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Biomedical subjects

A Vaheri

Publications and source records attributed to A Vaheri.

At least 19 recordsLinked to original sources
PubMed1992

Heparin binding to the urokinase kringle domain.

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.

Amino Acid Sequence↗
PubMed1992

Urokinase binding to laminin-nidogen. Structural requirements and interactions with heparin.

Recently we have shown that heparin and related sulfated polyanions are low-affinity ligands of the kringle domain in the amino-terminal region (ATF) of human urokinase (u-PA), and proposed that this may facilitate loading of u-PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin-nidogen) for u-PA binding, and found that laminin-nidogen is also a ligand of the u-PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin-nidogen showed that there are u-PA binding sites in fragment E4 of laminin as well as in nidogen. The long-arm terminal domain of laminin (fragment E3), which contains a heparin-binding site, competed for binding of u-PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u-PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy-terminal domain. Direct binding assays confirmed that u-PA binds to nidogen through a site in the u-PA ATF. We conclude that u-PA binds to laminin-nidogen by interactions involving the ATF region of u-PA, the E4 domain of laminin and the rod or amino-terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u-PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.

Binding, Competitive↗
PubMed1992

Comparison of the deduced gene products of the L, M and S genome segments of hantaviruses.

The amino acid sequences deduced from all currently available nucleotide sequences of hantaviruses are compared. Comparisons of three large (L), eight medium (M) and five small (S) genome segments are included. A consensus sequence is provided, allowing easy identification of conserved and unique gene regions. The viruses included in this report represent four serologically distinct hantaviruses which are capable of causing severe, moderate, mild or no human disease.

Amino Acid Sequence↗
PubMed1992

Cloning and sequencing of Puumala virus Sotkamo strain S and M RNA segments: evidence for strain variation in hantaviruses and expression of the nucleocapsid protein.

The prototype Puumala virus (PV) Sotkamo strain small (S) and medium (M) RNA genome segments were amplified by the polymerase chain reaction (PCR), cloned and sequenced. The S segment is 1830 nucleotides long with an open reading frame coding for 433 amino acids. The identity to the PV Hällnäs strain was 83% at the nucleotide and 96% at the amino acid level. The M segment in the Sotkamo strain is 3616 nucleotides long and contains one open reading frame of 1148 amino acids with 83% nucleotide and 94% amino acid identity to the Hällnäs strain. Most amino acid changes were conservative and the five predicted glycosylation sites are identical. The amino acid identity to the prototype hantavirus, Hantaan virus, was 62 and 54% for S and M segments, respectively. The coding region of the S segment was further amplified by PCR, ligated to pEX vectors and expressed in Escherichia coli as a beta-galactosidase fusion protein and was seen to be specifically detected by nephropathia epidemica sera in immunoblotting.

Amino Acid Sequence↗
PubMed1992

Activation of integrated human immunodeficiency virus type 1 in human neuroblastoma cells by the cytokines tumour necrosis factor alpha and interleukin-6.

Human immunodeficiency virus type 1 (HIV-1) infection was studied in two different human neuroblastoma cell lines, SK-N-MC and SH-SY5Y. Results from immunofluorescence analysis indicate that SK-N-MC cells express a 68K neurofilament, and SH-SY5Y cells express additionally a 160K to 200K neurofilament complex and thus represent a more differentiated state. HIV-1 infection in these cell lines was demonstrated by nested polymerase chain reaction and further characterized by in situ hybridization, which showed that about 50% of SK-N-MC cells and 20% of SH-SY5Y cells were infected by HIV-1 and contained integrated proviral HIV-1 DNA. Among the cytokines and growth factors studied, tumour necrosis factor alpha (TNF-alpha) enhanced virus production in both cell lines, but to a differing extent, according to our mRNA and p24 antigen capture assay. In SK-N-MC cells the enhancement of HIV-1 mRNA was detected after 24 h of stimulation, and declined to the control level by 48 h. In SH-SY5Y cells a clear-cut stimulation was seen at both time points. By contrast, interleukin-6 (IL-6) enhanced the virus replication only in SK-N-MC cells, as shown at the mRNA level. Immunochemical staining showed no differences in the proportion of HIV-1-positive cells after 48 h of stimulation by TNF-alpha or IL-6 when compared to the control cells. In addition, based on a thymidine incorporation assay, TNF-alpha inhibited, but IL-6 strongly increased, the DNA synthesis in SK-N-MC cells, whereas in the SH-SY5Y cell line no such differences were seen. We discuss the possibility that developing, less-differentiated neurons may be more readily infected by HIV-1 than fully differentiated neurons, and that cytokines such as TNF-alpha and IL-6, which are elevated in HIV-1-infected individuals, may enhance HIV production.

