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anti-CD20 and anti-CD3 immunofluorescence costaining of rat spleen follicles. Anti-CD20-positive B cells are in the B cell zone of splenic follicles (yellow), and anti-CD3-positive T cells in the T cell zone of the splenic follicles (red), also shown are proliferating Ki67-positive cells in splenic follicles (green). (PDF 3198 kb)

CXCR4 and CXCL12 expression. This figure shows quantitative RT-PCR analysis of chemokine and chemokine receptor expression from renal grafts after syngeneic or allogeneic transplantation treated with CyA for 6 or 28 days, or CyA on alternating days until d28. mRNA expression of target genes was normalized to the house-keeping gene HPRT and x-fold expression in comparison to syngeneic Tx d6 is shown. (A) CXCR4 (B) CXCL12. Data is shown as mean ± SEM. Groups consisted of at least 6 animals. Statistical analysis is shown (Mann-Whitney U Test). Significance is shown as *p

Background Non-adherence has been associated with reduced graft survival. The aim of this study was to investigate the immunological mechanisms underlying chronic renal allograft rejection using a model of non-adherence to immunosuppressive therapy. We used a MHC (major histocompatibility complex) -mismatched rat model of renal transplantation (Brown Norway to Lewis), in which rats received daily oral cyclosporine A. In analogy to non-adherence to therapy, one group received cyclosporine A on alternating days only. Rejection was histologically graded according to the Banff classification. We quantified fibrosis by trichrome staining and intra-graft infiltration of T cells, B cells, and monocytes/macrophages by immunohistochemistry. The distribution of B lymphocytes was assessed using immunofluorescence microscopy. Intra-graft chemokine, chemokine receptor, BAFF (B cell activating factor belonging to the TNF family), and immunoglobulin G transcription levels were analysed by RT-PCR. Finally, we evaluated donor-specific antibodies (DSA) and complement-dependent cytotoxicity using flow cytometry. Results After 28 days, cellular rejection occurred during non-adherence in 5/6 animals, mixed with humoral rejection in 3/6 animals. After non-adherence, the number of T lymphocytes were elevated compared to daily immunosuppression. Monocyte numbers declined over time. Accordingly, lymphocyte chemokine transcription was significantly increased in the graft, as was the transcription of BAFF, BAFF receptor, and Immunoglobulin G. Donor specific antibodies were elevated in non-adherence, but did not induce complement-dependent cytotoxicity. Conclusion Cellular and humoral rejection, lymphocyte infiltration, and de novo DSA are induced in this model of non-adherence. Electronic supplementary material The online version of this article (doi: 10.1186/s12865-017-0236-6) contains supplementary material, which is available to authorized users.

Nonalcoholic fatty liver disease (NAFLD) covers a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Free fatty acids (FFA) induce steatosis and lipo-toxicity and correlate with severity of NAFLD. In this study we aimed to investigate the role of exogenous and endogenous ALR (augmenter of liver regeneration) for FFA induced ER (endoplasmatic reticulum) - stress and lipoapoptosis. Primary human hepatocytes or hepatoma cells either treated with recombinant human ALR (rhALR, 15kDa) or expressing short form ALR (sfALR, 15kDa) were incubated with palmitic acid (PA) and analyzed for lipo-toxicity, -apoptosis, activation of ER-stress response pathways, triacylglycerides (TAG), mRNA and protein expression of lipid metabolizing genes. Both, exogenous rhALR and cytosolic sfALR reduced PA induced caspase 3 activity and Bax protein expression and therefore lipotoxicity. Endogenous sfALR but not rhALR treatment lowered TAG levels, diminished activation of ER-stress mediators C-Jun N-terminal kinase (JNK), X-box binding protein-1 (XBP1) and proapoptotic transcription factor C/EBP-homologous protein (CHOP), and reduced death receptor 5 protein expression. Cellular ALR exerts its lipid lowering and anti-apoptotic actions by enhancing FABP1, which binds toxic FFA, increasing mitochondrial beta-oxidation by elevating the mitochondrial FFA transporter CPT1 alpha, and decreasing ELOVL6, which delivers toxic FFA metabolites. We found reduced hepatic mRNA levels of ALR in a high fat diet mouse model, and of ALR and FOXA2, a transcription factor inducing ALR expression, in human steatotic as well as NASH liver samples, which may explain increased lipid deposition and reduced beta-oxidation in NASH patients. Present study shows that exogenous and endogenous ALR reduce PA induced lipoapoptosis. Furthermore, cytosolic sfALR changes mRNA and protein expression of genes regulating lipid metabolism, reduces ER-stress finally impeding progression of NASH.

