Rat Experimental Autoimmune Gastritis Model.

BACKGROUND: Autoimmune gastritis (AIG) is an autoimmune disease of the stomach characterized by the destruction of the oxyntic mucosa, which stops producing acid and becomes both functionally and morphologically atrophic. The pathogenic mechanisms behind the disease are still poorly understood. There is no early diagnosis and specific AIG therapy. To elucidate the pathogenesis of AIG, to search for early diagnostic markers, as well as to test new therapeutic approaches, an adequate and easily reproducible experimental model for autoimmune gastritis (EAG) is needed. Existing EAG models have some limitations, including slow development of signs, absence of advanced gastritis, irrational use of animals to obtain antigen. The aim was to find out whether it is possible to cause autoimmune gastritis similar to human disease in Wistar rats through immunization with a homologous gastric mucosa extract. METHODS: Wistar rats were immunized with gastric mucosa extract. Histology studies and evaluation of serological parameters were performed 56 and 91 days later. RESULTS: Destruction of oxyntic glands by infiltrating T lymphocytes were detected in rats on 56 and 91 days after initial immunization with gastric mucosa extract. Hyperplasia of enterochromaffin-like (ECL) cells was detected on the 91st day. Antral mucosa remained unchanged. CONCLUSION: Wistar rats, immunized with gastric mucosa extract, developed EAG similar to human AIG. The advantages of received EAG model are the ease of obtaining, the rapid development of oxyntic mucosa damage, which may progress to ECL cell hyperplasia.

Treatment with IgG Fc fragments bearing regRF epitopes prevents destruction of the gastric mucosa in experimental autoimmune gastritis model.

Autoimmune gastritis (AIG) is the autoimmune disease of the stomach characterized by the destruction of the oxyntic mucosa, which stops producing acid and becomes both functionally and morphologically atrophic. There is no specific treatment for AIG. Previously, we identified a new immunoregulatory factor (regulatory rheumatoid factor (regRF)), the stimulation production of which reduces certain experimental autoimmune diseases. Epitopes specific to the regulatory rheumatoid factor (regRF epitopes) can be obtained on IgG Fc fragments. In the rat AIG model, the therapeutic efficacy of IgG Fc fragments bearing regRF epitopes was tested. Treatment with IgG Fc fragments bearing regRF epitopes reduced T lymphocytic infiltration of oxyntic mucosa and prevented its damage in the AIG rat model, while in rats treated with placebo, T lymphocytic infiltration of the mucosa, loss of parietal cells, including severe were observed. Therefore, IgG Fc fragments bearing regRF epitopes are a potential therapeutic agent for treating autoimmune gastritis in its early stages.

Regulatory Rheumatoid Factor is Specific to PD-1 and Uses PD-1 Pathway to Control CD4 T Lymphocytes.

BACKGROUND: We have earlier discovered a new factor of autoimmunity downregulation, called regulatory rheumatoid factor (regRF). Being anti-idiotypic antibodies, regRF restricts the expansion of CD4+ T lymphocytes to the idiotype of which it is specific, according to the negative feedback principle. It has been shown that only activated CD4+ T lymphocytes are the target of regRF. However, it is still not clear the way regRF distinguishes activated cells from naive ones. RegRF molecules, apart from individual paratopes specific to unique sequences of B- and T-cell receptors, have a shared paratope. We assume that regRF by means of a shared paratope recognizes one of the surface activation molecules of CD4+ T lymphocytes and initiates the cell death. Programmed death-1 (PD-1) has been tested as a potential receptor of the shared regRF paratope and transmitter of the negative regRF signal into activated CD4+ T lymphocytes. METHODS: The specificity of the shared regRF paratope to PD-1 was determined by ELISA. T cell activation was performed with immobilized anti-CD3ε antibodies. Flow cytometry was used to study the effect of regRF on PD-1+CD4+ lymphocytes. RESULTS: We found that regRF binds to PD-1. IgG Fc fragments carrying epitopes specific to the shared paratope of regRF compete with PD-1 for binding to regRF. It follows that regRF recognizes specifically PD-1 by means of a shared paratope. RegRF-containing serum reduced the number of PD-1+CD4+ lymphocytes in proportion to their increase resulting from the action of anti-CD3ε antibodies. CONCLUSION: RegRF uses PD-1 pathway to control activated CD4+ T lymphocytes.