DNA↗
PubMed1992

Anti-endothelial cell antibodies in nephropathia epidemica and other viral diseases.

Increased capillary permeability is a central feature of the severe forms of haemorrhagic fever with renal syndrome (HFRS) and occurs also, though less frequently, in nephropathia epidemica (NE), one of the milder forms of this syndrome, caused by Puumala virus. We therefore searched for antiendothelial cell antibodies (AECA) in patients with NE and in those with other presumed or serologically proven acute viral illnesses. By enzyme immunoassay, using human umbilical vein endothelial cells (HUVEC) as the substrate, IgG class AECA were detected significantly more frequently in patients with NE and with influenza A than in Red Cross blood donors. A lesser degree of reactivity could be shown with a human alveolar cell carcinoma line and with human and mouse embryonic fibroblasts. Pretreatment of HUVEC with interferon-gamma (IFN-gamma), but not with IL-1 or tumour necrosis factor-alpha (TNF-alpha), increased their ability to bind IgG of sera from patients with NE and acute febrile illnesses. We conclude that, although AECA can be demonstrated in NE, they occur also in other acute viral illnesses and, unless cytopathic by a mechanism not shared by the AECA of these other illnesses, are unlikely to be casually related to the capillary leak in HFRS.

Acute Disease↗
PubMed1992

Retinal pigment epithelial cells secrete urokinase-type plasminogen activator and its inhibitor PAI-1.

Secretion of plasminogen activators and their inhibitors was examined in cultures of human retinal pigment epithelial (RPE) cells. The methods employed were zymography and reverse zymography, solid-phase immunocapture assay, metabolic labeling followed by immunoprecipitation, and immunofluorescence. The results showed that these cells produce urokinase-type plasminogen activator (u-PA) and a plasminogen activator inhibitor (PAI) which is immunologically and biochemically similar to PAI-1. Tissue-type plasminogen activator activity (t-PA) was not detected, but we detected small amounts of t-PA in an inactive complex with inhibitor in RPE cell-conditioned media. We conclude that RPE cells have the potential to utilize u-PA-catalyzed plasminogen activation which is subject to regulation by PAI-1. These results may have a bearing on the pathogenesis of proliferative retinal diseases.

Antibodies, Monoclonal↗
PubMed1992

Tear plasmin activity with contact lens wear.

Plasmin, a proteolytic enzyme, has been detected in the tears of patients experiencing anterior ocular disease, and during contact lens wear. Using a radial caseinolysis procedure, we examined tear plasmin levels in 66 patients who were wearing soft and rigid lenses for daily and extended wear. Compared to non-contact lens wearers, patients wearing soft and rigid lenses for extended wear were significantly more likely to exhibit tear plasmin activity. Eight hours of open-eye thick HEMA lens wear did not induce tear plasmin activity in a group of 10 subjects. However, significant increases in tear plasmin activity were recorded after short-term (1 hour) eye closure with and without lens wear, and following overnight (8 hours) eye closure without lens wear. Overnight eye closure also resulted in significantly increased numbers of epithelial cells and leucocytes in the tear fluid. Our results suggest that increased tear plasmin activity during extended contact lens wear may be attributable to the effects of eye closure rather than hypoxia or the presence of the contact lens per se.

Adult↗
PubMed1992

Human papillomavirus infection of the female genital tract: correlation of HPV DNA with cytologic, colposcopic, and natural history findings.

Although molecular biology evidence suggests a strong link between HPVs and anogenital neoplasias, evidence from clinical studies is still less convincing. Thus, the recognition of both overt and subclinical HPV infection has become increasingly important. We studied the correlation between HPV DNA and cytologic, colposcopic and natural history findings in a defined group of women. The study population consisted of 150 HPV DNA positive cases (mean age 25.7 years, SD 7.4) and 69 randomly selected HPV DNA negative controls (mean age 27.3 years, SD 7.9) enrolled in an ongoing study of the natural history of genital HPV infections. All cases and controls had normal cervicovaginal cytology at the enrollment. A commercial dot-blot technique hybridization test (Virapap and Viratype, Digene Diagnostics, USA) was used for HPV DNA testing of cervicovaginal scrapes. Five percent of the patients had HPV 16/18, 31% had HPV 31/33/35, 15% had more than one of the three HPV DNA groups ("mixed"), and 15% had untypable HPV DNA. Cases and controls were followed by repeat examinations every 4th month. The mean follow-up time was 12.2 months (SD 8.7) for the cases, and 12.8 months (SD 6.9) for the controls. The study endpoint was defined as the presence of cytologic changes consistent with CIN. The overall prevalence of atypical transformation zone (ATZ) findings was 45% in the controls and 56% in the cases, with no significant differences between the specific HPV DNA groups. Although vulvar or vaginal abnormalities (acetowhite epithelium, squamous papillomatosis, filaments, satellite lesions, fissures, papules or exophytic condylomas) were more commonly seen in the cases than in the controls, the difference was significant only for condylomas and fissures.(ABSTRACT TRUNCATED AT 250 WORDS)

Colposcopy↗
PubMed1991

Rapid diagnosis of hantavirus disease with an IgG-avidity assay.

