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An interview with functional medicine healthcare provider Dr. Datis Kharrazian is presented. Topics of the interview include the start of Kharrazian's interest in being a chiropractor and a nutritionist, his stand on the move of some reviewers in medical journals to reject research paper without any useful commentary, and his clinical research fellowship at Harvard University.

Enhancement or inhibition of cytokine signaling and corresponding immune cells responses are critical factors in various disease treatments. Cytokine signaling may be inhibited by cytokine-neutralizing antibodies (CNAs), which prevents further activation of cytokine receptors. However, CNAs may result in enhanced—instead of inhibitory—cytokine signaling (an “agonistic effect”) in various in vitro and in vivo experiments. This may lead to lack of efficacy or adverse events for cytokine-inhibiting based medicines. Alternatively, cytokine-antibody complexes may produce stronger signaling vs. cytokine alone, thereby increasing the efficacy of stimulating cytokine-based drugs, at equal or lower cytokine doses. In this paper, the effect of cytokine signaling enhancement by a CNA was studied in a generic mathematical model of interleukin-4 (IL-4) driven T-cell proliferation. The occurrence of the agonistic effect depends upon the antibody-to-cytokine binding affinity and initial concentrations of antibody and cytokine. Model predictions were in agreement with experimental studies. When the cytokine receptor consists of multiple subunits with substantially differing affinities (e.g., IL-4 case), the choice of the receptor chain to be blocked by the antibody is critical, for the agonistic effect to appear. We propose a generic mechanism for the effect: initially, binding of the CNA to the cytokine reduces free cytokine concentration; yet, cytokine molecules bound within the cytokine-CNA complex—and released later and over time—are “rescued” from earlier clearance via cellular internalization. Hence, although free cytokine-dependent signalling may be less potent initially, it will also be more sustained over time; and given non-linear dynamics, it will lead ultimately to larger cellular effector responses, vs. the same amount of free cytokine in the absence of CNA. We suggest that the proposed mechanism is a generic property of {cytokine, CNA, receptor} triads, both in vitro and in vivo, and can occur in a predictable fashion for a variety of cytokines of the immune system. [ABSTRACT FROM AUTHOR]

Zukamu granules (ZKMG), as the preferred drug for the treatment of colds in Uygur medical theory, has been used for 1500 years. It is also widely used in China and included in the National Essential Drugs List (2018 edition). It has unique anti-inflammatory, antitussive and analgesic effects. Aiming at the research of traditional Chinese medicine (TCM) with the characteristics of overall regulation of body diseases and the immune regulation mechanism with the concept of integrity, this paper put forward the integrated application of network composite module analysis and animal experiment verification to study the immune regulation mechanism of TCM. The active components and targets of ZKMG were predicted, and network module analysis was performed to explore their potential immunomodulatory mechanisms. Then acute lung injury (ALI) mice and idiopathic pulmonary fibrosis (IPF) rats were used as pathological models to observe the effects of ZKMG on the pathological conditions of infected ALI and IPF rats, determine the contents of Th1, Th2 characteristic cytokines and immunoglobulins, and study the intervention of GATA3/STAT6 signal pathway. The results of network composite module analysis showed that ZKMG contained 173 pharmacodynamic components and 249 potential targets, and four key modules were obtained. The immunomodulatory effects of ZKMG were related to T cell receptor signaling pathway. The validation results of bioeffects that ZKMG could carry out bidirectional immune regulation on Th1/Th2 cytokines in the stage of ALI and IPF, so as to play the role of regulating immune homeostasis and organ protection. The network composite module analysis and verification method is an exploration to study the immune regulation mechanism of TCM by combining the network module prediction analysis with animal experiments, which provides a reference for subsequent research. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction Maternal antibody interference of the infant's humoral immune responses raises some concern to the strategy of maternal Tdap (tetanus, diphtheria, acellular pertussis [aP]) vaccination. This study assessed the impact of maternal Tdap antibodies on the infant's pertussis-specific T lymphocyte responses following infant vaccination with an aP containing vaccine, in a term and preterm born cohort. Methods Heparin samples (±0.5 mL) were conveniently drawn from infants of a Belgian prospective cohort study (N = 79, NCT02511327), including Tdap vaccinated (Boostrix®) and nonvaccinated women (no Tdap vaccine in the last 5 years) that delivered at term or prematurely. Sampling was performed before and 1 month after primary (8-12-16 weeks) and booster vaccination (13 or 15 months) with DTaP-IPV-HB-PRP~T vaccine (Hexyon®). Pertussis toxin (PT)-specific CD3+, CD3+ CD4+ and CD3+ CD8+ lymphoblasts and their cytokine secretions were measured using a flow cytometric assay on whole blood (FASCIA) and multiplex technology (Meso Scale Discovery), respectively. Results In total, 57% of all infants were considered PT-specific CD3+ CD4+ lymphoblasts responders after primary and booster vaccination, whereas 17% were CD3+ CD8+ lymphoblast responders. Interferon (IFN)-γ, interleukin (IL)-13, IL-17A, and IL-5 cytokine secretions after primary and booster vaccination were indicative of a mixed T helper (Th) 1/Th2/Th17 cell profile. Lymphoblast and cytokine levels were comparable between term and preterm infants. Nonresponders for IL-13 after booster vaccination had higher maternal PT immunoglobulin G (IgG) levels at birth when compared to responders. Conclusions Term and preterm born infants are capable of inducing Th1, Th2, and Th17 responses after aP vaccination, yet maternal vaccination modulate these responses. Evaluation of this effect in larger trials is needed. [ABSTRACT FROM AUTHOR]

Regulatory B cells (Breg) are widely regarded as immunomodulatory cells which play an immunosuppressive role. Breg inhibits pathological autoimmune response by secreting interleukin-10 (IL-10), transforming growth factor-β (TGF-β), and adenosine and through other ways to prevent T cells and other immune cells from expanding. Recent studies have shown that different inflammatory environments induce different types of Breg cells, and these different Breg cells have different functions. For example, Br1 cells can secrete IgG4 to block autoantigens. Idiopathic membranous nephropathy (IMN) is an autoimmune disease in which the humoral immune response is dominant and the cellular immune response is impaired. However, only a handful of studies have been done on the role of Bregs in this regard. In this review, we provide a brief overview of the types and functions of Breg found in human body, as well as the abnormal pathological and immunological phenomena in IMN, and propose the hypothesis that Breg is activated in IMN patients and the proportion of Br1 can be increased. Our review aims at highlighting the correlation between Breg and IMN and proposes potential mechanisms, which can provide a new direction for the discovery of the pathogenesis of IMN, thus providing a new strategy for the prevention and early treatment of IMN. [ABSTRACT FROM AUTHOR]

