Human responses in heat - comparison of the Predicted Heat Strain and the Fiala multi-node model for a case of intermittent work.

Two mathematical models of human thermal regulation include the rational Predicted Heat Strain (PHS) and the thermophysiological model by Fiala. The approaches of the models are different, however, they both aim at providing predictions of the thermophysiological responses to thermal environments of an average person. The aim of this study was to compare and analyze predictions of the two models against experimental data. The analysis also includes a gender comparison. The experimental data comprised of ten participants (5 males, 5 females, average anthropometric values were used as input) conducting an intermittent protocol of rotating tasks (cycling, stacking, stepping and arm crank) of moderate metabolic activities (134-291W/m2) with breaks in-between in a controlled environmental condition (34°C, 60% RH). The validation consisted of the predictions' comparison against experimental data from 2.5h of data of rectal temperature and mean skin temperature based on contact thermometry from four body locations. The PHS model over-predicted rectal temperatures during the first activity for males and the cooling effectiveness of sweat in the recovery periods, for both males and females. As a result, the PHS simulation underestimated the thermal strain in this context. The Fiala model accurately predicted the rectal temperature throughout the exposure. The fluctuation of the experimental mean skin temperature was not reflected in any of the models. However, the PHS simulation model showed better agreement than the Fiala model. As both models predicted responses more accurately for males than females, we suggest that in future development of the models it is important to take this result into account. The paper further discusses possible sources of the observed discrepancies and concludes with some suggestions for modifications.

Exposure to different lengths of sick leave and subsequent work absence among young adults.

BACKGROUND: Sweden has a public and easily accessible sickness insurance. Research shows, however, downsides to taking sick leave. Both short and longer periods of sick leave have been seen to increase the risk for subsequent work absence. The aim of this study was to investigate whether there was an association between sick leave claimed in 1993 and work absence in the subsequent 15 years, i.e. up to 2008. A further aim was to explore differences in this relation with regard to gender, origin and educational level at baseline. METHODS: Our cohort consisted of all immigrants aged 21-25 years in Sweden in 1993 and a control group of native Swedes in the same age group. RESULTS: Subsequent work absence increased from 313 days among persons with no days of claimed sick leave in 1993 to 567 days among persons with 1-7 days of claimed sick leave in 1993. Thereafter there was a lower, but steady increase in days of future work absence, to 611 days among persons with 8-14 days of sick leave claimed in 1993. There was an interaction between sick leave and gender, education and origin respectively regarding later work absence. CONCLUSION: Periods of sick leave claimed were associated with subsequent work absence. Immigrants, women and persons with low education had the most risk of future work absence after a period of sick leave.

Sickness absence at a young age and later sickness absence, disability pension, death, unemployment and income in native Swedes and immigrants.

BACKGROUND: Sickness absence with cash benefits from the sickness insurance gives an opportunity to be relieved from work without losing financial security. There are, however, downsides to taking sickness absence. Periods of sickness absence, even short ones, can increase the risk for future spells of sickness absence and unemployment. The sickness period may in itself have a detrimental effect on health. The aim of the study was to investigate if there is an association between exposure to sickness absence at a young age and later sickness absence, disability pension, death, unemployment and income from work. METHODS: Our cohort consisted of all immigrants aged 21-25 years in Sweden in 1993 (N = 38 207) and a control group of native Swedes in the same age group (N = 225 977). We measured exposure to sickness absence in 1993 with a follow-up period of 15 years. We conducted separate analyses for men and women, and for immigrants and native Swedes. RESULTS: Exposure to ≥60 days of sickness absence in 1993 increased the risk of sickness absence [hazard ratio (HR) 1.6-11.4], unemployment (HR 1.1-1.2), disability pension (HR 1.2-5.3) and death (HR 1.2-3.5). The income from work, during the follow-up period, among individuals with spells of sick leave for ≥60 days in 1993 was around two-thirds of that of the working population who did not take sick leave. CONCLUSIONS: Individuals on sickness absence had an increased risk for work absence, death and lower future income.

