Mutations in LRRC50 Predispose Zebrafish and Humans to Seminomas

Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis

Intratumoural and peripheral blood lymphocyte subsets in patients with metastatic renal cell carcinoma undergoing interleukin-2 based immunotherapy: association to objective response and survival

The aim of the present study was to analyse lymphocyte subsets in consecutive peripheral blood samples and consecutive tumour tissue core needle biopsies performed before and during interleukin-2 based immunotherapy, and to correlate the findings with objective response and survival. Twenty-six patients with metastatic renal cell carcinoma were treated with low dose s.c. interleukin-2, interferon-α and histamine. A total of 250 blood samples and 62 core needle biopsies from 23 and 19 of these patients, respectively, were analysed. After 2 weeks of treatment, a significant positive correlation between absolute number of peripheral blood lymphocytes (P=0.028), CD3 (P=0.017), CD57 (P=0.041) and objective response was demonstrated. There was no correlation between any peripheral blood leukocyte subsets and survival. Cytotoxicity of peripheral blood mononuclear cells was not correlated to objective response or survival. Within the tumour tissue at baseline, a significant positive correlation between CD4 (P=0.027), CD8 (P=0.028), CD57 (P=0.007) and objective response was demonstrated. After one month of immunotherapy, a significant positive correlation between intratumoral CD3 (P=0.026), CD8 (P=0.015), CD57 (P=0.009) and objective response was demonstrated. A significant positive correlation between intratumoral baseline CD4 (P=0.047), baseline CD57 (P=0.035), CD3 at one month (P=0.049) and survival was demonstrated. These data provide novel in vivo evidence of the possible contribution of lymphocyte subsets in the tumour reduction in responding patients during interleukin-2 based immunotherapy. Confirmation of the results requires further studies including a larger number of patients

External validation of the PAGE-B score for HCC risk prediction in people living with HIV/HBV coinfection

Background & Aims: HBV coinfection is common among people living with HIV (PLWH) and is the most important cause of hepatocellular carcinoma (HCC). While risk prediction tools for HCC have been validated in patients with HBV monoinfection, they have not been evaluated in PLWH. Thus, we performed an external validation of PAGE-B in people with HIV/HBV coinfection. Methods: We included data on PLWH from four European cohorts who were positive for HBsAg and did not have HCC before starting tenofovir. We estimated the predictive performance of PAGE-B for HCC occurrence over 15 years in patients receiving tenofovir-containing antiretroviral therapy. Model discrimination was assessed after multiple imputation using Cox regression with the prognostic index as a covariate, and by calculating Harrell's c-index. Calibration was assessed by comparing our cumulative incidence with the PAGE-B derivation study using Kaplan-Meier curves. Results: In total, 2,963 individuals with HIV/HBV coinfection on tenofovir-containing antiretroviral therapy were included. PAGE-B was <10 in 26.5%, 10–17 in 57.7%, and ≥18 in 15.7% of patients. Within a median follow-up of 9.6 years, HCC occurred in 68 individuals (2.58/1,000 patient-years, 95% CI 2.03–3.27). The regression slope of the prognostic index for developing HCC within 15 years was 0.93 (95% CI 0.61–1.25), and the pooled c-index was 0.77 (range 0.73–0.80), both indicating good model discrimination. The cumulative incidence of HCC was lower in our study compared to the derivation study. A PAGE-B cut-off of <10 had a negative predictive value of 99.4% for the development of HCC within 5 years. Restricting efforts to individuals with a PAGE-B of ≥10 would spare unnecessary HCC screening in 27% of individuals. Conclusions: For individuals with HIV/HBV coinfection, PAGE-B is a valid tool to determine the need for HCC screening. Impact and implications: Chronic HBV infection is the most important cause of hepatocellular carcinoma (HCC) among people living with HIV. Valid risk prediction may enable better targeting of HCC screening efforts to high-risk individuals. We aimed to validate PAGE-B, a risk prediction tool that is based on age, sex, and platelets, in 2,963 individuals with HIV/HBV coinfection who received tenofovir-containing antiretroviral therapy. In the present study, PAGE-B showed good discrimination, adequate calibration, and a cut-off of <10 had a negative predictive value of 99.4% for the development of HCC within 5 years. These results indicate that PAGE-B is a simple and valid risk prediction tool to determine the need for HCC screening among people living with HIV and HBV

Influenza Virus Respiratory Infection and Transmission Following Ocular Inoculation in Ferrets

While influenza viruses are a common respiratory pathogen, sporadic reports of conjunctivitis following human infection demonstrates the ability of this virus to cause disease outside of the respiratory tract. The ocular surface represents both a potential site of virus replication and a portal of entry for establishment of a respiratory infection. However, the properties which govern ocular tropism of influenza viruses, the mechanisms of virus spread from ocular to respiratory tissue, and the potential differences in respiratory disease initiated from different exposure routes are poorly understood. Here, we established a ferret model of ocular inoculation to explore the development of virus pathogenicity and transmissibility following influenza virus exposure by the ocular route. We found that multiple subtypes of human and avian influenza viruses mounted a productive virus infection in the upper respiratory tract of ferrets following ocular inoculation, and were additionally detected in ocular tissue during the acute phase of infection. H5N1 viruses maintained their ability for systemic spread and lethal infection following inoculation by the ocular route. Replication-independent deposition of virus inoculum from ocular to respiratory tissue was limited to the nares and upper trachea, unlike traditional intranasal inoculation which results in virus deposition in both upper and lower respiratory tract tissues. Despite high titers of replicating transmissible seasonal viruses in the upper respiratory tract of ferrets inoculated by the ocular route, virus transmissibility to naïve contacts by respiratory droplets was reduced following ocular inoculation. These data improve our understanding of the mechanisms of virus spread following ocular exposure and highlight differences in the establishment of respiratory disease and virus transmissibility following use of different inoculation volumes and routes

