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The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

 

On 9/25/2017, Aviva Lev-Ari, PhD, RN commissioned Dr. Larry H. Bernstein to write a short article on the following topic reported on 9/22/2017 in sciencemission.com

 

We are publishing, below the new article created by Larry H. Bernstein, MD, FCAP.

 

Background

During the period between 9/2015  and 6/2017 the Team at Leaders in Pharmaceutical Business Intelligence (LPBI)  has launched an R&D effort lead by Aviva Lev-Ari, PhD, RN in conjunction with SBH Sciences, Inc. headed by Dr. Raphael Nir.

This effort, also known as, “DrugDiscovery @LPBI Group”  has yielded several publications on EXOSOMES on this Open Access Online Scientific Journal. Among them are included the following:

 

QIAGEN – International Leader in NGS and RNA Sequencing, 10/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

cell-free DNA (cfDNA) tests could become the ultimate “Molecular Stethoscope” that opens up a whole new way of practicing Medicine, 09/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

Detecting Multiple Types of Cancer With a Single Blood Test (Human Exomes Galore), 07/02/2017

Reporter and Curator: Irina Robu, PhD

 

Exosomes: Natural Carriers for siRNA Delivery, 04/24/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI, 01/05/2017

Curator: Marzan Khan, B.Sc

 

SBI’s Exosome Research Technologies, 12/29/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

A novel 5-gene pancreatic adenocarcinoma classifier: Meta-analysis of transcriptome data – Clinical Genomics Research @BIDMC, 12/28/2016

Curator: Tilda Barliya, PhD

 

Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute, Micro and Nanotechnology Lab, 12/28/2016

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN

 

Exosomes – History and Promise, 04/28/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

Exosomes, 11/17/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Liquid Biopsy Assay May Predict Drug Resistance, 11/16/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Glypican-1 identifies cancer exosomes, 10/31/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Circulating Biomarkers World Congress, March 23-24, 2015, Boston: Exosomes, Microvesicles, Circulating DNA, Circulating RNA, Circulating Tumor Cells, Sample Preparation, 03/24/2015

Reporter: Aviva Lev-Ari, PhD, RN

 

Cambridge Healthtech Institute’s Second Annual Exosomes and Microvesicles as Biomarkers and Diagnostics Conference, March 16-17, 2015 in Cambridge, MA, 03/17, 2015

Reporter: Aviva Lev-Ari, PhD, RN

 

The newly created think-piece on the relationship between regulatory functions of Exosomes and Metabolic processes is developed conceptually, below.

 

The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

We have had more than a half century of research into the genetic code and transcription leading to abundant work on RNA and proteomics. However, more recent work in the last two decades has identified RNA interference in siRNA. These molecules may be found in the circulation, but it has been a challenge to find their use in therapeutics. Exosomes were first discovered in the 1980s, but only recently there has been a huge amount of research into their origin, structure and function. Exosomes are 30–120 nm endocytic membrane-bound extracellular vesicles (EVs)(1-23) , and more specifically multiple vesicle bodies (MVBs) by a budding process from invagination of the outer cell membrane that carry microRNA (miRNA), and have structures composed of protein and lipids (1,23-27 ). EVs are the membrane vesicles secreted by eukaryotic cells for intracellular communication by transferring the proteins, lipids, and RNA under various physiologic conditions as well as during the disease stage. EVs also act as a signalosomes in many biological processes. Inward budding of the plasma membrane forms small vesicles that fuse. Intraluminal vesicles (ILVs) are formed by invagination of the limiting endosomal membrane during the maturation process of early endosome.

EVs are the MVBs secreted that serve in intracellular communication by transferring a cargo consisting of proteins, lipids, and RNA under various physiologic conditions (4, 23). Exosome-mediated miRNA transfer between cells is considered to be necessary for intercellular signaling and exosome-associated miRNAs in biofluids (23). Exosomes carry various molecular constituents of their cell of origin, including proteins, lipids, mRNAs, and microRNAs (miRNAs) (. They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, hydrothoracic fluid, and ascitic fluid, as well as in culture medium of most cell types.Exosomes have also been shown to be involved in noncoding RNA surveillance machinery in generating antibody diversity (24). There are also a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) that accumulate R-loop structures upon RNA exosome ablation, thereby, resolving deleterious DNA/RNA hybrids arising from active enhancers and distal divergent eRNA-expressing elements (lncRNA-CSR) engaged in long-range DNA interactions (25). RNA exosomes are large multimeric 3′-5′ exo- and endonucleases representing the central RNA 3′-end processing factor and are implicated in processing, quality control, and turnover of both coding and noncoding RNAs. They are large macromolecular cages that channel RNA to the ribonuclease sites (29). A major interest has been developed to characterize of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids (1). Moreover, exosomes play an active role in tumorigenesis, metastasis, and response to therapy through the transfer of oncogenes and onco-miRNAs between cancer cells and the tumor stroma. Blood cells and the vascular endothelium is also exosomal shedding, which has significance for cardiovascular,   neurologicological disorders, stroke, and antiphospholipid syndrome (1). Dysregulation of microRNAs and the affected pathways is seen in numerous pathologies their expression can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers (30).

Exosomes are secreted by many cells like B lymphocytes and dendritic cells of hematopoietic and non-hematopoietic origin viz. platelets, Schwann cells, neurons, mast cells, cytotoxic T cells, oligodendrocytes, intestinal epithelial cells were also found to be releasing exosomes (4). They are engaged in complex functions like persuading immune response as the exosomes secreted by antigen presenting cells activate T cells (4). They all have a common set of proteins e.g. Rab family of GTPases, Alix and ESCRT (required for transport) protein and they maintain their cytoskeleton dynamics and participate in membrane fusion. However, they are involved in retrovirus disease pathology as a result of recruitment of the host`s endosomal compartments in order to generate viral vesicles, and they can either spread or limit an infection based on the type of pathogen and its target cells (5).

Upon further consideration, it is understandable how this growing biological work on exosomes has enormous significance for laboratory diagnostics (1, 3, 5, 6, 11, 14, 15, 17-20, 23,30-41) . They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, thoracic and abdominal effusions, and ascitic fluid (1). The involvement of exosomes in disease is broad, and includes: cancer, autoimmune and infectious disease, hematologic disorders, neurodegenerative diseases, and cardiovascular disease. Proteins frequently identified in exosomes include membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), heat shock proteins (e.g., HSC70), tetraspanins (e.g., CD9, CD63, and CD81), MVB biogenesis proteins (e.g., alix and TSG101), and lipid-related proteins and phospholipases. The exosomal lipid composition has been thoroughly analyzed in exosomes secreted from several cell types including DCs and mast cells, reticulocytes, and B-lymphocytes (1). Dysregulation of microRNAs of pathways observed in numerous pathologies (5, 10, 12, 21, 27, 35, 37) including cancers (30), particularly, colon, pancreas, breast, liver, brain, lung (2, 6, 17-20, 30, 33-36, 38, 39). Following these considerations, it is important that we characterize the content of exosomal cargo to gain clues to their biogenesis, targeting, and cellular effects which may lead to identification of biomarkers for disease diagnosis, prognosis and response to treatment (42).

We might continue in pursuit of a particular noteworthy exosome, the NLRP3 inflammasome, which is activated by a variety of external or host-derived stimuli, thereby, initiating an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1b maturation and secretion (43).
Inflammasomes are multi-protein signaling complexes that activate the inflammatory caspases and the maturation of interleukin-1b. The NLRP3 inflammasome is linked with human autoinflammatory and autoimmune diseases (44). This makes the NLRP3 inflammasome a promising target for anti-inflammatory therapies. The NLRP3 inflammasome is activated in response to a variety of signals that indicate tissue damage, metabolic stress, and infection (45). Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Heritable and acquired inflammatory diseases are both characterized by dysregulation of NLRP3 inflammasome activation (45).
Receptors of innate immunity recognize conserved moieties associated with either cellular damage [danger-associated molecular patterns (DAMPs)] or invading organisms [pathogen-associated molecular patterns (PAMPs)](45). Either chronic stimulation or overwhelming tissue damage is injurious and responsible for the pathology seen in a number of autoinflammatory and autoimmune disorders, such as arthritis and diabetes. The nucleotide-binding domain leucine-rich repeat (LRR)-containing receptors (NLRs) are PRRs are found intracellularly and they share a unique domain architecture. It consists of a central nucleotide binding and oligomerization domain called the NACHT domain that is located between an N-terminal effector domain and a C-terminal LRR domain (45). The NLR family members NLRP1, NLRP3, and NLRC4 are capable of forming multiprotein complexes called inflammasomes when activated.

The (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease because the activation results, in pro-IL-1β processing and the secretion of the proinflammatory cytokine IL-1β (46). It has been proposed that Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma (46). First, NLRP3 interacts with the adaptor protein ASC by sensing microbial pathogens and self-danger signals. Then pro-caspase-1 is recruited and the large protein complex called the NLRP3 inflammasome is formed. This is followed by autocleavage and activation of caspase-1, after which pro-IL-1β and pro-IL-18 are converted into their mature forms. Ion fluxes disrupt membrane integrity, and also mitochondrial damage both play key roles in NLRP3 inflammasome activation (47). Depletion of mitochondria as well as inhibitors that block mitochondrial respiration and ROS production prevented NLRP3 inflammasome activation. Futhermore, genetic ablation of VDAC channels (namely VDAC1 and VDAC3) that are located on the mitochondrial outer membrane and that are responsible for exchanging ions and metabolites with the cytoplasm, leads to diminished mitochondrial (mt) ROS production and inhibition of NLRP3 inflammasome activation (47). Inflammasome activation not only occurs in immune cells, primarily macrophages and dendritic cells, but also in kidney cells, specifically the renal tubular epithelium. The NLRP3 inflammasome is probably involved in the pathogenesis of acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal-related nephropathy (48). The inflammasome also plays a role in autoimmune kidney disease. IL-1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and β-hydroxybutyrate, may prove to have value in the treatment of inflammasome-mediated conditions.

Autophagosomes derived from tumor cells are referred to as defective ribosomal products in blebs (DRibbles). DRibbles mediate tumor regression by stimulating potent T-cell responses and, thus, have been used as therapeutic cancer vaccines in multiple preclinical cancer models (49). It has been found that DRibbles could induce a rapid differentiation of monocytes and DC precursor (pre-DC) cells into functional APCs (49). Consequently, DRibbles could potentially induce strong innate immune responses via multiple pattern recognition receptors. This explains why DRibbles might be excellent antigen carriers to induce adaptive immune responses to both tumor cells and viruses. This suggests that isolated autophagosomes (DRibbles) from antigen donor cells activate inflammasomes by providing the necessary signals required for IL-1β production.

The Hsp90 system is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function (50). The co-chaperones enable Hsp90 to chaperone structurally and functionally diverse client proteins. Sahasrabudhe et al. (50) show that the nature of the client protein dictates the contribution of a co-chaperone to its maturation. The study reveals the general importance of the cochaperone Sgt1 (50). In addition to Hsp90, we have to consider Hsp60. Adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins (21).

A new Protein Organic Solvent Precipitation (PROSPR) method efficiently isolates the EV repertoire from human biological samples. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes. In addition lipidomic characterization of enriched CNS vesicles identified previously reported EV-specific lipid families and novel lipid isoforms not previously detected in human EVs. The characterization of these structures from central nervous system (CNS) tissues is relevant to current neuroscience, especially to advance the understanding of neurodegeneration in amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD)(15). In addition, study of EVs in brain will enable characterization of the degenerative posttranslational modifications (DPMs) occurring in those proteins.
Neurodegenerative disease is characterized by dysregulation because of NLRP3 inflammasome activation. Alzheimer’s disease (AD) and Parkinson’s disease (PD), both neurodegenerative diseases are associated with the NLRP3 inflammasome. PD is characterized by accumulation of Lewy bodies (LB) formed by a-synuclein (aSyn) aggregation. A recent study revealed that aSyn induces synthesis of pro-IL-1b by an interaction with TLR2 and activates NLRP3 inflammasome resulting in caspase-1 activation and IL-1b maturation in human primary monocytes (43). In addition mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals. It is notable that in this aberrant activation mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals (43).

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von Willebrand Factor

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

FDA approves first recombinant von Willebrand factor to treat bleeding episodes

Dr. Anthony Melvin Castro

 

 

12/08/2015 02:44
The U.S. Food and Drug Administration today approved Vonvendi, von Willebrand factor (Recombinant), for use in adults 18 years of age and older who have von Willebrand disease (VWD). Vonvendi is the first FDA-approved recombinant von Willebrand factor, and is approved for the on-demand (as needed) treatment and control of bleeding episodes in adults diagnosed with VWD.
Company Baxalta Inc.
Description Recombinant human von Willebrand factor (vWF)
Molecular Target von Willebrand factor (vWF)
Mechanism of Action
Therapeutic Modality Biologic: Protein
Latest Stage of Development Registration
Standard Indication Bleeding
Indication Details Treat and prevent bleeding episodes in von Willebrand disease (vWD) patients; Treat von Willebrand disease (vWD)
Regulatory Designation U.S. – Orphan Drug (Treat and prevent bleeding episodes in von Willebrand disease (vWD) patients);
EU – Orphan Drug (Treat and prevent bleeding episodes in von Willebrand disease (vWD) patients);
Japan – Orphan Drug (Treat and prevent bleeding episodes in von Willebrand disease (vWD) patients)

 

The U.S. Food and Drug Administration today approved Vonvendi, von Willebrand factor (Recombinant), for use in adults 18 years of age and older who have von Willebrand disease (VWD). Vonvendi is the first FDA-approved recombinant von Willebrand factor, and is approved for the on-demand (as needed) treatment and control of bleeding episodes in adults diagnosed with VWD.

VWD is the most common inherited bleeding disorder, affecting approximately 1 percent of the U.S. population. Men and women are equally affected by VWD, which is caused by a deficiency or defect in von Willebrand factor, a protein that is critical for normal blood clotting. Patients with VWD can develop severe bleeding from the nose, gums, and intestines, as well as into muscles and joints. Women with VWD may have heavy menstrual periods lasting longer than average and may experience excessive bleeding after childbirth.

“Patients with heritable bleeding disorders should meet with their health care provider to discuss appropriate measures to reduce blood loss,” said Karen Midthun, M.D., director of the FDA’s Center for Biologics Evaluation and Research. “The approval of Vonvendi provides an additional therapeutic option for the treatment of bleeding episodes in patients with von Willebrand disease.”

The safety and efficacy of Vonvendi were evaluated in two clinical trials of 69 adult participants with VWD. These trials demonstrated that Vonvendi was safe and effective for the on-demand treatment and control of bleeding episodes from a variety of different sites in the body. No safety concerns were identified in the trials. The most common adverse reaction observed was generalized pruritus (itching).

The FDA granted Vonvendi orphan product designation for these uses.Orphan product designation is given to drugs intended to treat rare diseases in order to promote their development.

Vonvendi is manufactured by Baxalta U.S., Inc., based in Westlake Village, California.

 

von Willebrand Disease

Author: Eleanor S Pollak; Chief Editor: Srikanth Nagalla

Von Willebrand disease (vWD) is a common, inherited, genetically and clinically heterogeneous hemorrhagic disorder caused by a deficiency or dysfunction of the protein termed von Willebrand factor (vWF). Consequently, defective vWF interaction between platelets and the vessel wall impairs primary hemostasis.

vWF, a large, multimeric glycoprotein, circulates in blood plasma at concentrations of approximately 10 mg/mL. In response to numerous stimuli, vWF is released from storage granules in platelets and endothelial cells. It performs two major roles in hemostasis. First, it mediates the adhesion of platelets to sites of vascular injury. Second, it binds and stabilizes the procoagulant protein factor VIII (FVIII). (See Etiology.)

vWD is divided into three major categories: (1) partial quantitative deficiency (type I), (2) qualitative deficiency (type II), and (3) total deficiency (type III). vWD type II is further divided into four variants (IIA, IIB, IIN, IIM), based on characteristics of dysfunctional vWF. These categories correspond to distinct molecular mechanisms, with corresponding clinical features and therapeutic recommendations.

For discussion of vWD in children, see Pediatric Von Willebrand Disease.

http://emedicine.medscape.com/article/206996-overview

 

 

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Diagnostic Revelations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

New Liquid Biopsy Test Uses Platelet RNA as Cancer Diagnostic

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    Using platelet RNA, scientists have been able to detect the presence of cancer and pinpoint its primary location. [Best et al., 2015, Cancer Cell 28, 1–11]

    The age of fast, accurate, and noninvasive cancer screening is rapidly becoming reality. The power of next-generation sequencing has allowed molecular diagnostic techniques to sample small amounts of blood for the genetic hallmarks of tumorigenesis. These liquid biopsy procedures, as they have been dubbed, typically search for circulating tumor DNA (ctDNA) that has made its way into the systemic circulation from tumor cells that have died or enrich for circulating tumor cells (CTCs) that have broken off from the primary cancer site.

    Now, a team of researchers lead by scientists at Massachusetts General Hospital (MGH), have developed a new diagnostic test that analyzes the tumor RNA picked up in circulating platelets. The investigators believe this new method could become even more useful than other molecular technologies for diagnosing cancer since it can also determine the primary location of the tumor and provide insight to potential therapeutic approaches.

    “By combining next-generation-sequencing gene expression profiles of platelet RNA with computational algorithms we developed, we were able to detect the presence of cancer with 96 percent accuracy,” explains co-senior author Bakhos Tannous, Ph.D., associate professor Harvard Medical School and associate neuroscientist at MGH. “Platelet RNA signatures also provide valuable information on the type of tumor present in the body and can guide the selection of the most optimal treatment for individual patients.

    The findings from this study were published recently in Cancer Cell through an article entitled “RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics.”

    In the current study the research team describes finding that the RNA profiles of tumor-educated platelets (TEPs)—those that have taken up molecules shed by tumors—can distinguish among blood samples of healthy individuals and those of patients with six types of cancer, determine the location of the primary tumor, and identify tumors carrying mutations that can guide therapeutic decision-making.

    Over the past several years, the scientific literature has shown that in addition to their role in promoting blood clotting, platelets take up protein and RNA molecules from tumors, possibly playing a role in tumor growth and metastasis. Dr. Tannous and his colleagues set out to determine whether tumor RNA carried in platelets could be used to diagnose and classify common types of cancer.

    The investigators isolated platelets from blood samples taken from 55 healthy donors, 39 individual with early-stage cancer and 189 patients with advanced, metastatic cancer. Among those patients with cancer, they were diagnosed with non-small-cell lung cancer, colorectal cancer, glioblastoma, pancreatic cancer, hepatobiliary cancer, or breast cancer.

    The comparison of RNA profiles from the healthy donors to those of the cancer patients identified increased levels of approximately 1,500 RNA molecules—many involved in cancer-associated processes—and a reduction of almost 800 in samples from cancer patients. Using their novel algorithm, the MGH group was able to examine close to 1,000 RNAs from almost 300 individuals with 96% accuracy for the presence of cancer.

    Additionally, the platelet mRNA profiles were able to identify the particular type of cancer within each patient participant, including distinguishing among three types of gastrointestinal adenocarcinoma: colorectal cancer, pancreatic cancer, and hepatobiliary cancer. Platelets from patients with tumors driven by mutations in KRAS or EGFR proteins—biomarkers that can guide the use of drugs targeting those mutations—proved to have unique RNA profiles as well.

    The researchers were excited by their findings and emphasize the uniqueness of their approach as currently utilized liquid biopsy approaches have been unable to diagnose cancer while simultaneously pinpointing the location of the primary tumor.

    “We observed that the mRNA profiles of tumor-educated platelets have the sensitivity and specificity to detect cancer, even in early, non-metastasized tumors,” noted Dr. Tannous. “We are further assessing the potential of TEP-based screening for therapeutic decision making and also investigating how non-cancerous diseases may further influence the RNA repertoire of TEPs.”

  • RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics

Myron G. Best Nik Sol, Jihane Tannous, Bart A. Westerman, François Rustenburg, Pepijn Schellen, Heleen Verschueren, Edward Post, Jan Koster, Bauke Ylstra, Irsan Kooi, et al.
Highlights

Tumors “educate” platelets (TEPs) by altering the platelet RNA profile

TEPs provide a RNA biosource for pan-cancer, multiclass, and companion diagnostics

TEP-based liquid biopsies may guide clinical diagnostics and therapy selection

A total of 100–500 pg of total platelet RNA is sufficient for TEP-based diagnostics

mRNA Profiles of Tumor-Educated Platelets Are Distinct from Platelets of Healthy Individuals

Summary

Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, orPIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based “liquid biopsies”.

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Significance

Blood-based “liquid biopsies” provide a means for minimally invasive molecular diagnostics, overcoming limitations of tissue acquisition. Early detection of cancer, clinical cancer diagnostics, and companion diagnostics are regarded as important applications of liquid biopsies. Here, we report that mRNA profiles of tumor-educated blood platelets (TEPs) enable for pan-cancer, multiclass cancer, and companion diagnostics in both localized and metastasized cancer patients. The ability of TEPs to pinpoint the location of the primary tumor advances the use of liquid biopsies for cancer diagnostics. The results of this proof-of-principle study indicate that blood platelets are a potential all-in-one platform for blood-based cancer diagnostics, using the equivalent of one drop of blood.

Introduction

Cancer is primarily diagnosed by clinical presentation, radiology, biochemical tests, and pathological analysis of tumor tissue, increasingly supported by molecular diagnostic tests. Molecular profiling of tumor tissue samples has emerged as a potential cancer classifying method (Akbani et al., 2014, Golub et al., 1999, Han et al., 2014, Hoadley et al., 2014, Kandoth et al., 2013,Ramaswamy et al., 2001, Su et al., 2001). In order to overcome limitations of tissue acquisition, the use of blood-based liquid biopsies has been suggested (Alix-Panabières et al., 2012, Crowley et al., 2013, Haber and Velculescu, 2014). Several blood-based biosources are currently being evaluated as liquid biopsies, including plasma DNA (Bettegowda et al., 2014, Chan et al., 2013, Diehl et al., 2008, Murtaza et al., 2013, Newman et al., 2014, Thierry et al., 2014) and circulating tumor cells (Bidard et al., 2014, Dawson et al., 2013, Maheswaran et al., 2008, Rack et al., 2014). So far, implementation of liquid biopsies for early detection of cancer has been hampered by non-specificity of these biosources to pinpoint the nature of the primary tumor (Alix-Panabières and Pantel, 2014,Bettegowda et al., 2014).

It has been reported that tumor-educated platelets (TEPs) may enable blood-based cancer diagnostics (Calverley et al., 2010, McAllister and Weinberg, 2014,Nilsson et al., 2011). Blood platelets—the second most-abundant cell type in peripheral blood—are circulating anucleated cell fragments that originate from megakaryocytes in bone marrow and are traditionally known for their role in hemostasis and initiation of wound healing (George, 2000, Leslie, 2010). More recently, platelets have emerged as central players in the systemic and local responses to tumor growth. Confrontation of platelets with tumor cells via transfer of tumor-associated biomolecules (“education”) is an emerging concept and results in the sequestration of such biomolecules (Klement et al., 2009,Kuznetsov et al., 2012, McAllister and Weinberg, 2014, Nilsson et al., 2011,Quail and Joyce, 2013). Moreover, external stimuli, such as activation of platelet surface receptors and lipopolysaccharide-mediated platelet activation (Denis et al., 2005, Rondina et al., 2011), induce specific splicing of pre-mRNAs in circulating platelets (Power et al., 2009, Rowley et al., 2011, Schubert et al., 2014). Platelets may also undergo queue-specific splice events in response to signals released by cancer cells and the tumor microenvironment—such as stromal and immune cells. The combination of specific splice events in response to external signals and the capacity of platelets to directly ingest (spliced) circulating mRNA can provide TEPs with a highly dynamic mRNA repertoire, with potential applicability to cancer diagnostics (Calverley et al., 2010, Nilsson et al., 2011) (Figure 1A). In this study, we characterize the platelet mRNA profiles of various cancer patients and healthy donors and investigate their potential for TEP-based pan-cancer, multiclass cancer, and companion diagnostics.

  
Results

We prospectively collected and isolated blood platelets from healthy donors (n = 55) and both treated and untreated patients with early, localized (n = 39) or advanced, metastatic cancer (n = 189) diagnosed by clinical presentation and pathological analysis of tumor tissue supported by molecular diagnostics tests. The patient cohort included six tumor types, i.e., non-small cell lung carcinoma (NSCLC, n = 60), colorectal cancer (CRC, n = 41), glioblastoma (GBM, n = 39), pancreatic cancer (PAAD, n = 35), hepatobiliary cancer (HBC, n = 14), and breast cancer (BrCa, n = 39) (Figure 1B; Table 1; Table S1). The cohort of healthy donors covered a wide range of ages (21–64 years old, Table 1).

Table 1Summary of Patient Characteristics
PATIENT GROUP TOTAL (N) GENDER M (%)A AGE (SD)B METASTASIS (%) MUTATION PRESENCE (%)
TRAINING VALIDATION TRAINING VALIDATION TRAINING VALIDATION TRAINING VALIDATION TRAINING VALIDATION
HD 39 16 21 (54) 6 (38) 41 (13) 38 (16)
GBM 23 16 18 (78) 10 (63) 59 (16) 62 (14) 0 (0) 0 (0)
NSCLC 36 24 14 (39) 14 (58) 60 (11) 59 (12) 33 (92) 23 (96) KRAS 15 (42) 11 (46)
EGFR 14 (39) 7 (29)
MET-overexpression 5 (14) 3 (13)
CRC 25 16 13 (52) 9 (56) 59 (13) 63 (16) 20 (80) 15 (94) KRAS 7 (28) 8 (50)
PAAD 21 14 12 (57) 7 (50) 66 (9) 66 (10) 15 (71) 9 (64) KRAS 13 (62) 9 (64)
BrCa 23 16 0 (0) 0 (0) 59 (11) 59 (11) 16 (70) 9 (56) HER2+ 7 (30) 5 (31)
PIK3CA 6 (26) 2 (13)
triple negative 5 (22) 3 (19)
HBC 8 6 6 (75) 2 (33) 68 (13) 62 (16) 6 (75) 4 (67) KRAS 3 (38) 1 (17)

HD, healthy donors; GBM, glioblastoma; NSCLC, non-small cell lung cancer; CRC, colorectal cancer; PAAD, pancreatic cancer; BrCa, breast cancer; HBC, hepatobiliary cancer. See also Table S1.

aIndicated are number of male individuals.
bIndicated is mean age in years.

Platelet purity was confirmed by morphological analysis of randomly selected and freshly isolated platelet samples (contamination is 1 to 5 nucleated cells per 10 million platelets, see Supplemental Experimental Procedures), and platelet RNA was isolated and evaluated for quality and quantity (Figure S1A). A total of 100–500 pg of platelet total RNA (the equivalent of purified platelets in less than one drop of blood) was used for SMARTer mRNA amplification and sequencing (Ramsköld et al., 2012) (Figures 1C and S1A). Platelet RNA sequencing yielded a mean read count of ∼22 million reads per sample. After selection of intron-spanning (spliced) RNA reads and exclusion of genes with low coverage (seeSupplemental Experimental Procedures), we detected in platelets of healthy donors (n = 55) and localized and metastasized cancer patients (n = 228) 5,003 different protein coding and non-coding RNAs that were used for subsequent analyses. The obtained platelet RNA profiles correlated with previously reported mRNA profiles of platelets (Bray et al., 2013, Kissopoulou et al., 2013, Rowley et al., 2011, Simon et al., 2014) and megakaryocytes (Chen et al., 2014) and not with various non-related blood cell mRNA profiles (Hrdlickova et al., 2014) (Figure S1B). Furthermore, DAVID Gene Ontology (GO) analysis revealed that the detected RNAs are strongly enriched for transcripts associated with blood platelets (false discovery rate [FDR] < 10−126).

Among the 5,003 RNAs, we identified known platelet markers, such as B2M, PPBP, TMSB4X, PF4, and several long non-coding RNAs (e.g., MALAT1). A total of 1,453 out of 5,003 mRNAs were increased and 793 out of 5,003 mRNAs were decreased in TEPs as compared to platelet samples of healthy donors (FDR < 0.001), while presenting a strong correlation between these platelet mRNA profiles (r = 0.90, Pearson correlation) (Figure 1D). Unsupervised hierarchical clustering based on the differentially detected platelet mRNAs distinguished two sample groups with minor overlap (Figure 1E; Table S2). DAVID GO analysis revealed that the increased TEP mRNAs were enriched for biological processes such as vesicle-mediated transport and the cytoskeletal protein binding while decreased mRNAs were strongly involved in RNA processing and splicing (Table S3). A correlative analysis of gene set enrichment (CAGE) GO methodology, in which 3,875 curated gene sets of the GSEA database were correlated to TEP profiles (see Experimental Procedures), demonstrated significant correlation of TEP mRNA profiles with cancer tissue signatures, histone deacetylases regulation, and platelets (Table 2). The levels of 20 non-protein coding RNAs were altered in TEPs as compared to platelets from healthy individuals and these show a tumor type-associated RNA profile (Figure S1C).

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Tumor-Educated Platelet mRNA Profiling for Pan-Cancer Diagnostics

(A) Schematic overview of tumor-educated platelets (TEPs) as biosource for liquid biopsies.

(B) Number of platelet samples of healthy donors and patients with different types of cancer.

(C) TEP mRNA sequencing (mRNA-seq) workflow, as starting from 6 ml EDTA-coated tubes, to platelet isolation, mRNA amplification, and sequencing.

(D) Correlation plot of mRNAs detected in healthy donor (HD) platelets and cancer patients’ TEPs, including highlighted increased (red) and decreased (blue) TEP mRNAs.

(E) Heatmap of unsupervised clustering of platelet mRNA profiles of healthy donors (red) and patients with cancer (gray).

(F) Cross-table of pan-cancer SVM/LOOCV diagnostics of healthy donor subjects and patients with cancer in training cohort (n = 175). Indicated are sample numbers and detection rates in percentages.

(G) Performance of pan-cancer SVM algorithm in validation cohort (n = 108). Indicated are sample numbers and detection rates in percentages.

(H) ROC-curve of SVM diagnostics of training (red), validation (blue) cohort, and random classifiers, indicating the classification accuracies obtained by chance of the training and validation cohort (gray).

(I) Total accuracy ratios of SVM classification in five subgroups, including corresponding predictive strengths. Genes, number of mRNAs included in training of the SVM algorithm.

See also Figure S1 and Tables S1, S2, S3, and S4.

Table 2Pan-Cancer CAGE Gene Ontology
TOP 25 GO CORRELATIONS
# LOWESTA HIGHESTA
DOWN
Translation 10 −0.865 −0.890
Immune, T cell 5 −0.853 −0.883
Cancer-associated 2 −0.875 −0.887
Viral replication 2 −0.875 −0.878
IL-signaling 2 −0.869 −0.874
RNA processing 1 −0.886
Ago2-Dicer-silencing 1 −0.882
Protein metabolism 1 −0.879
Receptor processing 1 −0.869
UP
Cancer-associated 6 −0.783 −0.906
Infection 3 −0.798 −0.853
HDAC 3 −0.795 −0.852
Platelet 3 −0.837 −0.906
Cytoskeleton 2 −0.801 −0.886
Hypoxia 2 −0.763 −0.937
Protease 1 −0.854
Immunodeficiency 1 −0.812
Differentiation 1 −0.810
Immune differentiation 1 −0.801
Methylation 1 −0.778
Metabolism 1 −0.768

Top-ranking correlations of platelet-mRNA profiles with 3,875 Broad Institute curated gene sets. CAGE, Correlative Analysis of Gene Set Enrichment; GO, gene ontology; #, number of hits per annotation; IL, interleukin; HDAC, histone deacetylase.

aIndicated are lowest and highest correlations per annotation.

Next, we determined the diagnostic accuracy of TEP-based pan-cancer classification in the training cohort (n = 175), employing a leave-one-out cross-validation support vector machine algorithm (SVM/LOOCV, see Experimental Procedures; Figures S1D and S1E), previously used to classify primary and metastatic tumor tissues (Ramaswamy et al., 2001, Su et al., 2001, Vapnik, 1998, Yeang et al., 2001). Briefly, the SVM algorithm (blindly) classifies each individual sample as cancer or healthy by comparison to all other samples (175 − 1) and was performed 175 times to classify and cross validate all individuals samples. The algorithms we developed use a limited number of different spliced RNAs for sample classification. To determine the specific input gene lists for the classifying algorithms we performed ANOVA testing for differences (as implemented in the R-package edgeR), yielding classifier-specific gene lists (Table S4). For the specific algorithm of the pan-cancer TEP-based classifier test we selected 1,072 RNAs (Table S4) for the n = 175 training cohort, yielding a sensitivity of 96%, a specificity of 92%, and an accuracy of 95% (Figure 1F). Subsequent validation using a separate validation cohort (n = 108), not involved in input gene list selection and training of the algorithm, yielded a sensitivity of 97%, a specificity of 94%, and an accuracy of 96% (Figure 1G), with an area under the curve (AUC) of 0.986 to detect cancer (Figure 1H) and high predictive strength (Figure 1I). In contrast, random classifiers, as determined by multiple rounds of randomly shuffling class labels (permutation) during the SVM training process (see Experimental Procedures), had no predictive power (mean overall accuracy: 78%, SD ± 0.3%, p < 0.01), thereby showing, albeit an unbalanced representation of both groups in the study cohort, specificity of our procedure. A total of 100 times random class-proportional subsampling of the entire dataset in a training and validation set (ratio 60:40) yielded similar accuracy rates (mean overall accuracy: 96%, SD: ± 2%), confirming reproducible classification accuracy in this dataset. Of note, all 39 patients with localized tumors and 33 of the 39 patients with primary tumors in the CNS were correctly classified as cancer patients (Figure 1I). Visualization of 22 genes previously identified at differential RNA levels in platelets of patients with various non-cancerous diseases (Gnatenko et al., 2010, Healy et al., 2006, Lood et al., 2010,Raghavachari et al., 2007), revealed mixed levels in our TEP dataset (Figure S1F), suggesting that the platelet RNA repertoire in patients with non-cancerous disease is distinct from patients with cancer.