Unemployment at a young age and later sickness absence, disability pension and death in native Swedes and immigrants.

BACKGROUND: Youth unemployment is an increasing problem for societies around the world. Research has revealed negative health effects of unemployment, and this longitudinal register-based cohort study examined the relationship between unemployment and later sickness absence, disability pension and death among youth in Sweden. METHOD: The study group of 199,623 individuals comprised all immigrants born between 1968 and 1972 who immigrated before 1990 (25,607) and a random sample of native Swedes in the same age-range (174,016). The baseline year was 1992, and the follow-up period was from 1993 to 2007. Subjects with unemployment benefit in 1990-91, disability pension in 1990-92, severe disorders leading to hospitalization in 1990-92 and subjects who emigrated during follow-up were excluded. RESULTS: Those who were unemployed in 1992 had elevated risk of ≥60 days of sickness absence (OR 1.02-1.49), disability pension (HR 1.08-1.62) and all except native Swedish women had elevated risk of death (HR 1.01-1.65) during follow-up compared with non-unemployed individuals. The risk of future sickness absence increased with the length of unemployment in 1992 (OR 1.06-1.54), and the risk of sickness absence increased over time. A larger part of the immigrant cohort was unemployed at baseline than native Swedes. Selection to unemployment by less healthy subjects may explain part of the association between unemployment and the studied outcomes. CONCLUSION: Unemployment at an early age may influence the future health of the individual. To a society it may lead to increased burdens on the welfare system and productivity loss for many years.

Work and health among immigrants and native Swedes 1990-2008: a register-based study on hospitalization for common potentially work-related disorders, disability pension and mortality.

BACKGROUND: There are many immigrants in the Swedish workforce, but knowledge of their general and work-related health is limited. The aim of this register-based study was to explore whether documented migrant residents in Sweden have a different health status regarding receipt of a disability pension, mortality and hospitalization for lung, heart, psychiatric, and musculoskeletal disorders compared with the native population, and if there were variations in relation to sex, geographical origin, position on the labor market, and time since first immigration. METHODS: This study included migrants to Sweden since 1960 who were 28-47 years old in 1990, and included 243 860 individuals. The comparison group comprised a random sample of 859 653 native Swedes. These cohorts were followed from 1991 to 2008 in national registers. The immigrants were divided into four groups based on geographic origin. Hazard ratios for men and women from different geographic origins and with different employment status were analyzed separately for the six outcomes, with adjustment for age, education level, and income. The influence of length of residence in Sweden was analyzed separately. RESULTS: Nordic immigrants had increased risks for all investigated outcomes while most other groups had equal or lower risks for those outcomes than the Swedes. The lowest HRs were found in the EU 15+ group (from western Europe, North America, Australia and New Zealand). All groups, except Nordic immigrants, had lower risk of mortality, but all had higher risk of disability pension receipt compared with native Swedes. Unemployed non-Nordic men displayed equal or lower HRs for most outcomes, except disability pension receipt, compared with unemployed Swedish men. A longer time since first immigration improved the health status of men, while women showed opposite results. CONCLUSIONS: Employment status and length of residence are important factors for health. The contradictory results of low mortality and high disability pension risks need more attention. There is great potential to increase the knowledge in this field in Sweden, because of the high quality registers.

Sustainable Earnings among Immigrants, and the Role of Health Status for Self-Sufficiency: A 10-Year Follow-Up Study of Labour Immigrants and Refugees to Sweden 2000-2006.

This study aimed to investigate economic self-sufficiency for immigrants, and how health status affected self-sufficiency. The proportion of self-sufficiency during years 1-10 after receiving a residence permit is presented for all non-European labour immigrants (n = 1259) and refugees (n = 23,859), aged 18-54, who immigrated to Sweden 2000-2006, and compared to a control group of Swedish-born (n = 144,745). The risk of not being self-sufficient in year 10 was analysed with Cox regression models, and the results are presented as hazard ratios (HRs) with 95% confidence intervals (CIs). Moreover, the impact on the self-sufficiency of having a diagnosis from specialised health care during the first five years in Sweden was analysed. The results showed that half of the refugees and three-quarters of the labour immigrants were self-sufficient 10 years after residency. The adjusted risk of not being self-sufficient at year 10 was 80% higher among labour immigrants (HR = 1.8; CI = 1.6-2.0) and more than two-fold among refugees (HR = 2.7; CI = 2.6-2.8) compared to the Swedish-born. Having a diagnosis from specialised health care during the first five years in Sweden had an impact on self-sufficiency in all groups; however, the impact of having a diagnosis did not differ between refugees and Swedish-born. Measures must be taken to increase immigrants' work participation.

