A rapid solution hybridization method for detection of human papillomaviruses

Nucleic acid hybridization methods in routine diagnosis of micro-organisms have been limited by the tedious assay procedures. We have previously described the sandwich hybridization method which allows convenient testing of biological specimens. In this paper we describe the adaptation of the solution hydridization method into the microtitre plate format using 35S-isotope as label. Using 3-hour hybridization followed by 2-hour collection of the hybrids a sensitivity of 5 x 10(5) target DNA molecules was achieved. The method was applied for identification of human papillomaviruses in crude gynaecological specimens. A simple 1-day assay protocol was achieved with high HPV type specificity. The specificity was confirmed by testing a variety of unrelated micro-organisms, none of which gave a positive signal in the test. Results, obtained as numerical values, were easy to interpret; positive and negative samples gave clearly distinguishable signals.     

Lymphocytes and Langerhans cells in patch tests

The distribution of immunocompetent cells was analysed in allergic (nickel) and irritant (dithranol) patch tests using conventional transmission electron microscopy and labelling with monoclonal antibodies in an avidin-biotin immunoperoxidase study. The biopsies were taken 24 or 48 h after the allergen/irritant application. In allergic and irritant reactions, most inflammatory cells were OKT11 positive (pan T lymphocytes). The majority of these cells were also OKT4 positive (helper/inducer T lymphocytes), while the minority were OKT8 positive (suppressor/cytotoxic T lymphocytes). NK9 positive cells (natural killer cells) were observed in small numbers. The number of dendritic OKT6 and OKIal positive cells (Langerhans cells) in the epidermis was unaffected in allergic reactions. In irritant reactions, a normal number of OKT6 positive Langerhans cells was observed, while the number of OKIal positive cells had increased in the epidermis. Dithranol caused prominent fine structural changes in the mitochondria of the Langerhans cells, while the keratinocytes appeared largely unaffected. The present study indicates that allergic and irritant patch tests cannot be differentiated reliably using current immunohistopathological or electron microscopic techniques, in spite of the small differences observed.     

Adolescents’ Attitudes on Smoking Are Related to Experimentation with Smoking,Daily Smoking and Best Friends’ Smoking in Two Karelias in Finland and in Russia

Purpose

Becoming a smoker usually starts during adolescence and is a dynamic process involving experimentation before the establishment of daily smoking. It has been suggested that adolescents who smoke differ from those who do not in their attitudes to smoking. The purpose of this study was to find out whether attitudes related to smoking legislation and restrictions, social pressures in smoking and image of smokers are associated with smoking experimentation, daily smoking and best friends’ smoking.

Method

The data were gathered with a self-administered questionnaire in North Karelia, Eastern Finland and in the Pitkyaranta district, Republic of Karelia, Russia. The respondents were 15-year-old 9th grade pupils in local schools. In Pitkyaranta, the data analyses covered pupils in all eight schools in the area (n?=?179). In North Karelia, the data analyses comprised of selected eight schools (n?=?601). Data were analysed with exploratory factor analysis.

Results

The models revealed that attitudes related to restrictions and social pressure were significantly associated with experimenting with smoking [OR (95 % CI) 7.923 (5.787–10.847)], daily smoking [OR (95 % CI) 9.575 (6.727–13.628)] and the likelihood of best friends’ smoking [OR (95 % CI) 3.154 (2.579–3.858)]. The stronger the young peoples’ attitudes and opinions, for example, towards restrictions and with more difficulties in refusing smoking, the higher the likelihood for smoking experimentations, daily smoking as well as the likelihood for their best friends’ smoking. The country and factor interactions were not associated with smoking experimentations, daily smoking or best friends’ smoking.

Conclusion

Regardless of cultural background, adolescents who smoke have more positive attitudes to smoking, and perceive more social support for smoking, than do adolescents who do not smoke. The study stresses the similarity of the results in both Karelia’s despite the enormous differences in culture, economy and public policy.

     

Combination of gemcitabine and Ad5/3-Delta24, a tropism modified conditionally replicating adenovirus, for the treatment of ovarian cancer

Conditionally replicating adenoviruses (CRAds) represent a novel approach for the treatment of cancers resistant to conventional therapies. The efficacy of CRAds might be further improved by using chemotherapeutic agents in a multimodal antitumor approach. We have evaluated the use of Ad5/3-Delta24, a serotype 3 receptor targeted Rb/p16 pathway selective CRAd, in combination with gemcitabine against human ovarian adenocarcinoma. The combination of these agents showed synergistic cell killing in vitro compared to single treatments. However, the effect was dependent on dose and sequencing of the agents. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Delta24 replication without affecting the total amount of virus produced. Possible reasons for synergy between Ad5/3-Delta24 and gemcitabine include the chemosensitizing activity of E1A and/or altered replication kinetics. In an orthotopic murine model of peritoneally disseminated ovarian cancer, the combination increased the survival of mice over either agent alone, and almost 60% of treated mice were cured. Sequencing of the agents was critical for toxicity versus efficacy. Mice remained free from intraperitoneal disease, but some succumbed to treatment-related hepatic or bone marrow toxicity. This suggests that improved efficacy may uncover treatment-related toxicity, which needs to be monitored closely in clinical trials.     