Tumor-Specific Educational Program of Blood Platelets Allows for Multiclass Cancer Diagnostics

In addition to the pan-cancer diagnosis, the TEP mRNA profiles also distinguished healthy donors and patients with specific types of cancer, as demonstrated by the unsupervised hierarchical clustering of differential platelet mRNA levels of healthy donors and all six individual tumor types, i.e., NSCLC, CRC, GBM, PAAD, BrCa, and HBC (Figures 2A, all p < 0.0001, Fisher’s exact test, and S2A; Table S5), and this resulted in tumor-specific gene lists that were used as input for training and validation of the tumor-specific algorithms (Table S4). For the unsupervised clustering of the all-female group of BrCa patients, male healthy donors were excluded to avoid sample bias due to gender-specific platelet mRNA profiles (Figure S2B). SVM-based classification of all individual tumor classes with healthy donors resulted in clear distinction of both groups in both the training and validation cohort, with high sensitivity and specificity, and 38/39 (97%) cancer patients with localized disease were classified correctly (Figures 2B and S2C). CAGE GO analysis showed that biological processes differed between TEPs of individual tumor types, suggestive of tumor-specific “educational” programs (Table S6). We did not detect sufficient differences in mRNA levels to discriminate patients with non-metastasized from patients with metastasized tumors, suggesting that the altered platelet profile is predominantly influenced by the molecular tumor type and, to a lesser extent, by tumor progression and metastases.

 We next determined whether we could discriminate three different types of adenocarcinomas in the gastro-intestinal tract by analysis of the TEP-profiles, i.e., CRC, PAAD, and HBC. We developed a CRC/PAAD/HBC algorithm that correctly classified the mixed TEP samples (n = 90) with an overall accuracy of 76% (mean overall accuracy random classifiers: 42%, SD: ± 5%, p < 0.01,Figure 2C). In order to determine whether the TEP mRNA profiles allowed for multiclass cancer diagnosis across all tumor types and healthy donors, we extended the SVM/LOOCV classification test using a combination of algorithms that classified each individual sample of the training cohort (n = 175) as healthy donor or one of six tumor types (Figures S2D and S2E). The results of the multiclass cancer diagnostics test resulted in an average accuracy of 71% (mean overall accuracy random classifiers: 19%, SD: ± 2%, p < 0.01,Figure 2D), demonstrating significant multiclass cancer discriminative power in the platelet mRNA profiles. The classification capacity of the multiclass SVM-based classifier was confirmed in the validation cohort of 108 samples, with an overall accuracy of 71% (Figure 2E). An overall accuracy of 71% might not be sufficient for introduction into cancer diagnostics. However, of the initially misclassified samples according to the SVM algorithms choice with strongest classification strength the second ranked classification was correct in 60% of the cases. This yields an overall accuracy using the combined first and second ranked classifications of 89%. The low validation score of HBC samples can be attributed to the relative low number of samples and possibly to the heterogenic nature of this group of cancers (hepatocellular cancers and cholangiocarcinomas).
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Tumor-Educated Platelet mRNA Profiles for Multiclass Cancer Diagnostics

(A) Heatmaps of unsupervised clustering of platelet mRNA profiles of healthy donors (HD; n = 55) (red) and patients with non-small cell lung cancer (NSCLC; n = 60), colorectal cancer (CRC; n = 41), glioblastoma (GBM; n = 39), pancreatic cancer (PAAD, n = 35), breast cancer (BrCa; n = 39; female HD; n = 29), and hepatobiliary cancer (HBC; n = 14).

(B) ROC-curve of SVM diagnostics of healthy donors and individual tumor classes in both training (left) and validation (right) cohort. Random classifiers, indicating the classification accuracies obtained by chance, are shown in gray.

(C) Confusion matrix of multiclass SVM/LOOCV diagnostics of patients with CRC, PAAD, and HBC. Indicated are detection rates as compared to the actual classes in percentages.

(D) Confusion matrix of multiclass SVM/LOOCV diagnostics of the training cohort consisting of healthy donors (healthy) and patients with GBM, NSCLC, PAAD, CRC, BrCa, and HBC. Indicated are detection rates as compared to the actual classes in percentages.

(E) Confusion matrix of multiclass SVM algorithm in a validation cohort (n = 108). Indicated are sample numbers and detection rates in percentages. Genes, number of mRNAs included in training of the SVM algorithm.

See also Figure S2 and Tables S4, S5, and S6.

Companion Diagnostics Tumor Tissue Biomarkers Are Reflected by Surrogate TEP mRNA Onco-signatures

Blood provides a promising biosource for the detection of companion diagnostics biomarkers for therapy selection (Bettegowda et al., 2014, Crowley et al., 2013,Papadopoulos et al., 2006). We selected platelet samples of patients with distinct therapy-guiding markers confirmed in matching tumor tissue. Although the platelet mRNA profiles contained undetectable or low levels of these mutant biomarkers, the TEP mRNA profiles did allow to distinguish patients with KRASmutant tumors from KRAS wild-type tumors in PAAD, CRC, NSCLC, and HBC patients, and EGFR mutant tumors in NSCLC patients, using algorithms specifically trained on biomarker-specific input gene lists (all p < 0.01 versus random classifiers, Figures 3A–3E ; Table S4). Even though the number of samples analyzed is relatively low and the risk of algorithm overfitting needs to be taken into account, the TEP profiles distinguished patients with HER2-amplified, PIK3CA mutant or triple-negative BrCa, and NSCLC patients with MET overexpression (all p < 0.01 versus random classifiers, Figures 3F–3I).

 We subsequently compared the diagnostic accuracy of the TEP mRNA classification method with a targeted KRAS (exon 12 and 13) and EGFR (exon 20 and 21) amplicon deep sequencing strategy (∼5,000× coverage) on the Illumina Miseq platform using prospectively collected blood samples of patients with localized or metastasized cancer. This method did allow for the detection of individual mutant KRAS and EGFR sequences in both plasma DNA and platelet RNA (Table S7), indicating sequestration and potential education capacity of mutant, tumor-derived RNA biomarkers in TEPs. Mutant KRAS was detected in 62% and 39%, respectively, of plasma DNA (n = 103, kappa statistics = 0.370, p < 0.05) and platelet RNA (n = 144, kappa statistics = 0.213, p < 0.05) of patients with a KRAS mutation in primary tumor tissue. The sensitivity of the plasma DNA tests was relatively poor as reported by others (Bettegowda et al., 2014, Thierry et al., 2014), which may partly be attributed to the loss of plasma DNA quality due to relatively long blood sample storage (EDTA blood samples were stored up to 48 hr at room temperature before plasma isolation). To discriminate KRAS mutant from wild-type tumors in blood, the TEP mRNA profiles provided superior concordance with tissue molecular status (kappa statistics = 0.795–0.895, p < 0.05) compared to KRAS amplicon sequencing analysis of both plasma DNA and platelet RNA (Table S7). Thus, TEP mRNA profiles can harness potential blood-based surrogate onco-signatures for tumor tissue biomarkers that enable cancer patient stratification and therapy selection.
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Tumor-Educated Platelet mRNA Profiles for Molecular Pathway Diagnostics

Cross tables of SVM/LOOCV diagnostics with the molecular markers KRAS in (A) CRC, (B) PAAD, and (C) NSCLC patients, (D) KRAS in the combined cohort of patients with either CRC, PAAD, NSCLC, or HBC, (E) EGFR and (F) MET in NSCLC patients, (G) PIK3CA mutations, (H) HER2-amplification, and (I) triple negative status in BrCa patients. Genes, number of mRNAs included in training of the SVM algorithm. See alsoTables S4 and S7.

TEP-Profiles Provide an All-in-One Biosource for Blood-Based Liquid Biopsies in Patients with Cancer

Unequivocal discrimination of primary versus metastatic nature of a tumor may be difficult and hamper adequate therapy selection. Since the TEP profiles closely resemble the different tumor types as determined by their organ of origin—regardless of systemic dissemination—this potentially allows for organ-specific cancer diagnostics. Hence we selected all healthy donors and all patients with primary or metastatic tumor burden in the lung (n = 154), brain (n = 114), or liver (n = 127). We performed “organ exams” and instructed the SVM/LOOCV algorithm to determine for lung, brain, and liver the presence or absence of cancer (96%, 91%, and 96% accuracy, respectively), with cancer subclassified as primary or metastatic tumor (84%, 93%, and 90% accuracy, respectively) and in case of metastases to identify the potential organ of origin (64%, 70%, and 64% accuracy, respectively). The platelet mRNA profiles enabled assignment of the cancer to the different organs with high accuracy (Figure 4). In addition, using the same TEP mRNA profiles we were able to again indicate the biomarker status of the tumor tissues (90%, 82%, and 93% accuracy, respectively) (Figure 4).

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Organ-Focused TEP-Based Cancer Diagnostics

SVM/LOOCV diagnostics of healthy donors (n = 55) and patients with primary or metastatic tumor burden in the lung (n = 99; totaling 154 tests), brain (n = 62; totaling 114 tests), or liver (n = 72; totaling 127 tests), to determine the presence or absence of cancer, with cancer subclassified as primary or metastatic tumor, in case of metastases the identified organ of origin, and the correctly identified molecular markers. Of note, at the exam level of mutational subtypes some samples were included in multiple classifiers (i.e., KRAS, EGFR, PIK3CA,HER2-amplification, MET-overexpression, or triple negative status), explaining the higher number in mutational tests than the total number of included samples. TP, true positive; FP, false positive; FN, false negative; TN, true negative. Indicated are sample numbers and detection rates in percentages.

Discussion

The use of blood-based liquid biopsies to detect, diagnose, and monitor cancer may enable earlier diagnosis of cancer, lower costs by tailoring molecular targeted treatments, improve convenience for cancer patients, and ultimately supplements clinical oncological decision-making. Current blood-based biosources under evaluation demonstrate suboptimal sensitivity for cancer diagnostics, in particular in patients with localized disease. So far, none of the current blood-based biosources, including plasma DNA, exosomes, and CTCs, have been employed for multiclass cancer diagnostics (Alix-Panabières and Pantel, 2014, Bettegowda et al., 2014, Skog et al., 2008), hampering its implementation for early cancer detection. Here, we report that molecular interrogation of blood platelet mRNA can offer valuable diagnostics information for all cancer patients analyzed—spanning six different tumor types. Our results suggest that platelets may be employable as an all-in-one biosource to broadly scan for molecular traces of cancer in general and provide a strong indication on tumor type and molecular subclass. This includes patients with localized disease possibly allowing for targeted diagnostic confirmation using routine clinical diagnostics for each particular tumor type.

Since the discovery of circulating tumor material in blood of patients with cancer (Leon et al., 1977) and the recognition of the clinical utility of blood-based liquid biopsies, a wealth of studies has assessed the use of blood for cancer diagnostics, prognostication and treatment monitoring (Alix-Panabières et al., 2012, Bidard et al., 2014, Crowley et al., 2013, Haber and Velculescu, 2014). By development of highly sensitive targeted detection methods, such as targeted deep sequencing (Newman et al., 2014), droplet digital PCR (Bettegowda et al., 2014), and allele-specific PCR (Maheswaran et al., 2008, Thierry et al., 2014), the utility and applicability of liquid biopsies for clinical implementation has accelerated. These advances previously allowed for a pan-cancer comparison of various biosources and revealed that in >75% of cancers, including advanced stage pancreas, colorectal, breast, and ovarian cancer, cell-free DNA is detectable although detection rates are dependent on the grade of the tumor and depth of analysis (Bettegowda et al., 2014). Here, we show that the platelet RNA profiles are affected in nearly all cancer patients, regardless of the type of tumor, although the abundance of tumor-associated RNAs seems variable among cancer patients. In addition, surrogate RNA onco-signatures of tissue biomarkers, also in 88% of localized KRAS mutant cancer patients as measured by the tumor-specific and pan-cancer SVM/LOOCV procedures, are readily available from a minute amount (100–500 pg) of platelet RNA. As whole blood can be stored up to 48 hr on room temperature prior to isolation of the platelet pellet, while maintaining high-quality RNA and the dominant cancer RNA signatures, TEPs can be more readily implemented in daily clinical laboratory practice and could potentially be shipped prior to further blood sample processing.

Blood platelets are widely involved in tumor growth and cancer progression (Gay and Felding-Habermann, 2011). Platelets sequester solubilized tumor-associated proteins (Klement et al., 2009) and spliced and unspliced mRNAs (Calverley et al., 2010, Nilsson et al., 2011), whereas platelets do also directly interact with tumor cells (Labelle et al., 2011), neutrophils (Sreeramkumar et al., 2014), circulating NK-cells (Palumbo et al., 2005, Placke et al., 2012), and circulating tumor cells (Ting et al., 2014, Yu et al., 2013). Interestingly, in vivo experiments have revealed breast cancer-mediated systemic instigation by supplying circulating platelets with pro-inflammatory and pro-angiogenic proteins, supporting outgrowth of dormant metastatic foci (Kuznetsov et al., 2012). Using a gene ontology methodology, CAGE, we correlated TEP-cancer signatures with publicly available curated datasets. Indeed, we identified widespread correlations with cancer tissues, hypoxia, platelet-signatures, and cytoskeleton, possibly reflecting the “alert” and pro-tumorigenic state of TEPs. We observed strong negative correlations with RNAs implicated in RNA translation, T cell immunity, and interleukin-signaling, implying diminished needs of TEPs for RNAs involved in these biological processes or orchestrated translation of these RNAs to proteins (Denis et al., 2005). We observed that the tumor-specific educational programs in TEPs are predominantly influenced by tumor type and, to a lesser extent, by tumor progression and metastases. Although we were not able to measure significant differences between non-metastasized and metastasized tumors, we do not exclude that the use of larger sample sets could allow for the generation of SVM algorithms that do have the power to discriminate between certain stages of cancer, including those with in situ carcinomas and even pre-malignant lesions. In addition, different molecular tumor subtypes (e.g., HER2-amplified versus wild-type BrCa) result in different effects on the platelet profiles, possibly caused by different “educational” stimuli generated by the different molecular tumor subtypes (Koboldt et al., 2012). Altogether, the RNA content of platelets in patients with cancer is dependent on the transcriptional state of the bone-marrow megakaryocyte (Calverley et al., 2010, McAllister and Weinberg, 2014), complemented by sequestration of spliced RNA (Nilsson et al., 2011), release of RNA (Clancy and Freedman, 2014, Kirschbaum et al., 2015, Rak and Guha, 2012, Risitano et al., 2012), and possibly queue-specific pre-mRNA splicing during platelet circulation. Partial or complete normalization of the platelet profiles following successful treatment of the tumor would enable TEP-based disease recurrence monitoring, requiring the analysis of follow-up platelet samples. Future studies will be required to address the tumor-specific “educated” profiles on both an (small non-coding) RNA (Laffont et al., 2013, Landry et al., 2009, Leidinger et al., 2014, Lu et al., 2005) and protein (Burkhart et al., 2014,Geiger et al., 2013, Klement et al., 2009) level and determine the ability of gene ontology, blood-based cancer classification.

In conclusion, we provide robust evidence for the clinical relevance of blood platelets for liquid biopsy-based molecular diagnostics in patients with several types of cancer. Further validation is warranted to determine the potential of surrogate TEP profiles for blood-based companion diagnostics, therapy selection, longitudinal monitoring, and disease recurrence monitoring. In addition, we expect the self-learning algorithms to further improve by including significantly more samples. For this approach, isolation of the platelet fraction from whole blood should be performed within 48 hr after blood withdrawal, the platelet fraction can subsequently be frozen for cancer diagnosis. Also, future studies should address causes and anticipated risks of outlier samples identified in this study, such as healthy donors classified as cancer patients. Systemic factors such as chronic or transient inflammatory diseases, or cardiovascular events and other non-cancerous diseases may also influence the platelet mRNA profile and require evaluation in follow-up studies, possibly also including individuals predisposed for cancer.

References   

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Akbani, R., Ng, P.K.S., Werner, H.M.J., Shahmoradgoli, M., Zhang, F., Ju, Z., Liu, W., Yang, J.-Y., Yoshihara, K., Li, J. et al. A pan-cancer proteomic perspective on The Cancer Genome Atlas.

Nat. Commun. 2014; 5: 3887

Alix-Panabières, C. and Pantel, K. Challenges in circulating tumour cell research.

Nat. Rev. Cancer. 2014; 14:623–631

Alix-Panabières, C., Schwarzenbach, H., and Pantel, K. Circulating tumor cells and circulating tumor DNA.

Annu. Rev. Med. 2012; 63:199–215

Bettegowda, C., Sausen, M., Leary, R.J., Kinde, I., Wang, Y., Agrawal, N., Bartlett, B.R., Wang, H., Luber, B., Alani, R.M. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies.

Sci. Transl. Med. 2014; 6:224ra24

Bidard, F.-C., Peeters, D.J., Fehm, T., Nolé, F., Gisbert-Criado, R., Mavroudis, D., Grisanti, S., Generali, D., Garcia-Saenz, J.A., Stebbing, J. et al. Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data.

Lancet Oncol. 2014; 15: 406–414

Bray, P.F., McKenzie, S.E., Edelstein, L.C., Nagalla, S., Delgrosso, K., Ertel, A., Kupper, J., Jing, Y., Londin, E., Loher, P. et al. The complex transcriptional landscape of the anucleate human platelet.

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Burkhart, J.M., Gambaryan, S., Watson, S.P., Jurk, K., Walter, U., Sickmann, A., Heemskerk, J.W.M., and Zahedi, R.P.  What can proteomics tell us about platelets?.

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Calverley, D.C., Phang, T.L., Choudhury, Q.G., Gao, B., Oton, A.B., Weyant, M.J., and Geraci, M.W. Significant downregulation of platelet gene expression in metastatic lung cancer.

Clin. Transl. Sci. 2010; 3:227–232

Chan, K.C.A., Jiang, P., Chan, C.W.M., Sun, K., Wong, J., Hui, E.P., Chan, S.L., Chan, W.C., Hui, D.S.C., Ng, S.S.M. et al. Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing.

Proc. Natl. Acad. Sci. USA.2013; 110: 18761–18768

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Chi-Ping Day, Glenn Merlino, Terry Van Dyke
Cell, Vol. 163, Issue 1, p39–53
Published in issue: September 24, 2015
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Katherine A. Hoadley, Christina Yau, Denise M. Wolf, Andrew D. Cherniack, David Tamborero, Sam Ng, Max D.M. Leiserson, Beifang Niu, Michael D. McLellan, Vladislav Uzunangelov, Jiashan Zhang, Cyriac Kandoth, Rehan Akbani, Hui Shen, Larsson Omberg, Andy Chu, Adam A. Margolin, Laura J. van’t Veer, Nuria Lopez-Bigas, Peter W. Laird, Benjamin J. Raphael, Li Ding, A. Gordon Robertson, Lauren A. Byers, Gordon B. Mills, John N. Weinstein, Carter Van Waes, Zhong Chen, Eric A. Collisson, The Cancer Genome Atlas Research Network, Christopher C. Benz, Charles M. Perou, Joshua M. Stuart
Cell, Vol. 158, Issue 4, p929–944
Published online: August 7, 2014

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Pau Creixell, Erwin M. Schoof, Craig D. Simpson, James Longden, Chad J. Miller, Hua Jane Lou, Lara Perryman, Thomas R. Cox, Nevena Zivanovic, Antonio Palmeri, Agata Wesolowska-Andersen, Manuela Helmer-Citterich, Jesper Ferkinghoff-Borg, Hiroaki Itamochi, Bernd Bodenmiller, Janine T. Erler, Benjamin E. Turk, Rune Linding
Cell, Vol. 163, Issue 1, p202–217
Published online: September 17, 2015
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Published in issue: September 26, 2013

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Corina E. Antal, Andrew M. Hudson, Emily Kang, Ciro Zanca, Christopher Wirth, Natalie L. Stephenson, Eleanor W. Trotter, Lisa L. Gallegos, Crispin J. Miller, Frank B. Furnari, Tony Hunter, John Brognard, Alexandra C. Newton
Cell, Vol. 160, Issue 3, p489–502
Published online: January 22, 2015
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Cell, Vol. 160, Issues 1-2, p7
Published in issue: January 15, 2015
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Amelia J. Johnston, Kate T. Murphy, Laura Jenkinson, David Laine, Kerstin Emmrich, Pierre Faou, Ross Weston, Krishnath M. Jayatilleke, Jessie Schloegel, Gert Talbo, Joanne L. Casey, Vita Levina, W. Wei-Lynn Wong, Helen Dillon, Tushar Sahay, Joan Hoogenraad, Holly Anderton, Cathrine Hall, Pascal Schneider, Maria Tanzer, Michael Foley, Andrew M. Scott, Paul Gregorevic, Spring Yingchun Liu, Linda C. Burkly, Gordon S. Lynch, John Silke, Nicholas J. Hoogenraad
Cell, Vol. 162, Issue 6, p1365–1378
Published in issue: September 10, 2015
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Levi A. Garraway, Eric S. Lander
Cell, Vol. 153, Issue 1, p17–37
Published in issue: March 28, 2013

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Cell, Vol. 163, Issue 1, p28–30
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Silk Biomaterials Produced from 3D Bone Marrow Generate Platelets

Reported by: Irina Robu, PhD

The team used silk protein scaffolds that silk is a very biocompatible material that is amenable to many manipulations to customize it for a specific use, while also avoiding any cell-specific signaling. They formed silk scaffolds with thickness ranging from 2 to 5 micrometers and stiffness combined with growth factors, to test the success of megakaryocyte adhesion and the formation of pro-platelets—the parts of the megakaryocytes that fragment into platelets.  After determining the best combination of scaffolds with appropriate thickness and stiffness, the researchers attached the silk scaffolds to a plastic framework to guide the growth of cells. The next step is to grow endothelial primary cells on one side of the silk scaffold and megakaryocytes on the other side, partly because endothelial primary cells are known to secrete growth factors that help megakaryocytes mature.

In order to mimic the microvasculature and environment, the  researchers form silk sponges around the porous microtubes. The culture media with necessary nutrients is being pumped to mimic the flow of blood which leads to higher numbers of platelets generated than was previously possible; and most importantly, the platelets were functional.This is the first time researchers were able to create the complete micro environment where platelets are formed.

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Mature cells can be reprogrammed to become pluripotent – John Gurdon and Shinya Yamanaka

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Innovation

Series E: 2; 7.1

In 1962, John B. Gurdon successfully cloned frogs. He took the nucleus of an adult frog cell – the part of the cell that holds the DNA – and put it into a frog egg cell. The egg was able to develop into a normal tadpole. These experiments showed that an adult, specialised cell still had the information needed to form a new tadpole. The same technique was later used to produce the famous cloned sheep, Dolly.

In 2006, Shinya Yamanaka’s work again took the scientific community by surprise and changed the way researchers think about how cells develop.Yamanaka showed that adult, fully specialised mouse cells could be reprogrammed to become cells that behave like embryonic stem cells – so-called induced pluripotent stem cells, which can develop into all types of cells in the body.

Gurdon and Yamanaka’s work is celebrated and explained in the award-winning documentary, Stem Cell Revolutions, by Clare Blackburn and Amy Hardie. The short clip above is taken from the film and links Gurdon and Yamanaka’s work (click the red button on the image above to watch the clip). Amy Hardie, who directed the film, commented: “So many scientists have said that Shinya Yamanaka has overturned our understanding of basic developmental biology. And he has – with the discovery of iPS cells. What Shinya Yamanaka himself points out and we were able to show in our film, Stem Cell Revolutions, is the lineage from John Gurdon who cloned frogs in Cambridge. Shinya’s groundbreaking discovery would not have been possible without Gurdon’s pioneering work.

Proc Natl Acad Sci U S A. 2013 Apr 9; 110(15): 5740–5741.

Published online 2013 Mar 28. doi:  10.1073/pnas.1221823110

Sir John Bertrand Gurdon, FRS, FMedSci (born 2 October 1933), is an English developmental biologist. He is best known for his pioneering research in nuclear transplantation[2][3][4] and cloning.[1][5][6][7] He was awarded the Lasker Award in 2009. In 2012, he and Shinya Yamanaka were awarded the Nobel Prize for Physiology or Medicine for the discovery that mature cells can be converted to stem cells.[8]

The Nobel Prize in Physiology or Medicine 2012
Sir John B. Gurdon, Shinya Yamanaka

ohn Bertrand Gurdon (JBG), born 2 October 1933, was brought up in a comfortable home by his parents (fig.1) on the Surrey/Hampshire border in a village, Frensham in South England, endowed with a large amount of National Trust heathland and ponds. His mother, Marjorie Byass, was from an East Yorkshire farming family. Brought up on a farm, and educated in that region, she became a physical training teacher working for some time in an American private school. When her son and daughter (Caroline, who trained as a nurse) had been raised, she gave much time to the regional administration of the “Women’s Institute,” a voluntary organisation for educating women.

His father, William Gurdon, was from a longstanding Suffolk family whose ancestors go back to 1199 (fig. 2; Muskett, 1900; Cunnington, 2008); with the family motto “virtus viget in arduis” [virtue flourishes in adversity].

Paternal lineage of JBG.

Many of them had distinguished careers in government and as regional administrators, including Sir Adam Gurdon [Muskett, 1900]. JBG’s ancestors lived in a stately home, Assington Hall, in West Suffolk (fig. 3).

His grandfather had to leave the family home through lack of money to maintain it, due to repeal of the Corn Laws (1846) so that tenant farmers could no longer pay their rent, because of foreign imports. Assington Hall was requisitioned by the army during World War II, and was burnt down in a supposedly accidental fire in 1957. The remaining part of the house was partly restored and part of the original home, including its minarets, is still present in Assington. One of JBG’s ancestors married again after his first wife died and the outcome of a second marriage yielded a distinguished lawyer who accepted the hereditary title of Baron Cranworth. JBG’s father left school at the age of 16 and took a position in a rice broking firm in Burma. He was an early volunteer in the First World War and was decorated with the Distinguished Conduct Medal (DCM) before being commissioned to an officer rank. After that he led a career in banking in Assam and East India. He retired, in his forties, and in retirement, he gave much time to the transcribing of professional textbooks (especially legal) into Braille for the blind as voluntary work.

World War II started in 1939 when JBG was aged six. It was a time of austerity. Limited rations of food were managed by his mother, and the garden was used to raise chickens. He did not see luxuries like a banana or an orange until well after the end of the war. At the age of eight he was sent to a local private school, Frensham Heights. In an intelligence test at that age, he was asked to draw an orange. He started drawing the stalk by which the orange would hang from a tree, reasoning that an orange would not exist in space. The teacher tore up the piece of paper and reported to his parents that he was mentally subnormal and would need special teaching. The teacher meant to say, draw a circle. He was moved to another private school in the village, namely Edgeborough, where he thrived. At that age he had an intense interest in plants and insects. In most of his spare time he collected butterflies and moths and raised their caterpillars.

At the age of 13, he started school at Eton as a boarder. He found life there intensely uncomfortable, because senior boys acted as despots, administering punishments for trivial misdemeanours. As a means of survival, he took up squash, and as a result of hard work rather than ability, he became eventually the school captain in this sport. While at school he continued his interest in Lepidoptera, raising large numbers of moths from their larval stage.

Gurdon attended Edgeborough and then Eton College, where he ranked last out of the 250 boys in his year group at biology, and was in the bottom set in every other science subject. A schoolmaster wrote a report stating “I believe he has ideas about becoming a scientist; on his present showing this is quite ridiculous.”[9] Gurdon explains it is the only document he ever framed; Gurdon also told a reporter “When you have problems like an experiment doesn’t work, which often happens, it’s nice to remind yourself that perhaps after all you are not so good at this job and the schoolmaster may have been right.”[10]

It was during his first term of being taught Science at the school, at the age of 15, that he received a totally damning report from the Biology master (fig. 4). This report resulted from JBG being placed in the bottom position of the lowest form in a group of 250 students of the same age. The report, sent to his housemaster, resulted in him being taken off any further study of Science of any kind at the school. For the rest of his school days, for the next three years, he was given no Science teaching and was placed in a class which studied Ancient Greek, Latin and a modern language, a course intended for those judged to be unsuited for studying any subject in depth.

Eton school report for JBG from Biology master, 1949.

 

Entrance to University was a problem: having sat the Entrance examination in Latin and Greek, the Admissions tutor at Christ Church Oxford University told JBG that he would be accepted for Entrance on condition that he did not plan to study the subject in which he took the Entrance (Classics). Later the Admissions tutor admitted that he had under-filled the college and had his mind on other things; he was Hugh Trevor-Roper, later Lord Dacre, and author of The Last Days of Hitler. In due course it emerged that JBG’s acceptance for Christ Church involved a complicated arrangement between JBG’s uncle, at that time a Fellow of Christ Church, JBG’s school housemaster and a friend of his uncle, Sir John Masterman, who was Master of Worcester College, Oxford and in charge of the wartime Enigma operation at Bletchley, agreeing to accept the housemaster’s son. Such a manoeuvre, and admission to Oxford on those terms, could never happen now. At that time, 1952, it was not very easy to fill a college with paying students. Before entering University, JBG had to take a year off to learn elementary Biology with a private tutor, generously funded by his parents who had already paid several years of Eton fees. He was told that he could formally enter the Department of Zoology course at Oxford if he passed the elementary exams in Physics, Chemistry and Biology in a preliminary year. He survived this and started the course in Zoology at Oxford in 1953. The course was extremely oldfashioned, by today’s standards. A major part of the teaching involved learning Palaeontology, and the names of skeletal parts of dinosaurs. JBG later became a personal friend of Sir Alister Hardy, the Head of that department, through his Oxford aunt (see later).

As the Zoology course came to an end, JBG enquired about the possibility of doing a PhD in Entomology, in accord with his continuing interest in insects. While still a student, he had got permission to go to Oxford University’s nature reserve, namely Wytham Woods, with his butterfly net. No butterflies were to be seen, but he caught the only moving thing, which was a kind of fly. He used the taxonomic reference works to try to identify this “fly.” Having realised that the fly was a Hymenopteron, he was still unable to identify it. He therefore went to the Natural History Museum in London for help. They pronounced that it was in fact a species of sawfly new to Britain. This must have been intensely irritating to the Professor of Entomology, whose main research project was to identify animals and plants in Wytham Woods. JBG was later rejected for PhD work in Entomology. This was a great blessing because the work he would have done in Entomology was not well regarded and had very little, if any, analytical component to it. By his immense good fortune, he was invited to do a PhD with the Oxford University lecturer who taught Developmental Biology, Dr Michael Fischberg.

Fischberg was born in St Petersburg, Russia, in 1919. He was educated in Switzerland and was a PhD student of E. Hadorn. Hadorn in turn was a student of F. Baltzer, who was a student of H. Spemann, himself a student of T. Boveri. This German-Swiss lineage of eminent Developmental Biologists turns out to be the background of a great many of the successful Developmental Biologists of the mid-1950s. Most of those that did not have this background can trace their own training back to R. G. Harrison (1870–1959) of the USA, who pioneered cell culture. Having finished his PhD with Hadorn, Fischberg took a position in the Institute of Animal Genetics under Waddington in Edinburgh, from where he accepted his appointment in the Oxford Zoology department, headed by Professor Sir Alister Hardy, an eminent marine biologist [Royal Society memoirs].