Human papillomavirus and Papanicolaou tests to screen for cervical cancer.

BACKGROUND: Screening for cervical cancer based on testing for human papillomavirus (HPV) increases the sensitivity of detection of high-grade (grade 2 or 3) cervical intraepithelial neoplasia, but whether this gain represents overdiagnosis or protection against future high-grade cervical epithelial neoplasia or cervical cancer is unknown. METHODS: In a population-based screening program in Sweden, 12,527 women 32 to 38 years of age were randomly assigned at a 1:1 ratio to have an HPV test plus a Papanicolaou (Pap) test (intervention group) or a Pap test alone (control group). Women with a positive HPV test and a normal Pap test result were offered a second HPV test at least 1 year later, and those who were found to be persistently infected with the same high-risk type of HPV were then offered colposcopy with cervical biopsy. A similar number of double-blinded Pap smears and colposcopies with biopsy were performed in randomly selected women in the control group. Comprehensive registry data were used to follow the women for a mean of 4.1 years. The relative rates of grade 2 or 3 cervical intraepithelial neoplasia or cancer detected at enrollment and at subsequent screening examinations were calculated. RESULTS: At enrollment, the proportion of women in the intervention group who were found to have lesions of grade 2 or 3 cervical intraepithelial neoplasia or cancer was 51% greater (95% confidence interval [CI], 13 to 102) than the proportion of women in the control group who were found to have such lesions. At subsequent screening examinations, the proportion of women in the intervention group who were found to have grade 2 or 3 lesions or cancer was 42% less (95% CI, 4 to 64) and the proportion with grade 3 lesions or cancer was 47% less (95% CI, 2 to 71) than the proportions of control women who were found to have such lesions. Women with persistent HPV infection remained at high risk for grade 2 or 3 lesions or cancer after referral for colposcopy. CONCLUSIONS: The addition of an HPV test to the Pap test to screen women in their mid-30s for cervical cancer reduces the incidence of grade 2 or 3 cervical intraepithelial neoplasia or cancer detected by subsequent screening examinations. (ClinicalTrials.gov number, NCT00479375 [ClinicalTrials.gov].).

Detection of genomic amplification of the human telomerase gene TERC, a potential marker for triage of women with HPV-positive, abnormal Pap smears.

The vast majority of invasive cervical carcinomas harbor additional copies of the chromosome arm 3q, resulting in genomic amplification of the human telomerase gene TERC. Here, we evaluated TERC amplification in routinely collected liquid based cytology (LBC) samples with histologically confirmed diagnoses. A set of 78 LBC samples from a Swedish patient cohort were analyzed with a four-color fluorescence in situ hybridization probe panel that included TERC. Clinical follow-up included additional histological evaluation and Pap smears. Human papillomavirus status was available for all cases. The correlation of cytology, TERC amplification, human papillomavirus typing, and histological diagnosis showed that infection with high-risk human papillomavirus was detected in 64% of the LBC samples with normal histopathology, in 65% of the cervical intraepithelial neoplasia (CIN)1, 95% of the CIN2, 96% of the CIN3 lesions, and all carcinomas. Seven percent of the lesions with normal histopathology were positive for TERC amplification, 24% of the CIN1, 64% of the CIN2, 91% of the CIN3 lesions, and 100% of invasive carcinomas. This demonstrates that detection of genomic amplification of TERC in LBC samples can identify patients with histopathologically confirmed CIN3 or cancer. Indeed, the proportion of TERC-positive cases increases with the severity of dysplasia. Among the markers tested, detection of TERC amplification in cytological samples has the highest combined sensitivity and specificity for discernment of low-grade from high-grade dysplasia and cancer.

