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Posts Tagged ‘Immunology’


Approach to Controlling Pathogenic Inflammation in Arthritis

Curator: Larry H Bernstein, MD, FCAP

A network approach to controlling pathogenic inflammation: Sequence sharing pattern peptides downregulate experimental arthritis

a new approach to network regulation of inflammation based on

Chai Ezerzer, Raanan Margalit and Irun R. Cohen

Aberrant inflammation probably results from aberrant regulation of the molecules that mediate inflammation; the actual molecules mediating inflammation –

  • chemokines,
  • cytokines, and
  • growth factors and their receptors –
    • would appear to be normal in their chemical structure.

If faulty regulation is indeed the problem,

  • a reasonable approach to alleviating inflammatory diseases might be to influence the interactions
  • within the network of connectivity of the disease-associated proteins (DAPs).
Aberrant inflammation appears to be a pathogenic factor in autoimmune diseases and other noxious inflammatory
conditions in which the inflammatory process
  1. is misapplied,
  2. exaggerated,
  3. recurrent or chronic.
The protein molecules involved in pathogenic inflammation—
disease-associated proteins (DAP )
  1. chemokines,
  2. cytokines, and
  3. growth factors and their receptors,
  • appear normal; their networks of interaction are at fault.

These researchers asked the question – 

  • whether shared amino acid sequence motifs among DAPs
  • might identify novel peptide treatments for regulating inflammation.

We aligned the sequences of 37 DAPs previously discovered to be associated with arthritis

  • to uncover shared sequence motifs.

We focused on chemokine receptor molecules because

  • chemokines and chemokine receptors play important roles in directing the migration of inflammatory cells into sites of tissue inflammation.
  •  different chemokine receptors shared amino acid sequence motifs in their extra-cellular loop domains (ECL2);
  • the ECL2 loop is outside of the known ligand binding site.

These shared sequence motifs established what we term a sequence-sharing network (SSN). SSN motifs exhibited very low E-values,

  • indicating their preservation during evolution.
This study demonstrates a new
  • approach to network regulation of inflammation based on peptide sequence motifs
  • shared by the second extra-cellular loop (EC L2) of different chemokine receptors;
  • previously known chemokine receptor binding sites have not involved the EC L2 loop.
These motifs of 9 amino acids, which were detected by sequence alignment, manifest very low E-values
  • compared with slightly modified sequence variations,
  • indicating that they were not likely to have evolved by chance.
To test whether this shared sequence network (SSN) might serve a regulatory function,
  • theysynthesized 9-amino acid SSN peptides from the EC L2 loops of three different chemokine receptors.
Theye administered these peptides to rats during the
Two of the peptides significantly downregulated the arthritis; one of the peptides
  • synergized with non-specific anti-inflammatory treatment with dexamethasone.
These findings suggest that
  • the SSN peptide motif reported here is likely to have adaptive value in controlling inflammation.
  • detection of SSN motif peptides could provide a network-based approach to immune modulation.
administering a highly connected chemokine receptor peptide motif , as done here, induced
  • the downregulation of inflammation in a rat model of arthritis.
Thus, study of the SSN provides a new network approach toward modulating inflammation
English: Typical chemokine receptor structure ...

English: Typical chemokine receptor structure showing seven transmembrane domains and a chanracteristic “DRY” motif in the second intracelluar domain. (Photo credit: Wikipedia)

Structure of Chemokines

Structure of Chemokines (Photo credit: Wikipedia)

Chemokine receptor

Chemokine receptor (Photo credit: Wikipedia)

 

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Lipoxin A4 Regulates Natural Killer Cell in Asthma

Reporter: Larry H Bernstein, MD, FCAP

Lipoxin A4 Regulates Natural Killer Cell and Type 2 Innate Lymphoid Cell Activation in Asthma
 C Barnig, M Cernadas, S Dutile,…BR Levy.
Sci Transl Med  27 Feb 2013. ; 5(174): p. 174ra26  SciTranslMed.             http://dx.doi.org/10.1126/scitranslmed.3004812
Asthma is a prevalent disease of chronic inflammation in which
  • endogenous counterregulatory signaling pathways are dysregulated.
Recent evidence suggests that innate lymphoid cells (ILCs), including
  • natural killer (NK) cells and
  • type 2 ILCs (ILC2s),
    • can participate in the regulation of allergic airway responses,
    • in particular airway mucosal inflammation.
Sci Transl Med 27 February 2013:  5(174) 174ra26        http://dx.doi.org/10.1126/scitranslmed.3004812
Both NK cells and ILC2s expressed
  • the pro-resolving ALX/FPR2 receptors.
Lipoxin A4, a natural pro-resolving ligand for ALX/FPR2 receptors, significantly
  • increased NK cell–mediated eosinophil apoptosis and
  • decreased IL-13 release by ILC2s.
Together, these findings indicate that ILCs are targets for lipoxin A4
  • to decrease airway inflammation and mediate the catabasis of eosinophilic inflammation

