Cytotoxic immune responses in the lungs correlate to disease severity in patients with hantavirus infection.

Hantavirus infections may cause severe and sometime life-threatening lung failure. The pathogenesis is not fully known and there is an urgent need for effective treatment. We aimed to investigate the association between pulmonary viral load and immune responses, and their relation to disease severity. Bronchoscopy with sampling of bronchoalveolar lavage (BAL) fluid was performed in 17 patients with acute Puumala hantavirus infection and 16 healthy volunteers acting as controls. Lymphocyte subsets, granzyme concentrations, and viral load were determined by flow cytometry, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Analyses of BAL fluid revealed significantly higher numbers of activated CD8(+) T cells and natural killer (NK) cells, as well as higher concentrations of the cytotoxins granzymes A and B in hantavirus-infected patients, compared to controls. In patients, Puumala hantavirus RNA was detected in 88 % of BAL cell samples and correlated inversely to the T cell response. The magnitude of the pulmonary cytotoxic lymphocyte response correlated to the severity of disease and systemic organ dysfunction, in terms of need for supplemental oxygen treatment, hypotension, and laboratory data indicating renal failure, cardiac dysfunction, vascular leakage, and cell damage. Regulatory T cell numbers were significantly lower in patients compared to controls, and may reflect inadequate immune regulation during hantavirus infection. Hantavirus infection elicits a pronounced cytotoxic lymphocyte response in the lungs. The magnitude of the immune response was associated with disease severity. These results give insights into the pathogenesis and possibilities for new treatments.

Seroprevalence of Sindbis virus and associated risk factors in northern Sweden.

Mosquito-borne Sindbis virus (SINV) cause disease characterized by rash, fever and arthritis which often leads to long-lasting arthralgia. To determine the seroprevalence of SINV and associated risk factors in northern Sweden, a randomly selected population aged between 25 and 74 years were invited to join the MONICA study. Serum from 1611 samples were analysed for specific IgG antibodies. Overall, 2·9% had IgG against SINV. More men (3·7%) than women (2·0%) were SINV seropositive (P = 0·047) and it was more common in subjects with a lower educational level (P = 0·013) and living in small, rural communities (P < 0·001). Seropositivity was associated with higher waist circumference (P = 0·1), elevated diastolic blood pressure (P = 0·037), and history of a previous stroke (P = 0·011). In a multiple logistic regression analysis, adjusting for known risk factors for stroke, seropositivity for SINV was an independent predictor of having had a stroke (odds ratio 4·3, 95% confidence interval 1·4-13·0, P = 0·011).

Viral load and humoral immune response in association with disease severity in Puumala hantavirus-infected patients--implications for treatment.

Hantaviruses are the causative agents of haemorrhagic fever with renal syndrome (HFRS) in Eurasia and of hantavirus cardiopulmonary syndrome (HCPS) in the Americas. The case fatality rate varies between different hantaviruses and can be up to 40%. At present, there is no specific treatment available. The hantavirus pathogenesis is not well understood, but most likely, both virus-mediated and host-mediated mechanisms are involved. The aim of the present study was to investigate the association among Puumala hantavirus (PUUV) viral RNA load, humoral immune response and disease severity in patients with HFRS. We performed a study of 105 PUUV-infected patients that were followed during the acute phase of disease and for up to 1-3 months later. Fifteen of the 105 patients (14%) were classified as having moderate/severe disease. A low PUUV-specific IgG response (p <0.05) and also a higher white blood cell count (p <0.001) were significantly associated with more severe disease. The PUUV RNA was detected in a majority of patient plasma samples up to 9 days after disease onset; however, PUUV RNA load or longevity of viraemia were not significantly associated with disease severity. We conclude that a low specific IgG response was associated with disease severity in patients with HFRS, whereas PUUV RNA load did not seem to affect the severity of HFRS. Our results raise the possibility of passive immunotherapy as a useful treatment for hantavirus-infected patients.

Acoustofluidics 5: Building microfluidic acoustic resonators.

Acoustophoresis is getting more attention as an effective and gentle non-contact method of manipulating cells and particles in microfluidic systems. A key to a successful assembly of an acoustophoresis system is a proper design of the acoustic resonator where aspects of fabrication techniques, material choice, thickness matching of involved components, as well as strategies of actuation, all have to be considered. This tutorial covers some of the basics in designing and building microfluidic acoustic resonators and will hopefully be a comprehensive and advisory document to assist the interested reader in creating a successful acoustophoretic device.

