IgA-secreting Cells Research Articles

The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease.

BackgroundClonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes.MethodsWe constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores’ surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson’s trichrome.ResultsThe B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores.Conclusions Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic specific immune responses were elicited by oral administration of B.s-CotC-CsCP spores. The spores effectively promoted intestinal health by inducing secretion of acidic mucins, with no other side effects to the liver or intestine. Oral administration of spores expressing CsCP could provide effective protection against C. sinensis. This study may be a cornerstone for development of antiparasitic agents or vaccines against clonorchiasis based on B. subtilis spore expressing CsCP on the surface.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1928-0) contains supplementary material, which is available to authorized users.

Pulmonary α-1,3-Glucan–Specific IgA-Secreting B Cells Suppress the Development of Cockroach Allergy

There is a higher incidence of allergic conditions among children living in industrialized countries than those in developing regions. One explanation for this is reduced neonatal exposure to microbes and the consequent lack of immune stimulation. Sensitivity to cockroach allergen is highly correlated with the development of severe asthma. In this study, we determined that an Ab to microbial α-1,3-glucan binds an Enterobacter species and cockroach allergen. Neonatal, but not adult, mice immunized with this α-1,3-glucan-bearing Enterobacter (MK7) are protected against cockroach allergy. Following exposure to cockroach allergen, α-1,3-glucan-specific IgA-secreting cells are present in the lungs of mice immunized with MK7 as neonates but not in the lungs of those immunized as adults. Mice that are unable to generate anti-α-1,3-glucan IgA Abs were immunized with MK7 as neonates and were no longer protected against cockroach allergy. Thus, neonatal, but not adult, exposure to α-1,3-glucan results in suppressed development of cockroach allergy via pulmonary α-1,3-glucan-specific IgA-secreting cells.

Pulmonary anti-α-1,3-glucan IgA-secreting B cells are protective against cockroach-induced allergic disease

Abstract Currently, an estimated 20% of the population worldwide suffers from an allergic disorder. Children born in industrialized countries have a higher risk of acquiring allergies and asthma than those in developing countries. This difference has been attributed to lower exposure to microbial antigens during early life and the consequent lack of stimulation for the developing immune system. We have previously demonstrated that bacteria and allergens can bear similar epitopes. German cockroach allergen and an enteric Enterobacter species isolated from mice bear similarly-recognized α-1,3-glucan epitopes. Neonatal mice have a large frequency of anti-α-1,3-glucan-IgA secreting cells that significantly decrease by 5 weeks of age. Following challenge with cockroach allergen, neonatal, but not adult, mice immunized with α-1,3-glucan-bearing Enterobacter had a significantly decreased frequency of eosinophils and T cells in the pulmonary parenchyma, antigen-specific memory T cells in the mediastinal lymph node, and IgE in the blood compared to mice immunized with α-1,3-glucan-deficient Enterobacter. A unique population of anti-α-1,3-glucan IgA-secreting cells were identified in the lungs of mice immunized with α-1,3-glucan-bearing Enterobacter as neonates, but not in those immunized as adults. IgA-deficient mice immunized with α-1,3-glucan-bearing Enterobacter as neonates were no longer protected against allergic disease development. We suggest that early immunization with α-1,3-glucan selects neonatal anti-α-1,3-glucan-IgA secreting clones for incorporation in to the adult repertoire such that these B cells have the ability to suppress the development of cockroach-induced allergic disease during adult life.

