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Biomedical subjects

G Virella

Publications and source records attributed to G Virella.

At least 109 records · Page 6Linked to original sources

Inhibition of platelet-activating factor by rabbit C-reactive protein.

Incubation of 0.5 nM platelet-activating factor (PAF) with 123 micrograms/ml C-reactive protein (CRP) for 60 min at room temperature in the presence of 2 mM calcium resulted in total inhibition of the platelet-aggregating capacity of 0.5 nM PAF. There was no inhibition of platelet aggregation if PAF and CRP were added to the platelets simultaneously, without prior incubation of the mixture, or if calcium was not present during the incubation of CRP and PAF. The inhibitory effect of CRP (123 micrograms/ml) was dependent upon the time of incubation with PAF (1 nM). We observed 0, 42, and 85% inhibition of platelet aggregation with incubations of 0, 20, and 120 min, respectively. The inhibitory effect of CRP was also concentration dependent. After incubations of 20 min at room temperature with 0.5 nM PAF there was no inhibition of PAF-induced platelet aggregation using 12.3 micrograms/ml CRP with the inhibition increasing to 73% using 123 micrograms/ml CRP. Pretreatment of the platelets with CRP (123 micrograms/ml) for 60 min before addition of PAF did not affect the induction of platelet aggregation by PAF. These observations indicate that CRP inhibits PAF and strongly suggest that binding of CRP to PAF, presumably to the phosphocholine moiety of PAF, is required for this inhibition.

Animals↗

Solid-phase enzymoimmunoassay of antitetanus toxoid antibodies.

A solid-phase enzymoimmunoassay (EIA) for antitetanus toxoid antibodies has been developed using activated agarose beads as the solid phase to which tetanus toxoid was covalently attached. The resulting EIA proved to be remarkably free of interference due to nonspecific absorption of reactants to the solid phase. The lower limit of detection for the assay was 0.008 U/ml of antibody. Reproducibility studies showed a satisfactory degree of consistency with coefficients of variation of 4.8% (within run) and 6.6% (run-to-run). This assay has been applied successfully to the evaluation of the humoral response to tetanus toxoid in both normal and immunocompromised individuals and it has sufficient sensitivity to determine serum levels of antitetanus antibody above the accepted protective limit of 0.01 U/ml.

Adolescent↗

Validation of the PEG-IgG screening test for soluble immune complexes by longitudinal studies in experimental acute serum sickness.

A wide variety of tests for the detection of circulating immune complexes (IC) has been proposed by different authors, but there is very little to no information concerning the performance of IC screening assays in samples known to contain in vivo-formed IC. The purpose of our investigation was to compare the behavior of a non-specific assay, the PEG-IgG screening test for IC, with an antigen-specific assay in serum samples sequentially obtained from rabbits to which we induced acute serum sickness. Five animals were used in the study; we were able to detect an increase of IC constituted by the heterologous antigen (human serum albumin) and corresponding antibodies in all, and in 4 animals the results of the PEG-IgG assay closely correlated with the results of the antigen-specific assay (rho values between 0.975 and 1.00). The 4 animals in which IC showed a definite peak by both assays developed proteinuria and IC deposits at the glomerular level, while the animal that failed to develop IC detectable by the PEG-IgG test remained normal throughout the study. These results demonstrate the ability of the PEG-IgG test to detect in vivo-formed IC and suggest that the IC detected by this test have pathogenic potential.

Acute Disease↗

Immune complexes and antinuclear, antinucleolar, and anticentromere antibodies in scleroderma.

Forty-one patients with various forms of systemic sclerosis (scleroderma) and positive antinuclear antibodies of nucleolar (ten patients), speckled (eleven patients), or centromere pattern (twenty patients) were selected for study of immune complexes by the radioisotope labeled Clq binding and the radioisotope labeled protein A binding methods. The presence of immune complexes was found by the Clq binding assay in sixteen patients (39%) and by a protein A binding assay in eight patients (20%). Overall, 46% of patients (19/41) had immune complexes. A lower incidence of organ involvement and fewer positive results in the screening of serum immune complexes were observed in patients with centromere antibody (35%) than in patients with nucleolar (60%) or speckled pattern (55%). Patients with immune complexes had higher frequencies of kidney, heart, and muscle involvement and digital ulceration than did patients with no detectable immune complexes, but the differences were not statistically significant. Diffuse skin involvement was not related to the presence of immune complexes.

