[A simple method for determination of antibodies to AIDS associated virus (HTLV-III)].
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
A Vaheri
Publications and source records attributed to A Vaheri.
A rapid and highly sensitive solid-phase enzyme immunoassay specific for human fibronectin using a characterized monoclonal antibody.
A solid-phase enzyme immunoassay (EIA) was developed for the quantitation of human fibronectin in body fluids and cell culture media. In the assay a human fibronectin-specific murine monoclonal IgG1 (f-33) was used as capture antibody and polyclonal rabbit anti-fibronectin as detector antibody. The antibody showed no reactivity to purified monkey, dog, rabbit, horse, sheep, mouse, bovine or chicken fibronectins. The determinant of the monoclonal antibody was mapped to the cell-binding region of the fibronectin molecule. This localization was based on the use of purified fragments of fibronectin, immunoblotting, EIA and inhibition of fibroblast adhesion and spreading by the antibody. The detection limit of the fibronectin assay was 2 ng/ml. The assay was used for the quantitation of fibronectin in human plasma, urine and cerebrospinal fluid specimens and culture media of human cells.
Animals↗
Laminin interacts with plasminogen and its tissue-type activator.
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Agglutination Tests↗
Tissue type plasminogen activator, but not urokinase, exerts transformation-enhancing activity.
Previous studies have established that plasma cryoprecipitates of tumor patients, culture media of transformed cells and defined proteolytic fragments of fibronectin enhance the morphological cell transformation ( TEF activity) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We now report that purified human tissue type plasminogen activator (t-PA), but not urokinase (u-PA), has a similar TEF activity, at doses as low as 2 ng/ml (30 pM). Specific antibodies effectively neutralized the activity. No significant contamination (less than or equal to 1%) between the preparations of t-PA and fibronectin (FN) or its fragments ( FNdp ) was detected. The results suggest that t-PA may have a direct role in the process of morphological cell transformation in vitro.
Cell Transformation, Neoplastic↗
Plasminogen activators, activation inhibitors and alpha 2-macroglobulin produced by cultured normal and malignant human cells.
In the present paper we have characterized the plasminogen activators (PA) synthesized by 25 different human cell lines. Technically easy methods were adopted for concentration and immunological characterization of the activators even in the presence of PA inhibitors. Most cell lines produced u-PA (mol. wt 55,000), melanoma and HeLa cells t-PA (mol. wt 66,000) and two carcinoma cell lines and normal skin fibroblasts produced no detectable PA. The classical 125I-fibrin method was compared to a caseinolytic assay and some of the discrepancies between results obtained with the two methods were shown to be due to cell-derived NaDodSO4-sensitive proteinase inhibitors in culture media. Additionally, synthesis and uptake by the cells of the wide-spectrum proteinase inhibitor alpha-2-macroglobulin ( alpha 2M ) were studied by radioimmunoassay and immunofluorescence. No production of alpha 2M could be measured in any of the malignant cell lines. In normal cells no correlation existed between the production of alpha 2M and the observed inhibition of PA activity, which indicates that other proteinase inhibitors are produced by the cells.
Cells, Cultured↗
Monoclonal antibody to human T-cell leukemia virus p19 defines polypeptide antigen in human choriocarcinoma cells and syncytiotrophoblasts of first-trimester placentas.
In previous studies we detected retrovirus RD114 p30-related antigen in human placental syncytiotrophoblasts and antibodies in cord blood sera. We now report that a monoclonal IgG antibody specific for the p19 protein of human T-cell leukemia virus (HTLV) reacts with human syncytiotrophoblastic antigen. When used in the immunoperoxidase tissue-staining procedure, the monoclonal antibody reacted with the syncytiotrophoblastic cells in sections of all early placentas (less than 15 weeks of gestation), blighted ova and benign (hydatidiform mole) or malignant placental tumors (destructive mole, choriocarcinoma). In all cases the reactivity was mainly confined to the cytoplasm of the cells with syncytiotrophoblastic morphology. Normal embryonal or adult tissues tested were negative. In immunoblotting of cultured choriocarcinoma cells, anti-HTLV p19 detected a single polypeptide at mol.wt 28,000 from proteins separated by electrophoresis.
