given the early recognition, diagnosis, and treatment of this condition. The most common presenting symptoms are nonspecific including weakness, lethargy, and arthralgia.

The liver is a major site of iron storage in healthy individuals and as such it is the organ that is universally affected in HFE-associated hemochromatosis. Elevation of liver aminotransferases indicative of hepatocyte injury is the most common mode of presentation and it can be indistinguishable from other causes of hepatitis. Approximately 15%–40% of patients with HFE-associated hemochromatosis have other liver conditions, including chronic viral hepatitis B or C infection, nonalcoholic fatty liver disease, and alcoholic liver disease.

 

The liver in haemochromatosis

Rune J. Ulvik
Journal of Trace Elements in Medicine and Biology xxx (2014) xxx–xxx
http://dx.doi.org/10.1016/j.jtemb.2014.08.005

The review deals with genetic, regulatory and clinical aspects of iron homeostasis and hereditary hemochromatosis. Hemochromatosis was first described in the second half of the 19th century as a clinical entity characterized by excessive iron overload in the liver. Later, increased absorption of iron from the diet was identified as the pathophysiological hallmark. In the 1970s genetic evidence emerged supporting the apparent inheritable feature of the disease. And finally in 1996 a new “hemochromato-sis gene” called HFE was described which was mutated in about 85% of the patients. From the year2000 onward remarkable progress was made in revealing the complex molecular regulation of iron trafficking in the human body and its disturbance in hemochromatosis. The discovery of hepcidin and ferroportin and their interaction in regulating the release of iron from enterocytes and macrophages to plasma were important milestones. The discovery of new, rare variants of non-HFE-hemochromatosis was explained by mutations in the multicomponent signal transduction pathway controlling hepcidin transcription. Inhibited transcription induced by the altered function of mutated gene products, results in low plasma levels of hepcidin which facilitate entry of iron from enterocytes into plasma. In time this leads to progressive accumulation of iron and subsequently development of disease in the liver and other parenchymatous organs. Being the major site of excess iron storage and hepcidin synthesis the liver is a cornerstone in maintaining normal systemic iron homeostasis. Its central pathophysiological role in HFE-hemochromatosis with downgraded hepcidin synthesis, was recently shown by the finding that liver transplantation normalized the hepcidin levels in plasma and there was no sign of iron accumulation in the new liver.

Gastrointestinal

Decoding the enigma of necrotizing enterocolitis in premature infants

Roberto Murgas TorrazzaNan Li, Josef Neu
Pathophysiology 21 (2014) 21–27
http://dx.doi.org/10.1016/j.pathophys.2013.11.011

Necrotizing enterocolitis (NEC) is an enigmatic disease that affects primarily premature infants. It often occurs suddenly and when it occurs, treatment attempts at treatment often fail and results in death. If the infant survives, there is a significant risk of long term sequelae including neurodevelopmental delays. The pathophysiology of NEC is poorly understood and thus prevention has been difficult. In this review, we will provide an overview of why progress may be slow in our understanding of this disease, provide a brief review diagnosis, treatment and some of the current concepts about the pathophysiology of this disease.

Necrotizing enterocolitis (NEC) has been reported since special care units began to house preterm infants .With the advent of modern neonatal intensive care approximately 40 years ago, the occurrence and recognition of the disease markedly increased. It is currently the most common and deadly gastro-intestinal illness seen in preterm infants. Despite major efforts to better understand, treat and prevent this devastating disease, little if any progress has been made during these 4 decades. Underlying this lack of progress is the fact that what is termed “NEC” is likely more than one disease, or mimicked by other diseases, each with a different etiopathogenesis.

Human gut microbiome

Human gut microbiome

Term or near term infants with “NEC” when compared to matched controls usually have occurrence of their disease in the first week after birth, have a significantly higher frequency of prolonged rupture of membranes, chorio-amnionitis, Apgar score <7 at 1 and 5 min, respiratory problems, congenital heart disease, hypoglycemia, and exchange transfusions. When a “NEC” like illness presents in term or near term infants, it should be noted that these are likely to be distinct in pathogenesis than the most common form of NEC and should be differentiated as such.

The infants who suffer primary ischemic necrosis are term or near term infants (although this can occur in preterms) who have concomitant congenital heart disease, often related to poor left ventricular output or obstruction. Other factors that have been associated with primary ischemia are maternal cocaine use, hyperviscosity caused by polycythemia or a severe antecedent hypoxic–ischemic event. Whether the dis-ease entity that results from this should be termed NEC can be debated on historical grounds, but the etiology is clearly different from the NEC seen in most preterm infants.

The pathogenesis of NEC is uncertain, and the etiology seems to be multifactorial. The “classic” form of NEC is highly associated with prematurity; intestinal barrier immaturity, immature immune response, and an immature regulation of intestinal blood flow (Fig.). Although genetics appears to play a role, the environment, especially a dysbiotic intestinal microbiota acting in concert with host immaturities predisposes the preterm infant to disruption of the intestinal epithelia, increased permeability of tight junctions, and release of inflammatory mediators that leads to intestinal mucosa injury and therefore development of necrotizing enterocolitis.

NEC is a multifactorial disease

NEC is a multifactorial disease

What causes NEC? NEC is a multifactorial disease with an interaction of several etiophathologies

It is clear from this review that there are several entities that have been described as NEC. What is also clear is that despite having some overlap in the final parts of the pathophysiologic cascade that lead to necrosis, the disease that is most commonly seen in the preterm infant is likely to have an origin that differs markedly from that seen in term infants with congenital heart disease or severe hypoxic–ischemic injury. Thus, epidemiologic studies will need to differentiate these entities, if the aim is to dissect common features that are most highly associated with development of the disease. At this juncture, we areleft with more of a population based preventative approach, where the use of human milk, evidence based feeding guide-lines, considerations for microbial therapy once these are proved safe and effective and approved as such by regulatory authorities, and perhaps even measures that prevent prematurity will have a major impact on this devastating disease.

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines, including thymic stromal lymphoprotein (TSLP), transforming growthfactor (TGF), and interleukin-10 (IL-10), that can influence pro-inflammatory cytokine production by dendritic cells (DC) and macrophages present in the laminapropria (GALT) and Peyer’s patches. Signals from commensal organisms may influence tissue-specific functions, resulting in T-cell expansion and regulation of the numbers of Th-1,
Th-2, and Th-3 cells. Also modulated by the microbiota, other IEC derived factors, including APRIL (a proliferation-inducing ligand),B-cell activating factor (BAFF), secretory leukocyte peptidase inhibitor (SLPI), prostaglandin E2(PGE2), and other metabolites, directly regulate functions ofboth antigen presenting cells and lymphocytes in the intestinal ecosystem. NK: natural killer cell; LN: lymph node; DC: dendritic cells.Modified from R. Sharma, C. Young, M. Mshvildadze, J. Neu, Intestinal microbiota does it play a role in diseases of the neonate? NeoReviews 10 (4) (2009)e166, with permission

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Current Issues in the Management of Necrotizing Enterocolitis

Marion C. W. Henry and R. Lawrence Moss
Seminars in Perinatology, 2004; 28(3): 221-233
http://dx.doi.org:/10.1053/j.semperi.2004.03.010

Necrotizing enterocolitis is almost exclusively a disease of prematurity, with 90% of all cases occurring in premature infants and 90% of those infants weighing less than 2000 g. Prematurity is the only risk factor for necrotizing enterocolitis consistently identified in case control studies and the disease is rare in countries where prematurity is uncommon such as Japan and Sweden. When necrotizing enterocolitis does occur in full-term infants, it appears to by a somewhat different disease, typically associated with some predisposing condition.

NEC occurs in one to three in 1,000 live births and most commonly affects babies born between 30-32 weeks. It is most often diagnosed during the second week of life and occurs more often in previously fed infants. The mortality from NEC has been cited as 10% to 50% of all NEC cases. Surgical mortality has decreased over the last several decades from 70% to between 20 and 50%. The incremental cost per case of acute hospital care is estimated at $74 to 186 thousand compared to age matched controls, not including additional costs of long term care for the infants’ with lifelong morbidity. Survivors may develop short bowel syndrome, recurrent bouts of catheter-related sepsis, malabsorption, malnutrition, and TPN induced liver failure.

Although extensive research concerning the pathophysiology of necrotizing enterocolitis has occurred, a complete understanding has not been fully elucidated. The classic histologic finding is coagulation necrosis; present in over 90% of specimens. This finding suggests the importance of ischemia in the pathogenesis of NEC. Inflammation and bacterial overgrowth also are present. These findings support the assumptions by Kosloske that NEC occurs by the interaction of 3 events:

  • intestinal ischemia,
  • colonization by pathogenic bacteria and
  • excess protein substrate in the intestinal lumen.

Additionally, the immunologic immaturity of the neonatal gut has been implicated in the development of NEC. Reparative tissue changes including epithelial regeneration, formation of granulation tissue and fibrosis, and mixed areas of acute and chronic inflammatory changes suggest that the pathogenesis of NEC may involve a chronic process of injury and repair.

Premature newborns born prior to the 32nd week of gestational age may have compromised intestinal peristalsis and decreased motility. These motility problems may lead to poor clearance of bacteria, and subsequent bacterial overgrowth. Premature infants also have an immature intestinal tract in terms of immunologic immunity.

There are fewer functional B lymphocytes present and the ability to produce sufficient secretory IgA is reduced. Pepsin, gastric acid and mucus are also not produced as well in prematurity. All of these factors may contribute to the limited proliferation of intestinal flora and the decreased binding of these flora to mucosal cells (Fig).

Role of nitric oxide in the pathogenesis of NEC

Role of nitric oxide in the pathogenesis of NEC

Role of nitric oxide in the pathogenesis of NEC.

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis.

As understanding of the pathophysiology of necrotizing enterocolitis continues to evolve, a unifying concept is emerging. Initially, there is likely a subclinical insult leading to NEC. This may arise from a brief episode of hypoxia or infection. With colonization of the intestines, bacteria bind to the injured mucosa eliciting an inflammatory response which leads to further inflammation.

Intestinal Microbiota Development in Preterm Neonates and Effect of Perinatal Antibiotics

Silvia Arboleya, Borja Sanchez,, Christian Milani, Sabrina Duranti, et al.
Pediatr 2014;-:—).  http://dx.doi.org/10.1016/j.jpeds.2014.09.041

Objectives Assess the establishment of the intestinal microbiota in very low birth-weight preterm infants and to evaluate the impact of perinatal factors, such as delivery mode and perinatal antibiotics.
Study design We used 16S ribosomal RNA gene sequence-based microbiota analysis and quantitative polymerase chain reaction to evaluate the establishment of the intestinal microbiota. We also evaluated factors affecting the microbiota, during the first 3 months of life in preterm infants (n = 27) compared with full-term babies (n = 13).
Results Immaturity affects the microbiota as indicated by a reduced percentage of the family Bacteroidaceae during the first months of life and by a higher initial percentage of Lactobacillaceae in preterm infants compared with full term infants. Perinatal antibiotics, including intrapartum antimicrobial prophylaxis, affects the gut microbiota, as indicated by increased Enterobacteriaceae family organisms in the infants.

Human gut microbiome

Human gut microbiome

Conclusions Prematurity and perinatal antibiotic administration strongly affect the initial establishment of microbiota with potential consequences for later health.

