Arpad Zsigmond Barabas

Production of a new model of slowly progressive Heymann nephritis

Summary.  A slowly progressive autoimmune kidney disease was induced in Sprague Dawley rats by subcutaneous injection of a chemically modified kidney antigen (rKF3), incorporated into Alum and Distemper complex vaccine, followed by subcutaneous injections of an aqueous preparation of the same antigen. Pathogenic autoantibodies developed, which reacted with fixed glomerular nephritogenic antigen. Subsequently, immunopathological events lead to chronic progressive immune complex glomerulonephritis and proteinuria.

Down-regulation of pathogenic autoantibody response in a slowly progressive Heymann nephritis kidney disease model

In the present article, we describe an antigen‐specific down‐regulation of a pathogenic autoantibody (aab)‐mediated disease process in an experimental autoimmune kidney disease in rats called slowly progressive Heymann nephritis (SPHN). This autoimmune disease is initiated and maintained by pathogenic immunoglobulin G (IgG) autoantibodies (aabs), which cause an immune‐complex (IC) glomerulonephritis associated with proteinuria. We achieved down‐regulated pathogenic aab response in SPHN rats by injections of an IC containing the native nephritogenic antigen and specific high‐titred nonpathogenic IgM aabs, in antigen excess. The injected IC increased the level of circulating nonpathogenic IgM aabs; the increased levels of specific IgM aabs in turn facilitated the removal of the injected altered nephritogenic and liberated autoantigens from the renal tubules and greatly diminished the production of pathogenic aabs and the build up of immune deposits in the glomeruli. While animals treated early had advantages over rats whose kidney disease was well established before treatment; animals treated late into the disease still manifested noticeable improvements in similar areas, i.e. with lessened proteinuria, kidney lesion reduction and a decreased pathogenic aab response. At the end of the experiment at 29 weeks, 80% of all the treated rats had insignificantly low levels of circulating IgG aabs, indicating cessation of pathogenic aab production and corresponding termination of the disease process. In contrast, most untreated rats with the kidney disease still had high levels of circulating pathogenic aabs at the end of the experiment, which maintained disease progression.

Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats.

Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular‐fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double‐staining techniques of rat kidney sections obtained from normal and rabbit anti‐FX1A‐injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti‐FX1A antibody. Western blot analysis have shown that the rabbit anti‐rat FX1A IgG and the rat anti‐rat KF3 IgM antibodies are directed against the same renal tubular‐derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.

Production of Heymann nephritis by a chemically modified renal antigen

An autoimmune kidney disease morphologically and functionally similar to Heymann nephritis (HN) was induced in mature male Sprague Dawley rats by repeated weekly IP injections of a chemically modified azo sonicated ultracentrifuged (u/c) rat kidney fraction 3 (rKF3) antigen in an aqueous medium. The experiment was terminated 15 weeks after the first injection of the chemically altered antigen. Serum samples collected and analysed by an indirect fluorescent antibody test on normal rat kidney sections during the course of the experiment showed a gradual rise in circulating pathogenic autoantibodies directed against the proximal tubular brush border regions. Proteinuria was present and significantly increased in the urine of two of eight rats. The arising immune‐complex glomerulonephritis (ICGN) revealed typical HN kidney disease lesions in 70% of the rats in histological, direct fluorescent antibody and electron‐microscopical examinations. Control rats injected similarly with the an unmodified version of the same antigen did not develop the HN‐characteristic morphological and functional changes. To our knowledge, this is the first time that the autoimmune kidney disease designated as an active HN has been produced by the administration of a chemically altered renal antigen in an aqueous solution and not by the usual presentation of the nephritogenic renal antigen in an adjuvant.

Downregulation of a pathogenic autoantibody response by IgM autoantibodies directed against the nephritogenic antigen in slowly progressive Heymann nephritis

The purpose of the study was to find out if a new modified vaccination technique would be effective in downregulating immunopathological events during the course of an experimental autoimmune kidney disease (which is morphologically and functionally similar to Heymann nephritis) called ‘slowly progressive Heymann nephritis’ (SPHN). We have shown that the pathogenic IgG autoantibody (aab)‐induced experimental autoimmune kidney disease process can be downregulated early on as well as during the chronic progressive phase, when rats were restimulated. The IgM aab, resulting from stimulation by immune complexes made up of rat kidney fraction 3 (rKF3) antigen and rat anti‐rKF3 IgM antibody in antigen excess (MIC), can greatly diminish pathogenic aab production by removing or blocking nephritogenic antigens. Reduced IgG aab production limits the formation of damaging immune complexes (IC) in the glomeruli and development of proteinuria. At the end of the experiment 60% and 80% of the MIC‐treated groups had no pathogenic IgG aab in their circulation, while all the untreated SPHN rats had high levels of IgG aab associated with disease progression manifesting in increased proteinuria and severe immune complex glomerulonephritis.