Mouse and Human Liver Contain Immunoglobulin A–Secreting Cells Originating From Peyer's Patches and Directed Against Intestinal Antigens

The liver receives blood from the gastrointestinal tract through the portal vein, and thereby is exposed continuously to dietary antigens and commensal bacteria. Alcoholic liver disease (ALD) is associated with intestinal dysbiosis, increased intestinal permeability, release of microbes into the portal circulation, and increased serum levels and liver deposits of IgA. We characterized B-cell production of IgA in livers of mice at homeostasis, after oral immunization, in a mouse model of ALD and in human liver samples. We performed studies with Balb/c and C57BL/6-Ly5.1 mice, as well as transgenic mice (quasimonoclonal, activation-induced [cytidine] deaminase-Cre-tamoxifen-dependent estrogen receptor 2 [ERT2], Blimp-1-green fluorescent protein [GFP]). C57BL/6-Ly5.1 mice were fed chronic plus binge ethanol to create a model of ALD. Some mice also were given repeated injections of FTY720, which prevents egress of IgA-secreting cells from Peyer's patches. We obtained nontumor liver tissues from patients with colorectal carcinoma undergoing surgery for liver metastases or hepatocellular carcinoma. B cells were isolated from mouse and human liver tissues and analyzed by flow cytometry and enzyme-linked ImmunoSpot (ELISpot). In wild-type and transgenic mice, we traced newly generated IgA-secreting cells at steady state and after oral immunization with 4-hydroxy-3-nitrophenylacetyl (NP)-Ficoll or cholera toxin. IgA responses were also evaluated in our model of ALD. Livers of control mice contained proliferative plasmablasts that originated from Peyer's patches and produced IgAs reactive to commensal bacteria. After oral immunization with cholera toxin or a thymus-independent antigen, a substantial number of antigen-specific IgA-secreting cells was found in the liver. Mice fed ethanol had features of hepatitis and increased numbers of IgA-secreting cells in liver, compared with mice given control diets, as well as higher levels of serum IgA and IgA deposits in liver sinusoids. Injection of FTY720 during ethanol feeding reduced liver and serum levels of IgA and IgA deposits in liver and prevented liver injury. Human liver tissues contained a significant proportion of IgA-producing plasma cells that shared phenotypic and functional attributes with those from mouse liver, including reactivity to commensal bacteria. Based on studies of mice and human liver tissues, we found the liver to be a site of IgA production by B cells, derived from gut-associated lymphoid tissues. These IgAs react with commensal bacteria and oral antigens. Livers from mice with ethanol-induced injury contain increased numbers of IgA-secreting cells and have IgA deposits in sinusoids. IgAs in the liver could mediate clearance of gut-derived antigens that arrive through portal circulation at homeostasis and protect these organs from pathogens.

CpG DNA facilitate the inactivated transmissible gastroenteritis virus in enhancing the local and systemic immune response of pigs via oral administration

Transmissible gastroenteritis virus (TGEV) replicates in the small intestine and induces enteritis and watery diarrhea. Establishment of local immunity in the intestine would thus prevent TGEV transmission. CpG DNA has been reported as a promising mucosal adjuvant in some animals. The effects of oral immunization of CpG DNA together with inactivated TGEV (ITGEV) were investigated in this study. Pigs (6 weeks old) were orally immunized with ITGEV plus CpG DNA. The TGEV-specific IgA level in the intestinal tract and the TGEV-specific IgG level in serum significantly increased following immunization with ITGEV plus CpG DNA (P≤0.05). Moreover, populations of IgA-secreting cells, CD3+ T lymphocytes and intraepithelial lymphocytes (IELs), in the intestine increased significantly after immunization with ITGEV plus CpG DNA (P≤0.05). Furthermore, the expression of IL-6, IL-12 and interferon-γ (IFN-γ) in ligated intestine segments increased significantly after injection with ITGEV plus CpG DNA (P≤0.05). Taken together, these data suggest that oral immunization of ITGEV plus CpG DNA elicits a local immune response. Further studies are required to determine whether this immunity provides protection against TGEV in pigs.

