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The pathogenesis of excess cardiovascular risk in type 1 diabetes is unclear. LDL cholesterol is only weakly predictive, and its concentration is often normal in type 1 diabetes. We therefore examined whether markers of LDL oxidation such as antibodies to oxidized LDL (Ab-OxLDL) and LDL-containing immune complexes, rather than LDL concentration, were predictive of coronary artery disease (CAD) in type 1 diabetes. This nested case-control study from an epidemiologic cohort study included 49 incident cases of myocardial infarction (MI), angina, or CAD death and 49 age-, sex-, and duration-matched control subjects. Ab-OxLDL was measured by enzyme immunoassay and the apolipoprotein B (ApoB) content of immune complexes (ApoB-IC) precipitated by polyethylene glycol by immunoelectrophoresis in baseline stored samples. Ab-OxLDL was inversely, and ApoB-IC directly, related to subsequent CAD. In multivariate analyses, Ab-OxLDL remained a significant independent predictor along with previously recognized predictors, hypertension and Beck depression score. In conclusion, oxidation of LDL and the immune response it elicits may play a role in predicting the development of CAD in type 1 diabetes and explain at least some of the enhanced CAD risk in type I diabetes. [ABSTRACT FROM AUTHOR]

We investigated the hypothesis that modified lipoproteins trigger an immune response leading to the production of autoantibodies and subsequently to the formation of atherogenic immune complexes (IC). We recruited 20 type 2 diabetic patients with macrovascular disease, 14 nondiabetic patients with coronary artery disease (CAD), and 34 healthy control subjects matched for age, sex, and race. Serum antibodies to oxidized and glycated LDL did not differ significantly among the 3 groups. Serum IC contained variable, but not statistically different, amounts of IgG, IgM, and IgA. In contrast, the content of cholesterol in IC isolated from diabetic patients was significantly higher than that in IC isolated from control subjects, and the content of apolipoprotein (apo)-B was significantly higher than that in IC isolated from control subjects and patients with CAD. Cholesteryl ester accumulation in human monocyte-derived macrophages incubated with IC, a measure of the atherogenic potential of IC, was significantly higher in macrophages incubated with red blood cell-adsorbed IC isolated from diabetic patients compared with IC isolated from control subjects (P < 0.03) or from patients with CAD (P < 0.04) and was strongly correlated with the content of apoB (r = 0.68, P < 0.001) and cholesterol (r = 0.61, P < 0.001) in IC. LDL from diabetic patients was more susceptible to oxidation in vitro, was significantly smaller, and contained significantly less alpha-tocopherol than LDL isolated from subjects in the other groups. In addition, the n-3 polyunsaturated fatty acid content of phospholipids and cholesteryl esters in LDL isolated from diabetic patients was significantly increased (P < 0.05) compared with that from patients with CAD or from control subjects. We postulate that LDL size, susceptibility to oxidation, and lipid fatty acid composition may play a critical role in the production of antibodies to oxidized LDL and consequently in the formation of LDL-containing IC in patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, C1q 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. [ABSTRACT FROM AUTHOR]

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, β 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. [ABSTRACT FROM AUTHOR]

The existence of soluble insulin-anti-insulin immune complexes in the serum of patients with diabetes mellitus was investigated. Formation of such immune complexes in vitro was studied by adding radioiodinated insulin to the sera of patients with anti-insulin antibodies; immune complexes were formed readily, but apparently differed from patient to patient. Immune complexes formed in vitro were precipitated with 5% polyethylene glycol. They eluted in the high-molecular weight fractions when the precipitated material was fractionated by get filtration, and they remained bound at neutral pH when the high-molecular weight Tractions were submitted to affinity chromatography on protein A-Sepharose. When the bound immune complexes were recovered by acid elution and immediately filtered through a Sephadex G-50 column equilibrated with the same acid buffer, free antigen (radiolabelled insulin) and antibody were recovered. This antibody, after neutralization, showed binding capacity when remixed with radioiodinated insulin. When this protocol was applied to a serum that gave positive results in several screening methods for soluble immune complexes, insulin was detected by radioimmunoassay in the high-molecular weight fractions separated from the 5% polyethylene glycol precipitate and in the fractions retained by protein A; however, no free insulin was detected after gel filtration in Sephadex G-50, perhaps due to excessive dilution. The high-molecular weight fraction did have binding capacity for radioiodinated insulin. No insulin-binding protein could be recovered with a similar procedure from a serum negative by all screening tests for soluble immune complexes. These results prove that soluble immune complexes can be formed easily in sera containing anti-insulin antibodies and can be recovered from sera of diabetic patients that show positive results in screening techniques for soluble immune complexes. [ABSTRACT FROM AUTHOR]

