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R Kulhavy

Publications and source records attributed to R Kulhavy.

At least 19 recordsLinked to original sources
PubMed2000

False positivity of enzyme-linked immunosorbent assay for measurement of secretory IgA antibodies directed at HIV type 1 antigens.

We have determined that polymeric IgA in saliva of HIV-1-uninfected individuals binds in varying degrees to components of culture supernatants containing HIV-1 recombinant proteins when ELISA is used for the determination. This finding did not extend to salivary IgG antibodies. Further, such problems were not encountered in Western blot. Binding did not appear to be mediated by salivary proteins known to bind to IgA, including secretory component, amylase, lactoferrin, lysozyme, galactosyl transferase, or secretory leukocyte protease inhibitor, and was not influenced by blocking reagents or by changes in secondary anti-IgA antibodies. Although these findings will not likely impact on the use of saliva as a diagnostic fluid for HIV-1 infection (the HIV-1 response in saliva is mostly of the IgG isotype), they indicate that assessments of this secretion as an indicator of IgA mucosal immune responses to HIV-1 vaccines should be undertaken with caution.

Animals↗
PubMed2000

Heterogeneity of O-glycosylation in the hinge region of human IgA1.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was applied to studies of the molecular heterogeneity of desialylated human IgA1 hinge region glycopeptides released with two IgA1 proteases. Typically, the hinge region of an alpha1 chain contains three to five O-linked glycan chains. Variants of the hinge region peptides released from IgA1(Kni) myeloma protein carrying 0, 1, 2, or 3 GalNAc residues were observed in the mass spectra as well as the nonglycosylated peptide. Variable numbers of Gal residues indicated additional heterogeneity in O-glycosylation of IgA1. In the hinge region preparation from normal human serum IgA1, glycopeptides carrying 2, 3, 4, or 5 GalNAc residues with variable numbers of Gal residues were detected. In conclusion, our new approach using the site-specific cleavage with two IgA1 proteases allowed precise and sensitive MALDI-TOF mass spectrometric analysis of O-glycosylation heterogeneity in IgA1 hinge region.

Amino Acid Sequence↗
PubMed1994

Carbohydrate heterogeneity of human myeloma proteins of the IgA1 and IgA2 subclasses.

Comparative studies of the N-linked carbohydrate chains of human myeloma proteins of the IgA1 and IgA2 subclasses were performed. The N-linked carbohydrate chains were released by hydrazinolysis from the polypeptide backbone, converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation and separated into one neutral and two acidic fractions by paper electrophoresis. The acidic oligosaccharides were completely converted to neutral oligosaccharides by sialidase treatment, indicating that they were sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that human myeloma IgA proteins contained significant amounts of biantennary complex-type carbohydrate chains in addition to a small amount of the high mannose-type. The results indicated that the oligosaccharide structures of human IgA1 and IgA2 display a high degree of heterogeneity not only in the number of carbohydrate chains, but also in their composition.

Anions↗
PubMed1993

Interactions of cell-surface galactosyltransferase with immunoglobulins.

Detection of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of beta-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of beta-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface beta-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of beta-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-beta-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn2+ and UDP-[3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[3H]-galactose, beta-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > alpha chain > mu chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, beta-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the beta-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, alpha-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37 degrees C, the apparent affinity constants and association rate constants of interaction between cell surface beta-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and alpha 2 chain or monomeric IgA1 were in the range from 7.1 x 10(7) to 4.6 x 10(8) M-1 and from 1 x 10(5) to 3 x 10(6) M-1 s-1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 x 10(-2) to 5.0 x 10(-4) s-1 and from 33 to 1380 s, respectively. The number of alpha 2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 x 10(5) to 1.3 x 10(6). The binding of IgA by the cell surface beta-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.

Animals↗
PubMed1993

Interactions of galactosyltransferase with serum and secretory immunoglobulins and their component chains.

Assay of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of beta-1,4-GT activity have shown that beta-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of beta-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in beta-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the beta-1,4-GT activity was detected in fractions that contained lactoferrin and alpha-lactalbumin. Interactions of beta-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and alpha-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the beta-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of beta-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the beta-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral beta-1,4-GT in the presence of Mn2+. Beta-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely alpha, mu and J chains, bound beta-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1992

Transforming growth factor-beta inhibits the production of IgG, IgM, and IgA in human lymphocyte cultures.