Reduction in experimental autoimmune thyroiditis by IgG Fc fragments bearing regRF epitopes.

Previously, we identified a new immunoregulatory factor, the production of which provides rats with resistance to certain experimental autoimmune diseases. It has been named regulatory rheumatoid factor (regRF). RegRF inhibits the expansion of CD4 T lymphocytes by killing activated cells. CD4 T cells are essential for antibody production against a majority of antigens and for the generation of cytotoxic T cells; therefore, regRF is an attractive therapeutic biotarget for T-cell and antibody-mediated autoimmune diseases. RegRF is anti-idiotypic antibodies that have a shared paratope in addition to an individual paratope. Epitopes specific to the shared regRF paratope (regRF epitopes) can be obtained on conformers of IgG Fc fragments. Immunization with Fc fragments carrying regRF epitopes reduces rat collagen-induced arthritis and diminishes experimental autoimmune encephalomyelitis. The aim of this study was to determine whether IgG Fc fragments bearing regRF epitopes suppress experimental autoimmune thyroiditis (EAT). Four weeks after EAT induction, rats were immunized with IgG Fc fragments exhibiting regRF epitopes. Histology studies of the thyroid were performed 4 weeks later. Thyroid function and other parameters were also evaluated. Treatment of rats with Fc fragments bearing regRF epitopes decreased the number of rats affected by EAT, significantly decreased the extent of thyroid damage, prevented thyroid metaplasia, and restored normal thyroid hormone production. Therefore, RegRF is a promising biotarget in autoimmune thyroiditis, and Fc fragments bearing regRF epitopes are a potential therapeutic agent for that condition.

Fourier-transform infrared spectroscopy of human IgG Fc fragments, which are a promising drug for the treatment of autoimmune diseases.

IgG Fc fragments that expose regulatory rheumatoid factor epitopes (regRF epitopes) have emerged as a promising immunosuppressive drug. Immunization of rats with such Fc fragments reduced symptoms of experimental autoimmune diseases. The immunosuppressive effect of Fc fragments is based on stimulating the production of regRF, which kills activated CD4 T lymphocytes. The formation of regRF epitopes on Fc fragments was previously shown to be associated with a reduction in disulfide bonds in the fragments' hinge region. However, the structure of Fc fragments that bear regRF epitopes remained largely unclear. Infrared spectra were compared for lyophilized Fc fragments displaying regRF epitopes and Fc fragments without such epitopes. FTIR spectroscopy found no differences in the amide I, amide II, and amide III bands, indicating that there are no distinctive features in the secondary structure of Fc fragments bearing regRF epitopes. The distinctive feature of Fc fragments bearing regRF epitopes, irrespective of whether the free SH groups in the hinge were preserved or lost after lyophilization, is the presence of a band or a fine structure in the region containing the bending vibrations of the SH groups. Furthermore, the Fc fragments with regRF epitopes differ from those without in that they have a band in the absorption region of aromatic amino acid rings. Taken together, these facts suggest that the appearance of regRF epitopes results from changes in the tertiary structure of the hinge and the domains that occur when the hinge is reduced, and they also indicate that these conformational changes are resistant to subsequent changes in the state of cysteine residues in the hinge. Bands in the regions of aromatic amino acids and the bending vibrations of SH groups are markers of the presence of regRF epitopes on IgG Fc fragments. FTIR spectroscopy can be used to detect these epitopes.

Regulatory and other rheumatoid factors in rheumatoid arthritis patients with active disease or in remission.