Ischemia and necrotizing enterocolitis: where, when, and how

Philip T. Nowicki
Seminars in Pediatric Surgery (2005) 14, 152-158
http://dx.doi.org:/10.1053/j.sempedsurg.2005.05.003

While it is accepted that ischemia contributes to the pathogenesis of necrotizing enterocolitis (NEC), three important questions regarding this role subsist. First, where within the intestinal circulation does the vascular pathophysiology occur? It is most likely that this event begins within the intramural microcirculation, particularly the small arteries that pierce the gut wall and the submucosal arteriolar plexus insofar as these represent the principal sites of resistance regulation in the gut. Mucosal damage might also disrupt the integrity or function of downstream villous arterioles leading to damage thereto; thereafter, noxious stimuli might ascend into the submucosal vessels via downstream venules and lymphatics. Second, when during the course of pathogenesis does ischemia occur? Ischemia is unlikely to the sole initiating factor of NEC; instead, it is more likely that ischemia is triggered by other events, such as inflammation at the mucosal surface. In this context, it is likely that ischemia plays a secondary, albeit critical role in disease extension. Third, how does the ischemia occur? Regulation of vascular resistance within newborn intestine is principally determined by a balance between the endothelial production of the vasoconstrictor peptide endothelin-1 (ET-1) and endothelial production of the vasodilator free radical nitric oxide (NO). Under normal conditions, the balance heavily favors NO-induced vasodilation, leading to a low resting resistance and high rate of flow. However, factors that disrupt endothelial cell function, eg, ischemia-reperfusion, sustained low-flow perfusion, or proinflammatory mediators, alter the ET-1:NO balance in favor of constriction. The unique ET-1–NO interaction thereafter might facilitate rapid extension of this constriction, generating a viscous cascade wherein ischemia rapidly extends into larger portions of the intestine.

Schematic representation of the intestinal microcirculation

Schematic representation of the intestinal microcirculation

Schematic representation of the intestinal microcirculation. Small mesenteric arteries pierce the muscularis layers and terminate in the submucosa where they give rise to 1A (1st order) arterioles. 2A (2nd order) arterioles arise from the 1A. Although not shown here, these 2A arterioles connect merge with several 1A arterioles, thus generating an arteriolar plexus, or manifold that serves to pressurize the terminal downstream microvasculature. 3A (3rd order) arterioles arise from the 2A and proceed to the mucosa, giving off a 4A branch just before descent into the mucosa. This 4A vessel travels to the muscularis layers. Each 3A vessel becomes the single arteriole perfusing each villus.

Collectively, these studies indicate that disruption of endothelial cell function has the potential to disrupt the normal balance between NO and ET-1 within the newborn intestinal circulation, and that such an event can generate significant ischemia. In this context, it is important to note that NO and ET-1 each regulate the expression and activity of the other. An increased [NO] within the microvascular environment reduces ET-1 expression and compromises ligand binding to the ETA receptor (thus decreasing its contractile efficacy), while ET-1 compromises eNOS expression. Thus, factors that upset the balance between NO and ET-1 will have an immediate and direct effect on vascular tone, but also exert an additional indirect effect by extenuating the disruption of balance between these two factors.

It is not difficult to construct a hypothesis that links the perturbations of I/R and sustained low-flow perfusion with an initial inflammatory insult. Initiation of an inflammatory process at the mucosal–luminal interface could have a direct impact on villus and mucosal 3A arterioles, damaging arteriolar integrity and disrupting villus hemodynamics. Ascent of proinflammatory mediators to the submucosal 1A–2A arteriolar plexus could occur via draining venules and lymphatics, generating damage to vascular effector systems therein; these mediators might include cytokines and platelet activating factor, as these elements have been recovered from human infants with NEC. This event, coupled with a generalized loss of 3A flow throughout a large portion of the mucosal surface, could compromise flow rate within the submucosal arteriolar plexus.

Necrotizing enterocolitis: An update

Loren Berman, R. Lawrence Moss
Seminars in Fetal & Neonatal Medicine 16 (2011) 145e150
http://dx.doi.org:/10.1016/j.siny.2011.02.002

Necrotizing enterocolitis (NEC) is a leading cause of death among patients in the neonatal intensive care unit, carrying a mortality rate of 15e30%. Its pathogenesis is multifactorial and involves an over reactive response of the immune system to an insult. This leads to increased intestinal permeability, bacterial translocation, and sepsis. There are many inflammatory mediators involved in this process, but thus far none has been shown to be a suitable target for preventive or therapeutic measures. NEC usually occurs in the second week of life after the initiation of enteral feeds, and the diagnosis is made based on physical examination findings, laboratory studies, and abdominal radiographs. Neonates with NEC are followed with serial abdominal examinations and radiographs, and may require surgery or primary peritoneal drainage for perforation or necrosis. Many survivors are plagued with long term complications including short bowel syndrome, abnormal growth, and neurodevelopmental delay. Several evidence-based strategies exist that may decrease the incidence of NEC including promotion of human breast milk feeding, careful feeding advancement, and prophylactic probiotic administration in at-risk patients. Prevention is likely to have the greatest impact on decreasing mortality and morbidity related to NEC, as little progress has been made with regard to improving outcomes for neonates once the disease process is underway.

Immune Deficiencies

Primary immunodeficiencies: A rapidly evolving story

Nima Parvaneh, Jean-Laurent Casanova,  LD Notarangelo, ME Conley
J Allergy Clin Immunol 2013;131:314-23.
http://dx.doi.org/10.1016/j.jaci.2012.11.051

The characterization of primary immunodeficiencies (PIDs) in human subjects is crucial for a better understanding of the biology of the immune response. New achievements in this field have been possible in light of collaborative studies; attention paid to new phenotypes, infectious and otherwise; improved immunologic techniques; and use of exome sequencing technology. The International Union of Immunological Societies Expert Committee on PIDs recently reported on the updated classification of PIDs. However, new PIDs are being discovered at an ever-increasing rate. A series of 19 novel primary defects of immunity that have been discovered after release of the International Union of Immunological Societies report are discussed here. These new findings highlight the molecular pathways that are associated with clinical phenotypes and suggest potential therapies for affected patients.

Combined Immunodeficiencies

  • T-cell receptor a gene mutation: T-cell receptor ab1 T-cell depletion

T cells comprise 2 distinct lineages that express either ab or gd T-cell receptor (TCR) complexes that perform different tasks in immune responses. During T-cell maturation, the precise order and efficacy of TCR gene rearrangements determine the fate of the cells. Productive β-chain gene rearrangement produces a pre-TCR on the cell surface in association with pre-Tα invariant peptide (β-selection). Pre-TCR signals promote α-chain recombination and transition to a double-positive stage (CD41CD81). This is the prerequisite for central tolerance achieved through positive and negative selection of thymocytes.

  • Ras homolog gene family member H deficiency: Loss of naive T cells and persistent human papilloma virus infections
  • MST1 deficiency: Loss of naive T cells

New insight into the role of MST1 as a critical regulator of T-cell homing and function was provided by the characterization of 8 patients from 4 unrelated families who had homozygous nonsense mutations in STK4, the gene encoding MST1. MST1 was originally identified as an ubiquitously expressed kinase with structural homology to yeast Ste. MST1 is the mammalian homolog of the Drosophila Hippo protein, controlling cell growth, apoptosis, and tumorigenesis. It has both proapoptotic and antiapoptotic functions.

  • Lymphocyte-specific protein tyrosine kinase deficiency: T-cell deficiency with CD41 lymphopenia

Defects in pre-TCR– and TCR-mediated signaling lead to aberrant T-cell development and function (Fig). One of the earliest biochemical events occurring after engagement of the (pre)-TCR is the activation of lymphocyte-specific protein tyrosine kinase (LCK), a member of the SRC family of protein tyrosine kinases. This kinase then phosphorylates immunoreceptor tyrosine-based activation motifs of intracellular domains of CD3 subunits. Phosphorylated immunoreceptor tyrosine-based activation motifs recruit z-chain associated protein kinase of 70 kDa, which, after being phosphorylated by LCK, is responsible for activation of critical downstream events. Major consequences include activation of the membrane-associated enzyme phospholipase Cg1, activation of the mitogen-activated protein kinase, nuclear translocation of nuclear factor kB (NFkB), and Ca21/Mg21 mobilization. Through these pathways, LCK controls T-cell development and activation. In mice lacking LCK, T-cell development in the thymus is profoundly blocked at an early double-negative stage.

TCR signaling

TCR signaling

TCR signaling. Multiple signal transduction pathways are stimulated through the TCR. These pathways collectively activate transcription factors that organize T-cell survival, proliferation, differentiation, homeostasis, and migration. Mutant molecules in patients with TCR-related defects are indicated in red.

  • Uncoordinated 119 deficiency: Idiopathic CD41 lymphopenia

Idiopathic CD41 lymphopenia (ICL) is a very heterogeneous clinical entity that is defined, by default, by persistent CD41 T-cell lymphopenia (<300 cells/mL or <20% of total T cells) in the absence of HIV infection or any other known cause of immunodeficiency.

Well-Defined Syndromes with Immunodeficiency

  • Wiskott-Aldrich syndrome protein–interacting protein deficiency: Wiskott-Aldrich syndrome-like phenotype

In hematopoietic cells Wiskott-Aldrich syndrome protein (WASP) is stabilized through forming a complex with WASP interacting protein (WIP).

  • Phospholipase Cg2 gain-of-function mutations: Cold urticaria, immunodeficiency, and autoimmunity/autoinflammatory

This is a unique phenotype, sharing features of antibody deficiency, autoinflammatory diseases, and immune dysregulatory disorders, making its classification difficult. Two recent studies validated the pleiotropy of genetic alterations in the same gene.

Predominantly Antibody Defects

  • Defect in the p85a subunit of phosphoinositide 3-kinase: Agammaglobulinemia and absent B cells
  • CD21 deficiency: Hypogammaglobulinemia
  • LPS-responsive beige-like anchor deficiency:
  • Hypogammaglobulinemia with autoimmunity and

early colitis

Defects Of Immune Dysregulation

  • Pallidin deficiency: Hermansky-Pudlak syndrome type 9
  • CD27 deficiency: Immune dysregulation and
  • persistent EBV infection

Congenital Defects Of Phagocyte Number, Function, Or Both

  • Interferon-stimulated gene 15 deficiency: Mendelian susceptibility to mycobacterial diseases

Defects In Innate Immunity

  • NKX2-5 deficiency: Isolated congenital asplenia
  • Toll/IL-1 receptor domain–containing adaptor inducing IFN-b and TANK-binding kinase 1 deficiencies: Herpes simplex encephalitis
  • Minichromosome maintenance complex component 4 deficiency: NK cell deficiency associated with growth retardation and adrenal insufficiency

Autoinflammatory Disorders

  • A disintegrin and metalloproteinase 17 deficiency: Inflammatory skin and bowel disease

 

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Cross-talk between monocyte/macrophage cells and T/NK lymphocytes. Genes in the IL-12/IFN-g pathway are particularly important for protection against mycobacterial disease. IRF8 is an IFN-g–inducible transcription factor required for the induction of various target genes, including IL-12. The NF-kB essential modulator (NEMO) mutations in the LZ domain impair CD40-NEMO–dependent pathways. Some gp91phox mutations specifically abolish the respiratory burst in monocyte-derived macrophages. ISG15 is secreted by neutrophils and potentiates IFN-g production by NK/T cells. Genetic defects that preclude monocyte development (eg, GATA2) can also predispose to mycobacterial infections (not shown). Mutant molecules in patients with unusual susceptibility to infection are indicated in red.

The field of PIDs is advancing at full speed in 2 directions. New genetic causes of known PIDs are being discovered (eg, CD21 and TRIF). Moreover, new phenotypes qualify as PIDs with the identification of a first genetic cause (eg, generalized pustular psoriasis). Recent findings contribute fundamental knowledge about immune system biology and its perturbation in disease. They are also of considerable clinical benefit for the patients and their families. A priority is to further translate these new discoveries into improved diagnostic methods and more effective therapeutic strategies, promoting the well-being of patients with PIDs.

Primary immunodeficiencies

Luigi D. Notarangelo
J Allergy Clin Immunol 2010; 125(2): S182-194
http://dx.doi.org:/10.1016/j.jaci.2009.07.053

In the last years, advances in molecular genetics and immunology have resulted in the identification of a growing number of genes causing primary immunodeficiencies (PIDs) in human subjects and a better understanding of the pathophysiology of these disorders. Characterization of the molecular mechanisms of PIDs has also facilitated the development of novel diagnostic assays based on analysis of the expression of the protein encoded by the PID-specific gene. Pilot newborn screening programs for the identification of infants with severe combined immunodeficiency have been initiated. Finally, significant advances have been made in the treatment of PIDs based on the use of subcutaneous immunoglobulins, hematopoietic cell transplantation from unrelated donors and cord blood, and gene therapy. In this review we will discuss the pathogenesis, diagnosis, and treatment of PIDs, with special attention to recent advances in the field.

