Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine

Intestinal immunoglobulin A (IgA) ensures host defense and symbiosis with our commensal microbiota. Yet previous studies hint at a surprisingly low diversity of intestinal IgA, and it is unknown to what extent the diverse Ig arsenal generated by somatic recombination and diversification is actually used. In this study, we analyze more than one million mouse IgA sequences to describe the shaping of the intestinal IgA repertoire, its determinants, and stability over time. We show that expanded and infrequent clones combine to form highly diverse polyclonal IgA repertoires with very little overlap between individual mice. Selective homing allows expanded clones to evenly seed the small but not large intestine. Repertoire diversity increases during aging in a dual process. On the one hand, microbiota-, T cell-, and transcription factor RORγt-dependent but Peyer's patch-independent somatic mutations drive the diversification of expanded clones, and on the other hand, new clones are introduced into the repertoire of aged mice. An individual's IgA repertoire is stable and recalled after plasma cell depletion, which is indicative of functional memory. These data provide a conceptual framework to understand the dynamic changes in the IgA repertoires to match environmental and intrinsic stimuli.

Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.

The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine

The nuclear receptor liver receptor homologue-1 (LRH-1, NR5A2) is a crucial transcriptional regulator of many metabolic pathways. In addition, LRH-1 is expressed in intestinal crypt cells where it regulates the epithelial cell renewal and contributes to tumorigenesis through the induction of cell cycle proteins. We have recently identified the intestinal epithelium as an important extra-adrenal source of immunoregulatory glucocorticoids. We show here that LRH-1 promotes the expression of the steroidogenic enzymes and the synthesis of corticosterone in murine intestinal epithelial cells in vitro. Interestingly, LRH-1 is also essential for intestinal glucocorticoid synthesis in vivo, as LRH-1 haplo-insufficiency strongly reduces the intestinal expression of steroidogenic enzymes and glucocorticoid synthesis upon immunological stress. These results demonstrate for the first time a novel role for LRH-1 in the regulation of intestinal glucocorticoid synthesis and propose LRH-1 as an important regulator of intestinal tissue integrity and immune homeostasis.

Quantitative mRNA analysis of muscarinic acetylcholine receptors in the intestine of dairy cows with spontaneous caecal dilatation-dislocation

Muscarinic receptors mediate acetylcholine-induced muscular contractions. In this study, mRNA levels of muscarinic receptor subtypes 2 and 3 (M(2) and M(3)) in the ileum, caecum, proximal loop of the ascending colon (PLAC) and external loop of the spiral colon (ELSC) were determined by quantitative polymerase chain reaction in seven cows with caecal dilatation-dislocation (CDD) and seven healthy control cows. Levels of M(2) were significantly lower in the caecum, PLAC and ELSC and levels of M(3) were significantly lower in the ileum, caecum, PLAC and ELSC of cows with CDD compared to healthy cows (P<0.05). Down-regulation of M(3) may play a role in the pathogenesis of CDD.

Construct Validity of the Pig Intestine Model in the Simulation of Laparoscopic Urethrovesical Anastomosis

Introduction: Laparoscopic training models are increasingly important in urology to allow trainees to improve their laparoscopic skills prior to going to the operating room. For a training model to be valid, it must correlate with performance in a real case. The model must also discriminate between experienced and inexperienced subjects. [See PDF for complete abstract]

Upregulation of defensins in burn sheep small intestine.

