Immunological Context Research Articles

Control of cytokine production by human fc gamma receptors: implications for pathogen defense and autoimmunity.

Control of cytokine production by immune cells is pivotal for counteracting infections via orchestration of local and systemic inflammation. Although their contribution has long been underexposed, it has recently become clear that human Fc gamma receptors (FcγRs), which are receptors for the Fc region of immunoglobulin G (IgG) antibodies, play a critical role in this process by controlling tissue- and pathogen-specific cytokine production. Whereas individual stimulation of FcγRs does not evoke cytokine production, FcγRs cell-type specifically interact with various other receptors for selective amplification or inhibition of particular cytokines, thereby tailoring cytokine responses to the immunological context. The physiological function of FcγR-mediated control of cytokine production is to counteract infections with various classes of pathogens. Upon IgG opsonization, pathogens are simultaneously recognized by FcγRs as well as by various pathogen-sensing receptors, leading to the induction of pathogen class-specific immune responses. However, when erroneously activated, the same mechanism also contributes to the development of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. In this review, we discuss control of cytokine production as a novel function of FcγRs in human innate immune cells in the context of homeostasis, infection, and autoimmunity and address the possibilities for future therapeutic exploitation.

Phagocytosis of immunoglobulin-coated emulsion droplets

Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. The size of targets to be eliminated ranges from small particles as bacteria to large objects as cancerous or senescent cells. Most of our current quantitative knowledge on phagocytosis is based on the use of solid polymer microparticles as model targets that are well adapted to the study of phagocytosis mechanisms that do not involve any lateral mobility of the ligands, despite the relevance of this parameter in the immunological context. Herein we designed monodisperse, IgG-coated emulsion droplets that are efficiently and specifically internalized by macrophages through in-vitro FcγR-mediated phagocytosis. We show that, contrary to solid polymeric beads, droplet uptake is efficient even for low IgG densities, and is accompagnied by the clustering of the opsonins in the zone of contact with the macrophage during the adhesion step. Beyond the sole interest in the design of the material, our results suggest that lateral mobility of proteins at the interface of a target greatly enhances the phagocytic uptake.

High preexisting serological antibody levels correlate with diversification of the influenza vaccine response.

Reactivation of memory B cells allows for a rapid and robust immune response upon challenge with the same antigen. Variant influenza virus strains generated through antigenic shift or drift are encountered multiple times over the lifetime of an individual. One might predict, then, that upon vaccination with the trivalent influenza vaccine across multiple years, the antibody response would become more and more dominant toward strains consistently present in the vaccine at the expense of more divergent strains. However, when we analyzed the vaccine-induced plasmablast, memory, and serological responses to the trivalent influenza vaccine between 2006 and 2013, we found that the B cell response was most robust against more divergent strains. Overall, the antibody response was highest when one or more strains contained in the vaccine varied from year to year. This suggests that in the broader immunological context of viral antigen exposure, the B cell response to variant influenza virus strains is not dictated by the composition of the memory B cell precursor pool. The outcome is instead a diversified B cell response. Vaccine strategies are being designed to boost broadly reactive B cells present in the memory repertoire to provide universal protection to the influenza virus. It is important to understand how past exposure to influenza virus strains affects the response to subsequent immunizations. The viral epitopes targeted by B cells responding to the vaccine may be a direct reflection of the B cell memory specificities abundant in the preexisting immune repertoire, or other factors may influence the vaccine response. Here, we demonstrate that high preexisting serological antibody levels to a given influenza virus strain correlate with low production of antibody-secreting cells and memory B cells recognizing that strain upon revaccination. In contrast, introduction of antigenically novel strains generates a robust B cell response. Thus, both the preexisting memory B cell repertoire and serological antibody levels must be taken into consideration in predicting the quality of the B cell response to new prime-boost vaccine strategies.

Host-pathogen interactions and immune evasion strategies in Francisella tularensis pathogenicity.

Francisella tularensis is an intracellular Gram-negative bacterium that causes life-threatening tularemia. Although the prevalence of natural infection is low, F. tularensis remains a tier I priority pathogen due to its extreme virulence and ease of aerosol dissemination. F. tularensis can infect a host through multiple routes, including the intradermal and respiratory routes. Respiratory infection can result from a very small inoculum (ten organisms or fewer) and is the most lethal form of infection. Following infection, F. tularensis employs strategies for immune evasion that delay the immune response, permitting systemic distribution and induction of sepsis. In this review we summarize the current knowledge of F. tularensis in an immunological context, with emphasis on the host response and bacterial evasion of that response.

