Sorption of Interleukins 12 and 23 Using the Developed Magnetocontrollable Ustekinumab-Based Sorbent.

The aim of the study was to assess the sorption capacity of Ustekinumab, a drug based on interleukins 12 (IL-12) and 23 (IL-23), when using it with a magnetocontrollable sorbent. MATERIALS AND METHODS: To reduce the concentration of proinflammatory cytokines (IL-12, IL-23), the blood of patients with psoriatic arthritis was passed through magnetocontrollable polyacrylamide granules (MPG) of spherical shape with the particle size of 10-100 µm, obtained by emulsion polymerization. Perfusion was performed through a 10 ml column equipped with an electromagnet, into which MPG with immobilized antibodies to IL-12 and IL-23 was added. Ustekinumab, a commercial drug with concentration of monoclonal antibodies equaling 0.2 mg per 1 ml of saline was used as the source of antibodies to these interleukins. The specific sorption capacity of MPG was determined using a column filled with the granules in a volume of 0.2 ml and inducing their subsequent interaction with 1 ml of IL-12 and IL-23 solutions with increasing concentrations.The heparinized blood of 10 patients with psoriatic arthritis of various degrees of activity, who had no parenteral administration of IL-12/IL-23 inhibitors (Ustekinumab) for 12 months, was subjected to in vitro treatment. The blood of 10 healthy donors was used as the control; the perfusion procedure was similar. RESULTS: Digital parameters were measured for each sample before and after the interaction between the sorbent and blood plasma. There was a significant decrease in cytokines from the baseline - by 99.8% (in donors) and by 99.9% (in patients). When using a carbon sorbent, their concentration decreases by 92.6% from the baseline. As a result of sorption, blood cell counts did not change reliably, which is an additional positive aspect of this procedure. CONCLUSION: The maximum possible decrease in the level of cytokines has great practical importance, since they play a leading role in the pathogenesis of psoriatic arthritis. The use of a synthesized Ustekinumab-based sorbent is a highly effective way of removing them simultaneously from blood plasma.

[Techniques of modifying lipid antigens for synthesis of diagnostic and therapeutic preparations in rheumatology.]

The objective of the study is to enhance sorption capacity of diagnostic agents by using cardiolipin antigens for antiphospholipid syndrome in patients with systemic lupus erythematosus (SLE). A technique of emulsion polimerization was used. Having integrated antigen nanoobjects we developed immobilized magnetocontrollable antigen nanosystems and put them to an evaluation test. The nanosystems are polyacrylamide granules with a built in antigen. To obtain stable immobilized multi-use biopharmaceuticals with targeted properties (shape, particle diameter, pore size, density) we used a modified version of emulsion polymerization method using polyacrylamide carrier gel. This method permitted a greater sorptive capacity, preserving the antigen in maximum native state, and opened up the possibility of controllable modification of nanoobjects. Cardiolipin was used as the antigen in question. Following the method described above we performed sorption of anticardiolipin antibodies from blood plasma of SLE patients who showed clinical presentations of antiphospholipid syndrome. All SLE patoents with signs of antiphospholipid syndrome showed reliably higher levels of cardiolipin antibodies compared with SLE patients without antiphospholipid syndrome signs; the antibody level was 0.365 ± 0.026 and 0.075 ± 0.003 on average, correspondingly (p < 0.001). Blood serum from 10 apparently healthy individuals served as control. The level of cardiolipin antibodies was determined before and after sorption by indirect solid phase immunoenzyme method. In the eluate we estimated total protein by Lowry method. In vitro testing showed that the obtained antigen nanosystems based on immobilized cardiolipin could effectively remove cardiolipin antibodies from whole blood of SLE patients with clinical presentations of APS to achieve the values of healthy individuals (before sorption cardiolipin antibodies 0.328 ± 0.0289; after sorption 0.059 ± 0.0170; p<0,001; sorption capacity 8.00 ± 0.390 mg/ml). The method of emulsion polymerization with consideration to hydrophobic and hydrophilic properties of lipid molecules permits obtaining and modifying biomolecules with certain properties, in a controlled fashion.

Anti-DNase I antibodies in systemic lupus erythematosus: diagnostic value and share in the enzyme inhibition.

Diagnostic accuracy of anti-DNase I antibodies measurement in a differentiation between SLE and other autoimmune rheumatic diseases was evaluated. The share of anti-DNase I and actin in the DNase I activity decrease in SLE was established. Serum samples were obtained from 54 patients with verified SLE, 52 control patients with other autoimmune rheumatic diseases, and 44 healthy persons. Anti-DNase I concentrations were measured by ELISA. Free and actin inhibited DNase I activities were evaluated in the fresh serum samples. The appraisal of antibodies and actin effects on DNase I activity was made using multiple regression. Anti-DNase I antibodies were positive in 35 SLE and 8 control patients, without significant difference between the mean antibody concentrations. Sensitivity of this test was 64.81 %, and specificity-84.62 %. Mean free DNase I activity in SLE was somewhat lower than in the control group as a result of augmented frequency of extremely low enzyme activities. On the contrary, after the exclusion of the latter cases we have revealed elevated mean free DNase I activity in the other SLE patients comparing to the similar control subgroup. Unlike the controls, low serum DNase I activity in SLE arose not only from actin and antibody action, but also, in half of the cases, from unidentified factor, related to active SLE. The accuracy of the anti-DNase I antibodies measurement is approximate to the present reference standard of SLE diagnostics. We first demonstrated that neither antibodies nor actin caused DNase I activity decrease in SLE.