Fermented milk with probiotic Lactobacillus rhamnosus S1K3 (MTCC5957) protects mice from salmonella by enhancing immune and nonimmune protection mechanisms at intestinal mucosal level

We investigated the mechanism by which an Indian indigenous probiotic culture, Lactobacillus rhamnosus S1K3, could overcome the pathogenic strain Salmonella enterica with an emphasis on the response at the intestinal mucosal level after long-term (30days) consumption. S1K3 was able to produce antimicrobial compounds against the pathogens. The probiotic adhered strongly to intestinal epithelium and maintained its integrity in presence of Salmonella through stimulation of tight junction and antimicrobial peptide genes in vitro. Mice prefed for 30days with S1K3-fermented milk exhibited low incidence of pathogenic Salmonella at mucosal and systemic levels. The probiotic induced TLRs transcripts at the Peyer's patches, followed by an increase in the Secretory-IgA in intestinal fluid, the IgA-secreting cells in lamina propria of small intestine and the IgA level in serum. Moreover, S1K3 maintained the protein level of IL-12, increased the IL-4 and reduced the TGF-β level in intestinal fluid/serum at the later stage of infection. All these actions concurred to lower the count of Salmonella in feces, its invasion in spleen, liver and intestine tissues and improved the health status of probiotic-fed group. In view of this performance, S1K3 appears to be a suitable candidate for the development of nutraceutical food.

Immunology of the gut and oral tolerance

The pathogens and harmless antigens from the bacterial flora and food constantly expose the mucosal surface of the gastrointestinal tract. The mucosal epithelial cells act not only as a physical barrier, but also as a local immune system, which plays a vital role in defense and self-tolerance. The gut mucosal immune system comprises several compartments: Peyer’s patches and lymphoid follicles in the colonic mucosa, and lymphocytes in the lamina propria and intraepithelial lymphocytes. Peyer’s patches mediate antigen uptake via specialized epithelial cells (M cells) and are rich in B cells for class switching into IgA-secreting cells. IgA secretion is one of the primary defenses against pathogens at mucosal surfaces. The lamina propria contains a high proportion of activated and memory T cells that allows rapid immune response against pathogens. In the physiological situation, mucosally encountered antigens induce tolerance of lamina propria and intraepithelial lymphocytes by modified antigen presentation, antigen-induced anergy, or deletion of T cells, or regulation of effector T cells by regulatory or suppressor T cells. Costimulatory molecules mediate cellular interaction and induce regulatory cytokines. While the absence of gut immune privilege to food results in food allergy, the consequences of immune privilege collapse to commensal gut flora is Inflammatory Bowel Disease (IBD). Hence, the knowledge of the homeostatic regulation of the intestinal immune system paves the way for the development of the new immunomodulatory drugs in the therapy of IBD. Moreover, the generation of immune mediated cells through orally fed antigens could be the area of research in the treatment of certain autoimmune diseases.Journal of Advances in Internal Medicine 2015;04(01):16-24

Effects of Lactobacillus plantarum NCU116 on Intestine Mucosal Immunity in Immunosuppressed Mice.

The effects of Lactobacillus plantarum (L. plantarum) NCU116 isolated from pickled vegetables on intestine mucosal immunity in cyclophosphamide treated mice were investigated. Animals were divided into six groups: normal group (NIM), immunosuppression group (IM), immunosuppression plus L. plantarum NCU116 groups with three different doses (NCU-H, NCU-M, and NCU-L), and plus Bifidobacterium BB12 as positive control group (BB12). Results showed that the thymus indexes of the four treatment groups were significantly higher than that of the IM group (2.02 ± 0.16) (p < 0.05) and close to the index of the NIM group (2.61 ± 0.37) at 10 days. The level of immune factor IL-2 notably increased (IM, 121 ± 9.0) (p < 0.05) and was close to 65% of NIM group's level (230 ± 10.7). The levels of other immune factors (IFN-γ, IL-10, IL-12p70, and sIgA), the gene expression levels of IL-2 and IFN-γ, and the number of IgA-secreting cells showed similar patterns (p < 0.05). However, the level of immune factor IL-4 remarkably decreased (IM, 128 ± 10.2) (p < 0.05) and was only approximately 50% of the NIM group (154 ± 18.2). The levels of other immune factors (IL-6 and IgE) and the gene expression level of IL-6 at 10 days exhibited similar changes (p < 0.05) but showed a slight recovery at 20 days, accompanied by the altered protein expression levels of T-bet and GATA-3 in the small intestine. These findings suggest that L. plantarum NCU116 enhanced the immunity of the small intestine in the immunosuppressed mice.