 

 

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Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker

 

Co-Author of the FIRST Article: Larry H. Bernstein, MD, FCAP

Reviewer and Curator of the SECOND and of the THIRD Articles: Larry H. Bernstein, MD, FCAP

and

Article Architecture Curator: Aviva Lev-Ari, PhD, RN

This article presents Advances in the Treatment using Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe) for IMPROVEMENT of Severe and Resistant Congestive Heart Failure and its resultant Anemia.  The Leading Biomarker for Congestive Heart Failure is an Independent Predictor identified as an Elevated N-terminal proBNP.

NT-proBNP schematic diagram-Copy.pdf_page_1

FIRST ARTICLE

Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3,  LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6,  John F. Heitner, MD, FACC7
1Cardiology Fellow,  2  Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology,  Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript)  Poster Presentation

SECOND ARTICLE

The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7

http://content.onlinejacc.org/article.aspx?articleid=1127229

THIRD ARTICLE

The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6

http://content.onlinejacc.org/article.aspx?articleid=1126474

Perspective

This THREE article sequence is related by investigations occurring by me, a very skilled cardiologist and his resident, and my premedical student at New York Methodist Hospital-Weill Cornell, in Brooklyn, NY, while a study had earlier been done applying the concordant discovery, which the team in Israel had though was knowledge neglected.  There certainly was no interest in the problem of the effect of anemia on the patient with severe congestive heart failure, even though erythropoietin was used widely in patients with end-stage renal disease requiring dialysis, and also for patients with myelofibrosis.  The high cost of EPO was only one factor, the other being a guideline to maintain the Hb concentration at or near 11 g/dl – not higher.  In the first article, the authors sought to determine whether the amino terminal pro– brain type natriuretic peptide (NT-pro BNP) is affected by anemia, and to determine that they excluded all patients who had renal insufficiency and/or CHF, since these were associated with elevated NT-proBNP.  It was already well established that this pro-peptide is secreted by the heart with the action as a urinary sodium retention hormone on the kidney nephron, the result being an increase in blood volume.  Perhaps the adaptation would lead to increased stroke volume from increased venous return, but that is not conjectured.  However, at equilibrium, one would expect there to be increased red cell production to maintain the cell to plasma volume ratio, thereby, resulting in adequate oxygen exchange to the tissues.  Whether that is always possible is uncertain because any reduction in the number of functioning nephrons would make the kidney not fully responsive at the Na+ exchange level, and the NT-pro BNP would rise.  This introduces complexity into the investigation, requiring a removal of confounders to establish the effect of anemia.

The other two articles are related studies by the same group in Israel.  They surmised that there was evidence that was being ignored as to the effect of anemia, and the treatment of anemia was essential in addition to other treatments.  They carried out a randomized trial to determine just that, a benefit to treating the anemia.  But they also conjectured that an anemia with a Hb concentration below 12 g/dl has an deleterious effect on the targeted population.  Treatment by intermittent transfusions could potentially provide the added oxygen-carrying capacity, but the fractionation of blood, the potential for transfusion-transmitted disease and transfusion-reactions, combined with the need for the blood for traumatic blood loss made EPO a more favorable alternative to packed RBCs.  The proof-of-concept is told below.  Patients randomized to receive EPO at a lower than standard dose + iron did better.

 

Introduction

In this article, Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker we provides a summary of three articles on the topic and we shading new light on the role that Anemia Hb < 12 g%  plays as a Biomarker for CHF and for

  • prediction of elevated BNP, known as an indicator for the following Clinical Uses:
Clinical Use
  • Rule out congestive heart failure (CHF) in symptomatic individuals
  • Determine prognosis in individuals with CHF or other cardiac disease
  • Maximize therapy in individuals with heart failure by the use of Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe)
Evaluation of BNP and NT-proBNP Clinical Performance
Study Sensitivity(%) Specificity(%) PPV(%) NPV(%)
Diagnose impaired LVEF3
BNP 73 77 70 79
NT-proBNP 70 73 61 80
Diagnose LV systolic dysfunction after MI2
BNP 68 69 56 79
NT-proBNP 71 69 56 80
Diagnose LV systolic dysfunction after MI12
BNP 94 40 NG 96
NT-proBNP 94 37 NG 96
Prognosis in newly diagnosed heart failure patients: prediction of mortality/survival1
BNP 98 22 42 94
NT-proBNP 95 37 47 93
Prognosis post myocardial infarction: prediction of mortality2
BNP 86 72 39 96
NT-proBNP 91 72 39 97
Prognosis post myocardial infarction: prediction of heart failure2
BNP 85 73 54 93
NT-proBNP 82 69 50 91
PPV, positive predictive value; NPV, negative predictive value; LVEF, left ventricular ejection fraction; NG, not given.
Reference Range
BNP: < 100 pg/mL13
proBNP, N-terminal: 300 pg/mL
The NT-proBNP reference range is based on EDTA plasma. Other sample types will produce higher values.
Interpretive Information
Symptomatic patients who present with a BNP or NT-proBNP level within the normal reference range are highly unlikely to have CHF. Conversely, an elevated baseline level indicates the need for further cardiac assessment and indicates the patient is at increased risk for future heart failure and mortality.BNP levels increase with age in the general population, with the highest concentrations seen in those greater than 75 years of age.14 Heart failure is unlikely in individuals with a BNP level <100 pg/mL and proBNP level ≤300 pg/mL. Heart failure is very likely in individuals with a BNP level >500 pg/mL and proBNP level ≥450 pg/mL who are <50 years of age, or ≥900 pg/mL for patients ≥50 years of age. Patients in between are either hypertensive or have mild ischemic or valvular disease and should be observed closely.15BNP is increased in CHF, left ventricular hypertrophy, acute myocardial infarction, atrial fibrillation, cardiac amyloidosis, and essential hypertension. Elevations are also observed in right ventricular dysfunction, pulmonary hypertension, acute lung injury, subarachnoid hemorrhage, hypervolemic states, chronic renal failure, and cirrhosis.NT-proBNP levels are increased in CHF, left ventricular dysfunction, myocardial infarction, valvular disease, hypertensive pregnancy, and renal failure, even after hemodialysis.Although levels of BNP and NT-proBNP are similar in normal individuals, NT-proBNP levels are substantially greater than BNP levels in patients with cardiac disease due to increased stability (half-life) of NT-proBNP in circulation. Thus, results from the two tests are not interchangeable.
References
  1. Cowie MR and Mendez GF. BNP and congestive heart failure. Prog Cardiovasc Dis. 2002;44:293-321.
  2. Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin. New neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circulation. 1998:97:1921-1929.
  3. Hammerer-Lercher A, Neubauer E, Muller S, et al. Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction. Clin Chim Acta. 2001;310:193-197.
  4. McDonagh TA, Robb SD, Murdoch DR, et al. Biochemical detection of left-ventricular systolic dysfunction. Lancet. 1998;351:9-13.
  5. Mukoyama Y, Nakao K, Hosoda K, et al. Brain natriuretic peptide as a novel cardiac hormone in humans: Evidence for an exquisite dual natriuretic peptide system, ANP and BNP. J Clin Invest. 1991;87:1402-1412.
  6. Hunt PJ, Richards AM, Nicholls MG, et al. Immunoreactive amino-terminal pro-brain natriuretic peptide (NT-PROBNP): a new marker of cardiac impairment. Clin Endocrinol. 1997;47:287-296.
  7. Davis M, Espiner E, Richards G, et al. Plasma brain natriuretic peptide in assessment of acute dyspnoea. Lancet. 1994;343:440-444.
  8. Kohno M, Horio T, Yokokawa K, et al. Brain natriuretic peptide as a cardiac hormone in essential hypertension. Am J Med. 1992;92:29-34.
  9. Bettencourt P, Ferreira A, Pardal-Oliveira N, et al. Clinical significance of brain natriuretic peptide in patients with postmyocardial infarction. Clin Cardiol. 2000;23:921-927.
  10. Jernberg T, Stridsberg M, Venge P, et al. N-terminal pro brain natriuretic peptide on admission for early risk stratification of patients with chest pain and no ST-segment elevation. J Am Coll Cardiol. 2002;40:437-445.
  11. Richards AM, Troughton RW. Use of natriuretic peptides to guide and monitor heart failure therapy. Clin Chem. 2012;58:62-71.
  12. Pfister R, Scholz M, Wielckens K, et al. The value of natriuretic peptides NT-pro-BNP and BNP for the assessment of left-ventricular volume and function. A prospective study of 150 patients.Dtsch Med Wochenschr. 2002;127:2605-2609.
  13. Siemens ADVIA Centaur® BNP directional insert; 2003.
  14. Redfield MM, Rodeheffer RJ, Jacobsen SJ, et al. Plasma brain natriuretic peptide concentration: impact of age and gender. J Am Coll Cardiol. 2002;40:976-982.
  15. Weber M, Hamm C. Role of B-type natriuretic peptid (BNP) and NT-proBNP in clinical routine.Heart. 2006;92:843-849.

SOURCE

B-type Natriuretic Peptide and proBNP, N-terminal

http://www.questdiagnostics.com/testcenter/testguide.action?dc=TS_BNP_proBNP

FIRST ARTICLE

Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3,  LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6,  John F. Heitner, MD, FACC7
1Cardiology Fellow,  2  Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology,  Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript)  Poster Presentation:

Anemia as an Independent Predictor of Elevated N-Terminal proBNP Levels in
Patients without Evidence of Heart Failure and Normal Renal Function.

Haq SA, Alam ME, Bernstein L, Banko LB, Saul BI, Lee LY, Sacchi TJ, Heitner JF.

Table 1.  Patient Characteristics

Variable No Anemia(n=138) Anemia(n=80)
Median Age (years) 63 76
Men (%) 35 33
Creatinine (mg/dl) 0.96 1.04
Hemoglobin (g/dl) 13.7 10.2
LVEF (%) 67 63
Median NT-proBNP (pg/ml) 321.6 1896.0

Poster-ProBNP_final[1]

A series of slide showing the determination of the representation of normal NT-proBNP range
after removal of patient confounders.

Slide1

Slide10

Slide5

Slide8

ABSTRACT

Introduction

N-terminal proBNP (NT-proBNP) has emerged as a primary tool for diagnosing congestive heart failure (CHF). Studies have shown that the level of

  • NT-proBNP is affected by renal insufficiency (RI) and age, independent of the diagnosis of CHF.

There is some suggestion from recent studies that

  • anemia may also independently affect NT-proBNP levels.

Objective

To assess the affect of anemia on NT-proBNP independent of CHF, RI, and age.

Methods

We evaluated 746 consecutive patients presenting to the Emergency Department (ED) with shortness of breath and underwent evaluation with serum NT-proBNP.

All patients underwent a trans-thoracic echocardiogram (TTE) and clinical evaluation for CHF. Patients were included in this study if they had a normal TTE (normal systolic function, mitral inflow pattern and left ventricular (LV) wall thickness) and no evidence of CHF based on clinical evaluation. Patients were excluded if they had RI (creatinine > 2 mg/dl) or a diagnosis of sepsis. Anemia was defined using the World Health Organization (W.H.O.) definition of

  • hemoglobin (hgb) < 13 g/dl for males and hgb < 12 g/dl for females.

Results

Of the 746 consecutive patients, 218 patients (138 anemia, 80 no anemia) met the inclusion criteria. There was a markedly significant difference between

  • NT- proBNP levels based on the W.H.O. diagnosis of anemia.

Patients with anemia had a

  • mean NT- proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p=0.0001).

There was a markedly

  • significant difference in patients who had a hgb > 12 (median 295 pg/ml) when compared to
  • both patients with an hgb of 10.0 to 11.9 (median 2,102 pg/ml; p = 0.0001) and
  • those with a hgb < 10 (median 2,131 pg/ml; p = 0.001).

