Microbiota and Food Allergy.

Emerging evidence suggests that the increasing prevalence of food allergies is associated with compositional and functional changes in our gut microbiota. Microbiota-host interactions play a key role in regulating the immune system. Development of a healthy gut microbiota and immune system occurs early in life and is largely shaped by exposure to maternal microbes through vaginal/natural delivery and breast milk, whereas use of antibiotics can disrupt gut homeostasis and significantly raise the risk of allergic diseases. Thus, changes in the quantity or diversity of gut microbes affect oral toleranace through interations of microbial molecules with pattern recognition receptors on immune cells and confer susceptibility to food allergies. On the other hand, short chain fatty acids which are fermentation end products of insoluble fibers by intestinal micoorganisms have been shown to confer protective effects on food allergy. As a preventive and therapeutic treatment for food allergies, probiotics have gained widespread attention in recent years. Reintroducing certain commensal microbes, such as Clostridia, both in animal models and clinical trials led to the prevention or resolution of allergic symptoms. This review highlights recent progress in our understanding of the gut microbiota's role in food allergy. However, mechanistic details underlying the anti-allergic effects of probiotics and the interaction between the gut microbiota and the immune system remain circumstantial and are not fully understood. Future studies should address possible factors and underlying mechanisms for microbiota-host interactions and gut immunity, as well as the efficacy, safety, and appropriate use of probiotics in establishing a standard treatment regimen for food allergies.

Low-Dose Allergen-Specific Immunotherapy Induces Tolerance in a Murine Model of Shrimp Allergy.

BACKGROUND: The efficacy and safety of allergen-specific immunotherapy (AIT) are highly dose-dependent. METHODS: We investigated the dosage effects of AIT and the underlying mechanisms in a murine model of shrimp hypersensitivity. BALB/c mice were sensitized with recombinant shrimp allergen rMet e 1 and challenged orally with a high dose of rMet e 1 to elicit an allergic response. These sensitized mice were then treated with a low (0.01 mg), medium (0.05 mg), or high dosage (0.1 mg) of rMet e 1 intraperitoneally before receiving a second oral challenge. The allergic responses and immunological changes in the gut were compared between animals receiving different dosages. RESULTS: We found that all sensitized mice that received rMet e 1 immunotherapy were desensitized, regardless of the dosage, and protected at the second oral challenge. Nevertheless, the mice in the high-dosage group experienced severe systemic reactions during the treatment phase. In contrast, regulatory T (Treg) cell-associated genes were upregulated only in the low- and medium-dosage groups, and Foxp3+ cells were more abundant in the gut lymphoid tissues than in the high-dosage group. CONCLUSIONS: Our results demonstrate that low-dosage immunotherapy favors the induction of local Foxp3+ Treg cells and the upregulation of regulatory cytokines. The safety advantages and long-term efficacy of low-dosage immunotherapy should be taken into consideration when developing immunotherapy dose schedules.

Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one-compound peptide libraries.

The one-bead-one-compound (OBOC) combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E (IgE) epitopes of the major shellfish allergen tropomyosin. OBOC peptide libraries with 8-12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.Cellular & Molecular Immunology advance online publication, 14 september 2015; doi:10.1038/cmi.2015.83.

Gastrointestinal Immune Response to the Shrimp Allergen Tropomyosin: Histological and Immunological Analysis in an Animal Model of Shrimp Tropomyosin Hypersensitivity.

BACKGROUND: Shellfish hypersensitivity is among the most common food allergies. A murine model of IgE-mediated shrimp allergy has been established in our laboratory. The aim of this study is to determine the intestinal histological changes and cytokine expression profile of this model sensitized with the major shellfish allergen tropomyosin. METHODS: Female Balb/c mice orally sensitized and challenged with recombinant tropomyosin were sacrificed. Continuous sections of duodenum, jejunum and ileum were prepared using the Swiss roll technique for histological and immunological analysis. Duodenal epithelial cell apoptosis and migration were examined. mRNA expression of IL-4, IL-6, IL-10, IL-13, IL-18 and IFN-γ in intestinal tissue was measured via RT-PCR. RESULTS: In tropomyosin-sensitized and challenged mice, an increased number of eosinophils, mast cells and goblet cells was found 24 h after challenge. There were also increased mast cell and goblet cell numbers at 72 h after challenge, but the level of eosinophils decreased. Differences compared with control mice are most prominent at the duodenum compared to the distal regions. In addition, TUNEL assay indicates a significantly higher apoptosis rate in sensitized mice sacrificed 72 h after challenge, and mRNA expression showed a biased Th2/Th1 cytokine profile and a higher level of murine mast cell protease 1. CONCLUSIONS: This study documented a multitude of histological and immunological changes in the gut in a murine model of shrimp allergy. Even without repetitive intragastric challenge, shrimp tropomyosin induces an increase in the number of inflammatory cells to varying degrees within the small intestine. This model provides an important tool for testing new therapeutic interventions.

Gene Therapy for Autoimmune Disease.

