Search PubMed⌕ Search

Biomedical subjects

V A DeBari

Publications and source records attributed to V A DeBari.

At least 37 records · Page 2Linked to original sources
PubMed1990

The development of a membrane-based screening method to detect antibodies to intermediate filament proteins.

Autoantibodies directed against the intermediate filament proteins (IF) arise in a variety of disease states. The authors have investigated the binding of the IF to solid membrane supports in a dot blot format in an attempt to develop a simple procedure to detect antibodies (ab) to IF. Commercially obtained, purified IF were utilized. These were: vimentin (VIM), cytokeratin 8 (CYK), glial fibrillary acidic protein (GFA), desmin (DES), and the neurofilament triplet proteins (68, 160, and 200 KDa, respectively designated LMW, MMW, and HMW). Murine monoclonal antibody (mAb) probes were used to detect the presence and immunoreactivity of IF. The mAb were visualized with HRP-anti-mouse conjugates using alpha-chloronaphthol/H 2O 2 as substrate. The membranes studied were nitrocellulose (NC), and two of modified nylon. Nitrocellulose provided the most reproducible binding; no advantage was found to ensue from the use of the other membranes with regard either to quantitative binding or improved capping. Among the IF studied, VIM, GFA, LMW, MMW, and HMW bound well to NC; optimal mass/dot was 1 mug. Filtered, non-fat dry milk is a better capping agent than either albumin or fetal calf serum, but interferes with ab binding to GFA. Binding of CYK and DES is weak at neutral pH. Standard densitometric techniques provide the possibility of quantitation. We conclude that dot and slot blot assays may be practical methods to detect ab to IF antigens.

Autoantibodies↗
PubMed1989

Interaction of anti-DNA antibodies with synthetic polyanionic antigens.

Autoantibodies to DNA (anti-DNAab), found primarily in systemic lupus erythematosus (SLE), cross-react with a variety of antigens. The binding of these antibodies to naturally occurring mucopolysaccharides such as heparan and chondroitin sulfates has led to the suggestion that anti-DNAab have specificity for a polyanionic epitope. In this study, to avoid the use of endogenous immunogens to which humans may have become sensitized, we have used the synthetic polyanions, dextran sulfate (DS), polyvinyl sulfate (PVS) and the semi-synthetic antigen, pectic acid (PA) to evaluate this hypothesis using SLE sera (n = 15) and sera from healthy individuals (controls; n = 15, age and sex matched to SLE group). Inhibition of binding with 125I-DNA was optimal at 1 mg/ml for DS and PVS, and resulted in significant inhibition of binding by both SLE and control sera of native DNA (P less than 0.01, each group); no inhibition was observed with PA, nor was a significant inhibition observed with any antigen on binding of SLE or control sera groups to denatured DNA. We conclude that while on quantitative grounds reaction of anti-DNAab with polyanions may not be clinically relevant, it is clear that, unlike polycarboxylates (e.g. PA), polysulfated polymers, whether aliphatic, as in the case of PVS, or glycosidic, such as DS, react with a subpopulation of anti-DNAab in such a manner as to block significantly the ability of these antibodies to bind DNA.

Analysis of Variance↗
PubMed1988

Autoantibodies and circulating immune complexes in subjects infected with human immunodeficiency virus.

We wish to report the results of a retrospective study of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) as well as a group of asymptomatic human immunodeficiency virus (HIV)-infected individuals, in whom we examined markers of systemic autoimmune disease including anti-cell antibodies, immunoglobulin (Ig) and complement (C3 and C4) profiles as well as circulating immune complexes (CIC). Antibodies to cytoplasmic (but not nuclear) antigens were significantly elevated in the AIDS and ARC groups when compared with a population of normal individuals (for AIDS, P = 0.0002 vs. control; for ARC, P = 0.0004 vs. control). The non-AIDS/ARC, HIV+ group did not demonstrate significance (P = 0.054). Each of the three study groups also demonstrated increased Ig (P less than 0.05 for each immunoglobulin class). CIC, as determined by a Clq-binding enzyme immunoassay, were higher in all three study groups (P less than 0.05) when compared with controls. C3 and C4 were not significantly lower than control subjects (P greater than 0.05), but C4 did demonstrate a significant (P = 0.01) inverse correlation with CIC. These findings strengthen the hypothesized autoimmune aspect of AIDS and ARC, and extend this concept to include HIV-infected individuals without frank disease.

AIDS-Related Complex↗
PubMed1988

Relationships between the structure and function of lipopolysaccharide chemotypes with regard to their effects on the human polymorphonuclear neutrophil.