BACKGROUND: Previously, we identified a regulatory rheumatoid factor (regRF), the production of which provides rats with resistance to collagen-induced arthritis (CIA). Immunization with conformers of IgG Fc fragments carrying epitopes specific to regRF reduces symptoms of CIA. The aim of this study was to determine whether there is a link between regRF levels and rheumatoid arthritis (RA) activity in humans in order to assess the potential of regRF as a therapeutic biotarget in RA. The variability of rheumatoid factor (RF) specificities present in the blood of RA patients was also studied. METHODS: The regRF were studied in RA patients with active disease and in remission. Variability in the specificities of RF associated with RA was studied by concurrent inhibition of RF latex fixation by variants of modified IgG. RESULTS: Patients in remission had regRF levels higher than in healthy subjects. The regRF in remission was characterized by tight binding to its antigen, as in healthy subjects. The regRF levels in patients with active RA varied dramatically, and regRF binding to its antigen was weak. The exacerbation of Still's disease coincided with low regRF levels and affinity, while an improvement in patient condition was associated with an increase in regRF levels and affinity. The RF specific to RA, which was detected by the RF latex-fixation method, was a nonhomogeneous population of antibodies that included RF to lyophilized IgG, to IgG immobilized on polystyrene, and to rabbit IgG. CONCLUSION: Stimulating regRF production might enable improved RA therapy.

Suppression of experimental autoimmune encephalomyelitis by IgG Fc fragments bearing regRF epitopes.

Previously we identified a rheumatoid factor, the production of which provides rats with resistance to experimental autoimmune diseases. It has been named regulatory rheumatoid factor (regRF). Immunization with conformers of IgG Fc fragments carrying epitopes specific to regRF reduces rat collagen-induced arthritis. The aim of this study was to determine whether IgG Fc fragments bearing regRF epitopes suppress experimental autoimmune encephalomyelitis, and to evaluate the potential of a strategy of stimulating production of regRF to treat multiple sclerosis. Two days after myelin basic protein injection, rats were immunized with Fc fragments exhibiting regRF epitopes, as well as with Fc fragments without those epitopes. The effect of Fc immunization on clinical signs of EAE and immunological parameters was evaluated. Stimulation of regRF production by IgG Fc fragments bearing regRF epitopes diminished EAE symptoms in rats, while immunization with Fc fragments without those epitopes worsened EAE. The improvement of EAE symptoms in rats treated with Fc fragments bearing regRF epitopes was associated with regRF production and with the relatively low number of blood CD4 T lymphocytes during disease development. In experiments involving immunizing intact rats and lymph node mononuclear cell cultures, Fc fragments bearing regRF epitopes decreased the CD4 T lymphocyte population indirectly, via regRF production. RegRF is a promising biotarget in MS, and Fc fragments bearing regRF epitopes are a potential therapeutic agent for MS.

Comparison of the specificity of rheumatoid factor detected by latex fixation with that of regulatory rheumatoid factor.

BACKGROUND: Rheumatoid factor (RF), originally defined as pathological autoantibodies to IgG that are detected in rheumatoid arthritis, turned out to be multi-specific antibodies, some of which exhibit immunoregulatory properties. Recently, we identified a RF, the production of which confers resistance to experimental autoimmune diseases and is associated with the remission of autoimmune diseases. To differentiate the RF, we discovered from the one associated with rheumatic disease onset or progression and to reflect its immunoregulatory properties, we named it regulatory rheumatoid factor (regRF). Immunization with conformers of Fc fragments that expose regRF neoepitopes reduces collagen-induced arthritis in rats. Certain information about the specificity of classical RF and regRF indicates that these populations may be one and the same. Therefore, the aim of this study was to determine whether there is a difference between the classical RF and regRF. METHODS: Classical RF was measured in diseased blood by the latex fixation method, and regRF was detected by the agglutination of human IgG-loaded tanned erythrocytes. Competitive analysis was used to determine the specificity of rheumatoid factors. RESULTS: It was found that regRF and pathology-associated RF constitute different antibody populations. Pathology-associated RF is specific for lyophilized IgG. RegRF does not interact with IgG. RegRF is specific to conformers of IgG Fc fragments that have a reduced hinge. In latex-positive rheumatoid arthritis sera, regRF may be present in addition to pathology-associated RF. The latex fixation method detects both rheumatoid factor populations. CONCLUSION: RegRF and classical pathology-associated RF have different specificity.

Аtherosclerosis-like changes in the rabbit aortic wall induced by immunization with native high-density lipoproteins.

