Genomic landscape of retinoblastoma in Rb−/−p130−/− mice resembles human retinoblastoma

Several murine retinoblastoma models have been generated by deleting the genes encoding for retinoblastoma susceptibility protein pRb and one of its family members p107 or p130. In Rb^?/?p107^?/? retinoblastomas, somatic copy number alterations (SCNAs) like Mdm2 amplification or Cdkn2a deletion targeting the p53‐pathway occur, which is uncommon for human retinoblastoma. In our study, we determined SCNAs in retinoblastomas developing in Rb^?/?p130^?/? mice and compared this to murine Rb^?/?p107^?/? tumors and human tumors. Chimeric mice were made by injection of 129/Ola‐derived Rb^?/?p130^?/? embryonic stem cells into wild type C57BL/6 blastocysts. SCNAs of retinoblastoma samples were determined by low‐coverage (～0.5×) whole genome sequencing. In Rb^?/?p130^?/? tumors, SCNAs included gain of chromosomes 1 (3/23 tumors), 8 (1/23 tumors), 10 (1/23 tumors), 11 (2/23 tumors), and 12 (4/23 tumors), which could be mapped to frequently altered chromosomes in human retinoblastomas. While the altered chromosomes in Rb^?/?p130^?/? tumors were similar to those in Rb^?/?p107^?/? tumors, the alteration frequencies were much lower in Rb^?/?p130^?/? tumors. Most of the Rb^?/?p130^?/? tumors (16/23 tumors, 70%) were devoid of SCNAs, in strong contrast to Rb^?/?p107^?/? tumors, which were never (0/15 tumors) SCNA‐devoid. Similarly, to human retinoblastoma, increased age at diagnosis significantly correlated with increased SCNA frequencies. Additionally, focal loss of Cdh11 was observed in one Rb^?/?p130^?/? tumor, which enforces studies in human retinoblastoma that identified CDH11 as a retinoblastoma suppressor. Moreover, based on a comparison of genes altered in human and murine retinoblastoma, we suggest exploring the role of HMGA1 and SRSF3 in retinoblastoma development. © 2016 Wiley Periodicals, Inc.     

Time-resolved fluorimetric immunoassay of calprotectin: technical and clinical aspects in diagnosis of inflammatory bowel diseases.

BACKGROUND: Growing evidence in the literature shows the clinical importance of the calprotectin assay in faeces, especially for the differential diagnosis and monitoring of patients with inflammatory bowel diseases. METHODS: We developed a time-resolved fluorimetric immunoassay for calprotectin to extend the limited measuring range of the commercially available ELISA method currently used in our laboratory. Together with the introduction of a non-enzymatic label, this new method offers the advantages of better precision and higher sensitivity. RESULTS: The new assay shows a dynamic measuring range extended by a factor four, which reduces the number of samples with concentrations outside the measuring range from 30% to only 4%. The value of the assay was confirmed in various patient groups suffering from active and inactive gastrointestinal diseases. We suggest that the frequent coincidence of a high calprotectin concentration with intestinal blood loss is not the consequence of mere blood loss, but can be ascribed to neutrophil infiltration and subsequent shedding into the intestinal lumen as a result of intestinal inflammation or malignancy. CONCLUSION: We expect that in the near future faecal calprotectin will be used as a non-invasive routine diagnostic marker and an effective laboratory parameter to monitor patients with inflammatory bowel disease.     

Inter- and intramolecular epitope spreading in equine recurrent uveitis

PURPOSE: To test the hypothesis that inter- and intramolecular spreading to S-antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP)-derived epitopes occurs in a spontaneous model of recurrent uveitis in the horse. METHODS: The immune response of eight horses with equine recurrent uveitis (ERU) was compared with that of five control horses with healthy eyes. Lymphocytes derived from peripheral blood (PBLs) were tested every 8 weeks for their reactivity against S-Ag and various S-Ag and IRBP-derived peptides for 12 to 39 months (median, 22 months). During uveitic episodes, additional blood samples were analyzed. RESULTS: Intermolecular epitope spreading was detectable in all ERU cases during the study. Intramolecular spreading occurred in seven (of eight) horses with ERU. Fourteen relapses were analyzed during the observation period. Ten uveitic episodes were accompanied by neoreactivity to S-Ag or IRBP-derived peptides during the relapse. Shifts in the immune response profile were also detectable without any clinical signs of inflammation. Eye-healthy control horses were negative at all time points in the in vitro proliferation assays. CONCLUSIONS: Inter- and intramolecular spreading was detectable in a spontaneous model of recurrent uveitis. The shifts in immunoreactivity could account for the remitting-relapsing character of the disease.     

Effects and interaction of 7-hydroxy methotrexate and methotrexate in leukaemic cells ex vivo measured by the thymidylate synthase inhibition assay

In high dose therapy with methotrexate (MTX) the main metabolite 7-hydroxy-methotrexate (7-OH MTX) exceeds the plasma concentration of MTX achieving about tenfold higher levels. To investigate the interaction between 7-OH MTX and MTX ex vivo, the thymidylate synthase inhibition assay was used to quantify antifolate effects in patient blast samples, measuring the inhibition of the key enzyme thymidylate synthase (TS). In 18 leukemic samples (7 ALL, 11 AML) no dose-dependent TS inhibition was observed for 7-OH MTX. However, a statistically significant increase of TS inhibition (p<0.05) was observed for a 1:1 mixture of MTX and 7-OH MTX as compared to the effect of MTX alone. The half-maximal inhibitory concentrations in the short-exposure assay were 0.857 M for MTX alone versus 0.088 M for the 1:1 mixture with 7-OH MTX, respectively (p0.05). This interaction was not observed with an excess of 7-OH MTX. Similar results were obtained in long exposure experiments. We conclude that there is a dose-dependent interaction between 7-OHMTX and MTX, despite the lack of TS inhibitory effects of the metabolite alone.