Antibodies, Monoclonal↗
Binding of fibronectin by the acute phase reactant C-reactive protein.
Following tissue injury, the concentration of C-reactive protein (CRP) is known to increase in plasma rapidly, while that of fibronectin often decreases. We now report that CRP immobilized onto polystyrene surfaces binds soluble plasma fibronectin (Kd = 1.5 X 10(-8) M). The binding of fibronectin by CRP was relatively sensitive to ionic conditions, being maximal at physiological NaCl concentrations. A decrease of pH from neutral to 5-6 greatly enhanced the binding of fibronectin by CRP. Ca2+ ions at greater than 1 mM inhibited binding. No binding was observed between fibronectin and CRP in soluble phase. CRP was found also to bind fibrinogen, which competed with fibronectin for CRP-binding sites. This was shown to explain why fibronectin was effectively bound from serum but not from plasma by immobilized CRP. The amount of CRP immobilized was critical in binding fibronectin; a too dense molecular layer of CRP inhibited the binding, as did the postsaturation of free surfaces with albumin, which itself was not bound by CRP. Soluble fibronectin agglutinated CRP-coated latex particles. Most or all of the CRP-binding activity in the fibronectin molecule was localized to the 120-140-kilodalton fragment, which also contains cell-binding and heparin-binding domains of fibronectin. The results provide a link between acute phase response and tissue repair.
C-Reactive Protein↗
Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans.
The pericellular matrix fibers of cultured human fibroblasts contain fibronectin, other glycoproteins, and heparan and chondroitin sulfate proteoglycans. In the present study, cell-free pericellular matrices were isolated from metabolically labeled fibroblast cultures. The isolated matrices were digested with heparinase from Flavobacterium heparinum, and then analyzed for sulfated glycosaminoglycans (GAGs). Nitrous acid degradation was used to distinguish the N-sulfated GAGs (heparan sulfate) from chondroitin sulfate. Fibronectin and the other major matrix polypeptides were studied using gel electrophoresis, enzyme immunoassay and immunofluorescence. Upon heparinase digestion, greater than 95% of sulfated GAGs were degraded in the matrix without detectable release of fibronectin or other matrix polypeptides or alteration of the fibrillar matrix structure. We conclude that in fibroblast cultures the integrity of the fibrillar matrix is independent of sulfated GAGs. Together with earlier observations, this suggests that filamentous polymerization of fibronectin forms the backbone of early connective tissue matrix.
Cells, Cultured↗
Laminin is induced in astrocytes of adult brain by injury.
Laminin is a high mol. wt. non-collagenous matrix glycoprotein, confined in adult tissues to basement membranes. In normal rat brain we found laminin mainly in vessel walls but, after injury, induced by stereotaxic injection of a neurotoxin, laminin immunoreactivity appeared also in reactive astrocytes, which are characteristically positive for the glial fibrillary acidic protein (GFAP). Laminin was first detected in GFAP-immunoreactive glial cells 24 h after injury. Four days later the majority of reactive astrocytes in the gray matter were positive for laminin and the laminin immunoreactivity, but not that of GFAP, gradually subsided within a month. Fibronectin, the other major matrix glycoprotein, was found only in capillary structures both in normal and lesioned brain tissue. The results indicate that mature astrocytes have the potential to produce laminin and suggest a role for this glycoprotein in brain regeneration.
Animals↗
Altered hemolysis in single radial hemolysis from a single serum sample as an indicator of recent primary rubella virus infection.
