Increased intestinal permeability and tight junction disruption by altered expression and localization of occludin in a murine graft versus host disease model

<p>Abstract</p> <p>Background</p> <p>Hematopoietic stem cell transplantation is increasingly performed for hematologic diseases. As a major side effect, acute graft versus host disease (GvHD) with serious gastrointestinal symptoms including diarrhea, gastrointestinal bleeding and high mortality can be observed. Because surveillance and biopsies of human gastrointestinal GvHD are difficult to perform, rare information of the alterations of the gastrointestinal barrier exists resulting in a need for systematic animal models.</p> <p>Methods</p> <p>To investigate the effects of GvHD on the intestinal barrier of the small intestine we utilized an established acute semi allogenic GvHD in C57BL/6 and B6D2F1 mice.</p> <p>Results</p> <p>By assessing the differential uptake of lactulose and mannitol in the jejunum, we observed an increased paracellular permeability as a likely mechanism for disturbed intestinal barrier function. Electron microscopy, immunohistochemistry and PCR analysis indicated profound changes of the tight-junction complex, characterized by downregulation of the tight junction protein occludin without any changes in ZO-1. Furthermore TNF-α expression was significantly upregulated.</p> <p>Conclusions</p> <p>This analysis in a murine model of GvHD of the small intestine demonstrates serious impairment of intestinal barrier function in the jejunum, with an increased permeability and morphological changes through downregulation and localization shift of the tight junction protein occludin.</p

Influence of oral beclomethasone dipropionate on early non-infectious pulmonary outcomes after allogeneic hematopoietic cell transplantation: results from two randomized trials.

Early non-infectious pulmonary complications represent a significant cause of mortality after hematopoietic cell transplantation (HCT). We tested the hypothesis that oral beclomethasone dipropionate (BDP) is effective for preventing early non-infectious pulmonary complications after allogeneic HCT. We retrospectively reviewed the medical records of 120 patients, 60 in each treatment arm, to identify non-infectious and infectious pulmonary events and pulmonary function test results from all patients who participated in two randomized trials of oral BDP for treatment of acute gastrointestinal GVHD. 17-Beclomethasone monopropionate (17-BMP), the active metabolite of BDP, was evaluated in blood from the right atrium in four patients. Thirty-three of 42 (79%) placebo-treated patients experienced a decrease of the DL(CO) from pretransplant to day 80 after transplant, compared with 27 of 49 (55%) BDP-treated patients (P=0.02). In the first 200 days after randomization, there were no cases of non-infectious pulmonary complications in BDP-treated patients, vs four cases among placebo-treated patients (P=0.04). Levels of 17-BMP were detected in atrial blood at steady state. Delivery of a potent glucocorticoid such as 17-BMP to the pulmonary artery after oral dosing of BDP may be useful in modulating pulmonary inflammation and preventing the development of non-infectious pulmonary complications after allogeneic HCT.Bone Marrow Transplantation advance online publication, 29 June 2009; doi:10.1038/bmt.2009.129

Genome-Wide Data-Mining of Candidate Human Splice Translational Efficiency Polymorphisms (STEPs) and an Online Database

Variation in pre-mRNA splicing is common and in some cases caused by genetic variants in intronic splicing motifs. Recent studies into the insulin gene (INS) discovered a polymorphism in a 5' non-coding intron that influences the likelihood of intron retention in the final mRNA, extending the 5' untranslated region and maintaining protein quality. Retention was also associated with increased insulin levels, suggesting that such variants--splice translational efficiency polymorphisms (STEPs)--may relate to disease phenotypes through differential protein expression. We set out to explore the prevalence of STEPs in the human genome and validate this new category of protein quantitative trait loci (pQTL) using publicly available data.Gene transcript and variant data were collected and mined for candidate STEPs in motif regions. Sequences from transcripts containing potential STEPs were analysed for evidence of splice site recognition and an effect in expressed sequence tags (ESTs). 16 publicly released genome-wide association data sets of common diseases were searched for association to candidate polymorphisms with HapMap frequency data. Our study found 3324 candidate STEPs lying in motif sequences of 5' non-coding introns and further mining revealed 170 with transcript evidence of intron retention. 21 potential STEPs had EST evidence of intron retention or exon extension, as well as population frequency data for comparison.Results suggest that the insulin STEP was not a unique example and that many STEPs may occur genome-wide with potentially causal effects in complex disease. An online database of STEPs is freely accessible at http://dbstep.genes.org.uk/

Platelet Toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-alpha production in vivo