Molecular biology for formyl peptide receptors in human diseases
Yongsheng Li , 

Leukocytes accumulate at sites of inflammation and immunological reaction in response to locally existing chemotactic mediators. The first chemotactic factors structurally defined were N-formyl peptides. Subsequently, numerous ligands were identified

FPRs interact with this menagerie of structurally diverse pro- and anti-inflammatory ligands to possess important regulatory effects in multiple diseases, including

  1. inflammation,
  2. amyloidosis,
  3. Alzheimer’s disease,
  4. prion disease,
  5. acquired immunodeficiency syndrome,
  6. obesity,
  7. diabetes, and
  8. cancer.

How these receptors recognize diverse ligands and how they contribute to disease pathogenesis and host defense are basic questions currently under investigation that

    • would open up new avenues for the future management of inflammation-related diseases.

FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexin-derived peptide-stimulated phagocytosis 
PMaderna, DC Cottell, T Toivonen, N Dufton, J Dalli, M Perretti and C Godson
The FASEB Journal Nov 2010; 24 (11): 4240-4249      Published online June 22, 2010, http://dx.doi.org/10.1096/fj.10-159913

Lipoxins (LXs) are endogenously produced eicosanoids with well-described anti-inflammatory and proresolution activities,

  • stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages.

LXA4 and the glucocorticoid-derived annexin A1 peptide (Ac2–26) bind to a common G-protein-coupled receptor, termed FPR2/ALX. However, direct evidence of the involvement of FPR2/ALX in the anti-inflammatory and proresolution activity of LXA4 is still to be investigated. Here we describe FPR2/ALX trafficking in response to LXA4 and Ac2–26 stimulation. We have transfected cells with HA-tagged FPR2/ALX and studied receptor trafficking in unstimulated, LXA4 (1–10 nM)- and Ac2–26 (30 μM)-treated cells using multiple approaches that include immunofluorescent confocal microscopy, immunogold labeling of cryosections, and ELISA and investigated receptor trafficking in agonist-stimulated phagocytosis. We conclude that PKC-dependent internalization of FPR2/ALX is required for phagocytosis. Using bone marrow-derived macrophages (BMDMs) from mice in which the FPR2/ALX ortholog Fpr2 had been deleted, we observed

  • the nonredundant function for this receptor in LXA4 and Ac2–26 stimulated phagocytosis of apoptotic neutrophils.
  1. LXA4 stimulated phagocytosis 1.7-fold above basal (P<0.001) by BMDMs from wild-type mice, whereas no effect was found on BMDMs from Fpr2−/− mice.
  2. Ac2–26 stimulates phagocytosis by BMDMs from wild-type mice 1.5-fold above basal (P<0.05), but  Ac2–26 failed to stimulate phagocytosis by BMDMs isolated from Fpr2−/− mice.

These data reveal novel and complex mechanisms of the FPR2/ALX receptor trafficking and functionality in the resolution of inflammation.—
Maderna, P., Cottell, D. C., Toivonen, T., Dufton, N., Dalli, J., Perretti, M., Godson, C.
http://www.FASEB.j.org/FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexin-derived peptide-stimulated phagocytosis.
We have transfected cells with HA-tagged FPR2/ALX and studied receptor trafficking in unstimulated, LXA4 (1–10 nM)- and Ac2–26 (30 μM)-treated cells using multiple approaches and conclude that PKC-dependent internalization of FPR2/ALX is required for phagocytosis. Using bone marrow-derived macrophages (BMDMs) from mice in which the FPR2/ALX ortholog Fpr2 had been deleted,

  • we observed the nonredundant function for this receptor in LXA4 and Ac2–26 stimulated phagocytosis of apoptotic neutrophils.

LXA4 stimulated phagocytosis 1.7-fold above basal (P<0.001) by BMDMs from wild-type mice,

  • whereas no effect was found on BMDMs from Fpr2−/− mice.

Ac2–26 stimulates phagocytosis by BMDMs from wild-type mice 1.5-fold above basal (P<0.05)

  •  Ac2–26 failed to stimulate phagocytosis by BMDMs isolated from Fpr2−/− mice relative to vehicle.

These data reveal novel and complex mechanisms of the FPR2/ALX receptor trafficking and functionality in the resolution of inflammation.
The lipoxin receptor ALX: potent ligand-specific and stereoselective actions in vivo.
Chiang, N., Serhan, CN, Dahlen, SE, Drazen, JM, Hay, DW, Rovati, GE, et al.
Pharmacol. Rev. 2006; 58, 463–487.      http://www.PharmacolRev.com/The_lipoxin_receptor_ALX:_potent_ligand_specific_and_stereoselective_actions_in_vivo/

Asthma Obstruction of the lumen of the bronchi...