Nephropathia epidemica (NE), due to Puumala virus, is endemic in eastern Europe and Scandinavia. Serodiagnosis of NE relies on conventional indirect immunofluorescence to detect IgG against Puumala virus. However, in conventional serology, most patients with acute NE have raised but stable non-diagnostic antibody titres. For better serodiagnosis of NE, we have developed a test that measures the avidity (functional affinity) of IgG antibodies against Puumala virus. This new assay was highly specific and sensitive; a diagnosis of NE could be confirmed or excluded rapidly from an early single serum sample. With this test we have now verified the diagnosis of NE in more than 1300 Finnish patients during 22 months in 1989-91. Our findings point to an incidence of confirmed hantavirus disease much higher than previously shown.

Antibodies, Viral↗
PubMed1991

Epidemiology of encephalitis in children: a 20-year survey.

Four hundred five children from the Helsinki area who were 1 month to 16 years old were treated for acute encephalitis at the Children's Hospital, University of Helsinki, from January 1968 through December 1987. Encephalitis occurred most commonly in children 1 to 1.9 years of age, among whom the incidence was 16.7 per 100,000 child-years. The incidence remained quite high until the age of 10 years, and then gradually declined to 1.0 per 100,000 child-years at the age of 15 years. Since 1983, when mumps, measles, and rubella vaccination eradicated the encephalitides associated with these microbes, the major associated agents have been varicella-zoster, Mycoplasma pneumoniae, and respiratory and enteroviruses. In infants younger than 1 year of age, the major agents were enteroviruses, herpes simplex virus, and the group of "others," whereas in older children, respiratory viruses and Mycoplasma pneumoniae, as well as varicella-zoster virus, dominated. In children aged 1 to 11 months, the causal agent could not be identified in one-half of all cases, whereas in children who were at least 10 years old, the etiology remained unknown in only one-fourth of cases. Male dominance was most evident in the 4- to 9-year age group. The difference in etiology between males and females was significant (p = 0.02); mumps and varicella were more common in boys, and adenovirus and Mycoplasma pneumoniae were more common in girls. The overall male-to-female ratio was 1.4:1. Characteristic seasonal variation occurred in encephalitides associated with mumps, measles, and entero- and respiratory viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1991

Persistent inapparent HIV-1 infection of human neuroblastoma cells.

We have studied human immunodeficiency virus type 1 (HIV-1) infection in three different human neuroblastoma cell lines; SK-N-MC, IMR-32 and SH-SY5Y. In all of these cell lines the infection became productive. However, the virus expression was different as determined by the p24 antigen capture assays from culture supernatants and immunochemical (APAAP) staining of cells. The medium of SK-N-MC cells contained approximately 300 pg p24 antigen per 10(6) cells, 0.1-1% of the cells were p24 antigen-positive and characteristic genomic and subgenomic HIV mRNA species were seen in Northern blotting. In infected IMR-32 and SH-SY5Y cell cultures, the HIV-1 production was below the level of detection. However, infectious virus was found by inoculating cultures of the lymphoid cell C8166 with the cell-free supernatant fluid from the neuroblastoma cultures. The lymphoid cells became positive within one week. Moreover, phytohemagglutinin-stimulated normal human lymphocytes produced virus, if cocultured with any of the three infected neuroblastoma cell lines. The infection was persistent and has been followed, using the above techniques, for 4 months in the case of SK-N-MC and IMR-32 cells and 6 months in the case of SH-SY5Y cells. During this period, no alterations in cell morphology, viability, or proliferative capacity were seen. All three neuroblastoma lines were negative for the CD4 receptor mRNA according to Northern hybridization and RNase protection assays. We conclude that HIV-1 produces persistent and inapparent infection in human neuroblastoma cells, using a CD 4-independent mechanism of entry to the cells.

Blotting, Northern↗
PubMed1991

Genotyping of herpes simplex viruses by polymerase chain reaction.

