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Gulf War Illness (GWI) is a debilitating condition marked by chronic fatigue, cognitive problems, pain, and gastrointestinal (GI) complaints in veterans who were deployed to the 1990-1991 Gulf War. Fatigue, GI complaints, and other chronic symptoms continue to persist more than 30 years post-deployment. Several potential mechanisms for the persistent illness have been identified and our prior pilot study linked an altered gut microbiome with the disorder. This study further validates and builds on our prior preliminary findings of host gut microbiome dysbiosis in veterans with GWI. Using stool samples and Multidimensional Fatigue Inventory (MFI) data from 89 GW veteran participants (63 GWI cases and 26 controls) from the Boston biorepository, recruitment, and integrative network (BBRAIN) for Gulf War Illness, we found that the host gut bacterial signature of veterans with GWI showed significantly different Bray-Curtis beta diversity than control veterans. Specifically, a higher Firmicutes to Bacteroidetes ratio, decrease in Akkermansia sp., Bacteroides thetaiotamicron , Bacteroides fragilis , and Lachnospiraceae genera and increase in Blautia , Streptococcus , Klebsiella , and Clostridium genera, that are associated with gut, immune, and brain health, were shown. Further, using MaAsLin and Boruta algorithms, Coprococcus and Eisenbergiella were identified as important predictors of GWI with an area under the curve ROC predictive value of 74.8%. Higher self-reported MFI scores in veterans with GWI were also significantly associated with an altered gut bacterial diversity and species abundance of Lachnospiraceae and Blautia . These results suggest potential therapeutic targets for veterans with GWI that target the gut microbiome and specific symptoms of the illness.

Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4 ). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [ 3 H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation. NEW & NOTEWORTHY This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation.

Gulf War (GW) veterans show gastrointestinal disturbances and gut dysbiosis. Prolonged antibiotic treatments commonly employed in veterans, especially the use of fluoroquinolones and aminoglycosides, have also been associated with dysbiosis. This study investigates the effect of prolonged antibiotic exposure on risks of adverse renal pathology and its association with gut bacterial species abundance in underlying GWI and aims to uncover the molecular mechanisms leading to possible renal dysfunction with aging. Using a GWI mouse model, administration of a prolonged antibiotic regimen involving neomycin and enrofloxacin treatment for 5 months showed an exacerbated renal inflammation with increased NF-κB activation and pro-inflammatory cytokines levels. Involvement of the high mobility group 1 (HMGB1)-mediated receptor for advanced glycation end products (RAGE) activation triggered an inflammatory phenotype and increased transforming growth factor-β (TGF-β) production. Mechanistically, TGF-β- induced microRNA-21 upregulation in the renal tissue leads to decreased phosphatase and tensin homolog (PTEN) expression. The above event led to the activation of protein kinase-B (AKT) signaling, resulting in increased fibronectin production and fibrosis-like pathology. Importantly, the increased miR-21 was associated with low levels of Lachnospiraceae in the host gut which is also a key to heightened HMGB1-mediated inflammation. Overall, though correlative, the study highlights the complex interplay between GWI, host gut dysbiosis, prolonged antibiotics usage, and renal pathology via miR-21/PTEN/AKT signaling.

Biotin is an essential vitamin and critical cofactor in several metabolic pathways, and its deficiency has been linked to several disorders including inflammatory bowel disease (IBD). We previously reported that biotin deficiency (BD) in mice, whether modeled through intestine-specific deletion of biotin transporter (SMVT-icKO) or through a biotin-deficient diet, resulted in intestinal inflammation consistent with an IBD-like phenotype. To assess whether the gut microbiome is associated with these BD-induced changes, we collected stool and intestinal samples from both of these mouse models and utilized them for 16S rRNA gene sequencing. We find that both diet-mediated and deletion-mediated BD result in the expansion of opportunistic microbes including Klebsiella , Enterobacter , and Helicobacter , at the expense of mucus-resident microbes including Akkermansia . Additionally, microbiome dysbiosis resulting from diet-mediated BD precedes the onset of the IBD-like phenotypic changes. Lastly, through the use of predictive metagenomics, we report that the resulting BD-linked microbiome perturbations exhibit increased biotin biosynthesis in addition to several other perturbed metabolic pathways. Altogether, these results demonstrate that biotin deficiency results in a specific microbiome composition, which may favor microbes capable of biotin synthesis and which may contribute to intestinal inflammation.