Time to revise the paradigm of hantavirus syndromes? Hantavirus pulmonary syndrome caused by European hantavirus.

Hantaviruses have previously been recognised to cause two separate syndromes: hemorrhagic fever with renal syndrome in Eurasia, and hantavirus pulmonary syndrome (HPS) in the Americas. However, increasing evidence suggests that this dichotomy is no longer fruitful when recognising human hantavirus disease and understanding the pathogenesis. Herein are presented three cases of severe European Puumala hantavirus infection that meet the HPS case definition. The clinical and pathological findings were similar to those found in American hantavirus patients. Consequently, hantavirus infection should be considered as a cause of acute respiratory distress in all endemic areas worldwide.

Review of cell and particle trapping in microfluidic systems.

The ability to obtain ideal conditions for well-defined chemical microenvironments and controlled temporal chemical and/or thermal variations holds promise of high-resolution cell response studies, cell-cell interactions or e.g. proliferation conditions for stem cells. It is a major motivation for the rapid increase of lab-on-a-chip based cell biology research. In view of this, new chip-integrated technologies are at an increasing rate being presented to the research community as potential tools to offer spatial control and manipulation of cells in microfluidic systems. This is becoming a key area of interest in the emerging lab-on-a-chip based cell biology research field. This review focuses on the different technical approaches presented to enable trapping of particles and cells in microfluidic system.

Factors associated with development of stroke long-term after myocardial infarction: experiences from the LoWASA trial.

OBJECTIVE: To describe factors associated with the development of stroke during long-term follow-up after acute myocardial infarction (AMI) in the LoWASA trial. PATIENTS: Patients who had been hospitalized for AMI were randomized within 42 days to receive either warfarin 1.25 mg plus aspirin 75 mg daily or aspirin 75 mg alone. DESIGN: The study was performed according to the probe design, that is open treatment and blinded end-point evaluation. SETTING: The study was performed in 31 hospitals in Sweden. The mean follow-up time was 5.0 years with a range of 1.7-6.7 years. RESULTS: In all, 3300 patients were randomized in the trial, of which 194 (5.9%) developed stroke (4.2% nonhaemorrhagic, 0.5% haemorrhagic and 1.3% uncertain. The following factors appeared as independent predictors for an increased risk of stroke: age, hazard ratio and 95% confidence interval (1.07; 1.05-1.08), a history of diabetes mellitus (2.4; 1.8-3.4), a history of stroke (2.3; 1.5-3.5), a history of hypertension (2.0; 1.5-2.7) and a history of smoking (1.5;1.1-2.0). Most of these factors were also predictors of a nonhaemorrhagic stroke whereas no predictor of haemorrhagic stroke was found. CONCLUSION: Risk indicators for stroke long-term after AMI were increasing age, a history of either diabetes mellitus, stroke, hypertension or smoking.

A novel and rapid PCR-based method for genotyping human papillomaviruses in clinical samples.

Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5(+)-GP6(+) consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil N-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a "T-sequencing ladder" was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.

Expression of the alpha6 integrin confers papillomavirus binding upon receptor-negative B-cells.

Papillomaviruses (PV) bind to a wide range of cell lines in a specific and saturable manner. We have recently identified a candidate receptor for papillomavirus as the alpha6 integrin (Evander et al., J. Virol. 71, 2449-2456, 1997). We have further investigated the role the alpha6 integrin plays in PV binding. Here we show that the cells expressing the alpha6 integrin, partnered with either the beta4 integrin or the beta1 integrin, are equally able to bind PV HPV6b L1 virus-like particles, indicating that the beta partner does not play a major role in virus binding. In order to provide definitive evidence that the alpha6 integrin is required for PV binding we undertook to genetically complement the receptor-negative B-cell line DG75 by expressing the human alpha6A gene. The transduction of the alpha6 integrin gene into DG75 cells results in the cell surface expression of the alpha6 protein and this expression confers upon DG75 cells the ability to bind laminin, a normal ligand for alpha6 integrin. Furthermore, the alpha6 protein is partnered with the beta1 integrin in DG75 cells. Finally, we show that the DG75-alpha6 cells were able to bind papillomavirus VLPs and this binding was inhibited by a functionally blocking anti-alpha6 antibody. Together these data indicate that the alpha6 integrin is a primary cell receptor for papillomaviruses and is both necessary and sufficient for PV binding.