OBJECTIVE: The aim of this study was to visualize and localize the sheep antimicrobials, beta-defensins 1, 2, and 3, (SBD-1, SBD-2, SBD-3), sheep neutrophil defensin alpha (SNP-1), and the cathelicidin LL-37 in sheep small intestine after burn injury, our hypothesis being that these compounds would be upregulated in an effort to overcome a compromised endothelial lining. Response to burn injury includes the release of proinflammatory cytokines and systemic immune suppression that, if untreated, can progress to multiple organ failure and death, so protective mechanisms have to be initiated and implemented. METHODS: Tissue sections were probed with antibodies to the antimicrobials and then visualized with fluorescently labeled secondary antibodies and subjected to fluorescence deconvolution microscopy and image reconstruction. RESULTS: In both the sham and burn samples, all the aforementioned antimicrobials were seen in each of the layers of small intestine, the highest concentration being localized to the epithelium. SBD-2, SBD-3, and SNP-1 were upregulated in both enterocytes and Paneth cells, while SNP-1 and LL-37 showed increases in both the inner circular and outer longitudinal muscle layers of the muscularis externa following burn injury. Each of the defensins, except SBD-1, was also seen in between the muscle layers of the externa and while burn caused slight increases of SBD-2, SBD-3, and SNP-1 in this location, LL-37 content was significantly decreased. CONCLUSION: That while each of these human antimicrobials is present in multiple layers of sheep small intestine, SBD-2, SBD-3, SNP-1, and LL-37 are upregulated in the specific layers of the small intestine.

Suppression by {\it Trypanosoma brucei\/} of anaphylaxis-mediated ion transport in the small intestine of rats

Most tissue-invasive parasitic helminths prime for type 1 hypersensitivity or anaphylaxis during some phase of their life cycles. A prototype in this regard is the nematode Trichinella spiralis. Blood protozoa capable of tissue invasion, such as Trypanosoma brucei, might also be expected to prime for the expression of anaphylaxis. However, this response is usually absent in protozoal infections. The hypothesis tested was that failure of hosts infected with T.brucei to express anaphylaxis is related to this parasite's ability to selectively down-regulate immunoglobulin E (IgE) production, and not to an innate lack of allergenicity on the part of T.brucei-derived antigens. This hypothesis was tested by studying in the intestine of rats, antigen-induced Cl$\sp-$ secretion, which results from a local anaphylactic response mediated by IgE and mucosal mast cells. The Cl$\sp-$ secretory response can be primed either by infection with T.spiralis or by the parenteral administration of antigen. Anaphylaxis-induced Cl$\sp-$ secretion is expressed in vitro, and can be quantified electrophysiologically, as a change in transmural short-circuit current when sensitized intestine is mounted in Ussing chambers and challenged with the sensitizing antigen.^ Rats injected parenterally with trypanosome antigen elicited intestinal anaphylaxis in response to antigenic challenge. In contrast, the intestine of rats infected with T.brucei failed to respond to challenge with trypanosome antigen. Infection with T.brucei also suppressed antigen-induced Cl$\sp-$ secretion in rats sensitized and challenged with various antigens, including T.spiralis antigen. However, T.brucei infection did not inhibit the anaphylactic response in rats concomitantly infected with T.spiralis. Relative to the anaphylactic mediators, T.brucei infection blocked production of IgE in rats parenterally injected with antigen but not in T.spiralis-infected hosts. Also, the mucosal mastocytosis normally associated with trichinosis was unaffected by the trypanosome infection. These results support the conclusion that the failure to express anaphylaxis-mediated Cl$\sp-$ secretion in T.brucei infected rats, is due to this protozoan's ability to inhibit IgE production and not to the lack of allergenicity of trypanosome antigens. ^

The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4(+) T cells and/or CD4(-) cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69(-/-) CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS)-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69(-/-) CD4 T cell accumulation in colonic lamina propria (cLP) was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69(-/-) mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69(-/-) CD45RB(high) CD4 T cells into RAG(-/-) hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis.

IMAGING DIAGNOSIS-MUSCULAR HYPERTROPHY OF THE SMALL INTESTINE AND PSEUDODIVERTICULA IN A HORSE.

A 14-year-old Thoroughbred gelding was presented for chronic colic and weight loss. Transcutaneous and transrectal abdominal ultrasonography revealed distended, thickened small intestine with primary thickening of the muscularis and a focally more thickened loop with an echoic structure crossing the wall from the mucosa to the serosa. Visualization of diffuse thickening of the muscularis (muscular hypertrophy of the small intestine) and a focal lesion (pseudodiverticulum) helped clinicians make informed decisions. This case illustrates the importance of transabdominal and transrectal ultrasonography in horses with chronic colic and the relevance of considering the abnormalities in layering pattern of the intestinal wall.

Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine

Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N′-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine . By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.