Work environment and production development in Swedish manufacturing industry.

Swedish manufacturing industry has previous held a leading position regarding the development of attractive industrial work environments, but increasing market competition has changed the possibilities to maintain the position. The purpose of this literature study is therefore to describe and analyze how Swedish manufacturing industry manages work environment and production development in the new millennium. The description and analysis is based on recently reported Swedish research and development. The gathered picture of how production systems generally are developed in Sweden strongly contrasts against the idealized theoretical and legal view of how production systems should be developed. Even if some of the researchers' and authorities' ambitions and demands may seem unrealistically high today, there still is a very large potential for improving the processes and tools for designing production systems and work environment.

Follow-up after treatment of cervical intraepithelial neoplasia by human papillomavirus genotyping.

OBJECTIVE: To assess the use of human papillomavirus genotyping in cervical intraepithelial neoplasia posttreatment follow-up. STUDY DESIGN: Prospective observational study. Ninety women underwent cytologic testing and human papillomavirus genotyping at the follow-up visit after conization. Cones were retrospectively genotyped. A second cytologic follow-up was performed. RESULTS: Margin status and presence of cervical intraepithelial neoplasia 3+ in the cone were poor predictors of treatment outcome (sensitivity, < 50%; diagnostic odds ratio,

Healthy migrant effect in the Swedish context: a register-based, longitudinal cohort study.

OBJECTIVES: Studies have found a 'healthy-migrant effect' (HME) among arriving migrants, that is, a better health status compared with others in the home country, but also in comparison with the population in the host country. The aims were to investigate whether the HME hypothesis is applicable to the Swedish context, that is, if health outcomes differed between a group of mainly labour migrants (Western migrants) and a group of mainly refugee/family reunion migrants (non-Western migrants) compared with the native Swedish population, and if there were any correlations between labour market attachment (LMA) and these health outcomes. DESIGN: Register-based, longitudinal cohort study. PARTICIPANTS: The cohort was defined on 31 December 1990 and consisted of all migrants aged 18-47 years who arrived in Sweden in 1985-1990 (n=74 954) and a reference population of native Swedes (n=1 405 047) in the same age span. They were followed for three consecutive 6-year periods (1991-1996, 1997-2002 and 2003-2008) and were assessed for five measures of health: hospitalisation for cardiovascular and psychiatric disorders, mortality, disability pension, and sick leave. RESULTS: Western migrants had, compared with native Swedes, lower or equal HRs for all health measures during all time periods, while non-Western migrants displayed higher or equal HRs for all health measures, except for mortality, during all time periods. Age, educational level, occupation and LMA explained part of the difference between migrants and native Swedes. High LMA was associated with higher HRs for cardiovascular disorders among Western migrants, higher HRs of psychiatric disorders among non-Western migrants and higher HRs of mortality among both migrant groups compared with native Swedes. CONCLUSIONS: There were indications of a HME among Western migrants, while less proof of a HME among non-Western migrants. Stratification for LMA and different migrant categories showed some interesting differences, and measurements of the HME may be inconclusive if not stratified by migrant category or other relevant variables.

Expression of p16INK4a and MIB-1 in relation to histopathology and HPV types in cervical adenocarcinoma.

A total of 101 primary cervical adenocarcinomas were analyzed for the presence of p16INK4a and MIB-1 expression in correlation with the presence of 'high-risk' types of human papillomavirus (HR-HPV) and clinical outcome. We found that adenocarcinoma grading showed a significant negative correlation to p16INK4a levels (p=0.001): i.e. we found less intense p16 staining in poorly differentiated tumors than in more highly differentiated tumors as well as a highly significant correlation between HPV infection and higher levels of p16INK4a staining (p=0.00), which was similar for different HPV-types. Tumor suppressor protein p16INK4a levels were higher in HPV positive than in HPV negative tumors. Higher levels of the proliferation marker MIB-1 were associated with poorer outcome. Higher MIB-1 levels were seen in tumors with a lower grade and higher stage at diagnosis. Moreover, MIB-1 levels seem to be higher in tumors due to infection with HPV 16 and 18 compared with HPV 45. MIB-1 may be a helpful marker in grading adenocarcinoma: a high level of expression of MIB-1 indicates a low grade of tumor, whereas high expression of p16INK4a indicates a highly differentiated of the tumor. Thus, immunostaining for p16INK4a appears to be a useful diagnostic tool for cervical adenocarcinoma.

