Chronic serum sickness glomerulonephritis: modification of the immune response influences the rate of removal of mesangial electron-dense deposits.

We have used a chronic serum sickness model of glomerulonephritis to investigate whether gross interference with the immune system can influence the rate of removal of antigen and established electron-dense deposits from the glomerulus. Radio-labelled cationized bovine serum albumin (BSA) was used as antigen. During the 2 weeks after the cessation of injections, the rate of removal of antigen from isolated glomeruli and from renal cortex, liver, spleen, and lung was measured. The rate of removal of mesangial and subepithelial deposits was assessed by point-counting. Urinary excretion of free and protein-bound isotope was also measured. Having quantified the rate of removal of antigen and deposits from the glomerulus, we attempted to influence the rate of removal by interfering with the immune response in the course of recovery. Contrary to our expectations, stimulation of the immune system with antigen in Freund's complete adjuvant, 4 days after the last injection of antigen, inhibited the removal of antigen and mesangial electron-dense deposits. Prednisolone had no detectable effect, but large doses of a non-nephritogenic form of the antigen (native BSA) enhanced removal. Removal of antigen and mesangial deposits was inversely correlated with the levels of circulating anti-BSA antibody, suggesting that specific antibody, circulating through the mesangium, inhibits the removal of antigen which is already trapped at that site. None of the forms of intervention applied during recovery produced a detectable change in the rate of removal of subepithelial deposits.

[Sorption properties of polystyrene plates used in immunoenzyme analyses]. / Izuchenie sorbtsionnykh svoistv polistirolovykh planshetov, ispol'zuemykh v immunofermentnom analize.

The capacity of polystyrene carriers used in the enzyme immunoassay (EIA) for adsorbing 131I-labeled human serum albumin under different conditions has been studied, and the comparison of the plates manufactured by Dynatech AG (Switzerland) and by the Leningrad Works of Medical Polymers has been made. At the first stages of the reaction the antigen is separated from the carrier and the amount of the desorbed antigen depends on its initial dose and the dilution of the assayed sera. The irregular desorption of the antigen leads to misinterpretation of the results. Comparison of the polystyrene plates has shown that each plate is characterized by individual adsorption capacity, which impedes at present the standardization of EIA-based test systems.

Influence of circulating maternal antibody on the transfer of dietary antigen to neonatal mice via milk.

125I-BSA injected intravenously into lactating mice persisted in the circulation for 4 h, entered the mammary gland, and was transferred to the neonate via the milk. When injected with anti-BSA antibodies, it was rapidly cleared from the circulation. Although animals receiving anti-BSA antibodies transferred more radioactivity to the neonate via the milk, the radioactivity was not TCA-precipitable or bound to staphylococcal protein A, nor was it immunoreactive. These findings suggest that circulating maternal antibody can limit the transfer of specific protein antigen from mother to newborn.

Immune complex formation analysed by high-performance size exclusion chromatography (HPLC-SEC) using either 125I-labelled antigen or enzyme-linked immunosorbent assay (ELISA) for detection.

A physical/immunochemical method has been developed for the analysis of soluble immune complexes. Human serum with high amount of antibody against bovine serum albumin (BSA) was incubated at 37 degrees with a wide range of 125I-labelled BSA and separated according to size by high-performance size exclusion chromatography (HPLC-SEC) on a TSK G 6000 PW column. Fractions were collected onto microplates and analysed either by gamma counting or by an enzyme-linked immunosorbent assay (ELISA) technique detecting anti-BSA antibody. The size of the immune complexes were the same when analysed by the two methods. The heaviest immune complexes were seen in moderate antibody excess where the majority of the complexes were eluted in a broad peak around Mr of 7 X 10(6). In large antibody excess the immune complexes eluted at around 3 X 10(6). Around equilibrium two peaks were seen corresponding to 3 X 10(6) and 5 X 10(5). In antigen excess the peak at 3 X 10(6) is diminished and free antigen appears. No changes in the distribution of the complexes were observed when the serum was made 20 mM in EDTA before incubation with BSA.

The effect of complement-depletion or mannosyl receptor blockade on the in vivo clearance of antigen during a primary immune response in rabbits.

The effect of complement-depletion mediated by cobra venom factor or mannosyl receptor blockade induced by ovalbumin injections on the blood clearance of in vivo-generated soluble immune complexes were analysed in 2 variants of a primary immune elimination model system in rabbits. In the first variant, animals were immunized with particulate antigen to elicit high titre IgM antibodies, and received radiolabelled antigen on the first day of immunization. In the second variant, radiolabelled antigen was introduced on Day 8 of the immunization schedule. The results showed that (a) immune clearance of antigen (Days 6-8) was not inhibited by prior decomplementation of the animals with cobra venom factor, and (b) injection of ovalbumin just prior to immune clearance did not significantly alter the clearance kinetics of the resulting immune complexes. Thus, the results indicate that clearance of soluble immune complexes generated during a primary immune response is not mediated via C3b-C3b receptor interaction or via mannosyl-mannosyl receptor interaction in the reticuloendothelial system, but proceeds through other mechanisms perhaps involving Fc receptors.

Antigen-induced arthritis: inflammation and antigen handling after two different doses of intra-articularly injected antigen.