Linear regression analysis comparing hgb with log NT-proBNP was statistically significant (r = 0.395; p = 0.0001). MANOVA demonstrated that

  • elevated NT- proBNP levels in patients with anemia was independent of age.

Conclusion

This study shows that NT-proBNP is associated with anemia independent of CHF, renal insufficiency, sepsis or age.

INTRODUCTION

B-type natriuretic peptide (BNP) is secreted from the myocardium in response to myocyte stretch. 1-2 BNP is released from the myocytes as a 76 aminoacid N-terminal fragment (NT-proBNP) and a 32-amino acid active hormone (BNP). 3 These peptides have emerged as a primary non-invasive modality for the diagnosis of congestive heart failure (CHF). 4- 7 In addition, these peptides have demonstrated prognostic significance in patients with invasive modality for the diagnosis of

  • congestive heart failure (CHF). 4- 7
  • heart failure 8-9,
  • stable coronary artery disease 10, and
  • in patients with acute coronary syndromes. 11-14

Studies have shown that the level of NT- proBNP is affected by

  • age and renal insufficiency (RI) independent of the diagnosis of CHF. 15,16

There is some suggestion from the literature that

  • anemia may also independently affect NT-proBNP levels. 17-20

Willis et al. demonstrated in a cohort of 209 patients without heart failure that anemia was associated with an elevated NT- proBNP. 17 Similarly, in 217 patients undergoing cardiac catheterization, blood samples were drawn from the descending aorta prior to contrast ventriculography for BNP measurements and

  • anemia was found to be an independent predictor of plasma BNP levels. 18

The objective of this study is to assess the effect of anemia on NT-proBNP independent of CHF, sepsis, age or renal insufficiency.

METHODS

Patient population

The study population consisted of 746 consecutive patients presenting to the emergency room who underwent NT-proBNP evaluation for the evaluation of dyspnea. Transthoracic echocardiogram (TTE) was available on 595 patients. Patients were included in this study if they had a normal TTE, which was defined as normal systolic function (left ventricular ejection fraction [LVEF] > 45%), normal mitral inflow pattern and normal LV wall thickness. CHF was excluded based on thorough clinical evaluation by the emergency department attending and the attending medical physician. Patients with disease states that may affect the NT- proBNP levels were also excluded:

  1. left ventricular systolic dysfunction (LVEF < 45%),
  2. renal insufficiency defined as a creatinine > 2 mg/dl and
  3. sepsis (defined as positive blood cultures with two or more of the following systemic inflammatory response syndrome (SIRS) criteria: heart rate > 90 beats per minute;
  4. body temperature < 36 (96.8 °F) or > 38 °C (100.4 °F);
  5. hyperventilation (high respiratory rate) > 20 breaths per minute or, on blood gas, a PaCO2 less than 32 mm Hg;
  6. white blood cell count < 4000 cells/mm3 or > 12000 cells/mm³ (< 4 x 109 or > 12 x 109 cells/L), or greater than 10% band forms (immature white blood cells). 21

The study population was then divided into two groups, anemic and non- anemic. Anemia was defined using the world health organization (W.H.O.) definition of hemoglobin (hgb) < 13 g/dl for males and < 12 g/dl for females.The data was also analyzed by dividing the patients into three groups based on hgb levels i.e. hgb > 12, hgb 10 to 11.9 and hgb < 10.

Baseline patient data

Patient’s baseline data including age, gender, ethnicity, hemoglobin (hgb), hematocrit (hct), creatinine, NT- proBNP were recorded from the electronic medical record system in our institution. Chemistry results were performed on the Roche Modular System (Indianapolis, IN), with the NT- proBNP done by chemiluminescence assay. The hemogram was performed on the Beckman Coulter GenS. All TTE’s were performed on Sonos 5500 machine. TTE data collected included LVEF, mitral inflow pattern and LV wall thickness assessment.

Statistical analysis

The results are reported in the means with p < 0.05 as the measure of significance for difference between means. Independent Student’s t-tests were done comparing NT proBNP and anemia. Univariate ANOVAs and multivariate ANOVA (MANOVA) with post hoc tests using the Bonferroni method were used to compare NT- proBNP levels with varying ranges of hgb and age using SPSS 13.0 (SPSS, Chicago, IL). A linear regression analysis was performed using SYSTAT. Calculations included Wilks’Lamda, Pillai trace and Hotelling-Lawley trace. A GOLDMineR® plot was constructed to estimate the effects of age and anemia on NT- proBNP levels. The GOLDMineR® effects plot displays the odds-ratios for predicted NT-proBNP elevation versus the predictor values. Unlike the logistic regression, the ordinal regression, which the plot is derived from, can have polychotomous as well as dichotomous values, as is the case for NT-proBNP.

RESULTS

Of the 746 consecutive patients, 218 patients met the inclusion criteria (fig 1). Baseline characteristics of patients are listed in table 1. The median age for anemic patients was 76 years and 63 years for patients without anemia. One third of patients in both groups were men. The mean hemoglobin for

  • anemic patients was 10.2 g/dl as compared to 13.7 g/dl for non-anemic patients.
  • The mean LVEF of patients with anemia was 64% as compared to 67% for non-anemic patients.

Based on the WHO definition of anemia, 138 patients were determined to be anemic while 80 patients were diagnosed as non-anemic. There was a markedly  significant difference between NT-proBNP levels based on the WHO diagnosis of anemia.

Patients with anemia had a

  • mean NT-proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p = 0.0001).

Of the 218 patients in the study, 55 patients had a hgb of < 10 g/dl. Analysis using

  • hgb < 10 g/dl for anemia demonstrated a statistically significant difference in the NT-proBNP values.

Patients with a hgb < 10 g/dl had a mean NT- proBNP of 5,130 pg/ml

  • compared to 2,882 pg/ml in patients with a hgb of > 10 g/dl (p = 0.01)

The groups were also divided into three separate categories of hgb for subset analysis:

  • hgb > 12 g/dl,
  • hgb 10 to 11.9 g/dl and
  • hgb < 10 g/dl.

There was a markedly significant difference in

  •  the NT- ProBNP levels of patients who had a hgb > 12 g/dl (median 295 pg/ml) when
  • compared to those with a hgb range of 10.0 g/dl to 11.9 g/dl (median 2,102 pg/ml) (p = 0.0001),

and also a significant difference in

  • NT- proBNP levels of patients with a hgb > 12 g/dl (median 295 pg/ml) when
  • compared to a hgb of < 10 g/dl (median 2,131 pg/ml) (p = 0.001).

However, there was no statistically significant difference in NT-proBNP levels of patients with hgb 10 g/dl to 11.9 g/dl

  • when compared to those with a hgb of < 10 g/dl (p = 1.0).

A scatter plot comparing hgb with log NT-proBNP and fitting of a line to the data by ordinary least squares regression was significant (p = 0.0001) and demonstrated

  • a correlation between anemia and NT-proBNP levels (r = 0.395) (fig. 2).

MANOVA demonstrated that elevated NT- proBNP levels in patients with anemia was independent of age (Wilks’ Lambda [p = 0.0001]). In addition, using GOLDMineR® plots (figure 3a and 3b) with a combination of age and hb scaled as predictors of elevated NT-proBNP,

  • both age and hgb were required as independent predictors.

What about the effect of anemia? The GOLDminer analysis of ordinal regression was carried out in a database from which renal insufficiency and CHF were removed. The anemia would appear to have an independent effect on renal insufficiency. Figure 4 is a boxplot comparison of NT – proBNP, the age normalized function NKLog (NT- proBNP)/eGFR formed from taking 1000*Log(NT- proBNP) divided by the MDRD at eGFR exceeding 60 ml/min/m2 and exceeding 30 ml/min/m2. The transformed variable substantially makes the test independent of age and renal function. The boxplot shows the medians, 97.5, 75, 25 and 2.5 percentiles. There appears to be no significance in the NKLog(NT pro-BNP)/MDRD plot. Table II compares the NT-proBNP by WHO criteria at eGFR 45, 60 and 75 ml/mln/m2 using the t-test with unequal variance assumed, and the Kolmogorov-Smirnov test for nonparametric measures of significance. The significance at 60 ml/min/m2 is marginal and nonexistent at 75 ml/min/m2. This suggests that the contribution from renal function at above 60 ml/min2 can be ignored. This is consistent with the findings using the smaller, trimmed database, but there is an interaction between

  •  anemia, and
  •  eGFR at levels below 60 ml/min/m2

DISCUSSION

The findings in this study indicate that

  • anemia was associated with elevated NT-proBNP levels independent of CHF, renal insufficiency, sepsis or age.

These findings have been demonstrated with NT-proBNP in only one previous study. Wallis et al. demonstrated that after adjusting for age, sex, BMI, GFR, LVH and valvular disease;

  1. only age,
  2. valvular disease and
  3. low hemoglobin

were significantly associated with increased NT-proBNP. 18.

In our study, CHF was excluded based on both a normal TTE and a thorough clinical evaluation. In the only other study directly looking at NT- proBNP levels in anemic patients without heart failure

  • only 25% of patients had TTEs, with one patient having an LVEF of 40%. 17

BNP, the active molecule released after cleavage along with NT- proBNP, has also been studied in relation to blood hemoglobin levels. 18 In 263 patients undergoing cardiac catheterization  blood samples were drawn from the descending aorta prior to contrast ventriculography to determine the value of BNP. Anemia was present in 217 patients. Multivariate linear regression model adjusting for

  1.  age,
  2.  gender,
  3.  body mass index,
  4.  history of myocardial infarction,
  5.  estimated creatinine clearance, and
  6.  LVEF
  • found hgb to be an independent predictor of BNP levels.

In our study, patients with anemia were slightly older than those without anemia. However, both MANOVA and GOLDMineR® plot demonstrated that

  • elevated NT-proBNP levels in patients with anemia was independent of age.

Other studies have found that BNP is dependent on renal insufficiency and age. Raymond et al. randomly selected patients to complete questionnaires regarding CHF and

  1. then underwent pulse and blood pressure measurements,
  2.  electrocardiogram (ECG),
  3.  echocardiography and
  4.  blood sampling. 15

A total of 672 subjects were screened and 130 were determined to be normal, defined as

  • no CHF or ischemic heart disease,
  • normal LVEF,
  • no hypertension,
  • diabetes mellitus,
  • lung disease, and
  • not on any cardiovascular drugs.

They found

  1. older age,
  2. increasing dyspnea,
  3. high plasma creatinine and a
  4. LVEF < 45%

to be independently associated with an elevated NT-proBNP plasma level by multiple linear regression analysis. In another study, McCullough et al. evaluated the patients from the Breathing Not Properly Multinational Study

  • looking at the relationship between BNP and renal function in CHF. 16

Patients were excluded if they were on hemodialysis or had a estimated glomerular filteration rate (eGFR) of < 15 ml/min. They found that the BNP levels correlated significantly with the eGFR, especially in patients without CHF, suggesting

  1. chronic increased blood volume and
  2. increased left ventricular wall tension as a possible cause. 16

Our study was designed to exclude patients with known diseases such as CHF and renal insufficiency in order to demonstrate

  • the independent effect of anemia on elevated NT-proBNP levels.

The mechanism for elevated NT-proBNP levels in patients with anemia is unknown. Some possible mechanisms that have been reported in the literature include

  • hemodilution secondary to fluid retention in patients with CHF 18,
  • decreased oxygen carrying capacity with accompanying tissue hypoxia which
  • stimulates the cardio-renal compensatory mechanism leading to increased release of NT-proBNP. 17

The findings from our study suggest that

  •  NT-proBNP values should not be interpreted in isolation of hemoglobin levels and
  • should be integrated with other important clinical findings for the diagnosis of CHF.

Further studies are warranted

  1.  to assess the relationship between anemia and plasma natriuretic peptides, and
  2. possibly modify the NT-proBNP cutoff points for diagnosing acutely decompensated CHF in patients with anemia.

CONCLUSION

This study shows that elevated NT-proBNP levels are associated with anemia independent of

  •   CHF,
  •  renal insufficiency,
  •  sepsis and
  •  age.

NT-proBNP levels should be interpreted with caution in patients who have anemia.