Purpose: Bisphenol A (BPA) is found in many plastic products and is thus a common environmental endocrine disruptor. Plastic-related health problems, including allergic diseases, are attracting increasing attention. However, few experimental studies have explored the effect of BPA on allergic rhinitis (AR). We explore whether BPA was directly related to the allergic inflammation induced by ovalbumin (OVA) in AR mice.Methods: We first constructed OVA-induced mouse model, and after BPA administration, we evaluated nasal symptoms and measured the serum OVA-specific IgE levels by ELISA. Th2 and Treg-related cytokines of nasal mucosa were measured by cytometric bead array. Th2 and Treg-specific transcription factor levels were assayed by PCR. The proportions of CD3+CD4+IL-4+Th2 and CD4+Helios+Foxp3+ T cells (Tregs) in spleen tissue were determined by flow cytometry.Results: Compared to OVA-only-induced mice, BPA addition increased nasal symptoms and serum OVA-specific IgE levels. OVA and BPA coexposure significantly increased IL-4 and IL-13 protein levels compared to those after OVA exposure alone. BPA plus OVA tended to decrease the IL-10 protein levels compared to those after OVA alone. Coexposure to OVA and BPA significantly increased the GATA-3-encoding mRNA level, and decreased the levels of mRNAs encoding Foxp3 and Helios, compared to those after OVA exposure alone. BPA increased the Th2 cell proportion, and decreased that of Tregs, compared to the levels with OVA alone.Conclusion: BPA exerted negative effects by exacerbating AR allergic symptoms, increasing serum OVA-specific IgE levels, and compromising Th2 and Treg responses. [ABSTRACT FROM AUTHOR]

Bifidobacterium animalis subsp. lactis IPLA 20020 and Lactobacillus gasseri IPLA 20212, two strains isolated from human samples, were evaluated for safety and influence over the intestinal microbiota and cytokine production by the intestinal tissue of adult BALB/c mice. Mice were divided into four groups receiving during 8 days PBS or a suspension of each strain, prepared fresh or lyophilized (bifidobacteria), at an amount of 4x108 viable cells/day. This dose could be comparable to the probiotic intake of a human adult who consumed about 100-200 mL of functional fermented milk per day, considering the usual level of probiotics in commercial products. No microbial translocation to liver or alterations in food intake, weight, and behavior were observed in treated mice. Intestinal content of secretory immunoglobulin A (s-IgA) was not affected, discarding any adverse effect on the mucosa-associated immunity. The profile of intestinal proinflammatory/regulatory cytokines after intervention evidenced that the microbial strain administered and its cellular state (fresh or lyophilized) as well as the host tissue analyzed (small or large intestine) influenced the immune response and suggests a moderate shift towards a T helper 1 profile (Th1) in the large intestine after the administration of both strains. Changes on relative levels of some intestinal microbial groups were evidenced after intervention. It is noteworthy that butyrate was positively associated with a balanced pro-Th1 immune response. Therefore, B. animalis subsp. lactis IPLA20020 and L. gasseri IPLA 20212 could be considered potential probiotic candidates to be included in functional foods for balancing the intestinal immune response. [ABSTRACT FROM AUTHOR]

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whether i.v. butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins, and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ), and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10, and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/pathogenic genera, which might contribute to its overall beneficial effect. [ABSTRACT FROM AUTHOR]

Anti-CTLA-4 mAb is efficacious in enhancing tumor immunity in humans. CTLA-4 is expressed by conventional T cells upon activation and by naturally occurring FOXP3+CD4+ Treg cells constitutively, raising a question of how anti-CTLA-4 mAb can differentially control these functionally opposing T cell populations in tumor immunity. Here we show that FOXP3high potently suppressive effector Treg cells were abundant in melanoma tissues, expressing CTLA-4 at higher levels than tumor-infiltrating CD8+ T cells. Upon in vitro tumor-antigen stimulation of peripheral blood mononuclear cells from healthy individuals or melanoma patients, Fc-region-modified anti-CTLA-4 mAbwith high antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) activity selectively depleted CTLA-4+FOXP3+ Treg cells and consequently expanded tumorantigen-specific CD8+T cells. Importantly, the expansion occurred only when antigen stimulation was delayed several days from the antibody treatment to spare CTLA-4+ activated effector CD8+T cells from mAb-mediated killing. Similarly, in tumor-bearing mice, high-ADCC/ADCP anti-CTLA-4 mAb treatment with delayed tumor-antigen vaccination significantly prolonged their survival and markedly elevated cytokine production by tumor-infiltrating CD8+ T cells, whereas antibody treatment concurrent with vaccination did not. Anti-CTLA-4 mAb modified to exhibit a lesser or no Fc-binding activity failed to show such timing-dependent in vitro and in vivo immune enhancement. Thus, high ADCC anti-CTLA-4 mAb is able to selectively deplete effector Treg cells and evoke tumor immunity depending on the CTLA-4-expressing status of effector CD8+ T cells. These findings are instrumental in designing cancer immunotherapy with mAbs targeting the molecules commonly expressed by FOXP3+ Treg cells and tumor-reactive effector T cells. [ABSTRACT FROM AUTHOR]

Purpose: The present study was designed to determine the effects of both choline form and availability on maternal immune function during lactation.Methods: Sprague-Dawley rats were randomized to one of the three diets 24-48 h before parturition and fed ad libitum until 21 days postnatal: 1 g/kg choline as free choline (C, n = 11), the current form, and amount of choline in commercial diets; 1 g/kg choline as phosphatidylcholine (PC1, n = 11); or 2.5 g/kg choline as PC (PC2.5, n = 8). Choline metabolites in offspring stomach contents were quantified. At 21 days, lymphocytes from mothers’ mesenteric lymph nodes and spleens were isolated and phenotypes and ex vivo cytokine production after mitogen exposure were determined.Results: There was a higher proportion of choline and a lower proportion of lyso-PC in stomach contents (representing dam’s milk) of C pups compared to PC1. In the mesenteric lymph nodes, feeding PC1 compared to C led to a higher IL-2 production after Concanavalin A (ConA) stimulation and a higher proportion of T cells (CD3+) and a lower proportion of B cells [immunoglobulin (Ig)κ, CD45RA+, and IgM+; P < 0.05]. Splenocytes from the PC1 group produced more IL-6 and TNF-α after lipopolysaccharides stimulation compared to C (P < 0.05). Splenocytes from the PC2.5 group produced more IL-2 and IL-6 after ConA stimulation compared to PC1 (P < 0.05).Conclusions: Feeding choline as PC in the maternal diet improved the ability of immune cells to respond ex vivo to mitogens and increasing the amount of PC in the diet further improved T cell proliferation. [ABSTRACT FROM AUTHOR]

Objective. The relevance of the Th17 pathway in primary SS (pSS) is unclear. Published studies have relied on restimulating circulating CD161+ T cells in vitro for quantitation of IL-17-producing cells. While CD161 marks all IL-17+ T cells, it is also expressed by other Th subsets. The aim of this study was to directly analyse retinoic acid receptor-related orphan nuclear receptor (ROR)-γ expressing and non-expressing subsets of CD161+ T cells to determine the relevance of the Th17 pathway in pSS. Methods. We quantitated the frequencies of both CD161- and RORγ-expressing T cells by comparative flow cytometry in peripheral blood mononuclear cells from a well-stratified cohort of pSS patients and control subjects. We also analysed the expression of antigen D-related HLA (HLA-DR) and CD161 in labial salivary glands from nine subjects undergoing a diagnostic biopsy. Results. While the frequencies of both RORγ+ and RORγ- subsets of CD161+ CD4+ T cells were increased in peripheral blood from pSS patients, the increase in the RORγ+ subset positively correlated with humoral manifestations of the disease (anti-SSA/SSB autoantibodies and hypergammaglobulinaemia), but not with disease activity, and vice versa for the RORγ- subset. An increased frequency of HLA-DR+ CD161+CD4+ T cells was observed in labial salivary gland biopsies from pSS patients, suggesting chronic activation of CD161+CD4+ T cells in the target tissue of the disease. Conclusion. In addition to pointing to CD161 as a marker of a pathogenic subset of CD4+ T cells in pSS patients, our data indicate that even though the RORγ+ (Th17) CD161 + subset might contribute to humoral manifestations of the disease, the RORγ- (non-Th17) CD161+ subset is the one associated with disease activity in pSS patients. [ABSTRACT FROM AUTHOR]

