The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted V H gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1-2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification. The Journal of Immunology, 2000, 165: 2012-2019.T he rabbit is one of a few vertebrate species known to make limited use of combinatorial joining of multiple V H, D H , and J H gene segments during Ig heavy chain gene rearrangement. Although there are more than 100 V H gene segments available within the rabbit Ig heavy chain locus, many of which appear to be potentially functional (1), the 3Ј-most V H gene segment, V H 1, is utilized in 80 -90% of VDJ gene rearrangements (2). Most of the remaining 10 -20% of VDJ gene rearrangements utilize only two other V H gene segments, V H x and V H y (3). Rabbit B cells diversify their VDJ genes in gut-associated lymphoid tissue (GALT) 4 at 1-2 mo of age through two targeted mutational processes: a somatic gene conversion-like mechanism that transfers tracts of nucleotide sequence from upstream V H gene segment donors into the rearranged V H gene segment (4), and somatic hypermutation that distributes point mutations throughout the entire VDJ gene (5-7). At that time, the B cell population is also expanded through proliferation in GALT. B cell proliferation and Ig VDJ gene diversification in GALT results in a large population of B cells that express a wide range of Ab specificities.Several studies have established the importance of GALT in B cell proliferation and VDJ gene diversification. Weinstein et al. (7), for example, determined VDJ gene nucleotide...

Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as non-bloody diarrhea may be Stx-independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria to the cecum and colon. Greater numbers of EHEC were also cultured from stool of streptomycin-pretreated mice, as compared to those that received no antibiotic. Transmission electron microscopy demonstrated that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in colonic tissues of infected animals. Increased mucosal permeability to 4KDa FITC-Dextran was also observed in colonic tissues of infected mice. Immunofluorescence microscopy revealed that EHEC infection resulted in redistribution of the tight junction proteins occludin and claudin-3 and increased expression of claudin-2 while ZO-1 localization remained unaltered. Quantitative real-time PCR revealed that EHEC altered mRNA transcription of Ocln, Cldn2 and Cldn3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of tight junction proteins plays a role in the increase in intestinal permeability.

Lactoferrin (LF), a pleiotropic iron-binding glycoprotein, is known to modulate the humoral immune response. However, its exact role in Ig synthesis has yet to be elucidated. In this study, we investigated the effect of LF on Ig production by mouse B cells and its underlying mechanisms. LF, like transforming growth factor (TGF)-β1, stimulated B cells to produce IgA and IgG2b, while downregulating other isotypes. Using limiting dilution analysis, LF was shown to increase the frequency of IgA-secreting B-cell clones. This was paralleled by an increase in Ig germ-line α (GLα) transcripts, indicating that LF plays a role as an IgA switch factor. Interestingly, LF directly interacted with betaglycan (TGF-β receptor III, TβRIII) and in turn induced phosphorylation of TβRI and Smad3 through formation of the TβRIII/TβRII/TβRI complex, leading to IgA isotype switching. Peroral administration of LF increased intestinal/serum IgA production as well as number of IgA plasma cells in lamina propria. Finally, we found that LF has an adjuvant activity when nontoxigenic Salmonella typhimurium was inoculated perorally, conferring protection against intragastrical infection of toxigenic S. typhimurium. These results suggest that LF has an important effect on the mucosal/systemic IgA response and can contribute to protection against intestinal pathogens.

BackgroundPiperine is a compound comprising 5-9% of black pepper (Piper nigrum), which has a variety of biological roles related to anticancer activities. Helicobacter pylori has been classified as a gastric carcinogen, because it causes gastritis and gastric cancer by injecting the virulent toxin CagA and translocating VacA. The present study investigated the inhibitory action of piperine on H. pylori growth and adhesion.MethodsInhibition of H. pylori growth was determined by the broth macrodilution method, and adhesion to gastric adenocarcinoma cells validated by urease assay. Motility test was performed by motility agar and the expression of adhesion gene and flagellar gene in response to the piperine treatment was assessed by RT-PCR and immunoblotting.ResultsAdministrated piperine suppressed the level of H. pylori adhesion to gastric adenocarcinoma cells in a dose dependent manner and the inhibition was statistically significant as determined by Student’s t-test. In addition, piperine treatment effects on the flagellar hook gene flgE and integral membrane component of the export apparatus gene flhA expression to be suppressed and piperine diminished the H. pylori motility.ConclusionsflhA, encodes an integral membrane component of the export apparatus, which is also one of the regulatory protein in the class 2 genes expression and flgE is one of them that encodes hook part of the flagella. Suppression of both genes, leads to less motility results in the organism attracted less towards to the gastric epithelial cells might be the possible reason in the adhesion inhibition. To our knowledge, this is the first report published on the inhibitory effects of piperine against the adhesion of H. pylori to gastric adenocarcinoma cells.

Enterotoxigenic Bacteroides fragilis (ETBF) is a human gut commensal bacteria that causes inflammatory diarrhea and colitis. ETBF also promotes colorectal tumorigenesis in the Min mouse model. The key virulence factor is a secreted metalloprotease called B. fragilis toxin (BFT). BFT induces E-cadherin cleavage, cell rounding, activation of the β-catenin pathway and secretion of IL-8 in colonic epithelial cells. However, the precise mechanism by which these processes occur and how these processes are interrelated is still unclear. E-cadherin form homophilic interactions which tethers adjacent cells. Loss of E-cadherin results in detachment of adjacent cells. Prior studies have suggested that BFT induces IL-8 expression by inducing E-cadherin cleavage; cells that do not express E-cadherin do not secrete IL-8 in response to BFT. In the current study, we found that HT29/C1cells treated with dilute trypsin solution induced E-cadherin degradation and IL-8 secretion, consistent with the hypothesis that E-cadherin cleavage causes IL-8 secretion. However, physical damage to the cell monolayer did not induce IL-8 secretion. We also show that EDTA-mediated disruption of E-cadherin interactions without E-cadherin degradation was sufficient to induce IL-8 secretion. Finally, we determined that HT29/C1 cells treated with LiCl (β-catenin activator) induced IL-8 secretion in a dose-dependent and time-dependent manner. Taken together, our results suggest that BFT induced IL-8 secretion may occur by the following process: E-cadherin cleavage, disruption of cellular interactions, activation of the β-catenin pathway and IL-8 expression. However, we further propose that E-cadherin cleavage per se may not be required for BFT induced IL-8 secretion.