Adult↗

Isolation and characterization of an outer membrane protein of Salmonella paratyphi B: a mitogen and polyclonal activator of human B lymphocytes.

Salmonella paratyphi B (S. paratyphi B) has been previously characterized as a human T-independent polyclonal B cell activator. To define further the nature of the bacterial structure responsible for these properties, we studied the effects of autoclaving and enzyme treatment of S. paratyphi B on its stimulatory capacity. We found that both autoclaving and papain treatment decreased the ability of S. paratyphi B to induce B cell activation, while trypsin treatment did not affect this capacity. Neither type of treatment affected the binding of S. paratyphi B to lymphocytes, suggesting that binding and B cell stimulation are mediated by different structures. The observation that B cell stimulation was significantly reduced by papain treatment led us to attempt to purify membrane proteins so that we could investigate whether they shared the stimulating capacity of S. paratyphi B. A water-insoluble, 43-45,000 mol. wt. protein, rich in aspartic acid, glutamine, glycine, alanine and leucine, similar in mol. wt. and physicochemical chemical properties to the porins of other gram negative bacteria, was isolated and designated as outer membrane protein (OMP). This protein was equally efficient to S. paratyphi B in inducing T-independent B cell activation. By performing time-course studies of [3H]-thymidine incorporation we observed a burst of mitogenic activity after stimulation of PBL or purified B cells with both S. paratyphi B and OMP peaking at 48-96 hr of culture (compared to 96-120 hr for the PWM proliferation peak), and with a magnitude of roughly 10% of that observed after PWM stimulation. Given the fact that the proportion of B lymphocytes in PBL is 4-12%, it appears likely that the proliferation burst seen with S. paratyphi B and OMP corresponds to a mitogenic effect mainly restricted to the B cell population.

Amino Acids↗

Human platelets aggregate during antigen-antibody reactions at different antigen-antibody ratios.

The effects of immune complex formation on human platelets were tested in vitro using tetanus-toxoid and rabbit anti-tetanus toxoid for immune complex formation and washed human platelets. The antigen-antibody reactions took place at 37 degrees C in a stirred cuvette, constantly monitored. To this cuvette we added initially a platelet suspension and a dilution of antibody, and after this mixture reached equilibrium, antigen was added. Platelets aggregated as a consequence of immune reactions taking place at antigen-antibody ratios ranging from four times antigen excess to twenty times antibody excess (relative to equivalence). Platelets also aggregated as a result of reactions in which heat-inactivated antiserum was used, although in a more narrow range of Ag:Ab concentrations, suggesting that complement is not essential for their stimulation but may help in stabilizing the Ag:Ab reaction. Our observations provide indirect evidence in support of the involvement of human platelets in the pathogenesis of immune complex-mediated hypersensitivity reactions.

Animals↗

Statistical analysis of five immune complex screening assays: patterns of detection in patients with rheumatoid arthritis, systemic lupus erythematosus, infectious endocarditis, and diabetes mellitus.

A comparative study of four nonspecific screening techniques (direct nephelometry, PEG-C4, PEG-IgG, and radiolabeled Clq binding) for immune complexes (IC) and of a technique specific for the detection of insulin-anti-insulin IC was undertaken in four groups of patients with diagnosis of infectious endocarditis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and diabetes. The highest frequency of positive results was given by the PEG-IgG test in RA, the Clq-binding test in SLE, the insulin-anti-insulin IC screening test in diabetes, and the PEG-IgG and Clq-binding tests in infectious endocarditis. Of the four nonspecific tests, the PEG-C4 assay appeared to be the least discriminative, since it failed to show significant differences between any group of patients and the group of controls. Direct nephelometry, PEG-IgG, and radiolabeled Clq binding gave consistently higher results in RA than in other diseases, and in this disease the rates of agreement between these tests were highly significant. Significant agreements between the rates of positivity of Clq binding and PEG-IgG tests were seen in all groups of patients studied. Spearman's analysis of rank showed the best correlations among tests based on similar principles (ie, PEG precipitation), and also a strong correlation between Clq binding and the PEG-IgG test in RA. The PEG-IgG test appears to be a reliable IC screening test for general use with the advantage of not involving radioisotopes. In regard to antigen-specific tests, although their specificity and sensitivity may be high, their results may show no correlation with nonspecific screening tests nor with the presence or absence of clinical or laboratory abnormalities suggestive of IC deposition, as exemplified by the insulin-anti-insulin IC screening test in diabetic patients.

