†Authors contributed equally to this work.Small bowel transplantation can be a life-preserving procedure for patients with irreversible intestinal failure. Allograft rejection remains a major source of morbidity and mortality and its accurate diagnosis and treatment are critical. In this study, we used pyrosequencing of 16S ribosomal RNA gene tags to compare the composition of the ileal microbiota present during nonrejection, prerejection and active rejection states in small bowel transplant patients. During episodes of rejection, the proportions of phylum Firmicutes (p < 0.001) and the order Lactobacillales (p < 0.01) were significantly decreased, while those of the phylum Proteobacteria, especially the family Enterobacteriaceae, were significantly increased (p < 0.005). Receiveroperating characteristic analysis revealed that relative proportions of several bacterial taxa in ileal effluents and especially Firmicutes, could be used to discriminate between nonrejection and active rejection. In conclusion, the findings obtained during this study suggest that small bowel transplant rejection is associated with changes in the microbial populations in ileal effluents and support microbiota profiling as a potential diagnostic biomarker of rejection. Future studies should investigate if the dysbiosis that we observed is a cause or a consequence of the rejection process.

Reactive oxygen species (ROS) are increased in human abdominal aortic aneurysms (AAA). NADPH oxidases are the predominant source of superoxide anion (O 2 − ) in the vasculature. Inducible nitric oxide synthase (iNOS) produces significant amount of nitric oxide (NO) during inflammatory processes. We hypothesized that ROS produced by NADPH oxidases and iNOS play an important role in aneurysm formation. We examined this hypothesis using selective blockade of NADPH oxidases and iNOS in a murine model of AAA. Mice, including C57BL/6, iNOS knockout (iNOS −/− ) mice, and its background matched control (C57BL/6), underwent AAA induction by periaortic application of CaCl 2 . Aortic diameter was measured at aneurysm induction and harvest. Beginning 1 week prior to aneurysm induction and continuing to aortic harvest 6 weeks later, one group of the C57BL/6 mice were treated with orally administered apocynin (NADPH oxidase inhibitor). Control mice were given water. The mean diameter and change in diameter of each group were compared with concurrent controls. Aortic levels of the NO metabolite, NO x N(NO 2 and NO 3 ), are significantly increased in CaCl 2 -treated wild type mice. INOS −/− mice are resistant to aneurysm induction. This is associated with reduced expression of matrix metalloproteinase (MMP)-2 and MMP-9 and decreased production of NO x in the aortic tissues. Inhibition of NADPH oxidase by apocynin also blocked aneurysm formation. In conclusion, both iNOS deficiency and NADPH oxidase inhibition suppress aneurysm formation in association with decreased NO x levels. These studies suggest that both the NADPH oxidase and iNOS pathways contribute to ROS production and AAA development.

Human-specific HIV-1 and hepatitis co-infections significantly affect patient management and call for new therapeutic options. Small xenotransplantation models with human hepatocytes and hematolymphoid tissue should facilitate antiviral/antiretroviral drug trials. However, experience with mouse strains tested for dual reconstitution is limited, with technical difficulties such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities. The best animal strains for hepatocyte transplantation are not optimal for human hematopoietic stem cell (HSC) engraftment, and vice versa. We evaluated a new strain of highly immunodeficient nonobese diabetic/Shi-scid (severe combined immunodeficiency)/IL-2Rγc(null) (NOG) mice that carry two copies of the mouse albumin promoter-driven urokinase-type plasminogen activator transgene for dual reconstitution with human liver and immune cells. Three approaches for dual reconstitution were evaluated: i) freshly isolated fetal hepatoblasts were injected intrasplenically, followed by transplantation of cryopreserved HSCs obtained from the same tissue samples 1 month later after treosulfan conditioning; ii) treosulfan conditioning is followed by intrasplenic simultaneous transplantation of fetal hepatoblasts and HSCs; and iii) transplantation of mature hepatocytes is followed by mismatched HSCs. The long-term dual reconstitution was achieved on urokinase-type plasminogen activator-NOG mice with mature hepatocytes (not fetal hepatoblasts) and HSCs. Even major histocompatibility complex mismatched transplantation was sustained without any evidence of hepatocyte rejection by the human immune system.

SummaryDifferentiation and proliferation of haematopoietic progenitor cells occur in intimate contact with the bone marrow microenvironment which is composed of stromal cells and extracellular matrix proteins. MOP3 (also known as brain and muscle Arnt-like protein-1, BMAL1), a master regulator of circadian rhythm, plays important roles in the regulation of cell differentiation and general physical functions. In the present studies, MOP3-deficient mice had significantly reduced levels of B cells in the peripheral blood, spleen and bone marrow compared MOP3 +/-or MOP3

Elevated serum soluble (s) Suppressor of Tumorigenicity 2 (ST2) is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n=41) and SBTx recipient (n=18) sera. At times of biopsy-diagnosed HTx rejection (cellular and/or antibody-mediated), serum sST2 was elevated compared to rejection-free time points (1714±329 vs. 546.5±141.6 pg/ml; P=0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536±1561 vs. 2662±543.8 pg/ml; P=0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2>600 pg/ml could discriminate time points of HTx rejection and non-rejection (Area under the curve (AUC)=0.724±0.053; P=0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC=0.6921±0.0820; P=0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted TNF-α and IL-1β as upstream activators. In total, our data indicate that alloimmune-associated pro-inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the non-invasive diagnosis of rejection.