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Biomedical subjects

A Vaheri

Publications and source records attributed to A Vaheri.

At least 145 records · Page 8Linked to original sources
PubMed1996

Antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells.

Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes.

Animals↗
PubMed1996

Quasispecies in wild-type tula hantavirus populations.

Tula virus (TUL) is a recently detected hantavirus carried by European common voles. Reverse transcriptase PCR cloning was used to study TUL S segment/N protein quasispecies. Both the distribution and character of mutations observed in three mutant spectra indicated limited selection at the protein level. At least 8% of the mutations were neutral or well tolerated; fixation of such mutations may play a role in TUL evolution in its natural host.

Animals↗
PubMed1996

Cerebrospinal fluid activity of tissue plasminogen activator in patients with neurological diseases.

AIM: To study cerebrospinal fluid (CSF) activity of tissue plasminogen activator (tPA) in patients with neurological diseases. METHODS: CSF tPA and urokinase (uPA) activities were studied using an immunocapture assay and zymography in 44 patients with neurological disease and 20 reference subjects. The patient group comprised three patients with meningitis, 21 with encephalitis, nine with acute lymphoblastic (n = 7) and myeloid (n = 2) leukaemia, seven with multiple sclerosis, three with facial paresis, and one with polyradiculitis. RESULTS: Raised tPA activities were observed in patients with multiple sclerosis, leukaemia and encephalitis. In contrast, there were no differences in the mean activities of tPA in patients with meningitis or other diseases compared with the reference subjects. The highest tPA activities were found in patients with multiple sclerosis. The mean activity in patients with leukaemia was higher than in those with meningitis and polyradiculitis, but not encephalitis and facial paresis. Although the CSF tPA activity correlated positively with age in reference subjects, no correlation was observed in patients. Samples were qualitatively screened for both tPA and uPA activity by zymography and positive samples were quantitated. Some of the samples had quantifiable levels of uPA activity: three of seven multiple sclerosis samples, 10 of 21 samples from patients with encephalitis and five of nine leukaemic samples. The highest activities were recorded in patients with leukaemia. uPA was not detected in the CSF of the patients with meningitis, facial paresis or polyradiculitis. CONCLUSIONS: Plasminogen activator activity can be measured reliably in CSF and the assessment of tPA activity may be useful for studying the pathogenesis of neurological diseases.

Adolescent↗
PubMed1996

Herpes encephalitis is a disease of middle aged and elderly people: polymerase chain reaction for detection of herpes simplex virus in the CSF of 516 patients with encephalitis. The Study Group.

OBJECTIVE: To assess the diagnostic potential of the polymerase chain reaction (PCR) in herpes simplex virus (HSV) encephalitis. METHODS: Samples of CSF from 516 patients with encephalitis were studied for HSV-DNA by PCR. RESULTS: Samples taken one to 29 days from the onset of symptoms from 38 patients (7.4%) were positive, 32 (6.2%) for HSV-1 and six (1.2%) for HSV-2. At follow up, eight of 28 patients studied were still HSV-PCR positive. A diagnostic serum:CSF antibody ratio to HSV but not to other viruses was detected in 25 of the 38 HSV-PCR positive patients thus supporting the initial PCR findings. Patients positive by HSV-PCR were concentrated in the age group > or = 40 years, and especially in patients aged 60-64 years, of whom nine of 24 (37.5%) were positive. The HSV-PCR was negative in all other patients with encephalitis of known or unknown aetiology. This group included 34 patients with a diagnostic serum:CSF antibody ratio to other viruses. A dual infection, HSV and another microbe, was considered possible in seven patients. CONCLUSIONS: The HSV-PCR is a rapid and useful diagnostic method during the early phase of encephalitis. It may be useful in monitoring the efficacy of treatment and allowing the recognition of new features in the appearance of herpes encephalitis. The HSV-PCR test and antibody determinations from serum and CSF are complementary methods, which should both be applied in pursuit of clinical laboratory diagnosis of these conditions.