Antigen-Antibody Complex↗

Immunological diagnosis of systemic candidiasis by counterimmunoelectrophoresis and quantitative immunofluorescence using purified antigens.

The advantages of two different immunological approaches to the diagnosis of systemic candidiasis have been studied. Counterimmunoelectrophoresis (CIE) using purified cytoplasmic protein antigens and quantitative immunofluorescence (QIF) using purified polysaccharide antigens have proven to be approaches giving better discrimination between systemic and nonsystemic candidiasis. Using CIE and yeast cytoplasmic proteins we obtained nine positive reactions in 12 patients with systemic candidiasis, and none in 23 patients with other types of C albicans infection or in 30 normal healthy controls. Using quantitative immunofluorescence and mycelial polysaccharide antigen we obtained positive reactions in all tested sera from systemic candidiasis patients (14), but we also obtained positive reactions (although generally of lower magnitude) in 4 of 14 patients with localized or mucocutaneous candidiasis. It appears that although neither of the tests has 100% specificity and sensitivity, their use individually or in combination may result in valuable data to assist in establishing a diagnosis of systemic candidiasis.

Antibodies, Fungal↗

Non-complement-dependent adsorption of soluble immune complexes to human red cells.

Heat aggregated IgG and soluble immune complexes (IC) prepared by combining human serum albumin with rabbit anti-serum albumin and tetanus toxoid with rabbit antiserum to tetanus toxoid were shown to bind to human O+ RBC. The binding was greater for soluble IC prepared at antigen excess, and although it was usually maximal when IC were pre-opsonized, it could also be demonstrated using non-opsonized heat aggregated IgG or soluble IC prepared in the absence of complement. These observations suggest that two types of receptors may be involved in binding of soluble IC to human RBC: the classical C3b receptor, and a non-complement-dependent receptor, perhaps recognizing the Fc region of the immunoglobulin molecule exposed after heat aggregation or antigen-antibody reaction.

Adsorption↗

Circulating immune complexes in patients with Candida albicans infections.

The existence of circulating immune complexes in patients with candidiasis has been suggested but not confirmed. In this study we have used four immune complex screening techniques (direct nephelometry, Clq binding assay, PEG-IgG assay and PEG-C4 assay) and an immune complex score compiled from results of the individual tests to establish that elevated levels of immune complexes do exist in a series of 11 patients with systemic candidiasis as compared with a group of 18 normal controls. Isolation and characterization of high molecular weight fractions from the sera of five patients with candidiasis and further purification by affinity chromatography on Sepharose 4B-protein A substantiated the presence of anti-Candida antibody and/or Candida antigen in at least three of them. The presence of complement components and IgG in these fractions further supported the existence of immune complexes containing microbial antigen and antibody.

Antibodies, Fungal↗

Release of PAF by human polymorphonuclear leucocytes stimulated by immune complexes bound to Sepharose particles and human erythrocytes.

Human polymorphonuclear leucocytes (PMN) incubated with surface-bound immune complexes (IC) release a substance that induces platelet aggregation and serotonin-release. This substance was identified as platelet-activating factor (PAF) on the basis of its sensitivity to phospholipase A2 and of its purification by thin-layer chromatography in identical conditions to those used to purify zymosan-induced PAF. We used two types of substrates to absorb our IC:Sepharose particles to which we coupled human serum albumin, and which were later incubated with specific rabbit antiserum to form surface-bound immune complexes, and human erythrocytes, to which soluble IC can be passively adsorbed. Both types of surface-bound IC were found to stimulate the release of PAF by human PMN in the absence of complement. These results suggest that PMN may play a central role in the early stages of IC-induced inflammation: they recognize IC adsorbed to red cells or to any other cell able to adsorb IC, and they induce the activation of platelets and release of vasoactive amines, which leads to the increase of vascular permeability believed to be essential for extravascular IC deposition.

Animals↗

Development of a single numerical expression for the results of multiple screening tests for immune complexes in diabetic patients: use in statistical comparison of clinically and biochemically defined patient populations.

A series of 88 diabetic patients were studied for the presence of soluble immune complexes, proteinuria, microangiopathy, and diabetic complications. Results of the five different assays for immune complexes were analyzed individually, and four combinations of the individual results (i.e. four different immune complex "scores") were also analyzed. The only assay which consistently discriminated between the different patient groups was the PEG-IgG test, in which a ratio between the amount of IgG precipitated with 3% PEG 6000 and the serum concentration of IgG is determined. In contrast, all four of the immune complex "scores" detected significant differences between patients with and without the clinical or biochemical parameter in question. One combination, designated as the "weighted and corrected IC score", gave a particularly high probability of detecting differences between groups. These results indicate that proper compilation of the results of a battery of immune complex screening assays can provide definite advantages over the results of individual tests for the investigation of correlations between the presence of soluble immune complexes and the course and pathology of various diseases.

