Effect of normal and immune IgG, IgM and IgA on the intestinal microflora of mice with experimental dysbacteriosis

Human IgG, IgM and IgA produce a pronounced protective effect, preventing enterobacteria from penetration into the mucous membrane of the proximal section of the small intestine of mice in antibiotic-induced dysbacteriosis. Normal mouse IgG and IgM, in contrast to IgA, are effective against mucosal enterobacteria of the small intestine. Immune mouse IgG, IgM and IgA show greater activity in protecting the mucous membrane than normal immunoglobulins of these classes.     

Heterogeneity of binding of human IgA subclasses to protein A.

The ability of human IgA myeloma immunoglobulins to interact with protein A-containing Staphylococcus aureus was examined. Some IgA1 and IgA2 immunoglobulins bound to S. aureus although others of both subclasses failed to do so. These results were obtained by using both direct binding of radiolabeled immunoglobulins to S. aureus and with inhibition-type assays. Binding was dependent on the Fc fragment of IgA since there was no binding to S. aureus by an F(ab')2 fragment of IgA1. Nonprotein A-containing bacteria did not bind these immunoglobulins and isolated protein A interacted with radiolabeled immunoglobulins. This strongly suggested that protein A was responsible for the observed binding to S. aureus. These data indicate, in contrast to previous reports, that there is no simple relationship between IgA subclass and the capacity to bind to protein A.     

Determination of antiplatelet antibodies on the surface of platelets by direct radioimmune method in patients with different types of immune thrombocytopenia

Direct radioimmune assay (RIA) have been developed for detection of antibodies associated wild platelet membrane. Platelets from 12 patients with idiopathic thrombocytopenic purpura (ITP) and 27 patients with chronic lymphocytic leukemia (CLL) (platelet count (100,000 in 1 microliters) have been tested. Antibodies on platelets surface have been detected in all 12 patients with ITP and in 21 patients with CLL. In 6 CLL patients the number of immunoglobulins associated with platelets surface does not increase control level. It is possible, that in some CLL patients development of thrombocytopenia is mediated not only by platelet associated antibodies but by other mechanisms, one of which can be linked with the depression of megakaryocytes growth in bone marrow. Direct RIA for measurement of antibodies on platelet surface detect antiplatelet antibodies with higher frequency than indirect enzyme-linked-immunosorbent-assay (ELISA), developed earlier for assessment of antiplatelet antibodies in serum. Increase of platelet count in CLL patients after steroid and cytostatic treatment correlated with the decrease of platelet surface associated antibodies.     

Compactin (ML-236B) reduces the content of filipin-cholesterol complexes in the plasma membrane of chronic lymphocytic leukemia cells

The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15% delipidized horse serum or 15% normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.     

Dual binding specificities in MOPC 384 and 870 murine myeloma immunoglobulins.

Homogeneous murine myeloma immunoglobulins (IgA, kappa), M 384, and M 870, bind methyl alpha-D-galactopyranoside and phosphorylcholine at different subsites. Heterologous recombinant immunoglobulins of these two immunoglobulins with M 603 (a homogeneous IgA, kappa with known phosphorylcholine specificity) also bind phosphorylcholine.     

Unknown functions of immunoglobulins A

Traditionally, the function of immunoglobulins A (IgA), the major type of secreted antibodies, has been thought to be restricted to binding antigens outside the epithelium basal membrane. Therefore, effector mechanisms eliminating IgA-opsonized targets have not been investigated so far. However, some indirect observations of infectious agents penetrating into tissues and blood from the environment suggest such mechanisms (analogous to IgG/IgM-dependent activation of complement and natural killers). In the present review, we examine details of IgA structure that might contribute to elucidation of IgA-dependent effector functions in human and animal immunity. Special attention is given to a putative transduction of signal about antigen binding in the active center of IgA from the Fab- to the Fc-superdomain via intramolecular conformational rearrangements. Different structure of the IgA subclasses (IgA1 and IgA2) is examined taking into account probable divergence of their functions in immune response.     

Decrease in expression of human J-chain in lung squamous cell cancer and adenocarcinoma

Secretory polymeric immunoglobulins (IgA dimers and IgM pentamers) are unique in that, apart from L- and H-chains, they contain J-chains responsible for their oligomerization. These antibodies are part of the local adaptive immune system acting on mucosa membranes of the respiratory and digestive systems as the first protection barrier to potential infectious agents. Secretory polymeric immunoglobulins are produced by highly specific B-cells and actively transported to the surface of mucosa membrane through epithelium cells. Therefore, their synthesis and J-chain content are dependent upon epithelium translocation function and condition that are markedly affected by tumorous transformation. Here, we used RT-PCR and immunoblotting to study of the J-chain content and its mRNA expression level in normal and tumorous tissues in lung squamous cell cancer and adenocarcinoma at various stages of disease progression.     