Asthma Obstruction of the lumen of the bronchiole by mucoid exudate, goblet cell metaplasia, epithelial basement membrane thickening and severe inflammation of bronchiole. (Photo credit: Wikipedia)

Schematic diagram indicating the complementary...

Schematic diagram indicating the complementary activities of cytotoxic T-cells and NK cells. (Photo credit: Wikipedia)

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Immunoreactivity of Nanoparticles

Author: Tilda Barliya PhD

As nanotechnology progresses from research and development to commercialization and use, it is likely that manufactured nanomaterials and nanoproducts will be released into the environment.

Adverse effects of nanoparticles on human health depend on individual factors such as genetics and existing disease, as well as exposure, and nanoparticle chemistry, size, shape, agglomeration state, and electromagnetic properties. Animal and human studies show that inhaled nanoparticles are lessefficiently removed than larger particles by the macrophage clearance mechanisms in the lung,causing lung damage, and that nanoparticles can translocate through the circulatory, lymphatic, and nervous systems to many tissues and organs, including the brain.

The key to understanding the toxicity of nanoparticles is that their minute size, smaller than cells and cellular organelles, allows them to penetrate these basic biological structures, disrupting their normal function. Examples of toxic effects include tissue inflammation, and altered cellular redox balance toward oxidation, causing abnormal function or cell death. http://arxiv.org/ftp/arxiv/papers/0801/0801.3280.pdf

Some NPs happen to be toxic to biological systems, others are relatively benign, while others confer health benefits. As current knowledge of the toxicology of ‘bulk’ materials may not suffice in reliably predicting toxic forms of nanoparticles, ongoing and expanded study of ‘nanotoxicity’ will be necessary. For nanotechnologies with clearly associated health risks, intelligent design of materials and devices is needed to derive the benefits of these new technologies while limiting adverse health impacts.

Human skin, lungs, and the gastro-intestinal tract are in constant contact with the environment. While the skin is generally an effective barrier to foreign substances, the lungs and gastro-intestinal tract are more vulnerable. These three ways are the most likely points of entry for natural or anthropogenic nanoparticles. Injections and implants are other possible routes of exposure, primarily limited to engineered materials. Due to their small size, nanoparticles can translocate from these entry portals into the circulatory and lymphatic systems, and ultimately to body tissues and organs. Some nanoparticles, depending on their composition and size, can produce irreversible damage to cells by oxidative stress or/and organelle injury.

Are they biocompatible? Do the nanoparticles enter the lymphatic and circulatory systems? If not, do they accumulate in the skin and what are the long-term effects of accumulation? Do they produce inflammation? If they enter the lymphatic and circulatory system, is the amount significant? What are the long-term effects of this uptake? Related to the beneficial antioxidant properties of some nanomaterials, long-term effect need to be studied, in addition to the short-term antioxidant effect. What is the long-

term fate of these nanoparticles? Are they stored in the skin? Do they enter circulation? What happens when the nanoparticles undergo chemical reactions and lose their antioxidant properties?

For a full view of the questions needed to be addressed please visit. http://bdds.fudan.edu.cn/…/fdfa2aa9-df2b-4c9f-a2a5-a33ee29acb76.pdf

The answers to some of these questions are known, and will be presented in the chapter dedicated to nanoparticles toxicity, however most of the remaining questions still remain unanswered.

The immunostimulatory properties of nanoparticles discussed here include their antigenicity, adjuvant properties, inflammatory responses and the mechanisms through which nanoparticles are recognized by the immune system. Since this is a very complicated mechanism , the factors affecting the immune response are summaried here:

Size

  • Th1/Th2 stimulation
  • Adjuvent properties
  • Internalization/phagocytic uptake
  • Hapten properties
  • Particle clearance

Charge

  • Toxicity to immune cells
  • Binding plasma proteins
  • Particle clearance
  • Immune cell stimulation

Hydrophobicity

  • Interaction with plasma proteins
  • Internalization/phagocytic uptake
  • Immune cell stimulation
  • Particle clearance

Targeting

  • Immunogenicity

For example: In general, cationic (positively-charged) particles are more likely to induce inflammatory reactions than anionic (negativelycharged) and neutral species. For example, anionic generation- 4.5 PAMAM dendrimers did not cause human leukocytes (white blood cells) to secrete cytokines53 but cationic liposomes induced secretion of cytokines such as TNF, IL-12 and IFNγ. Systemic administration of another cationic nanoliposome alone or in combination with bacterial DNA did not induce cytokine production but increased the expression of DC surface markers, CD80/CD86, which are important in the inflammatory response.