Evaluation of a web-based ECG-interpretation programme for undergraduate medical students.

BACKGROUND: Most clinicians and teachers agree that knowledge about ECG is of importance in the medical curriculum. Students at Karolinska Institute have asked for more training in ECG-interpretation during their undergraduate studies. Clinical tutors, however, have difficulties in meeting these demands due to shortage of time. Thus, alternative ways to learn and practice ECG-interpretation are needed. Education offered via the Internet is readily available, geographically independent and flexible. Furthermore, the quality of education may increase and become more effective through a superior educational approach, improved visualization and interactivity. METHODS: A Web-based comprehensive ECG-interpretation programme has been evaluated. Medical students from the sixth semester were given an optional opportunity to access the programme from the start of their course. Usage logs and an initial evaluation survey were obtained from each student. A diagnostic test was performed in order to assess the effect on skills in ECG interpretation. Students from the corresponding course, at another teaching hospital and without access to the ECG-programme but with conventional teaching of ECG served as a control group. RESULTS: 20 of the 32 students in the intervention group had tested the programme after 2 months. On a five-graded scale (1- bad to 5 - very good) they ranked the utility of a web-based programme for this purpose as 4.1 and the quality of the programme software as 3.9. At the diagnostic test (maximal points 16) by the end of the 5-month course at the 6th semester the mean result for the students in the intervention group was 9.7 compared with 8.1 for the control group (p = 0.03). CONCLUSION: Students ranked the Web-based ECG-interpretation programme as a useful instrument to learn ECG. Furthermore, Internet-delivered education may be more effective than traditional teaching methods due to greater immediacy, improved visualisation and interactivity.

C-Peptide replacement therapy and sensory nerve function in type 1 diabetic neuropathy.

OBJECTIVE: C-peptide replacement in animals results in amelioration of diabetes-induced functional and structural abnormalities in peripheral nerves. The present study was undertaken to examine whether C-peptide administration to patients with type 1 diabetes and peripheral neuropathy improves sensory nerve function. RESEARCH DESIGN AND METHODS: This was an exploratory, double-blinded, randomized, and placebo-controlled study with three study groups that was carried out at five centers in Sweden. C-peptide was given as a replacement dose (1.5 mg/day, divided into four subcutaneous doses) or a dose three times higher (4.5 mg/day) during 6 months. Neurological examination and neurophysiological measurements were performed before and after 6 months of treatment with C-peptide or placebo. RESULTS: The age of the 139 patients who completed the protocol was 44.2 +/- 0.6 (mean +/- SE) years and their duration of diabetes was 30.6 +/- 0.8 years. Clinical neurological impairment (NIA) (score >7 points) of the lower extremities was present in 86% of the patients at baseline. Sensory nerve conduction velocity (SCV) was 2.6 +/- 0.08 SD below body height-corrected normal values at baseline and improved similarly within the two C-peptide groups (P < 0.007). The number of patients responding with a SCV peak potential improvement >1.0 m/s was greater in C-peptide-treated patients than in those receiving placebo (P < 0.03). In the least severely affected patients (SCV < 2.5 SD below normal at baseline, n = 70) SCV improved by 1.0 m/s (P < 0.014 vs. placebo). NIA score and vibration perception both improved within the C-peptide-treated groups (P < 0.011 and P < 0.002). A1C levels (7.6 +/- 0.1% at baseline) decreased slightly but similarly in C-peptide-and placebo-treated patients during the study. CONCLUSIONS: C-peptide treatment for 6 months improves sensory nerve function in early-stage type 1 diabetic neuropathy.