Polymorphonuclear neutrophils (PMN), harvested from healthy volunteers, were challenged with endotoxins from Salmonella minnesota smooth-strain and rough-strain mutants (Ra, Rb2, RcP-, Rd1P- and Re) as well as with lipid A in an effort to determine the relationship between lipopolysaccharide (LPS) structure and its ability to affect PMN chemotaxis (CT), random migration (RM) and luminol-dependent chemiluminescence (CL). In the presence of untreated autologous serum (UAS), as has been previously demonstrated for CL, CT in a modified Boyden chamber is stimulated but no significant differences among the chemotypes were observed. In the presence of heat inactivated autologous serum (IAS), significant variations among the LPS chemotypes occurred. Also noteworthy was the observation that, although the total number of cells migrating across the membrane was similar for both UAS and IAS, UAS promoted PMN migration beyond the membrane into the chamber, as opposed to IAS which caused the cells to remain adherent to the distal face of the membrane. In the absence of serum, RM was inhibited in a dose-dependent fashion by LPS from lipid A and the rough-strain mutants, with the degree of inhibition being progressively greater with increasing molecular complexity of the chemotype LPS. The smooth-strain LPS exerted an intermediate effect. In the case of CL, lipid A was the most potent stimulus, with the response decreasing as molecular complexity increased up to RcP-; the remaining core mutant LPS provided slightly greater responses. The LPS from the smooth-strain promoted a response similar to the response to Ra LPS. Based on these data, it is concluded that there are qualitative as well as quantitative effects of the carbohydrate moieties of LPS. These data also suggest that while LPS may provoke active migration in the circulation during endotoxemia, infection localized in tissue might bring about an inhibition of PMN migration while allowing the PMN to mount an oxidative response.

Adult↗
PubMed1988

Immunocytochemical localization of cytoskeletal antigens in KB and HEp-2 cells.

KB and HEp-2 are cell lines that are commonly used for autoantibody detection. We have used conventionally produced and monoclonal antibodies as probes for cytoskeletal antigens in these cells. The presence and distribution of microfilament, microtubule, and intermediate filament antigens (vimentin and keratin) have been established. We have also confirmed that anti-vimentin antibodies of one clone do crossreact with DNA on an immunoadsorbent column, although this antibody did not appreciably stain the nucleus of either cell type.

Actin Cytoskeleton↗
PubMed1987

The neutrophil response to polystyrene microspheres bearing defined surface functional groups.

Luminol-induced chemiluminescence (CL) and phagocytosis by human neutrophils was studied using polystyrene microsphere latices as particulate stimuli. Chemiluminescence and phagocytosis parameters were measured for particles bearing carboxyl, hydroxyl, and amino groups, as well as for the underivatized microspheres. The kinetic curves of CL were bimodal, and curve parameters were evaluated for both the early- and late-phase responses. Significant differences were found among the particle surfaces studied. Underivatized particles elicited the greatest response, particles with the amino group stimulated PMN the least, carboxyl- and hydroxyl-group-bearing particles elicited intermediate magnitudes of response. Phagocytosis data were in good agreement with that obtained from CL measurements. These data provide further evidence in favor of the hypothesis that, in protein-free systems, hydrophobic particles are more readily phagocytosed. Additionally they demonstrate that electrostatic interactions are not a significant factor for neutrophil-particle contact.

Adult↗
PubMed1987

Characteristics of antibodies adsorbed on the DNA immunoadsorbent, agarose poly-L-lysine-DNA.

Agarose-poly-L-lysine (Ag-(lys)n-DNA) has been used to bind DNA for assay of anti-DNA antibodies (ab). In this work, an algorithmic approach has been used to classify antinuclear ab (ANA) as being directed against native DNA (dsDNA), denatured DNA (ssDNA), DNA-protein complexes (deoxyribonucleoprotein; DNP), and against antigens which are independent of DNA (iDNA). These ab were subjected to Ag-(lys)n-DNA, and the selectivity of this adsorbent for the various specificities of ab was determined. The DNA on the columns was left untreated or treated with S1 nuclease, this being effected either by treating the DNA prior to introducing it onto the columns or by adding S1 nuclease to the columns after the DNA was bound. Ag-(lys)n-DNA adsorbs ab directed against ssDNA and DNP as well as ab to dsDNA; iDNA ab are not adsorbed. S1 nuclease treatment does not effectively remove ssDNA regions from the Ag-(lys)n-DNA, but it does result in the abolition of the adsorption of a population of ab which are in the anti-DNP sera and contribute to the total ANA load. While anti-iDNA ab are not adsorbed onto the columns, they do contribute to the ANA titer, unlike anti-ssDNA ab which are adsorbed onto the Ag-(lys)n-DNA but do not contribute to the ANA titer. We conclude that Ag-(lys)n-DNA bears antigenic sites for dsDNA, ssDNA, and DNP ab and suggest that our understanding of the characteristic ab-binding profile of this versatile immunoadsorbent may have applications in the study of autoimmune diseases.