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2. Wiese S, Breyer T, Dragu A, et al. Gene expression of brain natriuretic peptide  in isolated atrial and ventricular human myocardium: influence of angiotensin II and diastolic fiber length. Circ 2000; 102:3074-79.

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4.   Dao Q, Krishnaswamy P, Kazanegra R, et al. Utility of B-type natriuretic  peptide in the diagnosis of congestive heart failure in an urgent care setting. J Am  Coll Cardiol 2001; 37:379-85.

5. Morrison LK, Harrison A, Krishnaswamy P, Kazanegra R, Clopton P, Maisel A. Utility of rapid natriuretic peptide assay in differentiating congestive heart failure from lung  disease in patients presenting with dyspnea.
J Am Coll Cardiol  2003; 39:202-09.

6.  Maisel AS, Krishnaswamy P, Nowak RM, et al.  Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med 2002; 347:161-67.

7. Januzzi JL, Camargo CA, Anwaruddin S, et al. The N-terminal Pro-BNP investigation of dyspnea in the emergency department (PRIDE) study. Am J  Cardiol 2005; 95:948-954.

8.  Tsutamoto T, Wada A, Meada K, et al.   Attenuation of compensation of  endogenous cardiac natriuretic peptide system  in chronic heart failure: prognostic role  of plasma  brain natriuretic peptide concentration in patients with chronic  symptomatic  left ventricular dysfunction.
Circulation 1997; 96(2): 509-16.

9.  Anand IS, Fisher LD, Chiang YT, et al. Changes in brain natriuretic peptide and norepinephrine over time and mortality and morbidity in the Valsartan Heart Failure Trial (Val-HEFT). Circulation 2003; 107:1278-1283.

10. Omland T, Richards AM, Wergeland R and Vik-Mo H. B-type natriuretic peptide and long term survival in patients with stable coronary artery disease.
Am J Cardiol 2005; 95:24-28.

11. Omland T, Aakvaag A, Bonarjee VV. et al. Plasma brain natriuretic peptide as an indicator of left ventricular systolic dysfunction and long term prognosis after acute myocardial infarction. Comparison with plasma atrial natriuretic peptide and N-terminal proatrial natriuretic peptide.
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12. de Lemos JA, Morrow DA, Bently JH, et al. The prognostic value of B-type natriuretic peptide in patients with acute coronary syndromes. N Engl J Med 2001; 345:1014-1021.

13. Richards AM, Nicholls MG, Espiner EA, et al. B-type natriuretic peptides and  ejection fraction for prognosis after myocardial infarction. Circulation 2003; 107:2786-2792.

14. Sabatine MS, Morrow DA, de Lemos JA, et al.  Multimarker approach to risk  stratification in non-ST elevation acute coronary syndromes: simultaneous  assessment of troponin I, C-reactive protein and B-type natriuretic peptide.
Circulation 2002; 105:1760-1763.

15. Raymond I, Groenning BA, Hildebrandt PR, Nilsson JC, Baumann M, Trawinski   J, Pedersen F.  The influence of age, sex andother variables on the plasma level of N-terminal pro brain natriureticpeptide in a large sample of the general  population. Heart 2003; 89:745-751.

16. McCollough PA, Duc P, Omland T, McCord J, Nowak RM, Hollander JE, et al. B-type natriuretic peptide and renal function in the diagnosis of heartfailure:  an analysis from the  Breathing Not Properly Multinational Study.
Am J Kidney Dis 2003; 41:571-579.

17. Willis MS, Lee ES, Grenache DG. Effect of anemia on plasma concentrations of  NT-proBNP.
Clinica Chim Acta 2005; 358:175-181.

18. Wold Knudsen C, Vik-Mo H, Omland T. Blood hemoglobin is an independent  predictor of B-type natriuretic peptide.
Clin Sci 2005; 109:69-74.

19. Tsuji H, Nishino N, Kimura Y, Yamada K, Nukui M, et al. Haemoglobin level influences plasma brain natriuretic peptide concentration. Acta Cardiol 2004;59:527-31.

20. Wu AH, Omland T, Wold KC, McCord J, Nowak RM, et al. Relationship  of B-type natriuretic peptide and anemia  in patients withand without heart failure:  A substudy from the Breathing Not Properly(BNP) Multinational Study.
Am J  Hematol 2005; 80:174-80.

22. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, et al.  Definitions for sepsis and organ failure and guidelines for theuse of innovative therapies in sepsis.  The ACCP/SCCM Consensus Conference Committee. Chest. 1992;101(6):1644-55.

Table Legends

Table I. Clinical characteristics of the study population

Table II. Comparison of NT- proBNP means under WHO criteria at different GFR

Table I
Variable No Anemia(n=80) Anemia(n=138)
Median age (years) 63 76
Gender
    Men (%) 27 (34) 47 (34)
    Women (%) 53 (66) 91 (66)
Weight (kg) 82.9 80.1
Chest Pain 21 (26) 3 (2)
Hemoglobin (g/dl) 13.7 10.2
Hematocrit (%) 40.5 30.5
Mean Corpuscular Volume 97 87
Creatinine (mg/dl) 0.99 1.07
Median NT-proBNP (pg/ml) 321 1896
Medical History
    HTN (%) 12 (15) 51 (37)
    Prior MI (%) 11 (14) 5 (4)
    ACS (%) 16 (20) 3 (2)
    CAD (%) 2 (1) 3 (2)
     DM (%) 18 (22) 11 (8)
Medication
   Clopidogrel 58 (72) 15 (11)
   Beta Blockers 68 (85) 27 (20)
   Ace Inhibitors 45 (56) 18 (13)
   Statins 57 (71) 17 (12)
   Calcium Channel Blocker 17 (21) 8 (6)
LVEF (%) 67 64

HTN: Hypertension CAD: Coronary Artery Disease
MI: Myocardial Infarction DM: Diabetes Mellitus
ACS: Acute Coronary Syndrome LVEF: Left Ventricular Ejection Fraction

Table II
GFR WHO Mean P (F) N NPar
> 45 0 3267 0.022 (4.33) 661
1 4681
> 60* 0 2593 0.031 (5.11) 456 0.018
1 4145
> 60r 0 786 0.203 (3.63) 303 0.08
1 3880
> 75 0 2773 > 0.80 320 0.043
1 3048

*AF, valve disease and elevated troponin T included
r AF, valve disease and elevated troponin T removed

FIGURE LEGENDS

FIGURE 1. Study population flow chart. (see poster)
FIGURE 2. Relationship between proBNP and hemoglobin. (see above)
FIGURE 3. NT-proBNP levels in relation to anemia (see above)

Supplementary Material

Table based on LatentGOLD Statistical Innovations, Inc., Belmont, MA., 2000: Jeroen Vermunt & Jay Magidson)

4-Cluster Model

Number of cases                                   408
Number of parameters (Npar)             24

Chi-squared Statistics
Degrees of freedom (df)                          71                     p-value
L-squared (L²)                                    80.2033                    0.21
X-squared                                            80.8313                     0.20
Cressie-Read                                        76.6761                     0.30
BIC (based on L²)                          -346.5966
AIC3 (based on L²)                        -132.7967
CAIC (based on L²)                       -417.5966

Model for Clusters
 Intercept                Cluster1      Cluster2     Cluster3     Cluster4     Wald     p-value
————–           0.1544           0.1434        0.0115        -0.3093     1.1981     0.75
Cluster Size           0.2870          0.2838       0.2487          0.1805
(across)

LogNTpr
< 1.5                       0.0843           0.2457       0.0006          0.0084
1.6-2.5                   0.6179            0.6458       0.0709          0.2809
2.5-3.5                  0.2848           0.1067         0.5319          0.5883
> 3.5                      0.0130           0.0018         0.3966         0.1224
MDRD
> 90                     0.1341             0.7919         0.0063         0.6106
61-90                  0.6019            0.2040          0.1633         0.3713
41-60                  0.2099            0.0041          0.3317         0.0175
< 41                     0.0542            0.0001         0.4987        0.0006
age
under 51           0.0668           0.5646          0.0568        0.0954
51-70                 0.3462            0.3602          0.3271         0.3880
over 70             0.5870            0.0752          0.6161         0.5166
WHO
No anemia      0.7518             0.6556          0.2041         0.0998
Anemia            0.2482             0.3444          0.7959         0.9002

———          Cluster1          Cluster2      Cluster3      Cluster4
Overall           0.2870            0.2838         0.2487        0.1805
(down)

LogNTpro
< 1.5                0.2492              0.7379           0.0013         0.0116
1.6-2.5            0.4163               0.4243           0.0427        0.1167
2.6-3.5           0.2296               0.0887          0.3723        0.3095
> 3.5              0.0328                0.0023          0.7982        0.1666
MDRD
> 90              0.1001                0.5998           0.0043        0.2958
61-90           0.5198                 0.1716           0.1136         0.1950
41-60           0.3860                 0.0055          0.5847         0.0238
< 41             0.1205                  0.0002          0.8785         0.0008
 age
< 51            0.0720                 0.7458           0.0910          0.0912
51-70         0.3036                 0.3084           0.2013          0.1867
over 70     0.3773                  0.0409          0.3633           0.2186
 WHO
No anemia 0.4589              0.3957           0.1076           0.0378
Anemia     0.1342                 0.1844            0.3742           0.3073

Hemoglobin on NT proBNP 3

SECOND ARTICLE

The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7

http://content.onlinejacc.org/article.aspx?articleid=1127229

OBJECTIVES

This is a randomized controlled study of anemic patients with severe congestive heart failure (CHF) to assess the effect of correction of the anemia on cardiac and renal function and hospitalization.

BACKGROUND

Although mild anemia occurs frequently in patients with CHF, there is very little information about the effect of correcting it with erythropoietin (EPO) and intravenous iron.

METHODS

Thirty-two patients with moderate to severe CHF (New York Heart Association [NYHA] class III to IV)
who had a left ventricular ejection fraction (LVEF) of 40% despite maximally tolerated doses of CHF medications and
  • whose hemoglobin (Hb) levels were persistently between 10.0 and 11.5 g% were randomized into two groups.
Group A (16 patients) received subcutaneous EPO and IV iron to increase the level of Hb to at least 12.5 g%. In Group B (16 patients) the anemia was not treated. The doses of all the CHF medications were maintained at the maximally tolerated levels except for oral and intravenous (IV) furosemide, whose doses were increased or decreased according to the clinical need.

RESULTS

Over a mean of 8.2 +/- 2.6 months,
  • four patients in Group B and none in Group A died of CHF-related illnesses.
  • The mean NYHA class improved by 42.1% in A and worsened by 11.4% in B.
  • The LVEF increased by 5.5% in A and decreased by 5.4% in B.
  • The serum creatinine did not change in A and increased by 28.6% in B.
  • The need for oral and IV furosemide decreased by 51.3% and 91.3% respectively in A and increased by 28.5% and 28.0% respectively in B.
  • The number of days spent in hospital compared with the same period of time before entering the study decreased by 79.0% in A and increased by 57.6% in B.

