pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD. Keywords: OGR1 (GPR68) expression and function; fibroblasts; fibrosis; inflammatory bowel disease; pH-sensing GPCR

P53 Codon 72 (Arg72Pro) Polymorphism and Prostate Cancer Risk: Association between Disease Onset and Proline Genotype

The tumor suppressor gene p53 plays an important role in the stress response of the cell and is mutated in 50% of all human tumors. The p53 Arg72Pro single-nucleotide polymorphism (SNP) was found to be associated with an increased risk of various malignancies. Biochemical and biological differences between the 2 polymorphic variants of wild-type P53 might lead to distinct susceptibility to HPV- and non-HPV-induced tumors. For prostate cancer, only limited data are available, especially in the Caucasian pop-ulation. Therefore, we determined the distribution of the Arg72Pro SNP in a Caucasian case-control study including 118 prostate cancer patients and 194 male controls without any malignancy using restriction fragment length polymorphism analysis. A subset of 33 tumors was tested for HPV infection, and no HPV DNA was found. Cases and controls showed similar distributions of alleles in the SNP (p = 0.720). Regarding the onset of the disease, patients diagnosed at ≤60 years of age and older patients (>60 years of age) showed a significant difference in genotype distribution (p = 0.035); there was also an increased occurrence of risk allele Pro72 in cases aged ≤60 years (p = 0.045). A subset of 64 prostate tumors was stained immunohistochemically for P53. 5 of 64 prostate tumors (7.8%) were positive for P53 expression, indicating integrity of the protein in the majority of cases. Genotype distribution showed no association with the Gleason score or additional histopathological characteristics. This study shows that the overall risk of prostate cancer was not associated with Arg72Pro SNP and HPV infection in our cohort. However, disease onset might be modulated by the p53 Pro72 allele, suggesting an important role of apoptosis regulation in prostate carcinogenesis

Characterization of Changes in Serum Anti-Glycan Antibodies in Crohn's Disease – a Longitudinal Analysis

INTRODUCTION: Anti-glycan antibodies are a promising tool for differential diagnosis and disease stratification of patients with Crohn's disease (CD). We longitudinally assessed level and status changes of anti-glycan antibodies over time in individual CD patients as well as determinants of this phenomenon. METHODS: 859 serum samples derived from a cohort of 253 inflammatory bowel disease (IBD) patients (207 CD, 46 ulcerative colitis (UC)) were tested for the presence of anti-laminarin (Anti-L), anti-chitin (Anti-C), anti-chitobioside (ACCA), anti-laminaribioside (ALCA), anti-mannobioside (AMCA) and anti-Saccharomyces cerevisiae (gASCA) antibodies by ELISA. All patients had at least two and up to eleven serum samples taken during the disease course. RESULTS: Median follow-up time for CD was 17.4 months (Interquartile range (IQR) 8.0, 31.6 months) and for UC 10.9 months (IQR 4.9, 21.0 months). In a subgroup of CD subjects marked changes in the overall immune response (quartile sum score) and levels of individual markers were observed over time. The marker status (positive versus negative) remained widely stable. Neither clinical phenotype nor NOD2 genotype was associated with the observed fluctuations. In a longitudinal analysis neither changes in disease activity nor CD behavior led to alterations in the levels of the glycan markers. The ability of the panel to discriminate CD from UC or its association with CD phenotypes remained stable during follow-up. In the serum of UC patients neither significant level nor status changes were observed. CONCLUSIONS: While the levels of anti-glycan antibodies fluctuate in a subgroup of CD patients the antibody status is widely stable over time

Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein Barr virus induced G protein coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in livers of NASH patients by liquid chromatography-mass spectrometry and tested the role of the EBI2-7α,25-diHC-system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared to controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype related differences were observed in Ebi2-/- animals and animals with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared to wildtype littermate controls,arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by enhanced level of 7α- hydroxycholest-4-en-3-one, and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH

Role of chromosome 8p deletions and loss of SFRP1-expression in human bladder cancer