Advances in understanding the immunological and molecular basis of autoimmune diseases have made gene therapy a promising approach to treat the affected patients. Gene therapy for autoimmune diseases aims to regulate the levels of proinflammatory cytokines or molecules and the infiltration of lymphocytes to the effected sites through successful delivery and expression of therapeutic genes in appropriate cells. The ultimate goal of gene therapy is to restore and maintain the immune tolerance to the relevant autoantigens and improve clinical outcomes for patients. Here, we summarize the recent progress in identifying genes responsible for autoimmune diseases and present examples where gene therapy has been applied as treatments or prevention in autoimmune diseases both in animal models and the clinical trials. Discussion on the advantages and pitfalls of gene therapy strategies employed is provided. The intent of this review is to inspire further studies toward the development of new strategies for successful treatment of autoimmune diseases.

Immunization with Hypoallergens of shrimp allergen tropomyosin inhibits shrimp tropomyosin specific IgE reactivity.

Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their in vitro reactivity towards IgE from shrimp allergy patients and Met e 1-sensitized mice, as well as considerable decrease in induction of mast cell degranulation as demonstrated in passive cutaneous anaphylaxis assay. Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy.

The changing geoepidemiology of food allergies.

The science of food allergy has been rapidly evolving before our eyes in the past half century. Like other allergic disorders, the prevalence of food allergies has dramatically increased, and coupled with the increased public awareness of anaphylaxis due to food allergy, this has driven an explosion in basic and clinical research in this extremely broad subject. Treatment of food allergies has evolved and practices such as food challenges have become an integral part of an allergy practice. The impact of the increase of food allergy has driven package labeling laws, legislation on emergency treatment availability in schools and other public places, and school policy. But to this day, our knowledge of the pathogenesis of food allergy is still incomplete. There are the most obvious IgE-mediated immediate hypersensitivity reactions, but then multiple previously unidentified conditions such as eosinophilic esophagitis, food protein-induced enterocolitis syndrome, milk protein allergy, food-induced atopic dermatitis, oral allergy syndrome, and others have complicated the diagnosis and management of many of our patients who are unable to tolerate certain foods. Many of these conditions are not IgE-mediated, but may be T cell-driven diseases. The role of T regulatory cells and immune tolerance and the newly discovered immunological role of vitamin D have shed light on the variable clinical presentation of food allergy and the development of new methods of immunotherapy in an example of bench-to-bedside research. Component-resolved diagnostic techniques have already begun to allow us to more precisely define the epitopes that are targeted in food allergic patients. The development of biological modulators, research on genomics and proteomics, and epigenetic techniques all offer promising avenues for new modes of therapy of food allergy in the twenty-first century.

Common methodologies in the evaluation of food allergy: pitfalls and prospects of food allergy prevalence studies.

Global and regional studies on the prevalence of food allergies are plagued by inconsistent methodologies, variations in interpretation of results, and non-standardized study design. Hence, it becomes difficult to compare the prevalence of food allergies in different communities. This information would be useful in providing critical data that will enhance research to elucidate the nature of food allergies, and the role of gene-environment interactions in the sensitization of children and adults to foods. Testing methodologies range from questionnaires to objective in vitro and in vivo testing, to the gold standard, double-blind placebo-controlled food challenge (DBPCFC). Although considered the most accurate and reliable method in detecting the prevalence of food allergy, DBPCFC is not always practical in epidemiological studies of food allergy. On the other hand, multiple logistic regression studies have been done to determine predictability of the outcome of food challenges, and it appears that skin prick testing and in vitro-specific serum IgE are the best predictors. Future studies directed towards confirming the validity of these methods as well as developing algorithms to predict the food challenge outcomes are required, as they may someday become accessory tools to complement DBPCFC.

Antimitochondrial antibody heterogeneity and the xenobiotic etiology of primary biliary cirrhosis.

UNLABELLED: Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8-bis(acetylthio) octanoic acid (SAc)-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme-linked immunosorbent assay (ELISA); SAc conjugate and rPDC-E2-specific affinity-purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late-stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC-E2 in early-stage versus late-stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. CONCLUSION: Specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e., by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host.

Development and validation of gene therapies in autoimmune diseases: Epidemiology to animal models.

Recent advancement in immunology, molecular biology, and bioinformatics has yielded extensive information on the pathophysiological mechanisms of autoimmunity, which has greatly facilitated the identification of potential therapeutic targets and the development of gene therapy in the treatment of autoimmune disease. Preclinical studies were carried out in animal models. This phenomenon is well illustrated in two prototypic animal models of autoimmune disease: the autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) and collagen-induced arthritis (CIA) model in rheumatoid arthritis (RA). Here we discuss the current data on the development and validation of gene therapy in autoimmunity in these two models. The success in preclinical animal model studies provides the proof-of-concept of gene therapy for potential future applications in the treatment of autoimmune diseases. Furthermore, the identification of risk factors from epidemiological studies reveals further potential therapeutic targets to be examined in animal models.

Cocoa flavanols and procyanidins can modulate the lipopolysaccharide activation of polymorphonuclear cells in vitro.

