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Background: To validate a novel method for post-transplant surveillance to detect kidney allograft rejection via a characteristic constellation of the urine metabolites alanine, citrate, lactate, and urea investigated by nuclear magnetic resonance (NMR) spectroscopy a first prospective, observational study was performed. Methods: Within the UMBRELLA study 986 urine specimens were collected from 109 consecutively enrolled renal transplant recipients, and metabolite constellations were analyzed. A metabolite rejection score was calculated and compared to histopathological results of corresponding indication and protocol allograft biopsies (n = 206). Findings: The metabolite constellation was found to be a useful biomarker to non-invasively detect acute allograft rejection (AUC = 0.75; 95% confidence interval (CI) 0.68–0.83; based on 46 cases and 520 control samples). Combined analysis of the metabolite rejection score and the estimated glomerular filtration rate (eGFR) at the time of urine sampling further improved the overall test performance significantly (AUC = 0.84; 95% CI 0.76–0.91; based on 42 cases and 468 controls). Regarding the time course analysis in patients without rejection episodes the test results remained well below a diagnostic threshold associated with high risk of acute rejection. In other cases, a marked increase above this threshold indicated acute allograft rejection already six to ten days before diagnostic renal biopsies were performed. Interpretation: A combination of an NMR-based urine metabolite analysis and eGFR is promising as a non-invasive test for post-transplant surveillance and to support decision making whether renal allografts need histopathological evaluation. Keywords: Kidney transplant rejection, Urinary metabolites, Biomarker, NMR-spectroscopy, Non-invasive test

Glycogenosis type Ib (GSD1B) causes not only hypoglycemia but also infections and “Crohn's disease like” inflammatory bowel disease (IBD) that can significantly impair patient's quality of life. We retrospectively evaluated infectious and digestive complications in 9 French patients (3 girls, 6 boys) diagnosed at 0.8 years on average, with a mean follow-up of 19.1 years. Infections occurred earlier than IBD, at mean ages of 1.7 and 3.8 years, respectively. The number of acute hospitalizations was 0.7/year due to infectious (0.4/year) or digestive symptoms (0.4/year). Clinical presentations allowed separating patients into mild (n = 5) and severe (n = 4) intestinal involvement. Patients in the severe group had more serious digestive symptoms but also earlier neutropenia (median 0.3 vs. 1.5 years, p =0 .046) with a tendency to a lower neutrophil count (NC) during follow-up, and a higher number of acute hospitalizations (median 1.3/year vs. 0.2/year, p =0 .014) due to digestive symptoms (median 0.6/year vs. 0.05/year, p = 0,012) and infections (median 0.8/year vs. 0.2/year, p =0 .014). Treatments included G-CSF and cotrimoxazole (n = 7), 5-aminosalicylic acid (n = 2), and a polymeric solution enriched in the anti-inflammatory cytokine TGF-β (n = 4, “severe” group), and immunomodulatory treatment (n = 1). In conclusion, infections and IBD are rare but severe complications in GSD1B. Neutropenia tended to be more prevalent in the severe IBD group than in the mild IBD group. Dietetic treatment with specific anti-inflammatory solutions seems particularly appropriate in these patients.

The prognosis of patients suffering from pancreatic cancer is still poor and novel therapeutic options are urgently needed. Recently, the transcription factor signal transducer and activator of transcription 5b (STAT5b) was associated with tumor progression in human solid cancer. Hence, we assessed whether STAT5b might serve as an anticancer target in ductal pancreatic adenocarcinoma (DPAC). We found that nuclear expression of STAT5b can be detected in approximately 50% of DPAC. Blockade of STAT5b by stable shRNA-mediated knockdown showed no effects on tumor cell growth in vitro. However, inhibition of tumor cell motility was found even in response to stimulation with epidermal growth factor or interleukin-6. These findings were paralleled by a reduction of prometastatic and proangiogenic factors in vitro. Subsequent in vivo experiments revealed a strong growth inhibition on STAT5b blockade in subcutaneous and orthotopic models. These findings were paralleled by impaired tumor angiogenesis in vivo. In contrast to the subcutaneous model, the orthotopic model revealed a strong reduction of tumor cell proliferation that emphasizes the meaning of assessing targets in an appropriate microenvironment. Taken together, our results suggest that STAT5b might be a potential novel target for human DPAC.

Non-alcoholic fatty liver disease (NAFLD) is among the most common liver diseases. Oxidative stress is one of the pathogenic mechanisms contributing to the progression of simple fatty liver to non-alcoholic steatohepatitis (NASH). Manganese superoxide dismutase (MnSOD) is a mitochondrial antioxidative enzyme and here its expression in rodent and human NAFLD has been analyzed. MnSOD is found reduced in the liver of male mice fed a high fat diet and male ob/ob mice. Female mice fed an atherogenic diet to induce NASH have MnSOD protein levels comparable to controls. In a cohort of 30 controls, 41 patients with fatty liver and 39 NASH patients, MnSOD mRNA is significantly lower in the steatotic and NASH liver. When analyzed in both genders separately reduction of MnSOD expression is only found in males. Here, MnSOD mRNA negatively correlates with steatosis grade but not with extent of fibrosis or inflammation. MnSOD is, however, not reduced in primary human hepatocytes (PHH) treated with palmitate or oleate to increase cellular triglycerides. Lipopolysaccharide, TNF, IL-6, TGFβ and leptin which are all raised in NAFLD do not affect MnSOD in PHH. Adiponectin which attenuates oxidative stress partly by increasing MnSOD in macrophages does not induce MnSOD in PHH. In summary, current data show that hepatic MnSOD is reduced in male but not female humans and rodents with NAFLD., (© 2013.)