Starting his PhD work in 1956, Fischberg suggested to JBG that he should try to carry out somatic cell nuclear transfer in Xenopus, a procedure for this having been recently published by Briggs and King (1952). The advisability and technical problems that arose at this point are described in the accompanying papers (Gurdon 2013 a,b). Once these technical obstacles had been overcome, largely as a result of good luck, JBG’s work proceeded extraordinarily fast; strongly motivated by early success, he became an intensely hard worker. By the end of his PhD he had succeeded in obtaining normal development of intestinal epithelium cell nuclei transplanted to enucleated eggs of Xenopus. When these tadpoles had eventually reached sexual maturity, he was able to publish a paper entitled “Fertile intestine nuclei.”This was the first decisive evidence that all cells of the body contain the same complete set of genes. This answered a long-standing and important question in the field of Developmental Biology. However it also showed very clearly, as was commented on in JBG’s papers at the time, the remarkable ability of eggs to reprogram somatic cell nuclei back to an embryonic state. Eventually this phenomenon attracted increasingly large interest, and led to the idea of cell replacement using accessible adult cells, such as skin. A key future discovery was that of Martin Evans (Nobel Prize, 2006) that a permanently proliferating embryonic stem cell line could be established from mouse embryos. Under appropriate conditions these cells could be caused to differentiate into all different cell types. The combination of somatic cell nuclear transfer and the derivation of embryonic stem cells in mammals made it realistic to think of cell replacement for human diseases. A huge boost for this idea was later provided by Takahashi and Yamanaka (2006), with their discovery that the overexpression of certain transcription factors can also yield embryonic stem cells from adult somatic tissue. The accompanying Nobel lecture provides more detail of the later scientific part of JBG’s career.

A visit by the Nobel Laureate George Beadle to the Fischberg Group in the Oxford Zoology department in 1960 led to an offer from the California Institute of Technology (CalTech) (previous chairman George Beadle) for JBG to do postdoctoral work there. Fischberg very wisely advised JBG to accept the CalTech offer of postdoctoral work rather than offers from other nuclear transplant labs. Stimulated by his mother’s adventurous spirit, JBG decided to buy a secondhand Chevrolet in New York and drive across the USA to California, using the famous Route 66 (now replaced). He gave lectures as he travelled across the USA and stopped at laboratories of Briggs and King, Alexander Brink (paramutation) etc. He had hoped to become a post-doctoral student of R. Dulbecco at CalTech (Nobel Prize), but the chairman of that department advised against this because JBG had no training in virology. Therefore JBG did his postdoctoral work with Robert Edgar on Bacteriophage Genetics. JBG found he had no aptitude at all for Phage Genetics and decided to return to Britain after one year at CalTech. Nevertheless, that year at CalTech was extremely formative because it provided some acquaintance with Molecular Biology, which had so far entirely escaped his training. During that year he met Sturtevant, a student of Morgan, who pioneered the whole field of Drosophila Genetics. He also got to know Ed Lewis (future Nobel Laureate). Thanks to James Ebert (director of the Department of Embryology, Carnegie Institute of Washington, in Baltimore) JBG visited various labs in the USA at the end of his post-doctoral period and met Donald Brown in Baltimore on that visit. Meantime, the success of the nuclear transfer work in Oxford had led to Michael Fischberg being offered a head of department professorship in Geneva, Switzerland. JBG was offered the teaching position in Oxford vacated by M. Fischberg. JBG returned from California to England via Japan and many other countries over a two-month period. One month of that time he spent in Japan and met Tokindo Okada and made other friends in Japan, including M. Furusawa and subsequently Koichiro Shiokawa.

While doing graduate and postdoctoral work in Oxford, JBG made other contacts and friendships. His mother’s sister lived in Oxford, and he spent much time at her house and visiting famous gardens, fostering a lifelong interest in plants. Through that connection he met Miriam Rothschild, and became a lifelong friend of hers (Van Emden and Gurdon, 2006). This friendship contained, through Miriam Rothschild’s generosity, ski mountaineering holidays based in her house in Wengen. JBG had achieved the British ski club’s Gold standard ski medal, again through relentless practice rather than any natural ability. Also, in accord with his interest in the open air and dogged determination, he became a reasonably accomplished ice figure skater.

Nobel Lecture by Sir John B. Gurdon (42 minutes)

Sir John B. Gurdon delivered his Nobel Lecture on 7 December 2012 at Karolinska Institutet in Stockholm. He was introduced by Professor Urban Lendahl, Chairman of the Nobel Committee for Physiology or Medicine.
Credits: Sveriges Television AB (production)

Copyright © Nobel Media AB 2012

The Nobel Prize in Physiology or Medicine 2012    Lecture (pdf)

Nuclear transfer

In 1958, Gurdon, then at the University of Oxford, successfully cloned a frog using intact nuclei from the somatic cells of a Xenopus tadpole.[14][15] This work was an important extension of work of Briggs and King in 1952 on transplanting nuclei from embryonic blastula cells[16] and the successful induction of polyploidy in fish Stickleback, Gasterosteus aculatus, in 1956 by Har Swarup reported in Nature.[17] However, he could not yet conclusively show that the transplanted nuclei derived from a fully differentiated cell. This was finally shown in 1975 by a group working at the Basel Institute for Immunology in Switzerland.[18] They transplanted a nucleus from an antibody-producing lymphocyte (proof that it was fully differentiated) into an enucleated egg and obtained living tadpoles.

Gurdon’s experiments captured the attention of the scientific community and the tools and techniques he developed for nuclear transfer are still used today. The term clone[19] (from the ancient Greek word κλών (klōn, “twig”)) had already been in use since the beginning of the 20th century in reference to plants. In 1963 the British biologist J. B. S. Haldane, in describing Gurdon’s results, became one of the first to use the word “clone” in reference to animals.

Messenger RNA expression

Gurdon and colleagues also pioneered the use of Xenopus (genus of highly aquatic frog) eggs and oocytes to translate microinjected messenger RNA molecules,[20] a technique which has been widely used to identify the proteins encoded and to study their function.

Recent research

Gurdon’s recent research has focused on analysing intercellular signalling factors involved in cell differentiation, and on elucidating the mechanisms involved in reprogramming the nucleus in transplantation experiments, including the role of histone variants,[21][22] and demethylation of the transplanted DNA.[23]

Reprogramming of Mature Cells

Our lives begin when a fertilized egg divides and forms new cells that, in turn, also divide. These cells are identical in the beginning, but become increasingly varied over time. As a result of this process, our cells become specialized for their location in the body – perhaps in a nerve, a muscle, or a kidney. It was long thought that a mature or specialized cell could not return to an immature state, but this has been proven incorrect.

In 1962, John Gurdon removed the nucleus of a fertilized egg cell from a frog and replaced it with the nucleus of a mature cell taken from a tadpole’s intestine. This modified egg cell grew into a new frog, proving that the mature cell still contained the genetic information needed to form all types of cells. In 2006, Shinya Yamanaka succeeded in identifying a small number of genes within the genome of mice that proved decisive in this process. When activated, skin cells from mice could be reprogrammed to immature stem cells, which, in turn, can grow into all types of cells within the body. In the long-term, these discoveries may lead to new medical treatments.

Shinya Yamanaka

A winding road to pluripotency

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/yamanaka-lecture.pdf

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/ypdfamanaka-lecture_slides.

Nobel Lecture

46 min.
by Shinya Yamanaka Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.
INTRODUC TION John Gurdon received recognition for his landmark achievement in 1962, which provided the first experimental evidence of reprogramming by the transplantation of amphibian somatic cell nuclei into enucleated oocytes [1]. This breakthrough in technology introduced a new paradigm; that each nucleus of a differentiated cell retains a complete set of blueprints for the whole body, while oocytes possess a certain potential for reprogramming. Inspired by this paradigm shift and subsequent research achievements, we identified four transcription factors that could induce pluripotency in somatic cells by their forced expression and successfully consolidated effective reprogramming methods in mouse cells in 2006 [2] and in human cells in 2007 [3]. The established reprogrammed cells were named “induced pluripotent stem (iPS) cells.” I would like to provide an overview focusing on the experimental background of the generation of iPS cells, and the future perspectives regarding iPS cell research, which has been developing rapidly.

Figure 1. My first experiment as a graduate student. Intravenous injection of a vasoactive molecule platelet activating factor (PAF) caused a transient decrease in blood pressure in dogs (upper panel). We hypothesized that this hypotension would be blocked by pretreatment with a thromboxane A2 inhibitor (lower left panel). Unexpectedly, we observed a profound hypotension (lower right panel).

In 1989, however, my life took a new turn from clinical medicine in orthopedic surgery to basic science research for two reasons. First, I found that I was not a very talented surgeon. Second, I saw many patients suffering from intractable diseases and injuries, which even highly talented surgeons and physicians were not able to cure. For example, I had encountered patients suffering from spinal cord injuries, amyotrophic lateral sclerosis and osteosarcomas. Furthermore, I lost my father due to liver cirrhosis during my residency. Basic medical research is the only way to find cures for these patients. For these reasons, I decided to go back to school. I became a Ph.D. student at Osaka City University Medical School in April of 1989.

Among the many departments at the school, I applied to the Department of Pharmacology, directed by Dr. Kenjiro Yamamoto.  Dr. Ikemoto repeatedly told me that we should not perform research that simply reproduced somebody else’s re-sults. Rather, we should do something unique and new. During my training as a scientist, I was very fortunate to have two types of teachers: namely, great men-tors and unexpected results from my experiments.
My direct mentor at the graduate school was Dr. Katsuyuki Miura. In my first few months as a Ph.D. student, Dr. Miura told me to read as many manuscripts as possible and propose new projects. I felt like I was given a blank canvas and told that I could draw whatever I wanted. This mentorship was very different from what I had experienced during my residency. At the hospital, I’d had little freedom, and had to follow instructions from senior physicians and textbooks. I thought “wow, I like this system!” Another thing that Dr. Miura often told me was that we were competing worldwide. Whatever project you chose, you will compete with other scientists throughout the world, mostly in the U.S. or Europe, on the same or similar projects. This was again very different from my experience at the hospital, where I was competing only with other residents at the same hospital. The idea of “worldwide” competition had never entered my mind when I was working at the hospital. For all of these reasons, I found that basic research was a more suitable career, based on my interests and temperament.
In the summer of 1989, I was still struggling to find my project. Dr. Miura proposed a simpler project to begin my research studies. He suggested that I examine the role of a vasoactive molecule, platelet activating factor (PAF), in dogs to study the regulation of blood pressure (Fig. 1). Because it was known that the intravenous injection of PAF into dogs caused a transient decrease in blood pressure (transient hypotension), Dr. Miura hypothesized that this decrease in blood pressure would be mediated by another vasoactive molecule, thromboxane A2. If that hypothesis was correct, then pretreatment with a thromboxane A2 inhibitor should block the PAF-induced transient decrease in blood pressure. My first experiment, where I treated dogs with an inhibitor of thromboxane A2, was performed based on his hypothesis, and I had expected no decrease in the blood pressure in the pretreated dogs. It should have been a simple experiment suitable for a beginner. However, the result was totally unexpected. In the beginning, the thromboxane A2 inhibitor did not seem to be effective, with subsequent PAF treatment inducing the normal transient decrease in the blood pressure. Surprisingly, however, a few minutes after the treatment, a profound and prolonged decrease in blood pressure was observed, which we had never observed following treatment with PAF alone (Fig. 1). I got so excited! I ran into Dr. Miura’s office to report this result excitedly. Although the result did not support his hypothesis, Dr. Miura responded with excitement, too, and encouraged me to explore the finding further. I spent another two years uncovering the mechanism responsible for this unexpected result [4, 5]. I was extremely lucky to obtain this kind of unexpected result in my very first experiment as a graduate student.

A scandal involving Japanese stem-cell research took a surprising turn Monday when the nation’s most revered researcher in the field, Nobel Prize laureate Shinya Yamanaka, apologized for what he described as poor record-keeping.

The apology came after months of soul-searching in Japan over research ethics. A researcher at the prestigious Riken institute, Haruko Obokata, apologized earlier this month after admitting errors in a paper in the journal Nature that described a possible new method of creating stem cells.

Last week, the head of the Riken panel investigating Dr. Obokata had to resign from the panel after admitting that a paper he co-authored used some of the same improper methods of cutting and pasting images that he had criticized in Dr. Obokata’s work.

On Monday evening, Dr. Yamanaka, a professor at Kyoto University, spoke at a news conference after questions arose about an image in a 2000 paper on which he was the lead author. In the paper, Dr. Yamanaka, then at Nara University, described a protein that played a role in turning embryo cells into cells specific to a part of the body.

The university said it conducted an investigation after Dr. Yamanaka informed administrators about allegations he discovered online that an image in the paper was doctored.

 

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Acute Lung Injury

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

 

 

Introduction

Acute lung injury is a serious phenomenon only recognized as having significant relevance to allogeneic blood transfusion in the last 15 years.  It is not limited to transfusion events, and is also related to SIRS and sepsis.  It is simulated in experimental models by lipoprotein, such as endotoxin.  It occurs in the pretransfused surgical patient, or in the medical patient as well.  Why it was not recognized earlier is a matter of conjecture.  The significant reduction in immune modulated blood type incompatibility reactions in Western countries is a factor.  The other factor is that the lipoprotein antigenic fractions involved are associated with component transfusions other than stored red cells. The following discussion will elaborate on what is increasingly recognized as a relevant issue in medicine today.
Transfusion Related Reaction

In medicinetransfusion related acute lung injury (TRALI) is a serious blood transfusion complication characterized by the acute onset of non-cardiogenic pulmonary edema following transfusion of blood products.[1]

Although the incidence of TRALI has decreased with modified transfusion practices, it is still the leading cause of transfusion-related fatalities in the United States from fiscal year 2008 through fiscal year 2012.

Transfusion Related Acute Lung Injury

TRALI-Hyaline_membranes_-_very_high_mag

TRALI-Hyaline_membranes_-_very_high_mag

Micrograph of diffuse alveolar damage, the histologic correlate of TRALI. H&E stain. Very high magnification micrograph of hyaline membranes, as seen in diffuse alveolar damage (DAD), the histologic correlate of acute respiratory distress syndrome (ARDS), transfusion related acute lung injury (TRALI), acute interstitial pneumonia (AIP).
http://upload.wikimedia.org/wikipedia/commons/thumb/c/c8/Hyaline_membranes_-_very_high_mag.jpg/1024px-Hyaline_membranes_-_very_high_mag.jpg

TRALI is defined as an acute lung injury that is temporally related to a blood transfusion; specifically, it occurs within the first six hours following a transfusion.[3]

It is typically associated with plasma components such as platelets and Fresh Frozen Plasma, though cases have been reported with packed red blood cells since there is some residual plasma in the packed cells. The blood component transfused is not part of the case definition. Transfusion-related acute lung injury (TRALI) is an uncommon syndrome that is due to the presence of leukocyte antibodies in transfused plasma. TRALI is believed to occur in approximately one in every 5000 transfusions. Leukoagglutination and pooling of granulocytes in the recipient’s lungs may occur, with release of the contents of leukocyte granules, and resulting injury to cellular membranes, endothelial surfaces, and potentially to lung parenchyma. In most cases leukoagglutination results in mild dyspnea and pulmonary infiltrates within about 6 hours of transfusion, and spontaneously resolves;

Occasionally more severe lung injury occurs as a result of this phenomenon and Acute Respiratory Distress Syndrome (ARDS) results. Leukocyte filters may prevent TRALI for those patients whose lung injury is due to leukoagglutination of the donor white blood cells, but because most TRALI is due to donor antibodies to leukocytes, filters are not helpful in TRALI prevention. Transfused plasma (from any component source) may also contain antibodies that cross-react with platelets in the recipient, producing usually mild forms of posttransfusion purpura or platelet aggregation after transfusion.

Another nonspecific form of immunologic transfusion complication is mild to moderate immunosuppression consequent to transfusion. This effect of transfusion is not completely understood, but appears to be more common with cellular transfusion and may result in both desirable and undesirable effects. Mild immunosuppression may benefit organ transplant recipients and patients with autoimmune diseases; however, neonates and other already immunosuppressed hosts may be more vulnerable to infection, and cancer patients may possibly have worse outcomes postoperatively.

http://en.wikipedia.org/wiki/Transfusion-related_acute_lung_injury

 

 

Perioperative transfusion-related acute lung injury: The Canadian Blood Services experience

Asim Alam, Mary Huang, Qi-Long Yi, Yulia Lin, Barbara Hannach
Transfusion and Apheresis Science 50 (2014) 392–398
http://dx.doi.org/10.1016/j.transci.2014.04.008

Purpose: Transfusion-related acute lung injury (TRALI) is a devastating transfusion-associated adverse event. There is a paucity of data on the incidence and characteristics of TRALI cases that occur perioperatively. We classified suspected perioperative TRALI cases reported to Canadian Blood Services between 2001 and 2012, and compared them to non-perioperative cases to elucidate factors that may be associated with an increased risk of developing TRALI in the perioperative setting. Methods: All suspected TRALI cases reported to Canadian Blood Services (CBS) since 2001 were reviewed by two experts or, from 2006 to 2012, the CBS TRALI Medical Review Group (TMRG). These cases were classified based on the Canadian Consensus Conference (CCC) definitions and detailed in a database. Two additional reviewers further categorized them as occurring within 72 h from the onset of surgery (perioperative) or not in that period (non-perioperative). Various demographic and characteristic variables of each case were collected and compared between groups. Results: Between 2001 and 2012, a total of 469 suspected TRALI cases were reported to Canadian Blood Services; 303 were determined to be within the TRALI diagnosis spectrum. Of those, 112 (38%) were identified as occurring during the perioperative period. Patients who underwent cardiac surgery requiring cardiopulmonary bypass (25.0%), general surgery (18.0%) and orthopedics patients (12.5%) represented the three largest surgical groups. Perioperative TRALI cases comprised more men (53.6% vs. 41.4%, p = 0.04) than non-perioperative patients. Perioperative TRALI patients more often required supplemental O2 (14.3% vs. 3.1%, p = 0.0003), mechanical ventilation (18.8% vs. 3.1%), or were in the ICU (14.3% vs. 3.7%, p = 0.0043) prior to the onset of TRALI compared to non-perioperative TRALI patients. The surgical patients were transfused on average more components than non-perioperative patients (6.0 [SD = 8.3] vs. 3.6 [5.2] products per patient, p = 0.0002). Perioperative TRALI patients were transfused more plasma (152 vs. 105, p = 0.013) and cryoprecipitate (51 vs. 23, p < 0.01) than non-perioperative TRALI patients. There was no difference between donor antibody test results between the groups. Conclusion: CBS data has provided insight into the nature of TRALI cases that occur perioperatively; this  group represents a large proportion of TRALI cases.

 

Transfusion-related acute lung injury: a clinical review

Alexander P J Vlaar, Nicole P Juffermans
Lancet 2013; 382: 984–94
http://dx.doi.org/10.1016/S0140-6736(12)62197-7

Three decades ago, transfusion-related acute lung injury (TRALI) was considered a rare complication of transfusion medicine. Nowadays, the US Food and Drug Administration acknowledge the syndrome as the leading cause of transfusion-related mortality. Understanding of the pathogenesis of TRALI has resulted in the design of preventive strategies from a blood-bank perspective. A major breakthrough in efforts to reduce the incidence of TRALI has been to exclude female donors of products with high plasma volume, resulting in a decrease of roughly two-thirds in incidence. However, this strategy has not completely eradicated the complication. In the past few years, research has identified patient-related risk factors for the onset of TRALI, which have empowered physicians to take an individualized approach to patients who need transfusion.

Development of an international consensus definition has aided TRALI research, yielding a higher incidence in specific patient populations than previously acknowledged Patients suffering from a clinical disorder such as sepsis are increasingly recognized as being at risk for development of TRALI. Thereby, from a diagnosis by exclusion, TRALI has become the leading cause of transfusion-related mortality. However, the syndrome is still under diagnosed and under-reported in some countries.

Although blood transfusion can be life-saving, it can also be a life-threatening intervention. Physicians use blood transfusion on a daily basis. Increased awareness of the risks of this procedure is needed, because management of patient-tailored transfusion could reduce the risk of TRALI. Such an individualized approach is now possible as insight into TRALI risk factors evolves. Furthermore, proper reporting of TRALI could prevent recurrence.

Absence of an international definition for TRALI previously contributed to underdiagnosis. As such, a consensus panel, and the US National Heart, Lung and Blood Institute Working Group in 2004, formulated a case definition of TRALI based on clinical and radiological parameters. The definition is derived from the widely used definition of acute lung injury (panel 1). Suspected TRALI is defined as fulfilment of the definition of acute lung injury within 6 h of transfusion in the absence of another risk factor (panel 1).

Although this definition seems to be straightforward, the characteristics of TRALI are indistinguishable from acute lung injury due to other causes, such as sepsis or lung contusion. Therefore, this definition would rule out the possibility of diagnosing TRALI in a patient with an underlying risk factor for acute lung injury who has also received a transfusion. To identify such cases, the term possible TRALI was developed.

Although the TRALI definition is an international consensus definition, surveillance systems in some countries, including the USA, France and the Netherlands, use an alternative in which imputability is scored. Imputability aims to identify the likelihood that transfusion is the causal factor. Imputability scores mostly imply that other causes of acute lung injury can be ruled out, so that diagnosis of TRALI is by exclusion. However, observational and animal studies suggest that risk factors for TRALI include other disorders, such as sepsis. Therefore, an imputability definition would result in underdiagnosis of TRALI. The consensus definition accommodates the uncertainty of the association of acute lung injury to the transfusion in possible TRALI. The conventional definition of TRALI uses a timeframe of 6 h in which acute lung injury needs to develop after a blood transfusion. In critically ill patients, transfusion increases the risk (odds ratio 2·13, 95% CI 1·75–2·52) for development of acute lung injury 6–72 h after transfusion.  However, whether the pathogenesis of delayed TRALI is similar to that of TRALI is unclear.

A two-hit hypothesis has been proposed for TRALI. The first hit is underlying patient factors, resulting in adherence of primed neutrophils to the pulmonary endothelium. The second hit is caused by mediators in the blood transfusion that activate the endothelial cells and pulmonary neutrophils, resulting in capillary leakage and subsequent pulmonary edema. The second hit can be antibody-mediated or non-antibody-mediated.

Panel 1: Definition of transfusion-related acute lung injury (TRALI)

Suspected TRALI

  • Acute onset within 6 h of blood transfusion
    • PaO2/FIO2<300 mm Hg, or worsening of P to F ratio
    • Bilateral infi ltrative changes on chest radiograph
    • No sign of hydrostatic pulmonary oedema (pulmonary arterial occlusion
    pressure ≤18 mm Hg or central venous pressure ≤15 mm Hg)
    • No other risk factor for acute lung injury

Possible TRALI
Same as for suspected TRALI, but another risk factor present for acute lung injury

Delayed TRALI
Same as for (possible) TRALI and onset within 6–72 h of blood transfusion

Pathophysiology of two-hit mediated transfusion-related acute lung injury (TRALI).  The pre-phase of the syndrome consists of a fi rst hit, which is mainly systemic. This first hit is the underlying disorder of the patient (eg, sepsis or pneumonia) causing neutrophil attraction to the capillary of the lung. Neutrophils are attracted to the lung by release of cytokines and chemokines from upregulated lung endothelium. Loose binding by L-selectin takes place. Firm adhesion is mediated by E-selectin and platelet-derived P-selectin and intracellular adhesion molecules (ICAM-1). In the acute phase of the syndrome, a second hit caused by mediators in the blood transfusion takes place. This hit results in activation of inflammation and coagulation in the pulmonary compartment. Neutrophils adhere to the injured capillary endothelium and marginate through the interstitium into the air space, which is filled with protein-rich edema fluid. In the air space, cytokines interleukin-1, -6, and -8, (IL-1, IL-6, and IL-8, respectively) are secreted, which act locally to stimulate chemotaxis and activate neutrophils resulting in formation of the elastase-α1-antitrypsin (EA) complex. Neutrophils can release oxidants, proteases, and other proinflammatory molecules, such as platelet-activating factor (PAF), and form neutrophil extracellular traps (NETs). Furthermore, activation of the coagulation system happens, shown by an increase in thrombin-antithrombin complexes (TATc), as does a decrease in activity of the fibrinolysis system, shown by a reduction in plasminogen activator activity. The influx of protein-rich edema fluid into the alveolus leads to the inactivation of surfactant, which contributes to the clinical picture of acute respiratory distress in the onset of TRALI. PAI-1 = plasminogen activator inhibitor-1.

Antibody-mediated TRALI is caused by passive transfusion of HLA or human neutrophil antigen (HNA) and corresponding antibodies from the donor directed against antigens of the recipient. Neutrophil activation occurs directly by binding of the antibody to the neutrophil surface (HNA antibodies) or indirectly, mainly by binding to the endothelial cells with activation of the neutrophil (HLA class I antibodies) or to monocytes with subsequent activation of the neutrophil (HLA class II antibodies). The antibody titer and the volume of antibody containing plasma both increase the risk for onset of TRALI. Although the role of donor HLA and HNA antibodies from transfused blood is widely accepted, not all TRALI cases are antibody mediated. In many patients, antibodies cannot be detected. Furthermore, many blood products containing antibodies do not lead to TRALI. This finding has led to development of an alternative hypothesis for the onset of TRALI, termed non-antibody-mediated TRALI.

Non-antibody-mediated TRALI is caused by accumulation of proinflammatory mediators during storage of blood products, and possibly by ageing of the erythrocytes and platelets themselves. Although most preclinical studies have noted a positive correlation between storage time of cell-containing blood products and TRALI, the mechanism is controversial. Two mechanisms have been suggested, including either plasma or the aged cells. In a small-case study and animal experiments, accumulation of bioactive lipids and soluble CD40 ligand (sCD40L) in the plasma layer of cell-containing blood products has been associated with TRALI. Bioactive lipids are thought to cause neutrophil activation through the G-protein coupled receptor on the neutrophil.

The two-hit model suggests that patients in a poor clinical state are at risk for development of TRALI. However, cases have been described of antibody-mediated TRALI developing in fairly healthy recipients. To explain this discrepancy, a threshold model has been suggested in which a threshold must be overcome to induce a TRALI reaction. The threshold is dependent both on the predisposition of the patient (first hit) and the quantity of antibodies in the transfusion (second hit). A large quantity of antibody that matches the recipient’s antigen can cause severe TRALI in a recipient with no predisposition.

Threshold model of antibody-mediated transfusion-related acute lung injury (TRALI). A specific threshold must be overcome to induce a TRALI reaction. To overcome a threshold, several factors act together: the activation status of the pulmonary neutrophils at the time of transfusion, the strength of the neutrophil-priming activity of transfused mediators (A), and the clinical status of the patient (B).

Panel 2: Clinical characteristics of transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO)

TRALI
• Dyspnea
• Fever
• Usually hypotension
• Hypoxia
• Leukopenia
• Thrombocytopenia
• Pulmonary edema on chest x-ray
• Normal left ventricular function*
• Normal pulmonary artery occlusion pressure

TACO
• Dyspnea
• Usually hypertension
• Hypoxia
• Pulmonary edema on chest radiographs
• Normal or decreased left ventricular function
• Increased pulmonary artery occlusion pressure
• Raised brain natriuretic peptide

Restrictive transfusion policy

The most effective prevention is a restrictive transfusion strategy. In a randomised clinical trial in critically ill patients, a restrictive transfusion policy for red blood cells was associated with a decrease in incidence of acute lung injury compared with a liberal strategy (7·7% vs 11·4%), suggesting that some of these patients might have had TRALI. The restrictive threshold was well tolerated and has greatly helped in guidance of red blood cell transfusion in the intensive-care unit.

Patient-tailored transfusion policy

Transfusion cannot be avoided altogether. A multivariate analysis in patients in intensive care showed that patient related risk factors contributed more to the onset of TRALI than did transfusion-related risk factors, suggesting that development of a TRALI reaction is dependent more on host factors then on factors in the blood product. Therefore, a patient-tailored approach aimed at reducing TRALI risk factors could be effective to alleviate the risk of TRALI.

Despite limitations of diagnostic tests, TRALI incidence seems to be high in at-risk patient populations. Therefore, TRALI is an underestimated health-care problem. Preventive measures, such as mainly male donor strategies, have been successful in reducing risk of TRALI. Identification of risk factors further improves the risk–benefit assessment of a blood transfusion. Efforts to further decrease the risk of TRALI needs increased awareness of this syndrome among physicians.

 

Transfusion-related acute lung injury: Current understanding and preventive strategies

A.P.J. Vlaar
Transfusion Clinique et Biologique 19 (2012) 117–124
http://dx.doi.org/10.1016/j.tracli.2012.03.001

Transfusion-related acute lung injury (TRALI) is the most serious complication of transfusion medicine. TRALI is defined as the onset of acute hypoxia within 6 hours of a blood transfusion in the absence of hydrostatic pulmonary edema. The past decades have resulted in a better understanding of the pathogenesis of this potentially life-threating syndrome. The present notion is that the onset of TRALI follows a threshold model in which both patient and transfusion factors are essential. The transfusion factors can be divided into immune and non-immune mediated TRALI. Immune-mediated TRALI is caused by the passive transfer of human neutrophil antibodies (HNA) or human leukocyte antibodies (HLA) present in the blood product reacting with a matching antigen in the recipient. Non-immune mediated TRALI is caused by the transfusion of stored cell-containing blood products. Although the mechanisms behind immune-mediated TRALI are reasonably well understood, this is not the case for non-immune mediated TRALI. The increased understanding of pathways involved in the onset of immune-mediated TRALI has led to the design of preventive strategies. Preventive strategies are aimed at reducing the risk to exposure of HLA and HNA to the recipient of the transfusion. These strategies include exclusion of “at risk” donors and pooling of high plasma volume products and have shown to reduce the TRALI incidence effectively.

Studies show that, in at risk patient populations, up to 8% of transfused patients may develop TRALI. Since the syndrome TRALI has been recognized, evidence on the pathogenesis of TRALI has been accumulating. The present notion is that the onset of TRALI follows a threshold model in which both patient and transfusion factors are essential in the development of TRALI. The transfusion factors can be divided into immune and non-immune mediated TRALI. Immune-mediated TRALI is caused by the passive transfer of human neutrophil antibodies (HNA) or human leukocyte antibodies (HLA) present in the blood product, reacting with a matching antigen in the recipient. Non-immune mediated TRALI is caused by the transfusion of stored cell-containing blood products. In recent years, many countries have successfully implemented preventive strategies resulting in a decrease of the incidence of TRALI.

Definition of transfusion-related acute lung injury (TRALI).

  • Acute onset within 6 hours after a blood transfusion
  • PaO2/FiO2 < 300 mmHg
  • Bilateral infiltrative changes on the chest X-ray
  • No sign of hydrostatic pulmonary edema (PAOP < 18 mmHg or CVP < 15 mmHg)
  • No other risk factor for acute lung injury present

Possible TRALI

  • Other risk factor for acute lung injury present

PAOP: pulmonary arterial occlusion pressure; CVP: central venous pressure

The first landmark report creating the basis for the understanding of the pathogenesis of TRALI was published by Popovsky et al. in 1983. They provided evidence on the association between the presence of leucocyte antibodies in the donor serum and onset of acute lung injury in the recipient of the transfusion. It was also recognized that multiparous blood donors whose plasma contained these antibodies represented a potential transfusion hazard. It was this research group that was the first to identify TRALI as a distinct clinical entity. Subsequently, many other authors reported on the association between the presence of HLA or HNA antibodies in donor blood and the onset of TRALI in the recipient.

Although the role of transfused blood donor HLA and HNA antibodies was widely accepted to be involved in the onset of TRALI, not all cases could be explained by this theory. A significant part of reported TRALI cases have no detectable antibodies. Also, many antibody-containing blood products fail to produce TRALI.

The alternative hypothesis proposed by the group of Silliman posed that TRALI is a “two hit” event. The “first hit” is the underlying condition of the patient, resulting in priming of the pulmonary neutrophil. The “second hit” is the transfusion of a blood product causing activation of the neutrophils in the pulmonary compartment, causing pulmonary edema finally resulting in TRALI. The transfusion factors causing the “second hit” are divided in two groups; immune and non-immune mediated TRALI.

The “second hit” is the transfusion itself and is either immune or non-immune mediated TRALI. The mechanisms behind immune-mediated TRALI are widely accepted and proven in both pre-clinical and clinical studies.  The mechanisms involved in non-immune mediated TRALI are less clear.

The role of stored cell-containing blood products in the onset of non-immune TRALI has extensively been studied in preclinical and clinical studies. Although most of the pre-clinical studies find a positive correlation between the transfusion of stored cell-containing blood products in the presence of a “first hit” and the onset of TRALI, the mechanism behind the onset is controversial.

TRALI management consists mainly of preventing future adverse reactions and providing proper incidence estimates. All suspected TRALI cases should be reported to the blood bank for immunologic work-up as it is impossible to distinguish immune-mediated TRALI from non-immune mediated TRALI at bedside. Immunologic work-up includes testing of incompatibility by cross-matching donor plasma against recipient’s leucocytes. A donor with antibodies which are incompatible with the patient is excluded from further donation of blood for transfusion products. Furthermore, it is important to stress that the absence of a positive serologic work-up does not exclude the diagnosis of TRALI. TRALI is a clinical diagnosis and the immunologic work-up can be supportive but is not part of the diagnosis of TRALI. the two-event hypothesis and threshold hypothesis do not exclude the role of antibodies in the occurrence of TRALI in the presence of an inflammatory condition. Thus any patient fulfilling the TRALI definition (including possible TRALI) should be reported to the blood bank for an immunologic work-up of the recipient and the implicated donors on the presence of HLA and HNA antibodies.