Interacting symbionts and immunity in the amphibian skin mucosome predict disease risk and probiotic effectiveness.

Pathogenesis is strongly dependent on microbial context, but development of probiotic therapies has neglected the impact of ecological interactions. Dynamics among microbial communities, host immune responses, and environmental conditions may alter the effect of probiotics in human and veterinary medicine, agriculture and aquaculture, and the proposed treatment of emerging wildlife and zoonotic diseases such as those occurring on amphibians or vectored by mosquitoes. Here we use a holistic measure of amphibian mucosal defenses to test the effects of probiotic treatments and to assess disease risk under different ecological contexts. We developed a non-invasive assay for antifungal function of the skin mucosal ecosystem (mucosome function) integrating host immune factors and the microbial community as an alternative to pathogen exposure experiments. From approximately 8500 amphibians sampled across Europe, we compared field infection prevalence with mucosome function against the emerging fungal pathogen Batrachochytrium dendrobatidis. Four species were tested with laboratory exposure experiments, and a highly susceptible species, Alytes obstetricans, was treated with a variety of temperature and microbial conditions to test the effects of probiotic therapies and environmental conditions on mucosome function. We found that antifungal function of the amphibian skin mucosome predicts the prevalence of infection with the fungal pathogen in natural populations, and is linked to survival in laboratory exposure experiments. When altered by probiotic therapy, the mucosome increased antifungal capacity, while previous exposure to the pathogen was suppressive. In culture, antifungal properties of probiotics depended strongly on immunological and environmental context including temperature, competition, and pathogen presence. Functional changes in microbiota with shifts in temperature provide an alternative mechanistic explanation for patterns of disease susceptibility related to climate beyond direct impact on host or pathogen. This nonlethal management tool can be used to optimize and quickly assess the relative benefits of probiotic therapies under different climatic, microbial, or host conditions.

TLR/NCR/KIR: Which One to Use and When?

By means of a complex receptor array, Natural killer (NK) cells can recognize variable patterns of ligands and regulate or amplify accordingly their effector functions. Such NK receptors include old, rather conserved, molecules, such as toll-like receptors (TLRs), which enable NK cells to respond both to viral and bacterial products, and newer and evolving molecules, such as killer Ig-like receptors and natural cytotoxicity receptors, which control NK cytotoxicity and are responsible for the elimination of virus-infected or tumor cells without damaging self-unaltered cells. In addition, to rapidly gain new functions NK cells also can acquire new receptors by trogocytosis. Thus, NK cells may have adapted their receptors to different functional needs making them able to play a key role in the modulation of critical events occurring in several compartments of human body (primarily in SLCs but also in decidua during pregnancy). In this review, we will discuss on how the various types of receptors can be used to address specific functions in different immunological contexts.

A human dendritic cell-based in vitro model to assess Mycobacterium tuberculosis SO2 vaccine immunogenicity.

Among the tuberculosis (TB) vaccine candidates, SO2 is the prototype of the first live-attenuated vaccine that recently entered into clinical trials. To investigate the capacity of SO2 to stimulate an appropriate immune response in vitro within a human immunological context, a comparative analysis of the effects promoted by SO2, the current Bacille Calmette-Guerin (BCG) vaccine and Mycobacterium tuberculosis (Mtb) was conducted in human primary dendritic cells (DC), which are critical modulators of vaccine-induced immunity. In particular, we found that SO2 promotes the expression of maturation markers similarly to BCG but at a lower extent than Mtb. Moreover, SO2-infected DC released higher levels of interleukin (IL)-23 than BCG-infected cells, which account for the expansion of interferon (IFN)-γ-producing T cells in an IL-12-independent manner. In the autologous mixed leukocyte reaction setting, the expansion of IL-17-producing T cells was also observed in response to SO2 infection. Interestingly, apoptosis and autophagic flux, events required for the antigen presentation within MHC class II complex, were not affected in DC infected with SO2, conversely to what observed upon Mtb stimulation. Collectively, our results indicate that SO2 represents a promising TB vaccine candidate, which displays an attenuated phenotype and promotes in DC a stronger capacity to stimulate the Th response than BCG vaccine. Interestingly, the data obtained by using the human DC-based experimental setting mirrored the results derived from studies in animal models, suggesting that this system could be used for an efficient and rapid down-selection of new TB vaccine candidates, contributing to achieve the "3Rs" objective.

