From Bioorganic Models to Cells.

I endeavor to share how various choices-some deliberate, some unconscious-and the unmistakable influence of many others shaped my scientific pursuits. I am fascinated by how two long-term, major streams of my research, DNA replication and purine biosynthesis, have merged with unexpected interconnections. If I have imparted to many of the talented individuals who have passed through my lab a degree of my passion for uncloaking the mysteries hidden in scientific research and an understanding of the honesty and rigor it demands and its impact on the world community, then my mentorship has been successful.

Catalytic Antibodies May Contribute to Demyelination in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Here we report preliminary data demonstrating that some patients with myalgic encephalomyelitis/chronic fatiguesyndrome (ME/CFS) may have catalytic autoantibodies that cause the breakdown of myelin basic protein (MBP). We propose that these MBP-degradative antibodies are important to the pathophysiology of ME/CFS, particularly in the occurrence of white matter disease/demyelination. This is supported by magnetic resonance imagining studies that show these findings in patients with ME/CFS and could explain symptoms of nerve pain and muscle weakness. In this work, we performed a series of experiments on patient plasma samples where we isolated and characterized substrate-specific antibodies that digest MBP. We also tested glatiramer acetate (copaxone), an FDA approved immunomodulator to treat multiple sclerosis, and found that it inhibits ME/CFS antibody digestion of MBP. Furthermore, we found that aprotinin, which is a specific serine protease inhibitor, specifically prevents breakdown of MBP while the other classes of protease inhibitors had no effect. This coincides with the published literature describing catalytic antibodies as having serine protease-like activity. Postpandemic research has also provided several reports of demyelination in COVID-19. Because COVID-19 has been described as a trigger for ME/CFS, demyelination could play a bigger role in patient symptoms for those recently diagnosed with ME/CFS. Therefore, by studying proteolytic antibodies in ME/CFS, their target substrates, and inhibitors, a new mechanism of action could lead to better treatment and a possible cure for the disease.

Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion.

AIMS: Arrhythmogenic cardiomyopathy (AC) is a severe heart disease predisposing to ventricular arrhythmias and sudden cardiac death caused by mutations affecting intercalated disc (ICD) proteins and aggravated by physical exercise. Recently, autoantibodies targeting ICD proteins, including the desmosomal cadherin desmoglein 2 (DSG2), were reported in AC patients and were considered relevant for disease development and progression, particularly in patients without underlying pathogenic mutations. However, it is unclear at present whether these autoantibodies are pathogenic and by which mechanisms show specificity for DSG2 and thus can be used as a diagnostic tool. METHODS AND RESULTS: IgG fractions were purified from 15 AC patients and 4 healthy controls. Immunostainings dissociation assays, atomic force microscopy (AFM), Western blot analysis and Triton X-100 assays were performed utilizing human heart left ventricle tissue, HL-1 cells and murine cardiac slices. Immunostainings revealed that autoantibodies against ICD proteins are prevalent in AC and most autoantibody fractions have catalytic properties and cleave the ICD adhesion molecules DSG2 and N-cadherin, thereby reducing cadherin interactions as revealed by AFM. Furthermore, most of the AC-IgG fractions causing loss of cardiomyocyte cohesion activated p38MAPK, which is known to contribute to a loss of desmosomal adhesion in different cell types, including cardiomyocytes. In addition, p38MAPK inhibition rescued the loss of cardiomyocyte cohesion induced by AC-IgGs. CONCLUSION: Our study demonstrates that catalytic autoantibodies play a pathogenic role by cleaving ICD cadherins and thereby reducing cardiomyocyte cohesion by a mechanism involving p38MAPK activation. Finally, we conclude that DSG2 cleavage by autoantibodies could be used as a diagnostic tool for AC.

A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule.

Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.

Cellular and Immunological Analysis of 2D2/Th Hybrid Mice Prone to Experimental Autoimmune Encephalomyelitis in Comparison with 2D2 and Th Lines.