The quality of hemolysis in the single radial hemolysis (SRH) test was observed to be altered in a small proportion (5.7%) of sera (N = 9628) studied for rubella rubella antibodies. Three different types of altered hemolysis were identified. Two of these types, the "soft margin" (SM) or the "soft zone" (SZ), occurred singly or in combination in 97% of paired sera (N = 321) taken 3-30 days after primary rubella infection (diagnostic seroconversion). For comparison, diagnostic increases of rubella antibodies (N = 77), including both primary and reinfections, contained these types of altered hemolysis in 80% of the cases. Of the remaining 20% of the samples (N = 15), rubella antibodies of IgM class were looked for in seven cases but not found. After primary rubella, SZ was always present in the first serum sample and disappeared rapidly within 20 days while SM persisted longer. The predictive value of the different types of hemolysis was estimated from 96 primary sera, which had altered hemolysis. In this material, SZ was followed by a diagnostic increase of antibodies in 87% of the cases; the prognostic value of the SM was significant but lower. These data show that the appearance of SM or SZ in a single serum sample is a useful marker of recent primary rubella. On the other hand, a normal hemolysis in SRH can be used to exclude recent primary rubella, but not reinfection, with a high degree of probability.
Antibodies, Viral↗
Neurons cultured from developing rat brain attach and spread preferentially to laminin.
Neurons, mechanically dissociated from newborn rat brain and identified by immunostaining for neurofilaments, attached preferentially to laminin-coated coverslips without need of an underlying glial monolayer. The most extensive neurite outgrowth was seen when 20-30 micrograms/ml of laminin was used to coat the coverslips. Higher concentrations of laminin (greater than 30 micrograms/ml) supported single neurons to spread on the coverslips. Fibronectin coating of the coverslips allowed glial cells to attach more rapidly than on uncoated surfaces, but it did not support neuronal spreading or neurite outgrowth. Spreading of neurons and neurite outgrowth were completely inhibited by preincubation of laminin-coated coverslips with laminin antibodies but were unaffected by fibronectin antibodies. These results indicate that laminin is an attachment and spreading factor for central neurons in culture and suggest the presence of a laminin receptor on the neuronal cell surfaces.
Animals↗
Herpes simplex virus encephalitis. Prolonged intrathecal IgG synthesis and cellular activity in the cerebrospinal fluid with transient impairment of blood-brain barrier.
Alterations of cerebrospinal fluid (CFS) proteins and cells and blood-brain barrier impairment were determined in 4 patients with proven and 2 patients with presumptive herpes simplex virus encephalitis ( HSVE ) using simultaneous nephelometric measurements of CSF and serum albumin and immunoglobulins and combined MilliporeR filtration-cytocentrifuge cytologic techniques. The follow-up period ranged from 17 to 855 days. All patients showed intrathecal IgG synthesis which in 1 case continued for 28.5 months (855 days). The daily production of IgG in the central nervous system ranged up to 1157 mg. CSF-IgA and -IgM were also elevated in the early phase of the disease. The impairment of the blood-brain barrier was variable being apt to develop during the first 2 months of the disease and diminishing thereafter. Pleocytosis, mainly due to lymphoid cells, varied from slight to severe (325 X 10(3) cells/ml) and was observed in the CSF of all cases during the first 2 months. Lymphoid reaction (increase of enlarged stimulated lymphoid cells) was persistent and was the most pronounced cellular alteration. The lymphoid reaction and intrathecal IgG synthesis indicated continuous immunoactivation of the CNS, which was most intensive during the first 2 months and appeared to persist for at least 16-28.5 months.
Adult↗
Retrovirus RD114 p30 is bound by the extracellular matrix proteins, fibronectin and laminin.
Purified retrovirus RD114 p30 was found to bind to the extracellular matrix proteins, fibronectin and laminin. The purified matrix proteins were immobilized onto polystyrene and the binding of p30 was quantitated using radioiodinated proteins and enzyme immunoassay (EIA). The dissociation constants were for the p30-fibronectin binding Kd = 5.3 X 10(-8) M and for the p30-laminin Kd = 7.3 X 10(-8) M. The molecular ratio in the binding from 4000 ng/ml of p30 was 1.9 mol per mol fibronectin and 2.5 mol per mol laminin. The interaction between fibronectin and RD114 retrovirus was also detected using a modified immunoblotting procedure.