How Cytokine Networks Fuel Inflammation: Toward a cytokine-based disease taxonomy

Discerning which mediators drive pathogenesis in chronic inflammatory diseases can be complex: immune cells can release various pathogenic cytokines, and numerous cytokines may either cause one specific disease or many. Human validation and mechanistic studies will be necessary to identify the key immune cells and cytokines for a given inflammatory disorder and to pinpoint which cytokine might be the appropriate target for tackling each disease. In 'Bedside to Bench', Georg Schett et al. discuss how human trials targeting different cytokines suggest the existence of a hierarchical framework of cytokines that defines groups of chronic inflamatory diseases rather differently from the homogenous molecular disease pattern previously assumed. In 'Bench to Bedside', Vijay Kuchroo and Dominique Baeten peruse the role of interleukin-17A as drug target in several autoimmune diseases to highlight how success in the clinic will need understanding of pathogenic pathways and the immunological and tissue context of each inflammatory disease.

Increased production of IFN-γ by natural killer cells triggered with bone marrow-derived dendritic cells cultured in the presence of retinoic acid

All-trans-retinoic acid (RA), a vitamin A metabolite, is beginning to be explored as an immunopharmacologic agent. While its effects on dendritic cells and the induction of regulatory T cells have been recognized, little is known about the effect of RA on dendritic cell–natural killer cell (DC–NK) crosstalk. DC–NK crosstalk is important in directing the innate immune response, as well as subsequent adaptive immune response during viral infection, cancer, pregnancy, as well as organ transplantation. Here we demonstrate with flow cytometry and cytokine bead array analysis, that bone marrow derived dendritic cells (BMDCs) cultured in the presence of ≥98% HPLC purified RA (RA-DCs) were suppressed in their ability to mature in response to TLR stimulation. 1µM of RA was found to be optimal without affecting the percentage of DCs in culture. Upregulation of MHCII and costimulatory molecule CD86, as well as IL-12 secretion were inhibited by RA treatment. RA-DCs differentially modulate NK cell function compared to BMDCs. In vitro co-culture of RA-DCs with NK cells reveal increased IFN-γ secretion. Increased production of IFN-γ in lung NK cells was also demonstrated when RA-DCs were injected into the tail vein. Our results suggest that RA-DCs exhibit a regulatory phenotype and function, which differentially modulates NK cell function. Furthermore, IFN-γ has various regulatory and immunological functions, depending on the immunological context. The effect of RA-DCs needs to be further explored in the context of a disease in order to understand the regulatory effects of retinoic acid.

P064 IL-21 neutralization in experimental colitis: regulation of cytokines, chemokines and colonic transcripts

Background: Interleukin 21 (IL-21), a member of the common gamma-chain family of cytokines, is secreted by activated T cells and NKT cells, and has a diverse range of immunomodulatory effects dependent upon the immunological context. IL-21 is produced in the inflamed intestine of patients with IBD, mostly by activated CD4+ helper T cells. Methods: Using a CD4+CD25 SCID adoptive transfer colitis model, we evaluated the effect of a murine monoclonal antibody against IL-21. Mice were treated with anti-IL-21 mAb 25mg/kg (i.p.) 3 × week in either a prophylactic (day 0 42) or a therapeutic (day 21 56) setup. Colon biopsies were analyzed using either luminex or deep sequencing technology. Mesenteric lymph node CD4+ T cells were isolated from mice with colitis and stimulated with different combinations of anti-CD3 and IL-21 or analyzed directly by intracellular FACS analysis. Results: IL-21 production by the transferred CD4+ T cells was found increased over time and correlated with clinical disease. Moreover, a significant reduction of clinical signs of colitis was shown in both anti-IL-21 mAb prophylactic and therapeutically treated mice, when compared to control Ig-treated mice. In anti-IL-21 mAb prophylactic treated mice, several cytokines (including TNF-a and IL-1b) were significantly down-regulated, revealed by deep sequencing analysis of colon biopsies. Additionally, in anti-IL-21 mAb treated mice, luminex analysis on colon biopsies showed a significant down regulation of CCL5, IL-17 and TNF-a. This is in agreement with an up-regulation of the same molecules when mesenteric lymph node CD4+ T cells isolated from mice with colitis were stimulated in vitro with anti-CD3 and IL-21. The percentage of IFN-g and IL-21 positive CD4+ T cells in mesenteric lymph nodes was also significantly down regulated in anti-IL-21 treated mice. Moreover, anti-IL-21 mAb treatment significantly down-regulated colon transcripts for metallo matrix proteases (MMPs), activation markers, adhesions molecules and chemokines (including MMP3, MMP9, CD44, Integrin a4b7, ICAM-1, CXCL13, CCL5, CCL20) and upregulated transcripts involved in mucosal barrier function (including Muc2, TFF3 and Occludin). Conclusions: These results suggest that IL-21 is a relevant target in experimental colitis regulating several pro-inflammatory cytokines and chemokines as well as key molecules in mucosal barrier function. Thus, blockade of this pathway may be of therapeutic benefit in patients with IBD.