INTRODUCTION: A high level of total cholesterol or low-density lipoprotein (LDL) cholesterol is considered the main cause of atherosclerosis and cardiovascular disease. For this reason, experimental atherosclerosis is induced by creating high blood cholesterol in animals. However, the hypothesis that atherosclerotic processes are mostly caused by immune (autoimmune) mechanisms has recently been gaining traction. At the same time, no experimental model has been developed that clearly demonstrates the autoimmune mechanism by which atherosclerosis develops and reproduces the full picture of atherosclerosis solely by means of an immune response, without resorting to additional interventions such as a high-cholesterol diet or the use of genetic models of hyperlipidemia. Previously, we were able to induce atherosclerosis-like lesions in the aorta and the development of pericardial fat in rats by immunizing them with human native lipoproteins. The purpose of this study was to test whether atherosclerosis can be induced in normocholesterolaemic rabbits by immunizing them with human native high-density lipoproteins (hnHDL). METHODS: Rabbits were immunized with hnHDL. Aortic wall structure, plasma cholesterol level, and antibodies against HDL were studied. RESULTS: Immunization with hnHDL was found to cause atherosclerosis-like lesions in the rabbit aorta such as adipocytic and chondrocytic metaplasia, proteoglycan deposits, leukocytic infiltration. Atherosclerosis-like lesions developed in the aorta of hnHDL-immunized rabbits against a background of normal blood LDL-cholesterol level. Therefore, a high plasma cholesterol level is not the sole cause of atherosclerosis. The immune response against HDL is an independent cause of atherogenesis. CONCLUSIONS: A rabbit model of atherosclerosis caused by immunization with hnHDL can be widely used to examine the mechanisms occurring during atherogenesis.

Immunoglobulin G amplifies the production of regulatory rheumatoid factor in vitro through idiotype-anti-idiotype interactions.

Immunoglobulin G can inhibit antibody response. The mechanism of immunosuppression by immunoglobulins remains unknown. Recently, we found a new factor of immunoregulation referred to as regulatory rheumatoid factor (regRF). RegRF prevents autoimmunity and reduces experimental autoimmune reactions. RegRF comprises a population of anti-idiotypic antibodies that have a unique paratope specific to the antigen-binding sites of the antibodies, and a shared paratope specific to neoepitopes of IgG Fc fragments. Given the specificity of regRF, we can anticipate that IgG would be able to induce regRF production, and consequently that the immunosuppressive effect of IgG may be mediated by regRF. We found that IgG induces regRF production in a culture of B lymphocytes obtained from the red bone marrow of intact rats. IgG does not expose neoepitopes recognized by the shared paratope of regRF, and does not acquire them in culture. Therefore, the stimulation of regRF production induced by IgG is not a result of the interaction between the shared paratope of regRF and the neoepitopes of IgG. Fc fragments of IgG are unable to stimulate regRF production. Fab fragments inhibit spontaneous regRF production. F(ab´)2 fragments stimulate regRF production in lymphocyte culture. We theorize that IgG activates regRF-producing lymphocytes through idiotype-anti-idiotype interactions.

Physicochemical characteristics of human IgG Fc fragments that expose regulatory rheumatoid factor neoepitopes and may show promise as antirheumatic agents.

Previously, we showed that immunoglobulin G (IgG) Fc fragments can expose neoepitopes specific to antibodies that were named regulatory rheumatoid factor (regRF). RegRF confers resistance to experimental autoimmune diseases. Immunization of rats with rat Fc fragments exposing neoepitopes recognized by regRF reduces the symptoms of collagen-induced arthritis. Therefore, IgG Fc fragments that expose neoepitopes recognized by regRF are promising antirheumatic agents and regRF-producing lymphocytes are potential therapeutic biotargets. The purpose of this study was to elucidate the physicochemical features of human IgG Fc fragments that are associated with the presence of immunosuppressive neoepitopes recognized by regRF. It was found that the acquisition of neoepitopes recognized by regRF is associated with a reduction of the hinge disulfide bonds and conformational changes in the Fc fragment domains. Alkylation of thiol groups in the hinge leads to loss of the epitopes. Therefore, the neoepitopes recognized by regRF may form directly in the hinge when interchain disulfide bonds are reduced or in the region of the CH2 domain as part of the conformational changes caused by the reduction of the interchain disulfide bonds in the hinge. Species-specificity studies of neoepitopes recognized by regRF revealed that human and rat neoepitopes recognized by regRF are cross-reactive.