CONCLUSIONS

When anemia in CHF is treated with EPO and IV iron, a marked improvement in cardiac and patient function is seen,
  • associated with less hospitalization and renal impairment and less need for diuretics. (J Am Coll Cardiol 2001;37:1775– 80)

Anemia of any cause is known to be capable of causing congestive heart failure (CHF) (1). In patients hospitalized with CHF the 

  • mean hemoglobin (Hb) is about 12 g% (2,3),

which is considered the lower limit of normal in adults (4). Thus, anemia appears to be

common in CHF. Recently, in 142 patients in our special CHF outpatient clinic, we found that

  • as the CHF worsened, the mean Hb concentration decreased, from 13.7 g% in mild CHF (New York Heart Association [NYHA] class I) to 10.9 g% in severe CHF (NYHA 4), and
  • the prevalence of a Hb 12 g% increased from 9.1% in patients with NYHA 1 to 79.1% in those with NYHA 4 (5).
The Framingham Study has shown that anemia is an
  • independent risk factor for the production of CHF (6).
Despite this association of CHF with anemia,
  • its role is not mentioned in the 1999 U.S. guidelines for the diagnosis and treatment of CHF (7), and
  • many studies consider anemia to be only a rare contributing cause of hospitalization for CHF (8,9).
Recently, we performed a study in which the anemia of severe CHF that was resistant to maximally tolerated doses of standard medications
  • was corrected with a combination of subcutaneous (sc) erythropoietin (EPO) and intravenous iron (IV Fe) (5).
We have found this combination to be safe, effective and additive
  • in the correction of the anemia of chronic renal failure (CRF) in both
  • the predialysis period (10) and the dialysis period (11).
The IV Fe appears to be more effective than oral iron (12,13). In our previous study of CHF patients (5), the treatment resulted in
  • improved cardiac function,
  • improved NYHA functional class,
  • increased glomerular filtration rate,
  • a marked reduction in the need for diuretics and
  • a 92% reduction in the hospitalization rate
compared with a similar time period before the intervention. In the light of these positive results, a prospective randomized study was undertaken
  • to determine the effects of the correction of anemia in severe symptomatic CHF resistant to maximally tolerated CHF medication.

Abbreviations and Acronyms

CABG coronary artery bypass graft
CHF congestive heart failure
CRF chronic renal failure
EPO erythropoietin
%Fe Sat percent iron saturation
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IU international units
IV intravenous
LVEF left ventricular ejection fraction
NYHA New York Heart Association
PA pulmonary artery
sc subcutaneous
SOLVD Studies Of Left Ventricular Dysfunction

MATERIALS AND METHODS

Patients.Thirty-two patients with CHF were studied. Before the study, the patients were treated for least six months in the CHF clinic with

  • maximally tolerated doses of angiotensin-converting enzyme inhibitors, the beta-blockers bisoprolol or carvedilol, aldospirone, long-acting nitrates, digoxin and oral and intravenous (IV) furosemide.

In some patients these agents could not be given because of contraindications and in others they had to be stopped because of side effects. Despite this maximal treatment

  • the patients still had severe CHF  (NYHA classIII), with  fatigue and/or shortness of breath  on even mild exertion or at rest.  All had levels of
  • Hb in the range of 10 to 11.5 g%  on at least three consecutive visits over a three-week period.
  • All had a LVEF of 40%.

Secondary causes of anemia including hypothyroidism, and folic acid and vitamin B12 deficiency were ruled out and

  • there was no clinical evidence of gastrointestinal bleeding.

The patients were randomized consecutively into two groups:

  • Group A, 16 patients, was treated with sc EPO and IV Fe to achieve a target Hb of at least 12.5 g%.
  • Group B, 16 patients, did not receive the EPO and IV Fe.

Treatment protocol for correction of anemia.

All patients in Group A received the combination of sc EPO and IV Fe. The EPO was given once a week at a starting dose of 4,000 international units (IU) per week  and
the dose was increased  to two  or  three  times a week or decreased to once every few weeks as  necessary

  • to achieve and maintain a target Hb of 12.5 g%.

The IV Fe (Venofer-Vifor International, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every two weeks

  • until the serum ferritin reached 400 g/l or
  • the %Fe saturation (%Fe Sat is serum iron/total iron binding capacity 100) reached 40% or
  • the Hb reached 12.5g%. 

The IV Fe was then given at longer intervals as needed to maintain these levels.

Investigations. 

Visits to the clinic were at two- to three week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before then,

  • potassium and ferritin and %Fe Sat were performed on every visit.
  • blood pressure was measured by an electronic device on every visit.
  • LVEF was measured initially and at four- to six-month intervals by MUGA radioisotope ventriculography.

This technique measures

  • the amount of blood in the ventricle at the end of systole and at the end of diastole, thus giving
  • a very accurate assessment of the ejection fraction.

It has been shown to be an accurate and reproducible method of measuring the ejection fraction (14).  Hospital records were reviewed at the end of the intervention period to compare

  • the number of days hospitalized during the study with 
  • the number of days hospitalized during a similar period 
    • when the patients were treated in the CHF clinic before the initial randomization and entry into the study.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study. The mean follow-up for patients was 8.2 +/-  2.7 months (range 5 to 12 months).  The study was done with the approval of the local ethics committee.Statistical analysis.

An analysis of variance with repeated measures (over time) was performed to compare the two study groups (control vs. treatment) and

  • to assess time trend and the interactions between the two factors.
  • A separate analysis was carried out for each of the outcome parameters.
  • The Mann-Whitney test was used to compare the change in NYHA class between two groups.

All the statistical analysis was performed by SPSS (version 10).

RESULTS

The mean age in Group A (EPO and Fe) was 75.3 +/-  14.6 years and in group B was 72.2 +/-  9.9 years. There were 11 and 12 men in Groups A and B, respectively.
Before the study the two groups were similar in
  1. cardiac function,
  2. comorbidities,
  3. laboratory investigations and
  4. medications
  • (Tables 1, 2 and 3), except for IV furosemide (Table 3),
which was higher in the treatment group. The mean NYHA class of Group A before the study was 3.8  0.4 and was 3.5  0.5 in Group B. The contributing factors to CHF in Groups A and B, respectively, are seen in Table 1 and were similar.
Table 1. Medical Conditions and Contributing Factors to Congestive Heart Failure in the 16 Patients Treated for the Anemia and in the 16 Controls

Table 1 medical conditions heart failure anemia

Table 2. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

Table 2 medications to treat heart failure anemia

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP  blood pressure; Fe Sat  iron saturation; Hb  hemoglobin; IV  intravenous; NS  not stated; Std Dev.  standard deviation.

The main contributing factors to CHF were considered to be

  • ischemic heart disease (IHD) in 11 and 10 patients respectively,
  • hypertension in two and two patients,
  • valvular heart disease in twoand two patients, and
  • idiopathic cardiomyopathy in one and two patients, respectively.

A significant change after treatment was observed in the two groups in the following parameters:

  • IV furosemide,
  • days in hospital,
  • Hb,
  • ejection fraction,
  • serum creatinine and
  • serum ferritin.
In addition, the interaction between the study group and time trend was significant in all measurements except for blood pressure and %Fe Sat. This interaction indicates that
  • the change over time was significantly different in the two groups.
Table 3. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

Table 3  CHF aneia EPO

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP  blood pressure; Fe Sat  iron saturation; Hb  hemoglobin; IV  intravenous; NS  not stated; Std Dev.  standard deviation.

We find in the comparisons of Tables 2 and 3:

  1. before treatment the level of oral furosemide was higher in the control group (136.2 mg/day) compared with the treatment group (132.2 mg/day).
  2. after treatment, while the dose of oral furosemide of the treated patients was reduced  to 64.4 mg/day
  • the dose of the nontreated patients was increased to 175 mg/day.

The same results of improvement in the treated group and deterioration in the control group are expressed in the following parameters:

  1. IV furosemide, days in hospital,
  2. Hb,
  3. ejection fraction and
  4. serum creatinine.

The NYHA class was

  • 3.8 +/- 0.4 before treatment and 2.2 +/- 0.7 after treatment in Group A  (delta mean = – 1.6) and
  • 3.5 +/-  0.7 before treatment and 3.9 +/- 0.3 after treatment in Group B. (delta mean = 0.4)

The improvement in NYHA class was significantly higher (p < 0.0001) in the treatment group compared with the control group (Table 4).

Table 4. Changes from Baseline to Final New York Heart Association (NYHA) Class
Initial minus final

Table 4  changes from NYHA baseline  CHF anemia

The improvement in NYHA class was statistically higher (p <  0.0001) in the treatment group compared with control.

There were no deaths in Group A and four deaths in Group B.

Case 1: A 71-year-old woman with severe mitral insufficiency and severe pulmonary hypertension  (a pulmonary artery [PA] pressure of 75 mm Hg) had persistent NYHA 4 CHF  and died during mitral valve surgery  seven months after onset of the study. She was hospitalized for 21 days  in the seven months before randomization and for 28 days  during the seven months after randomization.

Case 2:

A 62-year-old man with a longstanding history of hypertension complicated by IHD, coronary artery bypass graft (CABG) and atrial fibrillation had persistent NYHA 4 CHF  and a PA pressure of 35 mm Hg,  and died from pneumonia and septic shock eight months after onset of the study. He was hospitalized for seven days in the eight months before randomization and for 21 days during the eight months  after
randomization.

Case 3:
A 74-year old man with IHD, CABG, chronic obstructive pulmonary disease, a history of heavy smoking and diabetes had persistent NYHA 4 CHF and a PA pressure of  28 mm Hg, and died of pulmonary  edema and cardiogenic shock nine months after onset of the study. He was hospitalized for 14 days in the nine months before  randomization and for 41 days during the nine months after randomization.

Case 4:
A 74-year-old man with a history of IHD, CABG, diabetes, dyslipidemia, hypertension and atrial fibrillation, had persistent NYHA 4 CHF and a PA pressure of 30 mm Hg,  and died of pneumonia and septic shock   six months after onset of the study. He was hospitalized for five days in the six months before randomization and for 16 days during the nine months after randomization.

DISCUSSION

 Main findings.

The main finding of the present study is that the correction of

  • even mild anemia in patients with symptoms of very severe CHF despite being on maximally tolerated drug therapy
  • resulted in a significant improvement in their cardiac function and NYHA functional class.

There  was also a large

  • reduction in the number of days of  hospitalization compared with a similar period before the  intervention.
  • all this was achieved despite a marked reduction in the dose of oral and IV furosemide.

In the group in whom the anemia was not treated, four  patients died during the study. In all four cases

  • the CHF was unremitting and contributed to the deaths. 

In addition,  for the group as a whole, 

  • the LVEF, the NYHA class and  the renal function worsened.

There was also need for

  • increased oral and IV furosemide as well as increased  hospitalization.

Study limitations.

The major limitations of this study are

  1. the smallness of the sample size and
  2. the fact that randomization and treatment were not done in a blinded fashion.

Nevertheless, the two groups were almost identical in

  1. cardiac, renal and anemia status;
  2. in the types and doses of medication they were taking before and during the intervention and
  3. in the number of hospitalization days before the intervention.

Although the results of this study, like those of  our previous uncontrolled study (5), suggest that

  • anemia may play an important role in the mortality and morbidity of  CHF,
  • a far larger double-blinded controlled study should be carried out to verify our findings.

Anemia as a risk factor for hospitalization and death in CHF.

Our results are consistent with a recent analysis of 91,316 patients hospitalized with CHF (15). Anemia was found to be a stronger predictor of

  • the need for early rehospitalization than  was hypertension,  IHD or the presence of a previous CABG.  

A recent analysis of the Studies Of Left Ventricular Dysfunction (SOLVD) (16) showed that

  • the level of hematocrit (Hct) was an independent risk factor for mortality.

During a mean follow-up of 33 months the mortality was

  • 22%, 27% and 34% for those with a Hct of 40, 35 to 40 and 35 respectively.

The striking response of our patients to

  • correction of mild anemia suggests that the failing heart may be very susceptible to anemia.

It has, in fact, been found in both animal (17) and human studies (17–19) that

  • the damaged heart is more vulnerable to anemia and/or ischemia than is the normal heart.

These stimuli may result in a more marked reduction in cardiac function than occurs in the normal heart and may explain why,  in our study,

  • the patients were so resistant to high doses of CHF medications and
  • responded so well when the anemia was treated.

Our findings about the importance of anemia in CHF are not surprising when one considers that, in dialysis patients,

  • anemia has been shown to be associated with an increased prevalence and incidence of CHF (20) and that
  • correction of anemia in these patients is associated with improved
    • cardiac function (21,22),
    • less mortality (23,24) and
    • fewer hospitalizations (23,25).

Effect of improvement of CHF on CRF.

Congestive heart failure can cause progressive renal failure (26,27). Renal ischemia is found very early on

  • in patients with cardiac dysfunction (28,29), and
  • chronic ischemia may cause progression of renal failure (30). Indeed, the development of
  • CHF in patients with essential hypertension has been found to be one of the most powerful predictors of
  • the eventual development of end-stage renal disease (31).