Expression of the surface membrane receptors of neutrophils in myeloproliferative diseases

For 147 patients with myeloproliferative diseases, a study was made of the expression of Fc-receptors to immunoglobulins IgC, IgA, and that of FcH-receptor and receptors to C3-components of the complement in peripheral blood neutrophils. The data obtained show the lower level of neutrophils having membrane receptors in patients with acute myeloblastic leukemia and chronic granulocytic leukemia at the stage of blast transformation. A decrease in expression of membrane receptors of neutrophils was shown in patients with chronic granulocytic leukemia and benign subleukemic myelosis. The finding of a higher level of neutrophils having surface receptors revealed in patients with real polycythemia is close to the data obtained in the study of expression of membrane receptors in patients with chronic myeloproliferative diseases and healthy persons.     

Immunohistochemical localization of immunoglobulins A, G and M in the mouse female genital tract

The localization of immunoglobulins A, G and M (IgA, IgG, IgM) in the mouse genital tract was studied by immunoperoxidase techniques at oestrus, on the day of mating and at the time of implantation. In the horns and body of the uterus, IgA and IgG were located in plasma cells in the endometrium surrounding uterine glands and in the gland lumina. The numbers of these plasma cells increased markedly between Day 1 and Days 4 and 5 of pregnancy and the ratio of plasma cells containing IgA and IgG was about 3 or 4 to 1 at all stages. Area measurements indicated that the increased number of plasmacytes was not due to an increase in the amount of endometrial, myometrial or glandular tissues. Plasma cells were not detected in the cervix and vaginal fornix at oestrus and Day 1, but a few were present on Day 5. In the oviduct, plasma cells containing IgA and IgG were present only in the preampulla and both immunoglobulins were present in the extracellular space of the lamina propria only in this region. No IgM was detected in any part of the reproductive tract at any of the times studied. Uteri on Day 1 of pregnancy contained bacteria of several kinds, some of which were aggregated and coated with IgA. This suggests that the uterine lumen at this time may contain specific anti-bacterial IgA antibodies. Our observations indicate that the horns and body of the uterus and the preampulla of the oviduct are major sites of a local immune system in the female mouse genital tract.     

Specific immunoglobulin response in mice immunized with porins and challenged with Salmonella typhi

Specific antiporin antibody (IgG, IgM, and IgA) response was studied in control, infected, immunized-infected, and immunized mice. The activity of specific IgG immunoglobulins was found to be the highest in immunized and immunized-infected groups in which 87.5% protection had been observed by laboratory potency test in mice; the next-highest activities were those of IgM and IgA immunoglobulins. However, in the infected group the activity of specific IgM and IgG immunoglobulins was almost similar.     

Rat liver membrane secretory component is larger than free secretory component in bile: evidence for proteolytic conversion of membrane form to free form

Secretory component is a receptor for polymeric immunoglobulins on epithelial cells and hepatocytes that facilitates transport of polymeric immunoglobulins into external secretions. Little is known about the transcellular migration of secretory component-polymeric IgA complexes or the membrane forms of secretory component. We therefore examined rat bile and liver membranes to identify and compare the various molecular species of secretory component. Bile or liver membrane proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Protein profiles on blots were probed with antisecretory component antiserum, and the immunoreactive bands were visualized by indirect immunoperoxidase staining. Bile collected in the presence of proteolytic inhibitors showed an immunoreactive doublet band (Mr = 82,000 and 78,000) in the molecular weight range of free secretory component. By contrast, free secretory component in bile collected in the absence of proteolytic inhibitors and purified by affinity chromatography migrated as a single protein with an Mr = 70,000. Both components of the free secretory component doublet bound dimeric IgA when blots were probed with human dimeric IgA. Crude liver membranes prepared in the presence of proteolytic inhibitors showed two immunoreactive secretory component-containing bands, Mr = 107,000 and 99,000, whereas membranes prepared without proteolytic inhibitors showed two smaller immunoreactive bands; one of these proteolytically severed proteins comigrated with the 82,000-dalton free secretory component in bile. These results indicate that membrane forms of secretory component are present in rat liver. The observations that the membrane secretory component is larger than biliary free secretory component and yields biliary SC-like forms of secretory component upon proteolysis support the hypothesis that free secretory component in bile is a proteolytic product of larger liver membrane-associated secretory component.     

Anti-glycosyl antibodies in lipid rafts of the enterocyte brush border: a possible host defense against pathogens

The pig small intestinal brush border is a glycoprotein- and glycolipid-rich membrane that functions as a digestive/absorptive surface for dietary nutrients as well as a permeability barrier for pathogens. The present work was performed to identify carbohydrate-binding (lectinlike) proteins associated with the brush border. Chromatography on lactose-agarose was used to isolate such proteins, and their localization was studied biochemically and by immunofluorescence microscopy and immunogold electron microscopy. IgG and IgM were the two major proteins isolated, indicating that naturally occurring anti-glycosyl antibodies are among the major lectinlike proteins in the gut. IgG and IgM as well as IgA were localized to the enterocyte brush border, and a brief lactose wash partially released all three immunoglobulins from the membrane, indicating that anti-glycosyl antibodies constitute a major part of the immunoglobulins at the lumenal surface of the gut. The antibodies were associated with lipid rafts at the brush border, and they frequently (52%) coclustered with the raft marker galectin 4. A lactose wash increased the susceptibility of the brush border toward lectin peanut agglutin and cholera toxin B, suggesting that anti-glycosyl antibodies compete with other carbohydrate-binding proteins at the lumenal surface of the gut. Thus anti-glycosyl antibodies constitute a major group of proteins associated with the enterocyte brush border membrane. We propose they function by protecting the lipid raft microdomains of the brush border against pathogens.     