High titer of anti-β2-glycoprotein I antibodies (anti-β2GPI Ab) plays a pathogenic role in antiphospholipid syndrome (APS). Numerous studies have focused on the pathological mechanism in APS; however, little attention is paid to the immune mechanism of production of anti-β2GPI antibodies in APS. Our previous study demonstrated that Toll-like receptor 4 (TLR4) plays a vital role in the maturation of bone marrow-derived dendritic cells (BMDCs) from the mice immunized with human β2-glycoprotein I (β2GPI). TLR4 is required for the activation of B cells and the production of autoantibody in mice treated with β2GPI. However, TLR4 provides a third signal for B cell activation and then promotes B cells better receiving signals from both B cell antigen receptor (BCR) and CD40, thus promoting B cell activation, surface molecules expression, anti-β2GPI Ab production, and cytokines secretion and making B cell functioning like an antigen presenting cell (APC). At the same time, TLR4 also promotes B cells producing antibodies by upregulating the expression of B-cell activating factor (BAFF). In this paper, we aim to review the functions of TLR4 in B cell immune response and antibody production in autoimmune disease APS and try to find a new way for the prevention and treatment of APS. [ABSTRACT FROM AUTHOR]

Background and Objectives: Blinatumomab is a bispecific T-cell engager (BiTE(®)) antibody construct that transiently links CD19-positive B cells to CD3-positive T cells, resulting in induction of T-cell-mediated serial lysis of B cells and concomitant T-cell proliferation. Blinatumomab showed anti-leukemia activity in clinical trials and was approved by the US Food and Drug Administration for the treatment of Philadelphia chromosome-negative relapsed/refractory B-cell precursor acute lymphoblastic leukemia (r/r ALL). The objectives of this work were to characterize blinatumomab pharmacokinetics and pharmacodynamics and to evaluate dosing regimens.Methods: Data from six phase I and II trials in patients with r/r ALL, minimal residual disease-positive ALL, and non-Hodgkin's lymphoma (NHL) were analyzed. Blinatumomab pharmacokinetics was characterized by non-compartmental and population pharmacokinetic analyses and pharmacodynamics was described graphically.Results: Blinatumomab exhibited linear pharmacokinetics under continuous intravenous infusion for 4-8 weeks per cycle over a dose range of 5-90 µg/m(2)/day, without target-mediated disposition. Estimated mean (standard deviation) volume of distribution, clearance, and elimination half-life were 4.52 (2.89) L, 2.72 (2.71) L/h, and 2.11 (1.42) h, respectively. Pharmacokinetics was similar in patients with ALL and NHL and was not affected by patient demographics, supporting fixed dosing in adults. Although creatinine clearance was a significant covariate of drug clearance, no dose adjustment was required in patients with mild or moderate renal impairment. Incidence of neutralizing antidrug antibodies was <1 %. Blinatumomab pharmacodynamics featured T-cell redistribution and activation, B-cell depletion, and transient dose-dependent cytokine elevation. Blinatumomab did not affect cytochrome P450 enzymes directly; cytokines may trigger transient cytochrome P450 suppression with low potential for inducing drug interactions.Conclusions: Blinatumomab has unique pharmacokinetic and immunological features that require indication-dependent dosing regimens. Stepped dosing is required to achieve adequate efficacy and minimize cytokine release in diseases with high tumor burden. [ABSTRACT FROM AUTHOR]

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed. [ABSTRACT FROM AUTHOR]

B-cells not only produce immunoglobulins and present antigens to T-cells, but also additional key roles in the immune system. Current knowledge on the role of B-cells in infections caused by intracellular bacteria is fragmentary and contradictory. We therefore analysed the phenotypical and functional properties of B-cells during infection and disease caused by Mycobacterium tuberculosis (Mtb), the bacillus causing tuberculosis (TB), and included individuals with latent TB infection (LTBI), active TB, individuals treated successfully for TB, and healthy controls. Patients with active or treated TB disease had an increased proportion of antibodies reactive with mycobacteria. Patients with active TB had reduced circulating B-cell frequencies, whereas only minor increases in B-cells were detected in the lungs of individuals deceased from TB. Both active TB patients and individuals with LTBI had increased relative fractions of B-cells with an atypical phenotype. Importantly, these B-cells displayed impaired proliferation, immunoglobulin- and cytokine- production. These defects disappeared upon successful treatment. Moreover, T-cell activity was strongest in individuals successfully treated for TB, compared to active TB patients and LTBI subjects, and was dependent on the presence of functionally competent B-cells as shown by cellular depletion experiments. Thus, our results reveal that general B-cell function is impaired during active TB and LTBI, and that this B-cell dysfunction compromises cellular host immunity during Mtb infection. These new insights may provide novel strategies for correcting Mtb infection-induced immune dysfunction towards restored protective immunity. [ABSTRACT FROM AUTHOR]

While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis. [ABSTRACT FROM AUTHOR]

Background Although specific immunotherapy is the only aetiological treatment for allergic disorders, the underlying mechanisms are not fully understood. Specific immunotherapy induces changes in lymphocyte Th subsets from Th2 to Th1/Treg. Whether differences in immunological patterns underlie patient response to immunotherapy has not yet been established. Objectives We studied the immunological changes occurring during a 1-year period of Dermatophagoides pteronyssinus ( DP) immunotherapy and their relation with clinical outcome. Methods We included 34 patients with DP allergy who received subcutaneous specific immunotherapy ( SCIT) for 1 year. Following treatment, patients were classified as responders or non-responders. Fourteen allergic subjects who did not receive SCIT were included as controls. Peripheral blood was obtained at 0, 1, 3, 6 and 12 months and cultured with nDer p 1. Phenotypic changes, cytokine production and basophil response were analysed by flow cytometry; transcription factors were measured by mRNA quantification. Serum immunoglobulin levels were also measured. Results After 1 year of SCIT, 82% of cases showed improved symptoms (responders). Although increases in sIgG4 were observed, BAT reactivity was not modified in these patients. Increases in T- BET/ FOXP3 as well as nDer p 1-specific Th1/Treg frequencies were also observed, along with a decrease in Th2, Th9 and Th17. These changes corresponded to changes in cytokine levels. Conclusion Patients who respond well to DP- SCIT show immunological differences compared to non-responders. In responders, basal differences include a lower frequency of Th1 and higher frequencies of Th2, Th9 and Th17 cells. After 1 year of treatment, an increased production of sIgG4 was observed in responders, along with a change in Th2 response towards Th1/Treg. [ABSTRACT FROM AUTHOR]