Type-specific persistence of high-risk human papillomavirus infections in the New Independent States of the former Soviet Union Cohort Study.

BACKGROUND: Prospective follow-up studies have recently suggested that persistent high-risk human papillomavirus (HPV) infections play a key role in the progression of CIN lesions and in the development of cervical cancer. However, data on type-specific persistence, viral integration, and the role of multiple infections are scanty. MATERIALS AND METHODS: A cross-sectional/cohort study was conducted between 1998 and 2002 in three New Independent States of the former Soviet Union comprising a cohort of 3,187 women, of whom 854 women were followed up for a mean of 17 months (SD, 11.6). HPV genotyping was done with real-time PCR, detecting HPV types 16, 18/45, 31, 33/52/58, 35, and 39. The integration status of HPV16 was examined by using a novel Taqman-based PCR method. RESULTS: The mean clearance time for the individual high- risk-type infection was 16.5 months (range = 0.9-34.9 months). HPV16 and HPV31 were the most persistent infections (clearance times = 18.1 and 16.2 months, respectively), whereas HPV39 infections cleared most rapidly. The mean copies per cell in HPV18/45, HPV31, HPV33/52/58, and HPV39 infections were higher in persisting HPV infections than in HPV infections that cleared, but the difference was not significant. Integration of HPV16 was not found to correlate with HPV persistence. CONCLUSIONS: A large proportion of women remained high-risk HPV positive after 18 months. Coinfection with multiple HPV types, viral load, or integration status did not correlate with persistence of high-risk HPV infections.

Antisense RNA directed to the human papillomavirus type 16 E7 mRNA from herpes simplex virus type 1 derived vectors is expressed in CaSki cells and downregulates E7 mRNA.

BACKGROUND: Human papillomavirus (HPV) infection is known to be the most important etiologic factor of cervical cancer. There is no HPV specific therapy available for treatment of invasive squamous cell carcinoma of the cervix and its precursor lesions. The present study elucidates the potential to use herpes simplex virus (HSV) derived vectors for expression of antisense RNA to HPV -16 E7 oncogene. RESULTS: We have constructed replication competent, nonneuroinvasive HSV-1 vectors, deleted of the gamma134.5 gene. The vectors express RNA antisense to the first 100 nucleotides of the HPV-16 E7 gene. We assayed the ability of the antisense E7 vectors R5225 (tk-) and R5226 (tk+), to produce antisense RNA, as well as the consequent effects on E7 mRNA and protein levels in HPV-16 positive CaSki cells. Anti-E7 RNA was expressed by both constructs in a dose-dependent manner. Expression of HPV-16 E7 mRNA was downregulated effectively in CaSki cells infected with the tk- recombinant R5225 or with R5226. The tk+ recombinant R5226 was effective in downregulating E7 protein expression. CONCLUSION: We have shown that anti-E7 RNA expressed from an HSV vector could efficiently downregulate HPV-16 E7 mRNA and E7 protein expression in CaSki cells. We conclude that HSV vectors may become a useful tool for gene therapy of HPV infections.

Evaluation of multi-modal high salt binding ion exchange materials.

The performance and selectivity of novel cation and anion exchange multi-modal chromatographic materials were evaluated. Desorption profiles of 13 proteins possessing a range of properties (e.g. size, charge and hydrophobicity) were determined on the cation exchange materials. Batch experiments were carried out by loading individual proteins on each resin at low salt, and examining the desorption of the proteins during sequential washes with increasing salt concentrations. While all of the resins exhibited some binding of proteins at elevated salt concentrations, this effect was more pronounced on the resins with aromatic ligands as compared to the materials with aliphatic ligands. As expected, materials with higher ionic capacities exhibited higher binding at elevated salts. In addition, some proteins exhibited high binding at elevated salt concentrations to all of the resins. The combined effect of charge and other secondary interactions with these multi-modal chromatographic materials enables high salt binding of a range of proteins as well as unique selectivities for the recovery of certain classes of proteins. Since the anion exchange materials all exhibited high binding at elevated salt concentrations the work with these materials focused on a study of elution strategies to remove proteins from these aromatic based materials. After evaluating various elution protocols, a combined strategy of pH change and chaotropic salt were shown to minimize electrostatic and hydrophobic interactions and was found to be an effective elution strategy for this class of anion exchange materials using peanut lectin as a model protein.