Background & Aims: Biotin is a water-soluble vitamin that is indispensable for human health. Biotin deficiency can cause failure-to-thrive, immunodeficiency, alopecia, dermatitis, and conjunctivitis. We previously reported that biotin deficiency also can lead to severe colitis in mice, which is completely reversed with supplementation. Our aim in this study was to determine if high-dose biotin supplementation can provide a therapeutic benefit in a preclinical model for inflammatory bowel disease (IBD) and to identify the molecular mechanism by which this occurs., Methods: Mice were challenged with dextran sodium sulfate to induce colitis and were treated with 1 mmol/L biotin to induce or maintain remission. Clinical response was monitored by the Disease Activity Index and fecal calprotectin levels. The colon tissue was investigated for histology, length, as well as expression of inflammatory cytokines (interleukin 6, tumor necrosis factor-α, interleukin 1β), intestinal permeability, tight junctions (zonula occludens-1 and claudin-2), and the transcription factor nuclear factor-κB (NF-κB)., Results: Biotin therapy led to delayed onset and severity of colitis as well as accelerated healing. There was improvement in the Disease Activity Index, fecal calprotectin levels, colon length, and histology. In addition, biotin-treated mice had reduced expression of inflammatory cytokines, reduced intestinal permeability, and reduced activation of NF-κB., Conclusions: Oral supplementation with biotin provides benefit for maintenance and induction of remission in the dextran sodium sulfate preclinical model for IBD. Biotin does this by reducing the activation of NF-κB, which prevents the production of inflammatory cytokines and helps maintain the integrity of the intestinal barrier. Clinically, the NF-κB pathway is important in the development of IBD and this finding suggests that biotin may have therapeutic potential for patients with IBD., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The sodium-dependent multivitamin transporter (SMVT; SLC5A6 ) is involved in intestinal absorption of vitamin B7 (biotin). We have previously shown that mice with an embryonic intestinal-specific SMVT knockout (KO) develop biotin deficiency and severe spontaneous intestinal inflammation in addition to growth retardation, developmental delays, and death within the first 6-7 wk of life. The profound morbidity and mortality associated with the SMVT-KO has limited our ability to further characterize the intestinal inflammation and other sequelae of this deletion in adult mice with a mature gut microbiota. To overcome this limitation, we generated an intestine-specific, tamoxifen-inducible, conditional SMVT-KO (SMVT-icKO). Our results showed that adult SMVT-icKO mice have reduced body weight, biotin deficiency, shorter colonic length, and bloody diarrhea compared with age- and sex-matched control littermates. All SMVT-icKO mice also developed spontaneous intestinal inflammation associated with induction of calprotectin (S100a8/S100a9), proinflammatory cytokines (IL-1β, TNF-α, IFN-γ, and IL-6), and an increase in intestinal permeability. Additionally, the intestines of SMVT-icKO showed activation of the NF-κB pathway and the nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome. Notably, administration of broad-spectrum antibiotics reduced lethality and led to normalization of intestinal inflammation, proinflammatory cytokines, altered mucosal integrity, and reduced expression of the NLRP3 inflammasome. Overall, these findings support our conclusion that the biotin transport pathway plays an important role in the maintenance of intestinal homeostasis, and that NF-κB and the NLRP3 inflammasome, as well as gut microbiota, drive the development of intestinal inflammation when SMVT is absent. NEW & NOTEWORTHY This study demonstrates that deletion of the intestinal biotin uptake system in adult mice leads to the development of spontaneous gut inflammation and that luminal microbiota plays a role in its development.

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca 2+ , and a transgenic Ca 2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca 2+ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca 2+ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca 2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca 2+ transients ('sparkles') as cells migrate.

Hepatitis B virus (HBV) infection causes a tremendous clinical burden across the world with more than half a million people dying annually from HBV related disease. Significant advances have been made in HBV treatment in the past decade and several guidelines have been published by professional societies and expert panels. Although these recommendations have been valuable to help optimize HBV treatment, there is discordance in treatment criteria and many patients infected with HBV may fall outside of these recommendations. This paper systematically reviews the natural history of the disease and compares and contrasts the recommendations for initiation of treatment from the various societies. There is also discussion of special groups that require particular consideration and some of the open research questions and future research directions within the field.