Improved immune responses to a bivalent vaccine of Newcastle disease and avian influenza in chickens by ginseng stem-leaf saponins

Our previous investigation demonstrated that ginseng stem-leaf saponins (GSLS) derived from the stems and leaves of Panax ginseng C.A. Meyer promoted humoral and gut mucosal immunity in chickens vaccinated with live infectious bursa disease vaccine. The present study was designed to evaluate the effect of GSLS on the immune response to a bivalent inactive vaccine of Newcastle disease (ND) and avian influenza (AI) in chickens immunosuppressed by cyclophosphamide (Cy). One hundred and sixty-eight specific-pathogen-free (SPF) chickens were randomly divided into 7 groups, each containing 24 birds. Chickens in groups 3–7 received intramuscular injection of Cy at 100mg/kg BW for 3 days to induce immunosuppression. Groups 1 and 2 were injected with saline solution in the same way as groups 3–7. Following injection of Cy, groups 4–7 were orally administrated GSLS (2.5, 5 and 10mg/kg BW) or astragalus polysaccharide (APS) (200mg/L) in drinking water for 7 days; groups 1–3 were not medicated and served as control birds. After administration of GSLS or APS, groups 2–7 were subcutaneously injected with a bivalent inactive vaccine of ND and AI. After that, serum was sampled for detecting antibody titers by HI, spleen was collected for lymphocyte proliferation assay, and duodenum tissues were collected for measurement of IgA-secreting (IgA+) cells and intestinal intraepithelial lymphocytes (iIELs). The results showed that injection of Cy significantly suppressed immunity in chickens; oral administration of GSLS before immunization recovered splenocyte proliferation induced by ConA and LPS, and the numbers of IgA+ cells and iIELs as well as the specific antibody response to a bivalent inactive vaccine of ND and AIin immunosuppressed chickens treated with Cy. Therefore, GSLS may be the potential agent to improve vaccination in immunosuppressed chickens.

Immunogenicity of inactivated polio vaccine, given with or without mucosal adjuvants dmLT and TCTA1T in rat model (VAC9P.1102)

Abstract To eradicate polio, the use of oral polio vaccine (OPV) should be stopped to avoid circulation of vaccine-derived polioviruses (VDPVs). After OPV cessation, transition to inactivated polio vaccine (IPV) is recommended. This can only be accomplished when IPV induce better mucosal protection. This can be achieved by adding mucosal adjuvant to the IPV formulation. In the present study we used wistar rats to elucidate the adjuvant effect of double mutant heat-labile toxin (dmLT) of E. coli and recombinant cholera toxin (TCTA1T) on IPV. Rats were administered by intramuscular injection of 1.0-0.6-1.0 D-antigen units (DU) of Inactivated Polio type 1, 2 and 3 /rat. The E. coli dmLT and TCTA1T was used as adjuvant and was administered subcutaneously along with IPV. The control groups received IPV alone or PBS. Rats were immunized on days 0, 14 and 28 days. After last vaccination mice were sacrificed and spleen cells and blood were collected. IgA secreting cells specific for Polio Type1, 2 and 3 were measured by ELISPOT. Intramuscular immunization results in robust mucosal response in inactivated polio antigens + dmLT group compared to other groups. Further measurement of antibodies in serum doesn’t show significant difference among IPV and adjuvanted IPV groups. Thus the present data suggest that vaccination with IPV and dmLT results in specific induction of mucosal immunity, which will provide barrier to infection from oral fecal route.