Previously, we described the mechanisms of development of autoimmune encephalomyelitis (EAE) in 3-month-old C57BL/6, Th, and 2D2 mice. The faster and more profound spontaneous development of EAE with the achievement of deeper pathology occurs in hybrid 2D2/Th mice. Here, the cellular and immunological analysis of EAE development in 2D2/Th mice was carried out. In Th, 2D2, and 2D2/Th mice, the development of EAE is associated with a change in the differentiation profile of hemopoietic bone marrow stem cells, which, in 2D2/Th, differs significantly from 2D2 and Th mice. Hybrid 2D2/Th mice demonstrate a significant difference in these changes in all strains of mice, leading to the production of antibodies with catalytic activities, known as abzymes, against self-antigens: myelin oligodendrocyte glycoprotein (MOG), DNA, myelin basic protein (MBP), and five histones (H1-H4) hydrolyze these antigens. There is also the proliferation of B and T lymphocytes in different organs (blood, bone marrow, thymus, spleen, lymph nodes). The patterns of changes in the concentration of antibodies and the relative activity of abzymes during the spontaneous development of EAE in the hydrolysis of these immunogens are significantly or radically different for the three lines of mice: Th, 2D2, and 2D2/Th. Several factors may play an essential role in the acceleration of EAE in 2D2/Th mice. The treatment of mice with MOG accelerates the development of EAE pathology. In the initial period of EAE development, the concentration of anti-MOG antibodies in 2D2/Th is significantly higher than in Th (29.1-fold) and 2D2 (11.7-fold). As shown earlier, antibodies with DNase activity penetrate cellular and nuclear membranes and activate cell apoptosis, stimulating autoimmune processes. In the initial period of EAE development, the concentration of anti-DNA antibodies in 2D2/Th hybrids is higher than in Th (4.6-fold) and 2D2 (25.7-fold); only 2D2/Th mice exhibited a very strong 10.6-fold increase in the DNase activity of IgGs during the development of EAE. Free histones in the blood are cytotoxic and stimulate the development of autoimmune diseases. Only in 2D2/Th mice, during different periods of EAE development, was a sharp increase in the anti-antibody activity in the hydrolysis of some histones observed.

Autoantibodies-Abzymes with Phosphatase Activity in Experimental Autoimmune Encephalomyelitis Mice.

The exact mechanisms of MS (multiple sclerosis) evolution are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis simulating human MS) in C57BL/6 mice occurs due to the violation of bone marrow hematopoietic stem cell differentiation profiles, leading to the production of toxic for human autoantibody splitting MBP (myelin basic protein), MOG (mouse oligodendrocyte glycoprotein), five histones, DNA, and RNA. Here, we first analyzed the changes in the relative phosphatase activity of IgGs from C57BL/6 mice blood over time, corresponding to three stages of EAE: onset, acute, and remission. Antibodies have been shown to catalyze the hydrolysis of p-nitrophenyl phosphate at several optimal pH values, mainly in the range of 6.5-7.0 and 8.5-9.5. During the spontaneous development of EAE, the most optimal value is pH 6.5. At 50 days after the birth of mice, the phosphatase activity of IgGs at pH 8.8 is 1.6-fold higher than at pH 6.5. During spontaneous development of EAE from 50 to 100 days, an increase in phosphatase activity is observed at pH 6.5 but a decrease at pH 8.8. After mice were immunized with DNA-histone complex by 20 and 60 days, phosphatase activity increased respectively by 65.3 and 109.5 fold (pH 6.5) and 128.4 and 233.6 fold (pH 8.8). Treatment of mice with MOG at the acute phase of EAE development (20 days) leads to a maximal increase in the phosphatase activity of 117.6 fold (pH 6.5) and 494.7 fold (pH 8.8). The acceleration of EAE development after mice treatment with MOG and DNA-histone complex results in increased production of lymphocytes synthesizing antibodies with phosphatase activity. All data show that IgG phosphatase activity could be essential in EAE pathogenesis.

Direct conversion of a general antibody to its catalytic antibody and corresponding applications -Importance and role of Pro95 in CDR-3.