STAT6 plays a central role in IL-4-mediated allergic responses. Several studies indicate that regulatory T cells (Tregs) can be modulated by IL-4 in vitro. We previously showed that STAT6(-/-) mice are highly resistant to allergic lung inflammation even when wild-type Th2 effectors were provided and that they have increased numbers of Tregs. However, the role of STAT6 in modulating Tregs in vivo during allergic lung inflammation has not been thoroughly investigated. To examine Treg and STAT6 interaction during allergic inflammation, STAT6(-/-), STAT6xRAG2(-/-), and RAG2(-/-) mice were subjected to OVA sensitization and challenge following adoptive transfer of OVA-specific, wild-type Th2 effectors with or without prior Treg depletion/inactivation, using anti-CD25 (PC61). As expected, STAT6(-/-) mice were highly resistant to airway inflammation and remodeling. In contrast, allergic lung inflammation was partially restored in STAT6(-/-) mice treated with PC61 to levels observed in STAT6xRAG2(-/-) mice. In some cases, STAT6xRAG2(-/-) mice were also given natural Tregs along with Th2 effectors. Adoptive transfer of natural Tregs caused a substantial reduction in bronchoalveolar lavage eosinophil composition and suppressed airway remodeling and T cell migration into the lung in STAT6xRAG2(-/-) mice to levels comparable to those in STAT6(-/-) mice. These results demonstrate the STAT6-dependent suppression of Tregs in vivo to promote allergic airway inflammation.

Disseminated superficial actinic porokeratosis (DSAP) is a chronic disorder of keratinization characterized by numerous papules and plaques distributed over sun-exposed sites. Treatments are poorly standardized and several investigational therapies have demonstrated limited success in treating DSAP. To our knowledge, there have been no systematic reviews of the literature regarding the treatment of this disease. Herein, we review recent studies pertaining to the treatment of DSAP and evaluate the level of evidence for each of these therapeutic modalities.

Antigen-immunoglobulin fusion protein expressing B cells have been shown as excellent tolerogenic antigen-presenting cells in multiple disease models. Using efficient protein transduction by fusion with a HIV TAT protein transduction domain, we herein tested the TAT-fusion protein transduced B cells for their effects in antigen-specific tolerance induction in two animal models, experimental autoimmune encephalomyelitis (EAE) and type 1 diabetes. We demonstrated that transfer of TAT-MOG35-55 (myelin oligodendrocyte glycoprotein)-Ig 'transduced B cells' 10 days after EAE induction significantly protected mice from disease. Similarly, the onset of disease was delayed when NOD mice received insulin specific TAT-B9-23-B cells. Surprisingly, no protection against EAE was observed in a prophylactic protocol when transduced B cells were given before disease induction. Moreover, TAT-ovalbumin transduced cells were tolerogenic in primed but not naïve mice. Our results suggest that TAT-fusion protein transduced B cells were tolerogenic in antigen primed recipients, a condition clinically relevant to autoimmune diseases., (Published by Elsevier Inc.)

We herein tested the effect of B-cell depletion on tolerance induction to factor VIII (FVIII) in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential depletion effects were used. Thus, IgG1 anti-CD20 selectively depleted follicular B cells and spared marginal zone B cells, whereas IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice, a single dose of either IgG1 or IgG2a anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice by subsequent daily, high-dose FVIII intravenous injection as a model for immune tolerance induction. However, the IgG1, but not the IgG2a, anti-CD20 pretreatment led to a significant increase of regulatory T cells in the spleen. Importantly, 3 months after the partial B-cell depletion with IgG1 anti-CD20, the FVIII-specific hyporesponsive state remained. We suggest a tolerogenic role of the remaining marginal zone B cells as a potential mechanism for anti-CD20 therapy.

Hemophilia is a bleeding disorder that affects approximately 1 in 4000 males across populations worldwide. First-line therapy for the treatment of hemophilia is the intravenous administration of protein therapeutics to replace the deficient coagulation factor. However, in a significant number of patients, the immune system recognizes the therapeutic protein as 'dangerous' and mounts a humoral response that rejects the treatment and significantly increases the morbidity associated with this disease. Recent advances have been made in gene therapy in the field of hemophilia. Gene therapy provides the possibility of a cure for this disease; however, managing immunological tolerance to therapy is a challenge for this treatment modality. This review describes an approach in which gene therapy is used to deliver a tolerogenic construct to B-cells that can induce tolerance to protein therapy or to replacement gene therapy. Several other novel techniques to modulate immunity in patients with hemophilia, such as non-specific agents, mAbs and protein modifications, are at various stages of translation to the clinic and are also highlighted. The successful modulation of the immune system to accept treatment will significantly improve the quality of life for patients with hemophilia.