Analysis of Variance↗

Polymorphonuclear leucocytes release a factor(s) that induces platelet aggregation and ATP release after interaction with insoluble and surface-fixed immune complexes.

We have found that human polymorphonuclear leucocytes (PMN) can be stimulated by large aggregates (heat-aggregated IgG, chemically polymerized IgG, heavily aggregated human immune complexes) and by surface-bound immune complexes (IC) to release enzymes (lysozyme, beta glucuronidase) and a factor(s) able to induce platelet aggregation and ATP release from the platelets. Surface-bound IC were most effective in stimulating the release of this factor(s). We used several substrates for their preparation: plastic-adsorbed antigen. Sepharose-coupled antigen and polymerized antigen. The platelet-aggregating factor(s) released by IC-stimulated PMN and zymosan-stimulated PMN were compared for their susceptibility to inhibition by indomethacin. Both induced a first phase of platelet aggregation that was resistant to indomethacin, but the second phase of aggregation and the release of platelet ATP were inhibited to a variable degree, more pronounced in the case of the factor(s) released after PMN-IC interaction. The lack of inhibition of the early phases of aggregation induced by our factor(s) when platelets were simultaneously exposed to indomethacin suggests that the classical, phospholipid PAF is released under these experimental conditions. Although, further experiments will be necessary to fully characterize the factor(s) involved, our observations suggest a complex interrelationship between human PMN and platelet activation, which may play an important role in the sequence of events that mediate the tissue deposition of IC and appearance of inflammatory changes.

Adenosine Triphosphate↗

Polyclonal activation of human peripheral blood B lymphocytes by formaldehyde-fixed Salmonella paratyphi B. I. Immunoglobulin production without DNA synthesis.

A "new" polyclonal activator of human peripheral blood B cells, formaldehyde-fixed Salmonella paratyphi B, is described. This bacterium does not stimulate cell proliferation as measured by incorporation of tritiated thymidine but does stimulate a subpopulation of B cells to secrete large amounts of IgM, IgG, and IgA in 7-day cell cultures. The immunoglobulins (Ig) produced by cells responding to S. paratyphi B are not specific antibodies against the bacterial antigens. In comparison with other B cell activators (pokeweed mitogen, Staphylococcus aureus Cowan I, and lipopolysaccharide), S. paratyphi B stimulation produced greater amounts of IgM but less IgG than pokeweed mitogen (PWM) or S. aureus Cowan I; lipopolysaccharide failed to stimulate significant Ig production on day 7 in most cases. In addition, the response to S. paratyphi apparently did not require T cell collaboration. These results suggest that the B cell subpopulation(s) responding to S. paratyphi B may be more differentiated B cells than those responding to either PWM or S. aureus Cowan I. Peripheral blood mononuclear cells from five patients with common variable immunodeficiency without evidence of abnormal suppressor T cells or monocytes failed to respond to S. paratyphi B, whereas cells from two of the same patients responded well to S. aureus Cowan I and partially to PWM. Thus, S. paratyphi B appears to be superior to other B cell activators for studies of B cell function in normal and abnormal states.

B-Lymphocytes↗

Platelet aggregation and release of ATP after incubation with soluble immune complexes purified from the serum of diabetic patients.

Human platelets are known to have Fc receptors that are able to recognize soluble immune complexes and to respond to that stimulation by aggregating and releasing soluble factors. In diabetes, enhanced platelet aggregation has been proposed as one of the factors contributing to the development of microangiopathy. Soluble immune complexes isolated from seven diabetic patients were found to enhance ADP-induced platelet aggregation and release of ATP. This enhancement was proven not to be an artifact due to the isolation protocol, by comparison of purified immune complexes with nonspecific protein purified from normal sera by identical or slightly modified isolation protocols. Soluble immune complex appear to be the first well-characterized platelet aggregating factors form the sera of diabetic patients. The natural of the antigen involved in their formation does not appear relevant, since very similar results were obtained whether soluble immune complexes were purified from patients with insulin-anti-insulin complexes in their serum, or from those without such complexed but with positive results in nonspecific screening techniques.

Adenosine Diphosphate↗