Acetowhitening of the cervix and vulva as a predictor of subclinical human papillomavirus infection: sensitivity and specificity in a population-based study.

OBJECTIVE: To evaluate acetowhite changes of the cervix and vulva as a predictor of human papillomavirus (HPV) infection. METHODS: In this population-based study all women aged 19, 21, 23, and 25 years and registered as living in a primary health care area within the city of Umeå, Sweden were eligible for inclusion. Each participant underwent a gynecologic examination with sampling of epithelial cells for HPV-DNA detection and Papanicolaou smear. Colposcopy was performed 5 minutes after application of 5% acetic acid. A two-step polymerase chain reaction (PCR) technique was employed for HPV-DNA detection. RESULTS: Colposcopy and sampling of epithelial cells could be performed in 535 women. The sensitivity of detection of HPV infection by the acetowhitening of the cervix was 22% (95% confidence interval [CI] 18%, 26%). The specificity of detection of HPV infection by the acetowhitening of the cervix was 90% (95% CI 87%, 93%). The sensitivity of detection of HPV infection by cytology was 13% (95% CI 10%, 16%), and the specificity was 99% (95% CI 98%, 100%). The combination of acetowhitening and cytology did not improve the diagnostic value. CONCLUSION: Acetowhitening of the cervix and vulva has low sensitivity as a predictor of HPV infections as determined by PCR.

Presentation of the HPV16E7 protein by skin grafts is insufficient to allow graft rejection in an E7-primed animal.

The E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of FVB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the alpha A crystallin promoter (FVB alpha AcryHPV16E6E7). We have transferred skin from FVB alpha AcryHPV16E6E7 mice to naive or E7-primed syngeneic FVB recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB x C57BL/6J mice recipients of FVB alpha-AcryHPV16E6E7 x C57BL/6J skin generated humoral and DTH responses to E7 in vivo and E7-specific CTL precursors in the spleen, but failed to reject E7 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin.

Identification of the alpha6 integrin as a candidate receptor for papillomaviruses.

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction of human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [35S]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha6beta4 integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha6 or beta4 integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha6 integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta4 integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha6beta4 integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha6 integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha6 integrin subunit and that integrin complexes containing alpha6 integrin complexed with either beta1 or beta4 integrins may act as a receptor for PV binding and entry into epithelial cells.

Nonradioisotopic detection and typing of human papillomaviruses by use of polymerase chain reaction and single-strand conformation polymorphism.

The polymerase chain reaction (PCR), used to detect human papillomavirus (HPV), is finding increasing applications in clinical laboratories. The standard method of analysis to detect amplified PCR products is ethidium bromide gel electrophoresis combined with labor intensive blot hybridization. In this study, we describe single-strand conformation polymorphism (SSCP) to detect and genotype simultaneously general primer GP5+/GP6+ amplified HPV DNA using semiautomated electrophoresis on polyacrylamide gels (PAGE) combined with sensitive silver staining. To establish a standard for the band patterns of the various HPV types, we used HPV plasmid DNA, which allowed us to distinguish HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58, covering the most frequently recognized types. All the types tested are separated from each other, demonstrating diverse band patterns, HPV 16 being the most distinct. We also investigated PCR-SSCP for HPV detection and typing of 86 cervical biopsies diagnosed as cervical intraepithelial neoplasia (CIN) I-III and known to be HPV positive by PCR-slot blot hybridization and in situ hybridization. The correlation with SSCP was 91% for in situ hybridization and 98% for PCR-slot blot hybridization. SSCP is reproducible and specific. Its sensitivity is comparable to slot-blot hybridization. The interval to SSCP is approximately 2 h after PCR compared with several days' work when using conventional blot hybridization. We concluded that SSCP may be more advantageous than other PCR-based typing technologies.

Epithelial cells display separate receptors for papillomavirus VLPs and for soluble L1 capsid protein.