Expression of p16(INK4a) in relation to histopathology and viral load of 'high-risk' HPV types in cervical neoplastic lesions.

A total of 91 cervical archival biopsy series were analysed for the presence and viral load of 'high-risk' types of human papillomavirus (HR-HPV), and p16(INK4a) expression. The women had various degrees of CIN (cervical intraepithelial neoplasia). HPV 16 was the most prevalent type found, at 47% frequency. The frequency of HPV 16 increased with increasing immunoreactivity to p16(INK4a), from 39% to 44% at cases scored low to medium, to 65% at high reactivity. Thirty (33%) of the samples had negative p16(INK4a) analysis results, but were positive for HR-HPV. There was no significant correlation between viral load and the level of p16(INK4a) expression, while the grade of CIN correlated to such expressions. Thus, p16(INK4a) expression analysis yielded information which is consistent with results from the histopathology and might complement the HPV analysis in a clinical prognostic procedure in order to find women at risk for cervical cancer.

Long-term suppression of telomerase expression in HeLa cell clones, transfected with an expression vector carrying siRNA targeting hTERT mRNA.

HeLa cell cultures were used as model systems for small interfering RNA (siRNA) induced knockdown of mRNA expression of the human telomerase catalytic subunit, telomerase reverse transcriptase (hTERT). Four 21-bp siRNAs targeting different sites of the hTERT mRNA were designed, and the siRNA molecules produced by a T7 transcription system in vitro. In transient transfection assays on HeLa cells, only one of the tested siRNAs produced a potent knockdown effect on hTERT mRNA expression, associated with the suppression of telomerase activity (both reduced by approximately 50%). An expression vector encoding a hairpin siRNA against the effective hTERT mRNA target site was generated, and HeLa clones stably expressing hTERT-specific siRNA were created. Two clones showed extremely reduced hTERT mRNA expression, associated with unusually short telomeres, the inhibition of cell growth and the induction of senescence and apoptosis. Thus, there was obvious loss of viability in cells lacking hTERT expression and carrying short telomeres. This was most prominent in the clone that showed prolonged reductions (over two months) in both hTERT expression and telomerase activity. Thus, our data clearly show that long-term suppression of telomerase expression by siRNA is an attainable goal, at least in a HeLa cell model system.

Expression of E6/E7 mRNA from 'high risk' human papillomavirus in relation to CIN grade, viral load and p16INK4a.

Detection of E6/E7 mRNA expression with real-time nucleic acid sequence-based amplification assay (NASBA) method (PreTect HPV-Proofer) from high-risk types of human papillomaviruses (HR-HPV) were compared with the presence of viral load, determined with quantitative real-time PCR in 80 cervical samples. Results regarding positivity and typing were in agreement using the two methods. However, there was no correlation between viral loads for HPV 16 or 18/45 and oncogene expression. Among 15 women with low grade atypia detected at a population-based cytology screening, and scored as 'within normal limits' according to histopathology, 14% were positive for oncogene expression, whereas 71% were HR-HPV positive. A correlation was observed between HR-HPV oncogene expression and high scores of p16(INK4a) positivity. Since HPV-Proofer detects full-length E6/E7 mRNA, a positive result should correlate with presence of integrated HPV, loss of HPV replication and stabilized E6/E7 full-length mRNA expression. Such expression from integrated HR-HPV generates a high and stable expression of full-length E6 proteins, which explains why a positive HPV-Proofer result was independent of viral load and correlate with high expression of p16(INK4a). Thus, E6/E7 oncogene expression analysis yielded information, which is consistent with and will complement the results from a real-time PCR method in a clinical prognostic procedure.