1585 – Social adaptation and immune reactivity in schizophrenia

Study of social-clinical and biological components of adaptation in schizophrenia currently is of relevance. Identification of interrelationship immune reactivity with social adaptation types of schizophrenic patients. We examined 38 schizophrenic patients (F20.00, F20.01, F20.02). The scale SASS (Bosc M. et al., 1997) was used to estimate the social adaptation of schizophrenic patients. The immunological examination included the definition of phenotypes of surface receptors of immunocompetent cells, immunoglobulins, circulating immune complexes and phagocytosis levels. The research was carried out in two points: first - at admission, second - in 6 weeks of treatment. Three adaptation types have been revealed: normal type of social adaptation (15 patients, 39,47%), difficult social adaptation (18 persons), disadaptation (5 patients). By week 6 of treatment number of patients with normal social adaptation has heightened by 1,4 times (21 patients, 55,26%), improvement of indices of social adaptation was observed in 10 patients, type of social adaptation did not change in 28 patients. At admission T-immunodeficiency (CD2 + , CD3 + , CD16 + -lymphocytes decrease), increase CD8 + , HLADR + , CD20 + - lymphocytes, IgM and phagocytosis decrease were diagnosed among schizophrenic patients. During therapy, features of psychoneuroimmunomodulation were as follows: the normal type of social adaptation is relatively favorable in the immunological context. Patients with low level of social adaptation were characterized by clinical-immunological resistance toward therapy with deepening of T-cellular immune deficiency. Improvement of indices of social adaptation was accompanied by positive dynamic of immunological parameters. Thus, social differences between groups have been confirmed by data according to immune reactivity.

CD28 Aptamers as Powerful Immune Response Modulators

CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7), precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist) to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.

T-cell Electrophysiology Dynamics

Event Abstract Back to Event T-cell Electrophysiology Dynamics Christine D. Schmeitz1 and Michael Meyer-Hermann1* 1 Helmholtz Centre for Infection Research, Systems Immunology, Germany The presence of T-lymphocytes, in particular T-helper-cells, is in the context of great importance, but in some medical issues their immunological implication must be repressed. For example, the appearance of graft rejection is a major problem in transplantation medicine. In order to suppress the causative immunological reaction, the pharmaceuticals cyclosporin A (CsA) and tacrolimus (FK506) are in use as standard drugs. Both immunomodulators affect electrophysiological processes by blocking the relevant signaling cascade in activated T-cells and in this way also modulating the calcium influx passing through the calcium-release activated channel (CRAC) in the plasma membrane of T-lymphocytes. With regard to the significance of electrophysiology in coherence with immunological interactions and pharmacological issues, a mathematical modeling approach is demanded in order to quantitatively understand the associated ion dynamics in T-lymphocytes, embedded in a systems immunological context. Building on the results from electrophysiological measurements, the in silico T-cell model includes single transmembrane protein characteristics in order to derive ion dynamics patterns on the whole cell level with a particular focus on calcium. Beside of the most central CRAC, ionic pumps and channels for calcium, sodium and potassium, as well as the endoplasmatic reticulum acting as a calcium store are represented in the model. Starting with simulations of a naive T-cell, the model will then be used to analyse the electrophysiological changes following T-cell activation. Further, the modeling approach will finally provide a starting point for in silico studies relating to the therapeutic immune modulation of ion-conducting proteins in T-lymphocytes. Keywords: CRAC channel, calcium dynamics, mathematical modeling, T-cell electrophysiology, immunopharmacology, Calcium Channel Blockers, Transplantation, Immunsuppressive Treatments Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Schmeitz CD and Meyer-Hermann M (2013). T-cell Electrophysiology Dynamics. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00395 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Michael Meyer-Hermann, Helmholtz Centre for Infection Research, Systems Immunology, Braunschweig, Germany, michael.meyer-hermann@helmholtz-hzi.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Christine D Schmeitz Michael Meyer-Hermann Google Christine D Schmeitz Michael Meyer-Hermann Google Scholar Christine D Schmeitz Michael Meyer-Hermann PubMed Christine D Schmeitz Michael Meyer-Hermann Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