Antibodies, Antinuclear↗
PubMed1985

Chemiluminescence response of the human polymorphonuclear neutrophil to lipopolysaccharides.

Polymorphonuclear neutrophils (PMN) respond to a variety of stimuli with a sequence of reactions that lead to the production of "active oxygen" species, including H2O2, free radicals, such as superoxide (O2-.) and hydroxyl (HO.), and singlet molecular oxygen (1O2). Some of these can oxidize (5-amino-2,3-dihydrophthalazine 1,4-dione) (luminol) to the ground state aminophthalate ion; this reaction sequence is accompanied by the generation of a photon and forms the basis for the chemiluminescence (CL) response. In this work we used a dedicated photon counting instrument to record CL from PMN incubated with bacterial lipopolysaccharide (LPS). We have studied the CL response to the LPS from Escherichia coli strains 026:B6 and 055:B5, as well as Salmonella minnesota RE 595 and have determined that CL requires heat-labile serum factors, these most likely being intact components of the complement system.

Complement System Proteins↗
PubMed1985

Stimulation of the hexose monophosphate pathway in the human erythrocyte by Mn2+: evidence for a Mn2+-dependent NADPH peroxidase activity.

In human RBC hemolysates, Mn2+ was found to stimulate the HMP as determined by the release of 14CO2 from [1-14C]glucose, providing activities of 125, 200, and 300% of basal at Mn2+ concentrations of 1, 10, and 100 mM, respectively. To explore the possibility that this stimulatory effect upon the HMP is a result of redox recycling of NADPH, RBC hemolysates were used to study NADPH oxidation. Mn2+, alone or in combination with a free radical-generating system, did not enhance the ability of hemolysates to oxidize NADPH. However, hemolysates + 10 mM H2O2 brought about a 10-fold increase in NADPH oxidation (0.51 +/- 0.05 nmole/min to 5.67 +/- 0.84 nmole/min) and the addition of 10 mM Mn2+ to this system increased the rate of oxidation to 34.10 +/- 2.97 nmole/min. Boiled hemolysates, either in the presence or absence of Mn2+, had some residual catalytic activity.

Erythrocytes↗
PubMed1984

Water soluble vitamins in granulocytes, erythrocytes, and plasma obtained from chronic hemodialysis patients.

A granulocyte fraction, composed primarily of polymorphonuclear neutrophils, was harvested from six male and six female chronic hemodialysis patients receiving folate as their sole vitamin supplement. Cells were assayed for water soluble vitamins and levels compared with cells from 12 age- and sex-matched controls. Thiamin, riboflavin, and vitamin B6 and B12 levels were not significantly different from controls. In contrast, pantothenate and biotin were significantly increased while ascorbate and nicotinate were significantly reduced in dialysis patients. Neutrophil folylmonoglutamates were significantly increased in chronic hemodialysis patients, but after conjugase treatment no significant difference was observed. The erythrocytes and plasma from these two groups were also compared. In the erythrocytes of chronic hemodialysis patients, no vitamin deficiencies were observed. Significantly elevated levels of vitamin B12, riboflavin, biotin, and pantothenate were present in the patients, while thiamin, vitamin B6, nicotinate, and ascorbate were not significantly different from controls. In plasma, only ascorbate was significantly decreased in the patient group; biotin, riboflavin, and pantothenate were elevated. No significant difference was observed for vitamins B6, B12, and thiamin.

Adult↗
PubMed1984

Cyclic nucleotide metabolism in the human erythrocyte.

Erythrocytes, which show little or no guanylate or adenylate cyclase activity, take up cyclic nucleotides from blood. Studies were done by incubating human erythrocytes in isotonic medium with the dibutyryl derivatives of cAMP and cGMP and in a hypotonic medium in which the cells are partially hemolyzed and, therefore, freely permeable to cAMP and cGMP. At cAMP and cGMP concentrations of 50 microM and above, the amount of 14CO2 generated from 1-14C-glucose was decreased significantly. This effect was suppressed by 4.6 mM theophylline. Inosine and ribose, which are catabolites of cAMP and cGMP also decreased formation of 14CO2 from 1-14C-glucose. Accordingly, it is postulated that in the absence of theophylline, the activity of phosphodiesterase resulted in AMP and GMP formation. These mononucleotides enter into the purine salvage pathways to form ribose phosphate. Additionally, the production of lactate and 2,3-diphosphoglycerate (2,3-DPG) was measured in human erythrocytes after incubation with the dibutyryl derivatives of cAMP (bt2-cAMP) and cGMP (bt2-cGMP). At a concentration of 0.1 microM, bta2-cGMP inhibits lactate production at 60 min (p less than 0.01). Slight increases in 2,3-DPG were not statistically significant. Catabolism of cyclic nucleotides may prevent diffusion equilibria allowing for the erythrocyte's continuous uptake of cyclic nucleotides and providing a detoxification mechanism that could compensate for conditions in which elevations of these substances are observed.