Background: Experimental autoimmune encephalomyelitis (EAE) in the common marmoset monkey (Callithrix jacchus) is a relevant preclinical model for translational research into immunopathogenic mechanisms operating in multiple sclerosis (MS). Prior studies showed a core pathogenic role of T and B cells specific for myelin oligodendrocyte glycoprotein (MOG). However, in those studies, the quality of the response against MOG epitopes was strongly biased by bacterial antigens in the complete Freund's adjuvant (CFA), in which the immunizing recombinant human (rh) MOG protein had been formulated. In response to the need of a more refined EAE model, we have tested whether disease could also be induced with rhMOG in incomplete Freund's adjuvant (IFA).Method: Marmosets were immunized with rhMOG emulsified in IFA in the dorsal skin. Monkeys that did not develop neurological deficit were given booster immunizations at 28-day interval with the same antigen preparation. In a second experiment, three marmoset twin pairs were sensitized against MOG peptides in IFA to study a possibility for suppressive activity towards pathogenic T cells directed against the encephalitogenic epitope MOG40-48.Results: Despite the absence of strong danger signals in the rhMOG/IFA inoculum, all monkeys developed clinically evident EAE symptoms. Moreover, in all monkeys, demyelinated lesions were present in the white matter and in two cases also in the cortical grey matter. Immune profiling at height of the disease showed a dominant T cell response against the overlapping peptides 14-36 and 24-46, but reactivity against the pathogenically most relevant peptide 34-56 was conspicuously absent. In the second experiment, there was an indication for a possible suppressive mechanism.Conclusions: Immunization of marmoset monkeys with rhMOG in IFA elicits clinical EAE in all animals. Moreover, rhMOG contains pathogenic and regulatory epitopes, but the pathogenic hierarchy of rhMOG epitopes is strongly influenced by the adjuvant in which the protein is formulated. [ABSTRACT FROM AUTHOR]

Currently, many peptide vaccines are undergoing clinical studies. Most of these vaccines were developed to activate cytotoxic T cells; however, the response is not robust. Unlike vaccines, anti-cancer antibodies based on passive immunity have been approved as a standard treatment. Since passive immunity is more effective in tumor treatment, the evidence suggests that limited B cell epitope-based peptide vaccines may have similar activity. Nevertheless, such peptide vaccines have not been intensively developed primarily because humoral immunity is thought to be preferable to cancer progression. B cells secrete cytokines, which suppress immune functions. This review discusses the possibility of therapeutic antibody induction by a peptide vaccine and the role of active and passive B cell immunity in cancer patients. We also discuss the use of humanized mice as a pre-clinical model. The necessity of a better understanding of the activity of B cells in cancer is also discussed. [ABSTRACT FROM AUTHOR]

Th17 responses are critical to a variety of human autoimmune diseases, and therapeutic targeting with monoclonal antibodies against IL-17 and IL-23 has shown considerable promise. Here, we report data to support selective bromodomain blockade of the transcriptional coactivators CBP (CREB binding protein) and p300 as an alternative approach to inhibit human Th17 responses. We show that CBP30 has marked molecular specificity for the bromodomains of CBP and p300, compared with 43 other bromodomains. In unbiased cellular testing on a diverse panel of cultured primary human cells, CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines. CBP30 also inhibited IL-17A secretion by Th17 cells from healthy donors and patients with ankylosing spondylitis and psoriatic arthritis. Transcriptional profiling of human T cells after CBP30 treatment showed a much more restricted effect on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials. [ABSTRACT FROM AUTHOR]

Background: A large number of reports indicate the association of Epstein-Barr virus (EBV), and Mycobacterium avium subsp. paratuberculosis (MAP) with multiple sclerosis (MS). Objective: To gain a better understanding of the role of these two pathogens, we investigated the host response induced by selected antigenic peptides. Methods: We examined both humoral and cell-mediated responses against peptides deriving from EBV tegument protein BOLF1, the MAP_4027 and the human interferon regulatory factor 5 (IRF5424-434) homolog in several MS patients and healthy controls (HCs). Results: Antibodies against these peptides were highly prevalent in MS patients compared to HCs. Concerning MS patients, BOLF1305-320, MAP_402718-32 and IRF5424-434 peptides were able to induce mainly Th1-related cytokines secretion, whereas Th2-related cytokines were down-regulated. Flow cytometry analyses performed on a subset of MS patients highlighted that these peptides were capable of inducing the release of pro-inflammatory cytokines: IFN-γ and TNF-α by CD4+ and CD8+ T lymphocytes, and IL-6 and TNF-α by CD14+ monocyte cells. Conclusion: Our data demonstrated that both EBV and MAP epitopes elicit a consistent humoral response in MS patients compared to HCs, and that the aforementioned peptides are able to induce a T-cell-mediated response that is MS correlated. [ABSTRACT FROM AUTHOR]

Conclusion: This study demonstrated that the common immunological mechanism, which involves aberration of immunoglobulin and T-cell distribution in histologically distinctive tonsils, may be associated with the pathogenesis of tonsillar focal infection. Objectives: Tonsillar focal infection comprises a group of relatively common diseases combined with chronic tonsillar infection, is associated with unusual immune responses in tonsils, and may cause lesions in another distant target organ. This study aimed to investigate the distribution of inflammatory T cells and T-cell regulatory elements, such as programmed cell death-1 (PD-1) and Fork head box protein 3 (Foxp3), immunoglobulin production, and histological characteristics in tonsils from patients with tonsillar focal infection. Methods: Immunohistochemistry and reverse transcription-polymerase chain reaction (PCR) were used to compare the expression of CD8+ T cells, immunoglobulins, and cytokines associated with immunoglobulin production in the tonsils of patients with IgA nephropathy (IgAN), palmoplantar pustulosis (PPP), rheumatoid arthritis (RA), and chronic tonsillitis. Results: The overexpression of CD8+ T cells combined with decreased expression of Foxp3 and PD-1 and the aberration of immunoglobulin production, which may be due to the elevated expression of activation-induced deaminase (AID), B-cell-activating factor of the TNF family (BAFF), supporting isotype switching, and B-cell survival in the histologically distinctive tonsils. [ABSTRACT FROM AUTHOR]

Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28+, RNA+) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV – RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity. [ABSTRACT FROM AUTHOR]

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The interleukin-13 receptor alpha2 (IL13Rα2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13Rα2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13Rα2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13Rα2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13Rα2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13Rα2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect. Methods: Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using 3H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay. Results: TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67+) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages. Conclusions: IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation. [ABSTRACT FROM AUTHOR]