Oral delivery of Bacillus subtilis spore expressing enolase of Clonorchis sinensis in rat model: induce systemic and local mucosal immune responses and has no side effect on liver function.

Caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensis, human clonorchiasis remains a major public health problem in China. In previous study, we had expressed enolase from C. sinensis (CsENO) on the surface of Bacillus subtilis spore and the recombinant spore induced a pronounced protection in terms of reduced worm burden and eggs per gram feces, suggesting B. subtilis spore as an ideal vehicle for antigen delivery by oral treatment and CsENO as a promising vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgG and IgA levels both in serum and in intestinal mucus from rats orally administrated with B. subtilis spore surface expressing CsENO by ELISA. Lysozyme levels in serum and in intestinal mucus were analyzed too. In addition, IgA-secreting cells in intestine epithelium of the rats were detected by immunohistochemistry assay. The intestinal villi lengths of duodenum, jejunum, and ileum were also measured. Rats orally treated with B. subtilis spore or normal saline were used as controls. Our results showed that, compared with the control groups, oral administration of B. subtilis spore expressing CsENO induced both systemic and local mucosal immune response. The recombinant spores also enhanced non-specific immune response in rats. The spores had no side effect on liver function. Moreover, it might facilitate food utilization and digestion of the rats. Our work will pave the way to clarify the involved mechanisms of protective efficacy elicited by B. subtilis spore expressing CsENO and encourage us to carry out more assessment trails of the oral treated spore to develop vaccine against clonorchiasis.

Regulation of intestinal IgA responses.

The intestine harbors enormous numbers of commensal bacteria and is under frequent attack from food-borne pathogens and toxins. A properly regulated immune response is critical for homeostatic maintenance of commensals and for protection against infection and toxins in the intestine. Immunoglobulin A (IgA) isotype antibodies function specifically in mucosal sites such as the intestines to help maintain intestinal health by binding to and regulating commensal microbiota, pathogens and toxins. IgA antibodies are produced by intestinal IgA antibody-secreting plasma cells generated in gut-associated lymphoid tissues from naïve B cells in response to stimulations of the intestinal bacteria and components. Research on generation, migration, and maintenance of IgA-secreting cells is important in our effort to understand the biology of IgA responses and to help better design vaccines against intestinal infections.

Putting J Chain Back on the Map: How Might Its Expression Define Plasma Cell Development?

Joining chain (J chain) is a small polypeptide that regulates multimerization of secretory IgM and IgA, the only two mammalian Igs capable of forming multimers. J chain also is required for poly-Ig receptor-mediated transport of these Ig classes across the mucosal epithelium. It is generally assumed that all plasma cells express J chain regardless of expressed isotype, despite the documented presence of J chain(-) plasma cells in mammals, specifically in all monomeric IgA-secreting cells and some IgG-secreting cells. Compared with most other immune molecules, J chain has not been studied extensively, in part because of technical limitations. Even the reported phenotype of the J chain-knockout mouse is often misunderstood or underappreciated. In this short review, we discuss J chain in light of the various proposed models of its expression and regulation, with an added focus on its evolutionary significance, as well as its expression in different B cell lineages/differentiation states.

Classification of common variable immunodeficiencies using flow cytometry and a memory B-cell functionality assay