Catalytic antibodies possess unique features capable of both recognizing and enzymatically degrading antigens. Therefore, they are more beneficial than monoclonal antibodies (mAbs). Catalytic antibodies exhibit the ability to degrade peptides, antigenic proteins, DNA, and physiologically active molecules. However, they have a significant drawback in terms of their production. The production of a desired catalytic antibody has extensive costs, in terms of time and effort. We herein describe an evolutionary method to produce a desired catalytic antibody via conversion of a general antibody by the deletion of Pro95, which resides in complementarity-determining region-3. As over thousands of mAbs have been produced since 1975, using the novel technology discussed herein, the catalytic feature cleaving the antigen can be conferred to the mAb. In this review article, we discussed in detail not only the role of Pro95 but also the unique features of the converted catalytic antibodies. This technique will accelerate research on therapeutic application of catalytic antibodies.

Experimental Autoimmune Encephalomyelitis of Mice: IgGs from the Sera of Mice Hydrolyze miRNAs.

It was shown that the spontaneous development of experimental encephalomyelitis (EAE) in C57BL/6 mice occurs due to changes in the profile of bone marrow stem cells differentiation. This leads to the appearance of lymphocytes producing antibodies-abzymes that hydrolyze DNA, myelin basic protein (MBP), and histones. The activity of abzymes in the hydrolysis of these auto-antigens slowly but constantly increases during the spontaneous development of EAE. Treatment of mice with myelin oligodendrocyte glycoprotein (MOG) leads to a sharp increase in the activity of these abzymes with their maximum at 20 days (acute phase) after immunization. In this work, we analyzed changes in the activity of IgG-abzymes hydrolyzing (pA)23, (pC)23, (pU)23, and six miRNAs (miR-9-5p, miR-219a-5p, miR-326, miR-155-5p, miR-21-3p, and miR-146a-3p) before and after mice immunization with MOG. Unlike abzymes hydrolyzing DNA, MBP, and histones, the spontaneous development of EAE leads not to an increase but to a permanent decrease of IgGs activity of hydrolysis of RNA-substrates. Treatment of mice with MOG resulted in a sharp but transient increase in the activity of antibodies by day 7 (onset of the disease), followed by a sharp decrease in activity 20-40 days after immunization. A significant difference in the production of abzymes against DNA, MBP, and histones before and after mice immunization with MOG with those against RNAs may be since the expression of many miRNAs decreased with age. This can lead to a decrease in the production of antibodies and abzymes that hydrolyze miRNAs with age mice.

Autoantibody-Abzymes with Catalase Activity in Experimental Autoimmune Encephalomyelitis Mice.

The exact mechanisms of the evolution of multiple sclerosis are still unknown. At the same time, the development in C57BL/6 mice of experimental autoimmune encephalomyelitis (EAE, simulating human multiple sclerosis) happens as a result of the violation of bone marrow hematopoietic stem cell differentiation profiles integrated with the production of toxic auto-antibodies splitting the basic myelin protein, myelin oligodendrocyte glycoprotein (MOG), histones, and DNA. It has been shown that IgGs from the plasma of healthy humans and autoimmune patients oxidize many different compounds due to their peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent) activities. Here, we first analyzed the changes in the relative catalase activity of IgGs from C57BL/6 mice blood plasma over time at different stages of the EAE development (onset, acute, and remission phases). It was shown that the catalase activity of IgGs of 3-month-old mice is, on average, relatively low (kcat = 40.7 min-1), but it increases during 60 days of spontaneous development of EAE 57.4-fold (kcat = 2.3 × 103 min-1). The catalase activity of antibodies increases by a factor of 57.4 by 20 days after the immunization of mice with MOG (kcat = 2.3 × 103 min-1), corresponding to the acute phase of EAE development, and 52.7-fold by 60 days after the treatment of mice with a DNA-histone complex (kcat = 2.1 × 103 min-1). It is the acceleration of the EAE development after the treatment of mice with MOG that leads to the increased production of lymphocytes synthesizing antibodies with catalase activity. All data show that the IgGs' catalase activity can play an essential role in reducing the H2O2 concentration and protecting mice from oxidative stress.