Problem: The T-cell immunoglobulin and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important negative regulator of Th1 immunity and tolerance induction. Data about the TIM-3/Gal-9 pathway in the pathogenesis of human diseases is emerging, but their possible role during human pregnancy is not precisely known. The aim of our study was to investigate the number, phenotype and functional activity of TIM-3+ peripheral blood mononuclear cells during healthy human pregnancy. Methods of Study: 57 healthy pregnant women [first trimester (n = 16); second trimester (n = 19); third trimester (n = 22)] and 30 non-pregnant controls were enrolled in the study. We measured the surface expression of TIM-3 by cytotoxic T cells, NK cells and NK cell subsets as well as Galectin-9 expression by regulatory T cells by flow cytometry. We analyzed the cytokine production and cytotoxicity of TIM3+ and TIM3- CD8 T and NK cells obtained from non-pregnant and healthy pregnant women at different stages of pregnancy by flow cytometry. Serum Galectin-9 levels were measured by ELISA. Results: Our results show that the numbers of peripheral NK and cytotoxic T cells and their TIM-3 expression do not change between the first, second and third trimesters of pregnancy. Compared to non-pregnant individuals, regulatory T cells show higher level of Galectin-9 expression as pregnancy proceeds, which is in line with the level of Galectin-9 in the patients sera. Cytotoxic T cells, NK cells and NK cell subsets expressing TIM-3 molecule show altered cytokine production and cytotoxicity during pregnancy compared to non-pregnant individuals. Conclusion: Our results indicate that Galectin-9 expressing regulatory T cells, TIM-3+ cytotoxic T cells and NK cells could play an important role in the maintenance of healthy pregnancy. [ABSTRACT FROM AUTHOR]

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation. [ABSTRACT FROM AUTHOR]

TGFβ is reportedly responsible for accumulation of CD4+Foxp3+ regulatory T cells (Tregs) in tumor. Thus, we treated mouse 4T1 mammary carcinoma with 1D11, a neutralizing anti-TGFβ (1,2,3) antibody. The treatment delayed tumor growth, but unexpectedly increased the proportion of Tregs in tumor. In vitro, 1D11 enhanced while TGFβ potently inhibited the proliferation of Tregs. To enhance the anti-tumor effects, 1D11 was administered with cyclophosphamide which was reported to eliminate intratumoral Tregs. This combination resulted in long term tumor-free survival of up to 80% of mice, and the tumor-free mice were more resistant to re-challenge with tumor. To examine the phenotype of tumor infiltrating immune cells, 4T1-tumor bearing mice were treated with 1D11 and a lower dose of cyclophosphamide. This treatment markedly inhibited tumor growth, and was accompanied by massive infiltration of IFNγ-producing T cells. Furthermore, this combination markedly decreased the number of splenic CD11b+Gr1+ cells, and increased their expression levels of MHC II and CD80. In a spontaneous 4T1 lung metastasis model with resection of primary tumor, this combination therapy markedly increased the survival of mice, indicating it was effective in reducing lethal metastasis burden. Taken together, our data show that anti-TGFβ antibody and cyclophosphamide represents an effective chemoimmunotherapeutic combination. [ABSTRACT FROM AUTHOR]

Background: Humans with asthma display considerable heterogeneity with regard to T helper (Th) 2-associated eosinophilic and Th17-associated neutrophilic inflammation, but the impact of the environment on these different forms of asthma is poorly understood. Objective: We studied the nature and longevity of asthma-like responses triggered by inhalation of allergen together with environmentally relevant doses of inhaled lipopolysaccharide (LPS). Methods: Ovalbumin (OVA) was instilled into the airways of mice together with a wide range of LPS doses. Following a single OVA challenge, or multiple challenges, animals were assessed for pulmonary cytokine production, airway inflammation, and airway hyperresponsiveness (AHR). Results: Mice instilled with OVA together with very low doses (≤ 10-3 μg) of LPS displayed modest amounts of Th2 cytokines, with associated airway eosinophilia and AHR after a single challenge, and these responses were sustained after multiple OVA challenges. When the higher but still environmentally relevant dose of 10-1 μg LPS was used, mice initially displayed similar Th2 responses, as well as Th17-associated neutrophilia. After multiple OVA challenges, however, the 10-1 μg LPS animals also accumulated large numbers of allergen-specific T regulatory (Treg) cells with high levels of inducible co-stimulatory molecule (ICOS). As a result, asthma-like features in these mice were shorter-lived than in mice sensitized using lower doses of LPS. Conclusions: The nature and longevity of Th2, Th17, and Treg immune responses to inhaled allergen are dependent on the quantity of LPS inhaled at the time of allergic sensitization. These findings might account in part for the heterogeneity of inflammatory infiltrates seen in lungs of asthmatics. [ABSTRACT FROM AUTHOR]

Adoptive T-cell therapy recently achieved impressive efficacy in early phase trials, in particular in hematologic malignancies, strongly supporting the notion that the immune system can control cancer. A current strategy of favor is based on ex vivo-engineered patient T cells, which are redirected by a chimeric antigen receptor ( CAR) and recognize a predefined target by an antibody-derived binding domain. Such CAR T cells can substantially reduce the tumor burden as long as the targeted antigen is present on the cancer cells. However, given the tremendous phenotypic diversity in solid tumor lesions, a reasonable number of cancer cells are not recognized by a given CAR, considerably reducing the therapeutic success. This article reviews a recently described strategy for overcoming this shortcoming of the CAR T-cell therapy by modulating the tumor stroma by a CAR T-cell-secreted transgenic cytokine like interleukin-12 ( IL-12). The basic process is that CAR T cells, when activated by their CAR, deposit IL-12 in the targeted tumor lesion, which in turn attracts an innate immune cell response toward those cancer cells that are invisible to CAR T cells. Such TRUCKs, T cells redirected for universal cytokine-mediated killing, exhibited remarkable efficacy against solid tumors with diverse cancer cell phenotypes, suggesting their evaluation in clinical trials. [ABSTRACT FROM AUTHOR]

Carriage of the genetic combination encoding a high expression inhibitory Killer Immunoglobulin-like Receptor (KIR)3DL1 with its ligand, HLA-B*57 (*h/*y+B*57) is associated with slower time to AIDS and better HIV viral load control than being a Bw6 homozygote (Bw6hmz). Natural Killer (NK) cells from *h/*y+B*57 carriers receive potent educational signals through HLA-B*57 KIR3DL1 ligation leading to high functional potential. NK cells from Bw6hmz are not educated through KIR3DL1 because Bw6 antigens do not interact with this inhibitory receptor. To better understand the impact of KIR/HLA combinations on NK cell mediated anti-viral activity we measured NK cell mediated inhibition of HIV replication in autologous infected CD4 (iCD4) cells by assessing the frequency of p24 positive CD4 targets and supernatant levels of HIV p24 longitudinally in the presence versus absence of NK cells. Forty-seven HIV uninfected subjects were studied, including carriers of *h/*y+B*57, a low expression KIR3DL1 genotype with HLA-B*57 termed *l/*x+B*57, a genotype designated 3DS1+*80I and Bw6hmz. NK cells from *h/*y+B*57 carriers, like those from 3DS1+*80I subjects, inhibited HIV replication in autologous iCD4 cells better than those from Bw6hmz and *l/*x+B*57 carriers. Cell contact between NK and iCD4 cells activated NK cells to inhibit viral replication in a non-contact dependent fashion through secretion of CC-chemokines. iCD4 stimulated NK cells from *h/*y+B*57 and 3DS1+*80I carriers produced higher levels of CC-chemokines than those from Bw6hmz or *l/*x+B*57 carriers. Higher levels of CC-chemokines were produced by KIR3DL1+ than KIR3DL1− NK cells. We conclude that NK-mediated inhibition of viral replication in autologous iCD4 cells is partially due to a block at the level of HIV entry into new targets by secreted CC-chemokines. [ABSTRACT FROM AUTHOR]