Prevention of immune-mediated TRALI is achieved by exclusion of donors proven to have HLA or HNA antibodies in their plasma present or donors “at risk” to have these antibodies present.

  1. Exclusion of HLA or HNA positive donors
  2. Exclusion of donors “at risk” of being HLA or HNA positive
    Female donors – more specifically, multiparous donors
  3. Testing donors for HLA or HNA antibodies
  4. Multiple plasma pooling
    solvent/detergent plasma is produced from multiple donations, leading to an at least 500-fold dilution of a single plasma unit;
    neither HNA nor HLA antibodies are detectable in solvent/detergent fresh frozen plasma.
  5. To prevent non-immune mediated TRALI, the use of fresh blood only has been suggested

Strategies to prevent the onset of TRALI include the exclusion of female plasma donors and the pooling of plasma products. These strategies have already been implemented in some countries resulting in a reduction of the incidence of TRALI.
Transfusion-related immunomodulation (TRIM): An update

Eleftherios C. Vamvakas, Morris A. Blajchman
Blood Reviews (2007) 21, 327–348
http://dx.doi.org:/10.1016/j.blre.2007.07.003

Allogeneic blood transfusion (ABT)-related immunomodulation (TRIM) encompasses the laboratory immune aberrations that occur after ABT and their established or purported clinical effects. TRIM is a real biologic phenomenon resulting in at least one established beneficial clinical effect in humans, but the existence of deleterious clinical TRIM effects has not yet been confirmed. Initially, TRIM encompassed effects attributable to ABT by immunomodulatory mechanisms (e.g., cancer recurrence, postoperative infection, or virus activation). More recently, TRIM has also included effects attributable to ABT by pro-inflammatory mechanisms (e.g., multiple-organ failure or mortality). TRIM effects may be mediated by: (1) allogeneic mononuclear cells; (2) white-blood-cell (WBC)-derived soluble mediators; and/or (3) soluble HLA peptides circulating in allogeneic plasma. This review categorizes the available randomized controlled trials based on the inference(s) that they permit about possible mediator(s) of TRIM, and examines the strength of the evidence available for relying on WBC reduction or autologous transfusion to prevent TRIM effects.

Allogeneic blood transfusion (ABT) may either cause alloimmunization or induce tolerance in recipients. ABTs introduce a multitude of foreign antigens into the recipient, including HLA-DR antigens found on the donor’s dendritic antigen presenting cells (APCs). The presence or absence of recipient HLA-DR antigens on the donor’s white blood cells (WBCs) plays a decisive role as to whether alloimmunization or immune suppression will ensue following ABT. In general, allogeneic transfusions sharing at least one HLA-DR antigen with the recipient induce tolerance, while fully HLA-DR-mismatched transfusions lead to alloimmunization.

In addition to the degree of HLA-DR compatibility between donor and recipient, the immunogenicity of cellular or soluble HLA antigens associated with transfused blood components depends on the viability of the donor dendritic APCs and the presence of co-stimulatory signals for the presentation of the donor antigens to the recipient’s T cells. Nonviable APCs and/or the absence of the requisite co-stimulatory signals result in T-cell unreponsiveness.  Thus, when a multitude of antigens is introduced into the host by an ABT, the host response to some of these antigens is often decreased, and immune tolerance ensues. ABT has been shown to cause decreased helper T-cell count, decreased helper/suppressor T-lymphocyte ratio, decreased lymphocyte response to mitogens, decreased natural killer (NK) cell function, reduction in delayed-type hypersensitivity, defective antigen presentation, suppression of lymphocyte blastogenesis, decreased cytokine (IL-2, interferon-c) production, decreased monocyte/macrophage phagocytic function, and increased production of antiidiotypic and anticlonotypic antibodies.

All these laboratory immune aberrations that indicate immune suppression and occur in transfused patients could potentially be associated with clinically-manifest ABT effects. Thus a variety of beneficial or deleterious clinical effects, potentially attributable to ABT-related immunosuppression, have been described over the last 30 years. The constellation of all such ABT-associated laboratory and clinical findings is known as ABT-related immunomodulation (TRIM). Initially, TRIM encompassed effects attributable to ABT by means of immunologic mechanisms only; however more recently, the term has been used more broadly, to encompass additional effects that could be related to ABT by means of ‘‘proinflammatory’’ rather than ‘‘immunomodulatory’’ mechanisms.

Over 30 years ago, it was reported that pre-transplant ABTs could improve renal-allograft survival in patients who had undergone renal transplantation.  This beneficial immunosuppressive effect of ABT has been confirmed by animal data, observational clinical studies, and clinical experience worldwide, although it has not been proven in randomized controlled trials (RCTs). Before the advent of the AIDS pandemic, it had become standard policy in many renal units to deliberately expose patients on transplant waiting lists to one or more red blood cell (RBC) transfusions.

All the available data considered together indicate that TRIM is most likely a real biologic phenomenon, which results in at least one established beneficial clinical effect in humans, although the available evidence has not yet confirmed  the existence and/or magnitude of the deleterious clinical TRIM effects. In fact, the debate over the existence of such deleterious clinical TRIM effects has been long and sometimes acrimonious.

Many studies tended to indicate that patients receiving perioperative transfusion (compared with those not needing transfusion) almost always had a higher risk of developing postoperative bacterial infection. The studies also indicated that patients receiving ABT differed from those not receiving a transfusion in several prognostic factors that predisposed to adverse clinical outcomes.

The specific constituent(s) of allogeneic blood that mediate(s) either or both the immunomodulatory and the pro-inflammatory effect(s) of ABT remain
(s) unknown, and the published literature suggests that these TRIM effects
may be mediated by: (1) allogeneic mononuclear cells; (2) soluble biologic response modifiers released in a time dependent manner from WBC granules or membranes into the supernatant fluid of RBC or platelet concentrates
during storage; and/or  (3) soluble HLA class I peptides that circulate in allogeneic plasma. If each of these mediators do cause TRIM effects, ABT effects mediated by allogeneic mononuclear cells would be expected to be preventable by WBC reduction (performed either before or after storage of cellular blood components), as well as by autologous transfusion. The ABT effects mediated by soluble HLA peptides circulating in allogeneic plasma would be expected to be preventable only by autologous transfusion.

BENEFICIAL TRIM EFFECTS

  1. Enhanced survival of renal allografts
  2. Reduced recurrence rate of Crohn’s disease

DELETERIOUS

  1. Increased recurrence rate of resected malignancies
  2. Increased incidence of postoperative bacterial infections
  3. Activation of endogenous CMV or HIV infection
  4. Increased short-term (up to 3-month) mortality

Possible mechanisms and mediators of TRIM effects

Although the mechanisms of TRIM have been debated extensively, the exact mechanism(s) of this phenomenon has yet to be elucidated. A number of putative mechanisms have been postulated. The three major mechanisms accounting for much of the experimental data include:

  • clonal deletion,
  • induction of anergy, and
  • immune suppression.

Conceptually, clonal deletion refers to the inactivation and removal of alloreactive lymphocytes that would, for example, cause the rejection of an allograft; anergy implies immunologic nonresponsiveness; and immune suppression suggests that the responding cell is being inhibited of doing so by a cellular mechanism or by a cytokine. Antiidiotypic antibodies, which are predominantly of the VH6 gene family, have also been demonstrated in the sera of ABT recipients and in patients with long-term functioning renal allografts.

To date, no RCT has enrolled patients with sarcomas—tumors whose growth is stimulated by TGF-β—or patients with tumors for which the immune response plays a major role. (These would include skin tumors—such as melanomas, keratoacanthomas, squamous and basal-cell carcinomas—and certain virus-induced tumors—notably Kaposi’s sarcoma and certain lymphomas.) Instead, the 3 available RCTs of ABT and cancer recurrence enrolled patients with colorectal cancer—a tumor that is not sufficiently antigenic to render an impairment of host immunity capable of facilitating tumor growth, and a tumor whose cells have not been shown to be stimulated by TGF-β.

Fig not shown. Randomized controlled trials (RCTs) investigating the association of WBC-containing allogeneic blood transfusion (ABT) with cancer recurrence. For each RCT, the figure shows the odds ratio (OR) of cancer recurrence in recipients of non-WBC-reduced allogeneic versus autologous or WBC-reduced allogeneic RBCs, as calculated from an intention-to-treat analysis. A deleterious effect of ABT (and thus a benefit from autologous transfusion or WBC reduction) exists when the OR is greater than 1 as well as statistically significant. (In the figure, each OR is surrounded by its 95% confidence interval [CI]; if the 95% CI of the OR includes the null value of 1, the TRIM effect is not statistically significant [p > 0.05]).

Fig not shown. Randomized controlled trials (RCTs) investigating the association of WBC-containing allogeneic blood transfusions with postoperative infection (n = 17). For each RCT, the figure shows the odds ratio (OR) of postoperative infection in recipients of non-WBC reduced allogeneic versus autologous or WBC-reduced allogeneic RBCs, as calculated from an intention-to-treat analysis. A deleterious effect of ABT (and thus a benefit from autologous transfusion or WBC reduction) exists when the OR is greater than 1 as well as statistically significant. (In the figure, each OR is surrounded by its 95% confidence interval [CI]; if the 95% CI of the OR includes the null value of 1, the TRIM effect is not statistically significant [p > 0.05]).

The totality of the evidence from RCTs does not demonstrate a TRIM effect manifest across all clinical settings and transfused RBC products. Instead, WBC-containing ABT is associated with an increased risk of short-term (up to 3-month post transfusion) mortality from all causes combined specifically in cardiac surgery. The additional deleterious TRIM effect detected by the latest meta-analysis (i.e., the effect on postoperative infection prevented by poststorage filtration) contradicts current theories about the pathogenesis of TRIM, because it is not accompanied by a similar or larger effect prevented by prestorage filtration.

Thus, only in cardiac surgery (Fig. 5 – not shown) are the findings of RCTs pertaining to a deleterious TRIM effect consistent. Even in this setting, however, the reasons for the excess deaths attributed to WBC containing ABT remain elusive. The initial hypothesis suggested that WBC-containing ABT may predispose to MOF which, in turn, may predispose to mortality. However, hitherto, no cardiac-surgery RCT has demonstrated an association between WBC-containing ABT and MOF, and no other cause of death specifically attributed to WBC-containing ABT has been proposed.

The TRIM effect seen in cardiac surgery deserves further study to pinpoint the cause(s) of the excess deaths, but-now that the majority of transfusions in Western Europe and North America are WBC reduced- the undertaking of further RCTs comparing recipients of non-WBC-reduced versus WBC reduced allogeneic RBCs in cardiac surgery is unlikely. For countries that have not yet converted to universal WBC reduction, whether to opt for WBC reduction of all cellular blood components transfused in cardiac surgery-in the absence of information on the specific cause(s) of death ascribed to WBC-containing ABT-is a policy decision that will have to be made based on the hitherto available data.

 

Regulation of alveolar fluid clearance and ENaC expression in lung by exogenous angiotensin II

Jia Denga, Dao-xin Wanga, Wang Deng, Chang-yi Li, Jin Tong, Hilary Ma
Respiratory Physiology & Neurobiology 181 (2012) 53– 61
http://dx.doi.org:/10.1016/j.resp.2011.11.009

Angiotensin II (Ang II) has been demonstrated as a pro-inflammatory effect in acute lung injury, but studies of the effect of Ang II on the formation of pulmonary edema and alveolar filling remains unclear. Therefore, in this study the regulation of alveolar fluid clearance (AFC) and the expression of epithelial sodium channel (ENaC) by exogenous Ang II was verified. SD rats were anesthetized and were given Ang II with increasing doses (1, 10 and 100 [1]g/kg per min) via osmotic minipumps, whereas control rats received only saline vehicle. AT1 receptor antagonist ZD7155 (10 mg/kg) and inhibitor of cAMP degeneration rolipram (1 mg/kg) were injected intraperitoneally 30 min before administration of Ang II. The lungs were isolated for measurement of alveolar fluid clearance. The mRNA and protein expression of ENaC were detected by RT-PCR and Western blot. Exposure to higher doses of Ang II reduced AFC in a dose-dependent manner and resulted in a non-coordinate regulation of α-ENaC vs the regulation of β- and ϒ-ENaC, however Ang II type 1 (AT1) receptor antagonist ZD7155 prevented the Ang II-induced inhibition of fluid clearance and dysregulation of ENaC expression. In addition, exposure to inhibitor of cAMP degradation rolipram blunted the Ang II-induced inhibition of fluid clearance. These results indicate that through activation of AT1 receptor, exogenous Ang II promotes pulmonary edema and alveolar filling by inhibition of alveolar fluid clearance via downregulation of cAMP level and dysregulation of ENaC expression.

Effects of angiotensin II (Ang II) receptor antagonists and rolipram  on AFC

Effects of angiotensin II (Ang II) receptor antagonists and rolipram on AFC

Effects of angiotensin II (Ang II) receptor antagonists and rolipram on rat alveolar fluid clearance (AFC). Then AFC was measured 1 h after fluid instillation (4 mL/kg). Amiloride (100 [1]M), Ang II (10−7 M), ZD7155 (10−6 M), and rolipram (10−5 M) were added to the instillate as indicated (n = 10 per group). Mean values ± SEM. p < 0.01 vs control. p < 0.01 vs Ang II + ZD7155.
p < 0.05 vs amiloride. p < 0.05 vs Ang II.

Effects of angiotensin II (Ang II) on cyclic adenosine monophosphate (cAMP)

Effects of angiotensin II (Ang II) on cyclic adenosine monophosphate (cAMP)

Effects of angiotensin II (Ang II) on cyclic adenosine monophosphate (cAMP) concentration in lung. Rats were given saline or Ang II (1, 10 and 100 µg/kg per min) for 6 h, and cAMP in lung was determined by RIA (n = 30 per group). Mean values ± SEM. p < 0.01 vs control. p < 0.05 vs 10 µg/kg Ang II.

Histological examination of lung

Histological examination of lung

Histological examination of lung. Rats were given saline or Ang II (10 µg/kg per min) by osmotic minipump for 6 h. ZD7155 (10 mg/kg) was injected intraperitoneally 30 min before administration of Ang II. Shown are representative lung specimens obtained from the control (A), Ang II (B) and Ang II + ZD7155 (C) groups. All photographs are at 100× magnification. Interstitial edema and inflammatory cell infiltration were seen in Ang II group, but reduced in Ang II + ZD7155 group.
The present results demonstrate that Ang II infusion is associated with pulmonary edema and alveolar filling. Three important findings were observed:

(1) high doses of Ang II led to reduction of alveolar fluid clearance, and this effect was blunted by an AT1 receptor antagonist.
(2) Ang II infusion increased the abundance of α-ENaC, whereas decreased the abundance ofβ and ϒ-ENaC, and these effects were reversed in response to an AT1 receptor antagonist.
(3) Ang II infusion decreased cAMP concentration in lung tissue, and an inhibitor of cAMP degradation prevented inhibition of alveolar fluid clearance by Ang II, but had no effect on the dysregulation of ENaC.

Our data indicate that Ang II results in pulmonary edema by inhibition of alveolar fluid clearance via down-regulation of cellular cAMP level and dysregulation of the abundance of ENaC, whereas these effects are prevented by an AT1 receptor antagonist.

The renin-angiotensin system is a major regulator of body fluid and sodium balance, predominantly through the actions of its main effector Ang II. Several previous experimental studies demonstrated that plasma Ang II levels vary in both physiological and pathological conditions. In the kidney, Ang II added to the peritubular perfusion has a biphasic action with stimulation of sodium reabsorption at low doses (10−12–10−10M) and inhibition at high doses (10−7–10−6M) (Harris and Young, 1977). In vitro, Ang II also exerts a dose-dependent dual action on intestinal absorption (Levens, 1985). The evidence shows that the effect of Ang II on sodium and water absorption is dose-dependent. Our results showed that low intravenous doses of Ang II (<1 µg/kg per min) had no effect on alveolar fluid clearance which represents the sodium and water reabsorption in alveoli. However, with high intravenous doses, Ang II decreased alveolar fluid clearance. This finding suggests that the effect of Ang II on fluid absorption in lung is also dose-dependent.

 

Rat models of acute lung injury: Exhaled nitric oxide as a sensitive,noninvasive real-time biomarker of prognosis and efficacy of intervention

Fangfang Liu, Wenli Lib, Jürgen Pauluhn, Hubert Trübel, Chen Wang
Toxicology 310 (2013) 104– 114
http://dx.doi.org/10.1016/j.tox.2013.05.016

Exhaled nitric oxide (eNO) has received increased attention in clinical settings because this technique is easy to use with instant readout. However, despite the simplicity of eNO in humans, this endpoint has not frequently been used in experimental rat models of septic (endotoxemia) or irritant acute lung injury (ALI). The focus of this study is to adapt this method to rats for studying ALI-related lung disease and whether it can serve as instant, non-invasive biomarker of ALI to study lung toxicity and pharmacological efficacy. Measurements were made in a dynamic flow of sheath air containing the exhaled breath from spontaneously breathing, conscious rats placed into a head-out volume plethysmograph. The quantity of eNO in exhaled breath was adjusted (normalized) to the physiological variables (breathing frequency, concentration of exhaled carbon dioxide) mirroring pulmonary perfusion and ventilation. eNO was examined on the instillation/inhalation exposure day and first post-exposure day in Wistar rats intratracheally instilled with lipopolysaccharide (LPS) or single inhalation exposure to chlorine or phosgene gas. eNO was also examined in a Brown Norway rat asthma model using the asthmagen toluene diisocyanate (TDI). The diagnostic sensitivity of adjusted eNO was superior to the measurements not accounting forthe normalization of physiological variables. In all bioassays – whether septic, airway or alveolar irritant or allergic, the adjusted eNO was significantly increased when compared to the concurrent control. The maximum increase of the adjusted eNO occurred following exposure to the airway irritant chlorine. The specificity of adjustment was experimentally verified by decreased eNO following inhalation dosing ofthe non-selective nitric oxide synthase inhibitor amoni-guanidine. In summary, the diagnostic sensitivity of eNO can readily be applied to spontaneously breathing, conscious rats without any intervention or anesthesia. Measurements are definitely improved by accounting for the disease-related changes inexhaled CO2and breathing frequency. Accordingly, adjusted eNO appears to be a promising methodological improvement for utilizing eNO in inhalation toxicology and pharmacological disease models
with fewer animals.

 

Role of p38 MAP Kinase in the Development of Acute Lung Injury

J Arcaroli, Ho-Kee Yum, J Kupfner, JS Park, Kuang-Yao Yang, and E Abraham
Clinical Immunology 2001; 101(2):211–219
http://dx.doi.org:/10.1006/clim.2001.5108

Acute lung injury (ALI) is characterized by an intense pulmonary inflammatory response, in which neutrophils play a central role. The p38 mitogen-activated protein kinase pathway is involved in the regulation of stress-induced cellular functions and appears to be important in modulating neutrophil activation, particularly in response to endotoxin. Although p38 has potent effects on neutrophil functions under in vitro conditions, there is relatively little information concerning the role of p38 in affecting neutrophil driven inflammatory responses in vivo. To examine this issue, we treated mice with the p38 inhibitor SB203580 and then examined parameters of neutrophil activation and acute lung injury after hemorrhage or endotoxemia. Although p38 was activated in lung neutrophils after hemorrhage or endotoxemia, inhibition of p38 did not decrease neutrophil accumulation in the lungs or the development of lung edema under these conditions. Similarly, the increased production of proinflammatory cytokines and activation of NF-kB in lung neutrophils induced by hemorrhage or endotoxemia was not diminished by p38 inhibition. These results indicate that p38 does not have a central role
in the development of ALI after either hemorrhage or endotoxemia.

 

The coagulation system and pulmonary endothelial function in acute lung injury

James H. Finigan
Microvascular Research 77 (2009) 35–38
http://dx.doi.org:/10.1016/j.mvr.2008.09.002

Acute lung injury (ALI) is a disease marked by diffuse endothelial injury and increased capillary permeability. The coagulation system is a major participant in ALI and activation of coagulation is both a consequence and contributor to ongoing lung injury. Increased coagulation and depressed fibrinolysis result in diffuse alveolar fibrin deposition which serves to amplify pulmonary inflammation. In addition, existing evidence demonstrates a direct role for different components of coagulation on vascular endothelial barrier function. In particular, the pro-coagulant protein thrombin disrupts the endothelial actin cytoskeleton resulting in increased endothelial leak. In contrast, the anti-coagulant activated protein C (APC) confers a barrier protective actin configuration and enhances the vascular barrier in vitro and in vivo. However, recent studies suggest a complex landscape with receptor cross-talk, temporal heterogeneity and pro-coagulant/anticoagulant protein interactions. In this article, the major signaling pathways governing endothelial permeability in lung injury are reviewed with a particular focus on the role that endothelial proteins, such as thrombin and APC, which play on the vascular barrier function.

Acute lung injury (ALI) is a devastating illness with an annual incidence of approximately 200,000 and a mortality of 40%. Most commonly seen in the setting of sepsis, ALI is a complex inflammatory syndrome marked by increased vascular permeability resulting in tissue edema and organ dysfunction. The vascular endothelium is a key target and critical participant in the pathogenesis of sepsis-induced organ dysfunction and disruption of the endothelial barrier is central to the pathophysiology of both sepsis and ALI. Sepsis and acute lung injury (ALI) are syndromes marked by diffuse inflammation with a key feature being endothelial cell barrier disruption and increased vascular permeability resulting in widespread organ dysfunction. The endothelial cytoskeleton has been identified as a critical regulator of vascular barrier integrity with a current model of endothelial barrier regulation suggesting a balance between barrier-disrupting cellular contractile forces and barrier-protective cell–cell and cell–matrix forces. These competing forces exert their opposing effects via manipulation of the actin-based endothelial cytoskeleton and associated endothelial regulatory proteins. Endothelial cells generate tension via an actomyosin motor, and focally distributed changes in tension/relaxation can be accomplished by spatially-defined regulation of the phosphorylation of the regulatory 20 kDa myosin light chain (MLC) catalyzed by the Ca2+/calmodulin (CaM)-dependent enzyme myosin light chain kinase (MLCK).

Thrombin is the proto-typical coagulation protein with direct effects on the endothelial barrier via alterations in the cytoskeleton. In the coagulation cascade, thrombin converts fibrinogen to fibrin in the final step of thrombus formation and also activated platelets. In addition, this multifunctional protease is present at sites of vascular inflammation and induces barrier dysfunction. Through its receptor, protease-activated receptor-1 (PAR1), thrombin initiates a series of events which includes MLC phosphorylation, dramatic cytoskeletal reorganization and stress fiber formation, increased cellular contractility, paracellular gap formation, and enhanced fluid and protein transport. Similarly, thrombin exposure results in increased pulmonary edema in vivo, a finding which is also seen after treatment with a PAR1 activating peptide and attenuated in PAR1 knockout mice.

Disruptions in the coagulation system have long been recognized to be an integral part of inflammation, sepsis and ALI. In 1969, Saldeen demonstrated that thrombin infusion produced canine respiratory insufficiency which was linked pathologically to emboli in the pulmonary microcirculation, a condition he labeled the “Microembolism Syndrome” (Saldeen, 1979). Elemental to the pathophysiology of sepsis and ALI is a shift towards a pro-coagulant state. Bronchoalveolar (BAL) fluid from patients with ALI reflects this increase in procoagulant activity with elevated levels of fibrinopeptide A, factor VII and d-dimer. Concomitantly, there is a decrease in fibrinolytic activity, as shown by depressed BAL levels of urokinase and increased levels of the fibrinolysis inhibitors plasminogen activator inhibitor (PAI) and α2-antiplasmin.

Given that APC is a vascular endothelial protein which interacts with other coagulation proteins such as thrombin, it seems logical that it might have an effect on endothelial integrity. In cultured human pulmonary endothelial cells, while thrombin results in decreased electrical resistance, a reflection of increased permeability, pre- or post-exposure to physiologic concentrations of APC significantly attenuates this thrombin-induced drop in resistance. These APC-mediated alterations in barrier function are associated with MLC phosphorylation as well as activation of the endothelial protein Rac, and cytoskeletal re-arrangement in a barrier protective configuration all findings very reminiscent of the barrier protective signaling induced by the bioactive lipid, S1P. Interestingly, APC appears to activate sphingosine kinase and mediate its barrier protective effects through PI3 kinase and AKT-dependent ligation of the S1P receptor, S1P1. Moreover, the endothelial barrier-protective effects of APC have been observed in other tissues including brain and kidney. The barrier protection in these beds appears independent of any anti-coagulant effect of APC and is associated with decreased endothelial apoptosis.

Recently, the endothelial protein C receptor (EPCR) has been identified as a crucial participant in the protein C pathway. Structurally similar to the major histocompatibility class I/CD1 family of molecules, EPCR binds protein C, presenting it to the thrombin/TM complex, thereby increasing the activation of protein C by ∼20 fold. Importantly, APC can also bind EPCR, and while the bound form of APC loses its extra-cellular anti-coagulant activity, increasing evidence indicates that much, if not all, of APC intra-cellular signaling requires EPCR. APC-mediated increases in endothelial phosphor-MLC and activated Rac are all EPCR-dependent and APC-induced endothelial barrier protection requires ligation of EPCR.

Sepsis and ALI are significant causes of morbidity and mortality in the intensive care unit and are marked by zealous activation of the coagulation system. While this could conceivably confer certain benefits, such as enclosing and spatially controlling an infection, it is clear that this pro-coagulant environment participates in the pathophysiology of ALI, particularly via exacerbating endothelial damage and augmenting endothelial permeability. However, the biology of coagulation in ALI is incompletely understood and trials of new therapies specifically targeting coagulation in patients with ALI have been disappointing. Despite this, recent advances in the knowledge of the dynamic interplay between inflammation and coagulation in ALI as well as endothelial receptor-ligand binding and receptor cross talk have stimulated promising research and identified novel therapeutic targets for patients with ALI.

 

Phosphatidylserine-expressing cell by-products in transfusion: A pro-inflammatory or an anti-inflammatory effect?

  1. Saas, F. Angelot, L. Bardiaux, E. Seilles, F. Garnache-Ottou, S. Perruche
    Transfusion Clinique et Biologique 19 (2012) 90–97
    http://dx.doi.org/10.1016/j.tracli.2012.02.002

Labile blood products contain phosphatidylserine-expressing cell dusts, including apoptotic cells and microparticles. These cell by-products are produced during blood product process or storage and derived from the cells of interest that exert a therapeutic effect (red blood cells or platelets). Alternatively, phosphatidylserine-expressing cell dusts may also derived from contaminating cells, such as leukocytes, or may be already present in plasma, such as platelet-derived microparticles. These cell by-products present in labile blood products can be responsible for transfusion induced immunomodulation leading to either transfusion-related acute lung injury (TRALI) or increased occurrence of post-transfusion infections or cancer relapse. In this review, we report data from the literature and our laboratory dealing with interactions between antigen-presenting cells and phosphatidylserine-expressing cell dusts, including apoptotic leukocytes and blood cell-derived microparticles. Then, we discuss how these phosphatidylserine-expressing cell by-products may influence transfusion.

Potential consequences of phosphatidylserine-expressing cell by-products in transfusion

Potential consequences of phosphatidylserine-expressing cell by-products in transfusion

Potential consequences of phosphatidylserine-expressing cell by-products in transfusion. Interactions of phosphatidylserine-expressing cell dusts (apoptotic cells or microparticles) may lead to antigen-presenting cell activation or inhibition. Antigen-presenting cell activation may trigger inflammation and be involved in transfusion-related acute lung injury (TRALI), while antigen-presenting cell inhibition may exert transient immunosuppression or tolerance. Blood product process or storage may influence the generation of phosphatidylserine-expressing cell dusts. PtdSer: phosphatidylserine; APC: antigen-presenting cell.

Several publications report the presence of phosphatidylserine-expressing cell by-products in blood products. These cell by-products may be generated during the blood product process, such as filtration, or during storage (either cold storage for red blood cells or between 20–24 ◦C for platelets). Alternatively, they may be limited by filtration. Phosphatidylserine-expressing cell by-products can be apoptotic cells. Apoptotic cells have been found in different blood products: red blood cell units and platelet concentrates. These apoptotic cells correspond to dying cells of interest: red blood cells or platelets, both enucleated cells that can undergo apoptosis.

Immunomodulatory effects of apoptotic leukocytes

Immunomodulatory effects of apoptotic leukocytes

Immunomodulatory effects of apoptotic leukocytes. Early during the apoptotic program, phosphatidylserine-exposure occurs leading to apoptotic cell removal by macrophages or conventional dendritic cells. This uptake by antigen-presenting cells induces the production of anti-inflammatory factors and concomitantly inhibits the synthesis of inflammatory cytokines. These antigen-presenting cells are refractory to TLR activation. This leads to a transient immunosuppressive microenvironment. If antigen-presenting cells from this microenvironment migrate to secondary lymphoid organs, naive T cells are converted into inducible regulatory T cells. This leads to tolerance against apoptotic cell-derived antigens. M[1]: macrophage; cDC: conventional dendritic cells; PtdSer: phosphatidylserine; Treg: regulatory T cells; Th1: helper T cells; HGF: hepatocyte growth factor; IL-: interleukin; NO: nitrite oxide; PGE-2: prostaglandin-E2; TGF: transforming growth factor; TNF: tumor necrosis factor; TLR: Toll-like receptor.

Implication of phosphatidylserine in the inhibition of both inflammation and specific immune responses has been further demonstrated using  phosphatidylserine-expressing liposomes and is sustained by the following observations:

  • phosphatidylserine-dependent ingestion of apoptotic cells induces TGF-β secretion and resolution of lung inflammation;
  • inhibition of phosphatidylserine recognition through annexin-V enhances the immunogenicity of irradiated tumor cells in vivo;
  • masking of phosphatidylserine inhibits apoptotic cell engulfment and induces autoantibody production in mice.

Based on data from our group and Peter Henson’s group, some authors have speculated that apoptotic leukocytes present in blood products may be responsible for transfusion-related immunosuppression.

The first consequences of phosphatidylserine-expressing apoptotic cells in blood products may be a transient immunosuppression−responsible for an increase in infection rate and of cancer relapse−or tolerance induction− as observed after donor-specific transfusion − when Treg have been generated. However, apoptotic leukocytes become secondarily necrotic in the absence of phagocytes. This may certainly occur in blood product bags. Necrotic cells, through the release of damage-associated molecular patterns, may become immunogenic. The same process may occur for platelets. Necrotic platelets may represent the procoagulant form of platelets. Thus, hemostatic activation of platelets or their by-products may link thrombosis and inflammation to amplify lung microvascular damage during nonimmune TRALI.

What are the next steps to answer the question on the role of phosphatidylserine-expressing cell dusts in the modulation of immune responses after transfusion?

The next steps are to characterize or identify factors involved in the triggering of inflammation or its inhibition and produced during blood product storage or process. Several factors influence the immune responses against dying cells. We can speculate on some factors, including:

  • the number of phosphatidylserine-expressing cell byproducts contained per blood product, as the immunogenicity of apoptotic cells may be proportional to their number;
  • the occurrence of secondary necrosis and so the passive release of intracellular damage-associated molecular patterns that overpasses the inhibitory signals delivered by phosphatidylserine. One of these damage associated molecular patterns can be the heme released from stored red blood cells which signals via TLR4;
  • the size of cell by-products and especially microparticles, since these latter exert different functions according to their size. Moreover, antigen-presenting cells, such as plasmacytoid dendritic cells, respond only to lower size synthetic particles. This may explain the different responses observed between “amateur” phagocytes (plasmacytoid dendritic cells) versus professional phagocytes (conventional dendritic cells/macrophages) after incubation with microparticles. The size of cell by-products diminishes during plasma filtration, as assessed by dynamic light scattering from 101 to 464 nm in unfiltered fresh-frozen plasma versus 21 to 182 nm after 0.2 µm filtration process;
  • expression of the recently described phosphatidylserine receptors on different antigen-presenting cell subsets may also explain the different responses between plasmacytoid dendritic cells versus conventional dendritic cells/macrophages and may impact on the overall immune response.

 

Peroxisome proliferator-activated receptors and inflammation

Leonardo A. Moraes, Laura Piqueras, David Bishop-Bailey
Pharmacology & Therapeutics 110 (2006) 371 – 385
http://dx.doi.org:/10.1016/j.pharmthera.2005.08.007

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptors family. PPARs are a family of 3 ligand-activated transcription factors: PPARa (NR1C1), PPARh/y (NUC1; NR1C2), and PPARg (NR1C3). PPARα, -h/y, and -ϒ are encoded by different genes but show substantial amino acid similarity, especially within the DNA and ligand binding domains. All PPARs act as heterodimers with the 9-cis-retinoic acid receptors (retinoid X receptor; RXRs) and play important roles in the regulation of metabolic pathways, including those of lipid of biosynthesis and glucose metabolism, as well as in a variety of cell differentiation, proliferation, and apoptosis pathways. Recently, there has been a great deal of interest in the involvement of PPARs in inflammatory processes. PPAR ligands, in particular those of PPARα and PPARϒ, inhibit the activation of inflammatory gene expression and can negatively interfere with proinflammatory transcription factor signaling pathways in vascular and inflammatory cells. Furthermore, PPAR levels are differentially regulated in a variety of inflammatory disorders in man, where ligands appear to be promising new therapies.