Immune Response to Native Lipoproteins Induces Visceral Obesity and Aortic Wall Injury in Rats: The Role of Testosterone.

OBJECTIVES: The underlying mechanism of atherosclerosis and visceral obesity remains unknown.The purpose of this study was to test the hypothesis that atherosclerosis and visceral obesity are caused by an immune response to native plasma lipoproteins, and the atherogenic and adipogenic effects of the antibodies to native lipoproteins stem from the androgen deficiency that is created. METHODS: Wistar rats were immunized with native human (nh) low-density (LDL) or high-density lipoproteins (HDL). Visceral fat, aortic wall structure, and testosterone levels were studied. RESULTS: Immunization with nhLDL or nhHDL induced in rats increased visceral abdominal fat and perivascular adipose tissue volume, the appearance of epicardial fat, and atherosclerosis-like changes in the aortic wall: accumulation of leucocytes, destruction of the intima, and disruption of the media structure. Immunized rats produced antibodies to native plasma lipoproteins, while there was no difference between immunized and adjuvant-injected rats with regard to the level of antibodies to oxidized LDL. The immune response to nhHDL caused testosterone disturbances, but it is not associated with visceral obesity and atherosclerosis. CONCLUSION: The immune response to native lipoproteins is atherogenic and adipogenic and testosterone is not involved in the atherogenic and adipogenic effects of antibodies to lipoproteins.

The Role of Neutrophil Proteases in LPS-Induced Production of Regulatory Rheumatoid Factor that Suppresses Autoimmunity.

BACKGROUND: We recently identified a regulatory rheumatoid factor (regRF), the production of which is associated with autoimmune disease resistance and remission. In studies of regRF in the blood of healthy rats, spontaneous increases in the regRF level were noted. We suggest that in the normal state, a mechanism exists for maintaining the activity of the pool of regRF-producing lymphocytes at a level that makes it possible to control the expansion of autoreactive lymphocytes. OBJECTIVE: The purpose of this study was to test the hypothesis that the endogenous stimulator of regRF production is Fc fragments of IgG that are formed upon exposure to the proteases of neutrophils. RESULTS: Injection of Salmonella typhi LPS caused neutrophilic leukocytosis in the rats, followed by elevated level of regRF. Neutrophils were obtained from LPS-treated rats and then treated with LPS in vitro to degranulate them to form pre-split IgG that exposes antigenic determinants for regRF. A condition required for Fc fragments to be formed by neutrophils is that the pre-split IgG must be treated with a thiol reducing agent. Antigenic determinants for regRF were retained by Fc fragments of IgG. CONCLUSION: Thus, the pre-split IgG and Fc fragments of IgG formed by LPS-activated neutrophils are the potential physiological activators of regRF production.

Fc fragments of immunoglobulin G are an inductor of regulatory rheumatoid factor and a promising therapeutic agent for rheumatic diseases.

We recently identified rheumatoid factor, the production of which neither predicts nor exacerbates experimental autoimmune disease, but the opposite, namely it is associated with autoimmune disease resistance and remission. We have named it regulatory rheumatoid factor (regRF). The aim of this study was to determine whether rat Fc fragments and human Fc fragments are an antigen for regRF, and to determine the conditions for obtaining them. The presence of an antigenic determinant for regRF on IgG fragments was inferred from the fragments' ability to inhibit the agglutination caused by regRF and to induce regRF production in vivo. It was found that antigenic determinants for both human regRF and rat regRF are absent from native IgG and can be induced in the hinge region of Fc fragments of homologous IgG by papain digestion. The rat Fc fragments are susceptible to spontaneous reconfiguration, which results in loss of the antigenic determinants for regRF. Reconfiguration can be observed by SDS-PAGE. Immunization of arthritic rats with Fc fragments of rat IgG that carry antigenic determinants for rat regRF reduces the symptoms of collagen-induced arthritis. The Fc fragments can be viewed as the basis for a therapeutic vaccine to suppress autoimmune responses.