The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7highCXCR5highCCR6highPD-1high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells. [ABSTRACT FROM AUTHOR]

Expression of the programmed death 1 (PD-1) receptor and its ligands are implicated in the T cell exhaustion phenotype which contributes to the persistence of several chronic viral infections, including human hepatitis C virus (HCV). The antiviral potential of BMS-936558 (MDX-1106) – a fully human anti-PD-1 monoclonal immunoglobulin-G4 that blocks ligand binding – was explored in a proof-of-concept, placebo-controlled single-ascending-dose study in patients (N = 54) with chronic HCV infection. Interferon-alfa treatment-experienced patients (n = 42) were randomized 5∶1 to receive a single infusion of BMS-936558 (0.03, 0.1, 0.3, 1.0, 3.0 mg/kg [n = 5 each] or 10 mg/kg [n = 10]) or of placebo (n = 7). An additional 12 HCV treatment-naïve patients were randomized to receive 10 mg/kg BMS-936558 (n = 10) or placebo (n = 2). Patients were followed for 85 days post-dose. Five patients who received BMS-936558 (0.1 [n = 1] or 10 mg/kg) and one placebo patient achieved the primary study endpoint of a reduction in HCV RNA ≥0.5 log10 IU/mL on at least 2 consecutive visits; 3 (10 mg/kg) achieved a >4 log10 reduction. Two patients (10 mg/kg) achieved HCV RNA below the lower limit of quantitation (25 IU/mL), one of whom (a prior null-responder) remained RNA-undetectable 1 year post-study. Transient reductions in CD4+, CD8+ and CD19+ cells, including both naïve and memory CD4+ and CD8+ subsets, were observed at Day 2 without evidence of immune deficit. No clinically relevant changes in immunoglobulin subsets or treatment-related trends in circulating cytokines were noted. BMS-936558 exhibited dose-related exposure increases, with a half-life of 20–24 days. BMS-936558 was mostly well tolerated. One patient (10 mg/kg) experienced an asymptomatic grade 4 ALT elevation coincident with the onset of a 4-log viral load reduction. Six patients exhibited immune-related adverse events of mild-to-moderate intensity, including two cases of hyperthyroidism consistent with autoimmune thyroiditis. Further investigation of PD-1 pathway blockade in chronic viral disease is warranted. Trial Registration: ClinicalTrials.gov NCT00703469 NCT00703469 [ABSTRACT FROM AUTHOR]

Previous studies have indicated that Il21r−/− mice chronically infected with Toxoplasma gondii display a defect in serum IgG; however, the basis for this antibody defect was not defined and questions remain about the role of IL-21 in promoting the production of IL-10, which is required to limit infection-induced pathology during toxoplasmosis. Therefore, Il21−/− mice were challenged with T. gondii to determine whether IL-21 impacts the parasite-specific CD8+ T cell response, its contribution to thymus-dependent antibody production after infection, and balance between protective and pathogenic responses. Whereas IL-21 has been implicated in the differentiation of IL-10 producing CD4+ T cells no immune-mediated pathology was evident in Il21−/− mice during the acute response, nor was there a defect in the development of this population in chronically infected Il21−/− mice. However, Il21−/− mice displayed a defect in IgG production after infection that correlated with a decrease in GC B cell numbers, the CD4+ and CD8+ T cell numbers in the brain were reduced over the course of the chronic infection leading to a decrease in total IFN-γ production and an increase in parasite numbers associated with susceptibility to toxoplasmic encephalitis. Together, these results identify a key role for IL-21 in shaping the humoral and cellular response to T. gondii, but indicate that IL-21 has a limited role in regulating immunopathology. [ABSTRACT FROM AUTHOR]

: Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8+ T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8+ T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4+ T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism. [ABSTRACT FROM AUTHOR]

It is currently unknown how mucosal adjuvants cause induction of secretory immunoglobulin A (IgA), and how T cell-dependent (TD) or -independent (TI) pathways might be involved. Mucosal dendritic cells (DCs) are the primary antigen presenting cells driving TI IgA synthesis, by producing a proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), Retinoic Acid (RA), TGF-β or nitric oxide (NO). We hypothesized that the mucosal adjuvant Cholera Toxin subunit B (CTB) could imprint non-mucosal DCs to induce IgA synthesis, and studied the mechanism of its induction. In vitro, CTB-treated bone marrow derived DCs primed for IgA production by B cells without the help of T cells, yet required co-signaling by different Toll-like receptor (TLR) ligands acting via the MyD88 pathway. CTB-DC induced IgA production was blocked in vitro or in vivo when RA receptor antagonist, TGF-β signaling inhibitor or neutralizing anti-TGF-β was added, demonstrating the involvement of RA and TGF-β in promoting IgA responses. There was no major involvement for BAFF, APRIL or NO. This study highlights that synergism between CTB and MyD88-dependent TLR signals selectively imprints a TI IgA-inducing capacity in non-mucosal DCs, explaining how CTB acts as an IgA promoting adjuvant. [ABSTRACT FROM AUTHOR]

The immune responses of Angiostrongylus cantonensis infection are closely relevant to the host's self-protection and the nematode's pathogenesis. In the present study, BALB/c mice were randomly divided into uninfected control group, infection group 1, and infection group 2. The infection group 1 and infection group 2 were infected with 20 and 40 third-stage larvae of A. cantonensis per mouse, respectively. The splenocytes from the mice were collected and cultured on the 19th and 25th days post-infection; the subtypes of T cells in splenocytes were detected by flow cytometry with fluorescence staining method, and the cytokines in cultured supernatants of splenocytes were assayed by the method of ELISA. The specific IgG and IgE antibodies in sera of the mice were periodically detected by ELISA. The results showed that the percentages of CD4 and CD4 IL-4 T cells in splenocytes of infected mice were much higher ( P < 0.05) than those in control mice; however, the percentages of CD4 IL-17 and CD4 IFN-γ T cell were much lower( P < 0.01) after the infection. The levels of CD8 T cells in infected mice also rose, but differences between control mice and infected mice were not significant. In comparison with control mice, the concentration of IL-4 in the cultured supernatants of splenocytes in infected mice increased significantly ( P < 0.05), but that of IL-17 decreased significantly ( P < 0.01). In addition, the number of larvae infected and days after infection may influence levels of the T cell subtypes and the cytokines in spleen, too ( P > 0.05). On humoral immunity, the levels of specific IgG antibodies in sera rose a bit at the fifth day post-infection, and reached a peak at the 20th day post-infection; the specific IgE antibodies gradually heightened during first 10 days post-infection; then, it showed a downward trend during the 15th to 25th days post-infection. It is evident that the percentages of CD4 T lymphocytes of spleen in the mice infected with A. cantonensis markedly increase and polarize to Th2 phenotypes, and the function of Th17 cells is inhibited. In addition, the elevation of specific IgG antibodies in sera of the infected mice is more significant than that of specific IgE antibodies. [ABSTRACT FROM AUTHOR]