Fig. not shown.  Structure and transcriptional activation of PPARs. (A) Generic schematic of the structure of the PPAR family of nuclear receptors. Indicated are the N–C terminal regions subdivided in to 4 domains: the A/B, N terminal domain [also called the activation function (AF)-1 domain]; C, the DNA binding domain; D, the F hinge_region; and E, the ligand binding domain (AF-2). (B) Generic scheme for the activation of a PPAR receptor as a transcription factor. PPAR activation leads to heterodimerization with RXR and an accumulation in the nucleus. Ligand activation of PPAR results in a change from a repressed binding protein complex which may contain histone deacetylases (HDAC), the nuclear receptor corepressor (NCo-R), and the silencing mediator of retinoid and thyroid signaling (SMRT) to an activation complex that may contain the histone acetylases, steroid receptor co-activator-1 (SRC-1), the PPAR binding protein (PBP), cAMP response element binding protein (CBP/p300), TATA box binding proteins, and RNA polymerase (RNA pol) III. The activated PPAR–RXR heterodimer complex binds to DNA sequences called PPAR response elements (PPRE) in target genes initiation their transcription.

Although the nature of true endogenous PPAR ligands are still not known (Bishop-Bailey & Wray, 2003), PPARs can be activated by a wide variety of F endogenous or pharmacological ligands. PPARα activators include a variety of endogenously present fatty acids, LTB4 and hydroxyeicosatetraenoic acids (HETEs), and clinically used drugs, such as the fibrates, a class of first-line drugs in the treatment of dyslipidemia. Similarly, PPARg can be activated by a number of ligands, including docosahexaenoic acid, linoleic acid, the anti-diabetic glitazones, used as insulin sensitizers, and a number of lipids, including oxidized LDL, azoyle-PAF, and eicosanoids, such as 5,8,11,14-eicosatetraynoic acid and the prostanoids PGA1, PGA2, PGD2, and its dehydration products of the PGJ series of cyclopentanones (e.g., 15 deoxy-D12,14-PGJ2). Dyslipidemia and insulin-dependent diabetes are commonly found existing together as part of the metabolic X syndrome.

Because PPARa and PPARg ligands independently are useful clinical drugs in the treatment of these respective disorders, synthetic dual PPARα/ϒ ligands have recently been developed and show a combined clinical efficacy. PPAR h/y activators include fatty acids and prostacyclin and synthetic compounds L-165,041, GW501516, compound F and L-783,483. Unlike PPARα or-ϒ, there are no PPAR h/y drugs in the clinic, although ligands are in phase II clinical trials for dyslipidemia (http://www.science.gsk.com/pipeline). Indeed, part of the challenge in determining the function of PPARh/y has been the identification and availability of new ligands with more potency and selectivity for use as pharmacological tools.

Fig. not shown. Mechanisms of the anti-inflammatory effects of PPARα. PPARα ligands inhibit the activities of NF-nB, AP-1, and T-bet within cells. In sites of local inflammation, tissue and endothelial cell activity is inhibited, and expressions of adhesion molecules (ICAM-1 and VCAM-1), pro-inflammatory cytokines (IL-1, -6, -8, -12, and TNFα), vasoactive mediators (inducible cyclo-oxygenase, inducible nitric oxide synthase, and endothelin-1; COX-2, iNOS, and ET-1), and proteases (MMP-9) are decreased. The inflammatory responses in leukocytes are also diminished. Monocyte/macrophage activity is decreased, and lipid metabolizing pathways increased, T- and B-lymphocyte proliferation and differentiation are inhibited, and T-lymphocyte and eosinophil chemotaxis reduced. Bold italic text indicates positive regulation by the PPAR, all other text indicates a negative regulation.

Fig. not shown. Mechanisms of the anti-inflammatory effects of PPAR h/y. PPAR h/y ligands inhibit the activities of NF-nB and release the suppressor BCL-6 from PPAR h/y. In sites of local inflammation, endothelial cell adhesion molecule (VCAM-1) and chemokine (MCP-1) are reduced. PPAR h/y and its endogenous ligand(s) are induced during the inflammatory response in keratinocytes, which then promotes cell survival (integrin-linked kinase—Akt pathway) and wound healing. The inflammatory responses in monocyte/ macrophages are modulated. In the absence of ligand, PPAR h/y sequesters BCL-6 and induces MCP-1, MCP-3, and IL-1h. When PPAR h/y ligand is given, BCL-6 is released and MCP-1, -3, and IL-1h levels are reduced. Bold italic text indicates positive regulation by the PPAR, all other text indicates a negative regulation.

Fig. not shown. Mechanisms of the anti-inflammatory effects of PPARg. PPARg ligands can inhibit the activities of NF-nB, AP-1, STAT-1, N-FAT, Erg-1, Jun, and GATA-3 within cells. In sites of local inflammation, tissue and endothelial cell activity is inhibited, and expression of adhesion molecules (ICAM-1), proinflammatory cytokines (IL-8, -12, and TNFα), chemokines (MCP-1, MCP-3, IP-10, Mig, and I-TAC), vasoactive mediators (inducible nitric oxide synthase and endothelin-1; iNOS and ET-1), and proteases (MMP-9) are decreased. The inflammatory responses in leukocytes are also diminished. Monocyte/ macrophage activity is decreased, T- and B-lymphocyte proliferation and differentiation are inhibited, and T-lymphocyte and eosinophil chemotaxis reduced. Platelet activity is inhibited and dendritic cell production of IL-12, and expression of CCL3, CCL5, and CD80 is reduced, so pro-inflammatory TH1 lymphocytes maturation is inhibited. Bold italic text indicates positive regulation by the PPAR, all other text indicates a negative regulation.

The PPARs are one of the most intensely studied members of the nuclear receptor gene family, and since their initial discovery just over decade ago, the PPARs have attracted an increasing amount of experimental and clinical research by investigators from different scientific areas. PPARs through their central roles in regulating energy homeostasis regulate physiological function in many cell types, tissues, and organ systems. Many disease states from carcinogenesis to inflammation have been linked to abnormalities in the function of PPAR-regulated transcription factors. PPARs are expressed or regulate pathophysiology of diverse human disorders including atherosclerosis, inflammation, obesity, diabetes, and the immune response. PPARs have beneficial effects in many inflammatory conditions, where they regulate cytokine production, adhesion molecule expression, fibrinolysis cell proliferation, apoptosis, and differentiation. Further studies and development of novel PPAR ligands and their selective modulators may lead to novel therapeutic agents in the many conditions associated with inflammatory processes.

 

Regulators of endothelial and epithelial barrier integrity and function in acute lung injury

Rudolf Lucas, Alexander D. Verin, Stephen M. Black, John D. Catravas
Biochemical Pharmacology 77 (2009) 1763–1772
http://dx.doi.org:/10.1016/j.bcp.2009.01.014

Pulmonary permeability edema is a major complication of acute lung injury (ALI), severe pneumonia and ARDS. This pathology can be accompanied by

(1) a reduction of alveolar liquid clearance capacity, caused by an inhibition of the expression of crucial sodium transporters, such as the epithelial sodium channel (ENaC) and the Na+-K+-ATPase,
(2) an epithelial and endothelial hyperpermeability and
(3) a disruption of the epithelial and endothelial barriers, caused by increased apoptosis or necrosis.

Since, apart from ventilation strategies, no standard treatment exists for permeability edema, the following chapters will review a selection of novel approaches aiming to improve these parameters in the capillary endothelium and the alveolar epithelium.

Apoptosis is an essential physiological process for the selective elimination of cells. However, the dysregulation of apoptotic pathways is thought to play an important role in the pathogenesis of ALI. Both delayed neutrophil apoptosis and enhanced endothelial/epithelial cell apoptosis have been identified in ALI/ARDS. In the case of neutrophils, which contribute significantly to ALI/ ARDS, studies in both animals and ARDS patients suggest that apoptosis is inhibited during the early stages (<2 h) of inflammation.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, that includes receptors for steroid hormones, thyroid hormones, retinoic acid, and fat-soluble vitamins. Since their discovery in 1990, increasing data has been published on the role of PPARs in diverse processes, including lipid and glucose metabolism, diabetes and obesity, atherosclerosis, cellular proliferation and differentiation, neurological diseases, inflammation and immunity. PPARs have both gene-dependent and gene-independent effects. Gene-dependent functions involve the formation of heterodimers with the retinoid X-receptor. Activation by PPAR ligands results in the binding of the heterodimer to peroxisome proliferator response elements, located in the promoter regions of PPAR-regulated genes. Gene independent effects involve the direct binding of PPARs to transcription factors, such as NF-kB, which then alters their binding to DNA promoter elements. PPARs can also bind and sequester various cofactors for transcription factors, and thus further alter gene expression. Importantly, the precise effects of PPARs vary greatly between cell types. To date, three subtypes of PPAR have been identified: α, β, and ϒ. There is increasing data suggesting that PPAR signaling may play an important role in the pathobiology of systemic vascular disease. However, there is less data implicating PPAR signaling in diseases of the lung.

A role for PPARs in the control of inflammation was first evidenced for PPARα, where mice deficient in PPARα exhibited an increased duration of ear-swelling in response to the proinflammatory mediator, LTB4. More recently, a number of studies in mice and in humans have shown that PPAR agonists exhibit anti-inflammatory effects under a wide range of conditions. There are two main mechanisms by which PPARs exert their anti-inflammatory effect. The first involves complex formation, and the inhibition of transcription factors that positively regulate the transcription of pro-inflammatory genes. These include nuclear factor-kB (NF-kB), signal transducers and activators of transcription (STATs), nuclear factor of activated T cells (NF-AT), CAAT/enhancer binding protein (C/EBP) and activator protein 1 (AP-1). These transcription factors are the main mediators of the major proinflammatory cytokines, chemokines, and adhesion molecules involved in inflammation. The second PPAR-mediated anti-inflammatory pathway is mediated by the sequestration of rate limiting, but essential, co-activators or co-repressors.

Recent studies have shown that PPAR signaling can attenuate the airway inflammation induced by LPS in the mouse. It was shown that mice treated with the PPARα agonist, fenofibrate, had decreases in both inflammatory cell infiltration and inflammatory mediators. Conversely, PPARα -/- mice have been shown to have a greater number of neutrophils and macrophages, and increased levels of inflammatory mediators in bronchoalveolar lavage fluids (BALF). Other PPAR agonists, such as rosiglitazone or SB 21994 have also been shown to reduce LPS-mediated ALI in the mouse lung. PPARϒ signaling has also been shown to be protective in regulating pulmonary inflammation associated with fluorescein isothiocyanate (FITC)-induced lung injury, with the PPARϒ ligand pioglitazone decreasing neutrophil infiltration. Collectively, these data suggest that therapeutic agents that activate either or both PPARα and PPARϒ could be beneficial for the treatment of ALI.

Permeability edema is characterized by a reduced alveolar liquid clearance capacity, combined with an endothelial hyperpermeability. Various signaling pathways, such as those involving reactive oxygen species (ROS), Rho GTPases and tyrosine phosphorylation of junctional proteins, converge to regulate junctional permeability, either by affecting the stability of junctional proteins or by modulating their interactions. The regulation of junctional permeability is mainly mediated by dynamic interactions between the proteins of the adherens junctions and the actin cytoskeleton. Actin-mediated endothelial cell contraction is the result of myosin light chain (MLC) phosphorylation by MLC kinase (MLCK) in a Ca2+/calmodulin-dependent manner. RhoA additionally potentiates MLC phosphorylation, by inhibiting MLC phosphatase activity through its downstream effector Rho kinase (ROCK). As such, actin/myosin-driven contraction will generate a contractile force that pulls VE-cadherin inward. This contraction will force VE-cadherin to dissociate from its adjacent partner, as such producing interendothelial gaps.

Vascular endothelial cells can be regulated by nucleotides released from platelets. During vascular injury, broken cells are also the source of the extracellular nucleotides. Furthermore, endothelium may provide a local source of ATP within vascular beds. Primary cultures of human endothelial cells derived from multiple blood vessels release ATP constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists (ionomycin and thapsigargin) stimulate ATP release in a reversible and regulated manner. Enhanced release of pharmacologically relevant amounts of ATP was observed in endothelial cells under such stimuli as shear stress, lipopolysaccharide (LPS), and ATP itself. Pearson and Gordon demonstrated that incubation of aortic endothelial and smooth muscle cells with thrombin resulted in the specific release of ATP, which was converted to ADP by vascular hydrolases. Yang et al. showed that endothelial cells isolated from guinea pig heart release nucleotides in response to bradykinin, acetylcholine, serotonin and ADP. Nucleotide action is mediated by cell surface purinoreceptors. Once released from endothelial cells, ATP may act in the blood vessel lumen at P2 receptors on nearby endothelium downstream from the site of release. ATP is also degraded rapidly and its metabolites have also been recognized as signaling molecules, which can initiate additional receptor-mediated functions. These include ADP and the final hydrolysis product adenosine.

Signal transduction pathways implicated in ATP-mediated endothelial barrier enhancement

Signal transduction pathways implicated in ATP-mediated endothelial barrier enhancement

Signal transduction pathways implicated in ATP-mediated endothelial barrier enhancement

During the course of ALI, the alveolar space, as well as the interstitium, are sites of intense inflammation, leading to the local production of pro-inflammatory cytokines, such as IL-1β, TGF-β and TNF. The latter pleiotropic cytokine is a 51 kDa homotrimeric protein, binding to two types of receptors, i.e. TNF-R1 and TNF-R2 and which is mainly produced by activated macrophages and T cells. Soluble TNF, as well as the soluble TNF receptors 1 and 2, are generated upon cleavage of membrane TNF or of the membrane associated receptors, respectively, by the enzyme TNF-α convertase (TACE). TNF-R1, but not TNF-R2, contains a death domain, which signals apoptosis upon the formation of the Death Inducing Signaling Complex (DISC). In spite of its lack of a death domain, TNF-R2 can nevertheless be implicated in apoptosis induction, since its activation causes degradation of TNF Receptor Associated Factor 2 (TRAF2), an inhibitor of the TNF-R1-induced DISC formation. Moreover, apoptosis induction of lung microvascular endothelial cells by TNF was shown to require activation of both TNF receptors. TNF-R2 was also shown to be important for ICAM-1 upregulation in endothelial cells in vitro and in vivo, an activity important in the sequestration of leukocytes in the microvessels. Moreover, lung microvascular endothelial cells isolated from ARDS patients express significantly higher levels of TNF-R2 and of ICAM-1 than cells isolated from patients who had undergone a lobectomy for lung carcinoma, used as controls. These findings therefore suggest that ICAM-1 and TNF-R2 may have a particular involvement in the pathogenesis of acute lung injury.

Dichotomous activity of TNF in alveolar liquid clearance and barrier protection

Dichotomous activity of TNF in alveolar liquid clearance and barrier protection

Dichotomous activity of TNF in alveolar liquid clearance and barrier protection during ALI. TNF, which is induced during ALI, causes a downregulation of ENaC expression in type II alveolar epithelial cells, upon activating TNF-R1. Moreover, TNF increases permeability, by means of interfering with tight junctions (TJ) in both alveolar epithelial (AEC) and capillary endothelial cells (MVEC). ROS, the generation of which is frequently increased during ALI, were also shown to downregulate ENaC and Na+-K+-ATPase expression and moreover also lead to decreased endothelial barrier integrity. The TIP peptide, mimicking the lectin-like domain of TNF, is able to increase sodium uptake in alveolar epithelial cells and to restore endothelial barrier integrity, as such providing a significant protection against the development of permeability edema (red lines: inhibition, green arrows: activation).

Proposed mechanism of action for the anti-inflammatory and barrier-protective actions of hsp90 inhibitors.

Proposed mechanism of action for the anti-inflammatory and barrier-protective actions of hsp90 inhibitors.

Proposed mechanism of action for the anti-inflammatory and barrier-protective actions of hsp90 inhibitors.

Permeability edema represents a life-threatening complication of acute lung injury, severe pneumonia and ARDS, characterized by a combined dysregulation of pulmonary epithelial and endothelial apoptosis, endothelial barrier integrity and alveolar liquid clearance capacity. As such, it is likely that several of these parameters have to be targeted in order to obtain a successful therapy. This review focuses on a selection of recently discovered substances and mechanisms that might improve ALI therapy. As such, we have discussed the inhibition of apoptosis and necrosis occurring during ALI, by means of the restoration of Zn2+ homeostasis. PPARα and ϒ agonists can represent therapeutically  promising molecules, since they inhibit transcription factors as well as essential co-activators involved in the activation of pro-inflammatory cytokines, chemokines and adhesion molecules, all of which are implicated in ALI. Apart from inducing a potent inhibition of inflammation upon interfering with NF-kB activation, hsp90 inhibitors were shown to prevent and restore endothelial barrier integrity. These agents are able to significantly improve survival and lung function during LPS-induced ALI. A restoration of endothelial barrier integrity during ALI can also be obtained upon increasing extracellular levels of ATP or adenosine, which activate the purinoreceptors P2Y and P1A2, respectively, leading to a decrease in myosin light chain phosphorylation and an increase in MLC phosphatase 1 activity. The pro-inflammatory cytokine TNF is involved in endothelial apoptosis and hyperpermeability, as well as in the reduction of alveolar liquid clearance, upon activating its receptors. However, apart from its receptor binding sites, TNF harbors a lectin-like domain, which can be mimicked by the TIP peptide. This peptide has been shown to increase alveolar liquid clearance and moreover induces endothelial barrier protection. As such, TNF can be considered as a moonlighting cytokine, combining both positive and negative activities for permeability edema generation within one molecule.

 

The protective effect of CDDO-Me on lipopolysaccharide-induced acute lung injury in mice

Tong Chen, Yi Moua, Jiani Tan, LinlinWei, Yixue Qiao, Tingting Wei, et al.
International Immunopharmacology 25 (2015) 55–64
http://dx.doi.org/10.1016/j.intimp.2015.01.011

ALI is a clinical syndrome characterized by a disruption of epithelial integrity, neutrophil accumulation, noncardiogenic pulmonary edema, severe hypoxemia and an intense pulmonary inflammatory response with a wide array of increasing severity of lung parenchymal injury. Previous studies have shown that lots of pathogenesis contribute to ALI, such as oxidant/antioxidant dysfunction, dysregulation of inflammatory/anti-inflammatory pathway, upregulation of chemokine production and adhesion molecules. However, to date there is no effective medicine to control ALI. Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram negative bacteria. It has been reported to activate toll like receptors 4 (TLR4) and to stimulate the release of inflammatory mediators inducing ALI-like symptoms. Intratracheal administration of LPS has been used to construct animal models of ALI.

The biological importance of naturally occurring triterpenoids has long been recognized. Oleanolic acid, exhibiting modest biological activities, has been marketed in China as an oral drug for the treatment of liver disorders in humans. Among its derivatives, bardoxolonemethyl (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methylester) CDDO-Me, had completed a successful phase I clinical trial for the treatment of cancer and started a phase II trial for the treatment of patients with pulmonary arterial hypertension. For its broad spectrum antiproliferative and anti-tumorigenic activities, CDDO-Me has also been reported to possess a number of pharmacological activities such as antioxidant, anti-tumor and anti-inflammatory effects. However, the mechanisms by which CDDO-Me exerted its anti-inflammatory effects on macrophage were insufficiently elucidated. More importantly, there is no available report to evaluate its therapeutic effect on acute lung injury.

CDDO-Me, initiated in a phase II clinical trial, is a potential useful therapeutic agent for cancer and inflammatory dysfunctions, whereas the therapeutic efficacy of CDDO-Me on LPS-induced acute lung injury (ALI) has not been reported as yet. The purpose of the present study was to explore the protective effect of CDDO-Me on LPS-induced ALI in mice and to investigate its possible mechanism. BalB/c mice received CDDO-Me (0.5 mg/kg, 2 mg/kg) or dexamethasone (5 mg/kg) intraperitoneally 1 h before LPS stimulation and were sacrificed 6 h later. W/D ratio, lung MPO activity, number of total cells and neutrophils, pulmonary histopathology, IL-6, IL-1β, and TNF-α in the BALF were assessed. Furthermore, we estimated iNOS, IL-6, IL-1β, and TNF-α mRNA expression and NO production as well as the activation of the three main MAPKs, AkT, IκB-α and p65. Pretreatment with CDDO-Me significantly ameliorated W/D ratio, lung MPO activity, inflammatory cell infiltration, and inflammatory cytokine production in BALF from the in vivo study. Additionally, CDDO-Me had beneficial effects on the intervention for pathogenesis process at molecular, protein and transcriptional levels in vitro. These analytical results provided evidence that CDDO-Me could be a potential therapeutic candidate for treating LPS-induced ALI.

Effects of CDDO-Me on LPS-mediated lung changes

Effects of CDDO-Me on LPS-mediated lung histopathologic changes in lung tissues. (A) The lung section from the control mice; (B) the lung section from the mice administered with LPS (8 mg/kg); (C) the lung section from the mice administered with dexamethasone (5 mg/kg) and LPS (8 mg/kg); (D) the lung section from the mice administered with CDDO-Me (0.5mg/kg) and LPS (8mg/kg); (E) the lung section from the mice administered with CDDO-Me (2mg/kg) and LPS (8mg/kg); (hematoxylin and eosin staining, magnification 200×). Control group: the green arrow indicated alveolar wall, no hyperemia. All the other groups: The black arrow indicated the inflammatory cell infiltration; the green arrow indicated alveolar wall hyperemia.

 

The impact of cardiac dysfunction on acute respiratory distress syndrome and mortality in mechanically ventilated patients with severe sepsis and septic shock: An observational study

Brian M. Fuller, Nicholas M. Mohr, Thomas J. Graetz, et al.
Journal of Critical Care 30 (2015) 65–70
http://dx.doi.org/10.1016/j.jcrc.2014.07.027

Purpose: Acute respiratory distress syndrome (ARDS) is associated with significant mortality and morbidity in survivors. Treatment is only supportive, therefore elucidating modifiable factors that could prevent ARDS could have a profound impact on outcome. The impact that sepsis-associated cardiac dysfunction has on ARDS is not known. Materials and Methods: In this retrospective observational cohort study of mechanically ventilated patients with severe sepsis and septic shock, 122 patients were assessed for the impact of sepsis-associated cardiac dysfunction on incidence of ARDS (primary outcome) and mortality. Results: Sepsis-associated cardiac dysfunction occurred in 44 patients (36.1%). There was no association of sepsis-associated cardiac dysfunction with ARDS incidence (p= 0.59) or mortality, and no association with outcomes in patients that did progress to ARDS after admission. Multivariable logistic regression demonstrated that higher BMI was associated with progression to ARDS (adjusted OR 11.84, 95% CI 1.24 to 113.0, p= 0.02). Conclusions: Cardiac dysfunction in mechanically ventilated patients with sepsis did not impact ARDS incidence, clinical outcome in ARDS patients, or mortality. This contrasts against previous investigations demonstrating an influence of nonpulmonary organ dysfunction on outcome in ARDS. Given the frequency of ARDS as a sequela of sepsis, the impact of cardiac dysfunction on outcome should be further studied.

 

Suppression of NF-κβ pathway by crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in mice

Ruhui Yang, Lina Yang, Xiangchun Shen, Wenyuan Cheng, et al.
European Journal of Pharmacology 674 (2012) 391–396
http://dx.doi.org:/10.1016/j.ejphar.2011.08.029

Crocetin, a carotenoid compound, has been shown to reduce expression of inflammation and inhibit the production of reactive oxygen species. In the present study, the effect of crocetin on acute lung injury induced by lipopolysaccharide (LPS) was investigated in vivo. In the mouse model, pretreatment with crocetin at dosages of 50 and 100 mg/kg reduced the LPS-induced lung edema and histological changes, increased LPS-impaired superoxide dismutase (SOD) activity, and decreased lung myeloperoxidase (MPO) activity. Furthermore, treatment with crocetin significantly attenuated LPS-induced mRNA and the protein expressions of interleukin-6 (IL-6), macrophage chemoattractant protein-1 (MCP-1), and tumour necrosis factor-α (TNF-α) in lung tissue. In addition, crocetin at different dosages reduced phospho-IκB expression and NF-κB activity in LPS-induced lung tissue alteration. These results indicate that crocetin can provide protection against LPS-induced acute lung injury in mice.

 

Sauchinone, a lignan from Saururus chinensis, attenuates neutrophil pro-inflammatory activity and acute lung injury

Hui-Jing Han, Mei Li, Jong-Keun Son, Chang-Seob Seo, et al.
International Immunopharmacology 17 (2013) 471–477
http://dx.doi.org/10.1016/j.intimp.2013.07.011

Previous studies have shown that sauchinone modulates the expression of inflammatory mediators through mitogen-activated protein kinase (MAPK) pathways in various cell types. However, little information exists about the effect of sauchinone on neutrophils, which play a crucial role in inflammatory process such as acute lung injury (ALI). We found that sauchinone decreased the phosphorylation of p38 MAPK in lipopolysaccharide (LPS)-stimulated murine bone marrow neutrophils, but not ERK1/2 and JNK. Exposure of LPS-stimulated neutrophils to sauchinone or SB203580, a p38 inhibitor, diminished production of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2 compared to neutrophils cultured with LPS. Treatment with sauchinone decreased the level of phosphorylated ribosomal protein S6 (rpS6) in LPS-stimulated neutrophils. Systemic administration of sauchinone to mice led to reduced levels of phosphorylation of p38 and rpS6 in mice lungs given LPS, decreased TNF-α and MIP-2 production in bronchoalveolar lavage fluid, and also diminished the severity of LPS-induced lung injury, as determined by reduced neutrophil accumulation in the lungs, wet/dry weight ratio, and histological analysis. These results suggest that sauchinone diminishes LPS-induced neutrophil activation and ALI.

In the present study, the systemic administration of sauchinone decreased the phosphorylation of p38 MAPK and rpS6 in mice lungs subjected to LPS and diminished the severity of LPS-induced ALI. Neutrophils play an important role in acute inflammatory processes, such as ALI, which was demonstrated by various experimental models. Previous reports suggested that p38 MAPK inhibition of murine neutrophils could lead to the loss of chemotaxis toward MIP-2, as well as the loss of TNF-αandMIP-2 production in response to LPS, and also attenuated neutrophil accumulation in LPS-induced ALI models. Therefore, the beneficial effects of sauchinone on LPS-induced ALI are likely associated with decreases in the production of pro-inflammatory mediators by neutrophils, consistent with our in vitro experiments. However, we cannot exclude that the effects of sauchinone on reducing the release of TNF-α and MIP-2 in mice lungs subjected to LPS, with the resultant prevention of ALI, could be affected by various pulmonary cell populations, such as alveolar macrophages. Also, the inhibitory effects of sauchinone on NF-κB activation through various pulmonary cell populations (Supplemental Fig. S2), in addition to p38MAPK activity in mouse lungs given LPS, might enhance the anti-inflammatory action of sauchinone in mouse lungs subjected to LPS. In conclusion, we found that sauchinone significantly diminished the release of inflammatory mediators in isolated neutrophils and lungs subjected to LPS. The anti-inflammatory action of sauchinone was associated with the prevention of p38 MAPK and rpS6 activation. These findings suggest that sauchinone may be an appropriate pharmacological candidate for the treatment of ALI as well as other neutrophil driven acute inflammatory diseases.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.intimp.2013.07.011

 

Protective effect of dexmedetomidine in a rat model of α-naphthylthiourea- induced acute lung injury

Volkan Hancı, Gamze Yurdakan, Serhan Yurtlu, et al.
J Surg Res 178 (2012):424-430
http://dx.doi.org:/10.1016/j.jss.2012.02.027

Background: We assessed the effects of dexmedetomidine in a rat model of a-naphthylthiourea (ANTU)einduced acute lung injury.  Methods: Forty Wistar Albino male rats weighing 200e240 g were divided into 5 groups (n = 8 each), including a control group. Thus, there were one ANTU group and three dexmedetomidine groups (10-, 50-, and 100-mg/kg treatment groups), plus a control group. The control group provided the normal base values. The rats in the ANTU group were given 10 mg/kg of ANTU intraperitoneally and the three treatment groups received 10, 50, or 100 mg/kg of dexmedetomidine intraperitoneally 30 min before ANTU application. The rat body weight (BW), pleural effusion (PE), and lung weight (LW) of each group were measured 4 h after ANTU administration. The histopathologic changes were evaluated using hematoxylin-eosin staining. Results: The mean PE, LW, LW/BW, and PE/BW measurements in the ANTU group were significantly greater than in the control groups and all dexmedeto-midine treatment groups (P < 0.05). There were also significant decreases in the mean PE, LW, LW/BW and PE/BW values in the dexmedetomidine 50-mg/kg group compared with those in the ANTU group (P < 0.01). The inflammation, hemorrhage, and edema scores in the ANTU group were significantly greater than those in the control or dexmedetomidine 50-mg/kg group (P < 0.01). Conclusion: Dexmedetomidine treatment has demonstrated  a potential benefit by preventing ANTU-induced acute lung injury in an experimental rat model. Dexmedetomidine could have a potential protective effect on acute lung injury in intensive care patients.

 

Protective effects of Isofraxidin against lipopolysaccharide-induced acute lung injury in mice

Xiaofeng Niu, YuWang, Weifeng Li, Qingli Mu, et al.
International Immunopharmacology 24 (2015) 432–439
http://dx.doi.org/10.1016/j.intimp.2014.12.041

Acute lung injury (ALI) is a life-threatening disease characterized by serious lung inflammation and increased capillary permeability, which presents a high mortality worldwide. Isofraxidin (IF), a Coumarin compound isolated from the natural medicinal plants such as Sarcandra glabra and Acanthopanax senticosus, has been reported to have definite anti-bacterial, anti-oxidant, and anti-inflammatory activities. However, the effects of IF against lipopoly-saccharide-induced ALI have not been clarified. The aim of the present study is to explore the protective effects and potential mechanism of IF against LPS-induced ALI in mice. In this study, We found that pretreatment with IF significantly lowered LPS-induced mortality and lung wet-to-dry weight (W/D) ratio and reduced the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in serum and bronchoalveolar lavage fluid (BALF). We also found that total cells, neutrophils and macrophages in BALF,MPO activity in lung tissues were markedly decreased. Besides, IF obviously inhibited lung histopathological changes and cyclooxygenase-2 (COX-2) protein expression. These results suggest that IF has a protective effect against LPS induced ALI, and the protective effect of IF seems to result from the inhibition of COX-2 protein expression in the lung, which regulates the production of PGE2.

Ingestion of LPS stimulates vascular permeability, promotes inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from blood into lung tissues and activates numerous inflammatory cells such as neutrophils and macrophages. In macrophages, LPS challenge induces the transcription of gene encoding pro-inflammatory protein, which leads to cytokine release and synthesis of enzymes, such as cyclo-oxygenase-2 (COX-2). COX-2 usually can’t be found in normal tissues, but widely induced by pro-inflammatory stimuli, such as cytokines, endotoxins, and growth factors. COX-2 plays a vital role in the regulation of inflammatory process by modulating the production of prostaglandin E2 (PGE2). PGE2, induced by cytokines and other initiator, is an inflammatory mediator which is produced in the regulation of COX-2. Previous researches demonstrated that inhibition of COX-2 produced a dramatically anti-inflammatory effect with little gastrointestinal toxicity. Therefore, inhibition of COX-2 protein expression has far-reaching significance in the treatment of ALI.

effects of IF on LPS-induced mortality in ALI mice

effects of IF on LPS-induced mortality in ALI mice

The effects of IF on LPS-induced mortality in ALI mice (n = 12/group). IF (5, 10, 15 mg/kg, i.p.) or DEX (5 mg/kg, i.p.) were given to mice 1 h prior to LPS challenge. The mortalities were observed at 0, 12, 24, 36, 48, 60, and 72 h. ###P = 0.001 when compared with the control group; *P = 0.05, **P = 0.01, and ***P = 0.001 when compared with the LPS group.