anti-CD11b/c and anti-CD68 FACS costain of rat splenocytes. (A) shows unstained cells, (B) shows anti-CD11b/c-PE antibody only, and (C) shows anti-anti-CD11b/c-PE antibody and anti-CD68-APC antibody co-stain of rat splenocytes. (PDF 82 kb)

anti-CD20, anti-CD3, and anti-CD68 immunofluorescence costaining of rat spleen follicle. (A) shows anti-CD20-positive B cells in the B cell zone of a splenic follicle (green), anti-CD3-positive T cells in T cell zone of splenic follicle (red), and CD68-positive macrophages (yellow) in the splenic red pulp, also shown in (B) without costaining. (PDF 588 kb)

Objective: Recently, recurrent mutations within the core promoter of the human telomerase reverse transcriptase (TERT) gene generating consensus binding sites for ETS transcription factor family members were described in melanomas and other malignancies (e.g. bladder cancer, hepatocellular carcinoma). These mutations were discussed as early drivers for malignant transformation. In prostate cancer (PrCa) TERT expression has been associated with a poor prognosis and higher risk for disease recurrence. The underlying mechanisms for high TERT expression in PrCa have still not been clarified. To date, data on TERT promoter mutation analysis in PrCa are sparse. Therefore, we performed sequence analysis of the core promoter region of the TERT gene in an unselected cohort of prostate tumors. Methods: Sections from 167 formalin-fixed, paraffin-embedded and cryopreserved prostate tumors were microdissected and used for DNA isolation. The mutation hotspot region within the TERT core promoter (-260 to +60) was analyzed by direct Sanger sequencing or SNaPshot analysis. Results: All cases were analyzed successfully. Mutations within the core promoter of the TERT gene were not detected in any of the cases with all tumors exhibiting a wild-type sequence. Conclusion: TERT core promoter mutations reported from several other malignancies were not detected in our unselected cohort of PrCa. These data indicate that alterations within the core promoter of the TERT gene do not play an important role in prostate carcinogenesis.

The FGF/FGFR-system plays an important role in embryogenesis, tissue homeostasis and carcinogenesis. Mutational activation of FGFR2 resulting in aberrant FGFR2 signaling activation is known from both hereditary germ line alterations and somatic mutations in various malignancies (e.g. breast, gastric or ovarian cancer). FGFR2 mutations are mainly located within the hinge between Ig-like domains (exon 7), around the 3rd Ig-like domains and within the kinase domain. For bladder cancer only sparse data on FGFR2 mutations are available. Most interestingly a case of early-onset papillary carcinoma of the bladder showing a FGFR2 p.Pro253Arg mutation in exon 7 in a patient with Apert Syndrome was reported recently. To further evaluate the importance of FGFR2 exon 7 alterations in bladder cancer a cohort of 254 bladder tumors (cohort 1: unselected cases: n=139; cohort 2: early-onset bladder cancer cases (age at time of diagnosis ≤45 years): n=115) was analyzed. Sections from formalin-fixed, paraffin-embedded bladder tumors were used for DNA isolation. After precise microdissection exon 7 of the FGFR2 gene was analyzed by direct Sanger sequencing. All cases could be analyzed successfully. Mutations in exon 7 of FGFR2 could not be detected in any of the cases. All tumors showed wild type sequence. Our data demonstrate that the recently reported association between early-onset papillary carcinoma of the bladder with germ line FGFR2 p.Pro253Arg mutation could not be found in our cohorts of sporadic bladder tumors. These results indicate that FGFR2 gene mutations might only play a minor role in bladder carcinogenesis.

Objective: Human mouse double minute 2 (Mdm2) is essential in degrading p53 by acting as an ubiquitin ligase and therefore plays a vital role in cell cycle and survival. The G-variant of the Mdm2 SNP309, which is located within the promoter of the Mdm2 gene, increases expression of Mdm2 and thereby inhibits the p53 pathway. Several studies have investigated the influence of this SNP on disease risk and onset of various malignancies. The impact of Mdm2 SNP309 on bladder cancer is still to be established due to inconsistent data. Methods: In a case-control study we determined the distribution of Mdm2 SNP309 genotypes in 111 patients with an early-onset bladder cancer (diagnosis