The population of patients with common variable immunodeficiency (CVID) comprises a heterogeneous group of patients with different causes of hypogammaglobulinemia predisposing to recurrent infections, higher incidence of autoimmunity, and malignancy. Although memory B cells (memBcs) are key players in humoral defense and their numbers are commonly reduced in these patients, their functionality is not part of any current classification. We established and validated a memBc enzyme-linked immunosorbent spot (ELISpot) assay that reveals the capacity of memBcs to develop into antibody-secreting cells and present an idea for a new classification based on this functional capacity. The memBc ELISpot assay, combined with flow cytometry, was applied to patients with confirmed CVID in comparison with age-matched healthy control subjects. Ex vivo frequency of IgG-, IgM-, and IgA-secreting plasmablasts was significantly diminished by 27.2-, 2.4-, and 23.3-fold, respectively, compared with that seen in healthy control subjects. Moreover, in vitro differentiation of memBcs into antibody-secreting cells was 6.1-, 2.6-, and 3.7-fold significantly reduced for IgG-, IgM-, and IgA-secreting cells, respectively. Proliferation of memBcs correlates inversely to immunoglobulin-secreting capacity, suggesting compensatory hyperproliferation. Furthermore, patients with no serum IgA can still have a detectable IgA ELISpot assay result in vitro. Most importantly, the large heterogeneity of memBc function in patients with CVID homogenously grouped by means of fluorescence-activated cell sorting allowed additional subclassification based on memBc/plasmablast function. These data suggest almost normal memBc/immunoglobulin-secreting plasmablast functionality in some patients if sufficient stimulatory signals are delivered, which might open up opportunities for new therapeutic approaches.

Dietary rice bran protects against rotavirus diarrhea and promotes Th1-type immune responses to human rotavirus vaccine in gnotobiotic pigs.

Rice bran (RB) contains a distinct stoichiometry of phytochemicals that can promote gut mucosal immune responses against enteric pathogens. The effects of RB on rotavirus diarrhea and immunogenicity of an attenuated human rotavirus (HRV) vaccine were evaluated in gnotobiotic pigs. The four treatment groups studied were RB plus vaccine, vaccine only, RB only, and mock control. Pigs in the RB groups were fed the amount of RB that replaced 10% of the pigs' total daily calorie intake from milk starting from 5 days of age until they were euthanized. Pigs in the vaccine groups were orally inoculated with two doses of the attenuated HRV vaccine. A subset of pigs from each group was orally challenged with the homologous virulent HRV on postinoculation day 28. Diarrhea and virus shedding were monitored daily from postchallenge day 0 to day 7. RB feeding significantly protected against diarrhea upon virulent HRV challenge and enhanced the protective rate of the vaccine against rotavirus diarrhea. Consistent with protection, RB significantly increased gamma interferon (IFN-γ)-producing CD4(+) and CD8(+) T cell responses in intestinal and systemic lymphoid tissues. Furthermore, RB also increased the number of total IgM- and IgA-secreting cells, total serum IgM, IgG, and IgA titers, and HRV-specific IgA titers in intestinal contents. RB reduced the numbers of intestinal and systemic HRV-specific IgA and IgG antibody-secreting cells and reduced serum HRV-specific IgA and IgG antibody titers before the challenge. These results demonstrate clear beneficial effects of RB in protection against rotavirus diarrhea and stimulation of nonspecific and HRV-specific immune responses, as well as its biased Th1-type adjuvant effect for the vaccine.

Adding crude glycerin to nursery pig diet: Effect on nutrient digestibility, metabolic status, intestinal morphology and intestinal cytokine expression