 

Protective effects of intranasal curcumin on paraquot induced acute lung injury (ALI) in mice

Namitosh Tyagi, Asha Kumaria, D. Dash, Rashmi Singh
Environment  Toxicol  & Pharmacol  38 (2014) 913–921
http://dx.doi.org/10.1016/j.etap.2014.10.003

Paraquot (PQ) is widely and commonly used as herbicide and has been reported to be hazardous as it causes lung injury. However, molecular mechanism underlying lung toxicity caused by PQ has not been elucidated. Curcumin, a known anti-inflammatory molecule derived from rhizomes of Curcuma longa has variety of pharmacological activities including free-radical scavenging properties but the protective effects of curcumin on PQ-induced acute lung injury (ALI) have not been studied. In this study, we aimed to study the effects of curcumin on ALI caused by PQ in male parke’s strain mice which were challenged acutely byPQ (50 mg/kg, i.p.) with or without curcumin an hour before (5 mg/kg, i.n.) PQ intoxication. Lung specimens and the bronchoalveolar lavage fluid (BALF) were isolated for pathological and biochemical analysis after 48 h of PQ exposure. Curcumin administration has significantly enhanced superoxide dismutase (SOD) and catalase activities. Lung wet/dry weight ratio, malondialdehyde (MDA) and lactate dehydrogenase (LDH) content, total cell number and myeloperoxidase (MPO) levels in BALF as well as neutrophil infiltration were attenuated by curcumin. Pathological studies also revealed that intranasal curcumin alleviate PQ-induced pulmonary damage and pro-inflammatory cytokine levels like tumor necrosis factor-α (TNF-α) and nitric oxide (NO). These results suggest that intranasal curcumin may directly target lungs and curcumin inhalers may prove to be effective in PQ-induced ALI treatment in near future.

 

Phillyrin attenuates LPS-induced pulmonary inflammation via suppression of MAPK and NF-κB activation in acute lung injury mice

Wei-ting Zhong, Yi-chun Wu, Xian-xing Xie, Xuan Zhou, et al.
Fitoterapia 90 (2013) 132–139
http://dx.doi.org/10.1016/j.fitote.2013.06.003

Phillyrin (Phil) is one of the main chemical constituents of Forsythia suspensa (Thunb.), which has shown to be an important traditional Chinese medicine. We tested the hypothesis that Phil modulates pulmonary inflammation in an ALI model induced by LPS. Male BALB/c mice were pretreated with or without Phil before respiratory administration with LPS, and pretreated with dexamethasone as a control. Cytokine release (TNF-α, IL-1β, and IL-6) and amounts of inflammatory cell in bronchoalveolar lavage fluid (BALF) were detected by ELISA and cell counting separately. Pathologic changes, including neutrophil infiltration, interstitial edema, hemorrhage, hyaline membrane formation, necrosis, and congestion during acute lung injury in mice were evaluated via pathological section with HE staining. To further investigate the mechanism of Phil anti-inflammatory effects, activation of MAPK and NF-κB pathways was tested by western blot assay. Phil pretreatment significantly attenuated LPS-induced pulmonary histopathologic changes, alveolar hemorrhage, and neutrophil infiltration. The lung wet-to-dry weight ratios, as the index of pulmonary edema, were markedly decreased by Phil retreatment. In addition, Phil decreased the production of the proinflammatory cytokines including (TNF-α, IL-1β, and IL-6) and the concentration of myeloperoxidase (MPO) in lung tissues. Phil pretreatment also significantly suppressed LPS-induced activation of MAPK and NF-κB pathways in lung tissues. Taken together, the results suggest that Phil may have a protective effect on LPS-induced ALI, and it potentially contributes to the suppression of the activation of MAPK and NF-κB pathways. Phil may be a new preventive agent of ALI in the clinical setting.

A mass of studies have been reported basically on alleviating LPS-induced acute lung injury in models. Phillyrin (Fig. 1), a lignin, is one of the main chemical constituents of Forsythia suspensa (Thunb.), which is an important traditional Chinese medicine (“Lianqiao” in Chinese), and has long been used for gonorrhea, erysipelas, inflammation, pyrexia and ulcer. Previous studies indicated that Phil significantly inhibited NO production in LPS-activated macrophage cells. But there is not much evidence showing the anti-inflammatory properties of phillyrin. In the present study, we sought to investigate the effects of phillyrin on LPS-induced pulmonary inflammation in mice.

Fig. not shown. A: Effects of Phil on histopathological changes in lung tissues in LPS-induced ALI mice. Mice were given an intragastric administration of Phil (10 and 20 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Then mice were anesthetized and lung tissue samples were collected at 6 h after LPS challenge for histological evaluation. These representative histological changes of the lung were obtained from mice of different groups (hematoxylin and eosin staining, original magnification 200×, Scale bar: 50 μm). B: Effects of Phil on LPS-induced lung morphology. The slides were histopathologically evaluated using a semi-quantitative scoring method. Lung injury was graded from 0 (normal) to 4 (severe) in four categories: congestion, edema, interstitial inflammation and inflammatory cell infiltration. The total lung injury score was calculated by adding up the individual scores of each category. The values presented are the means ± S.E.M. (n = 4–6 in each group). ##P b 0.01 vs. the control group, **P b 0.01 vs. the LPS group. Cont: control group; LPS: LPS group; Phil + LPS: Phil + LPS group; Dex + LPS: Dex + LPS group.

In summary, the present study indicated that Phil has a protective effect on LPS-induced acute lung injury. Phil significantly attenuated histopathological changes initiated by LPS via reducing over inflammatory responses. We also demonstrated that MAPK and NF-κB signaling pathways are the important targets of Phil to perform its actions. Phil acts by preventing NF-κB translocation to the nucleus or inhibiting the activation of MAPKs directly or indirectly, which is to be investigated in further studies. All these results suggest that Phil may be a new therapeutic agent for the prevention of inflammation during acute lung injury.

 

 

 

 

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The History of Hematology and Related Sciences

Curator: Larry H. Bernstein, MD, FCAP

 

The History of Hematology and Related Sciences: A Historical Review of Hematological Diagnosis from 1880 -1980

 

Blood Description: The Analysis of Blood Elements a Window into Diseases

Diagnosing bacterial infection (BI) remains a challenge for the attending physician. An ex vivo infection model based on human fixed polymorphonuclear neutrophils (PMNs) gives an autofluorescence signal that differs significantly between stimulated and unstimulated cells. We took advantage of this property for use in an in vivo pneumonia mouse model and in patients hospitalized with bacterial pneumonia. A 2-fold decrease was observed in autofluorescence intensity for cytospined PMNs from broncho-alveolar lavage (BAL) in the pneumonia mouse model and a 2.7-fold decrease was observed in patients with pneumonia when compared with control mice or patients without pneumonia, respectively. This optical method provided an autofluorescence mean intensity cut-off, allowing for easy diagnosis of BI. Originally set up on a confocal microscope, the assay was also effective using a standard epifluorescence microscope. Assessing the autofluorescence of PMNs provides a fast, simple, cheap and reliable method optimizing the efficiency and the time needed for early diagnosis of severe infections. Rationalized therapeutic decisions supported by the results from this method can improve the outcome of patients suspected of having an infection.

Monsel A, Le´cart S, Roquilly A, Broquet A, Jacqueline C, et al. (2014) Analysis of Autofluorescence in Polymorphonuclear Neutrophils: A New Tool for Early Infection Diagnosis. PLoS ONE 9(3): e92564.
http://dx.doi.org:/10.1371/journal.pone.0092564

This study was designed to validate or refute the reliability of total lymphocyte count (TLC) and other hematological parameters as a substitute for CD4 cell counts. Participants consisted of two groups, including 416 antiretroviral naive (G1) and 328 antiretroviral experienced (G2) patients. CD4+ T cell counts were performed using a Cyflow machine. Hematological parameters were analyzed using a hematology analyzer. The median ± SEM CD4 count (range) of participants in G1 was 199 ± 10.9 (5–1840 cells/μL) and the median ± SEM TLC (range) was 1. 61 ± 0.05 (0.07–6.63 × 103/μL). The corresponding values among G2 were 421 ± 15.8 (13–1801) and 2.13 ± 0.04 (0.06–5.58), respectively. Using a threshold value of 1.2 × 103/μL for TLC alone, the sensitivity of G1 was 88.4% (specificity (SP) 67.4%, the positive predictive value (PPV) 53.5% and negative predictive value (NPV) of 93.2% for CD4 , 200 cells/μL, the sensitivity for G2 was 83.3%, SP 85.3%, PPV 23.8%, and NPV of 93.2%. Using multiple parameters, including TLC , 1.2 × 103/μL, hemoglobin , 10 g/dL, and platelets , 150 × 103/L, the sensitivity increased to 96.0% (SP, 82.7%; PPV, 80%; NPV, 96.7%) among G1, while no change was observed in the G2 cohort. TLC , 1.2 × 103/μL alone is an insensitive predictor of CD4 count of , 200 cells/μL. Incorporating hemoglobin , 10 g/dL, and platelets , 150 × 103/L enhances the ability of TLC , 1.2 × 103/μL to predict CD4 count , 200 cells/μL among the antiretroviral-naïve cohort. We recommend the use of multiple, inexpensively measured hematological parameters in the form of an algorithm for predicting CD4 count level.

Evaluating Total Lymphocyte Counts and Other Hematological Parameters as a Substitute for CD4 Counts in the Management of HIV Patients in Northeastern Nigeria. BA Denue, AU Abja, IM Kida, AH Gabdo, AA Bukar and CB Akawu.
Retrovirology: Research and Treatment 2013:5 9–16 http://dx.doi.org:/10.4137/RRT.S11562

Sepsis is a syndrome that results in high morbidity and mortality. We investigated the delta neutrophil index (DN) as a predictive marker of early mortality in patients with gram-negative bacteremia. Retrospective study. The DN was measured at onset of bacteremia and 24 hours and 72 hours later. The DN was calculated using an automatic hematology analyzer. Factors associated with 10-day mortality were assessed using logistic regression. A total of 172 patients with gram-negative bacteremia were included in the analysis; of these, 17 patients died within 10 days of bacteremia onset. In multivariate analysis, Sequental organ failure assessment scores (odds ratio [OR]: 2.24, 95% confidence interval [CI]: 1.31 to 3.84; P = 0.003), DN-day 1 ≥ 7.6% (OR: 305.18, 95% CI: 1.73 to 53983.52; P = 0.030) and DN-day 3 ≥ DN day 1 (OR: 77.77, 95% CI: 1.90 to 3188.05; P = 0.022) were independent factors associated with early mortality in gram-negative bacteremia. Of four multivariate models developed and tested using various factors, the model using both DN-day 1 ≥ 7.6% and DN-day 3 ≥ DN-day 1 was most predictive early mortality. DN may be a useful marker of early mortality in patients with gram-negative bacteremia. We found both DN-day 1 and DN trend to be significantly associated with early mortality.

Delta Neutrophil Index as a Prognostic Marker of Early Mortality in Gram Negative Bacteremia. HW Kim, JH Yoon, SJ Jin, SB Kim, NS Ku, SJ Jeong,
et al. Infect Chemother 2014;46(2):94-102. pISSN 2093-2340·eISSN 2092-6448
http://dx.doi.org/10.3947/ic.2014.46.2.94
Various indices derived from red blood cell (RBC) parameters have been described for distinguishing thalassemia and iron deficiency. We studied the microcytic to hypochromic RBC ratio as a discriminant index in microcytic anemia and compared it to traditional indices in a learning set and confirmed our findings in a validation set. The learning set comprised samples from 371 patients with microcytic anemia mean cell volume (MCV < 80 fL), which were measured on a CELL-DYN Sapphire analyzer and various discriminant functions calculated. Optimal cutoff values were established using ROC analysis. These values were used in the validation set of 338 patients. In the learning set, a microcytic to hypochromic RBC ratio >6.4 was strongly indicative of thalassemia (area under the curve 0.948). Green-King and England-Fraser indices showed comparable area under the ROC curve. However, the microcytic to hypochromic ratio had the highest sensitivity (0.964). In the validation set, 91.1% of microcytic patients were correctly classified using the M/H ratio. Overall, the microcytic to hypochromic ratio as measured in CELL-DYN Sapphire performed equally well as the Green-King index in identifying thalassemia carriers, but with higher sensitivity, making it a quick and inexpensive screening tool.
Differential diagnosis of microcytic anemia: the role of microcytic and hypochromic erythrocytes. E. Urrechaga, J.J.M.L. Hoffmann, S. Izquierdo, J.F. Escanero. Intl Jf Lab Hematology Aug 2014. http://dx.doi.org:/10.1111/ijlh.12290

Achievement of complete response (CR) to therapy in chronic lymphocytic leukemia (CLL) has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry [and polymerase chain reaction methods] can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10–4), using the above-mentioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD). Tumor burdens lower than 10–4 are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

Minimal Residual Disease Surveillance in Chronic Lymphocytic Leukemia by Fluorescence-Activated Cell Sorting. S Ringelstein-Harlev, R Fineman.
Rambam Maimonides Med J. Oct 2014   5 (4)  e0027. http://dx.doi.org:/10.5041/RMMJ.10161

Natural Killer cells (CD3-CD16+CD56+) are a major players in innate immunity, both as direct cytotoxic effectors as well as regulators for other innate immunity cell types. We have shown that, using the FlowCellect™ human NK cell characterization kit, one can achieve accurate phenotyping on a variety of sample types, including whole blood samples. Using the same kit to perform an NK cell cytotoxicity test, we demonstrate that unbound K562 target cells can be clearly distinguished from those that have been engaged by CD56+ NK cells, and each of these populations can be further investigated for viability using the eFluor 660® dye.

Analysis of NK cell subpopulations in whole blood

Analysis of NK cell subpopulations in whole blood

Analysis of NK cell subpopulations in whole blood

A

Proportion of K562 target cells bound to NK cells

Proportion of K562 target cells bound to NK cells

In a 5:1 effector cell:target cell population, 8% of the K562 cells were bound to NK cells (Figure 3B). 84% of the bound K562 cells were viable (Figure 3C) stained with fixable viability dye), while 96% of the unbound K562 cells were viable (Figure 3D). (B,C,D not shown)

Characterization of Natural Killer Cells Using Flow Cytometry.
EMD Millipore is a division of Merck KGaA, Darmstadt, Germany.

Red blood cell distribution width (RDW) is increased in liver disease. Its clinical significance, however, remains largely unknown. The aim of this study was to identify whether RDW was a prognostic index for liver disease. Retrospective: 33 patients with non-cirrhotic HBV chronic hepatitis, 125 patients with liver cirrhosis after HBV infection, 81 newly diagnosed primary epatocellular carcinoma (pHCC) patients, 17 alcoholic liver cirrhosis patients and 42 patients with primary biliary cirrhosis (PBC). Sixty-six healthy individuals represented the control cohort. The relationship between RDW on admission and clinical features: The association between RDW and hospitalization outcome was estimated by receiver operating curve (ROC) analysis and a multivariable logistic regression model. Increased RDW was observed in liver disease patients. RDW was positively correlated with serum bilirubin and creatinine levels, prothrombin time, and negatively correlated with platelet counts and serum albumin concentration. A subgroup analysis, considering the different etiologies, revealed similar findings. Among the patients with liver cirrhosis, RDW increased with worsening of Child-Pugh grade. In patients with PBC, RDW positively correlated with Mayo risk score. Increased RDW was associated with worse hospital outcome, as shown by the AUC [95% confidence interval (CI)] of 0.76 (0.67 – 0.84). RDW above 15.15% was independently associated with poor hospital outcome after adjustment for serum bilirubin, platelet count, prothrombin time, albumin and age, with the odds ratio (95% CI) of 13.29 (1.67 – 105.68). RDW is a potential prognostic index for liver disease.

Red blood cell distribution width is a potential prognostic index for liver disease
Z Hua , Y Suna , Q Wanga , Z Han , Y Huang , X Liu , C Ding, et al.
Clin Chem Lab Med 2013; 51(7):1403–1408.
http://dx.doi.org:/10.1515/cclm-2012-0704

Blood Plasma and Red Blood Cells

Whole blood consists of red and white blood cells, as well as platelets suspended in a liquid referred to as blood plasma. According to the American Red Cross, plasma is 92% water and makes up 55% of blood volume. The permeability of blood plasma is equal to 1.

Red blood cells make up slightly lower blood volume than blood plasma — about 45% of whole blood. As you probably already know, these types of blood cells contain hemoglobin, which in turn consists of iron that helps transport oxygen throughout the body. The permeability of red blood cells is slightly less than 1,
(1 – 3.9e-6). Or to put it in words, red blood cell particles are diamagnetic.

Due to their magnetic properties, red blood cells may be separated from the plasma via a magnetophoretic approach. If the blood were to be in a channel subject to a magnetophoretic force, we could control where the red blood cells and the plasma go within the channels. In other words, because the red blood cells have different permeability, they can be separated from the flow channel. However, such methodology is beyond the year 1980.

Timeline of Major Hematology Landmarks

1877 Paul Ehrlich develops techniques to stain blood cells to improve microscopic visualization.

1897 The Diseases of Infancy and Childhood contains a 20-page chapter on diseases of the blood and is the first American pediatric medical textbook to provide significant hematologic information.

1821–1902 Rudolph Virchow, during a long and illustrious career, demonstrates the importance of fibrin in the blood coagulation process, coins the terms embolism and thrombosis, identifies the disease leukemia, and theorizes that leukocytes are made in response to inflammation.

1901 Karl Landsteiner and colleagues identify blood groups of A, B, AB, and O.

1907 Ludvig Hektoen suggests that the safety of transfusion might be improved by crossmatching blood between donors and patients to exclude incompatible mixtures. Reuben Ottenberg performs the first blood transfusion using blood typing and crossmatching in New York. Ottenberg also observes the Mendelian inheritance of blood groups and recognizes the “universal” utility of group O donors.

1910 The first clinical description of sickle cell published in medical literature.

1914 Sodium citrate is found to prevent blood from clotting, allowing blood to be stored between collection and transfusion.

1924 Pediatrics is the first comprehensive American publication on pediatric hematology.

1925 Alfred P. Hart performs the first exchange transfusion.

1925 Thomas Cooley describes a Mediterranean hematologic syndrome of anemia, erythroblastosis, skeletal disorders, and splenomegaly that is later called Cooley’s anemia and now thalassemia.

1936 Chicago’s Cook County Hospital establishes the first true “blood bank” in the United States.

1938 Dr. Louis Diamond (known as the “father of American pediatric hematology”) along with Dr. Kenneth Blackfan describes the anemia still known as Diamond-Blackfan anemia.

1941 The Atlas of the Blood of Children is published by Blackfan, Diamond, and Leister.

1945 Coombs, Mourant, and Race describe the use of antihuman globulin (later known as the “Coombs Test”) to identify “incomplete” antibodies.

1954 The blood product cryoprecipitate is developed to treat bleeds in people with hemophilia.

1950s The “butterfly” needle and intercath are developed, making IV access easier and safer.

1961 The role of platelet concentrates in reducing mortality from hemorrhage in cancer patients is recognized.

1962 The first antihemophilic factor concentrate to treat coagulation disorders in hemophilia patients is developed through fractionation.

1969 S. Murphy and F. Gardner demonstrate the feasibility of storing platelets at room temperature, revolutionizing platelet transfusion therapy.

1971 Hepatitis B surface antigen testing of blood begins in the United States.

1972 Apheresis is used to extract one cellular component, returning the rest of the blood to the donor.

1974 Hematology of Infancy and Childhood is published by Nathan and Oski.

As I write today my hospital celebrates its 150th anniversary. Great Ormond Street Children’s Hospital was founded on 14 February 1852 by the visionary Dr Charles West followed his belief that hospital care allied to research in children’s diseases would reduce child mortality from above 50% by the age of 15 years. It is foolish to believe that we can progress in medicine without a knowledge of the past and that much of life is based upon experience. When putting together a series of articles on the history of haematology, initially published in BJH, this was the main raison d’être, along with the belief that the practice of medicine has become increasingly serious but should also be fun and interesting and even occasionally uplifting to the spirit.

The central problem of any survey of the history of haematology is usually the question of balance. Achieving a degree of balance among themes and topics that will be satisfactory to practicing haematologists/physicians with an interest in blood diseases is essentially impossible. Our preference has been for themes of general interest rather than those of a purely scientific view into a field that has led the way in understanding the molecular basis of human disease.

  1. M. Hann, London, 2002; O. P. Smith, Dublin, 2002.

Origins of the Discipline `Neonatal Haematology’, 1925-75

In every modern neonatal intensive care unit (NICU), haematological problems are encountered daily. Many of these problems involve varieties of anaemia, neutropenia or thrombocytopenia that are unique to NICU patients. A characteristic aspect of these unique problems is that, if the neonate survives, the haematological problem will remit and will not recur later in life, nor will it evolve into a chronic illness (although the problem might occur in a future newborn sibling). This characteristic comes about because the common haematological problems of NICU patients are not genetic defects but are environmental stresses (such as infection, alloimmunization or a variety of maternal illnesses) that are imposed on a developmentally immature haematopoietic system.

In the USA, and in some parts of Europe, the unique haematological problems that occur among NICU patients are diagnosed and treated by neonatologists, not by paediatric haematologists. Although these haematological conditions were generally first described by haematologists, the conditions occur, obviously, in neonates. Thus, the neonatologist, who is familiar with intensive care management of neonates, has also become familiar with the diagnosis and management of the neonate’s common haematological disorders. A growing number of neonatologists have sought specific additional training in haematology, with the goals of discovering the mechanisms underlying the unique haematological problems of NICU patients and improving the management and outcome of the patients who have these conditions. These physicians have remained as neonatologists and they do not practice paediatric haematology, although their research contributions certainly come under the purview of haematology, or more precisely under the discipline of `neonatal haematology’. In many places in Europe, it is the haematologists rather than the neonatologists who have an academic and clinical interest in neonatal haematology.

The roots of the discipline of neonatal haematology can be traced to the early application of haematological methods to animal and human embryos and fetuses, such as found in the reports of Maximow (1924) and Wintrobe & Schumacker (1936). The clinical underpinnings of this discipline include reports of anaemia (Fikelstein, 1911) and jaundice (Blomfeld, 1901; YlppoÈ, 1913) among neonates.

Before the 1930s, very few studies and very few published clinical case reports originated from premature nurseries. Such nurseries had dubious beginnings, which were criticized by some physicians as more resembling circus exhibitions than medical care wards (Bonar, 1932). These units generally had mortality rates greatly exceeding 50% on the day of admission, with the majority of the first-day survivors having late deaths or serious long-term morbidity.

It was not until publication of the review of premature nursery care at the Children’s Hospital of Michigan, in 1932, that it was clear that some units had instituted systematic attempts to monitor and improve outcomes. A special care nursery had been established at the Children’s Hospital in 1926 and, in 1932, Drs Marsh Poole and Thomas Cooley reported their experience in that unit (Poole & Cooley, 1932). The report included  incubator design with temperature and humidity control, growth curves of patients on various feeding practices, mortality statistics and attempts to determine causes of death.

At the time premature nursery care was beginning to merit academic credentials, reports were published of haematological problems that were unique to the neonate. These papers included the seminal publication on erythroblastosis fetalis by Drs Diamond (Fig 1), Blackfan and Baty (Diamond et al, 1932), and the report of sepsis neonatorum at the Yale New Haven Hospital by Ethyl C. Dunham (Fig 2) (Dunham,

1933).

The first major textbook devoted to clinical haematology, as well as the first textbook of neonatology, contained very little information about what are today’s common NICU haematological problems. For instance, in the first edition of Clinical Hematology by Dr Maxwell M. Wintrobe (Fig 3), of the Johns Hopkins University Hospital (Wintrobe, 1942), several topics related to paediatric haematology were reviewed, but discussions of the haematological problems of neonates were limited to three – erythroblastosis fetalis, haemorrhagic disease of the newborn and the `anaemia of prematurity’. Similarly, Premature Infants: A Manual for

Physicians, the original neonatology textbook, published in 1948 by Dr Ethyl C. Dunham (Fig 2; Dunham, 1948), had only a few pages devoted to haematological problems – the same three discussed by Dr Wintrobe. Also, the classic neonatology text book, `The Physiology of the Newborn Infant’, published in 1945 by Dr Clement A. Smith, contained almost no discussion of haematological problems (Smith, 1945). hrombocytopenia, which is now diagnosed among 25-30% of NICU patients, and neutropenia, now diagnosed in 8-10% of NICU patients, were not mentioned.

The first article published in Paediatrics (1948) dealing with a neonatal haematological problem was in volume two, in which Dr Diamond detailed his technique for performing a replacement transfusion (which later became known as an `exchange’ transfusion) as a treatment for erythroblastosis fetalis (Diamond, 1949). The second paper published by Paediatrics containing aspects of neonatal haematology was 1 year later, when Sliverman & Homan (1949) described leucopenia among neonates with sepsis. Most of the 25 infants they described, who were treated at Babies Hospital in New York over an 11-year period, had `late-onset’ sepsis, beginning after 3 days of life. They reported 14 neonates with Escherichia coli sepsis and four with streptococcal or staphylococcal sepsis, and observed that leucopenia occurred occasionally among these patients but was uncommon. (Indeed, today neutropenia remains uncommon in `late-onset’ sepsis, but common in congenital or `early onset’ sepsis.)

Louis K. Diamond, MD, at Children's Hospital, Boston,

Louis K. Diamond, MD, at Children’s Hospital, Boston,

Louis K. Diamond, MD, at Children’s Hospital, Boston, MA. , date unknown (obtained with the kind assistance of Charles F. Simmons, MD, Harvard University).

Diagnosing neutropenia, anaemia or thrombocytopenia in a neonate obviously requires knowledge of the expected normal range for neutrophil concentration, haematocrit and platelet concentration in the appropriate reference population. Early contributions to neonatal haematology included the publications of these reference ranges. The landmark studies included the range of blood leucocyte and neutrophil concentrations in neonates published in 1935 by Dr Katsuji Kato from the Department of Paediatrics at the University of Chicago (Kato, 1935). He tabulated the leucocyte concentrations and differential counts of 1081 children, ranging from birth to 15 years of age. A striking finding of his report (Fig 4) was the very high neutrophil counts during the first hours and days of life. Blood neutrophil concentrations among neonates with infections were published during the early and mid-1970s by Dr Marietta Xanthou (Fig 5) at the Hammersmith Hospital in London (Xanthou, 1970, 1972), and by Drs Barbara Manroe and Charles Rosenfeld (Fig 6) at the University of Texas Southwestern Medical Center in Dallas, Texas (Manroe et al, 1977).

Normal values for haemoglobin, haematocrit, erythrocyte indices and leucocyte concentrations were refined by DeMarsh et al (1942, 1948), and in a series of publications in the early 1950s in Archives of Diseases of Children by Gairdner et al (1952a, b). These were followed by observations on human fetal haematopoiesis by Thomas and Yoffey in the British Journal of Haematology (Thomas & Yoffey, 1962, 1964), and by the work on blood volume during the 1960s (Usher et al, 1963, Usher & Lind, 1965; Yao et al, 1967, 1968). Normal ranges for blood platelet counts in ill and well preterm and term infants were published in the early 1970s (Sell et al, 1973; Corrigan, 1974).

The first publication addressing the problem of neutropenia accompanying fatal early onset bacterial sepsis was that of Tygstrup et al (1968). This was a report of a near-term male with congenital Listeria sepsis who lived for only 4 h. The platelet count was 80*109/l and the leucocyte count was 13´7*109/l, but no granulocytes were observed on the differential count, which consisted of 84% lymphocytes, 8% monocytes and 8% leucocyte precursors. A sternal marrow aspirate was taken of the infant shortly before death that revealed myeloblasts, promyelocytes and myelocytes, but no band or segmented neutrophils.

An important advance in understanding the blood neutrophil count during neonatal sepsis occurred with the back-to-back papers in Archives of Diseases of Childhood in 1972 by Dr Marietta Xanthou of Hammersmith Hospital, London (Xanthou, 1972), and Drs Gregory and Hey of Babies’ Hospital, Newcastle upon Tyne (Gregory & Hey, 1972). Both papers reported that neonates who had life threatening (or indeed fatal) infections became neutropenic prior to death. Dr Xanthou reported 35 ill preterm and term babies within their first 28 d of life. Twenty-four were ill but not infected, and these had normal blood neutrophil concentrations and morphology. However, among the 11 who were ill with a bacterial infection, neutrophilia was observed in the survivors, but neutropenia, a `left shift’, and toxic granulation were observed in the non-survivors. Consistent with this observation, Gregory and Hey reported three neonates who died with overwhelming bacterial sepsis and noted that all had profound neutropenia. Neutrophilia was common among the survivors and neutropenia, a “left shift’, and specific neutrophil morphological changes were seen among those who subsequently died.

A pivotal publication that launched the search for mechanistic information and successful treatments was that of Dr Barbara Manroe, a fellow in Neonatal Medicine, and her mentor Dr Charles Rosenfeld (Fig 6) from the University of Texas, South-western, Parkland Hospital in Dallas, Texas (Manroe et al, 1977). They evaluated 45 neonates who had culture-proven group B streptococcal infection and found that 39 had abnormal leucocyte counts: 25 neutrophilia and 14 neutropenia, and that 41 had a `left shift’. This paper was the first to quantify the `left shift’ using a method that has since become popular in neonatology – the ratio of immature neutrophils to total neutrophils on the differential cell count.

From these beginning, hundreds of studies using experimental models and clinical observations and trials were published, detailing the kinetic and molecular mechanisms accounting for this common variety of neutropenia. Marked improvements in the survival of neonates with this condition have come about through combined efforts, including early maternal screening for GBS carriage, early anti-microbial administration to ill neonates, non-specific antibody administration and a variety of measures to improve supportive care of neonates with early onset sepsis.

In the early 1930s, Dr Helen Mackay worked as a paediatrician in Mother’s Hospital, a maternity hospital located in the north-east section of London. Acting on the observation of Lichtenstein (1921) that infants of subnormal birth weight regularly became anaemic in the first months of life, she measured and reported serial heel-stick haemoglobin levels on 150 infants during their first 6 months. Thirty-nine of these infants weighed under five pounds at birth (six were under four pounds), 52 weighed five to six pounds, and 59 weighed six pounds and upwards. She showed that babies of the lightest birth weights had the most rapid fall in haemoglobin and that these fell to lower levels than those of babies of heavier birth weight (MacKay et al, 1935). Figure 7 contrasts this fall in babies weighing `3-4 lbs odd at birth’ with those weighing `5 lbs odd at birth’.

Her attempts to prevent the anaemia of prematurity failed,  but her work constituted the first clear definition of the `anaemia of prematurity’ and showed that iron administration did not prevent this condition. In the early 1950s, Douglas Gairdner, John Marks and Janet D. Roscoe, of the Department of Pathology of Cambridge Maternity Hospital, published pioneering studies in blood formation in infancy (Gairdner et al, 1952a, b). Studying 105 blood samples and 102 bone marrow samples, they concluded that `erythropoiesis ceases when the oxygen saturation just after birth increases from about 65% in the umbilical vein to .95% just after birth’. Publications by Dr Irving Schulman, in the mid- to late 1950s, defined three phases of the anaemia of prematurity and provided a mechanistic explanation for the anaemia (Schulman & Smith, 1954; Schulman, 1959). His work illustrated that the early and intermediate phases of this anaemia occur in the face of relative iron excess and are unaffected by prophylactic iron administration.

Haemoglobin levels during the first 25 weeks of life among

Haemoglobin levels during the first 25 weeks of life among

Haemoglobin levels during the first 25 weeks of life among neonates in London [by permission; Archives Diseases of Children, (MacKay, 1935)].

In 1963, Dr Sverre Halvorsen of the Department of Paediatrics at Rikshospatalet in Oslo, Norway (Fig 9), provided an underlying explanation for the observations made by MacKay, Gairdner and Schulman (Halvorson, 1963). He observed that, compared with the blood of healthy adults, umbilical cord blood of healthy neonates had a high erythropoietin concentration, but the concentration was considerably higher in the plasma of severely erythroblastotic, anaemic infants. Among the healthy infants, erythropoietin levels fell to unmeasurably low concentrations after delivery, but levels remained elevated in hypoxic and cyanotic infants. Dr Per Haavardsholm Finne, also of the Children’s Department, Paediatric Research Institute and Department of Obstetrics and Gynaecology at Rikshospitalet in Oslo, observed high oncentrations of erythropoietin in the amniotic fluid and the umbilical cord blood after fetal hypoxia (Finne, 1964, 1967).

In subsequent studies, Dr Halvorsen observed lower plasma erythropoietin concentrations in the cord blood of preterm infants at delivery than in term neonates at delivery (Halvorsen & Finne, 1968). These observations supported the concept of Gairdner et al (1952a, b) that the postnatal fall in erythropoiesis (the `physiologic anaemia’ of neonates) is as a result of an increase in oxygen delivery to tissues following birth and is mediated by a fall in circulating erythropoietin concentration. The observations gave rise to the postulate that the `anaemia of prematurity’ was an exaggeration of this physiological anaemia and involved a limitation of preterm infants to appropriately increase erythropoietin production.

Many landmark reports of haematological findings of neonates that were published between 1925 and 1975 were not detailed in this review because they were outside the restricted topics selected.

Robert D. Christensen, MD, Gainesville, FL
Brit J Haem 2001; 113: 853-860

Towards Molecular Medicine; Reminiscences of the Haemoglobin Field

When historians of medicine in the twentieth century start to piece together the complex web of events that led from a change of emphasis of medical research from studies of patients and their organs to disease at the levels of cells and molecules they will undoubtedly have their attention drawn to the haemoglobin field, particularly the years that followed Linus Pauling’s seminal paper in 1949 which described sickle-cell anaemia as a `molecular disease’. These are personal reminiscences of some of the highlights of those exciting times, and of those who made them happen.