Bisher fehlen noch immer verlässliche prognostische und progressions-relevante Marker für das papilläre Urothelkarzinom der Harnblase, um eine bessere Risikoabschätzung für den Patienten zu gewährleisten. In der hier vorliegenden Arbeit wurde deshalb ein vielversprechender Marker auf Chromosom 8p12, SFRP1, funktionell in papillären Harnblasenkarzinomlinien untersucht, sowie in einer prospektiven Studie papillärer Tumoren validiert. Außerdem wurde der Einfluss von SFRP1 auch in fortgeschrittenen Tumoren untersucht. Um auch evolutionsbiologische Faktoren in die individuelle Risikoabschätzung zu involvieren, wurden außerdem zwei SNPs in SFRP1, rs3242 und rs921142, in zwei großen Blasenkrebskohorten analysiert und mit der gesunden Kontrollgruppe verglichen. Es zeigte sich, dass SFRP1 das maligne Potential (z.B. Migration und Viabilität) in BFTC905 verringern konnte, der kanonische Wnt-Signalweg aber scheinbar nicht den biologischen Mechanismus dahinter darstellt. Das Gesamtüberleben der Patienten mit papillären Tumoren wurde maßgeblich, wenn auch nicht signifikant, durch SFRP1 beeinflusst. Patienten mit SFRP1-Expression lebten länger. Eine Assoziation mit der Progression konnte aber nicht nachgewiesen werden. Bei fortgeschrittenen Blasenkarzinomen spielte SFRP1 keine Rolle. Der SFRP1-SNP rs3242 beeinflusste das Risiko von Patienten, die im Altern von ≤45 Jahren an Blasenkrebs erkrankten, maßgeblich. Bei der Gruppe der Erkrankten konnte ein signifikant gehäuftes Auftreten des Risikoallels T beobachtet werden. Eine Erklärung hierfür könnte die verstärkte Expression der mikroRNA-3646 bei jungen Blasenkrebspatienten sein. Diese bindet möglicherweise SFRP1 im Bereich des rs3242-SNPs und beeinflusst so die mRNA-Stabilität. Der SNP rs921142 hatte keinen Einfluss auf das Erkrankungsrisiko. Diese Studie war, nach unserem Kenntnisstand die erste, die eine Assoziation von SNPs in SFRP1 bei Krebs untersuchte. Neben der Untersuchung von SFRP1, sollten auch andere Kandidatengene auf Chromosom 8p identifiziert werden, die zu einer individuellen Risikoabschätzung beim nicht-invasiven, papillären Urothelkarzinom beitragen könnten. Dazu wurden 9 pTa und 10 papilläre pT1-Blasentumoren mittels Array-basierter genomischer Hybridisierung (aCGH) verglichen. Dieses hochauflösende Deletionsmapping konnte verschiedenen Zielregionen in pTa-Tumoren identifizieren, in denen Mikrodeletionen auf Chromosom 8p aufgetreten waren und in denen mögliche Kandidatengene lokalisiert sind. Betroffene Gene waren unter anderem SLC7A2, PDGFL, MTUS1, FGL1, PCM1, UNC5D und GFRA. MTUS1, der microtubule-associated tumor suppressor 1, auf 8p22 wurde anschließend für weitere Analysen ausgewählt und sowohl funktionell in Zelllinien, als auch prospektivisch in den verschiedenen Blasenkrebskohorten untersucht. Ähnlich wie bei SFRP1 konnte gezeigt werden, dass MTUS1 das maligne Potential in einer Blasenkrebszelllinien, RT112, herabsetzte. Bei papillären Tumoren konnte hingegen keine Assoziation mit dem Überleben und der Progression beobachtet werden. Es bestand aber eine inverse Korrelation zwischen MTUS1-Expression und den histopathologischen Parametern Grade, Stage, sowie mit der Ki67 und CK20-Expression. MTUS1 scheint das maligne Potential in papillären Tumoren zu erhöhen. In fortgeschrittenen Tumoren, hingegen wirkt MTUS1 scheinbar als klassischer Tumorsuppressor: ein Expressionsverlust ging mit einem schlechteren Gesamt- und tumorspezifischen Überleben einher.Due to a lack of molecular progression- and prognostic markers it is still difficult to predict individual patient risk and outcome in transitional cell carcinoma (TCC) of the urinary bladder. The aim of this project was to validate the promising marker and known Wnt-signaling antagonist at chromosome 8p, SFRP1, in cell culture experiments as well as in different tumor samples. In addition new progression-associated target genes at chromosome 8p should be identified. Therefore it was analyzed, if SFRP1-expression influences malignant potential of the tumor cells, Wnt-signaling activity and patient survival. To include epidemiological and evolutionary aspects into disease- and risk-prediction, genotypes of two SNPs in SFRP1 (rs3242 and rs921142) were determined and compared within a group of bladder cancer patients as well as in a healthy control group. To identify new progression-related target genes at chromosome 8p, an array-based comparative genomic hybridization (aCGH) was performed with nine non-invasive (pTa) and ten invasive (pT1) papillary TCCs. One potential progression-associated target gene, MTUS1, was chosen for functional and immunohistochemical analysis. SFRP1 decreased the malignant potential of the cells, however canonical Wnt-signaling does not seem to be the responsible biological mechanism beyond. SFPR1 expression loss was correlated with worse overall-survival, but not with progression in papillary bladder tumor patients. In advanced bladder cancer patients SFRP1 did not play any role for patient survival. SNP analysis revealed that presence of at least one T-allele in the rs3242 SNP of SFRP1 significantly increases bladder cancer risk in patients aged ≤45 years. One explanation could be the amplification and the altered binding capacity of hsa-miR-3646 at 3’UTR of SFRP1 mRNA. Genotypes of rs921142 did not influence bladder cancer risk. MTUS1 reduced the malignant potential of papillary RT112 bladder cancer cells, however, did not influence survival of papillary bladder cancer patients. In advanced bladder tumors, MTUS1-expression loss was associated with worse overall and tumor-specific survival. Thus, MTUS1 might act as a classical tumor suppressor in advanced bladder cancer. Taken together, SFRP1 and MTUS1 are apparently no new reliable progression markers in TCC of the bladder. However, SFRP1 might be considered as predictive indicator for papillary, muscle-invasive bladder cancer, whereas MTUS1 could act as prognostic factor in advanced bladder cancer