Antigen-specific tolerance induction using autologous B-cell gene therapy is a potential treatment to eliminate undesirable immune responses. For example, we have shown that experimental autoimmune encephalomyelitis (EAE) and type 1 diabetes in NOD mice can be ameliorated using antigen-Ig fusion protein transduced B cells. However, it is well established that auto-reactive antigen-specific B cells are activated in many autoimmune diseases and can contribute to pathogenesis. While syngeneic B cells from immunized or autoimmune mice can serve as tolerogenic antigen-presenting cells (APC), this observation begs the question of whether the antigen-specific B cells per se can be transduced as tolerogenic APC. To test this, we employed two model systems employing B cell receptor (BCR) transgenic or wild type (wt) mice as B-cell donors. While adoptively transferred MOG-Ig transduced wt C57Bl/6 B cells were highly tolerogenic and ameliorated EAE, MOG-Ig transduced anti-MOG B cells from BCR transgenic mice were not. This phenomenon was reproduced in the NOD diabetes model in which pro-insulin-Ig transduced polyclonal wt NOD B cells were protective, whereas similarly transduced anti-insulin BCR B cells were not. Since the frequency of antigen-specific B cells in an immunized animal is quite low, we wished to determine the threshold numbers of BCR transgenic B cells that could be present in an effective transduced population. Therefore, we "spiked" polyclonal wt C57Bl/6 B cells with different numbers of anti-MOG BCR transgenic B cells. In the EAE model, we found protection when BCR B cells were present at 1%, but they prevented tolerance induction at 10%. Antigen-specific B cells expressed normal levels of co-stimulatory molecules and were tolerogenic when transduced with an irrelevant antigen (OVA). Thus, the presence of a BCR specific for the target autoantigen may interfere with the tolerogenic process to that antigen, but BCR-specific B cells are not intrinsically defective as tolerogenic APC. Taken together, these data suggest that antigen-specific tolerance induction can be achieved in the presence of a limited number of antigen-specific B cells, but higher numbers of pathogenic B cells may mask this induction. This observation should guide future development of therapies using autologous B cells to treat patients with autoimmune diseases., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Although plasmacytoid dendritic cells (pDCs) express major histocompatibility complex class II (MHCII) molecules, and can capture, process, and present antigens (Ags), direct demonstrations that they function as professional Ag-presenting cells (APCs) in vivo during ongoing immune responses remain lacking. We demonstrate that mice exhibiting a selective abrogation of MHCII expression by pDCs develop exacerbated experimental autoimmune encephalomyelitis (EAE) as a consequence of enhanced priming of encephalitogenic CD4(+) T cell responses in secondary lymphoid tissues. After EAE induction, pDCs are recruited to lymph nodes and establish MHCII-dependent myelin-Ag-specific contacts with CD4(+) T cells. These interactions promote the selective expansion of myelin-Ag-specific natural regulatory T cells that dampen the autoimmune T cell response. pDCs thus function as APCs during the course of EAE and confer a natural protection against autoimmune disease development that is mediated directly by their ability to present of Ags to CD4(+) T cells in vivo.

Previous reports have shown that B-cell-mediated gene therapy can induce tolerance in several animal models for autoimmune diseases and inhibitory antibody formation in hemophilia A mice. We know from our previous work that the induction of tolerance following B-cell therapy is dependent upon CD25(+) regulatory T cells (Tregs). To extend these studies and identify the effects of this gene therapy protocol on the target CD4 T cells, we have adapted in vitro suppression assays using Tregs isolated from treated and control mice. Using carboxyfluorescein succinimidyl ester (CFSE) dilution as a measure of T-cell responsiveness to FVIII, we show that CD25(+) Tregs from treated mice are more suppressive than those from control animals. To monitor the induction of antigen-specific Tregs, we repeated these studies in ovalbumin (OVA) peptide-specific DO11.10 T-cell receptor (TCR) transgenic mice. Tregs from DO11.10 mice treated with a tolerogenic OVA-Ig construct are better than polyclonal Tregs at suppressing the proliferation of responder cells stimulated with OVA peptide 323-339 (pOVA). Furthermore, we show that following B-cell therapy, there is an increase in antigen-specific FoxP3(+) Tregs, and there is also a distinct decrease in antigen-specific CD4(+) effector T cells. These changes in the lymphocyte population shift the balance away from effector function toward a tolerogenic phenotype.