One of my first patients serving the RAMC was a Nepalese Ghurka child who was kept alive from the first few months of life with regular blood transfusion without a diagnosis. Henry Kunkel published a paper which described how, using electrophoresis in slabs of starch, he had found a minor component of human haemoglobin (Hb), Hb A2, the proportion of which was elevated in some carriers of thalassaemia. After several weeks spent knee deep in potato starch, we found that the Ghurka child’s parents had increased Hb A2 levels and, hence, that she was likely to be homozygous for thalassaemia. I was hauled up before the Director General of Medical Services for the Far East Land Forces and told that I could be court marshalled for not getting permission from the War House (Office) to publish information about military personnel. `And, in any case’, he added, `it is bad form to tell the world that one of our pukka regiments has bad genes; don’t do it again’.

Just before the end of my National Service I arranged to go to Johns Hopkins Hospital in Baltimore to train in genetics and haematology. I was told that I was wasting my time working on haemoglobin because there was `nothing left to do’. `Start exploring red cell enzymes’, he suggested. On arriving in Baltimore in 1960 it turned out that human genetics, and the haemoglobin field in particular, were bubbling with excitement and potential. The only lessons for those contemplating careers in medical research from this chapter of academic and military gaffs are that, regardless of the working conditions, when there are sick people there are always interesting research questions to be asked.

The excitement of the haemoglobin field in 1960 reflected the chance amalgamation of several disciplines in the 1950s, particularly X-ray crystallography, protein chemistry, human genetics and haematology.

From the early 1930s the structure of proteins became one of the central problems of biochemistry. At that time, the only way of tackling this problem was by X-ray crystallography. In 1937 Felix Haurowitz suggested to Max Perutz (Fig 1) that an X-ray study of haemoglobin might be a good subject for his doctoral thesis. He was given some large crystals of horse methaemoglobin which gave excellent Xray diffraction patterns.

Max Perutz

Max Perutz

However, there was a major snag; an X-ray diffraction pattern provided only half the information required to solve the structure of a protein, that is the amplitudes of diffracted rays, while the other half, their phases, could not be determined. But in 1953, they discovered that it could be solved in two dimensions by comparison of the diffraction patterns of a crystal of native haemoglobin with that of haemoglobin reacted with mecuribenzoate, which combines with its two reactive sulphydryl groups. In short, to solve the structure in three dimensions required the comparison of the diffraction patterns of at least three crystals, one native and two with heavy atoms combined with different sites on the haemoglobin molecule. In 1959 this approach yielded the first three-dimensional model of haemoglobin, at 5´5 AÊ resolution.

Protein chemistry evolved side-by-side with X-ray crystallography during the 1950s. In 1951 Fred Sanger solved the structure of insulin, a remarkable tour de force which showed that proteins have unique chemical structures and amino acid sequences. Sanger had perfected methods for fractionation and characterization of small peptides by paper chromatography or electrophoresis. In 1956 Vernon Ingram (Fig 2), who, like Max Perutz, was a refugee from Germany, was set the task of studying the structure of haemoglobin from patients with sickle-cell anaemia. Ingram separated the peptides produced after globin had been hydrolysed with the enzyme trypsin, which cuts only at lysine and arginine residues. Although these amino acids accounted for 60 residues per mol of haemoglobin, only 30 tryptic peptides were obtained, indicating that haemoglobin consists of two identical half molecules. Re-examination of the amino-terminal sequences of haemoglobin by groups in the United States and Germany showed 2 mols of valine ± leucine and 2 mols of valine ± histidine ± leucine per mol of globin. These findings, which were in perfect agreement with the X-ray crystallographic results, suggested that haemoglobin is a tetramer composed of two pairs of unlike peptide chains, which were called α and β.

A seminal advance, and one which was to mark the beginning of molecular medicine, was the chance result of an overnight conversation on a train journey between Denver and Chicago. Linus Pauling, the protein chemist, and William Castle (Fig 3), one of the founding fathers of experimental haematology, were returning from a meeting in Denver and Castle mentioned to Pauling that he and his colleagues had noticed that when red cells from patients with sickle-cell anaemia are deoxygenated and sickle they show birefringence in polarized light.

Five generations of Boston haematology. Seated is William Castle. Standing (left to right) are Stuart Orkin, David Nathan and Alan Michelson. The picture on the left is of Dean David Edsall of Harvard Medical School who established the Thorndyke Laboratory at the Boston City Hospital. He was succeeded by Dean Peabody, who recruited both George Minot, who won the Nobel Prize for his work on pernicious anaemia, and William Castle, who should have also received it.

Pauling guessed that this might reflect a structural difference between normal and sickle-cell haemoglobin which could be detected by a change in charge. He gave this problem to one of his postdoctoral students, a young medical graduate called Harvey Itano. At that time they knew that a Swede, Arne Tiselius, had invented a machine for separating proteins according to their charge by electrophoresis. As there was no machine of this kind in Pauling’s laboratory, Itano and his colleagues set to and built one. Eventually they found that the haemoglobin of patients with sickle-cell anaemia behaves differently to that of normal people in an electric field, indicating that it must have a different amino acid composition. Even better, the haemoglobin of sickle-cell carriers was a mixture of both types of haemoglobin. This work was published in Science in 1949, under the title `Sickle-cell anaemia: a molecular disease’.

Perutz and Crick suggested to Ingram that he should apply Sanger’s techniques of peptide analysis to see if he could find any difference between normal and sickle cell haemoglobin. After digesting haemoglobin with trypsin, Ingram separated the peptides by electrophoresis and chromatography in two dimensions to produce what he later called `fingerprints’. He recalls that his first efforts looked like a watercolour that had been left out in the rain. But gradually things improved and he was able to show that the fingerprints of Hbs A and S were identical except for the position of one peptide. Using a method that had been developed a few years earlier by Pehr Edman, which allowed a peptide to be degraded one amino acid at a time in a stepwise fashion, Ingram found that this difference was due to the substitution of valine for glutamic acid at position 6 in the β chain of Hb S.

As well as demonstrating how a crippling disease can result from only a single amino acid difference in the haemoglobin molecule, this beautiful work had broader implications for molecular genetics. Although nothing was known about the nature of the genetic code at the time, the findings were compatible with the notion that the primary product of the β-globin gene is a peptide chain, a further development of the one-gene-one-enzyme concept, suggested earlier by Beadle and Tatum from their studies of Neurospora, and a prelude to the later studies of Yanofsky on Escherichia coli, which were to confirm this principle.

With the advent of simple filter paper electrophoresis, haemoglobin analysis became the province of clinical research laboratories during the 1950s and `new’ abnormal haemoglobins appeared almost by the week. Although many scientists were involved it was Hermann Lehmann (Fig 4) who became the father figure. Like Handel, Hermann was born in Halle and, also like the composer, made his home in Great Britain. He came to England as a refugee and at the beginning of the Second World War had a short period of internment as a `friendly alien’ at Huyton, close to Liverpool, an experience shared with many others, including Max Perutz. He travelled widely during his later war service in the RAMC and developed a wide international network which enabled him to discover 81 haemoglobin variants during his career.

Harvey Itano and Elizabeth Robinson showed that Hb Hopkins 2 is an a chain variant. Hence, it was now clear that there must be at least two unlinked loci involved in regulating haemoglobin production, a and b. The discovery of the λ and δ chains of Hbs F and A2, respectively, meant that there must be at least four loci involved. Subsequent family studies and analyses of unusual variants resulting from the production of δβ or λβ fusion chains led to the ordering of the non-α globin genes.

It had been known for some years that children with severe forms of thalassaemia might have persistent production of HbF and it was found later that some carriers might have elevated levels of Hb A2. The seminal observation in favour of this notion came from the study of patients who had inherited the sickle-cell gene from one parent and thalassaemia from the other. Sickle-cell thalassaemia was first described by Ezio Silvestroni and his wife Ida Bianco in 1946, although at the time they could not have known the full significance of their finding.  Phillip Sturgeon and his colleagues in the USA found that the pattern of haemoglobin production in patients with sickle-cell thalassaemia is quite different to that of heterozygotes for the sickle-cell gene; the effect of the thalassaemia gene is to reduce the amount of Hb A to below that of Hb S, i.e. exactly the  opposite to the ratio observed in sickle-cell carriers. As it was known that the sickle-cell mutation occurs in the β globin gene, it could be inferred that the action of the thalassaemia gene was to reduce the amount of β globin production from the normal allele. Indeed, from the few family studies available in 1960 there was a hint that this form of thalassaemia might be an allele of the β globin gene. Another major observation that was made in the mid-50 s was the association of unusual tetramer haemoglobins, β4 (Hb H) and λ4 (Hb Bart’s), with a thalassaemia phenotype. In 1959 Vernon Ingram and Tony Stretton proposed in a seminal article that there are two major classes, α and β, just as there are two major types of structural haemoglobin variants. They extended the ideas of Linus Pauling and Harvey Itano, who had suggested that defective globin synthesis in thalassaemia might be due to `silent’ mutations of the β globin genes, and postulated that the defects might lie outside the structural gene in the area of DNA in the connecting unit. work on the interactions of thalassaemia and haemoglobin variants in the late 1950s had moved the field to a considerably higher level of understanding than is apparent in the earlier papers of Pauling and Itano. In any case, in their paper Ingram and Stretton generously acknowledged the ideas of other workers, including Lehmann, Gerald, Neel and Ceppellini, that had allowed them to develop their conceptual framework of the general nature of thalassaemia. This interpretation of events, and the input of scientists from many different disciplines into these concepts, is supported by the published discussions of several conferences on haemoglobin held in the late 1950s.

Historical Review. Towards Molecular Medicine; Reminiscences of the Haemoglobin Field. D. J. Weatherall, Weatherall Institute of Molecular Medicine, University of Oxford. Brit J  Haem 115:729-738.

The Emerging Understanding of Sickle Cell Disease

The first indisputable case of sickle cell disease in the literature was described in a dental student studying in Chicago between 1904 and 1907 (Herrick, 1910). Coming from the north of the island of Grenada in the eastern Caribbean, he was first admitted to the Presbyterian Hospital, Chicago, in late December 1904 and a blood test showed the features characteristic of homozygous sickle cell (SS) disease. It was a happy coincidence that he was under the care of Dr James Herrick (Fig 1) and his intern Dr Ernest Irons because both had an interest in laboratory investigation and Herrick had previously presented a paper on the value of blood examination in reaching a diagnosis (Herrick, 1904-05). The resulting blood test report by Dr Irons described and contained drawings of the abnormal red cells (Fig 2) and the photomicrographs, showing irreversibly sickled cells.

People with positive sickle tests were divided into asymptomatic cases, `latent sicklers’, and those with features of the disease, `active sicklers’, and it was Dr Lemuel Diggs of Memphis who first clearly distinguished symptomatic cases called sickle cell anaemia from the latent asymptomatic cases which were termed the sickle cell trait (Diggs et al, 1933).

Prospective data collection in 29 cases of the disease showed sickling in all 42 parents tested (Neel, 1949), providing strong support for the theory of homozygous inheritance. A Colonial Medical Officer working in Northern Rhodesia (Beet, 1949) reached similar conclusions at the same time with a study of one large family (the Kapokoso-Chuni pedigree). The implication that sickle cell anaemia should occur in all communities in which the sickle cell trait was common and that its frequency would be determined by the prevalence of the trait did not appear to fit the observations from Africa. Despite a sickle cell trait prevalence of 27% in Angola, Texeira (1944) noted the active form of the disease to be `extremely rare’ and similar observations were made from East Africa. Lehmann and Raper (1949, 1956) found a positive sickling test in 45% of one community, from which homozygous inheritance would have predicted that nearly 10% of children had SS disease, yet not a single case was found. The discrepancy led to a hypothesis that some factor inherited from non-black ancestors in America might be necessary for expression of the disease (Raper, 1950).

The explanation for this apparent discrepancy gradually emerged. Working with the Jaluo tribe in Kenya, Foy et al (1951) found five cases of sickle cell anaemia among very young children and suggested that cases might be dying at an age before those sampled in surveys. A similar hypothesis was advanced by Jelliffe (1952) and was supported by data from the then Belgian Congo (Lambotte-Legrand Lambotte-Legrand, 1951, Lambotte-Legrand, 1952, Vandepitte, 1952). Although most cases were consistent with the concept of homozygous inheritance, exceptions continued to occur. Patients with a non-sickling parent of Mediterranean ancestry were later recognized to have sickle cell-β thalassaemia (Powell et al, 1950; Silvestroni & Bianco, 1952; Sturgeon et al, 1952; Neel et al, 1953a), a condition also widespread in African and Indian subjects that presents a variable syndrome depending on the molecular basis of the β thalassaemia mutation and the amount of HbA produced.

Phenotypically, there are two major groups in subjects of African origin, sickle cell-β+ thalassaemia manifesting 20-30% HbA and mutations at 229(A,G) or 288(C,T), and sickle cell-β0 thalassaemia with no HbA and mutations at IVS2-849(A,G) or IVS2-1(G,A). In Indian subjects, a more severe β thalassaemia mutation IVS1-5(G,C) results in a sickle cell-β+ thalassaemia condition with 3-5% HbA and a relatively severe clinical course.

Other double heterozygote conditions causing sickle cell disease include sickle cell-haemoglobin C (SC) disease, (Kaplan et al, 1951; Neel et al, 1953b), sickle cellhaemoglobin O Arab (Ramot et al, 1960), sickle cellhaemoglobin Lepore Boston (Stammatoyannopoulos & Fessas, 1963) and sickle cell-haemoglobin D Punjab (Cooke & Mack, 1934). The latter condition was first described in siblings in 1934, who were reinvestigated for confirmation of HbD (Itano, 1951), the clinical features reported (Sturgeon et al, 1955) and who were finally identified as HbD Punjab (Babin et al, 1964), representing a remarkable example of longitudinal observation and investigation in the same family over 30 years.

The maintenance of high frequencies of the sickle cell trait in the presence of almost obligatory losses of homozygotes in Equatorial Africa implied that there was either a very high frequency of HbS arizing by fresh mutations or that the sickle cell trait conveyed a survival advantage in the African environment. There followed a remarkable period in the 1950s when three prominent scientists were each addressing this problem in East Africa, Dr Alan Raper and Dr Hermann Lehmann in Uganda and Dr Anthony Allison in Kenya. It was quickly calculated that mutation rates were far too low to balance the loss of HbS genes from deaths of homozygotes (Allison, 1954a). An increased fertility of heterozygotes was proposed (Foy et al, 1954; Allison, 1956a) but never convincingly demonstrated. Raper (1949) was the first to suggest that the sickle cell trait might have a survival advantage against some adverse condition in the tropics and Mackey & Vivarelli (1952) suggested that this factor might be malaria. The close geographical association between the distribution of malaria and the sickle cell gene supported this concept (Allison, 1954b) and led to an exciting period in the history of research in sickle cell disease.

The first observations on malaria and the sickle cell trait were from Northern Rhodesia where Beet (1946, 1947) noted that malarial parasites were less frequent in blood films from subjects with the sickle cell trait. Allison (1954c) drew attention to this association, concluding that persons with the sickle cell trait developed malaria less frequently and less severely than those without the trait. This communication marked the beginning of a considerable controversy.Two studies failed to document differences in parasite densities between `sicklers’ and `non-sicklers’ (Moore et al, 1954; Archibald & Bruce-Chwatt, 1955) and Beutler et al (1955) were unable to reproduce the inoculation experiments of Allison (1954c). Raper (1955) speculated that some feature of Allison’s observations had accentuated a difference of lesser magnitude and postulated that the sickle cell trait might inhibit the establishment of malaria in non-immune subjects. The conflicting results in these and other studies appear to have occurred because the protective effect of the sickle cell trait was overshadowed by the role of acquired immunity. Examination of young children before the development of acquired immunity confirmed both lower parasite rates and densities in children with the sickle cell trait (Colbourne & Edington, 1956; Edington & Laing, 1957; Gilles et al, 1967) and it is now generally accepted that the sickle cell trait confers some protection against falciparum malaria during a critical period of early childhood between the loss of passively acquired immunity and the development of active immunity (Allison, 1957; Rucknagel & Neel, 1961; Motulsky, 1964). The mechanism of such an effect is still debated, although possible factors include selective sickling of parasitized red cells (Miller et al, 1956; Luzzatto et al, 1970) resulting in their more effective removal by the reticulo-endothelial system, inhibition of parasite growth by the greater potassium loss and low pH of sickled red cells (Friedman et al, 1979), and greater endothelial adherence of parasitized red cells (Kaul et al, 1994).

The occurrence of the sickle cell mutation and the survival advantage conferred by malaria together determine the primary distribution of the sickle cell gene. Equatorial Africa is highly malarial and the sickle cell mutation appears to have arisen independently on at least three and probably four separate occasions in the African continent, and the mutations were subsequently named after the areas where they were first described and designated the Senegal, Benin, Bantu and Cameroon haplotypes of the disease (Kulozik et al, 1986; Chebloune et al, 1988; Lapoumeroulie et al, 1992). The disease seen in North and South America, the Caribbean and the UK is predominantly of African origin and mostly of the Benin haplotype, although the Bantu is proportionately more frequent in Brazil (Zago et al, 1992). It is therefore easy to understand the common misconception held in these areas that the disease is of African origin.

However, the sickle cell gene is widespread around the Mediterranean, occurring in Sicily, southern Italy, northern Greece and the south coast of Turkey, although these are all of the Benin haplotype and so, ultimately, of African origin. In the Eastern province of Saudi Arabia and in central India, there is a separate independent occurrence of the HbS gene, the Asian haplotype. The Shiite population of the Eastern Province traditionally marry first cousins, tending to increase the prevalence of SS disease above that expected from the gene frequency (Al-Awamy et al, 1984). Furthermore, extensive surveys performed by the Anthropological Survey of India estimate an average sickle cell trait frequency of 15% across the states of Orissa, Madhya Pradesh and Masharastra which, with the estimated population of 300 million people, implies that there may be more cases of sickle cell disease born in India than in Africa. The Asian haplotype of sickle cell disease is generally associated with very high frequencies of alpha thalassaemia and high levels of fetal haemoglobin, both factors believed to ameliorate the severity of the disease.

The promotion of sickling by low oxygen tension and acid conditions was first recognized by Hahn & Gillespie (1927) and further investigated by others (Lange et al, 1951; Allison, 1956b; Harris et al, 1956). The morphological and some functional characteristics of irreversibly sickled cells were described (Diggs & Bibb, 1939; Shen et al, 1949), but the essential features of the polymerization of reduced HbS molecules had to await the developments of electron microscopy (Murayama, 1966; Dobler & Bertles, 1968; Bertles & Dobler, 1969; White & Heagan, 1970) and Xray diffraction (Perutz & Mitchison, 1950; Perutz et al, 1951). The early observations on the inducement of sickling by hypoxia led to the first diagnostic tests utilizing sealed chambers in which oxygen was removed by white cells (Emmel, 1917), reducing agents such as sodium metabisulphite (Daland & Castle, 1948) or bacteria such as Escherichia coli (Raper, 1969). These slide sickling tests are very reliable with careful sealing and the use of positive controls, but require a microscope and some expertise in its use. An alternative method of detecting HbS utilizes its relative insolubility in hypermolar phosphate buffers (Huntsman et al, 1970), known as the solubility test. Both the slide sickle test and the solubility test detect the presence of HbS, but fail to make the vital distinction between the sickle cell trait and forms of sickle cell disease. This requires the process of haemoglobin electrophoresis, which detects the abnormal mobility of HbS, HbC and many other abnormal haemoglobins within an electric field.

The contributions of several workers on the determinants of sickling (Daland & Castle, 1948), birefringence of deoxygenated sickled cells (Sherman, 1940) the lesser degree of sickling in very young children which implied that it was a feature of adult haemoglobin (Watson, 1948) led Pauling to perform Tiselius moving boundary electrophoresis on haemoglobin solutions from subjects with sickle cell anaemia and the sickle cell trait. The demonstration of electrophoretic and, hence, implied chemical differences between normal, sickle cell trait and sickle cell disease led to the proposal that it was a molecular disease (Pauling et al, 1949). The chance encounter between Castle and Pauling who shared a train compartment returning from a meeting in Denver in 1945, its background and implications, has passed into the folklore of medical research (Conley, 1980; Feldman & Tauber, 1997).

The nature of this difference was soon elucidated. The haem groups appeared identical, suggesting that the difference resided in the globin, but early chemical analyses revealed no distinctive differences (Schroeder et al, 1950; Huisman et al, 1955). Analyses of terminal amino acids also failed to reveal differences, although an excess of valine in HbS was noted but considered an experimental error (Havinga, 1953). The development of more sensitive methods of fingerprinting combining high voltage electrophoresis and chromatography allowed the identification of the essential difference between HbA and HbS. This method enabled the separation of constituent peptides and demonstrated that a peptide in HbS was more positively charged than in HbA (Ingram, 1956). This peptide was found to contain less glutamic acid and more valine, suggesting that valine had replaced glutamic acid (Ingram, 1957). The sequence of this peptide was shown to be Val-His-Leu-Thr-Pro-Val-Glu-Lys in HbS instead of the Val-His-Leu-Thr-Pro-Glu-Glu-Lys in HbA (Hunt & Ingram, 1958), a sequence which was subsequently identified as the amino-terminus of the b chain (Hunt & Ingram, 1959). This amino acid substitution was consistent with the genetic code and was subsequently found to be attributable to the nucleotide change from GAG to GTG (Marotta et al, 1977).

Haemolysis and anaemia. The presence of anaemia and jaundice in the first four cases suggested accelerated haemolysis, which was supported by elevated reticulocyte counts (Sydenstricker et al, 1923) and expansion of the bone marrow (Sydenstricker et al, 1923; Graham, 1924). The bone changes of medullary expansion and cortical thinning were noted in early radiological reports (Vogt & Diamond, 1930; LeWald, 1932; Grinnan, 1935). Drawing on a comparison of sickle cell disease and hereditary spherocytosis, Sydenstricker (1924) introduced the term `haemolytic crisis’ that has persisted in the literature to this day, despite the lack of evidence for such an entity in sickle cell disease. The increased requirements of folic acid and the consequence of a deficiency leading to megaloblastic change was not noted until much later (Zuelzer & Rutzky, 1953; Jonsson et al, 1959; MacIver & Went, 1960).

The haemoglobin level in SS disease of African origin is typically between 6 and 9 g/dl and is well tolerated, partly because of a marked shift in the oxygen dissociation curve (Scriver & Waugh, 1930; Seakins et al, 1973) so that HbS within the red cell behaves with a low oxygen affinity. This explains why patients at their steady state haemoglobin levels rarely show classic symptoms of anaemia and fail to benefit clinically from blood transfusions intended to improve oxygen delivery.

Graham R. Serjeant
Sickle Cell Trust, Kingston, Jamaica
Brit J Haem 2001; 112: 3-18

The Immune Haemolytic Anaemias

The growth in knowledge of the scientific basis of haemolytic anaemias, which have been a main interest of the author, has been remarkable, as have consequent advances in the practice of medicine since the mid-1930s. At that time, the cause and mechanism of important disorders such as the acquired antibody determined (immune) haemolytic anaemias, haemolytic disease of the newborn, hereditary spherocytosis and paroxysmal nocturnal haemoglobinuria were unknown or but partially understood.

According to Crosby (1952), William Hunter of London, in an article on pernicious anaemia published in 1888, was the first to use the term `haemolytic’ to denote an anaemia caused by excessive blood destruction. By the turn of the century, the term was being widely used in clinical literature. Peyton Rous, in his comprehensive review `Destruction of the red blood corpuscles in health and disease’ (Rous, 1923), concluded that the generally held view in the early 1930s was that about one-fifteenth of the erythrocyte mass was destroyed daily. Rous was aware of the pioneer work of Winifred Ashby (1919), who, by following the survival of serologically distinct but compatible transfused erythrocytes, had found that normal erythrocytes might live for up to 100 d in the recipients’ circulation. Subsequent work using radioactive chromium (51Cr) as an erythrocyte label, showed that Ashby’s data and conclusions were in fact correct, i.e. that normal erythrocytes in health circulate in the peripheral blood for approximately 110 d. Erythrocyte labelling with 51Cr also had a further advantage over the Ashby method in addition to enabling the life-span of the patients’ erythrocytes to be assessed in the circulation by surface counting, to detect and measure the accumulation of radioactivity in the spleen and liver, and thereby assess the organs’ role in haemolysis

In the first decade of the twentieth century Widal et al (1908a) and Le Gendre & Brulea (1909) reported that autohaemoagglutination was a striking finding in some cases of icteare heamolytique acquis, and also Chauffard & Trosier (1908) and Chauffard & Vincent (1909) had described the presence of haemolysins in the serum of patients suffering from intense haemolysis. The conclusion was that abnormal immune processes, i.e. the development of auto-antibodies damaging the patients’ own erythrocytes, might play a part in the genesis of some cases of acquired haemolytic anaemia. This was indeed antedated by the classic observations of Donath & Landsteiner (1904) and Eason (1906) on the mechanism of haemolysis in paroxysmal cold haemoglobinuria.

That blood might auto-agglutinate when chilled had been described by Landsteiner (1903) and that an unusual degree of the phenomenon might complicate some types of respiratory disease was reported by Clough & Richter (1918) and later by Wheeler et al (1939). A few years later Peterson et al (1943) and Horstmann & Tatlock (1943) reported that cold auto-agglutinins at high titres were frequently found in the serum of patients who had suffered from the then so called primary atypical pneumonia.

Stats & Wasserman’s (1943) review on cold haemagglutination was a valuable contribution to contemporary knowledge. They listed in a table as many as 94 references to papers published between 1890 and 1943 in which cold haemagglutination had been described. In 32 of the papers the patients referred to had suffered from increased haemolysis

Recognition that cold auto-antibodies played an important role in the pathogenesis of some cases of haemolytic anaemia led to the concept that auto-immune haemolytic anaemia (AIMA) might usefully be classified into warm antibody or cold-antibody types, according to whether the patient is forming (warm) antibodies which react (perhaps optimally) at body temperature or (cold) antibodies which react strongly at low temperatures (e.g. 48C) but progressively less well as the temperature is raised and are perhaps inactive at 37oC. The clinical syndrome suffered by the patient would depend not only on the amount of antibody produced but also on its temperature requirement. Another important advance in understanding has been the realization that both types of AIHA could develop in association with a wide range of underlying disorders (secondary AIHA) as well as `idiopathically’, i.e. for no obvious cause (primary AIHA). The author’s own experience was summarized in a review (Dacie & Worlledge, 1969): 99 out of 210 cases of warm AIHA were judged to be secondary as were 39 out of 85 cases of cold AIHA. Petz & Garratty (1980), summarized the data from six centres: 55% out of a total of 656 cases had been reported as secondary. They listed the disorders with which warm antibody AIHA had been associated as chronic lymphocytic leukaemia, Hodgkin’s disease, non-Hodgkin’s lymphomas, thymomas, multiple myeloma, Waldenstrom’s macroglobulinaemia, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, infectious disease/ childhood viral disorders, hypogammaglobulinaemia, dysglobulinaemias, other immune deficiency syndromes, and ulcerative colitis.

Conley (1981), in an interesting review of warm-antibody AIHA patients seen at the Johns Hopkins Hospital, emphasized how important it was to carry out a careful enquiry into the patient’s past history and also to undertake a prolonged follow-up. He stated that a retrospective review of 33 patients whose illnesses in the past have been designated `idiopathic” had revealed an associated immunologically related disorder in 19 of them. An additional three patients had developed a lymphoma 2±10 years after they had developed AIHA. As already referred to, warm-antibody AIHA is now known to complicate a wide range of underlying diseases, particularly malignant lymphoproliferative disorders, other auto-immune disorders and immune deficiency syndromes. What proportion of patients suffering from a lymphoproliferative disorder develop AIHA is an interesting question. Duehrsen et al (1987) stated that this had occurred in 12 out of 637 patients. Early data on the incidence of a positive DAT in SLE were provided by Harvey et al (1954) – in six out of 34 patients tested the DAT had been positive. Later, Mongan et al (1967), who had studied a large number of patients suffering from a variety of connective tissue disorders, reported that the DAT had been positive in 15 out of 23 patients with SLE, none of whom, however, had suffered from overt haemolytic anaemia. It has also been realized since the 1960s that warm-antibody AIHA may develop in patients suffering from a variety of immune deficiency syndromes, both congenital and acquired.

It was in the mid-1960s that it was realized that, in a significant proportion of patients thought to have `idiopathic’ warm-antibody AIHA, the development of the causal auto-antibodies had been triggered in some way by a drug the patient was taking. The first drug implicated was the antihypertensive drug a-methyldopa (Aldomet) (Carstairs et al, 1966a,b). Following the finding that treating hypertensive patients with a-methyldopa led to the formation of anti-erythrocyte auto-antibodies in a significant percentage of patients, renewed interest was taken in the possibility that other drugs might have the same effect. Two main hypotheses have been advanced in relation to how certain drugs in some patients appear to have caused the development of anti-erythrocyte auto-antibodies. One hypothesis was that the drug or its metabolites act on the immune system so as to impair immune tolerance; the other was that the drug affects antigens at the erythrocyte surface in such a way that a normally active immune system responds by developing anti-erythrocyte antibodies. Clearly, too, the patient’s individuality must be an important factor, for only a proportion of patients receiving the same dosage of the offending drug for the same period of time develop a positive DAT and only a small percentage develop overt AIHA.

An interesting development in the history of the immune haemolytic anaemias was the realization in the mid-1950s that, rather rarely, haemolysis was brought about by the patient developing antibodies that were directed against a drug the patient had been taking and that the erythrocytes were in some way secondarily involved. The first drug to be implicated was Fuadin (stibophen), which had been used to treat a patient with schistosomiasis (Harris, 1954, 1956). The patient’s serum contained an antibody that agglutinated his own or normal erythrocytes and/or sensitized them to agglutination by antiglobulin sera; however, this occurred only in the presence of the drug.

In the late 1940s, several accounts of patients with AIHA who had persistently low platelet counts were published, e.g. Fisher (1947) and Evans & Duane (1949); and it was suggested that the patients might have been forming autoantibodies directed against platelets. This concept was further developed by Evans et al (1951). Eight out of their 18 patients with AIHA were thrombocytopenic; four had clinically obvious purpura. Evans et al (1951) suggested that there exists `a spectrum-like relationship between acquired haemolytic anaemia and thrombocytopenic purpura’; also that `on the one hand, acquired haemolytic anaemia with sensitization of the red cells is often accompanied with thrombocytopenia, while, on the other hand, primary thrombocytopenic purpura is frequently accompanied with red cell sensitization with or without haemolytic anaemia’. Many further case reports of AIHA accompanied by severe thrombocytopenia have since been published

There are two features in the blood film of a patient with an acquired haemolytic anaemia which indicate that he or she is suffering from AIHA; one is auto-agglutination, the other is erythrophagocytosis. Spherocytosis, although often present to a marked degree, is of course found in other types of haemolytic anaemia.

The pioneer French observations on auto-agglutination already referred to were generally overlooked until the late 1930s, and serological studies seem seldom to have been undertaken until the publication of Dameshek & Schwartz’s (1938b) report in which they described the presence of `haemolysins’ in cases of acute apparently acquired haemolytic anaemia. Dameshek & Schwartz (1940) summarized contemporary knowledge in an extensive review. They concluded that it was not improbable that haemolysins of various types and `dosages’ were in fact responsible for many cases of human haemolytic anaemias, including congenital haemolytic anaemia, which they suggested might be caused by the `more or less continued action of an haemolysin’.

Six years were to pass before the concept that an abnormal immune mechanism played a decisive role in some cases of acquired haemolytic anaemia was clearly demonstrated by Boorman et al (1946), who reported that the erythrocytes of five patients with acquired acholuric jaundice had been agglutinated by an antiglobulin serum, i.e. that the newly described antiglobulin reaction or Coombs test (Coombs et al, 1945) was positive, while the test had been negative in 28 patients suffering from congenital acholuric jaundice. This work aroused great interest and was soon confirmed.

Until the 1950s, the auto-antibodies responsible for AIHA were generally concluded to be `non-specific’. According to Wiener et al (1953), `Red cell auto-antibodies react not only with the individual’s own red cells but also with the erythrocytes of all other human beings. The substances on the red blood cell envelope with which the auto-antibodies combine are agglutinogens like the ABO, MN and RhHr systems, except that, in the former case, the blood factors with which the auto-antibodies react are not type specific but are shared by all human beings.’ They suggested that the auto-antibodies might be directed to the `nucleus of the RhHr substance’. Earlier work had, however, indicated that the sensitivity of normal group-compatible erythrocytes to a patient’s auto-antibody might vary considerably (Denys & van den Broucke, 1947; Kuhns & Wagley, 1949). That auto-antibodies might have a clearly defined Rh specificity, e.g. anti-e, was described by Race & Sanger (1954) in the second edition of their book. Referring to Wiener et al (1953), they wrote: `This beautifully clear investigation made the present authors realize that a curious result obtained by one of them (Ruth Sanger) in 1953 in Australia had after all been true; the serum of a man who had died of a haemolytic anaemia 3000 miles away contained anti-e; his cells were clearly CDe-cde’. A similar finding, i.e. an auto-anti-e, was described by Weiner et al (1953).