As probiotics in the gut, Lactobacilli are believed to play important roles in the development and maintenance of both the mucosal and systemic immune system of the host. This study was aimed to investigate the immunomodulatory function of candiate lactobacilli on T cells. Lactobacilli were isolated from healthy human feces and the microbiological characteristics were identified by API 50 CHL and randomly amplified polymorphic DNA (RAPD) assays. Anti-CD3 antibody activated peripheral blood mononuclear cells (PBMCs) were treated by viable, heat-killed lactobacilli and genomic DNA of lactobacilli, and cytokine profiles were tested by ELISA. Isolated lactobacilli C44 and C48 were identified as L. acidophilus and L. paracacei, which have properties of acid and bile tolerance and inhibitor effects on pathogens. Viable and heat-killed C44 and C48 induced low levels of proinflammatory cytokines (TNF-α, IL-6 and IL-8) and high levels of IFN-γ and IL-12p70 in PBMCs. In anti- CD3 antibody activated PBMCs, viable and heat-killed C44 increased Th2 cytokine levels (IL-5, IL-6 and IL-10), and simultaneously enhanced Th1 responses by inducing IFN-γ and IL-12p70 production. Different from that of lactabacillus strains, their genomic DNA induced low levels of IL-12p70, IFN-γ and proinflammatory cytokines in PBMCs with or without anti-CD3 antibody activation. These results provided in vitro evidence that the genomic DNA of strains of C44 and C48, especially C44, induced weaker inflammation, and may be potentially applied for treating allergic diseases. [ABSTRACT FROM AUTHOR]

Background: Gami-Cheongyeul-Sodok-Eum (GCSE), an herbal formula of traditional Korean medicine, comprises nine herb components. GCSE has various biological activities such as anti-inflammatory, anti-bacterial and anti-viral activities. However, it is still unclear whether GCSE has any immunomodulatory effect on atopic dermatitis (AD). Methods: GCSE was treated to primary B cells and CD4+ T cells isolated from atopic mice to compare its inhibitory effects on IgE secretion and cytokine expression. Experimental AD was established by alternative treatment of 2, 4-dinitrochlorobenzene (DNCB) and house dust mite extract to the ears of BALB/c mice. GCSE was topically applied to ears of atopic mice every day for 3 weeks. AD progression was analyzed by measuring ear thickness, serum IgE level, histological examination of ear tissue by H&E staining and cytokine profile of CD4+ T cells and CD19+ B cells by real time PCR and ELISA. Results: Treatment of GCSE significantly reduced IgE production and expression of AD associated pathogenic cytokines such as IL-4, IL-5, IL-10, IL-13, IL-17, TNF-α, and IFN-γ by lymphocytes isolated from AD-induced mice. Topical application of GCSE on the ears of AD-induced mice significantly reduced ear thickness, clinical score and lymphocytes infiltration to ears as compared to control group. GCSE treatment also reduced serum IgE level and the levels of major pathogenic cytokines such as IL-4, IL-5, IL-10, IL-13 and IL-17. In addition, GCSE treatment significantly increased Foxp3 expression level. Conclusions: The protective effect of GCSE in experimental AD is mediated by inhibition of IgE production, by reduction in the levels of pathogenic cytokines and by induction of Foxp3, all of which are suggesting the beneficial effect of GCSE on modulating atopic dermatitis. [ABSTRACT FROM AUTHOR]

The candidate malaria vaccine RTS,S/AS01E provides significant but partial protection from clinical malaria. On in vitro circumsporozoite protein (CSP) peptide stimulation and intra-cellular cytokine staining of whole blood taken from 407 5-17 month-old children in a phase IIb trial of RTS,S/AS01E, we identified significantly increased frequencies of two CSP-specific CD4+ T cells phenotypes among RTS,S/AS01E vaccinees (IFNγ-IL2+TNF2 and IFNγ-IL2+TNF+ CD4+ T cells), and increased frequency of IFNγ-IL2-TNF+ CD4+ T cells after natural exposure. All these T cells phenotypes were individually associated with reductions in the risk of clinical malaria, but IFNγ-IL2-TNF+ CD4+ T cells independently predicted reduced risk of clinical malaria on multi-variable analysis (HR = 0.29, 95% confidence intervals 0.15-0.54, p<0.0005). Furthermore, there was a strongly significant synergistic interaction between CSP-specific IFNγ-IL2-TNF+ CD4+ T cells and anti-CSP antibodies in determining protection against clinical malaria (p = 0.002). Vaccination strategies that combine potent cellular and antibody responses may enhance protection against malaria. [ABSTRACT FROM AUTHOR]

Background: Dendritic cells (DC) play a key role in initiation and regulation of immune responses. Plasmacytoid DC (pDC), a small subset of DC, characterized as type-I interferon producing cells, are critically involved in anti-viral immune responses, but also mediate tolerance by induction of regulatory T cells (Treg). In this study, we compared the capacity of human pDC + and conventional DC (cDC) to modulate T cell activity in presence of Foxp3 Treg. Principal Findings: In coculture of T effector cells (Teff) and Treg, activated cDC overcome Treg anergy, abrogate their suppressive function and induce Teff proliferation. In contrast, pDC do not break Treg anergy but induce Teff proliferation even in coculture with Treg. Lack of Treg-mediated suppression is independent of proinflammatory cytokines like IFN-a, IL- 1, IL-6 and TNF-a. Phenotyping of pDC-stimulated Treg reveals a reduced expression of Treg activation markers GARP and CTLA-4. Additional stimulation by anti-CD3 antibodies enhances surface expression of GARP and CTLA-4 on Treg and consequently reconstitutes their suppressive function, while increased costimulation with anti-CD28 antibodies is ineffective. Conclusions/Significance: Our data show that activated pDC induce Teff proliferation, but are insufficient for functional Treg activation and, therefore, allow expansion of Teff also in presence of Treg. [ABSTRACT FROM AUTHOR]

Background: Most infections with human papillomavirus (HPV) are resolved without clinical intervention, but a minority evolves into chronic lesions of distinct grades, including cervical-uterine cancer. It is known that in most cases the immune system mediates elimination of HPV infection. However, the mechanism of immune evasion leading to HPV persistence and development of early cervical lesions is not fully understood. The aim of the present work was to evaluate the potential of peripheral blood leukocytes (PBL) from low-grade squamous intraepitheliallesions (LSIL) patients to be activated ex-vivo by vaccine antigens, the participation of cytotoxic lymphocytes and regulatory T cells, and to determine the secretion of Th1 and Th2 cytokines mediated by stimulation of T cell receptors.Results: We found that PBL from LSIL patients showed a significantly lower proliferation rate to vaccine antigens as compared to that of healthy donors, even though there was not a difference in the presence of antibodies to those antigens in sera from both groups. We did not find differences in either the frequency of CD4 + CD25 + FoxP3+ in PBL, or the levels of IL-4, IL-5 and IL-10 in plasma or conditioned media from PBL incubated with TcR agonistsin vitro, between the two groups. However, we detected a lower production of IL-2 and a higher proportion of CD8 + IFNγ + cells in PBL from LSIL patients as compared with PBL from normal donors. We also observed that PB L from patients infected by HPV-16 and -18 were not able to proliferate in the presence of soluble HPV antigens added to the culture; however, a high level of proliferation was attained when these antigens were presented by activated dendritic cells.Conclusions: Our results suggest that the immunodeficiency reported in LSIL patients could be due to the inability of specific cytotoxic T lymphocytes that for some unknown reason are present but unable to mount a response when challenged with their antigens, probably related to an in situ IL-2 production deficiency. [ABSTRACT FROM AUTHOR]