Bucladesine↗
PubMed1984

Differential immunoadsorption coupled with rate nephelometry for estimation of DNA-binding immunoglobulins.

We describe a technique for estimating the mass of anti-DNA antibodies by immunonephelometry of serum immunoglobulins (IgG, IgA, IgM) before and after adsorption onto DNA bound to agarose-polylysine columns. Sixteen patients with systemic lupus erythematosus and 16 age- and sex-matched controls were studied. Precision was determined for high-value (in 10 patients) and low-value (in nine controls) ranges for each of the immunoglobulins. Within-run CVs ranged from 3.0% (IgG, controls) to 11.8% (IgA, patients); between-run CVs ranged from 15.5% (IgG, patients) to 25.2% (IgM, patients). We found anti-DNA antibody concentrations (mean +/- SD) in systemic lupus erythematosus of 1.981 +/- 1.015 g/L for IgG (controls: 0.243 +/- 0.231, p less than 0.001), 0.257 +/- 0.215 g/L for IgA (controls: 0.038 +/- 0.035, p less than 0.001), and 0.282 +/- 0.234 g/L for IgM (controls: 0.191 +/- 0.165, p greater than 0.05). Sensitivity and linearity are such that fivefold dilutions of patients' serum with either a buffered albumin solution or control serum yielded values close to the expected values for IgG. Similarly diluted sera gave inordinately high values in the radiometric binding assay. Neither parametric (linear regression) nor nonparametric correlation methods (Spearman's rank and Kendall's tau) show a significant correlation between patients' data obtained by the present technique and that by a radiometric binding assay (p greater than 0.05), although combined data from patients and controls demonstrate a significant nonparametric correlation (p less than 0.005 for Spearman's and p less than 0.02 for Kendall's).

DNA-Binding Proteins↗
PubMed1984

KB cells for antinuclear antibody determination: comparison with HEp-2 cells and the Crithidia luciliae assay.

Fifty-seven sera from 53 patients were assayed simultaneously on KB and HEp-2 cells and compared with regard to pattern and titer. Additionally, KB cell fluorescent antinuclear antibody (FANA) titer and pattern in 310 sera from 194 patients were compared with regard to the presence of antinative DNA antibodies (anti-nDNA ab) as determined by the Crithidia luciliae assay. Sixty-five percent (37/57) of the sera had the same titer on both KB and HEp-2 cells; the remainder had higher titers using KB cells. Regression analysis yielded a highly significant, unbiased correlation between the substrates. Forty-four percent (25/57) of these sera gave identical patterns on both substrates, another 25 of the 57 sera (44%) gave different patterns on the two substrates and 12% (7/57) could not be compared because they were negative on HEp-2 cells. KB cells detected positive FANA in 30 of 30 (100%) diagnosed cases of systemic lupus erythematosus; HEp-2 cells detected 29/30 (97%). From the standpoint of sensitivity, these data indicate a slight advantage to the use of KB over HEp-2 cells. Seventeen percent (53/310) of the sera were positive for anti-nDNA ab. The highest percentage of these positive sera occurs at reciprocal FANA titers between 320 and 1280. No association was found between KB FANA patterns and a positive Crithidia luciliae assay.

Antibodies, Antinuclear↗
PubMed1982

Cyclic GMP in the human erythrocyte. Intracellular levels and transport in normal subjects and chronic hemodialysis patients.

We have studied the intracellular levels of cyclic GMP in the red blood cells of chronic hemodialysis patients before and after dialysis treatment and in normal subjects. Cyclic GMP is present at levels (mean +/- 1SD) of 19.6 +/- 4.7 and 18.4 +/- 5.8 nM/L before and after treatment, respectively. The levels in normal subjects are 3.6 +/- 0.9. The uptake of cyclic GMP was also studied and the data demonstrate that the red blood cell is slightly permeable to cyclic GMP, suggesting that cyclic GMP uptake could substitute for guanylate cyclase activity in the human erythrocyte.

Biological Transport↗