An N-Terminal Mutation of the Foxp3 Transcription Factor Alleviates Arthritis but Exacerbates Diabetes

Maintenance of lymphoid homeostasis in a number of immunological and inflammatory contexts is served by a variety of regulatory T (Treg) cell subtypes and depends on interaction of the transcription factor FoxP3 with specific transcriptional cofactors. We report that a commonly used insertional mutant of FoxP3 (GFP-Foxp3) modified its molecular interactions, blocking HIF-1α but increasing IRF4 interactions. The transcriptional profile of these Treg cells was subtly altered, with an overrepresentation of IRF4-dependent transcripts. In keeping with IRF4-dependent function of Treg cells to preferentially suppress Tcell help to B cells and Th2 and Th17 cell-type differentiation, GFP-FoxP3 mice showed a divergent susceptibility to autoimmune disease: protection against antibody-mediated arthritis in the K/BxN model, but greater susceptibility to diabetes on the NOD background. Thus, specific subfunctions of Treg cells and the immune diseases they regulate can be influenced by FoxP3's molecular interactions, which result in divergent immunoregulation.

Syndrome d’Evans : attention aux diagnostics par excès

IntroductionEvans syndrome (ES) is characterized by the coexistence of an autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP). Despite being frequently evocated in the simultaneous presence of anemia and thrombocytopenia, this rare disease only accounts for 0.8 to 3.7% of patients with ITP or AIHA. Case reportsWe report three suspected cases of ES, diagnosed in the presence of thrombocytopenia and hemolytic anemia association, with a positive direct Coombs test in two patients. Standard ES treatment failure and occurrence of aditional features subsequently led to correct diagnosis to thrombotic thrombocytopenic purpura, myelodysplastic syndrome with AIHA, and ITP with hemorrhagic anemia, respectively. ConclusionBicytopenias, even in an immunological context, are not sufficient to ascertain ES diagnosis. Our cases illustrate the diagnostic difficulties that may arise in daily practice, and induce over-diagnosis of this rare disease.

Serglycin: A Structural and Functional Chameleon with Wide Impact on Immune Cells

Among the different proteoglycans expressed by mammals, serglycin is in most immune cells the dominating species. A unique property of serglycin is its ability to adopt highly divergent structures, because of glycosylation with variable types of glycosaminoglycans when expressed by different cell types. Recent studies of serglycin-deficient animals have revealed crucial functions for serglycin in a diverse array of immunological processes. However, its exact function varies to a large extent depending on the cellular context of serglycin expression. Based on these findings, serglycin is emerging as a structural and functional chameleon, with radically different properties depending on its exact cellular and immunological context.

Inflammation as an Animal Development Phenomenon

Inflammation is a term that has been used throughout history in different contexts; it may represent a simple collection of clinical symptoms for which drugs are developed, a disease mechanism, or even a defense mechanism against microbes validating Pasteur's studies on bacteriology and Darwin's proposed struggle for survival. Thus, an explanation of this term must also consider the scientific questions addressed. In this study, I propose that several of the inflammatory events typically described in immunological, pathological, and pharmacological contexts can also be perceived as mechanisms of animal development. Thus, by recognizing that the generation of an animal form, its conservation, and its regeneration after tissue damage are phenomena of the same nature, inflammation can be addressed through the approach of developmental biology, thereby acquiring a much neglected physiological counterpart.