Flavanols and procyanidins isolated from cocoa have been reported to possess multiple activities potentially relevant to oxidant defenses, vascular function, and immune function. In a combination of in vivo and in vitro studies, we and others have observed that cocoa can be an anti-inflammatory modulator and that compounds in cocoa are capable of modulating eicosanoid production, platelet aggregation, and the pool size of nitric oxide. The present study extends these findings by examining the in vitro effects of cocoa procyanidins on polymorphonuclear cells (PMNs). PMNs, part of the innate arm of the immune system, represent 50-60% of the total peripheral white blood cells and are the first cells to be recruited to the sites of inflammation or injury secondary to bacterial infections. Herein, we demonstrate that certain flavanols and procyanidins isolated from cocoa can moderate a subset of signaling pathways derived from lipopolysaccharide (LPS) stimulation of PMNs, mainly, PMN oxidative bursts and activation markers, and they can influence select apoptosis mechanisms. We hypothesize that flavanols and procyanidins can decrease the impact of LPS on the N-formyl-Met-Leu-Phe-primed PMN ability to generate reactive oxygen species by partially interfering in activation of the mitogen-activated protein kinase pathway.

Natural killer T cells exacerbate liver injury in a transforming growth factor beta receptor II dominant-negative mouse model of primary biliary cirrhosis.

UNLABELLED: Primary biliary cirrhosis (PBC) is an organ-specific autoimmune liver disease characterized by the presence of antimitochondrial antibodies and the destruction of small intrahepatic bile ducts with portal inflammation. In previous studies, we reported that both CD1d expression and the frequency of CD1d-restricted natural killer T (NKT) cells were increased in the livers of patients with PBC. To define a specific role of CD1d-restricted NKT cells in the pathogenesis of PBC, particularly early events, we investigated the function of hepatic CD1d-restricted NKT cells in our transforming growth factor beta (TGF-beta) receptor II dominant-negative (dnTGFbetaRII) mouse model of PBC. We generated CD1d(-/-) and CD1d(+/-) dnTGFbetaRII mice and performed a comparative study of liver immunopathology. We report herein that these dnTGFbetaRII mice demonstrate a massive increase of hyperactive CD1d-restricted NKT cells within the hepatic tissues. CD1d(-/-)dnTGFbetaRII mice, which lack CD1d-restricted CD1d-restricted NKT cells, exhibit significantly decreased hepatic lymphoid cell infiltrates and milder cholangitis compared with CD1d(+/-)dnTGFbetaRII mice. Interestingly, there was a significant increase in the production of interferon-gamma in hepatic CD1d-restricted NKT cells activated by alpha-galactosylceramide in young but not older dnTGFbetaRII mice, suggesting an age-dependent role of CD1d-restricted NKT cells. CONCLUSION: These data demonstrate that CD1d-restricted NKT cells in dnTGFbetaRII mice are a critical factor in liver injury.

The current immune function of hepatic dendritic cells.

While only a small percentage of the liver as dendritic cells, they play a major role in the regulation of liver immunity. Four major types of dendritic cell subsets include myeloid CD8alpha(-)B220(-), lymphoid CD8alpha(+)B220(-), plasmacytoid CD8alpha(-)B220(+), and natural killer dendritic cell with CD8alpha(-)B220(-)NK1.1(+) phenotype. Although these subsets have slightly different characteristics, they are all poor naïve T cell stimulators. In exchange for their reduced capacity for allostimulation, hepatic DCs are equipped with an enhanced ability to secrete cytokines in response to TLR stimulation. In addition, they have increased level of phagocytosis. Both of these traits suggest hepatic DC as part of the innate immune system. With such a high rate of exposure to the dietary and commensal antigens, it is important for the hepatic DCs to have an enhanced innate response while maintaining a tolerogenic state to avoid chronic inflammation. Only upon secondary infectivity does the hepatic DC activate memory T cells for rapid eradication of recurring pathogen. On the other hand, overly tolerogenic characteristics of hepatic DC may be responsible for the increase prevalence of autoimmunity or liver malignancies.

The influence of cyclosporin A on lymphocyte attenuator expression.

B and T lymphocyte attenuator (BTLA), a recently identified immune inhibitory receptor, has been demonstrated to have the ability to maintain self-tolerance and transplant-tolerance in mice. However, little is known about the effects of immunosuppressive drugs on the expression of BTLA. In the present study, we observed that the immunosuppressive drug cyclosporin A (CsA) could significantly reduce BTLA but not CD25 and CD69 expression on CD4+ T cells during activation in vitro, while rapamycin (RPM) had little effect on it. Exogenous interleukin-2 (IL-2) failed to reverse the inhibitory effect that CsA had on BTLA expression. Furthermore, phorbol 12-myristate 13-acetate (PMA) or ionomycin alone could efficiently induce BTLA protein expression on CD4+ and CD8+ T cells, while CsA significantly suppressed BTLA expression in this system. The present data indicate that the regulation of BTLA expression on CD4+ T cells does not depend on IL-2 and T cell activation but depends on calcineurin-dependent and calcineurin-independent pathways. The observation that CsA significantly inhibits BTLA expression on CD4+ T cells during activation, suggests that CsA might block the immune tolerance induced by BTLA and potentially increase the susceptibility to autoimmune diseases and graft rejection.