EAE of Mice: Enzymatic Cross Site-Specific Hydrolysis of H2B Histone by IgGs against H1, H2A, H2B, H3, and H4 Histones and Myelin Basic Protein.

Histones have vital roles in chromatin functioning and gene transcription. At the same time, they are pernicious in intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin-proteolipid sheath. Antibody-abzymes with various catalytic activities are specific features of some autoimmune diseases. IgGs against five individual histones (H2B, H1, H2A, H3, and H4) and MBP were isolated from the blood of experimental autoimmune encephalomyelitis-prone C57BL/6 mice by affinity chromatography. Abzymes corresponding to various stages of EAE development, including spontaneous EAE, myelin oligodendrocyte glycoprotein (MOG)- and DNA-histone complex-accelerated onset, as well as acute and remission stages, were analyzed. IgG-abzymes against MBP and five individual histones showed unusual polyreactivity in complex formation and enzymatic cross-reactivity in the specific hydrolysis of H2B histone. All IgGs against MBP and individual histones in 3-month-old mice (zero time) demonstrated from 4 to 11 different H2B hydrolysis sites. Spontaneous development of EAE during 60 days led to a significant change in the type and number of H2B hydrolysis sites by IgGs against the five histones and MBP. Mouse treatment with MOG and DNA-histone complex changed the type and number of H2B hydrolysis sites compared to zero time. The minimum number (3) of different H2B hydrolysis sites was found for IgGs against H3 20 days after mouse immunization with DNA-histone complex, whereas the maximum number (33) for anti-H2B IgGs was found 60 days after mouse treatment with DNA-histone complex. Overall, this is the first study to demonstrate that at different stages of EAE evolution, IgG-abzymes against five individual histones and MBP could significantly differ in the specific sites and number of H2B hydrolysis sites. Possible reasons for the catalytic cross-reactivity and significant differences in the number and type of histone H2B cleavage sites were analyzed.

Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens.

A catalytic antibody has multiple functions compared with a monoclonal antibody because it possesses unique features to digest antigens enzymatically. Therefore, many catalytic antibodies, including their subunits, have been produced since 1989. The catalytic activities often depend on the preparation methods and conditions. In order to elicit the high catalytic activity of the antibodies, the most preferable methods and conditions, which can be generally applicable, must be explored. Based on this view, systematic experiments using two catalytic antibody light chains, #7TR and H34, were performed by varying the purification methods, pH, and chemical reagents. The experimental results obtained by peptidase activity tests and kinetic analysis, revealed that the light chain's high catalytic activity was observed when it was prepared under a basic condition. These data imply that a small structural modulation of the catalytic antibody occurs during the purification process to increase the catalytic activity while the antigen recognition ability is kept constant. The presence of NaCl enhanced the catalytic activity. When the catalytic light chain was prepared with these preferable conditions, #7TR and H34 hugely enhanced the degradation ability of Amyloid-beta and PD-1 peptide, respectively.

Catalytic Antibodies in Bipolar Disorder: Serum IgGs Hydrolyze Myelin Basic Protein.

The pathogenesis of bipolar affective disorder is associated with immunological imbalances, a general pro-inflammatory status, neuroinflammation, and impaired white matter integrity. Myelin basic protein (MBP) is one of the major proteins in the myelin sheath of brain oligodendrocytes. For the first time, we have shown that IgGs isolated from sera of bipolar patients can effectively hydrolyze human myelin basic protein (MBP), unlike other test proteins. Several stringent criteria were applied to assign the studied activity to serum IgG. The level of MBP-hydrolyzing activity of IgG from patients with bipolar disorder was statistically significantly 1.6-folds higher than that of healthy individuals. This article presents a detailed characterization of the catalytic properties of MBP-hydrolyzing antibodies in bipolar disorder, including the substrate specificity, inhibitory analysis, pH dependence of hydrolysis, and kinetic parameters of IgG-dependent MBP hydrolysis, providing the heterogeneity of polyclonal MBP-hydrolyzing IgGs and their difference from canonical proteases. The ability of serum IgG to hydrolyze MBP in bipolar disorder may become an additional link between the processes of myelin damage and inflammation.