Analytical And Clinical Validation Of A Diagnostic Test For Evaluation Of NK Cell Subsets And NKT Cells

RATIONALE: Natural killer (NK) cells have both cytotoxic and cytokine-producing effector functions. NK cells are not a homogeneous subset and the ability to quantitatively evaluate various NK cell subsets, and functional proteins associated with these cell populations is clinically relevant.METHODS: A multiparametric flow cytometric assay was developed and analytically validated. CD45, CD3, CD16 and CD56 were used for total NK/NKT cell and NK subset quantitation. Additional markers (CD27, CD69, CD107a/b, NKp46, NKG2D, Granzyme A, Granzyme B, Perforin and IFNγ) characterizing NK cell functions were also used.RESULTS: Most NK-specific markers are only stable for up to 24h at ambient temperature, necessitating prompt analysis of whole blood samples. The inter- and intra- assay precision for the various subsets were typically within a coefficient of variation (CV) of 20%, for well-represented populations, and less than 10 cells absolute difference for rare events. Reference values (mid 95 percentile) for all subsets were established using 127 healthy adult donors. The precision at the limits of detection (1 - 201103cells/μL) were established for total NK cells as 10.31% at lower limits and 5.48% at upper limits. The assay was clinically validated with 21 patients representing a spectrum of primary or secondary immunological disorders, with various NK cell defects.CONCLUSIONS: We have a developed a clinical diagnostic assay for the quantitation of various NK cell/NKT subsets, including expression of functionally relevant markers, whose analytical parameters have been established, and can be used to evaluate NK cell populations in a variety of specific clinical and immunological contexts. RATIONALE: Natural killer (NK) cells have both cytotoxic and cytokine-producing effector functions. NK cells are not a homogeneous subset and the ability to quantitatively evaluate various NK cell subsets, and functional proteins associated with these cell populations is clinically relevant. METHODS: A multiparametric flow cytometric assay was developed and analytically validated. CD45, CD3, CD16 and CD56 were used for total NK/NKT cell and NK subset quantitation. Additional markers (CD27, CD69, CD107a/b, NKp46, NKG2D, Granzyme A, Granzyme B, Perforin and IFNγ) characterizing NK cell functions were also used. RESULTS: Most NK-specific markers are only stable for up to 24h at ambient temperature, necessitating prompt analysis of whole blood samples. The inter- and intra- assay precision for the various subsets were typically within a coefficient of variation (CV) of 20%, for well-represented populations, and less than 10 cells absolute difference for rare events. Reference values (mid 95 percentile) for all subsets were established using 127 healthy adult donors. The precision at the limits of detection (1 - 201103cells/μL) were established for total NK cells as 10.31% at lower limits and 5.48% at upper limits. The assay was clinically validated with 21 patients representing a spectrum of primary or secondary immunological disorders, with various NK cell defects. CONCLUSIONS: We have a developed a clinical diagnostic assay for the quantitation of various NK cell/NKT subsets, including expression of functionally relevant markers, whose analytical parameters have been established, and can be used to evaluate NK cell populations in a variety of specific clinical and immunological contexts.

A Role for Serglycin Proteoglycan in Mast Cell Apoptosis Induced by a Secretory Granule-mediated Pathway

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.

Menstrual Function, Menstrual Suppression, and the Immunology of the Human Female Reproductive Tract

Analyses of menstrual function are important to our understanding of human evolution and can help to assess the risks of menstrual suppression, a practice increasingly recommended for women. Two evolutionary issues, however, are insufficiently appreciated in these analyses: the selection pressure infections pose to the human female reproductive system, and the variety of different-and possibly conflicting-immunological functions in the healthy human female reproductive and genital tract. Part of the reason why these issues are inadequately addressed is that reproduction is not sufficiently contextualized in evolutionary and immunological accounts. I argue that expanding the immunological context for menstrual function reinvigorates Margie Profet's (1993a) hypothesis that menstruation defends against sperm-borne pathogens. This expanded context also suggests that menstruation may have more than one function. Thus, until more is known about menstruation, we should proceed cautiously with regard to menstrual suppression.