Transforming growth factor beta (TGF beta) has potent immunoregulatory effects acting on both T and B cells. It strongly inhibits secretion of IgG and IgM in human and murine B cell cultures, but has been shown to have an enhancing effect on IgA production in the mouse. We have studied the effect of TGF beta on the production of IgA in human lymphocyte cultures. The addition of TGF beta to pokeweed-stimulated peripheral blood lymphocytes resulted in a suppression of IgA production of both subclasses, similar in magnitude to the suppression of IgG and IgM production. Membrane IgA expression was not increased by culturing tonsillar lymphocytes with TGF beta. In conclusion, we find no evidence for a selective enhancing effect of TGF beta on IgA synthesis in humans, in contrast to the findings reported in mice.

Adult↗
PubMed1992

Cytokine-induced immunoglobulin production in primary IgA nephropathy.

Increased IgA synthesis probably plays a role in the pathogenesis of IgA nephropathy (IgAN). We investigated whether an increased sensitivity to the effect of various growth factor combinations leads to increased immunoglobulin synthesis by peripheral blood mononuclear cells (PBMC) from IgAN patients, in comparison to healthy controls. Although none of the growth factors studied (pokeweed mitogen [PWM], interleukin [IL]-2, IL-6, transforming growth factor-beta [TGF-beta], and combinations) led to greater IgA synthesis in IgAN patients than in controls, the IgA subclass ratio was shifted in favor of IgA1. In controls, but not in IgAN patients, IL-2 enhanced the production of IgA and IgA1 compared with media alone. This possibly reflects previous in vivo activation by IL-2 in IgAN patients. The suppressive effect of TGF-beta on immunoglobulin synthesis was modestly greater in IgAN patients than in controls. Increased production of IL-2 and perhaps other cytokines by T cells in vivo may be responsible for the elevated IgA immune response in these patients.

Adolescent↗
PubMed1988

Receptor-mediated binding and uptake of immunoglobulin A by human liver.

We have studied the molecular mechanisms of the binding and uptake of secretory and serum immunoglobulin A (IgA) of both subclasses (1 and 2) and molecular forms (monomer and polymer) by the particulate fraction of human liver homogenate and by a human hepatoma cell line (HepG2). Inhibition by asialoorosomucoid and the requirement for the presence of calcium indicated that the binding of secretory IgA and polymeric IgA1 was mediated by the asialoglycoprotein receptor. Secretory component, which functions as a receptor for polymeric IgA in several animal species, was detected in the epithelial cells of bile ducts, but not in hepatocytes. Secretory IgA and all molecular forms and subclasses of serum IgA were bound by HepG2 cells, which do not express secretory component. The requirement for the presence of calcium, the presence of a terminal galactose residue in IgA, and the molecular weight of the major plasma membrane protein responsible for binding (41,700 daltons) indicated the involvement of asialoglycoprotein receptor. Immunoglobulin A proteins bound by HepG2 cells were endocytosed and catabolized.

Humans↗
PubMed1986

The stoichiometry of J chain in human secretory dimeric IgA.

Dimeric human secretory IgA was completely reduced with mercaptoethanol and alkylated with [14C]iodoacetamide. The component polypeptide chains were separated by high performance gel filtration in 5 M guanidine HCl into two fractions: one containing secretory component (SC) + heavy (H) chains; and the second containing light (L) + J chains. L and J chains were subsequently separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) or in alkaline urea. Calculations of the J chain stoichiometry in the dimeric secretory IgA (S-IgA) molecule were based on: the measurement of the ratio of radioactivities of SC + H chain and L + J chain-fractions or L chain- and J chain-fractions; the known stoichiometry of SC, H and L chains; and the known number of half-cystine residues in the component polypeptide chains of S-IgA molecule. The data demonstrated that one molecule of dimeric S-IgA contains approx. one J chain.

Chemical Phenomena↗
PubMed1985

Carbohydrate-mediated clearance of secretory IgA from the circulation.