Trace impurities within the nanomaterial formulation can also frequently induce an inflammatory response. Early studies suggest that carbon nanotubes induce inflammatory reactions, but a more recent study shows that they don’t when they are purified.

Another consideration in the inflammatory response is maintaining the Th1/Th2 response — the inflammatory reaction.  triggered by Th cells that direct and activate other immune cells such as B and T cells and macrophages to secrete different cytokines. This response is important for protecting against cancer cells and pathogens and to avoid hypersensitivity (undesirable and exaggerated immune response) reactions. Several studies have addressed the influence of nanoparticles on Th1 and Th2 responses. Large (>1 μm) industrialized particles induced the Th1 response, whereas smaller ones (<500 nm) were associated with Th2.

In contrast, some small engineered nanoparticles such as 500 nm PLGA, 270 nm PLGA65, 80 nm and 100 nm nanoemulsions, 95 nm and 112 nm PEG–PHDA nanoparticles, and 123 nm dendrosome induced the Th1 response, while 5mn 5th generation PAMAM dendrimers didn’t cause overall inflammatory reaction in vivo but weakly induced Th2 cytokine production.

Therefore, more structure–activity relationship studies are required to understand how size, surface modification and charge of engineered particles influence the Th1/Th2 balance

Particle stimulation of adaptive (acquired) immunity has also been described. For example, small (<100 nm) polystyrene particles promoted CD8 and CD4 T-cell responses and were associated with higher antibody levels than larger (>500 nm) particles. Understanding the mechanisms requires further investigation, and is important for nanovaccine formulation development.

Phagosome-mediated processing and presentation of nanoparticles may differ from that of ‘canonical’ antigens. Certain biodegradable nanoparticles can be taken up through conventional pathogen-specific routes and can stimulate inflammatory reactions just like pathogens

More mechanistic studies are required to understand how the immune system manages non-biodegradable components of nanoparticles (for example, metallic cores). Many questions remain regarding processing of multi-component and multi functional nanoparticles. Are the individual components (the coating, core, and so on) stable inside the phagosome or do they separate? Are the biodegradable and non-biodegradable components processed together or individually?

Immunotoxicological analysis of new molecular entities is not a straightforward process, and there is no universal guide for immunotoxicity.

Conclusions:

The mechanism of cellular uptake of nanoparticles and the biodistribution depend on the physico-chemical properties of the particles and in particular on their surface characteristics. Moreover, as particles are mainly recognized and engulfed by immune cells special attention should be paid to nano–immuno interactions. It is also important to use primary cells for testing of the biocompatibility of nanoparticles, as they are closer to the in vivo situation when compared to transformed cell lines.

Understanding the unique characteristics of engineered nanomaterials and their interactions with biological systems is key to the safe implementation of these materials in novel biomedical diagnostics and therapeutics.

The main challenge in immunological studies of nanomaterials is choosing an experimental approach that is free of falsepositive or false-negative readouts. The majority of the standard immunotoxicological methods are applicable to nanomaterials. However, as nanoparticles represent physically and chemically diverse materials, the classical methods cannot always be applied without modification, and novel approaches may be required. For example, many nanoparticles absorb in the UV–Vis range and some particles may catalyse enzyme reactions or quench fluorescent dyes commonly used as detection reagents in various end-point or kinetic assays. These and other methodological

challenges in preclinical evaluation of nanoparticles are reviewed in detail elsewhere.

Both ‘classical’ and novel imunotoxicological assessments of nanomaterials clearly need a scrupulous stepwise validation, standardization, and demonstration of their physiological relevance.

Industry, academics, and federal agencies are now collaborating to identify critical parameters in nanoparticles characterization and to establish acceptance criteria for nanomaterial-specific assays.

Ref.

1.Cristina Buzea, Ivan. I. Pacheco Blandino, and Kevin Robbie. Nanomaterials and nanoparticles:Sources and toxicity. Biointerphases vol. 2, issue 4 (2007) pages MR17 – MR172 http://arxiv.org/ftp/arxiv/papers/0801/0801.3280.pdf

2. Marina A. Dobrovolskaia* and Scott E. McNeil. Immunological properties of engineered nanomaterials. Nature Nanotechnology 2007; 2; 469-479.  http:// bdds.fudan.edu.cn/…/fdfa2aa9-df2b-4c9f-a2a5-a33ee29acb76.pdf

3.  Kunzmanna A,  Anderssonb B, Thurnherrc T, Krugc H, Scheyniusb A,  Fadeel B. Toxicology of engineered nanomaterials: Focus on biocompatibility, biodistribution and biodegradation. Biochimica et Biophysica Acta (BBA) – General Subjects. Volume 1810, Issue 3, March 2011, Pages 361–373 http://www.sciencedirect.com/science/article/pii/S0304416510001145

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