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.

Hydrolysis of Oligodeoxyribonucleotides on the Microarray Surface and in Solution by Catalytic Anti-DNA Antibodies in Systemic Lupus Erythematosus.

Anti-DNA antibodies are known to be classical serological hallmarks of systemic lupus erythematosus (SLE). In addition to high-affinity antibodies, the autoantibody pool also contains natural catalytic anti-DNA antibodies that recognize and hydrolyze DNA. However, the specificity of such antibodies is uncertain. In addition, DNA binding to a surface such as the cell membrane, can also affect its recognition by antibodies. Here, we analyzed the hydrolysis of short oligodeoxyribonucleotides (ODNs) immobilized on the microarray surface and in solution by catalytic anti-DNA antibodies from SLE patients. It has been shown that IgG antibodies from SLE patients hydrolyze ODNs more effectively both in solution and on the surface, compared to IgG from healthy individuals. The data obtained indicate a more efficient hydrolysis of ODNs in solution than immobilized ODNs on the surface. In addition, differences in the specificity of recognition and hydrolysis of certain ODNs by anti-DNA antibodies were revealed, indicating the formation of autoantibodies to specific DNA motifs in SLE. The data obtained expand our understanding of the role of anti-DNA antibodies in SLE. Differences in the recognition and hydrolysis of surface-tethered and dissolved ODNs need to be considered in DNA microarray applications.

Identification of Antibody-Mediated Hydrolysis Sites of Oligopeptides Corresponding to the SARS-CoV-2 S-Protein by MALDI-TOF Mass Spectrometry.

Antibodies recognizing RBD and the S-protein have been previously demonstrated to be formed in humans after SARS-CoV-2 infection and vaccination with the Sputnik V adenovirus vaccine. These antibodies were found to be active when hydrolyzing FITC-labeled oligopeptides corresponding to linear epitopes of the S-protein. The thin-layer chromatography method allows the relative accumulation of the reaction product to be estimated but cannot identify hydrolysis sites. This study used the MALDI-TOF MS method to establish oligopeptide hydrolysis sites. Using the MALDI-TOF MS method in combination with the analysis of known hydrolysis sites characteristic of canonical proteases allowed us to establish the unique hydrolysis sites inherent only to catalytically active antibodies. We have discovered two 12-mer oligopeptides to have six hydrolysis sites equally distributed throughout the oligopeptide. The other three oligopeptides were found to have two to three closely spaced hydrolysis sites. In contrast to trypsin and chymotrypsin proteases, the catalytically active antibodies of COVID-19 patients have their peptide bond hydrolyzed mainly after proline, threonine, glycine, or serine residues. Here, we propose a new high-throughput experimental method for analyzing the proteolytic activity of natural antibodies produced in viral pathology.

Milk Exosomes: Next-Generation Agents for Delivery of Anticancer Drugs and Therapeutic Nucleic Acids.

Exosomes are nanovesicles 40-120 nm in diameter secreted by almost all cell types and providing humoral intercellular interactions. Given the natural origin and high biocompatibility, the potential for loading various anticancer molecules and therapeutic nucleic acids inside, and the surface modification possibility for targeted delivery, exosomes are considered to be a promising means of delivery to cell cultures and experimental animal organisms. Milk is a unique natural source of exosomes available in semi-preparative and preparative quantities. Milk exosomes are highly resistant to the harsh conditions of the gastrointestinal tract. In vitro studies have demonstrated that milk exosomes have an affinity to epithelial cells, are digested by cells by endocytosis mechanism, and can be used for oral delivery. With milk exosome membranes containing hydrophilic and hydrophobic components, exosomes can be loaded with hydrophilic and lipophilic drugs. This review covers a number of scalable protocols for isolating and purifying exosomes from human, cow, and horse milk. Additionally, it considers passive and active methods for drug loading into exosomes, as well as methods for modifying and functionalizing the surface of milk exosomes with specific molecules for more efficient and specific delivery to target cells. In addition, the review considers various approaches to visualize exosomes and determine cellular localization and bio-distribution of loaded drug molecules in tissues. In conclusion, we outline new challenges for studying milk exosomes, a new generation of targeted delivery agents.

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.