Association of distinct allelic haplotypes of DISC1 with psychotic and bipolar spectrum disorders and with underlying cognitive impairments

Bipolar disorder (BPD) and schizophrenia (SCZ) have at least a partially convergent aetiology and thus may share genetic susceptibility loci. Multiple lines of evidence emphasize the role of disrupted-in-schizophrenia-1 (DISC1) gene in psychotic disorders such as SCZ. We monitored the association of allelic variants of translin-associated factor X (TSNAX)/DISC1 gene cluster using 13 single-nucleotide polymorphisms (SNPs) in 723 members of 179 Finnish BPD families. Consistent with an earlier finding in Finnish SCZ families, the haplotype T-A of rs751229 and rs3738401 at the 5' end of DISC1 was over-transmitted to males with psychotic disorder (P = 0.008; for an extended haplotype P = 0.0007 with both genders). Haplotypes at the 3' end of DISC1 associated with bipolar spectrum disorder (P = 0.0002 for an under-transmitted haplotype T-T of rs821616 and rs1411771, for an extended haplotype P = 0.0001), as did a two-SNP risk haplotype at the 5' end of TSNAX (P = 0.007). The risk haplotype for psychotic disorder also associated to perseverations (P = 0.035; for rs751229 alone P = 0.0012), and a protective haplotype G-T-G with rs1655285 in addition to auditory attention (P = 0.0059). The 3' end variants associated with several cognitive traits, with the most robust signal for rs821616 and verbal fluency and rs980989 and psychomotor processing speed (P = 0.011 for both). These results support involvement of DISC1 in the genetic aetiology of BPD and suggest that its distinct variants contribute to variation in the dimensional features of psychotic and bipolar spectrum disorders. Finding of alternative associating haplotypes in the same set of BPD families gives evidence for allelic heterogeneity within DISC1, eventually leading to heterogeneity in the clinical outcome as well.     

N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible

Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.     

Smoking,nicotine dependence and nicotine intake by socio-economic status and marital status

Introduction

Low socio-economic status (SES) is strongly related to smoking, but studies examining the association of SES with nicotine dependence (ND) are scarce. The aim of this study was to examine the associations of SES and marital status with smoking, multiple measures of ND, and cotinine as a nicotine intake biomarker.

Methods

The sample comprised 1746 ever smokers, sampled from the National FINRISK 2007 Study, who had completed a tobacco specific questionnaire in addition to the standard clinical examination. The Fagerström Test for Nicotine Dependence (FTND), the Heaviness of Smoking Index (HSI), the Nicotine Dependence Syndrome Scale (NDSS), and the Hooked On Nicotine Checklist (HONC) were assessed, while plasma cotinine was measured as a biomarker of nicotine exposure in daily smokers. Univariate and multivariate associations were assessed by linear regression and multinomial logistic regression.

Results

In multivariate models, lower education was associated with higher FTND and HSI, income with HSI, and occupation with HSI (men only), FTND, HONC and NDSS scores. Lower education was related to higher cotinine levels among daily smokers, although the association diminished slightly after adjusting for daily smoking amount. Living without a spouse was associated with daily smoking and higher ND.

Conclusion

In this cross-sectional study low SES was linked with higher ND among current smokers, while low SES was associated with higher cotinine levels among daily smokers. Living alone was linked with higher ND. Longitudinal studies are warranted to further explore these associations. As lower SES smokers are more addicted they may need more targeted cessation services to succeed in quitting smoking.     

Genetic association of SP-C with duration of preterm premature rupture of fetal membranes and expression in gestational tissues

Background. Surfactant protein (SP) C has been shown to be expressed also outside pulmonary alveoli. Certain SP-C gene (SFTPC) polymorphisms associate with lung diseases and very preterm birth.

Aims. We investigated the association of SFTPC single nucleotide polymorphism (SNP) rs4715 with factors affecting spontaneous preterm birth and characterized the SP-C expression in human and mouse gestational tissues.

Methods. The SFTPC SNP rs4715 polymorphism was genotyped in a homogeneous northern European population of mothers and infants in spontaneous preterm birth and term controls. The expression and protein of SP-C in gestational tissues was analyzed.

Results. SFTPC SNP rs4715 did not associate with spontaneous preterm birth. However, fetuses with short interval (<72 hours) between preterm premature rupture of fetal membranes (PPROM) and preterm birth had significant over-representation of the minor allele A, whereas in fetuses with prolonged PPROM (≥72 hours) the frequency was decreased. Maternal SFTPC did not associate with the duration of PPROM. SP-C mRNA and proprotein were detected in fetal membranes, placenta, and pregnant uterus.

Conclusion. SFTPC SNP rs4715 associates with the duration of PPROM, and SP-C is expressed in gestational tissues. We propose that fetal SFTPC moderates the inflammatory activation within the fetal extra-embryonic compartment.