Expression of cadherins and CD44 isoforms in ovarian endometrial cysts

We evaluated the immunohistochemical expression of cadherins and CD44 variants in 20 endometriomas, 20 cystadenomas, 20 borderline ovarian tumours as well as 20 ovarian carcinomas, and the serological and cystic fluid concentrations of soluble E-cadherin and soluble CD44 standard (sCD44sdt) in 20 endometriomas, 20 cystadenomas, six borderline and 11 carcinomas of the ovary. In endometriomas, immunostaining of E- and N-cadherin was negative (20 and 30% respectively). CD44 H, v3 and v6 immunostaining were detected in 63, 10 and 40% respectively. A difference in immunostaining for E-cadherin was found between endometriomas and cystadenomas (P < 0.001) and for N- cadherin between endometriomas and carcinomas (P < 0.001). A difference in CD44H immunostaining was observed between endometriomas and cystadenomas (P < 0.035) but not with borderline ovarian tumours and carcinomas. No difference in serum concentrations of soluble E- cadherins and CD44 standard was found between the four groups of tumours. Cystic fluid concentrations of E-cadherin were lower in endometriomas than in borderline tumours and ovarian carcinomas (P < 0.001). High concentrations of soluble CD44 standard cystic fluid were found in endometriomas than in other ovarian cysts. Endometriomas and borderline tumours share alterations of cadherins and CD44 isoforms which may help in the understanding of the aggressive and invasive potentials of endometriotic cells.      

The uses of poxviruses as vectors

Poxviruses have played an amazing role in the development of virology, immunology and vaccinology. In 1796, deliberate inoculation of cowpox virus to humans was proved by Dr. Edward Jenner to protect against the antigenically related smallpox virus (variola). This discovery founded the science of immunology and eventually led to smallpox eradication from the earth in 1980 after a world wide vaccination campaign with vaccinia virus (another poxvirus). Paradoxically, despite the eradication of smallpox, there has been an explosion of interest in vaccinia virus in the eighties. This interest has stemmed in part from the application of molecular genetics to clone and express foreign genes from recombinant vaccinia virus. The use of these recombinant vaccinia viruses as efficacious in vitro expression system and live vaccine has raised concerns about their safety. The work of the scientific community of the last 20 years has contributed to improve drastically the safety of poxvirus derived vectors. Firstly, the safety of vaccinia virus has been enhanced by production of genetically attenuated strains. Secondly, alternative poxvirus vectors, such as avipoxviruses, were proved to be extremely safe and efficacious non-replicating vectors when used in non avian species. In the present chapter, the basic concepts of poxvirus biology required to assess the safety of a poxvirus derived vector are provided. The principal poxvirus vectors available to date are described in regards to their biosafety.     

Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells

PURPOSE: The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro. METHODS: A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel. RESULTS: Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative. CONCLUSIONS: These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs.     

Real-Time PCR Quantification of Methanobrevibacter oralis in Periodontitis

A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis.     

A quality control program for mutation detection in KIT and PDGFRA in gastrointestinal stromal tumours

Background

Although most gastrointestinal stromal tumours (GIST) carry oncogenic mutations in KIT exons 9, 11, 13 and 17, or in platelet-derived growth factor receptor alpha (PDGFRA) exons 12, 14 and 18, around 10% of GIST are free of these mutations. Genotyping and accurate detection of KIT/PDGFRA mutations in GIST are becoming increasingly useful for clinicians in the management of the disease.

Method

To evaluate and improve laboratory practice in GIST mutation detection, we developed a mutational screening quality control program. Eleven laboratories were enrolled in this program and 50 DNA samples were analysed, each of them by four different laboratories, giving 200 mutational reports.

Results

In total, eight mutations were not detected by at least one laboratory. One false positive result was reported in one sample. Thus, the mean global rate of error with clinical implication based on 200 reports was 4.5%. Concerning specific polymorphisms detection, the rate varied from 0 to 100%, depending on the laboratory. The way mutations were reported was very heterogeneous, and some errors were detected.

Conclusion

This study demonstrated that such a program was necessary for laboratories to improve the quality of the analysis, because an error rate of 4.5% may have clinical consequences for the patient.     