Reduced Incidence of Slowly Progressive Heymann Nephritis in Rats Immunized With a Modified Vaccination Technique

A slowly progressive Heymann nephritis (SPHN) was induced in three groups of rats by weekly injections of a chemically modified renal tubular antigen in an aqueous medium. A control group of rats received the chemically unmodified version of the antigen in an aqueous solution. One group of SPHN rats were pre- and post-treated with weekly injections of IC made up of rKF3 and rarKF3 IgM antibody at antigen excess (MIC) (immune complexes [ICs] containing sonicated ultracentrifuged [u/c] rat kidney fraction 3 [rKF3] antigen and IgM antibodies specific against the antigen, at slight antigen excess). One group of SPHN rats were post-treated with MIC 3 weeks after the induction of the disease and one group of SPHN animals received no treatment. The control group of rats received pre- and post-treatment with sonicated u/c rKF3. The incidence of immune-complex glomerulonephritis (ICGN) in the untreated SPHN rats was 87%, in the pre- and post-treated animals 13%, and in the post-treated-only rats 20%. Rats receiving sonicated ultracentrifuged rKF3 antigen did not develop ICGN. The present experiment demonstrates that the development of SPHN can be not only prevented but also effectively terminated by our newly developed modified vaccination technique.

The role of autoimmunologists in investigating and treating autoimmune disorders

The role of an autoimmunologist is to investigate and cultivate knowledge of normal and abnormal immune responses against self, which includes developing practical know-how to manipulate autoimmune activity and direct positive autoimmune outcomes. Where a subject develops an abnormal immune response directed against normal self, resulting in an autoimmune disease, the specialist should be able to diagnose the problem and institute an appropriate treatment. Obversely, where a subject lacks an immune response against cells bearing antigens that are abnormal or not quite self, i.e., cancer cells, the specialist should ideally be able to institute a specific cancer cell killing regimen. Essentially there are two beneficial and two harmful aspects of autoimmunity autoimmunologists should be familiar with. The beneficial aspects are the immune responses that assist in the clearance of cellular breakdown products and the elimination of cancer cells. The harmful aspects consist of immune responses, or lack thereof, that manifest in autoimmune disorders, i.e., autoimmune diseases and cancer. Recent medical discoveries, especially the modified vaccination technique developed by the Barabas research group, show great promise in both preventing and curing autoimmune disorders by utilizing the immune systems natural abilities to re-establish normal health.

A Modified Vaccination Technique for the Prevention and Treatment of an Experimental Autoimmune Kidney Disease

Abstract:  The main purpose of this article is to introduce a promising new vaccination technique and to outline its efficacy and safety as demonstrated in an experimental autoimmune kidney disease. We have found that antigen (AG)‐specific downregulation and/or upregulation of immune responses can be achieved by injections of immune complexes (ICs) which contain prepackaged information. This result is attained with the new vaccination method, a method developed in our laboratory which we have called “modified vaccination technique” (MVT). This MVT not only enables the prevention of pathogenic autoimmune events leading to the development of an experimental autoimmune kidney disease; it also allows, with equal effectiveness, therapeutic intervention to terminate the disease. With an injected IC containing predetermined immune response‐inducing components, the process effectuates a specific antibody information transfer conferring advantages that go beyond its prophylactic and therapeutic applicability. Its specificity can induce a precise immune response to correct mishaps, for example, in conditions where the immune system overreacts to an autologous antigen or fails to recognize unwanted self (as in autoimmune disorders, cancer, etc.) Preformed ICs are nontoxic and nonirritant, evoke a predetermined antibody response without the use of adjuvants, cause no disturbance in the overall regulatory function of the immune system, and produce no side effects. We firmly believe that proper implementation of the MVT will be able to induce and maintain specific preventive and/or curative responses in a way that is both natural and more effective in patients with chronic ailments presently treatable only with drugs.

Correcting autoimmune anomalies in autoimmune disorders by immunological means, employing the modified vaccination technique.

Our research group has developed a new vaccination technique in experimental animals that has the potential of correcting autoimmune anomalies in humans such as autoimmune disorders, cancer, and chronic infections, both prophylactically and therapeutically. The vaccination method is called Modified Vaccination Technique (MVT). The MVT necessitates the introduction of a purified target antigen (ag) and a specific antibody (ab) against the target ag in the form of immune complex (IC) to evoke the desired immune response outcome by ab information transfer in the injected recipient. The injected IC produces the same class of ab in the host, with the same specificity against the target ag, as resides in the inoculum. The MVT promises to provide a means of upregulating beneficial immune events and downregulating undesirable immune responses in individuals, thereby re-establishing normalcy/tolerance to self.

Modified Vaccination Technique for Prophylactic and Therapeutic Applications to Combat Endogenous Antigen–Induced Disorders

Public health can be protected most effectively through vaccination programmes. However, while presently available vaccination techniques protects the individual by provoking immune responses against exogenous antigens (ags), such as those associated with certain bacteria and viruses, they cannot protect against or treat mishaps caused by endogenous ag. Recently, Barabas and colleagues have developed a new vaccination method, called modified vaccination technique (MVT), which allows the presentation of disease causing agents in such a way as to initiate and maintain desired immune response outcomes even in the context of mishaps associated with endogenous ag. For example, in an experimental autoimmune kidney disease, the MVT downregulated/terminated pathogenic immune responses that were causing morphological and functional changes of the kidney. The MVT promises, with appropriate case‐specific modifications, both preventative and curative applications for ailments, such as endogenous ag initiated mishaps (i.e. autoimmune diseases and cancer) and diseases caused by chronic infection, that are presently only treatable with drugs. To achieve specific immune responses, purified components of the vaccine (ag and antibodies) must be produced and assembled into immune complexes having the potential of inducing predetermined corrective immune response outcomes.