Radioiodinated human secretory IgA (sIgA) injected intravenously into mice was rapidly cleared from the circulation by the liver. A portion of the sIgA was transported as an intact molecule into the bile. However, this transport was less efficient than that of human serum polymeric IgA (pIgA). The clearance of sIgA from the circulation was inhibited by prior injection of asialofetuin, suggesting that its uptake is mediated by the hepatic binding protein (HBP) specific for asialoglycoproteins. Mouse pIgA did not inhibit the hepatic clearance of sIgA. Results of in vivo studies were confirmed by in vitro experiments. The binding of 125I-asialoorosomucoid to either the particulate fraction (2000 g pellet of the homogenate) or the plasma membrane fraction of mouse liver was inhibited by sIgA. When polypeptide components of sIgA were used as inhibitors, significant inhibition was obtained with secretory component (SC), while inhibition with light and J-chains was not statistically significant. Examination of the inhibitory activity of IgA1 and IgA2 myeloma proteins and heavy chains isolated from these proteins revealed that binding of polymeric IgA1 and alpha 1 heavy chains can also be mediated by HBP. However, these interactions appear to be of lower avidity than those with SC. The inhibitory activity of human IgA2 and alpha 2 heavy chains was not significant. The involvement of HBP in binding of sIgA was also confirmed by measuring the inhibition of binding of 125I-sIgA. The binding of this protein by the particulate fraction of the mouse liver homogenate was inhibited by asialoglycoproteins and SC while inhibition with IgA1 and alpha 1 heavy chains was not significant. These results suggest that the carbohydrate moieties recognized by HBP reside primarily in the SC portion of sIgA.

Animals↗
PubMed1985

Urinary immunoglobulins in healthy individuals and children with acute pyelonephritis.

Urine samples obtained from children with acute pyelonephritis and from healthy children and adults were analysed with regard to the molecular form and specific antibody activity of urinary immunoglobulins. The urinary IgA and IgG levels were quantified in unconcentrated urine by radioimmunoassay. The children with urinary tract infection had significantly higher levels of IgG and IgA than age-matched controls but not higher than healthy adults. After tenfold concentration, the urine was fractionated on an Ultrogel AcA 22 column, and the IgA, secretory IgA, and IgG in the fractions were determined by radioimmunoassay. IgA in urine from healthy adults was predominantly represented by polymeric IgA linked to secretory component; small quantities of monomeric IgA were also present. IgG eluted in the position of the serum standard. Increased proportions of IgG and monomeric IgA were found in the infected patients. Specific antibody activity of the IgG and IgA classes to antigens of the infecting Escherichia coli strain was detected in whole and in fractionated urine from children with acute pyelonephritis. The specific antibody activity in healthy adults and children was low.

Acute Disease↗
PubMed1983

Biosynthesis of J-chain in human lymphoid cells producing immunoglobulins of various isotypes.

The relationship between synthesis, secretion, and subcellular localization of J-chain, IgM, IgA, and IgG was investigated in cultures of PWM-stimulated human PBL and in lymphoblastoid cell lines. Cells were examined for surface, cytoplasmic, and secreted immunoglobulins (Igs) and J-chain by immunofluorescence and radioimmunoassay (RIA). By these techniques, J-chain was detected in cells that produce polymeric or monomeric Igs. In PWM-stimulated PBL the synthesis of J-chain paralleled the production of Igs. In both PWM-stimulated (for 2 days) and unstimulated PBL, equal proportions of free and disulfide-linked J-chain were found. Increased amounts of intracellular J-chain were produced at later stages in PWM-stimulated PBL and J-chain occurred mostly in a free form. In tissue culture fluids, J-chain was not secreted in a free form but was always disulfide-linked to polymeric Igs. In lymphoblastoid cell lines, J-chain was present in a disulfide-linked form in IgM and IGA producers, but in IgG cells and in an IgM cell line (DAUDI) that did not secrete IgM but expressed it on the cell membrane, intracellular J-chain was present in free form. Although various proportions of polymeric and monomeric IgA were seen in culture fluids from IgA-secreting cell lines, intracellular IgA occurred mostly in a monomeric form. Further studies revealed that the ability to produce polymers was not equally distributed among all cells and might vary according to their content of J-chain and stage of maturation. Subcellular fractionation and subsequent analyses for J-chain and Ig in PWM-stimulated PBL and in IgM or IgG-producing cell lines revealed that these proteins were associated with fractions that contained ribosomes, cell sap, and low molecular weight RNA. In lysates of IgG and J-chain producing cells grown in the presence of 3H-labeled amino acids, intracellular J-chain was not disulfide-linked to IgG.

Cell Line↗
PubMed1983

IgA-mediated elimination of antigens by the hepatobiliary route.