Background & Aims: The aim of this study was to examine the development of biliary epithelial damage between organ retrieval and transplantation and its clinical relevance for patients., Methods: Common bile duct samples during donor hepatectomy, after cold storage, and after reperfusion were compared to healthy controls by hematoxylin and eosin (H&E) staining and immunofluorescence for tight junction protein 1 and Claudin-1. A bile duct damage score to quantify biliary epithelial injury was developed and correlated with recipient and donor data and patient outcome., Results: Control (N=16) and donor hepatectomy bile ducts (N=10) showed regular epithelial morphology and tight junction architecture. After cold storage (N=37; p=0.0119), and even more after reperfusion (N=62; p=0.0002), epithelial damage, as quantified by the bile duct damage score, was markedly increased, and both tight junction proteins were detected with inappropriate morphology. Patients with major bile duct damage after cold storage had a significantly increased risk of biliary complications (relative risk 18.75; p<0.0001) and graft loss (p=0.0004)., Conclusions: In many cases, the common bile duct epithelium shows considerable damage after cold ischemia with further damage occurring after reperfusion. The extent of epithelial damage can be quantified by our newly developed bile duct damage score and is a prognostic parameter for biliary complications and graft loss. Possibly, in an intraoperative histological examination, this bile duct damage score may influence decision-making in transplantation surgery., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The innate receptor "triggering-receptor-expressed-on-myeloid-cells-1" (TREM-1) enhances downstream signaling of "pattern recognition receptor" (PRR) molecules implicated in inflammatory responses. However the mechanistic role of TREM-1 in chronic heart rejection has yet to be elucidated. We examined the effect of TREM-1(+) antigen-presenting cells (APC) on alloreactive CD4(+) lymphocytes. Bm12 donor hearts were transplanted into wild-type MHC-class-II-mismatched C57BL/6J recipient mice. Progressive allograft rejection of bm12-donor hearts with decreased organ function, severe vasculopathy and allograft fibrosis was evident within 4 weeks. TREM-1(+) CD11b(+) MHC-II(+) F4/80(+) CCR2(+) APC and IFNγ-producing CD4(+) cells were detected during chronic rejection. Peptide inhibition of TREM-1 attenuated graft vasculopathy, reduced graft-infiltrating leukocytes and prolonged allograft survival, while being accompanied by sustained low levels of CD4(+) and CD8(+) cell infiltration. Remarkably, temporary inhibition of TREM-1 during early immune activation was sufficient for long-term allograft survival. Mechanistically, TREM-1 inhibition leads to reduced differentiation and proliferation of IFNγ-producing Th1 cells. In conclusion, TREM-1 influences chronic heart rejection by regulating the infiltration and differentiation of CD4(+) lymphocytes., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis-associated carcinoma (UCC). To date, the negative regulation of STAT3 is poorly understood. Interestingly, intestinal epithelial cells of UCC in comparison to ulcerative colitis show high expression levels of anti-inflammatory death-associated protein kinase (DAPK) and low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3(IEC-KO) mice. To unravel a possible regulatory mechanism, we used an in vitro TNF-treated intestinal epithelial cell model. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK siRNA knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation, IL-6 mRNA expression, and secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in three-dimensional structural modeling. This model suggests that DAPK-induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Alternatively, pharmacological inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process as confirmed in an AOM+DSS colon carcinogenesis mouse model. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of ulcerative colitis and UCC., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The prognosis of patients suffering from pancreatic cancer is still poor and novel therapeutic options are urgently needed. Recently, the transcription factor signal transducer and activator of transcription 5b (STAT5b) was associated with tumor progression in human solid cancer. Hence, we assessed whether STAT5b might serve as an anticancer target in ductal pancreatic adenocarcinoma (DPAC). We found that nuclear expression of STAT5b can be detected in approximately 50% of DPAC. Blockade of STAT5b by stable shRNA-mediated knockdown showed no effects on tumor cell growth in vitro. However, inhibition of tumor cell motility was found even in response to stimulation with epidermal growth factor or interleukin-6. These findings were paralleled by a reduction of prometastatic and proangiogenic factors in vitro. Subsequent in vivo experiments revealed a strong growth inhibition on STAT5b blockade in subcutaneous and orthotopic models. These findings were paralleled by impaired tumor angiogenesis in vivo. In contrast to the subcutaneous model, the orthotopic model revealed a strong reduction of tumor cell proliferation that emphasizes the meaning of assessing targets in an appropriate microenvironment. Taken together, our results suggest that STAT5b might be a potential novel target for human DPAC.