The objective of this study was to determine the effect of crude glycerin (Gly) added to nursery pig diet on nutrient digestibility, the digestive and metabolic status, intestinal morphology and intestinal cytokine expression. A total of 18 male piglets (weaned at 23 days) were used. There were three dietary treatments that differed in the inclusion level of Gly (0, 9 and 18%). On day 14 of the experiment, the animals were weighed and plasma samples were collected before slaughtering. In addition urine, digesta content and intestinal tissues were sampled post mortem. No differences were observed among the tested diets as regards the coefficients of apparent ileal digestibility of DM and CP. The concentration of lactic acid decreased linearly (P<0.05) in the jejunum and ileum segments as the level of Gly increased, although the concentrations of volatile fatty acids in the cecum and colon were not affected. The plasma concentrations of glucose, fructosamine and IGF-1 were not affected. However, urinary glycerol concentrations increased (P<0.01) with increasing levels of Gly. In general, there were no differences in villus height, crypt depth, villus/crypt ratio or number of lymphocytes in the intestinal segments between the different treatments. Nevertheless, the control treatment produced a higher level of goblet cells in the ileum than either of the Gly treatments (P<0.01), while in the jejunum, the number of IgA-secreting cells in the 9% Gly group was higher (P<0.01) than in the control group. There were no differences among the experimental treatments concerning the gene expression of IL-10, IL-12 p40 and TNF-α. Gene expressions of TGF-β, IL-12 p35, IFN-γ and IFN-α were remained unaffected or increased, depending on the intestinal segment and level of Gly addition. In conclusion, the inclusion of Gly at 9 and 18% to the nursery pig diet did not affect nutrient digestibility or plasma metabolites but increased the levels of urinary glycerol, suggesting that metabolic pathways of glycerol utilization became saturated when high levels of Gly are used. In addition, the intestinal cell structure and intestinal cytokine expression might be affected when Gly is included in the feed.

Escherichia coli Nissle 1917 colonization ameliorates human rotavirus diarrhea and modulates B cell responses in a neonatal gnotobiotic pig disease model (VIR7P.1064)

Abstract Microbiota regulate gut immune homeostasis, but the role of gram positive (G+)/negative (G-) commensals/probiotics in modulating human rotavirus (HRV) diarrhea and immunity is largely undefined. Gnotobiotic (Gn) piglets, the only animal model susceptible to HRV diarrhea, were colonized with G- Escherichia coli Nissle 1917 (EcN), G+ Lactobacillus rhamnosus strain GG (LGG) or LGG+EcN and challenged with virulent HRV (VirHRV) to assess probiotic effects on HRV pathogenesis and immunity. EcN+VirHRV piglets had significantly lower mean peak virus shedding titers and significantly reduced mean cumulative fecal scores and duration of diarrhea compared to LGG+VirHRV or control (uncolonized)+VirHRV piglets. LGG+VirHRV pigs had lower, but statistically similar fecal scores compared to control+VirHRV pigs. Combining LGG+EcN did not moderate VirHRV shedding or diarrhea more than EcN alone. Coinciding with reduced HRV infection, EcN+VirHRV piglets had significantly lower small intestinal (SI) HRV IgA antibody titers and HRV IgA and IgG antibody secreting cells (SC) compared to control+VirHRV piglets. Though not significantly different, SI HRV IgA antibody titers were 2-4-fold lower in the LGG+EcN+VirHRV and LGG+VirHRV piglets. Notably EcN+VirHRV piglets had higher SI total IgA levels and IgA SC compared to LGG+VirHRV piglets. Our results indicate that EcN alone was more effective than LGG or LGG+EcN in ameliorating HRV diarrhea and modulating B cell responses in a Gn piglet disease model.

Generation of colonic IgA-secreting cells in the caecal patch

Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyer's patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyer's patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.

Does tonsillectomy have a role in the treatment of patients with immunoglobulin A nephropathy?