A simple and rapid polymerase chain reaction (PCR) procedure was developed for simultaneous detection and genotyping of herpes simplex viruses (HSV) from virus isolates. It was possible to detect and type HSV using only one primer pair from the HSV DNA polymerase coding genes. After PCR the type of HSV present was determined on the basis of size of the ethidium-bromide-stained band after agarose gel electrophoresis. A hybridization test with radioactive target oligonucleotide probes after PCR was also developed to confirm the typing result.

Animals↗
PubMed1991

Cervical human papillomavirus deoxyribonucleic acid and cytologic evaluations in gynecologic outpatients.

We studied the prevalence and cytologic manifestations of human papillomavirus deoxyribonucleic acid among 2668 gynecologic outpatients. The study population consisted of 1573 women who came into the emergency room, 623 women from an abortion clinic who were seen because of induced first-trimester abortion, and 472 women who had been referred to a colposcopy clinic because of an abnormal Papanicolaou smear finding. A dot blot technique was used for the detection of human papillomavirus types 6/11, 16/18, and 31/33/35. The prevalence of human papillomavirus deoxyribonucleic acid was 4.4% in the emergency room population, 8.8% in the abortion clinic population, and 31.4% in the colposcopy clinic population. Human papillomavirus type 16/18 was most prevalent (3.5%), followed by human papillomavirus type 31/33/35 (3.1%), and human papillomavirus type 6/11 was least prevalent (2.1%). Of all unselected human papillomavirus deoxyribonucleic acid-positive women who were enrolled from the emergency room or from the abortion clinic, 73% had normal cytologic evaluations. Twenty-five percent had benign atypia, and 2% had dyskaryotic changes. No human papillomavirus deoxyribonucleic acid type-specific differences were detected in the distribution of cytologic atypias.

Adolescent↗
PubMed1991

Procollagen-III in serum, plasminogen activation and fibronectin in bronchoalveolar lavage fluid during and following irradiation of human lung.

In the search for predictors of late radiation-induced lung injury we studied procollagen type III peptide concentration (P-III-P) in serum as well as fibronectin and plasminogen activation in bronchoalveolar lavage (BAL) fluid during and following irradiation of human lung. The patients received either high-dose hemithorax irradiation for pleural mesothelioma (11 patients) or high-dose irradiation with individually shaped fields for non-small cell lung cancer (12 patients). The severity of radiation fibrosis was assessed clinically from CT scans 6 months and 12 months after treatment. Four scores were used: severe, moderate, mild, or normal. Radiological lung injury varied from "severe" (9 patients) to near absence of injury-"normal" (6 patients). Serum levels of P-III-P, when measured weekly during the 5-week period of radiotherapy or at several time-points after treatment, did not show consistent changes, nor did the levels correlate with the score for radiation fibrosis as assessed by CT scanning. Changes in fibronectin levels or in markers of plasminogen activation in BAL fluid did not correlate with the development of late lung injury. The levels of BAL fluid plasmin and plasminogen activator as assessed zymographically, but not the free net enzyme values, showed a tendency to be elevated in patients with severe radiation-induced lung injury, suggesting a possible role for inhibitors of the plasminogen activation cascade in the process of radiation-induced lung injury.

Adult↗
PubMed1991

Stimulation of cell surface plasminogen activation by heparin and related polyionic substances.

The functional operation of the cell surface pro-u-PA and plasminogen activating system has previously been shown to depend on the assembly of u-PA receptors, plasminogen binding sites, and their respective ligands at the focal adhesions of cell extensions. We now show that additional factors operate that affect the persistence of functional activity and that evidently involve charge interactions mediated by polyanions, such as those found in the cell surface proteoglycans. Heparin-like compounds and protamine were identified as fast-acting stimulators of cell surface plasminogen activation. Heparin stabilized surface u-PA activity during plasminogen activation, and we propose that a heparin binding site exists in the kringle structure of u-PA. Heparin at 40 micrograms/ml could reduce u-PA loss to only 20% compared with 60% on control cells activating plasminogen. Protamine (25 micrograms/ml) exerted a strong stimulatory effect on the level of generated bound plasmin and notably prolonged the persistence of this activity, so that 100 minutes after addition of plasminogen the level of plasmin on protamine-treated cells was five times higher than on control-treated cells. The effect of protamine on plasmin clearance suggests that an unknown plasmin inhibitor may be produced by rhabdomyosarcoma cells, whose action is accelerated by endogenous polyanions, in an analogous manner to thrombin inactivation by antithrombin III and protease nexin on endothelial cells and fibroblasts, respectively. The stimulatory effects of heparin and protamine do not affect the inactivation of cell surface u-PA by recombinant PAI-2.

Fibrinolysin↗