Age Distribution↗
PubMed1996

Chlamydia pneumoniae associated with central nervous system infections.

We identified 15 patients with serological evidence of current Chlamydia pneumoniae infection when we studied 263 patients with central nervous system infections among an adult population of 3 million in 1993. In 9 of the 15 patients no other associated or etiological agents were found. One patient died. Sequelae appeared in 7 patients. In neurological infections, C. pneumoniae may be more prevalent as an associated agent than appreciated, and adequate antibiotic therapy may be life-saving.

Adolescent↗
PubMed1996

Stillbirths and maternal antibodies to Chlamydia trachomatis. A new EIA test for serology.

OBJECTIVE: The relationship between stillbirths and infections during pregnancy. METHODS: Antibodies to 13 different viruses and to Mycoplasma pneumoniae, Treponema pallidum, Toxoplasma gondii, and Chlamydia trachomatis, psittaci, and pneumoniae were studied from sera of 42 mothers with stillbirth and of matched case-control pregnancies. RESULTS: Elevated antibody levels to Chlamydia trachomatis and human parvovirus B19 were observed in seven mothers with stillbirth as compared with one in matched case controls (p-0.03 and p < 0.06, Fisher's exact one- and two-tailed p values). By a novel EIA test for C. trachomatis, based on synthetic polypeptide, 15 positive cases were detected in patients compared to seven in controls. Placental calcifications and fetal growth retardation appeared more often (p < 0.001 and p < 0.05, respectively) in the C. trachomatis- associated cases than in the others. There was no significant relation between stillbirth and the other antibodies. CONCLUSIONS: We suggest C. trachomatis as a hitherto unrecognized possible cause of still-births besides human parvovirus B19. A new EIA test for C. trachomatis, one covering all strains, is promising and makes the future assays more convenient, and may thus make it possible to reduce the rate of stillbirths.

Adult↗
PubMed1996

Ezrin expression in stromal cells of capillary hemangioblastoma. An immunohistochemical survey of brain tumors.

Ezrin is a cytoskeleton-associated protein that appears to link actin filaments to the plasma membrane. Immunocytochemical studies suggest that ezrin is expressed in epithelial cells but not in mesenchymal cells. In addition, ezrin is expressed by certain epithelial tumors, such as renal cell adenocarcinomas. Ezrin serves as a tyrosine kinase substrate, and is phosphorylated in epidermal growth factor-stimulated cells. Ezrin may thus mediate regulatory signals in different cell functions. We studied the distribution of ezrin in 104 cases of primary tumors of the central nervous system (CNS) by immunocytochemistry. Special interest was focused on capillary hemangioblastoma, owing to its resemblance to renal cell adenocarcinoma, and on malignant gliomas, owing to their frequent epidermal growth factor receptor amplification. The stromal cells of hemangioblastomas were found to be strongly positive for ezrin. No expression was detected in gliomas and, except for hemangioblastomas, ezrin expression was restricted to those few CNS tumors that show epithelial differentiation, ie, choroid plexus papillomas, craniopharyngiomas, ependymomas, and cysts. The diffuse cytoplasmic expression of ezrin in the stromal cells of capillary hemangioblastoma may indicate that stromal cells overexpress ezrin or express ezrin with deficient binding properties.

Antibodies, Monoclonal↗
PubMed1996

Keratinocyte growth factor enhances urokinase-type plasminogen activator activity in HPV16 DNA-immortalized human uterine exocervical epithelial cells.