Immunoglobulin A nephropathy (IgAN) is an immune-complex mediated glomerulonephritis characterized by deposition of polymeric IgA (mainly IgA1) in the mesangium of the kidney [1]. It is the most common form of primary glomerulonephritis worldwide and is prevalent among all ages and racial demographics [2]. Its pathogenesis has been under investigation for the last several decades and one of the key players is the aberrant glycosylation pattern of IgA1. There is now convincing evidence that the hinge region of IgA1 heavy chain has defective glycosylation with a reduction in the galactose and or sialic acid residues [3]. As a result, the N-acetylgalactosamine in the IgA1 hinge region is exposed and recognized by IgG antibodies. This in turn leads to formation of IgG-IgA immune complexes and their deposition in the mesangium and renal injury that subsequently ensues [4–7]. It should be noted that aberrantly glycosylated IgA1 can also be seen in circulation in normal subjects following an immune response triggered by exposure to mucosal antigens such as food, bacteria or viruses. Thus, other factors, including genetic predisposition, are likely to influence the pathogenesis of IgAN [8]. The renal outcomes of IgAN vary significantly between individuals ranging from minimal proteinuria and stable renal function to development of end-stage renal disease (ESRD) in up to 50% of the cases [9]. Markers of poor prognosis include impaired kidney function at presentation, hypertension and persistent proteinuria >1 g/day in adults, and 0.5 g/day in children [10–12]. In addition, the recent Oxford classification has identified MEST (mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis, tubular atrophy/interstitial fibrosis) as an independent renal biopsy indicator of poor renal outcome [13]. Treatments aiming to block the angiotensin II system, the use of corticosteroids, either alone or in combination with cytotoxic medications have been used in patients with IgAN with variable success [14–18]. In some patients, renal disease progresses despite treatment and thus a search for additional forms of therapy have been carried out including the use of tonsillectomy. What is the rationale behind performing tonsillectomy in patients with IgA nephropathy? First, recurrent tonsillitis is the most common extrarenal clinical manifestation in IgAN and there is a clear recognition that in some patients episodes of macroscopic hematuria and proteinuria are often associated with tonsillitis (synpharyngitic hematuria) [19, 20]. In fact, studies have shown that episodes of macroscopic hematuria can be reduced following tonsillectomy [21, 22]. Second, tonsils provide the first line of defense against inhaled pathogens. The foreign antigens, particularly bacterial polysaccharides, activate the Toll-like receptors (TLRs) of the cells of innate immunity, which ultimately results in activation of tonsillar B-cells and production of immunoglobulins particularly polymeric IgA1. Third, tonsils from patients with IgAN have an increased number of polymeric IgA1 compared with controls [23]. Forth, the polymeric IgA1 deposited in the kidney are, in part, of tonsillar origin. This was based on the evidence that IgA1 eluted from the mesangium of patients with IgAN specifically bound the nuclear regions of tonsillar cells obtained from the same patient and the binding was completely inhibited by the addition of antihuman IgA sera [24]. Fifth, abnormal IgAsecreting cells which predominantly produce polymeric IgA1 are found in the tonsillar tissue of patients with IgAN in contrast to IgG-secreting cells that predominate in tonsils of patients without IgAN [20, 25–27]. Taken together, it has been postulated that chronic tonsillitis in a susceptible patient may lead to increased production of aberrantly glycosylated IgA1, formation of IgG-IgA immune complexes and their deposition in the glomeruli triggering an inflammatory response [28]. As such, tonsillectomy has been suggested as a possible treatment modality in patients with IgAN. Several retrospective studies have evaluated the efficacy of tonsillectomy in treatment of IgAN with conflicting results as shown in Table 1 [21, 22, 29– 35]. Most of the studies, however, are from an era prior to IN F O C U S

Oral Administration of Lactobacillus plantarum Strain AYA Enhances IgA Secretion and Provides Survival Protection against Influenza Virus Infection in Mice

The mucosal immune system provides the first line of defense against inhaled and ingested pathogenic microbacteria and viruses. This defense system, to a large extent, is mediated by the actions of secretory IgA. In this study, we screened 140 strains of lactic acid bacteria for induction of IgA production by murine Peyer’s patch cells. We selected one strain and named it Lactobacillus plantarum AYA. We found that L. plantarum AYA-induced production of IL-6 in Peyer’s patch dendritic cells, with this production promoting IgA+ B cells to differentiate into IgA-secreting plasma cells. We also observed that oral administration of L. plantarum AYA in mice caused an increase in IgA production in the small intestine and lung. This production of IgA correlated strongly with protective ability, with the treated mice surviving longer than the control mice after lethal influenza virus infection. Our data therefore reveals a novel immunoregulatory role of the L. plantarum AYA strain which enhances mucosal IgA production and provides protection against respiratory influenza virus infection.