Final Results of a Non-Interventional Study Evaluating the Quality of Life in Second-line Treatment of Metastatic Renal Cell Carcinoma With Everolimus: The EVERPRO Study

BACKGROUND This study assessed the quality of life (QoL) and the implication of time effort of everolimus treatment in patients with metastatic renal cell carcinoma (mRCC). METHODS Adult patients with mRCC were eligible for everolimus treatment after first-line vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors or bevacizumab therapy. The primary end-point, QoL, was assessed by means of the NCCN-FACT FKSI-19 questionnaire. RESULTS In total, 202 patients (24% of female patients; median age, 71 years) were evaluable for QoL analyses. The median treatment duration was 4.4 months (95% CI, 3.8-5.3) and the median time to progression was 6 months (95% CI, 5.4-7.5). The median FKSI-19 total score remained stable during treatment (52.0 at therapy start, 55.0 at observation end). The median time effort spent on total therapy was 20 hours per patient. Most of the patients stated to have "no limitations," "a little" or "moderate" limitations in their daily, social, and professional lives. Two months after the start of treatment, 65 patients reported none or a little time burden due to therapy. CONCLUSIONS QoL was maintained during the everolimus therapy and limitations as well as time effort were acceptable for most of the patients. The study supports previous findings on switching mode of action after anti-VEGFR-targeted therapy to a mammalian target of rapamycin inhibitor

Development of non-alcoholic steatohepatitis is associated with gut microbiota but not with oxysterol enzymes CH25H, EBI2, or CYP7B1 in mice.

Liver steatosis is the most frequent liver disorder and its advanced stage, non-alcoholic steatohepatitis (NASH), will soon become the main reason for liver fibrosis and cirrhosis. The "multiple hits hypothesis" suggests that progression from simple steatosis to NASH is triggered by multiple factors including the gut microbiota composition. The Epstein Barr virus induced gene 2 (EBI2) is a receptor for the oxysterol 7a, 25-dihydroxycholesterol synthesized by the enzymes CH25H and CYP7B1. EBI2 and its ligand control activation of immune cells in secondary lymphoid organs and the gut. Here we show a concurrent study of the microbial dysregulation and perturbation of the EBI2 axis in a mice model of NASH.We used mice with wildtype, or littermates with CH25H-/-, EBI2-/-, or CYP7B1-/- genotypes fed with a high-fat diet (HFD) containing high amounts of fat, cholesterol, and fructose for 20 weeks to induce liver steatosis and NASH. Fecal and small intestinal microbiota samples were collected, and microbiota signatures were compared according to genotype and NASH disease state.We found pronounced differences in microbiota composition of mice with HFD developing NASH compared to mice did not developing NASH. In mice with NASH, we identified significantly increased 33 taxa mainly belonging to the Clostridiales order and/ or the family, and significantly decreased 17 taxa. Using an Elastic Net algorithm, we suggest a microbiota signature that predicts NASH in animals with a HFD from the microbiota composition with moderate accuracy (area under the receiver operator characteristics curve = 0.64). In contrast, no microbiota differences regarding the studied genotypes (wildtype vs knock-out CH25H-/-, EBI2-/-, or CYP7B1-/-) were observed.In conclusion, our data confirm previous studies identifying the intestinal microbiota composition as a relevant marker for NASH pathogenesis. Further, no link of the EBI2 - oxysterol axis to the intestinal microbiota was detectable in the current study