We recently demonstrated that keratinocyte growth factor (KGF), a fibroblast-derived growth factor, induced anchorage-independent growth of HPV16 DNA-immortalized human uterine exocervical epithelial cells (HCE16/3 cell line) in soft agarose assay. We have now studied whether KGF might also regulate the plasminogen activator (PA) system, another transformation parameter related to cell invasiveness. HCE16/3 cells were found to produce urokinase-type PA (uPA) and small amounts of tissue-type PA (tPA) as determined by immunocapture assay. Secretion of both uPA and tPA was increased when HCE16/3 cells were exposed to KGF for 4 h and continued to increase during the next 24 h. The early increase following KGF treatment was presumably mediated by binding of KGF to its receptors. Enhanced secretion of uPA in HCE16/3 cells by KGF was also seen by zymographic analysis and immunofluorescence. According to Northern blotting KGF upregulated the transcription of uPA gene in HCE16/3 cells; the upregulation occurred only after 12 h of KGF treatment, suggesting that stimulation of uPA production by KGF is a biphasic event. As proteolysis is a prerequisite for tumor cell invasion, taken together with our previous results, the present findings suggest that KGF may play an important role in the transition of immortalized uterine cervical epithelial cells from in situ to invasive carcinoma.

Cell Line↗
PubMed1995

Quantitative polymerase chain reaction of lysyl oxidase mRNA in malignantly transformed human cell lines demonstrates that their low lysyl oxidase activity is due to low quantities of its mRNA and low levels of transcription of the respective gene.

Lysyl oxidase (EC 1.4.3.13), an extracellular copper amino oxidase, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We developed here a polymerase chain reaction (PCR) method for the quantification of lysyl oxidase mRNA in which a synthetic RNA is used as an internal standard for coamplification with the targeted mRNA. The amount of lysyl oxidase mRNA when studied by Northern blot analysis and the number of lysyl oxidase mRNA molecules when determined by the quantitative PCR method were found to be markedly low in various malignantly transformed cell lines relative to control cell lines, quantitative PCR indicating values of about 2-10% of those in the controls. No difference was found in the number of beta-actin mRNA molecules between the transformed cells and the controls. Nuclear runoff experiments indicated that most if not all of the decrease in the number of lysyl oxidase mRNA molecules can be explained by diminished transcription of the respective gene.

Adult↗
PubMed1995

Human skin fibroblasts induce anchorage-independent growth of HPV-16-DNA-immortalized cervical epithelial cells.

Immortalized non-malignant cells do not grow in soft agarose. We found, however, that HPV-16-DNA-immortalized human uterine exocervical epithelial cells (HCE16/3 cell line) formed colonies when co-cultured with human embryonic skin fibroblasts. This appeared to be mediated by diffusible growth factors, and direct cell-cell contacts were not required. HCE16/3 cells from colonies remained unable to grow alone in soft agarose like the parental HCE16/3 cells, indicating that only transient phenotypic changes without genetic alterations had occurred in the co-cultures. In a modified soft-agarose assay, HCE16/3 cells grown as an underlying monolayer did not produce enough (transforming) growth factors which could confer an anchorage-independent phenotype to non-transformed fibroblasts. Lethally irradiated fibroblasts were able to stimulate DNA synthesis and proliferation of HCE16/3 cells. Northern-blot analysis showed that HPV-16-DNA expression of the immortalized cells was not altered in the presence of fibroblasts. The expression of the IL-I gene in the immortalized cells, however, was augmented by the co-cultured fibroblasts compared with immortalized cells alone. In organotypic collagen raft co-cultures, in which epithelial cells were grown on top of a collagen gel containing fibroblasts, HCE16/3 cells appeared to be chemotactic to fibroblasts and fibroblasts stimulated the growth of the immortalized epithelial cells. Our results suggest that fibroblasts may contribute to human epithelial carcinogenesis in vivo.

Blotting, Northern↗
PubMed1995

Effect of interferon-alpha and cell differentiation on Puumala virus infection in human monocyte/macrophages.