A prospective comparison of post-surgical behavioral pain scales in preschoolers highlighting the risk of false evaluations

Four behavioral rating scales (BRS) (CHEOPS, CHIPPS, FLACC and OPS) assessing postoperative pain in children aged 1–7 years were studied to compare their psychometric properties, sensitivity and specificity. One hundred and fifty children included in this prospective longitudinal study were videotaped to analyze retrospectively peri-operative behaviors. Pain and anxiety were evaluated by children or by their parents prospectively. At the end of the study, four observers rated the peri-operative videos using the four BRS. Because self-reporting cannot be used for all the children, facial expression of pain was analyzed from the videos to create a Facial Action Summary Score (FASS) which was considered as a reference for the study of validity of the four BRS. Internal validities were excellent but external validities were mixed. The FLACC seems to be better adapted to assess post-surgical pain in children between 1 and 7 years old. Nevertheless, it was significantly correlated with anxiety measures. Moreover, the analysis of sensitivity and specificity using both self-reporting of pain and FASS showed that some children were still under-evaluated. The multivariate analysis underlines silence as a high risk factor of misevaluating postoperative pain. In conclusion, this study highlights the difficulty of discriminating pain intensity from anxiety when using the four BRS and that postoperatively, nearly one child in 10 was misevaluated.     

p53 mutations have no additional prognostic value over stage in bladder cancer.

Evidence is accumulating that the tumour-suppressor gene p53 is involved in the development of bladder cancer. Therefore we studied p53 mutations in 47 bladder cancers obtained from 45 patients using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. Eight out of 24 invasive tumours appeared to have a p53 mutation, while no p53 mutations were found in the superficial tumours. All the p53 mutations were found in grade 3 tumours. The tumours with altered p53 showed a higher frequency of allelic loss (FAL) than the tumours without a mutation (55.8% vs 21.1%, P < 0.05, chi 2 test). This increase in FAL suggests a correlation between p53 mutations and genetic instability. A significant correlation between mutated p53 and poor survival in the whole group studied was found (P < 0.001, log-rank test). However, within the group of muscle-invasive tumours the occurrence of p53 mutations had no additional prognostic value. Therefore, even though p53 mutations were found in aggressive tumours, the clinical usefulness of its detection seems limited. Nevertheless, these results imply that p53 is involved in the clinical behaviour of bladder cancer; its role in the progression of superficial cancer to invasive disease merits further attention.     

Predictive value of biological markers for hepatocellular carcinoma patients treated with orthotopic liver transplantation.

PURPOSE: To help stratify candidates with hepatocellular carcinoma (HCC) for orthotopic liver transplantation (OLT), biomarkers are needed that are capable of predicting recurrence of disease (ROD). We investigated the prognostic role in this setting of immunohistochemical markers reported previously to predict poor prognosis in HCC patients treated with resection. EXPERIMENTAL DESIGN: Eighty-three patients with HCC who underwent OLT between 1987 and 2001 with a minimum clinical follow up of 12 months were included in this retrospective study. We analyzed immunohistochemical expression of the adhesion molecules E-cadherin and beta-catenin (membrane/nuclear localization), MIB-1 proliferative index and the cyclin-dependent kinase inhibitor p27, alongside the main clinical-pathological variables. RESULTS: At univariate analysis, vascular thrombosis, high MIB-1 index, lower membrane expression of E-cadherin and beta-catenin, and nuclear beta-catenin localization were associated with ROD. At multivariate analysis, only MIB-1 index, low equal E-cadherin (with respect to non-neoplastic surrounding tissue), and nuclear beta-catenin appeared as independent predictors of ROD. The logistic regression analysis model indicated that detection of any one parameter was associated with at least 88% estimated risk of ROD (up to 99% for all three). CONCLUSIONS: We propose these three molecular parameters as an additional tool for rational selection of OLT candidates among HCC patients (stratification according to the risk of ROD might help provide a similar life expectancy for cirrhotic candidates with and without HCC).     

Could induced apoptosis of human granulosa cells predict in vitro fertilization-embryo transfer outcome? A preliminary study of 25 women

OBJECTIVE: The aim of this study was to investigate the relationship between induced apoptosis of human luteinized granulosa cells (GCs) and in vitro fertilization (IVF) outcome. STUDY DESIGN: We induced apoptosis with interferon gamma and an anti-human Fas antibody in cultured GCs isolated from follicular fluids coming from 25 different women undergoing 25 consecutive IVF cycles. After examination of 1000 GCs stained by DAPI with a fluorescent microscope, we determined the percentages of apoptotic GCs. Ovarian, endometrial and IVF parameters were recorded for every woman. RESULTS: We classified the women according to their induced GCs apoptosis percentages in two groups. Group 1 with a low percentage of apoptotic GCs (11.6+/-4.8%) had a significantly higher pregnancy rate (P<0.05) than group 2 with a high percentage of apoptotic GCs (59.5+/-14.8%). No other statistically significant differences were observed. CONCLUSION: Resistance of human GCs to apoptosis might be implicated in IVF outcome.