A further development in the unravelling of a complicated story was the realization that some of the antibodies which appeared to be specific were reacting with more basic antigens, although showing a preference for specific antigens, i.e. some specific auto-antibodies appeared to be less specific than their allo-antibody counterparts. Moreover, some antibodies, reacting with specific antigens, have been shown to be partially or completely absorbable by antigen negative cells.

Many apparently `non-specific’ antidl antibodies have been shown to be not strictly `nonspecific’ but to react with antigens of very high frequency, e.g. to be anti-Wrb, anti-Ena, anti-LW or anti-U. Issitt et al (1980)) listed six additional very common antigens that had been identified as targets for anti-dl auto-antibodies, i.e. Hr, Hro, Rh34, Rh29, Kpb and K13.

In relation to human acquired haemolytic anaemia, the discovery in the late 1940s and 1950s that many cases were apparently brought about by the development of damaging anti-erythrocyte antibodies led to intense interest and speculation into the why and how of auto-antibody formation. Of seminal importance at the time were the experiments and theoretical arguments of Burnet (Burnet & Fenner, 1949; Burnet, 1957, 1959, 1972) and the studies on transplantation immunity of Medawar (Billingham et al, 1953; Medawar, 1961). Of particular interest, too, was the report by Bielschowsky et al (1959) of the occurrence of AIHA in an inbred strain of mice – the NZB/BL strain. Remarkably, by the time the mice were 9-months-old the DAT was positive in almost every mouse. Burnet (1963) referred to the gift of the mice to the Walter and Eliza Hall Institute of Medical Research, Melbourne as `the finest gift the Institute has ever received’.

Exactly how is it that auto-antibodies reacting with an erythrocyte surface antigen result in the cell’s premature destruction? The possible role of auto-agglutination in bringing about haemolysis was emphasized by Castle and colleagues as the result of a series of studies carried out in the 1940s and 1950s. As summarized by Castle et al (1950), an antibody which appears to be incapable of causing `lysis in vitro might bring about the following sequence of events in vivo. (1) Red cell agglutination in the peripheral blood; (2) red cell sequestration and separation from plasma in tissue capillaries; (3) ischaemic injury of tissue cells with release of substances that increase the osmotic and mechanical fragilities of red cells locally; (4) local osmotic lysis of red cells or subsequent escape of mechanically fragile red cells into the blood stream where the traumatic motion of the circulation causes their destruction’.

We can expect, as the years pass, that more and more will be known as to the intricate mechanisms that bring about self-tolerance and the mechanisms underlying the occurrence of auto-immune disorders in general, including the role of infectious agents, drugs and genetic factors. Patients with immune haemolytic anaemias can be expected to benefit from the new knowledge; for in parallel with a better understanding as to how immune self-tolerance breaks down will hopefully be the development of more effective drugs and therapies aimed at controlling the breakdown.

The Immune Haemolytic Anaemias: A Century of Exciting Progress in Understanding.  Sir John Dacie, Emeritus Professor of Haematology.
Brit J Haem 2001; 114: 770-785.

A History of Pernicious Anaemia

This is a review of the ideas and observations that have led to our current understanding of pernicious anaemia (PA). PA is a megaloblastic anaemia (MA) due to atrophy of the mucosa of the body of the stomach which, in turn, is brought about by autoimmune factors.

A case report by Osler & Gardner (1877) in Montreal could be that of PA. This anaemic patient had numbness of the fingers, hands and forearms; the red blood cells were large; at autopsy the gastric mucosa appeared atrophic and the marrow had large numbers of erythroblasts with finely granular nuclei. The increased marrow cellularity had also been noted by Cohnheim (1876).

Ehrlich (1880) (Fig 1) distinguished between cells he termed megaloblasts present in the blood in PA from normoblasts present in anaemia as a result of blood loss. Not only were large red blood cells noted in PA, but irregular red cells, ? poikilocytes, were reported in wet blood preparations by Quincke (1877). Megaloblasts in the marrow during life were first noted by Zadek (1921). Hypersegmented neutrophils in peripheral blood in PA were described by Naegeli (1923) and came to be widely recognized after Cooke’s study (Cooke, 1927). The giant metamyelocytes in the marrow were described by Tempka & Braun (1932).

Paul Ehrlich

Paul Ehrlich

Fig 1. Paul Ehrlich (Wellcome Institute Library, London).

The association between PA and spinal cord lesions was described by Lichtheim (1887) and a full account was published by Russell et al (1900), who coined the term `subacute combined degeneration of the spinal cord’ (SCDC) although they were not convinced of its relation to PA. Arthur Hurst at Guy’s Hospital, London, confirmed the association of the neuropathy with PA and added, too, the association of loss of hydrochloric acid in the gastric juice (Hurst & Bell, 1922). Cabot (1908) found that numbness and tingling of the extremities were present in almost all of his 1200 patients and 10% had ataxia. William Hunter (1901) noted the prevalence of a sore tongue in PA, which was present in 40% of Cabot’s series.

In 1934, the Nobel Prize in medicine and physiology was awarded to Whipple, Minot and Murphy. Was there ever an award more deserved? They saved the lives of their patients and pointed the way forward for further research. What was there in liver that was lacking in patients with PA? The effect of liver in restoring the anaemia in Whipple’s iron-deficient dogs was by supplying iron which is  abundant in liver.

Liver given by mouth also provides Cbl and folic acid. But patients with PA cannot absorb Cbl, although some 1% of an oral dose can cross the intestinal mucosa by passive diffusion; this, presumably, is what happened when large amounts of liver were eaten. Beef liver contains about 110 mg of Cbl per 100 g and about 140 mg of folate per 100 g. Cbl is stable and generally resistant to heat; folate is labile unless preserved with reducing agents. The daily requirement of Cbl by man is l-2 mg. The liver diet, if consumed, had enough of these haematinics to provide a response in most MAs.

George Richard Minot

George Richard Minot

George Richard Minot (Wellcome Institute Library, London).

The availability of liver extracts brought about interest in the nature of the haematological response. An optimal response required a peak rise of reticulocytes 5±7 d after the injection of liver extract and the height of the peak was greatest in those with severe anaemia; the flood of reticulocytes was as a result of a synchronous maturation of a vast number of megaloblasts into red cells. There is a steady rise in the red cell count to reach 3 x 1012/l in the 3rd week (Minot & Castle, 1935). Many liver extracts did not have enough antianaemic factor to achieve this and some assayed by the author had only 1-2 mg of Cbl.  It took another 22 years for a pure antianaemic factor to be isolated, although, admittedly, the Second World War intervened; in 1948, an American group led by Karl Folkers and an English group led by E. Lester-Smith published, within weeks of each other, the isolation of a red crystalline substance termed vitamin B12 and subsequently renamed cobalamin.

The structure of this red crystalline compound was studied by the nature of its degradation products and by X-ray crystallography. It soon became apparent that there was a cobalt atom at the heart of the structure and this heavy atom was of great aid to the crystallographers, so much so that, with additional information from the chemists, they were the first to come up with the complete structure. To quote Dorothy Hodgkin: `To be able to write down a chemical structure very largely from purely crystallographic evidence on the arrangement of atoms in space – and the chemical structure of a quite formidably large molecule at that – is for any crystallographer, something of a dream-like situation’. As Lester-Smith (1965) pointed out, it also required some 10 million calculations. In 1964, Dorothy Hodgkin was awarded the Nobel Prize for chemistry.

Barker et al (1958) published an account of the metabolism of glutamate by a Clostridium. The glutamate underwent an isomerization and an orange-coloured co-enzyme was involved that turned out to be Cbl with a deoxyadenosyl group attached to the cobalt.

This Cbl co-enzyme, deoxyadenosylCbl, is the major form of Cbl in tissues; it is also extremely sensitive to light, being changed rapidly to hydroxoCbl. DeoxyadenosylCbl is concerned with the metabolism of methylmalonic acid in man (Flavin & Ochoa, 1957). The other functional form of Cbl is methylCbl involved in conversion of homocysteine to methionine (Sakami & Welch, 1950). Both these pathways are impaired in PA in relapse.

Cbl consists of a ring of four pyrrole units very similar to that present in haem. These, however, have the cobalt atom in the centre instead of iron and the ring is called the corrin nucleus. The cobalamins have a further structure, a base, termed benzimidazole, set at right angles to the corrin nucleus and this may have a link to the cobalt atom (base on position).

By the time Cbl had been isolated from liver it was already known that it was also present in fermentation flasks growing bacteria such as streptomyces species. Other organisms gave higher yields so that kilogram quantities of pure Cbl were obtained; these sources have replaced liver in the production of Cbl. By adding radioactive form of cobalt to the fermentation flasks instead of ordinary cobalt, labelled Cbl became available (Chaiet et al, 1950). The importance of labelled Cbl is that it made it possible to carry out Cbl absorption tests in patients, to design isotope dilution assays for serum Cbl, to design ways of assaying intrinsic factor (IF), to detect antibodies to IF and even to measure glomerular filtratration rate, as free Cbl is excreted by the glomerulus without any reabsorption by the renal tubules.

William Castle at the Thorndike Memorial Laboratory, Boston City Hospital, devised experiments to explore the relationship between gastric juice, the anti-anaemic factor that Castle assumed, correctly, was also present in beef, and the response in PA. The question Castle asked was `Was it possible that the stomach of the normal person could derive something from ordinary food that for him was equivalent to eating liver?’.

The experiment in untreated patients with PA consisted of two consecutive periods of 10 d or more during which daily reticulocyte counts were made. During the first period of 10 d, the PA patient received 200 g of lean beef muscle (steak) each day. There was no reticulocyte response. During the second period, the contents of the stomach of a healthy man were recovered 1 h after the ingestion of 300 g of steak; about 100 g could not be recovered. The gastric contents were incubated for a few hours until liquefied and then given to the PA patient through a tube. This was done daily. On day 6 there was a rise in reticulocytes reaching a peak on day 10, followed by a rise in the red cell count. The response was similar to that obtained with large amounts of oral liver.

Thus, Castle concluded that a reaction was taking place between an unknown intrinsic factor (IF) in the gastric juice and an unknown extrinsic factor in beef muscle. Whereas Minot & Murphy (1926) found that 200-300 g of liver daily was needed to get a response in PA, 10 g liver was adequate when incubated with 10-20 ml normal gastric juice (Reiman & Fritsch, 1934). Castle’s extrinsic factor is the same as the anti-anaemic factor that is Cbl, and IF is needed for its absorption. Presumably the gastric juice in PA lacks IF.

The elegant studies of Hoedemaeker et al (1964) in Holland using autoradiography of frozen sections of human stomach incubated with [57Co]-Cbl showed that IF was produced in the gastric parietal cell. The binding of Cbl to

the parietal cell was abolished by first incubating the section with a serum containing antibodies to IF. The parietal cell in man is thus the source of both hydrochloric acid and IF. The parietal cell is the only source of IF in man as a total gastrectomy is invariably followed by a MA due to Cbl deficiency. IF is a glycoprotein with a molecular weight of 45 000.

Assay of protein fractions of serum after electrophoresis showed that endogenous Cbl is in the position of α-1 globulin. Chromatography of serum after addition of [57Co]-Cbl on Sephadex G-200 showed that Cbl was attached to two proteins, one eluting before the albumin termed transcobalamin I (TCI) and the other after the albumin termed transcobalamin II (TCII). Charles Hall showed that, when labelled Cbl given by mouth is absorbed, it first appears in the position of TCII and later in the position of TCI as well (Hall and Finkler, l965). They concluded that TCII is the prime Cbl transport protein carrying Cbl from the gut into the blood and then to the liver from where it is redistributed by both new TCII as well as TCI. Congenital absence of a functional TCII causes a severe MA in the first few months of life owing to an inability to transport Cbl. Most of the Cbl in serum is on TCI because it has a relatively long half-life of 9±10 d, whereas the half-life of TCII is about 1.5 h. Thus, in assaying the serum Cbl level, it is mainly TCI-Cbl that is being assayed.

With the availability of labelled Cbl, Cbl absorption tests began to be widely used in the 1950s. The commonest method was the urinary excretion test described by Schilling (1953). Here, an oral dose of radioactive Cbl is followed by an injection of 1000 mg of cyano-Cbl. The free cyano-Cbl is largely excreted into the urine over the next 24 h and carries with it about one third of the absorbed labelled Cbl.

Parietal cell antibodies (Taylor et al, 1962) are present in serum in 76-93% of different series of PAs and in the serum of 36% of the relatives of PA patients. The antibody is present in sera from 32% of patients with myxoedema, 28% of patients with Graves’ disease, 20% of relatives of thyroid patients and 23% of patients with Addison’s disease. Parietal cell antibodies are found in between 2-16% of controls, the high 16% figure being in elderly women. There is a higher frequency of PA in women, the female to male ratio being 1.7 to 1.0. The parietal cell antibody is probably important in the production of gastric atrophy. Thyroid antibodies are present in sera from 55% of PAs, in sera from 50% of PA relatives, in 87% of sera from myxoedema patients, in 53% of sera in Graves’ disease and in 46% of relatives of patients with thyroid disease.

There is a high frequency of PA among those disorders that have antibodies against the target organ. Thus, among 286 patients with myxoedema, 9.0% also had PA (Chanarin, 1979), as compared with a frequency of PA of about 1 per 1000 (0.01%) in the general population. Of 102 consecutive patients with vitiligo,
eight also had PA.

Patients with acquired hypogammaglobulinaemia are unable to make humoral antibodies; nevertheless, one third have PA as well. This cannot be as a result of action of IF antibodies and must be because of specific cell-mediated immunity. Tai & McGuigan (1969) demonstrated lymphocyte transformation in the presence of IF in six out of 16 PA patients and Chanarin & James (1974) found 10 out of 51 tests were positive.

Twenty-five patients with PA were tested for the presence of humoral IF antibody in serum and gastric juice and for cell-mediated immunity against IF. All but one gave positive results in one or more tests. It was concluded that these findings establish the autoimmune nature of PA and that the immunity is not merely an interesting byproduct.

Patients with PA treated with steroids show a reversal of the abnormal findings characterizing the disease. If they are still megaloblastic, the anaemia will respond in the first instance (Doig et al, 1957), but in the longer term Cbl neuropathy may be precipitated. The absorption of Cbl improves and may become `normal’ (Frost & Goldwein, 1958). There is a return of IF in the gastric juice (Kristensen and Friis, 1960) and a decline in the amount of IF antibody in serum (Taylor, 1959). In some patients there is return of acid in the gastric juice. Gastric biopsy shows a return of parietal and chief cells (Ardeman & Chanarin, 1965b; Jeffries, 1965). All this is as a result of suppression of cell-mediated immunity against the parietal cell and against IF. Withdrawal of steroids leads to a slow return to the status quo.

The author has dipped freely into the two volumes by the late M. M. Wintrobe. These are: Wintrobe, M.M. (1985) Hematology, the Blossoming of a Science. Lea & Febinge

A History of Pernicious Anaemia
I. Chanarin, Richmond, Surrey
Brit J Haem 111: 407-415
History of Folic Acid

1928 Lucy Wills studied macrocytic anaemia in pregnancy in Bombay, India

1932 Janet Vaughn studied macrocytic anemia associated with coeliac disease and idiopathic steatorrhea (1932) showed a response to marmite

1941 Folic acid extracted from spinach and is a growth factor for S. Faecalis

1941 pteroylglutamic acid synthesized at Amer Cyanamide – Pteridine ring, paraminobenzoic acid, glutamine –  PGA differed from natural compound in some respects

1945 PGA resolved the macrocytic anemia, but not the neuropathy

1979 Stokstad and associates at Berkeley obtained the first purified mammalian enzymes involved in synthesis

Folate antagonists inhibit tumor growth (Hitchings and Elion)(Nobel)

  • Misincorporation of uracil instead of thymine into DNA

Sidney Farber introduced Aminopterine and also Methotrexate for treatment of childhood lymphoblastic leukemia

  • MTX inhibits DHFR enzyme (dihydrofolate reductase) necessary for THF

Wellcome introduces trimethoprim (antibacterial), and also pyramethoprime (antimalarial)

Homocysteine isolated by Du Vineaud, but it was not noticed

Finkelstein and Mudd demonstrated the importance of remethylation for tHy and worked out the transsulfuration pathway

  1. Function of methyl THF is remethylation of homocysteine
  2. Synthesized by MTHFR
Metabolism of folate

Metabolism of folate

Metabolism of folate

Allosterically regulated by S-adenosyl methionine (Stokstad)

MTHF also inhibits glycine methyl transferase controlling excess SAM – transmethylation

JD Finkelstein

JD Finkelstein

James D Finkelstein

  • Homocysteinuria – mental retardation, skeletal malformation, thromboembolic disease; deficiency of cystathionine synthase (controls trans-sulfuration)
  • NTDs – pregnancy
  • Hyperhomocysteinemia and VD

AD Hoffbrand and DG Weir
Brit J Haem 2001; 113: 579-589

The History of Haemophilia in the Royal Families of Europe Queen Victoria.

On 17 July 1998 a historic ceremony of mourning and commemoration took place in the ancestral church of the Peter and Paul Fortress in St Petersburg. President Boris Yeltsin, in a dramatic eleventh-hour change of heart, decided to represent his country when the bones of the last emperor, Tsar Nicholas II, and his family were laid to rest 80 years to the day after their assassination in Yekaterinberg (Binyon, 1998). He described it as ‘ironic that the Orthodox Church, for so long the bedrock of the people’s faith, should find it difficult to give this blessing the country had expected’. ‘I have studied the results of DNA testing carried out in England and abroad and am convinced that the remains are those of the Tsar and his family’ (The Times, 1998a). Unfortunately, politicians and the hierarchy of the Russian Orthodox Church had argued about what to do with the bones previously stored in plastic bags in a provincial city mortuary. Politics, ecclesiastical intrigue, secular ambition, and emotions had fuelled the debate. Yeltsin and the Church wanted to honour a man many consider to be a saint, but many of the older generation are opposed to the rehabilitation of a family which symbolizes the old autocracy.

Our story starts, almost inevitably, with Queen Victoria of England who had nine children by Albert, Prince of Saxe-Coburg-Gotha. Victoria was certainly an obligate carrier for haemophilia as over 20 individuals subsequently inherited the condition (Figs 1 and 2). Princess Alice (1843–78) was Victoria’s third child and second daughter. Having married the Duke of Hesse at an early age, Alice went on to have seven children, one of whom, Frederick (‘Frittie’) was a haemophiliac who died at the age of 3 following a fall from a window.

Prince Leopold with Sir William Jenner at Balmoral in 1877

Prince Leopold with Sir William Jenner at Balmoral in 1877

Prince Leopold with Sir William Jenner at Balmoral in 1877. (Hulton Deutsch Collection Ltd.)

Alexandra was the sixth child and was only 6 years old when her mother and youngest sister died. ‘Sunny’, as she became known, was a favourite of Queen Victoria, who as far as possible directed her upbringing from across the channel: Alexandra (Alix) was forced to eat her baked apples and rice pudding with the same regularity as her English cousins. Alix visited her older sister Elizabeth (Ella) on her marriage to Grand Duke Serge and met Tsarevich Nicholas for the first time: she was 12 and not impressed. Five years later they met again and Alix fell in love, but by now she had been confirmed in the Lutheran Church and religion became the solemn core of her life.

Victoria had other aspirations for Alix. She hoped that she would marry her grandson Albert Victor (The Duke of Clarence) and the eldest son of the Prince of Wales (later Edward VII). The Duke was an unimpressive young man who was somewhat deaf and had limited intellectual abilities. If this arrangement had proceeded then Alix’s haemophilia carrier status would have been introduced into the British Royal Family and the possibility of a British monarch with haemophilia might have become a reality; however, the Duke died in 1892.

Nicholas and Alexandra. Alix and Nicholas were married in 1894 one week after the death of Nicholas’s father (Alexander III). In the same way that Victoria, with her personal aspirations of a marriage between Alix and the Duke of Clarence, had not considered the possibility of haemophilia, neither did the St Petersburg hierarchy consider a marriage to Nicholas undesirable. Haemophilia was already well recognized in Victoria’s descendants. Her youngest son, Leopold, had already died, as had Frittie her grandson. The inheritance of haemophilia had been known for some time since its description by John Conrad Otto (Otto, 1803). However, it was as late as 1913 before the first royal marriage was declined because of the risk of haemophilia, when the Queen of Rumania decided against an association between her son, Crown Prince Ferdinand, and Olga, the eldest daughter of Nicholas and Alexandra. The Queen of Rumania was herself a granddaughter of Queen Victoria and therefore a potential haemophilia carrier!

Alix was received into the Russian Orthodox Church, taking the name of Alexandra Fedorova. The first duty of a Tsarina was to maintain the dynasty and produce a male heir, but between 1895 and 1901 Alix produced four princesses, Olga, Tatiana, Maria and Anastasia. Failure to produce a son made Alix increasingly neurotic and she had at least one false pregnancy. However, in early 1904 she was definitely pregnant.

For a month or so all seemed well with little Alexis, but it was then noticed that the Tsarevitch was bleeding excessively from the umbilicus (a relatively uncommon feature of haemophilia). At first the diagnosis was not admitted by the parents, but eventually the truth had to be faced although even then only by the doctors and immediate family. Alix was grief stricken: ‘she hardly knew a day’s happiness after she realized her boy’s fate’. As a newly diagnosed haemophilia carrier she dwelt morbidly on the fact that she had transmitted the disease. These feelings are well known to some haemophiliac mothers but the situation was different in Russia in the early twentieth century. The people regarded any defect as divine intervention. The Tsar, as head of the Church and leader of the people, must be free of any physical defect, so the Tsarevich’s haemophilia was concealed. The family retreated into greater isolation and were increasingly dominated by the young heir’s affliction (Fig 3).

Up to a third of haemophiliac males do not have a family history of the condition. This is usually thought to be the result of a relatively high mutation rate occurring in either affected males or female carriers. None of Queen Victoria’s ancestors, for many generations, showed any evidence of haemophilia. Victoria was therefore either a victim of a mutation, or the Duke of Kent was not her father.The mutation is unlikely to have been in her mother, Victoire, who had a son and daughter by her first marriage, and there is no sign of haemophilia in their numerous descendants.

Victoire was under considerable pressure to produce an heir. The year before Victoria was born, Princess Charlotte, the only close heir to the throne, had died and the Duke of Kent had somewhat reluctantly agreed to marry Victoire with the aim of producing an heir. The postulate that the Queen’s gardener had a limp has not been substantiated!

The Duke of Kent had no evidence of haemophilia (he was 51 when Victoria was born) but did inherit another condition from his father (George III): porphyria. While a young man in Gibralter he suffered bilious attacks which were recognized as being similar to his father’s complaint.

Had Queen Victoria carried the gene for porphyria we might expect that she would have at least as many descendants with this condition as had haemophilia. Until recently only two possible cases of porphyria have been suggested amongst Victoria’s descendants: Kaiser Wilhelm’s sister and niece (MacAlpine & Hunter, 1969), but they could have inherited it from their Hohenzollern ancestor, Frederick the Great. A recent television programme (Secret History, 1998) claims to have identified two more cases in Victoria’s descendants, Princess Victoria, the Queen’s eldest daughter, and Prince William of Gloucester, nephew of George V. If these two cases are correct then they would tend to confirm that Victoria was indeed the daughter of the Duke of Kent, but the apparent lack of more cases in Victoria’s extended family is difficult to understand. The gene for acute intermittent porphyria has been isolated on chromosome 11. There is still plenty of scope for further genetic analysis on the European Royal Families!

We can only speculate as to the impact on European events over the last 150 years if the marriages within the Royal houses had been different. What is evident is the dramatic effect of haemophilia on the Royal Princes and their families.

Empress Alexandra at the Tsarevich’s bedside during a haemophiliac crisis

Empress Alexandra at the Tsarevich’s bedside during a haemophiliac crisis

Empress Alexandra at the Tsarevich’s bedside during a haemophiliac crisis in 1912. (Radio Times Hulton Picture Library.)

Richard F. Stevens
Royal Manchester Children’s Hospital
Brit J Haem 1999, 105, 25–32

`The longer you can look back ± the further you can look forward’: Winston Churchill in an address to The Royal College of Physicians, London 1944. At the time that Churchill was speaking in 1944, leukaemia was a fatal disease that had been identified 100 years before. The disease was described as the dreaded leukaemias, sinister and poorly understood.

Thomas Hodgkin chose a career in medicine and enrolled as a pupil at Guy’s Hospital in London. Being a Quaker, however, he could not enter the English universities of Oxford and Cambridge and decided to follow the medical courses at Edinburgh. At that times, Aristotelian and Hippocratic medicine were greatly influencing British physicians. Hodgkin, still a medical student, wrote a paper `On the Uses of the Spleen’ where he reported his beliefs on the purposes of the spleen: to regulate fluid volume, clean impurities from the body, supply expandability to the portal system. The subject was a presage of the disease that bears his name.

Hodgkin interrupted his studies at Edinburgh to spend a year in Paris where he met many people who had a great influence in his life and future activities. Among them, were Laennec (Hodgkin played an important role in bringing the stethoscope to Great Britain); Baron von Humboldt who introduced Hodgkin to the field of anthropology; Baron Cuvier, a distinguished anatomist and palaeontologist; and Thomas A. Bowditch, whose expeditions to Africa had a great impact on Hodgkin’s future activities.

In 1825, Thomas Hodgkin returned to London to join the staff at Guy’s Hospital, and in 1826 he was made `Inspector of the Dead’ and `Curator of the Museum of Morbid Anatomy’. In developing the museum he had accumulated, by 1829, over 1600 specimens demonstrating the effects of disease. The correlation of clinical disease to pathological material was quite new: from analyses of pathological specimens Hodgkin was able to describe appendicitis with perforation and peritonitis, the local spread of cancer to draining lymph nodes, noting that the tumour had similar characteristics at both sides, and features of other diseases.

In his historic paper `On Some Morbid Appearances of the Absorbent Glands and Spleen’ (Hodgkin, 1832), he briefly described the clinical histories and gross postmortem findings on six patients from the experience at Guy’s Hospital and included another case sent to him in a detailed drawing by his friend Carswell (Fig 2). In the very first paragraph he wrote: `The morbid alterations of structure which I am about to describe are probably familiar to many practical morbid anatomists, since they can scarcely have failed to have fallen under their observation in the course of cadaveric inspection’. Hodgkin’s studies had convinced him that he was dealing with a primary disease of the absorbent (lymphatic) glands. `This enlargement of the glands appeared to be a primitive affection of those bodies, rather than the result of an irritation propagated to them from some ulcerated surface or other inflamed texture – Unless the word inflammation be allowed to have a more indefinite and loose eaning, this affection – can hardly be attributed to that cause’ was stated on pages 85 and 86 of his 1832 paper. Hodgkin also mentioned that the first reference that he could find to this or similar disease was in fact by Malpighi in 1666.

Wilks (1865) described the disease in detail and, made aware by Bright that the first observations were done by Hodgkin, linked his name permanently to this new entity in a paper entitled `Cases of Enlargement of the Lymphatic Glands and Spleen (or Hodgkin’s Disease) with Remarks’ (Fig 3).

In 1837 Thomas Hodgkin was the outstanding candidate for the position of Assistant Physician at Guy’s Hospital in succession to Thomas Addison who had been promoted to Physician. After 10 years spent as Inspector of the Dead, he had published a great deal, including a two-volume work entitled The Morbid Anatomy of Serous and Mucous Membrane.

Hodgkin, acting in his other capacity, had sent Benjamin Harrison a report on the terrible consequences to native Indians of monopoly trading and on the inhuman treatment they received from officials of the Hudson Bay Company, of which Harrison was the financier. when the opportunity to appoint an Assistant Physician occurred, Harrison exercised an autocratic rule over the hospital and presided at the appointment made by the General Court. Thomas Hodgkin did not get the job and the next day he resigned all his appointments at Guy’s Hospital. Social medicine, medical problems associated with poverty, antislavery, concern for underpriviledged groups such as American Indians and Africans, as well as a strong sense of responsibility defined his life after this separation.

Sternberg (1898) and Reed (1902) are generally credited with the first definitive and thorough descriptions of the histopathology of Hodgkin’s disease. Based on the findings observed in her case series, Dorothy Reed concluded `We believe then, from the descriptions in the literature and the findings in 8 cases examined, that Hodgkin’s disease has a peculiar and typical histological picture and could thus rightly be considered a histopathological disease entity’.

During the successive decades, pathologists began to describe a broader spectrum of histological features. However, it was Jackson and Parker who, in scientific papers and in their well-known book Hodgkin’s Disease and Allied Disorders (Jackson & Parker, 1947), presented the first serious effort at a histopathological classification. They assigned the name `Hodgkin’s granuloma’ to the main body of typical cases. A much more malignant variant, usually characterized by a great abundance of pleomorphic and anaplastic Reed-Sternberg cells and seen in a relativelysmall number of cases was named `Hodgkin’s sarcoma’. A third, similarly infrequent, variant characterized by an extremely slow clinical evolution, a relative paucity of Reed-Sternberg cells and a great abundance of lymphocytes was termed `Hodgkin’s paragranuloma’. It was only approximately 20 years later that Lukes & Butler (1966) reported a characteristic subtype of the heterogeneous `granuloma’ category, to which they assigned the name `nodular sclerosis’. They also proposed a new histopathological classification, still in use to date, with an appreciably greater prognostic relevance and usefulness than the

previous Jackson-Parker classification.

The first human bone marrow transfusion was given to a patient with aplastic anemia in 1939.9 This patient received daily blood transfusions, and an attempt to raise her leukocyte and platelet counts was made using intravenous injection of bone marrow. After World War II and the use of the atomic bomb, researchers tried to find ways to restore the bone marrow function in aplasia caused by radiation exposure. In the 1950s, it was proven in a mouse model that marrow aplasia secondary to radiation can be overcome by syngeneic marrow graft.10 In 1956, Barnes and colleagues published their experiment on two groups of mice with acute leukemia: both groups were irradiated as anti-leukemic therapy and both were salvaged from marrow aplasia by bone marrow transplantation.

The topics of leukemias and lymphomas will not be discussed further in  this discussion.

The related references are:

Leukaemia – A Brief Historical Review from Ancient Times to 1950
British Journal of Haematology, 2001, 112, 282-292

The Story of Chronic Myeloid Leukaemia
British Journal of Haematology, 2000, 110, 2-11

Historical Review of Lymphomas
British Journal of Haematology 2000, 109, 466-476

Historical Review of Hodgkin’s Disease
British Journal of Haematology, 2000, 110, 504-511

Multiple Myeloma: an Odyssey of Discovery
British Journal of Haematology, 2000, 111, 1035-1044

The History of Blood Transfusion
British Journal of Haematology, 2000, 110, 758-767

Hematopoietic Stem Cell Transplantation—50 Years of Evolution and Future Perspectives. Henig I, Zuckerman T.
Rambam Maimonides Med J 2014;5 (4):e0028.
http://dx.doi.org/10.5041/RMMJ.10162

Landmarks in the history of blood transfusion.

1666 Richard Lower (Oxford) conducts experiments involving transfusion of blood from one animal to another

1667 Jean Denis (Paris) transfuses blood from animals to humans

1818 James Blundell (London) is credited with being the first person to transfuse blood from one human to another

1901 Karl Landsteiner (Vienna) discovers ABO blood groups. Awarded Nobel Prize for Medicine in 1930

1908 Alexis Carrel (New York) develops a surgical technique for transfusion, involving anastomosis of vein in the recipient with artery in the donor. Awarded Nobel Prize for Medicine in 1912

1915 Richard Lewinsohn (New York) develops 0.2% sodium citrate as anticoagulant

1921 The first blood donor service in the world was established in London by Percy Oliver

1937 Blood bank established in a Chicago hospital by Bernard Fantus

1940 Landsteiner and Wiener (New York) identify Rhesus antigens in man

1940 Edwin Cohn (Boston) develops a method for fractionation of plasma proteins. The following year, albumin produced by this method was used for the first time to treat victims of the Japanese attack on Pearl Harbour

1945 Antiglobulin test devised by Coombs (Cambridge), which also facilitated identification of several other antigenic systems such as Kell (Coombs et al, 1946), Duffy (Cutbush et al, 1950) and Kidd (Cutbush et al, 1950)

1948 National Blood Transfusion Service (NBTS) established in the UK

1951 Edwin Cohn (Boston) and colleagues develop the first blood cell separator

1964 Judith Pool (Palo Alto, California) develops cryoprecipitate for the treatment of haemophilia

1966 Cyril Clarke (Liverpool) reports the use of anti-Rh antibody to prevent haemolytic disease of the newborn

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