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 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Soluble immune complexes (IC) arc known to bind to human red blood cells (HRBC). Most authors have attributed this binding to the interaction between IC-bound C3b and it red cell CR1 receptor, but contradictory data has been published concerning the ability of IC to bind to HRBC in the absence of complement. Using soluble tetanus toxoid-rabbit anti-tetanus toxoid (TT-ATT) IC, we have shown that binding through the CR1 receptor takes place when IC are formed at antibody excess, while IC formed at antigen excess do not require complement for erythrocyte binding. Once absorbed to HRBC, IC are recognized by CR1 and/or Fc receptors on phagocytic cells. This interaction is not associated with red cell engulfment, but using radiolabelled S. aureus protein A as a probe, we have demonstrated the transfer of IC from HRBC to phagocytic cells. Such transfer without red blood cell (RBC) damage agrees with the postulated role of RBC in the elimination of soluble IC from circulation. However, we have also demonstrated that the interaction between HRBC-IC and phagocytic cells is associated with the release of mediators of inflammation. It is, therefore, not absolutely clear whether the interaction of RBC-adsorbed IC and phagocytic cells will always have beneficial consequences. [ABSTRACT FROM AUTHOR]

In this report we compare the effect of stimulation of peripheral mononuclear cells (PBMC) by using two monoclonal antibodies (MoAb) directed against the CD2 receptor on T cells or by using autologous erythrocytes (E) which express on their surface lymphocyte function-associated antigen 3 (LFA3), a natural ligand for CD2, The addition of autologous erythrocytes to pokeweed mitogen (PWM)-stimulated PBMC results in the enhancement of polyclonal immunoglobulin synthesis and of antigen-specific B-cell responses. Because B cells lack the CD2 molecule, it can be concluded that their enhanced activity is a consequence of the delivery of activating signals by activated T lymphocytes. When PBMC cultures were stimulated with a pair of anti-CD2 MoAb (Lcu5b and V1TI3) we were able to induce polyclonal immunoglobulin synthesis, particularly IgM, in cultures supplemented with interleukin 2 (IL-2), Specific responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) were also enhanced by the addition of autologous E to PWM-stimulated PBMC, Significant anti-TT responses were observed in cultures stimulated with E+I+TT+IL-2. In contrast, stimulation of PBMC with VIT13+I+Leu5b+IL-2-(-antigen was not effective in inducing anli-TT antibody and only weakly effective in inducing anti-KLH antibodies. Replacing Leu5b by anti-CD3 had no effect on the induction of specific antibody responses; in contrast, replacement of Leu5b by E enhanced anti- TT antibody production while the effect on polyclonal production of IgM was minimal. Therefore, it appears that the signal delivered by the association of CD2 with LFA3 is a better potentiating signal for specific B-cell responses than the signal delivered by pairs of MoAb to different epitopes orCD2 or to CD2 and CD3 epitopes, [ABSTRACT FROM AUTHOR]

We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear cells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH, and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (1:50 ratio of PBMNC/RBC). The cultures were harvested on day 11: immunoglobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for Calibration. While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-TT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in the presence of autologous RBC Similarly, anti-KLH responses were easier to induce with PBMNC from an immune donor; maximal response was observed after stimulation with PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pre-treatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-y). However, addition of IFN-γ in different doses and at different times to PWM-stimulated PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody. [ABSTRACT FROM AUTHOR]

Human lymphocytes were cultured under different conditions to determine the effects of technical variations on the response to pokeweed mitogen (PWM) and Staphylococcus aureus as measured by (a) quantitation of immunoglobulins (Ig) in the culture supernatants and (b) enumeration of Ig-secreting cells (ISC) by a reverse plaque technique. The highest numbers of ISC were measured when the cells were cultivated in standing tubes, without glutamine supplementation during the culture, and at a concentration of 1 × 106 cells/ml. The highest Ig concentrations were measured under similar conditions, except that somewhat higher values were obtained with cells cultivated at 1 × 106/ml in 2 ml cultures with PWM stimulation and at 2.5 ×106/ml in 1 ml cultures with S. aureus stimulation. In time-course studies, peak ISC responses occurred on day 5 with each mitogen, whereas extracellular Ig levels kept rising until day 7, possibly owing to accumulation of secreted Ig. Measurements of the numbers of ISC and of secreted Ig levels in simultaneous cultures of lymphocytes from the same donors showed no correlation: however, co-stimulation of cultures with PWM and concanavalin A (to stimulate suppressor cells) depressed both Ig levels and ISC numbers. These results suggest heterogeneity in the plaque-forming cell population with respect to rate of Ig secretion, but indicate that these two techniques both reflect B-cell activation. They should not, however, be considered interchangeable, and the two probably should be used in conjunction for complete characterization of B-cell activation. [ABSTRACT FROM AUTHOR]