Patients who develop CHF after a myocardial infarction experience a

  • fall in the glomerular filtration rate (GFR) of about 1 ml/min/month if the CHF is not treated (32).

In another recent analysis of the SOLVD study, treating the CHF with

  • both angiotensin-converting enzyme inhibitors and beta-blockers resulted in better preservation of the renal function than did
  • angiotensin-converting enzyme inhibitors alone (26),
suggesting that the more aggressive the treatment of the CHF, the better the renal function is preserved. In the present study, as in our previous one (5), we found that the deterioration of GFR was prevented with
  • successful treatment of the CHF, including correction of the anemia, whereas
  • the renal function worsened when the CHF remained severe

All these findings suggest that early detection and treatment of CHF and systolic and/or diastolic dysfunction from whatever cause could prevent

  • the deterioration not only of the cardiac function
  • but of the renal function as well.

This finding has very broad implications in the prevention of CRFbecause most patients with advanced CRF have

  • either clinical evidence of CHF or at least some degree of systolic dysfunction (33).

Systolic and/or diastolic dysfunction can occur early on in many  conditions, such as

  • essential hypertension (34),
  • renal disease of any cause (35,36) or
  • IHD, especially after a myocardial infarction (37).

The early detection and adequate treatment of this cardiac dysfunction, including correction of the anemia, could prevent this cardiorenal insufficiency. To detect cardiac dysfunction early on, one would need  at least an echocardiogram and MUGA radio-nucleotide ventriculography. These tests should probably be done not only in patients with signs and symptoms of CHF,   but in all patients where CHF or systolic  and/or diastolic dysfunction are suspected, such as those with a history of heart disease or suggestive changes of ischemia or hypertrophy on the electrocardiogram, or in patients with hypertension or renal disease.

Other positive cardiovascular effects of EPO treatment.

Another possible explanation for the improved cardiac function in this study may be the direct effect that EPO itself has on improving cardiac muscle function (38,39) and myocardial cell growth (39,40) unrelated to its  effect of the anemia. In fact EPO may be  crucial in the formation of the heart muscle in utero (40). It may also improve  endothelial function (41).  Erythropoietin may be superior to blood transfusions  not only  because adverse reactions to EPO are infrequent, but also because

  • EPO causes the production and release of young cells from the bone marrow into the blood.

These cells have an oxygen dissociation curve that is shifted to the right of the normal curve, causing the release of

  • much greater amounts of oxygen into the tissues than occurs normally (42).

On the other hand, transfused blood consists of older red cells with an oxygen dissociation curve that is

  • shifted to the left, causing the release of much less oxygen into the tissues than occurs normally (42).

The combination of IV Fe and EPO. The use of IV Fe along with EPO has been found to have an additive effect, 

  • increasing the Hb even more than would occur with EPO alone while at the same time
  • allowing the dose of EPO to be reduced (10 –13).
  • The lower dose of EPO will be cost-saving and also reduce the chances of hypertension developing (43).
 We used iron sucrose (Venofer) as our IV Fe medication because, in our experience, it is extremely well tolerated (10,11) and  
  • has not been  associated  with any serious side effects in more than 1,200 patients over six years.

Implications of treatment of anemia in CHF. The correction of anemia is not a substitute for the well-documented effective therapy of CHF but seems to be  an important, if not vital,  addition to the therapy. It is surprising, therefore,  that judging from  the  literature  on CHF,

  • such an obvious treatment for improving CHF is so rarely considered.

We believe that correction of the anemia will have an important role to play in

  • the amelioration of cardiorenal insufficiency, and that this improvement will have
  • significant economic  implications as well.

Acknowledgments

The authors thank Rina Issaky, Miriam Epstein, Hava Ehrenfeld and Hava Rapaport for their secretarial assistance.
Reprint requests and correspondence: Dr. D. S. Silverberg, Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv, 64239, Israel.

 THIRD ARTICLE

The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6

http://content.onlinejacc.org/article.aspx?articleid=1126474

OBJECTIVES

This study evaluated the prevalence and severity of anemia in patients with congestive heart failure (CHF) and

  • the effect of its correction on cardiac and renal function and hospitalization.

BACKGROUND

The prevalence and significance of mild anemia in patients with CHF is uncertain, and the role of erythropoietin with intravenous iron supplementation in treating this anemia is unknown.

METHODS

In a retrospective study, the records of the 142 patients in our CHF clinic were reviewed to find
  • the prevalence and severity of anemia (hemoglobin [Hb]12 g).
In an intervention study, 26 of these patients, despite maximally tolerated therapy of CHF for at least six months, still had had severe CHF and were also anemic. They were treated with
  • subcutaneous erythropoietin and intravenous iron sufficient to increase the Hb to 12 g%.
The doses of the CHF medications, except for diuretics, were not changed during the intervention period.

RESULTS

The prevalence of anemia in the 142 patients increased with the severity of CHF,
  • reaching 79.1% in those with New York Heart Association class IV.
In the intervention study, the anemia of the 26 patients was treated for a mean of 7.2 5.5 months.
  • The mean Hb level and mean left ventricular ejection fraction increased significantly.
  • The mean number of hospitalizations fell by 91.9% compared with a similar period before the study.
  • The New York Heart Association class fell significantly,
  • as did the doses of oral and intravenous furosemide.
  • The rate of fall of the glomerular filtration rate slowed with the treatment.

CONCLUSIONS

Anemia is very common in CHF and its successful treatment is associated with a significant improvement in
  • cardiac function,
  • functional class,
  • renal function and
  • in a marked fall in the need for diuretics and hospitalization.
Abbreviations and Acronyms
ACE Angiotensin-converting enzyme
CHF congestive heart failure
COPD chronic obstructive pulmonary disease
CRF chronic renal failure
CVA cerebrovascular accident
EPO erythropoietin
Fe iron
g% grams Hb /100 ml blood
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IV intravenous
LVEF left ventricular ejection fraction
LVH left ventriculr hypertrophy
NYHA New York Heart Association
%Fe Sat percent iron saturation
sc subcutaneous
TNF tumor becrosis factor
ACE Angiotensin-converting enzyme
CHF congestive heart failure
COPD chronic obstructive pulmonary disease
CRF chronic renal failure
CVA cerebrovascular accident
EPO erythropoietin
Fe iron
g% grams Hb /100 ml blood
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IV intravenous
LVEF left ventricular ejection fraction
LVH left ventriculr hypertrophy
NYHA New York Heart Association
%Fe Sat percent iron saturation
sc subcutaneous
TNF tumor becrosis factor

The mean hemoglobin (Hb) in patients with congestive heart failure (CHF) is about 12 g Hb per 100 ml blood (g%) (1–3), which is considered to be the lower limit of normal in adult men and postmenopausal women (4). Thus, many patients with CHF are anemic, and

  • this anemia has been shown to worsen as the severity of the CHF progresses (5,6).
Severe anemia of any cause can produce CHF, and treatment of the anemia can improve it (7). In patients with chronic renal failure (CRF) who are anemic,
  • treatment of the anemia with erythropoietin (EPO) has improved many of the abnormalities seen in CHF,
  • reducing left ventricular hypertrophy (LVH) (8 –10),
  • preventing left ventricular dilation (11) and,
    • in those with reduced cardiac function, increasing the left ventricular ejection fraction (LVEF)(8 –10),
    • the stroke volume (12) and
    • the cardiac output (12).
In view of the high prevalence of anemia in CHF, it is surprising that we could find no studies in which EPO was used in the treatment of the anemia of CHF, and the use of EPO is not included in U.S. Public Health Service guide-lines of treatment of the anemia of CHF (13). In fact, anemia has been considered
  • only a rare contributing factor to the worsening of CHF, estimated as contributing to
  • no more than 0% to 1.5% of all cases (14 –16).
Perhaps for this reason, recent guidelines for the prevention and treatment of CHF do not mention treatment of anemia at all (17). If successful treatment of anemia could improve cardiac function and patient function in CHF,
  • this would have profound implications, because,
  • despite all the advances made in the treatment of CHF, it is still a major and steadily increasing cause of hospitalizations, morbidity and mortality (18 –20).
The purpose of this study is to examine
  • the prevalence of anemia (Hb 12 g%) in patients with different levels of severity of CHF and
  • to assess the effect of correction of this anemia in severe CHF patients
  • resistant to maximally tolerated doses of CHF medication.
A combination of subcutaneous (SC) EPO and intravenous (IV) iron (Fe) was used. We have found this combination to be additive in improving the anemia of CRF (21,22).

METHODS 

Patients.

The medical records of the 142 CHF patients being treated in our special outpatient clinic devoted to CHF were reviewed to determine the prevalence and severity of anemia and CRF in these patients. These patients were referred to the clinic either from general practice or from the various wards in the hospital.

Intervention study.

Despite at least six months of treatment in the CHF clinic,
  • 26 of the above patients had persistent, severe CHF (New York Heart Association [NYHA] class III),
  • had a Hb level of 12 g% and were on
    • angiotensin-converting enzyme [ACE] inhibitors, the 
    • alpha-beta-blocker carvedilol,
    • long-acting nitrates,
    • digoxin, 
    • aldactone and
    • oral and IV furosemide.

These 26 patients participated in an intervention study. The mean age was 71.76  8.12 years. There were 21 men and 5 women. They  all had a

  • LVEF below 35%,
  • persistent fatigue and
  • shortness

    of breath on mild to moderate exertion and often at rest, and had

  • required hospitalizations at least once during their follow-up in the CHF clinic for pulmonary edema.
In 18 of the 26 patients, the CHF was associated with ischemic heart disease either
  • alone in four patients, or
  • with hypertension in six,
  • diabetes in four,
  • the two together in three, or with
  • valvular heart disease in one.
Of the remaining eight patients,
  • four had valvular heart disease alone and
  • four had essential hypertension alone.
Secondary causes of anemia including
  • gastrointestinal blood loss (as assessed by history and by three negative stool occult blood examinations),
  • folic acid and vitamin B12 deficiency and
  • hypothyroidism were ruled out.
Routine gastrointestinal endoscopy was not carried out. The study passed an ethics committee.
Table 1. Initial Characteristics of the 142 Patients With CHF Seen in the CHF Clinic
Age, yearsMale/female,  %Associated conditionsDiabetesHypertensionDyslipidemiaSmoking

Main cardiac diagnosis
Ischemic heart disease

Dilated CMP

Valvular heart disease

Hypertension

LVEF,  %

Left atrial area (n 15 cm2)

Pulmonary artery pressure  (15 mm Hg)

Previous hospitalizations/year

Serum Na, mEq/liter

Serum creatinine, mg%

Hemoglobin, g%

70.1 +/- 11.1

79/21

28%

64%

72%

40%

74%

15%

6%

5%

32.5 +/- 12.2

31.3  +/- 10.3

43.1  +/-14.9

3.2  +/- 1.5

139.8  +/- 4.0

1.6   +/-  1.1

11.9   +/- 1.5

CMP  cardiomyopathy; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association class.

Correction of the anemia.

All patients received the combination of SC EPO and IV Fe. The EPO was given once a week at a starting dose of 2,000 IU per week subcutaneously, and the dose was increased or decreased as necessary to achieve and maintain a target Hb of 12 g%. The IV Fe (Venofer-Vifor International, St. Gallen, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every week until the serum ferritin reached 400  g/liter or the percent Fe saturation (%Fe Sat: serum iron/total iron binding capacity   100) reached 40% or until the Hb reached 12 g%. The IV Fe was then given at longer intervals as needed to maintain these levels.

Medication dose.

Except for oral and IV furosemide therapy, the doses of all the other CHF medications, which were used in the maximum tolerated doses before the intervention, were kept unchanged during the intervention period.

Duration of the study.

The study lasted for a mean of 7.2  5.5 months (range four to 15 months).

Investigations.