We examined the distribution of putative receptors for papillomavirus (PV) capsid proteins on various cell types, using either Hexahis HPV6b L1 fusion protein or synthetic HPV6b virus-like particles (VLPs). Specific, saturable binding of VLPs to CV-1 cells was demonstrated using 35S-labeled VLPs, with an average receptor number of 1 x 10(4)/cell and a binding affinity constant (Ka) of 4 x 10(7) M. VLP binding was quantitated by flow cytometry using a monoclonal antibody to the L1 capsid protein. Intense staining of epithelial and mesenchymal cells was observed. Some immature bone marrow-derived cells bound VLPs weakly, while the majority of B lymphoma cells demonstrated no binding. Binding to 12 of 16 VLP receptor positive cell lines was abolished by trypsin pretreatment of cells. Removal of cellular sialic acid or O-linked oligosaccharides separately did not affect VLP binding, which was enhanced about 25% when cells were pretreated with both neuraminidase and O-glycosidase. Culture of cells with sufficient tunicamycin to inhibit Concanavalin A binding did not diminish the binding of VLPs. Denatured L1 protein, either from VLPs or expressed from Escherichia coli as a Hexahis fusion protein, bound to a trypsin-resistant structure on a range of cell types and did not block the binding of VLPs to cells. Dual-fluorescence assay with a Burkitt lymphoma line BL72 demonstrated that Hexahis L1 protein and VLPs bind to separate cell surface molecules on BL72 cells. We conclude that the first binding of PV virus to cells is via a widely distributed membrane protein receptor(s) and that subsequent processing of particles may involve other non-trypsin-sensitive structure(s) also displayed on the cell membrane.

The influence of sexual and social factors on the risk of Chlamydia trachomatis infections: a population-based serologic study.

BACKGROUND: Genital chlamydia infections often are asymptomatic, which promotes their spread in the population. In women, the possible consequences of infection are pelvic inflammatory disease and infertility. Most studies on the prevalence of Chlamydia trachomatis have been based on clinical series, and prevalences tend to vary with the clinical setting. Few seroepidemiologic studies have emerged from industrialized countries. GOAL OF THIS STUDY: To assess the prevalence of Chlamydia trachomatis using culture and serology, and its relationship with possible risk factors. STUDY DESIGN: This was a population-based study involving completion of a self-administered questionnaire, analysis of cervical samples for Chlamydia trachomatis, and serologic tests for Chlamydia trachomatis antibodies. RESULTS: The prevalence of Chlamydia trachomatis infection was 2.7%, and the seroprevalence was 24.7% among the sexually active women. Seropositivity was correlated with sexual behavior variables, and the incidence of serologic cross-reactivity with respiratory infections (strain TWAR) was low. Multivariate logistic regression analysis showed the number of sexual partners, age at first coitus, history of therapeutic abortion, and previous pelvic inflammatory disease to be independently correlated with seropositivity. CONCLUSION: Early sexual experience and multiple lifetime sexual partners are independent risk factors for Chlamydia trachomatis infection.

Can a test for E6/E7 transcripts of human papillomavirus type 16 serve as a diagnostic tool for the detection of micrometastasis in cervical cancer?

Tissue from 11 cases of cervical cancer positive for human papillomavirus (HPV) type 16 DNA and 69 pelvic lymph nodes from the same patients were examined for HPV 16 DNA and mRNA from the E6/E7 genes. Five of the tumors were squamous, 3 adeno- and 3 adenosquamous carcinoma. From the primary tumors and the extirpated lymph nodes DNA and RNA or mRNA was subjected to PCR and RT-PCR. Three transcription profiles (only E6*I, E6*I and E6*II or full-length E6-E7 plus both of the spliced transcripts) were found in all of the 11 HPV 16 DNA-positive primary tumors. From the total of 69 lymph nodes analyzed 28 were positive for mRNA. HPV 16 DNA was found in 7 additional samples. Cytokeratin was found in 19 of these lymph nodes, indicating epithelial origin of tumor cells. Only 1 patient had 2 metastases evidenced by histology. These were both positive for HPV DNA and mRNA. The finding of HPV DNA, mRNA and cytokeratin in lymph nodes of patients with cervical cancer should be an indication of lymphogenically driven micrometastases of the tumor. The HPV mRNA assay should offer higher specificity than the DNA test since mRNA can be found in live cells only, while HPV DNA also can originate from dead cell material sequestered in the lymph nodes.

The silent suffering women--a population based study on the association between reported symptoms and past and present infections of the lower genital tract.