Pathogenesis of hantavirus infections is poorly understood. Puumala virus (PUU) is the etiologic agent of nephropathia epidemica, a form of hemorrhagic fever with renal syndrome common in Europe. We have studied PUU infection in primary human monocyte/macrophages and specifically the role of interferon alpha (IFN-alpha) and cell differentiation in it. PUU infection proceeded at a low level in monocyte/macrophages, and nucleocapsid (N) protein accumulation started 2 days postinfection. IFN-induced antiviral MxA protein was detected 3 days postinfection, suggesting IFN-alpha production in culture. IFN-alpha titers remained low, proposing that PUU is a poor IFN inducer. However, the PUU-induced IFN had an inhibitory effect on virus production as was shown by the effect of anti-IFN-alpha. Pretreatment of cells with IFN-alpha caused a dose-dependent inhibition of PUU N accumulation and reduced the yield of infectious virus. Monocytic U-937 cells overexpressing MxA protein were susceptible to PUU, suggesting that, unlike in some other negative strand RNA virus infections, MxA does not mediate resistance to PUU infection. Differentiation of monocyte/macrophages in culture and treatment of THP-1 promonocytic cells with phorbol 12-myristate 13-acetate made the cells more susceptible to PUU. The increased susceptibility of mature macrophages to PUU suggests that after differentiation to tissue macrophages they might function in the spread of the virus during PUU infection.

Animals↗
PubMed1995

Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response.

Puumala virus (PUU) is a member of the Hantavi rus genus in the family Bunyaviridae and the etiologic agent of nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome (HFRS). In this study we compared the immunofluorescence patterns of NE sera and antibodies raised against recombinant PUU proteins and confirm that the nucleocapsid protein is the major target in the early IgG response of NE patients and provides the molecular basis for simple and rapid differentiation between acute illness and old immunity by granular vs. diffuse fluorescence staining in the indirect immunofluorescence test. The differential kinetics of B-cell responses to PUU nucleocapsid vs. envelope proteins was emphasized further by the endpoint titres of IgG antibodies to N, G1 and G2 proteins in NE patients. The granular fluorescence correlated with low IgG avidity in 99.8%, and diffuse fluorescence with high avidity in 100% of 617 NE sera studied. Epitope scanning with overlapping 14-mer peptides covering the whole nucleocapsid protein by a shift of 3 amino acids revealed six major antigenic epitopes recognized by sera from acute-phase NE patients. The epitopes clustered mainly in the hydrophilic regions, and two of them in a highly variable region which could probably serve as an antigen to distinguish serologically between infections of closely related hantaviruses, some apparently apathogenic, some causing lethal infections. The anti-peptide epitope pattern varied between different individuals and a collection of several pin-bound peptides was needed to be recognised by most NE sera studied.

Animals↗
PubMed1995

Nucleotide and deduced amino acid sequences of the M and S genome segments of a Swedish Puumala virus isolate.

The Swedish Puumala (PUU) virus strain Vindeln 83-L20, isolated from a bank vole trapped in 1983 near Vindeln, Västerbotten county, Sweden, was characterized by nucleotide sequence analysis. The coding region of the M segment was determined by PCR followed by direct sequencing and the entire S segment was characterized by cloning and nucleotide sequence analysis. The genomic organization was found to be very similar to that of other PUU virus strains regarding open reading frames, polypeptide sizes and potential glycosylation sites. According to phylogenetic analysis 83-L20 was found to represent a new lineage within the Puumala virus serotype in the Hantavirus genus. The M segment sequence of 83-L20 was found to be more closely related to the Finnish PUU virus strains than to strains from Central Europe or from Russia. The evolutionary origin of the S segment was not as clearly resolved since the branching points of all PUU virus strains in the phylogenetic tree were nearly the same.

Amino Acid Sequence↗
PubMed1995

Genetic variation in Tula hantaviruses: sequence analysis of the S and M segments of strains from Central Europe.