Polymeric IgA antibody mediates the transport of corresponding antigens, in the form of IgA immune complexes (IC), from the circulation into the bile, whereas monomeric IgA, IgG, and IgM are ineffective. Transport has been shown with dinitrophenyl albumin and bacterial polysaccharides such as pneumococcal SIII and C-substance. The process does not result in breakdown of the antigen, which can be detected in the bile in intact free and IgA-bound forms. Although IgG promotes clearance of antigen from the circulation mainly to the liver, only low levels of breakdown fragments are detectable in bile. In mice, the mechanism of IgA IC transport does not appear to involve Kupffer cells, the complement system, or the glycoprotein receptors on liver cells because attempts to block these systems failed to affect transport. The mechanism appears to be analogous to that for free IgA. Transport could be inhibited by antigen-nonspecific IgA, but not IgG or IgM. IgA IC were found in the bile but not in the saliva, milk, urine, or bronchial or intestinal washings. A similar pattern of appearance was seen when IgA alone was injected. Thus hepatobiliary transport appears to be the major pathway for the clearance of both IgA IC and free IgA from the circulation.

Animals↗
PubMed1981

Shared idiotypes among anti-albumin antibodies of different species.

A system designed to detect plasma cells that produce antibodies directed at autologous idiotypic determinants of anti-human serum albumin (HSA) antibodies in rabbits was used to determine whether anti-HSA antibodies of horse, goat, swine and chicken origin were cross-reactive with rabbit antibodies of the same specificity. Fluorochrome-tagged anti-HSA preparations of these diverse species were used to stain splenic plasma cells of HSA-immunized rabbits and a similarly immunized chicken. The degree of idiotypic cross-reactivity, as detected by binding of anti-HSA antibodies to anti-idiotype within plasma cells of HSA-immunized animals, was sometimes equal to autologous staining. However chicken anti-HSA, the most phylogenetically distant idiotype examined, was demonstrably less cross-reactive than that obtained from the other species. Likewise, chicken plasma cells usually did not bind mammalian anti-HSA antibodies to an appreciable degree, as compared with autologous staining. These findings provide evidence for serologic and possibly structural similarities of antibodies of the same specificity from different species.

Animals↗
PubMed1981

Inhibition of the pokeweed mitogen-induced response of normal peripheral blood lymphocytes by humoral components of colostrum.

Colostral whey at dilutions up to 1 : 100 inhibited both the uptake of tritium-labelled thymidine and the differentiation of pokeweed mitogen-stimulated peripheral blood lymphocytes from normal adults into immunoglobulin-containing plasma cells. Upon gel filtration (Sephadex G-200), inhibitory activity was associated with high molecular weight fractions. Secretory component, but not monomeric, polymeric or colostral IgA, IgM, IgG, lactoferrin, casein or alpha-lactalbumin, inhibited the response to mitogen. Supernatants from cultures of colostral cells did not induce the differentiation of adult peripheral or cord blood lymphocytes into IgA-containing cells and did not stimulate the uptake of tritium-labelled thymidine.

Cell Differentiation↗
PubMed1980

IgA1 proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Streptococcus sanguis: comparative immunochemical studies.

IgA1 proteases from H. influenzae, N. meningitidis, S. pneumoniae, and S. sanguis were compared with respect to site of cleavage in the IgA1 molecule and EDTA sensitivity. Proteases from S. sanguis and S. pneumoniae cleaved the Pro (227)-Thr (228) bond within the hinge region of the alpha 1 chain and were inhibited by EDTA. H. influenzae IgA1 protease cleaved the Pro (231)-Ser (232) peptide bond. The activity of IgA1 proteases from H. influenzae and N. meningitidis was unaffected by EDTA. Purified and denatured alpha 1 chain was cleaved only in the hinge region. Other component chains of secretory IgA (secretory component, light and J chains) were not susceptible. In addition to IgA1 protease, S. pneumoniae released exo- and endoglycosidases that removed a considerable portion of carbohydrate side chains of IgA1; this activity was absent from crude IgA1 protease preparations of the other three bacterial species. Association in vitro of polymeric IgA1 with SC did not inhibit the degradation of IgA1 proteases. The considerable resistance of secretory IgA to cleavage by IgA1 proteases may be explained in part by the presence of IgA1 protease-neutralizing antibodies in secretory IgA.

Amino Acid Sequence↗