Analysis of enzyme reactions in situ

Estimations of metabolic rates in cells and tissues and their regulation on the basis of kinetic properties of enzymes in diluted solutions may not be applicable to intact living cells or tissues. Enzymes often behave differently in living cells because of the high cellular protein content that can lead to homologous and heterologous associations of protein molecules. These associations often change the kinetics of enzymes as part of post-translational regulation mechanisms. An overview is given of these interactions between enzyme molecules or between enzyme molecules and structural elements in the cell, such as the cytoskeleton. Biochemical and histochemical methods are discussed that have been developed for in vivo and in situ analyses of enzyme reactions, particularly for the study of effects of molecular interactions. Quantitative (histochemical) analysis of local enzyme reactions or fluxes of metabolites has become increasingly important. At present, it is possible to calculate local concentrations of substrates in cells or tissue compartments and to express local kinetic parameters in units that are directly comparable with those obtained by biochemical assays of enzymes in suspensions. In situ analysis of the activities of a number of enzymes have revealed variations in their kinetic properties (Km and Vmax) in different tissue compartments. This stresses the importance of in vivo or in situ analyses of cellular metabolism. Finally, histochemical determinations of enzyme activity in parallel with immunohistochemistry for the detection of the total number of enzyme molecules and in situ hybridization of its messenger RNA allow the analysis of regulation mechanisms at all levels between transcription of the gene and post-translational activity modulatio

In situ kinetic parameters of glucose-6-phosphatase in the rat liver lobulus

Glucose-6-phosphatase activity has been determined in periportal and pericentral zones of the rat liver lobule using a quantitative histochemical method. The study was performed on unfixed cryostat sections of livers from fasted and fed female and male rats. Highest activity was found in periportal zones, and starvation caused a 2-3-fold increase of glucose-6-phosphatase activity in periportal and pericentral zones of both sexes. Unexpectedly, KM values were also significantly different in periportal and pericentral zones and were found to increase linearly with Vmax values, irrespective of sex and feeding condition. Because the cryofixation procedure was shown to permeabilize the biomembranes in the tissue sections, it can be concluded that the rise in KM and Vmax values has to be attributed to the catalytic unit of the glucose-6-phosphatase system. It is suggested that the enzyme exists in a high affinity configuration at low enzyme concentrations but that at high enzyme concentrations a hysteretic mechanism, as proposed by Berteloot et al. (Berteloot, A., Vidal, H., and Van de Werve, G. (1991) J. Biol. Chem. 266, 5497-5507), transforms the enzyme from a high to a low affinity configuration. The present study indicates that the concept of functional heterogeneity of liver parenchyma may be more complex than thus far assume

Case report. Een paraganglioom in de blaas

We report a case of a 48 -year-old female with an atypical bladder tumor. The patient has a history of familial paraganglioma. A catecholamine releasing bladder tumor could cause an adrenal crisis with sudden hypertension during surgery. Therefore, the catecholamine receptors were blocked prior to the resection of the bladder tumor. A transurethral resection of an 1 cm large bladder tumor was performed. Pathological examination confirmed the diagnosis; it indeed turned out to be a bladder pheochromocytoma

Growth-behavior of human pancreatic-carcinoma xenograft correlates with nuclear features

Two human pancreatic ductal adenocarcinomas with different growth rates were serially transplanted into nude mice. Feulgen-stained 4-mu-m sections and imprints from the xenografts were studied with a VICOM automated image analysis system. After pooling the results from two passages, with three mice in each passage, it was shown that of 23 nuclear parameters measured the following were correlated with a fast tumor growth rate: in sections, a decrease in heterogeneity of the chromatin and an increase in perimeter and nuclear area; in imprints, an increase in lesser diameter, in mean grey level difference between second neighboring pixels, and in total integrated optical density (DNA content). Several parameters differed significantly between passages, and between animals in the same passage. These findings suggest that the growth speed of pancreatic tumors may be predicted by nuclear parameters

Clinical and Radiologic Signs of Relapsed Ovarian Germ Cell Tumor: Tissue Is the Issue

Malignant ovarian germ cell tumor is a rare disease, but with current treatment strategies including surgery and platinum based chemotherapy survival is excellent. After treatment, intensive followup is indicated to encounter tumor relapse at an early stage. This case describes a 22-year-old female with a history of common variable immune deficiency (CVID) who underwent a resection of a large ovarian germ cell tumor followed by 4 cycles of cisplatin and etoposide resulting in clinical complete remission. During followup, she developed a mass at the umbilicus and ascites. Initially, the cytology of the ascites was interpreted as tumor positive, suspicious of relapse of the disease, but tumor markers remained negative. However, during laparoscopy it turned out to be a mature teratoma, which can develop after chemotherapy, the so called growing teratoma syndrome. In retrospect, the ascites was false positive. This case shows that current diagnostic tools are not sufficient to distinguish between vital tumor and mature teratoma and can be misleading. Tumor biopsy and/or laparoscopic inspection are therefore indicated