Toll-like receptors (TLRs) play a critical role in stimulating innate immunity by recognizing pathogen-associated molecular patterns (PAMPs) on invading microorganisms. Platelets also play a role in innate immunity, and we studied whether they express TLR. Results show that human and murine platelets variably expressed TLR2, TLR4, and TLR9 by flow cytometry and Western blotting. TLR4 expression was confirmed by demonstrating murine platelet binding to lipopolysaccharide (LPS). Thrombin activation of the platelets significantly enhanced the expression of TLR9, suggesting that at least some TLRs may derive from intracellular compartments. When LPS was administered to LPS-sensitive C3H/HeN and LPS-resistant C3H/HeJ mice, functional TLR4 expression in vivo was shown to be responsible for LPS-induced thrombocytopenia. However, when the C3H/HeN mice were first rendered thrombocytopenic by an antiplatelet antibody and then administered LPS, a significant reduction occurred in their ability to produce TNF-alpha. The decreased cytokine production in the thrombocytopenic mice was restored with platelet transfusion. These results suggest that platelets express various TLRs and that the functional significance of one of these, TLR4, appears to be a role in the modulation of LPS-induced thrombocytopenia and TNF-alpha production. This work implicates platelets as important mediators of innate immune responses against invading microorganisms.Investigation of pathogenesis of immune thrombocytopenia and efficacy and mechanism of action of IVIG in a novel mouse modelCanadian Institutes of Health Research / Canadian Blood Services11681

The role of interferon-γ, nitric oxide and lipopolysaccharide in intestinal graft-versus-host disease developing in F(1)-hybrid mice

(C57BL/6 × DBA/2) F(1)-hybrid mice injected with lymphoid cells from wild-type, C57BL/6 donors develop acute, lethal graft-versus-host disease (GVHD) in which the intestine is a major target. In its destructive phase intestinal GVHD is characterized by apoptosis of intestinal crypt epithelial cells and the development of endotoxaemia. Injection of as little as 10 μg endotoxin is lethal in mice with acute GVHD, and associated with the release of large amounts of tumour necrosis factor-α (TNF-α) into the serum. To explore the role of interferon-γ (IFN-γ) in the pathogenesis of intestinal GVHD we used IFN-γ gene knockout (gko) mice as donors. Recipients of grafts from these donors did not develop intestinal GVHD and, unlike recipients of wild-type grafts, did not die when injected with lipopolysaccharide (LPS). We also found that injection 10 μg LPS into recipients of wild-type grafts induced apoptosis of intestinal epithelial crypt cells and was associated with a burst of nitric oxide production in the intestine. Administration of N(ω)nitro l-arginine methyl ester blocked this response. In contrast, LPS did not induce either intestinal epithelial cell apoptosis or increased nitric oxide production in recipients of IFN-γ gko grafts. These findings indicate that donor-derived IFN-γ is instrumental for the development of intestinal GVHD. In a previous study we showed that recipients of IFN-γ gko grafts develop high levels of LPS-induced TNF-α release. When our current data are viewed in the context of this observation, they suggest that intestinal epithelial cell apoptosis in the parent→F(1)-hybrid model of acute GVHD is mediated primarily by nitric oxide rather than TNF-α, and that this depends on donor-derived IFN-γ

Murine graft-versus-host disease induced using interferon-γ-deficient grafts features antibodies to double-stranded DNA, T helper 2-type cytokines and hypereosinophilia

Acute, lethal graft-versus-host disease (GvHD) develops in B6D2F(1) hybrid recipients of wild-type, C57BL/6, parental strain grafts; however, when interferon-γ (IFN-γ) gene knockout (gko) donors are used, the disease is prolonged and associated with a higher level of engraftment, particularly of T cells. Lesions containing large, mixed cellular infiltrates develop in the skin, liver, pancreas, salivary gland, lung and kidney. In our current study, we wished to determine whether GvHD features a preponderance of T helper 2 (Th2) cytokines in the absence of donor-derived IFN-γ, and whether autoantibody production, commonly associated with chronic GvHD, also occurs. Because mitogen responsiveness is consistently suppressed in mice with acute GvHD, we wished to measure this response in recipients of IFN-γ gko grafts. Our findings indicate that spleen cells from the latter produce interleukin (IL)-4, IL-5 and IL-13 in culture, but respond poorly to concanavalin A (Con A) and lipopolysaccharide (LPS). Their sera contain anti-nuclear antibodies (ANA), some of which are specific for double-stranded (ds)DNA and are predominantly immunoglobulin (Ig)M and IgG1. We also noted the presence of numerous eosinophils in the infiltrates developing within the target organs. In some respects, this syndrome bears resemblance to both systemic lupus erythematosus (SLE) and chronic GvHD. However, histological evidence of glomerulonephritis is lacking and proteinuria fails to develop in recipients of IFN-γ gko grafts, suggesting that IFN-γ may be necessary for the development of lupus nephritis. On a broader scope, our findings underscore the importance of IFN-γ in the pathogenetic mechanism of GvHD, and demonstrate that the absence of this cytokine promotes the development of chronic GvHD and autoimmunity