Hantavirus carried by the European common vole Microtus arvalis from Moravia (Czech Republic) was analyzed by RT-PCR-sequencing and by reactivity with a panel of monoclonal antibodies (MAbs). Sequencing of the full-length S segment and the proximal part of the M segment showed that the virus belonged to genotype Tula (TUL) we discovered earlier in Microtus arvalis from Central Russia. This finding supported the concept of host dependence of hantaviruses. Phylogenetic analyses suggested a similar evolutionary history for S and M genes of TUL strains; thus far there is no evidence for reassortment in TUL. Geographic clustering of TUL genetic variants was observed and different levels of the genetic variability were revealed resembling those estimated for another hantavirus, Puumala (PUU). Comparison of the deduced N protein sequence from Russia and from Moravia showed that genetic drift in TUL occurred not only by accumulation of point mutations but also by the deletion of a nucleotide triplet. It encoded Ser252 which was located within a highly variable hydrophilic part of the N protein carrying B-cell epitopes and presumably forming a loop. Analysis of naturally expressed TUL N-antigen derived from lung tissue of infected voles with MAbs indicated antigenic heterogeneity among TUL strains.

Amino Acid Sequence↗
PubMed1995

Keratinocyte growth factor is a bifunctional regulator of HPV16 DNA-immortalized cervical epithelial cells.

Various factors are known to regulate cell growth and differentiation in epithelial-mesenchymal interactions. Keratinocyte growth factor (KGF), an epithelial-specific cytokine produced by dermal fibroblasts and other mesenchymal cells, appears to affect growth, migration, and differentiation in epithelial-mesenchymal interactions. We have previously shown that human embryonic skin fibroblasts induce anchorage-independent growth of HPV16 DNA-immortalized human uterine exocervical epithelial cells (HCE16/3 cell line) in cocultures of HCE16/3 cells and fibroblasts. Here we report that KGF may be a major factor influencing growth and behavior of HCE16/3 cells in the coculture system. KGF stimulated both DNA synthesis and proliferation of normal human cervical epithelial (HCE) cells and HCE16/3 cells and the increase was stronger in HCE16/3 cells than in HCE cells. SiHa cells, a cervical carcinoma cell line with integrated HPV16 DNA, did not respond to the KGF mitogen signal. KGF receptor (KGFR) studies suggested that the different responses to the KGF mitogen signal may be correlated with KGFR. In addition, KGF alone was able to induce anchorage-independent growth of HCE16/3 cells, suggesting a potential role for KGF in the transformation process of epithelial cells. However, the transcription of HPV16 early genes was suppressed by KGF in the immortalized HCE16/3 cells, and this appeared to be due to transcriptional repression rather than a posttranscriptional process according to nuclear run-on analysis. In contrast, viral gene expression was not affected by KGF in SiHa cells. Our results suggest that KGF is a bifunctional growth factor in the HPV-immortalized cells, a positive regulator of cell growth and negative regulator of HPV16 early gene expression.

Blotting, Northern↗
PubMed1995

Chlamydia trachomatis seropositivity during pregnancy is associated with perinatal complications.

Sera from the mothers of all children from the greater Helsinki area who were treated in the Neonatal Intensive Care Unit (NICU) of Children's Hospital (University of Helsinki) during a 22-month period were studied serologically, and antibody levels for mothers were compared with those for matched controls. IgM to Chlamydia trachomatis serotype GFK was detected more often in sera from mothers with children in the NICU than in that from controls; IgM was detected in sera from 39 of 264 mothers vs. 15 of 274 controls (P < .001; two-tailed test). The gestational age of children born to mothers in the IgM-seropositive group was 32.4 weeks whereas that of children born to mothers in the IgG/IgM-seronegative group was 34.3 weeks (all children were treated in the NICU). The frequency of signs of maternal infection (i.e., fever and vaginal discharge), the frequency of meconium-stained amniotic fluid, the frequency of chorioamnionitis, and the mortality rate were higher in the IgM-seropositive group than in the IgM-seronegative group; pneumonia, atelectasis, and pneumothorax occurred more frequently in the seronegative group. Thus, in terms of birth weight and perinatal infections, the outcome was better for children whose mothers did not have antibodies to C. trachomatis.

Adult↗