Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours

Background

Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT.

Methods

The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 μM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined.

Results

MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p < 0.05) lower expression of the MMR and pluripotency factors, as well as a reduced MMR activity and cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p < 0.001) reduced cisplatin sensitivity.

Conclusion

This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis.

     

Conceptual framework for a Danish human biomonitoring program

The aim of this paper is to present the conceptual framework for a Danish human biomonitoring (HBM) program. The EU and national science-policy interface, that is fundamental for a realization of the national and European environment and human health strategies, is discussed, including the need for a structured and integrated environmental and human health surveillance program at national level. In Denmark, the initiative to implement such activities has been taken. The proposed framework of the Danish monitoring program constitutes four scientific expert groups, i.e. i. Prioritization of the strategy for the monitoring program, ii. Collection of human samples, iii. Analysis and data management and iv. Dissemination of results produced within the program. This paper presents the overall framework for data requirements and information flow in the integrated environment and health surveillance program. The added value of an HBM program, and in this respect the objectives of national and European HBM programs supporting environmental health integrated policy-decisions and human health targeted policies, are discussed.In Denmark environmental monitoring has been prioritized by extensive surveillance systems of pollution in oceans, lakes and soil as well as ground and drinking water. Human biomonitoring has only taken place in research programs and few incidences of e.g. lead contamination. However an arctic program for HBM has been in force for decades and from the preparations of the EU-pilot project on HBM increasing political interest in a Danish program has developed.     

Parental Infections Before,During, and After Pregnancy as Risk Factors for Mental Disorders in Childhood and Adolescence: A Nationwide Danish Study

Background

Previous studies have shown associations between maternal infections during pregnancy and increased risks of schizophrenia and autism spectrum disorder in the offspring. However, large-scale studies investigating an association between parental infections both during and outside the pregnancy period and the risk of any mental disorder in the child are lacking.

Multiple myeloma and infections: a population-based study on 9253 multiple myeloma patients

Infections are a major cause of morbidity and mortality in patients with multiple myeloma. To estimate the risk of bacterial and viral infections in multiple myeloma patients, we used population-based data from Sweden to identify all multiple myeloma patients (n=9253) diagnosed from 1988 to 2004 with follow up to 2007 and 34,931 matched controls. Cox proportional hazard models were used to estimate the risk of infections. Overall, multiple myeloma patients had a 7-fold (hazard ratio =7.1; 95% confidence interval = 6.8–7.4) risk of developing any infection compared to matched controls. The increased risk of developing a bacterial infection was 7-fold (7.1; 6.8–7.4), and for viral infections 10-fold (10.0; 8.9–11.4). Multiple myeloma patients diagnosed in the more recent calendar periods had significantly higher risk of infections compared to controls (P<0.001). At one year of follow up, infection was the underlying cause in 22% of deaths in multiple myeloma patients. Mortality due to infections remained constant during the study period. Our findings confirm that infections represent a major threat to multiple myeloma patients. The effect on infectious complications due to novel drugs introduced in the treatment of multiple myeloma needs to be established and trials on prophylactic measures are needed.     

Recreational drug use at a major music festival: trend analysis of anonymised pooled urine

Objective: The spread of new psychoactive substances (NPS) has expanded rapidly in the last decade. The complexity of the pharmacological effects of NPS challenges the traditional treatment guidelines, and information of the emergence of new arrivals is valuable. Our knowledge on the actual range of recreational drugs used and NPS available in Denmark is limited as identification is possible only when consumers become patients in the healthcare system or through drug seizures. We aimed to detect classical recreational drugs and NPS in the urine of music festival attendees and evaluate if the use of NPS could have been predicted by comparing study data with drug seizure data from the previous year published by European and Danish health authorities.

Methods: In a cross-sectional study, 44 urine samples were collected from three urinals at Roskilde Festival 2016—the largest Danish music festival. Two urinals were placed at music stages with late-night concerts, and one urinal was placed at a camp site. Samples were prepared using enzymatic hydrolysis followed by cationic and anionic solid phase extraction, and analysed using ultra performance liquid chromatography-high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS). Data were processed using an in-house library of 467 target substances, including legal and illegal drugs and metabolites. Urine drug-screening immunoassays were also evaluated and results were compared to UPLC-HR-TOF-MS results.

Results: In total, 77 drugs, including metabolites, were qualitatively identified in the 44 urine samples. The recreational drugs identified were amphetamine (n?=?30), cocaine (n?=?44), MDA (n?=?40), MDMA (n?=?44), THC-COOH (n?=?19) and ketamine (n?=?17). No NPS were identified. Sample testing using the urine drug-screening immunoassays showed presence of cocaine (n?=?27), methamphetamine/MDMA (n?=?4), THC (n?=?7), “Spice” (n?=?7) and methylphenidate (n?=?1). These discrepancies might be caused by differences in cut-off values between the analytical methods, limited specificity or cross-reactivity of the urine drug-screening immunoassays compared to UPLC-HR-TOFMS results.

Conclusion: Widespread uses of classical recreational drugs were identified in pooled urine samples. The prevalence of NPS was not as comprehensive as expected based on the European and Danish health authorities reports on illegal drugs. Urine drug-screening immunoassays results are advised to be confirmed by chromatographic bioanalysis.     

Novel human prostate cell lines derived from the transition and peripheral zones of the prostate for carcinogenesis studies

Epithelial cell lines were established from the transition and peripheral zones of human prostate by transduction with cdk4 and hTERT. The properties of these lines were investigated using immunocytochemical markers, ability to generate anchorage-independent colonies and by spectral karyotyping (SKY). Cells were exposed to fractionated doses of gamma irradiation to investigate their ability to transform. Cell lines were established from the transition and peripheral zones of human prostate. The expression of CD133, CK5, CK14, CK18, p16, PSCA, p63 and c-myc varied between the lines from the two regions. The line derived from the peripheral zone exhibited properties of a tumour line. A similar pattern was observed in two separate transductions. It was thus unlikely to be an in vitro transformation event, which is very rarely observed with human cells in vitro, and thus more likely to be derived from the immortalisation of a quiescent tumour clone. Fractionated irradiation of the transition zone cell line resulted in forming of transformed colonies. The transformed and tumour line had marked chromosomal rearrangements as demonstrated by SKY analysis. Cell lines have been derived from different zones of human prostate for studies on radiation carcinogenesis. The unirradiated cell line derived from the peripheral zone exhibited chromosomal rearrangements similar to those observed in prostate carcinoma. The cell line derived from the transitional zone exhibited a near diploid karyotype and could be transformed following exposure to fractionated doses of gamma irradiation.     

A protein profile study to discriminate CIN lesions from normal cervical epithelium

Background

Cervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN.

Methods

Cervical punch biopsies from 55 women were incubated for 24 h at 4°C in RPMI1640 medium for protein analysis prior to usual FFPE processing and p16 and Ki67-supported histologic consensus diagnosis was assessed. The biopsy supernatants were subjected to surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) for identifying differentially expressed proteins. Binary logistic regression and classification and regression trees (CART) were used to develop a classification model.

Results

The age of the patients ranged from 26 to 40 years (median 29.7). The consensus diagnoses were normal cervical tissue (n?=?10) and CIN2-3 (n?=?45). The mean protein concentration was 1.00 and 1.09 mg/ml in the normal and CIN2-3 group, respectively. The peak detection and clustering process resulted in 40 protein peaks. Many of these peaks differed between the two groups, but only three had independent discriminating power. The overall classification results were 88%.

Conclusions

Water-soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and CIN2-3.