MicroRNAs of Milk in Cells, Plasma, and Lipid Fractions of Human Milk, and Abzymes Catalyzing Their Hydrolysis.

Human milk provides neonates with various components that ensure newborns' growth, including protection from bacterial and viral infections. In neonates, the biological functions of many breast milk components can be very different compared with their functions in the body fluids of healthy adults. Catalytic antibodies (abzymes) that hydrolyze peptides, proteins, DNAs, RNAs, and oligosaccharides were detected, not only in the blood sera of autoimmune patients, but also in human milk. Non-coding microRNAs (18−25 nucleotides) are intra- and extracellular molecules of different human fluids. MiRNAs possess many different biological functions, including the regulation of several hundred genes. Five of them, miR-148a-3p, miR-200c-3p, miR-378a-3p, miR-146b-5p, and let-7f-5p, were previously found in milk in high concentrations. Here, we determined relative numbers of miRNA copies in 1 mg of analyzed cells, lipid fractions, and plasmas of human milk samples. The relative amount of microRNA decreases in the following order: cells ≈ lipid fraction > plasma. IgGs and sIgAs were isolated from milk plasma, and their activities in the hydrolysis of five microRNAs was compared. In general, sIgAs demonstrated higher miRNA-hydrolyzing activities than IgGs antibodies. The hydrolysis of five microRNAs by sIgAs and IgGs was site-specific. The relative activity of each microRNA hydrolysis was very dependent on the milk preparation. The correlation coefficients between the contents of five RNAs in milk plasma, and the relative activities of sIgAs compared to IgGs in hydrolyses, strongly depended on individual microRNA, and changed from −0.01 to 0.80. Thus, it was shown that milk contains specific antibodies (abzymes) that hydrolyze microRNAs specific for human milk.

Autoimmune Diseases: Enzymatic cross Recognition and Hydrolysis of H2B Histone, Myelin Basic Protein, and DNA by IgGs against These Antigens

Human milk provides neonates with various components that ensure newborns' growth, including protection from bacterial and viral infections. In neonates, the biological functions of many breast milk components can be very different compared with their functions in the body fluids of healthy adults. Catalytic antibodies-abzymes hydrolyzing peptides, proteins, DNAs, RNAs, and oligosaccharides were detected not only in the blood sera of autoimmune patients but also in human milk. Non-coding microRNAs (18-25 nucleotides) are intra- and extra-cellular molecules of different human fluids. MiRNAs possess many different biological functions, including regulating several hundred genes. Five of them: miR-148a-3p, miR-200c-3p, miR-378a-3p, miR-146b-5p and let-7f-5p were previously found in milk in increased concentrations. Here, we determined number of copies of these miRNAs in 1 mg of analyzed cells, lipid fractions, and plasmas of human milk samples. The relative amount of microRNA decreases in the following order: cells ¼ lipid fraction > plasma. IgGs and sIgAs were isolated from milk plasma, and their activity in the hydrolysis of five microRNAs was compared. In general, sIgAs demonstrated higher miRNA-hydrolyzing activity than IgGs antibodies. The hydrolysis of five microRNAs by sIgAs and IgGs was site-specific. The relative activity of each microRNA hydrolysis was very dependent on the milk preparation. The correlation coefficients between the content of five RNAs in milk plasma and the relative activity of sIgAs than IgGs in their hydrolysis strongly depended on individual microRNA and changed from -0.01 to 0.80. Thus, it was shown that milk contains specific antibodies-abzymes hydrolyzing microRNAs specific for human milk.

Natural IgG against S-Protein and RBD of SARS-CoV-2 Do Not Bind and Hydrolyze DNA and Are Not Autoimmune.