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

Histones play vital roles in chromatin function and gene transcription; however, they are very harmful in the intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin-proteolipid sheath. Antibodies-abzymes with various catalytic activities are specific features of some autoimmune diseases. IgGs against individual histones (H2A, H1, H2B, H3, and H4) and MBP were isolated from the blood of experimental-autoimmune-encephalomyelitis-prone C57BL/6 mice by several affinity chromatographies. These Abs-abzymes corresponded to various stages of EAE development: spontaneous EAE, MOG, and DNA-histones accelerated the onset, acute, and remission stages. IgGs-abzymes against MBP and five individual histones showed unusual polyreactivity in the complex formation and enzymatic cross-reactivity in the specific hydrolysis of the H2A histone. All the IgGs of 3-month-old mice (zero time) against MBP and individual histones demonstrated from 4 to 35 different H2A hydrolysis sites. The spontaneous development of EAE over 60 days led to a significant change in the type and number of H2A histone hydrolysis sites by IgGs against five histones and MBP. Mice treatment with MOG and the DNA-histone complex changed the type and number of H2A hydrolysis sites compared to zero time. The minimum number (4) of different H2A hydrolysis sites was found for IgGs against H2A (zero time), while the maximum (35) for anti-H2B IgGs (60 days after mice treatment with DNA-histone complex). Overall, it was first demonstrated that at different stages of EAE evolution, IgGs-abzymes against individual histones and MBP could significantly differ in the number and type of specific sites of H2A hydrolysis. The possible reasons for the catalytic cross-reactivity and great differences in the number and type of histone H2A cleavage sites were analyzed.

Catalase Activity of IgGs of Patients Infected with SARS-CoV-2.

Coronavirus disease (COVID-19), caused by the SARS-CoV-2 coronavirus, leads to various manifestations of the post-COVID syndrome, including diabetes, heart and kidney disease, thrombosis, neurological and autoimmune diseases and, therefore, remains, so far, a significant public health problem. In addition, SARS-CoV-2 infection can lead to the hyperproduction of reactive oxygen species (ROS), causing adverse effects on oxygen transfer efficiency, iron homeostasis, and erythrocytes deformation, contributing to thrombus formation. In this work, the relative catalase activity of the serum IgGs of patients recovered from COVID-19, healthy volunteers vaccinated with Sputnik V, vaccinated with Sputnik V after recovering from COVID-19, and conditionally healthy donors were analyzed for the first time. Previous reports show that along with canonical antioxidant enzymes, the antibodies of mammals with superoxide dismutase, peroxidase, and catalase activities are involved in controlling reactive oxygen species levels. We here show that the IgGs from patients who recovered from COVID-19 had the highest catalase activity, and this was statistically significantly higher each compared to the healthy donors (1.9-fold), healthy volunteers vaccinated with Sputnik V (1.4-fold), and patients vaccinated after recovering from COVID-19 (2.1-fold). These data indicate that COVID-19 infection may stimulate the production of antibodies that degrade hydrogen peroxide, which is harmful at elevated concentrations.

Isolation of Extracellular Vesicles of Holothuria (Sea Cucumber Eupentacta fraudatrix).

Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.

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.

Secretory immunoglobulin A from human milk hydrolyzes 5 histones and myelin basic protein.

Mother's milk provides newborns with various nutrients (e.g., enzymes, proteins, peptides, hormones, antibodies) that help babies grow and protect them from bacterial and viral infections. The functions of many components of breast milk can be very different from their corresponding functions in body fluids of healthy adults. Catalytic antibodies (abzymes) that hydrolyze peptides, proteins, DNA, RNA, and oligosaccharides were detected not only in human milk, but also in the blood sera of autoimmune patients. However, abzymes with unexpected synthetic activities (lipids, oligosaccharides, and protein kinase activities) were revealed in milk that were not found in the blood of autoimmune patients. The nutrition of infants with fresh milk has a very specific role; newborns are well protected by antibodies of mother's milk (passive immunity). Protease abzymes were found in the blood sera of autoimmune patients, whereas healthy humans usually do not contain such autoantibodies. Here, we present the first evidence that the milk of healthy mothers contains secretory (s)IgA that effectively hydrolyze 5 histones (e.g., H1, H2A, H2B, H3, and H4) and myelin basic protein (MBP). Several rigid criteria were applied to show that protease activity is an intrinsic property of sIgA. Milk abzymes against 5 histones cannot hydrolyze different control proteins except histones and MBP, whereas autoantibodies against MBP split this protein and 5 histones. Antibodies against histones and MBP exhibit complexation polyreactivity as well as specific and unusual catalytic cross-reactivity. With some exceptions, the specific sites of hydrolysis of H1, H2A, and H2B by sIgA against histones do not coincide with the sites of hydrolysis by abzymes against MBP. On the whole, fresh human milk is a very specific source of many of the most unusual antibodies and abzymes.

Essential Protective Role of Catalytically Active Antibodies (Abzymes) with Redox Antioxidant Functions in Animals and Humans.