Administration of human factor VIII (FVIII) to FVIII knockout hemophilia mice is a useful small animal model to study the physiologic response in patients iatrogenically immunized to this therapeutic protein. These mice manifest a robust, T cell-dependent, antibody response to exogenous FVIII treatment, even when encountered through traditionally tolerogenic routes. Thus, FVIII given via these routes elicits both T- and B-cell responses, whereas a control, foreign protein, such as ovalbumin (OVA), is poorly immunogenic. When FVIII is heat inactivated, it loses function and much of its immunogenicity. This suggests that FVIII's immunogenicity is principally tied to its function and not its structure. If mice are treated with the anticoagulant warfarin, which depletes other coagulation factors including thrombin, there is a reduced immune response to FVIII. Furthermore, when mice are treated with the direct thrombin inhibitor, hirudin, the T-cell responses and the serum anti-FVIII antibody concentrations are again significantly reduced. Notably, when FVIII is mixed with OVA, it acts to increase the immune response to OVA. Finally, administration of thrombin with OVA is sufficient to induce immune responses to OVA. Overall, these data support the hypothesis that formation of thrombin through the procoagulant activity of FVIII is necessary to induce costimulation for the immune response to FVIII treatment.

Patients with hemophilia A are deficient in coagulation Factor VIII. This bleeding disorder can be treated with Factor VIII replacement therapy, but close to a third of patients will be immunized to the treatment and begin to form inhibitory antibodies known as "inhibitors". These inhibitors will render the treatment ineffective and represent the most severe complication in the treatment of hemophilia A. In this review, we highlight factors involved in inhibitor development and emphasize research being done to modulate the immune response to this life-saving therapy.

Tolerance must be maintained to prevent deleterious immune responses. Thus, when tolerance is lost, autoimmunity can result. A number of novel approaches to (re-) induce tolerance for potential clinical applications have been developed in the last decade. Our lab has implemented an immunoglobulin-based gene therapy approach, which may have powerful implications for the treatment of human conditions. These include a variety of autoimmune diseases, transplantation, and the immune response to therapeutic proteins (as in the treatment of hemophilia A) or gene therapy per se. We clone the target (immunogenic) protein in frame with an immunoglobulin heavy chain and deliver it via retrovirus to an activated B cell. In our system, we observe tolerance to multiple epitopes of the protein cloned. An important advantage of this regimen is that identification of the precise peptide epitopes of a target protein is not necessary since selection and presentation by the host's own antigen presenting cells (APC's) eliminates the issue of HLA polymorphism. Additionally, our data indicate that these tolerogenic B cells are stimulating an endogenous population of regulatory T cells, which are effective at suppressing the immune response in both naïve and primed hosts. Thus, this approach has potential for future clinical therapy.

The chromatographic purification of biological macromolecules requires a novel approach to overcome some of the pore size limitations of commercially available resins. Membrane adsorbers offer the potential for better resolution as well as productivity. Sharp peaks are gained by the rapid exchange rate with the adsorbing membranes associated with the convective flow path, in contrast to the pore diffusion requirement for resin exchange. The resolution advantage is preserved even when the very short bed heights of membranes are exploited for the purpose of exceptionally high flow rates and productivity. Breakthrough experiments were used to assess the membrane dynamic loading capacities of flexible macromolecules using supercoiled (SC) DNA as a model system. In contrast to reports for smaller biomolecules such as proteins and antibodies, the dynamic capacity for DNA was found to be highly dependent on flow rates and concentrations. Increasing flow rates induced DNA elongation, which increased the surface coverage and, in turn, lowered the capacity. Increasing concentrations beyond C*, the overlap concentration, led to exclusion-volume interactions, which reduced the size of DNA and increased the membrane adsorber capacity. In the chromatographic mode, membranes with a strongly positive charge were able to resolve various isoforms of DNA, surpassing the capabilities of analogous chromatographic resins. In this study, we found that the convective-flow-induced-structural behavior of DNA is responsible for the resolution in separation.