Since the onset of the COVID-19 pandemic, numerous publications have appeared describing autoimmune pathologies developing after a coronavirus infection, with several papers reporting autoantibody production during the acute period of the disease. Several viral diseases are known to trigger autoimmune processes, and the appearance of catalytic antibodies with DNase activity is one of the earliest markers of several autoimmune pathologies. Therefore, we analyzed whether IgG antibodies from blood plasma of SARS-CoV-2 patients after recovery could bind and hydrolyze DNA. We analyzed how vaccination of patients with adenovirus Sputnik V vaccine influences the production of abzymes with DNase activity. Four groups were selected for the analysis, each containing 25 patients according to their relative titers of antibodies to S-protein: with high and median titers, vaccinated with Sputnik V with high titers, and a control group of donors with negative titers. The relative titers of antibodies against DNA and the relative DNase activity of IgGs depended very much on the individual patient and the donor, and no significant correlation was found between the relative values of antibodies titers and their DNase activity. Our results indicate that COVID-19 disease and vaccination with adenoviral Sputnik V vaccine do not result in the development or enhancement of strong autoimmune reactions as in the typical autoimmune diseases associated with the production of anti-DNA and DNA hydrolyzing antibodies.

Experimental Autoimmune Encephalomyelitis of Mice: Enzymatic Cross Site-Specific Hydrolysis of H4 Histone by IgGs against Histones and Myelin Basic Protein.

Histones play vital roles in chromatin functioning and gene transcription, but in intercellular space, they are harmful due to stimulating systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the most important protein of the axon myelin-proteolipid sheath. Antibodies-abzymes with different catalytic activities are critical and specific features of some autoimmune diseases. Five IgG preparations against histones (H4, H1, H2A, H2B, and H3) and against MBP corresponding to different spontaneous, MOG (myelin oligodendrocyte glycoprotein of mice), and DNA-histones that accelerated onset, acute, and remission stages of experimental autoimmune encephalomyelitis (EAE; model of human multiple sclerosis) development were obtained from EAE-prone C57BL/6 mice by several affinity chromatographies. IgG-abzymes against five histones and MBP possess unusual polyreactivity in complexation and catalytic cross-reactivity in the hydrolysis of histone H4. IgGs against five histones and MBP corresponding to 3 month-old mice (zero time) in comparison with Abs corresponding to spontaneous development of EAE during 60 days differ in type and number of H4 sites for hydrolysis. Immunization of mice with MOG and DNA-histones complex results in an acceleration of EAE development associated with an increase in the activity of antibodies in H4 hydrolysis. Twenty days after mouse immunization with MOG or DNA-histones complex, the IgGs hydrolyze H4 at other additional sites compared to zero time. The maximum number of different sites of H4 hydrolysis was revealed for IgGs against five histones and MBP at 60 days after immunization of mice with MOG and DNA-histones. Overall, it first showed that at different stages of EAE development, abzymes could significantly differ in specific sites of H4 hydrolysis.

Antibodies-Abzymes with Antioxidant Activities in Two Th and 2D2 Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology.

The exact mechanisms of multiple sclerosis development are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis) in Th and 2D2 mice is associated with the infringement of the differentiation profiles of bone marrow hematopoietic stem cells which are bound with the production of compounds that are harmful for human autoantibodies-abzymes that hydrolyze myelin oligodendrocyte glycoprotein, myelin basic protein, and DNA. It showed that autoimmune patients' antioxidant IgG antibodies oxidise some compounds due to their peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent) activities more effectively than those in healthy humans can. It was interesting to identify whether the redox activities of the antibodies change during the development of autoimmune diseases. Here, we analyzed the change in these redox activities of the IgGs from the blood of Th and 2D2 mice, which corresponded to different stages of the EAE development. The peroxidase activity in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in the Th (4-fold) and 2D2 (2-fold) mice IgGs, on average, is higher than the oxidoreductase activity is. The peroxidase activity of the Th (1.9-fold) and 2D2 (3.5-fold) mice IgGs remarkably increased during the 40 days of the spontaneous development of EAE. Forty days after the immunization of the MOG peroxidase activity, the IgGs of the Th and 2D2 mice increased 5.6-6.0 times when they were compared with those that presented no increase (3 months of age). The mice IgGs were oxidized with 3,3'-diaminobenzidine (2.4-4.3-fold) and o-phenylenediamine (139-143-fold) less efficiently than they were with ABTS. However, the temper of the change in the IgG activity in the oxidation of these substrates during the spontaneous and MOG-induced development of EAE was close to that which occurred for ABTS. All of the data show that the IgG peroxidase and oxidoreductase activities of EAE mice can play an important role in their protection from toxic compounds and oxidative stress.