Visits were at weekly intervals initially and then at two- to three-week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before the intervention study.
  • A complete blood count, serum creatinine, serum ferritin and % Fe Sat were performed on every visit.
  •  An electronic device measured the blood pressure on every visit.
  • The LVEF was measured by a multiple gated ventricular angiography heart scan initially and at four- to six-month intervals.
Hospital records were reviewed to compare the number of hospitalizations during the time the patients were treated for the anemia with the number of hospitalizations
  • during a similar period of time that they were treated in the CHF clinic 
    before the anemia was treated.
Clinic records were reviewed to evaluate the types and doses of CHF medications used 
before and during the study.

Period of time that they were treated in the CHF clinic before the anemia was treated.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study.  The glomerular filtration rate (GFR) was calculated from the serum creatinine by the formula: 1/serum creatinine in mg% x 100 GFR in ml/min. The rate of change of the GFR before and during the intervention period was calculated by comparing the change in GFR per month in the year before the intervention with that during the intervention.

Statistical analysis.

Mean standard deviation was calculated. One-way analysis of variance (ANOVA) was performed to compare parameter levels between the four NYHA groups. Hochberg’s method of multiple comparisons (23) was used for pair-wise comparison between two groups. A p value of less than 0.05 was considered statistically significant. In the intervention study, the significance of the difference between the initial values and those at the end of the study for the individual parameters in the 26 treated patients was assessed by paired student’s t test; p < 0.05 was considered statistically significant. All the statistical analysis was performed by the SPSS program (Version 9, Chicago, Illinois).

 RESULTS

CHF: the whole study group.

The clinical, biochemical and hematological characteristics of the 142 patients seen in the clinic are shown in Tables 1 and 2.

  • Sixty-seven patients (47%) had severe CHF as judged by a NYHA class of IV (Table 2).
  • Seventy- nine of the 142 patients (55.6%) were anemic (Hb  12 g%).

The mean Hb level fell progressively from 13.73 +/- 0.83 g% in class I NYHA to 10.90 +/- 1.70 g% in class IV NYHA (p  0.01). The percentage of patients with Hb  12 g% increased from 9.1% in class I to 79.1% in class IV.
Fifty eight patients (40.8%) had CRF as defined as a serum creatinine  1.5 mg%. The mean serum creatinine increased from 1.18 +/_  0.38 mg% in class I NYHA, to 2.0 +/-    1.89 mg% in class IV NYHA, p  0.001. The percentage of patients with an elevated serum creatinine ( 1.5 mg%)      increased from 18.2% in class I to 58.2% in class IV.

The mean ejection fraction fell from 37.67 +/-  15.74% in class I to 27.72 +/-  9.68% (p  0.005) in class IV.

Table 2. LVEF and Biochemical and Hematological Parameters by NYHA Class in 142 Patients With CHF 
NYHA Class I II III IV  Significantly Different Pairs*

 *p  0.05 at least between the two groups by pair-wise comparison between groups.

†p  0.05 at least between the groups by ANOVA.

No. of patients

11

26  

38

67

(total 142) (%)

    (7.7)    (18.3)    (26.8)    (47.2)

Hb, g%†

13.73 (0.83)

13.38 (1.26)

11.95 (1.48)

10.90 (1.70) 

1–3, 1–4, 2–3, 2–4

Serum creatinine,

1.18

1.22

1.32

2.00

1–2, 1–3, 1–4

mg%†

    (0.38)     (0.29)      (0.38)     (1.89)

LVEF, %†

37.67 (15.74)

32.88 (12.41)

32.02 (10.99)

27.72 (9.68)

1–4, 2–4

Hb 12 g%,  (%)

1
(9.1)

5 (19.2) 

20 (52.6) 

53 (79.1)

Serum creatinine

    2      5     12     39

1.5 mg%,  (%) 

 (18.2)

(19.2)

(31.6)

 (58.2)

The intervention study: medications.

The percentage of patients receiving each CHF medication before and after the intervention period and the reasons for not receiving  them are seen in Table 3.

Table 3. Number (%) of the 26 Patients Taking Each Type of Medication Before and During the Intervention Period and the Reason Why the Medication Was Not Used

Medication    No. of Patients  (%)         Reason for Not Receiving the Medications (No. of Patients)
BP  blood pressure; CRF  chronic renal failure; IV  intravenous.

The main reason for not receiving:

  • 1) ACE inhibitors was the presence of reduced renal function;
  • 2) carvedilol was the presence of chronic obstructive pulmonary disease (COPD);
  • 3) nitrates was low blood pressure and aortic stenosis and
  • 4) aldactone was hyperkalemia.
Table 4. Mean Dose of Each Medication Initially and at the End of the Intervention Period in the 26 Patients

                                       No. of Patients                                 Initial Dose ^                 Final Dose^
Carvedilol (mg/day)                      20                                                        26.9 15.5                                   28.8 14.5
Captopril (mg/day)                          7                                                        69.6 40.0                                 70.7 40.4
Enalapril (mg/day)                        13                                                        25.7 12.5                                   26.9 12.6
Digoxin (mg/day)                          25                                                       0.10 0.07                                    0.10 0.07
Aldactone (mg/day)                       19                                                        61.2 49.2                                   59.9 47.1
Long-acting nitrates                      23                                                        53.2 13.2                                   54.1 14.4
Oral furosemide (mg/day)           26                                                      200.9 120.4                                78.3 41.3*
IV furosemide (mg/month)         26                                                      164.7 178.9                                  19.8 47.0*
*p  0.05 at least vs. before by paired Student’s t test.
^  +/-

The mean doses of the medications are shown in Table 4. 

The mean dose of oral furosemide was 200.9 +/-  120.4 mg/day before and 78.3 +/-  41.3 mg/day after the intervention (p   0.05). The dose of IV furosemide was 164.7 +/-  19.8,  178.9 mg/month before and  7.0 mg/month after the intervention (p  0.05).  

The doses of the other CHF medications were almost identical in the two periods.

Clinical results.

DEATHS.
There were three deaths during the intervention period. An 83-year-old man died after eight months of respiratory failure after many years of COPD, a 65-year-old man at eight months of a CVA with subsequent pneumonia and septic shock and a 70-year-old man at four months of septicemia related to an empyema that developed after aortic valve replacement.
HEMODIALYSIS.
Three patients, a 76-year-old man, an 85-year-old woman and a 72-year-old man, required chronic hemodialysis after six, 16 and 18 months, respectively. The serum creatinines of these three patients at onset of the anemia treatment were 4.2, 3.5 and 3.6 mg%, respectively. All three had improvement in their NYHA status but
  • their uremia worsened as the renal function deteriorated, demanding the initiation of dialysis.

In no cases, however, was pulmonary congestion an indication for starting dialysis.

Functional results (Table 5).

During the treatment period, the NYHA class fell from a mean of 3.66 +/- 0.47 to 2.66 +/- 0.70 (p 0.05), and
  • 24 had some improvement in their functional class.
The mean LVEF increased from 27.7 +/- 4.8 to 35.4  +/- 7.6% (p 0.001), an increase of 27.8%.
Compared with a similar period of time before the onset of the anemia treatment, the mean number of hospitalizations fell from 2.72 +/-  1.21 to 0.22 +/-  0.65 per patient (p   0.05)a decrease of 91.9%.
No significant changes were found in the mean systolic/diastolic blood pressure.

Hematological results (Table 5).

  • The mean hematocrit (Hct) increased from 30.14 +/- 3.12%) to 35.9  +/- 4.22% (p < 0.001).
  • The mean Hb increased from 10.16 +/- 0.95 g%) to 12.10 +/-  1.21 g% (p <  0.001).
  • The mean serum ferritin increased from 177.07 +/-  113.80  g/liter to 346.73 +/- 207.40 g/liter (p  0.005).
  • The mean serum Fe increased from 60.4 +/- 19.0 g% to 74 +/- .80  20.7 g% (p  0.005). 
  • The mean %Fe Sat increased from 20.05   6.04% to 26.14 =/- 5.23% (p  0.005).
  • The mean dose of EPO used throughout the treatment period was 5,227  +/- 455 IU per week, and
  • the mean dose of IV Fe used was 185.1 +/- 57.1 mg per month.
In four of the patients, the target Hb of 12 g% was maintained despite stopping the EPO for at least four months.

Renal results (Table 5).

The changes in serum creatinine were not significant. The estimated creatinine clearance fell at a rate of 0.95 + 1.31 ml/min/month before the onset of treatment of the anemia and increased at a rate of 0.85 + 2.77 ml/min/month during the treatment period.
Table 5. The Hematological and Clinical Data of the 26 CHF Patients at Onset and at the End of the Intervention Period

————–                                         Initial ^                                    Final^
Hematocrit, vol%                              30.14 3.12                            35.90 4.22*
Hemoglobin, g%                                10.16 0.95                              2.10 1.21*
Serum ferritin, g/liter                    177.07 113.80                       346.73  207.40*
Serum iron, g%                                  60.4 19.0                               74.8  20.7*
% iron saturation                              20.5 6.04                               26.14 5.23*
Serum creatinine, mg%                   2.59 0.77                                 2.73 1.55
LVEF, %                                              27.7 4.8                                   35.4  7.6*
No. hospitalizations/patient          2.72 1.21                                 0.22   0.65*
Systolic BP, mm Hg                       127.1 19.4                                128.9  26.4
Diastolic BP, mm Hg                       73.9 9.9                                   74.0   12.7
NYHA (0–4)                                     3.66 0.47                                2.66 0.70*
*p  0.05 at least vs before by paired Student’s t test.     ^ +/-
BP  blood pressure; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association.

DISCUSSION

The main findings in the present study are that anemia is common in CHF patients and becomes progressively more prevalent and severe as CHF progresses. In addition, for patients with resistant CHF, the treatment of the associated anemia causes a marked improvement in their

  1. functional status,
  2. ejection fraction and
  3. GFR.
        • All these changes were associated with a markedly
            • reduced need for hospitalization and
            • for oral and IV furosemide.

The effect of anemia on the ischemic myocardium.

We used the IV Fe together with EPO to avoid the Fe deficiency caused by the use of EPO alone (38,39).
The Fe deficiency will cause

  • a resistance to EPO therapy and
  • increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may increase the blood pressure excessively (41). The IV Fe reduces the dose of EPO needed to correct the anemia, because

  • the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42).

Oral Fe, however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain why

  • the blood pressure did not increase significantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

The Effect of Anemia Correction on Renal Function.

Congestive heart failure is often associated with some degree of CRF (1–3,27–29), and

  • this is most likely due to renal vasoconstriction and ischemia (28,29).

When the anemia is treated and the cardiac function improves,

  • an increase in renal blood flow and glomerular filtration is seen (7,28).

In the present study, renal function decreased as the CHF functional class worsened (Table 2). The rate of deterioration of renal function was slower during the intervention period. Treatment of anemia in CRF has been associated with

  • a rate of progression of the CRF that is either unchanged (30) or is slowed (31–33).

It is possible, therefore, that adequate treatment of the anemia in CHF may, in the long term, help slow down the progression of CRF.

Possible Adverse Effects of Correction of the Anemia.

There has been concern, in view of the recent Amgen study (34), that correction of the Hct to a mean 42% in hemodialysis patients might increase cardiovascular events in those receiving EPO compared with those maintained at a Hct of 30%. Although there is much uncertainty about how to interpret this study (35), there is a substantial body of evidence that shows

  • correction of the anemia up to a Hb of 12 g% (Hct 36%) in CRF on dialysis is safe and desirable (35–38), and
  • results in a reduction in mortality, morbidity and in the number and length of hospitalizations.

The same likely holds true for the anemia of CHF with or without associated CRF. Certainly, our patients’ symptoms were strikingly improved, as was their cardiac function (LVEF) and need for hospitalization and diuretics. It remains to be established

  • if correction of the anemia up to a normal Hb level of 14 g% might be necessary in order to further improve the patient’s clinical state.

The Role of Fe Deficiency and its Treatment in the Anemia of CHF.

We used the IV Fe together with EPO to avoid the Fe deficiency caused by the use of EPO alone (38,39). The Fe deficiency will cause

  • a resistance to EPO therapy and increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may

  • increase the blood pressure excessively (41).

The IV Fe reduces the dose of EPO needed to correct the anemia, because the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42). Oral Fe,  however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain

  • why the blood pressure did not increase significantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

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