Orientation and kinetics of surface denaturation of normal human and myeloma IgA in monolayers at the interface of air-NaCl aqueous solutions

Normal serum immunoglobulin A (IgA) and abnormal IgA isolated from serum of patients with multiple A-myeloma have been studied by monolayer technique at air--NaCl solution interfaces. Normal IgA analogous to human normal IgG and secretory IgA was shown to have horizontal orientation at air--water interface. Only some abnormal IgA were similar to myeloma IgG and differed from the normal ones by their orientation at phase border. Majority of myeloma IgA under study could not be distinguished from the normal ones by orientation and denaturation kinetics at interface. B-lymphocytes of the first group of patients were assumed to carry IgA-receptors at their surface, but B-lymphocytes of the second group of patients carried Ig receptors of some other class of immunoglobulins.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

The Interactions of Porcine and Ovine,Serum and Colostral Immunoglobulins with Staphylococcal Protein A

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produced by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG₁, IgG₂, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG₁ from most ewes possessed a low affinity for Protein A whereas ovine IgG₂ generally possessed a high affinity; 100% of the IgG₂ in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.     

Dynamics of the accumulation of the basic immunoglobulin classes in the intestinal contents in food toxinfections]

The work presents the data on the dynamics of accululation of the main classes of immunoglobulins (A, G and M) in coprofiltrates obtained from patients with alimentary toxicoinfections (bacteriologically confirmed salmonellosis and diseases of unknown etiology). The levels of immunoglobulins of all classes (mainly IgA and IgG) were shown to be elevated in the process of the disease. The dynamics of the increase in the level of IgA (both general and secretory) was supposed to indicate the formation of local immunity in the intestinal wall. The presence of serum IgA and the characteristic IgG dynamics seemed to be indicative of destructive processes occurring in the intestinal wall. Thus, the dynamics of accumulation of immunoglobulins in coprofiltrates obtained from patients with alimentary toxicoinfections reflects the main local pathological and immunological processes.     

IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde–Acetaldehyde Adduct,an Immunodominant Oxidation-Specific Epitope

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde–acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.     

Antilipoprotein autoimmune hyperlipidemia. The Ig-Lp test

In the antilipoprotein type of autoimmune hyperlipidemia (AIH), the immunoglobulins (Ig) are bound to lipoproteins by their antibody site and circulate as immune Ig-Lp complexes. In the earlier studies, the specific antibody activities were demonstrated in vitro by specific but rather sophisticated methods which were not suitable for the screening of antilipoprotein AIH in large populations. In the Ig-Lp test described here, the immunoglobulins bound to the low density lipoproteins (Ig-Lp) are detected by floating the complexes at D 1.10 in the ultracentrifuge in a physiological saline sucrose density gradient; delipidating them by ether in the presence of 0.2 M urea, and assaying the protein by radial immunodiffusion and laser immunonephelometry with antisera specific for IgG, IgA, IgM, low density lipoproteins and albumin. Radial immunodioffusion and immunonephelometry gave similar results. This Ig-Lp test was positive in 5 myelomas associated with hyperlipidemia, which were previously classified as AIH with specific methods. And the test was specific for the Ig type of the monoclonal antibody involved in each case (3 IgA, 1 IgG and 1 IgM). It was negative in 6 normolipidemic myelomas and also in 40 sera from healthy blood donors and one normal serum taken 4 hours after a fat meal. Although the Ig-Lp test is not specific for antilipoprotein antibodies, the results of this study allow to use if for the screening for AIH.     

Effect of immunoglobulins and IgG-fragments on the human erythrocyte aggregation, studied by a rheoscope combined with image analyzer

Using a rheoscope, combined with a TV image analyzer and a computer, the effects of immunoglobulins and IgG-fragments on the process of human erythrocyte aggregation were determined. The immunoglobulins accelerated the aggregation; the effect increased with their molecular weight, i.e., IgG congruent to gamma-globulin less than IgA less than IgM. An empirical relationship, expressing the dependence on the immunoglobulin concentrations, was proposed. F(ab')2-fragment accelerated the aggregation, more effectively than IgG, while Fab- and Fc-fragments did not. Therefore, two Fab-portions are presumably needed to form the aggregates, and the flexibility between two Fab-portions may be important. A case of multiple myeloma showed an increased aggregation, due to the increased myeloma protein.