Cell Differentiation and Proliferation in the Bone Marrow and Other Organs of 2D2 Mice during Spontaneous Development of EAE Leading to the Production of Abzymes.

The exact cellular and molecular mechanisms of multiple sclerosis and other autoimmune diseases have not been established. Autoimmune pathologies are known to be associated with faults in the immune system and changes in the differentiation profiles of bone marrow stem cells. This study analyzed various characteristics of experimental autoimmune encephalomyelitis (EAE) in 2D2 mice. Differentiation profiles of six hematopoietic stem cells of bone marrow were found to significantly differ in 2D2 male and female mice during the spontaneous development of EAE. In addition, we found various properties of B and T cells, CD4+ and CD8+ lymphocytes in blood and several organs (bone marrow, spleen, thymus, and lymph nodes) of 2D2 male and female mice to be considerably different. These changes in hematopoietic stem cells differentiation profiles and level of lymphocyte proliferation in various organs of 2D2 mice were found to induce the production of IgGs against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, increasing the number of autoantibodies hydrolyzing these substrates. We compared the changes of these immunological and biochemical parameters in 2D2 mice with those of mice of two other lines (Th and C57BL/6), also prone to spontaneous development of EAE. Some noticeable and even extreme variations were found in the time-related development of parameters between male and female mice of 2D2, Th, and C57BL/6 lines. Despite some differences, mice of all three lines demonstrated the changes in hematopoietic stem cells profiles, lymphocyte content, and production of catalytic autoantibodies. Given that these changes are harmful to mice, we believe them to cause the development of experimental autoimmune encephalomyelitis.

The association between EAE development in mice and the production of autoantibodies and abzymes after immunization of mice with different antigens.

We have previously shown that immunization of C57BL/6 mice, prone to spontaneous development of experimental autoimmune encephalomyelitis (EAE), with three antigens (MOG35-55 , DNA-histone complex or DNA-methylated BSA complex), alters the differentiation profiles of bone marrow haematopoietic stem cells (HSCs). These are associated with the production of autoantibodies (auto-Abs) against these antigens and the formation of abzymes hydrolysing DNA, MOG, myelin basic protein (MBP) and histones. Immunization of mice with antigens accelerates the development of EAE. This work is the first to analyse the ratio of auto-Abs without and with catalytic activities at different stages of EAE development (onset, acute and remission phases) after immunization of mice with the three specific antigens. Prior to immunization and during spontaneous in-time development of EAE, the concentration of auto-Abs against MBP, MOG, histones and DNA and activities of IgG antibodies in the hydrolysis of substrates increased in parallel; correlation coefficients = +0.69-0.94. After immunization with MOG, DNA-histone complex or DNA-met-BSA complex, both positive (from +0.13 to +0.98) and negative correlations (from -0.09 to -0.69) were found between these values. Our study is the first showing that depending on the antigen, the relative amount of harmful auto-Abs without and abzymes with low or high catalytic activities may be produced only at onset and in acute or remission phases of EAE. The antigen governs the EAE development rate, whereby the ratio of auto-Abs without catalytic activity and with enzymatic activities of harmful abzymes hydrolysing MBP, MOG, histones and DNA varies strongly between different disease phases.

Catalytic antibodies in the bone marrow and other organs of Th mice during spontaneous development of experimental autoimmune encephalomyelitis associated with cell differentiation.

Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.