The saliva of an individual with selective IgA deficiency was found to contain IgG and IgM, with some of the IgM linked to secretory component. Some specimens showed evidence of low molecular weight Immunoglobulin fragments, presumed to be the result of proteolysis. [ABSTRACT FROM AUTHOR]

An immunochemical characterization of salivary immunoglobulins from ten patients with multiple myeloma was undertaken. Seven of the patients had IgA monoclonal components and their transfer to saliva could be proved immunochemically in five. Three salivas containing monoclonal IgA were fractionated by gel filtration on Sephadex G-200, and secretory component could be detected in the same peaks as the monoclonal IgA. Molecular si/e studies by electrophoresis in sodium dodecyl sulphate-polyacrylamide gel showed that in most salivas oligomeric forms of IgA were exclusively or predominantly detected. These findings suggest that oligomeric IgA of systemic origin might be as effectively transferred to external secretions as oligomeric IgA of regional origin. Of the three remaining patients, two had IgG monoclonal proteins that could be detected in concentrated saliva, while the monoclonal component of the last patient was of light chain type. In this last patient no free light chains were detected in concentrated saliva, but normal IgA as well as an apparently increased amount of polyclonal IgG were evident. [ABSTRACT FROM AUTHOR]

This article focuses on the placental transfer of human immunoglobulin G (IgG) subclass. IgG is unique among the major immunoglobulin classes for its active transfer across the placenta. Since the description of four IgG subclasses determined by structural differences located on the heavy chains, attempts have been made to determine whether all four subclasses were equally transferred. IgG subclasses were titrated with the antisera. Results are expressed as titres corresponding to the last double dilution showing precipitation with subclass specific antisera.

Heavy chain subclasses of 121 IgG monoclonal proteins were found to be 74% IgG1, 10% IgG2, 12% IgG3 and 4% IgG4. Light chain subclasses showed a slight kappa excess among IgG1 proteins. Electrophoretic studies of these monoclonal proteins and of normal γ-globulin showed IgG1 had a wide range of mobility including the most slow (electronegative) proteins; IgG2 and IgG4 were among the Faster moving, while IgG3 showed medium to slow mobility. The above random distribution of IgG subclasses was found in eighteen monoclonal proteins causing hyperviscosity syndrome, whereas among fourteen monoclonal cryoglobulins there was an excess of IgG2 and IgG3 subclasses. [ABSTRACT FROM AUTHOR]

The serum of an IgA myeloma patient was found to contain at least 90% of the monoclonal protein as a 10-05S disulphide-bonded dimer. Small amounts of monomeric and higher oligomeric forms were also observed. Light chains were K type, disulphide-bonded to heavy chains and preferentially released upon reduction with dithioerithritol. Electron microscopy of the dimer and oligomeric forms showed structures suggesting linkage between Y-shaped monomer units via the terminus of the Fc fragment. The data is discussed in relation to secretion of the different oligomeric forms. [ABSTRACT FROM AUTHOR]

A semi-quantitative assay method was used to study the distribution of IgG heavy chain sub-classes in normal IgG and the fractions obtained from it by isoelectric focusing, and in the residual IgG from hypogammaglobulinaemic sera. The assay of the isoelectric focusing fractions proved that IgG2 was present in the fractions of lowest pH; IgG1, although detectable in all fractions, was maximal at high pH values, and was the only sub-class found in the pH 9.5 fraction, which was the highest pH found. IgG3 was detected in fractions with intermediate pH values. The assay of hypogammaglobulinaemic sera proved that the residual IgG present in these sera could have a grossly abnormal distribution of sub-classes mainly due to the variability of the IgG2 and IgG3 titres. [ABSTRACT FROM AUTHOR]

The action of 0.05 M DL-penicillamine (DL-P) and α-mercaptopropionylgIycine upon immunoglobulins, both in their native serum and after isolation, has been studied. IgM was always affected, apparently with breakdown of 19S polymers and release of 7S sub-units. This depolymerization leads both to a change in electrophoretic mobility and to increased diffusibility in gels, as well as to decrease in serum viscosity. IgG, as a rule, is not affected when studied in whole serum. The exception to the rule is provided by monoclonal proteins of the IgG3 subgroup. The observed are variable, and may consist of an increase in the electropositivity of the molecules, the release of light chains, or the splitting into Fab- and Fc-like fragments. IgA was irregularly affected. The results suggest that several types of action may be present: depolymerization of high molecular weight polymers, which seem to be more resistant to the action of DL-P than its IgM counterparts, unfolding of 7S molecules with increased electropositivity, and breakdown of 7S molecules into halves, each containing both H and L chains. Precipitation of cryoglobulins was inhibited by both thiols, both when added serum or isolated proteins. [ABSTRACT FROM AUTHOR]