1,3-β-Glucan was a major cell wall component of fungus. The existing studies showed that 1,3-β-glucan exposure could induce lung inflammation that involved both Th1 and Th2 cytokines. Regulatory T cells (Treg cells) played a critical role in regulating immune homeostasis by adjusting the Th1/Th2 balance. The role of Treg cells and regulatory mechanism in 1,3-β-glucan-induced lung inflammation is still unclear. In our study, mice were exposed to 1,3-β-glucan by intratracheal instillation. To investigate the role of Treg cells in response to 1,3-β-glucan, we generated Treg-depleted mice by intraperitoneal administration of anti-CD25 mAb. The Treg-depleted mice showed more inflammatory cells and severer pathological inflammatory change in lung tissue. Depletion of Treg cells led to increased Th1 cytokines and decreased Th2 cytokines. Treg-depleted mice showed a decreased expression of anti-inflammation cytokine and lower-level expression of CTLA-4. In all, our study indicated that Treg cells participated in regulating the 1,3-β-glucan-induced lung inflammation. Depletion of Treg cells aggravated the 1,3-β-glucan-induced lung inflammation, regulated the Th1/Th2 balance by enhancing Th1 response. Treg cells exerted their modulation function depending on both direct and indirect mechanism during the 1,3-β-glucan-induced lung inflammation. [ABSTRACT FROM AUTHOR]

Interleukin 9 (IL-9) is a cytokine linked to lung inflammation, but its cellular origin and function remain unclear. Here we describe a reporter mouse strain designed to map the fate of cells that have activated IL-9. We found that during papain-induced lung inflammation, IL-9 production was largely restricted to innate lymphoid cells (ILCs). IL-9 production by ILCs depended on IL-2 from adaptive immune cells and was rapidly lost in favor of other cytokines, such as IL-13 and IL-5. Blockade of IL-9 production via neutralizing antibodies resulted in much lower expression of IL-13 and IL-5, which suggested that ILCs provide the missing link between the well-established functions of IL-9 in the regulation of type 2 helper T cell cytokines and responses. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/- mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vβ gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/- mice using lentiviral SIN vectors. [ABSTRACT FROM AUTHOR]

The pivotal role of spleen CD4+ T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4+ T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4+ T cells generated in CD1d-/- mice are I-Ab- restricted (conventional cells), while their counterparts in I-Ab-/- mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4+ T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4+ T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-α and IFN-&γ production depend mostly on conventional CD4+ T cells. IFN-γ is produced simultaneously by non-conventional and conventional CD4+ T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4+ T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4+ T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-γ and parasite-specific antibody production. We concluded that conventional CD4+ T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with nonconventional CD4+ T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection. [ABSTRACT FROM AUTHOR]

HSV-1 is the leading cause of sporadic encephalitis in humans. HSV infection of susceptible 129S6 mice results in fatal encephalitis (HSE) caused by massive inflammatory brainstem lesions comprising monocytes and neutrophils. During infection with pathogenic microorganisms or autoimmune disease, IgGs induce proinflammatory responses and recruit innate effector cells. In contrast, high dose intravenous immunoglobulins (IVIG) are an effective treatment for various autoimmune and inflammatory diseases because of potent anti-inflammatory effects stemming in part from sialylated IgGs (sIgG) present at 1-3% in IVIG. We investigated the ability of IVIG to prevent fatal HSE when given 24 h post infection. We discovered a novel anti-inflammatory pathway mediated by low-dose IVIG that protected 129S6 mice from fatal HSE by modulating CNS inflammation independently of HSV specific antibodies or sIgG. IVIG suppressed CNS infiltration by pathogenic CD11b+ Ly6Chigh monocytes and inhibited their spontaneous degranulation in vitro.FcγRllb expression was required for IVIG mediated suppression of CNS infiltration by CD45+ Ly6Clow monocytes but not for inhibiting development high of Ly6Chigh monocytes. IVIG increased accumulation of T cells in the CNS, and the non-sIgG fraction induced a dramatic expansion of FoxP3+CD4+ T regulatory cells (Tregs) and FoxP3- ICOS+ CD4+ T cells in peripheral lymphoid organs. Tregs purified from HSV infected IVIG treated, but not control, mice protected adoptively transferred mice from fatal HSE. IL-10, produced by the ICOS+ CD4+ T cells that accumulated in the CNS of IVIG treated, but not control mice, was essential for induction of protective anti-inflammatory responses. Our results significantly enhance understanding of IVIG's anti-inflammatory and immunomodulatory capabilities by revealing a novel sIgG independent anti-inflammatory pathway responsible for induction of regulatory T cells that secrete the immunosuppressive cytokine IL-10 and further reveal the therapeutic potential of IVIG for treating viral induced inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: The fetal immune system is characterized by a Th2 bias but it is unclear how the Th2 predominance is established. Natural killer T (NKT) cells are a rare subset of T cells with immune regulatory functions and are already activated in utero. To test the hypothesis that NKT cells are part of the regulatory network that sets the fetal Th2 predominance, percentages of Vα24+Vβ11+ NKT cells expressing Th1/Th2-related chemokine receptors (CKR) were assessed in cord blood. Furthermore, IL-4 and IFN-γ secreting NKT cells were quantified within the single CKR+ subsets. Results: Cord blood NKT cells expressed the Th2-related CCR4 and CCR8 at significantly higher frequencies compared to peripheral blood NKT cells from adults, while CXCR3+ and CCR5+ cord blood NKT cells (Th1-related) were present at lower percentages. Within CD4negCD8neg (DN) NKT cells, the frequency of IL-4 producing NKT cells was significantly higher in cord blood, while frequencies of IFN-γ secreting DN NKT cells tended to be lower. A further subanalysis showed that the higher percentage of IL-4 secreting DN NKT cells was restricted to CCR3+, CCR4+, CCR5+, CCR6+, CCR7+, CCR8+ and CXCR4+ DN subsets in cord blood. This resulted in significantly decreased IFN-γ /IL-4 ratios of CCR3+, CCR6+ and CCR8+ cord blood DN NKT cells. Sequencing of VA24AJ18 T cell receptor (TCR) transcripts in sorted cord blood Vα24Vβ11 cells confirmed the invariant TCR alpha-chain ruling out the possibility that these cells represent an unusual subset of conventional T cells. Conclusions: Despite the heterogeneity of cord blood NKT cells, we observed a clear Th2-bias at the phenotypic and functional level which was mainly found in the DN subset. Therefore, we speculate that NKT cells are important for the initiation and control of the fetal Th2 environment which is needed to maintain tolerance towards self-antigens as well as non-inherited maternal antigens. [ABSTRACT FROM AUTHOR]