We studied (i) the inflammatory response induced by intra-articular (i.a.) injection of 2.5 and 0.5 mg 125I-labelled bovine serum albumin (125I-BSA) into the paired knee joints of rabbits immunized with BSA in Freund's complete adjuvant, and (ii) handling of these different doses of antigen by means of regular external radioactivity measurements. The joint inflammation was quantified by 99mTechnetium pertechnetate uptake measurements. More severe arthritis was seen in the 2.5 mg BSA-injected than in the paired 0.5 mg BSA-injected knee, both in the early and in the late phase of antigen-induced arthritis. External radioactivity measurements showed enhanced disappearance of radioactivity from the 2.5 mg 125I-BSA challenged knee joint, resulting in lower percentual retention of the dose injected than in the 0.5 mg injected knee joint. However the calculated absolute amount of long term retained BSA in the 2.5 mg BSA injected knee, 4 weeks after i.a. injection, was still about 2.8 times the quantity of BSA retained in the 0.5 mg BSA-injected paired knee joint. These data indicate that the severity of both the early and late phase of antigen-induced arthritis is i.a. antigen dose-dependent and, in addition, suggest that handling of i.a. antigen depends in part on the severity of joint inflammation.

Clearance of immune complexes formed in normal and leukaemic rats.

Immune complexes of 125I-HSA-rat anti-HSA formed in vivo under conditions of antibody excess were rapidly cleared from the circulation of both normal and leukaemic Hooded rats. In HSA-immune rats most of the 125I-HSA present in the blood was found to be cell-bound, but a proportion was present as circulating immune complexes that could be precipitated from plasma by 2.5% polyethylene glycol. There was no evidence that clearance of a soluble antigen was impaired in leukaemic animals.

Absorption of inhaled antigen into the circulation of isolated lungs from normal and immunized rabbits.

The purpose of this study was to determine whether the absorption of inhaled antigen (Ag) across the pulmonary air-blood barrier of the isolated perfused lung can be modulated by immunologic mechanisms. Lungs from immunized or nonimmunized rabbits were removed, ventilated, and perfused with autochthonous blood. Radioiodinated Ag (human serum albumin or ovalbumin) was introduced as an aerosol into the isolated lung for 15 min and blood samples were taken over a 4-h period. The results showed that radioactivity fom inhaled Ag entered the perfusing blood as two fractions. One fraction was precipitable by 5% trichloroacetic acid or antiserum. The TCA-soluble fraction chromatographed differently from iodide and may have represented metabolites of the Ag. Immunization specifically reduced the amount of antigenically intact protein entering the blood. On the other hand, the metabolite reached higher concentrations in the blood of immunized lungs. We conclude that the alveolar capillary barrier of the normal rabbit lung could provide a significant route of entry for inhaled antigen into the systemic circulation and that immunization reduces absorption via this route and enhances pulmonary metabolism of the Ag.

Analysis of the major histocompatibility complex in Syrian hamsters. I. Skin graft rejection, graft-versus-host reactions, mixed lymphocyte reactions, and immune response genes in inbred strains.

Five inbred strains of Syrian hamsters (Mesocricetus auratus) were examined for the presence of disparity at a genetic region similar to the major histocompatibility complex in other species. Vigorous reciprocal alloreactions developed in several strain combinations, which resulted in acute skin graft rejection, strong mixed lymphocyte reactions, and potent graft-versus-host reactions. In addition, we found evidence for an immune response gene which controls the antibody response to bovine serum albumin. Patterns of alloreactivity observed for skin graft rejection, graft-versus-host reactivity, and mixed lymphocyte reactivity are compatible with the hypothesis that hamsters possess a major histocompatibility complex, but the absence of discernable disparity at a serologically defined locus makes a definitive statement premature.

[Preparation of an iodinated penicillin derivative for radioimmunologic use]. / Préparation d'un dérivé iodé de la pénicilline utilisable en radio-immunologie

A 125I-BSA Penicilloyl conjugate was prepared by coupling Penicillin G to Bovine Serum Albumine previously labeled with Iodine-125. The reaction of fixation by covalent binding was made in alkaline solution without the use of carbodiimide. Immunoreactivity and specific activity of this labeled conjugate enable radioimmunoassay of penicilloyl groups.

The effect of various immunosuppressive agents on mouse peritoneal macrophages and on the in vitro phagocytosis of Escherichia coli O4:K3:H5 and degradation of 125I-labelled HSA-antibody complexes by these cells.

Large doses of hydrocortisone, cyclophosphamide, and methotrexate injected subcutaneously, and whole-body irradiation (500 rads) caused a reduction in the number of peritoneal cells (PE cells) obtained after intraperitoneal injection of the treated mice with proteose-peptone. The same dose of cyclophosphamide and irradiation induced morphological changes in PE macrophages. There were more giant cells in the peritoneal exudates from treated mice as compared to control mice. 'Pharmacological' and larger doses of hydrocortisone, methotrexate and azathioprine or anti-lymphocyte globulin had no effect on the in vitro phagocytic capacity of proteose-peptone-stimulated mouse PE macrophages. This also applied to doses of up to 50 mg/kg of cyclophosphamide. In contrast, whole-body irradiation (500 rad) and 100 mg/kg of cyclophosphamide decreased the phagocytic capacity of mouse macrophages in vitro and reduced the ability of PE cells to degrade 125I-labelled HSA-antibody complexes in vitro. The greatest effect was noted 4-5 days after whole-body irradiation or four to five subcutaneous injections of cyclophosphamide.