Fibronectins↗
Vaccination with a live RA 27/3 strain rubella vaccine (Rudivax), a follow-up study.
Thirteen-year-old Helsinki schoolgirls were screened for rubella antibodies using fingertip blood specimens and the single radial haemolysis (SRH) test. The seronegative individuals (326/1234; 26.4%) were immunized with the RA 27/3 strain of rubella vaccine (Rudivax). All 326 produced SRH and haemagglutinin inhibition (HAI) antibodies and 129 IgM antibodies (39.4%) detected by enzyme immunoassay in the two-month follow-up serum samples. The incidence and nature of side effects was low. In the twenty-four month follow-up serum samples all individuals had substantial levels of rubella antibodies and no clinical reinfections had occurred during the observation period.
Adolescent↗
Clinical and serological diagnosis of Nephropathia epidemica, the mild type of haemorrhagic fever with renal syndrome.
The serological diagnosis of Nephropathia epidemica (NE), the mild European type of haemorrhagic fever with renal syndrome (HFRS), was studied by means of an indirect immunofluorescent antibody technique (IFAT) in a large outbreak in Finland (morbidity 1.4/1000 population). Acetone-fixed sections of lung of bank voles (Clethrionomys glareolus) naturally infected with Puumala virus served as antigen. Altogether 65 patients were followed serologically for up to 20 months. Serological results obtained by NE-IFAT were compared with the clinical diagnosis of NE. Of the 41 clinically accepted NE cases 40 were serologically positive. Use of the NE-IFAT serological test allowed the diagnosis to be made in seven patients for whom the clinical data were not sufficient for diagnosing NE. The antibody response in NE often appears to be slower than that against Hantaan virus. Practical recommendations are given for the clinical and serological diagnosis of the milder forms of HFRS.
Adolescent↗
Human placental syncytiotrophoblastic Mr 75,000 polypeptide defined by antibodies to a synthetic peptide based on a cloned human endogenous retroviral DNA sequence.
Antibodies to a synthetic undecapeptide (NH2-Cys-Glu-Asn-Pro-Ser-Gln-Phe-Tyr-Glu-Arg-Leu-COOH), the sequence (except cysteine) of which was deduced from a previously reported cloned human retroviral gag-gene-related DNA sequence erv-1, were raised in rabbits. In immunohistochemical staining these antibodies reacted with normal human first-trimester placentas and with blighted ova and benign and malignant trophoblastic tumors (hydatidiform and destructive moles, choriocarcinomas) but not with any other normal embryonic or adult tissues tested. In all tissues the reactivity was mainly confined to cells with trophoblastic morphology. In immunoblotting the antibody detected an Mr 75,000 polypeptide in syncytiotrophoblasts isolated from first-trimester placentas and in three different lines of cultured choriocarcinoma cells. The undecapeptide blocked the reactivity of the antibody.
Antibodies↗
Distribution of different collagen types and fibronectin in neurofibromatosis tumours.
Collagen types I, III, IV and V and fibronectin were localized in neurofibromas from six patients with von Recklinghausen's neurofibromatosis (NF) using indirect immunofluorescence (IIF) and peroxidase anti-peroxidase (PAP) techniques. Type I and III collagens were abundantly and rather evenly present in the tumours and formed a continuous network, but were absent from the capillary endothelial walls and were sparse in the perineurium of the occasional nerve fascicles. The type III/type I + type III collagen ratio in neurofibromas varied from 17.4% to 37.3% when estimated with cyanogen bromide peptide analysis. Fibronectin was detected in areas where type I and III collagens were present but was most intensively stained in the vascular walls and perineurium. Type IV collagen was detected at the dermo-epidermal junction of the skin overlying the tumours, in the endothelial cells of the capillaries, the perineurium and endoneurium. Furthermore, in the tumourous stroma there was plenty of type IV collagen appearing as a discontinuous patchy pattern suggesting abundant basement membrane material associated with cells forming the tumours. Type V collagen distribution was very similar to that of type IV collagen.
Collagen↗