OBJECTIVES: To assess the prevalence of lower genital tract symptoms and the association between reported symptoms and past and present signs of sexually transmitted diseases (STD) in young women. DESIGN: All women belonging to the 19-, 21-, 23- and 25-year age cohorts and living in the catchment area of the community health centre, were invited by mail to take part in a population-based study. The participants answered a structured questionnaire and a gynaecologic examination was performed. Samples for wet smear, cervical Pap smear, HPV DNA determination and Chlamydia trachomatis culture were taken at the gynaecologic examination. The presence of genital warts was noted. A blood sample was analysed for antibodies against C trachomatis and HSV-2. SETTING: The community health care centre was located in Umeå, a city in Northern Sweden. RESULTS: Of the 886 women who were eligible, 611 (70%) participated in the investigation. One out of four women reported symptoms from the lower genital tract. The most commonly reported symptoms were itching, followed by discharge, and soreness. The most commonly reported STD was C trachomatis (15%). The most prevalent present STD was HPV infection (20%) whereas C trachomatis infection could be isolated from 2.7% of the women. Antibodies against C trachomatis and HSV-2 were present among 22% and 6% of the women, respectively. There was a significant correlation between the women's complaint of vaginal discharge and previous C trachomatis infection, lack of lactobacilli and presence of leucocytosis in wet smear. CONCLUSIONS: We have in a population-based study of young healthy women found that one out of four women had some kind of lower genital tract complaint. Itching was the most commonly reported symptom and was associated with pseudohyphae and acetowhite patches. Reported vaginal discharge and soreness were associated with the history of a past C trachomatis infection and signs of a disturbed vaginal flora.

Human papillomavirus infection is transient in young women: a population-based cohort study.

The prevalence of human papillomavirus (HPV) infection in cervical cell scrapes from a cohort of 276 young women was determined by a general two-step polymerase chain reaction. HPV infection fluctuated among young women during a 2-year interval. The total prevalence of HPV infection decreased from 21% to 8.3%. The most prevalent HPV types at enrollment were HPV-16 (3.3%) and HPV-6 (2.9%). At follow-up, the most common type was HPV-16 (2.9%), while no HPV-6 was detected. In 2 women only, the same HPV type persisted. Regression of HPV infection was found in 80% of the women. A new HPV type-specific infection was detected in 7.2% of the women and was independently associated with a new sex partner or an abnormal smear since enrollment.

Lifetime number of partners as the only independent risk factor for human papillomavirus infection: a population-based study.

BACKGROUND AND OBJECTIVES: Previous studies of relationships between genital human papillomavirus infection and tentative risk factors have yielded conflicting results, possibly because of inaccuracy of the viral detection methods used and differences in selection criteria. GOAL OF THIS STUDY: To determine human papillomavirus prevalence and identify risk factors in a group of young Swedish women. STUDY DESIGN: This was a population-based study involving completion of a structured questionnaire, analysis of cervical scrapings for human papillomavirus and Chlamydia trachomatis, and serologic tests for C. trachomatis and herpes simplex virus antibodies. RESULTS: The prevalence of human papillomavirus infection was 22% among the sexually active women and 4% among the virgins. A number of factors were associated with human papillomavirus prevalence in univariate analysis, but logistic regression analysis showed that lifetime number of male sexual partners was the only independent risk factor for human papillomavirus infection (adjusted odds ratio, 7.45; 95% CI, 2.79-19.92 for six or more partners vs. one partner). CONCLUSION: Human papillomavirus infection is a prevalent sexually transmitted disease among young Swedish women, and the lifetime number of male sexual partners is a major risk factor.

The absence of vaginal human papillomavirus 16 DNA in women who have not experienced sexual intercourse.

OBJECTIVE: To determine whether young women who have not experienced sexual intercourse may harbor genital human papillomavirus (HPV) infection in the vaginal-ectocervical mucosa. METHODS: We included 151 women, 10-25 years of age, attending two adolescent health care units (Stockholm and Uppsala) and one primary health care center (Umeå). The size of the hymenal orifice, use of tampons, and the habit of digital vaginal manipulation were registered. Samples of epithelial cells were collected from the vagina and analyzed for the presence of HPV using polymerase chain reaction. RESULTS: One hundred thirty of 154 samples contained an adequate number of cells. Two samples were HPV 6 DNA-positive. None were HPV 16 DNA-positive. None of the women had external genital warts. In 84%, the hymenal opening was 15 mm or less. Forty-eight percent of the women used tampons during periods. Fifty-four percent had inserted their own finger into the vagina and in 23%, a boyfriend's finger had penetrated the vagina. CONCLUSION: Human papillomavirus is rarely present vaginally in virginal women, even with the use of tampons or digital penetration.