During the life of aerobic organisms, the oxygen resulting from numerous reactions is converted into reactive oxygen species (ROS). Many ROS are dangerous due to their high reactivity; they are strong oxidants, and react with various cell components, leading to their damage. To protect against ROS overproduction, enzymatic and non-enzymatic systems are evolved in aerobic cells. Several known non-enzymatic antioxidants have a relatively low specific antioxidant activity. Superoxide dismutases, catalase, glutathione peroxidase, glutathione S-transferase, thioredoxin, and the peroxiredoxin families are the most important enzyme antioxidants. Artificial antibodies catalyzing redox reactions using different approaches have been created. During the past several decades, it has been shown that the blood and various biological fluids of humans and animals contain natural antibodies that catalyze different redox reactions, such as classical enzymes. This review, for the first time, summarizes data on existing non-enzymatic antioxidants, canonical enzymes, and artificial or natural antibodies (abzymes) with redox functions. Comparing abzymes with superoxide dismutase, catalase, peroxide-dependent peroxidase, and H2O2-independent oxidoreductase activities with the same activities as classical enzymes was carried out. The features of abzymes with the redox activities are described, including their exceptional diversity in the optimal pH values, dependency and independence on various metal ions, and the reaction rate constants for healthy donors and patients with different autoimmune diseases. The entire body of evidence indicates that abzymes with redox antioxidant activities existing in the blood for a long time compared to enzymes are an essential part of the protection system of humans and animals from oxidative stress.

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.

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.

Protease and DNase Activities of a Very Stable High-Molecular-Mass Multiprotein Complex from Sea Cucumber Eupentacta fraudatrix.

Only some human organs, including the liver, are capable of very weak self-regeneration. Some marine echinoderms are very useful for studying the self-regeneration processes of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix (holothurians) demonstrate complete restoration of all organs and the body within several weeks after their division into two parts. Therefore, these cucumbers are a prospective model for studying the general mechanisms of self-regeneration. However, there is no data available yet concerning biomolecules of holothurians, which can stimulate the processes of organ and whole-body regeneration. Investigation of these restoration mechanisms is very important for modern medicine and biology because it can help to understand which hormones, nucleic acids, proteins, enzymes, or complexes play an essential role in self-regeneration. It is possible that stable, polyfunctional, high-molecular-weight protein complexes play an essential role in these processes. It has recently been shown that sea cucumbers Eupentacta fraudatrix contain a very stable multiprotein complex of about 2000 kDa. The first analysis of possible enzymatic activities of a stable protein complex was carried out in this work, revealing that the complex possesses several protease and DNase activities. The complex metalloprotease is activated by several metal ions (Zn2+ > Mn2+ > Mg2+). The relative contribution of metalloproteases (~63.4%), serine-like protease (~30.5%), and thiol protease (~6.1%) to the total protease activity of the complex was estimated. Metal-independent proteases of the complex hydrolyze proteins at trypsin-specific sites (after Lys and Arg). The complex contains both metal-dependent and metal-independent DNases. Mg2+, Mn2+, and Co2+ ions were found to strongly increase the DNase activity of the complex.

Analysis of Proteins and Peptides of Highly Purified CD9+ and CD63+ Horse Milk Exosomes Isolated by Affinity Chromatography.

Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9+ and CD63+ exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.

Multiplex Analysis of Serum Cytokine Profiles in Systemic Lupus Erythematosus and Multiple Sclerosis.

Changes in cytokine profiles and cytokine networks are known to be a hallmark of autoimmune diseases, including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). However, cytokine profiles research studies are usually based on the analysis of a small number of cytokines and give conflicting results. In this work, we analyzed cytokine profiles of 41 analytes in patients with SLE and MS compared with healthy donors using multiplex immunoassay. The SLE group included treated patients, while the MS patients were drug-free. Levels of 11 cytokines, IL-1b, IL-1RA, IL-6, IL-9, IL-10, IL-15, MCP-1/CCL2, Fractalkine/CX3CL1, MIP-1a/CCL3, MIP-1b/CCL4, and TNFa, were increased, but sCD40L, PDGF-AA, and MDC/CCL22 levels were decreased in SLE patients. Thus, changes in the cytokine profile in SLE have been associated with the dysregulation of interleukins, TNF superfamily members, and chemokines. In the case of MS, levels of 10 cytokines, sCD40L, CCL2, CCL3, CCL22, PDGF-AA, PDGF-AB/BB, EGF, IL-8, TGF-a, and VEGF, decreased significantly compared to the control group. Therefore, cytokine network dysregulation in MS is characterized by abnormal levels of growth factors and chemokines. Cross-disorder analysis of cytokine levels in MS and SLE showed significant differences between 22 cytokines. Protein interaction network analysis showed that all significantly altered cytokines in both SLE and MS are functionally interconnected. Thus, MS and SLE may be associated with impaired functional relationships in the cytokine network. A cytokine correlation networks analysis revealed changes in correlation clusters in SLE and MS. These data expand the understanding of abnormal regulatory interactions in cytokine profiles associated with autoimmune diseases.

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.

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.