[Experimental modeling of nucleoprotein disposal disorders in systemic lupus erythematosus].

The objective of this research was to adapt the experimental model simulating the nucleoprotein disposal disorders in systemic lupus erythematosus (SLE) for further study of its extracorporeal correction, as well as to assess validity of the model by short-term experiment. Twenty to female Wistar rats were intraperitoneally injected with the chromatin-containing extract from bovine liver followed by intravenous administration of anti-DNA antibodies derived from SLE patients. After these procedures plasma concentrations of anti-dsDNA, circulating immune complexes and DNA became sharply increased, together with distinct elevation of leukocytes. On the contrary, changes in erythrocytes, platelets, total protein concentration, creatinine, asparagine and alanine aminotransferase activities, as well as blood coagulation time were changed insignificantly. Using direct immunofluorescence of cryosections, we detected human IgG deposition in rat kidneys treated in accordance with the simulation protocol. Thus, our model reproduces essential DNA disposal disorders in SLE without any animal death or the life-threatening changes in examined markers during short-term experiment.

[Extracorporeal treatment of nucleoprotein disposal disorders using the systemic lupus erythematosus model: evaluation of efficacy and safety in a short-term simulated experiment].

Efficacy and safety of the extracorporeal blood perfusion through DNase I- and C1q-containing magnetic beads have been evaluated using the experimental model simulating the nucleoprotein disposal disorders in systemic lupus erythematosus (SLE). The study was performed using 20 rats in which the essential impairments of nucleoprotein catabolism typical for SLE were modeled. The animals were randomized into the experimental group and the placebo perfusion control group. Rats of the experimental group were characterized by the statistically significant reduction of increased levels of circulating immune complexes and plasma DNA as well as diminished levels of plasma creatinine and kidney IgG deposition as compared with placebo controls. During short-term experiment there were neither animal deaths nor substantial blood cell destruction and hepatotoxicity signs.

Emulsive polymerization as a method of enzyme modification preserving biological properties of nanostructures.

The proposed method of emulsive polymerization provides the possibility of modifying and obtaining insoluble forms of superoxide dismutase (SOD), glutathione reductase, and streptolysin-O preserving nanoobjects (conformationally active centers and antigenic determinants) in their native states. Apart from enzymatic and immunological properties, the samples acquired some new features: resistance to high temperature, resistance to 3 M KCNS solution and buffer solutions with high concentration of hydrogen ions, and resistance to preserving solutions. Magnetic properties provide the possibility of simplifying enzyme-linked immunosorbent and immunofluorescence assays. In addition, sensitivity of these assays was by an order of magnitude higher and the specificity fully preserved. Taking all the facts into account, we prepared agents for long-term and repeated use. Due to preserved enzymatic properties, insoluble forms of SOD and glutathione reductase can be considered as a tool for correction of peroxide-antioxidant balance and associated immunological abnormalities.

[Clinical and pathogenetic significance of antibodies to antioxidative enzymes in patients with Raynaud's syndrome].

Patients presenting with systemic scleroderma were found to have antibodies to antioxidative enzymes the levels of which increased with activity of the disease. Taking into account the important role of immune disorders in the development of systemic sclerosis, it can be conjectured that antienzyme antibodies may cause dysregulation of enzymatic systems. Serum antibodies were detected by the original modification of indirect enzyme immunoassay using immobilized antigenic forms of enzymes. All patients with high antibody titers presented with class II-IV Raynaud"s syndrome (RS). Pathogenetic mechanisms of RS are complicated and poorly known, it is supposed to be a multifactor pathology associated with disturbances in neural, vascular, mediator, and immune systems. Reperfusion and free oxygen radicals may also contribute to the development of ischemia in RS. The antioxidative system is known to involve a number of enzymes that neutralize pathogenic effects of free oxygen species. Disturbance of equilibrium between aggressive and protective factors under pathological conditions leads to further aggravation of tissue lesions. The presence of antienzyme antibodies in the blood of patients with primary RS may be regarded as an unfavourable prognostic factor preceding a systemic disease of connective tissue.

[Interrelationship between antibodies to enzymes of antioxidant system and cardiac involvement in patients with systemic scleroderma].

In blood of patients with systemic scleroderma we detected antibodies to the antioxidant system enzymes. Level of specific immunoglobulins rose with increase of the disease activity. Antioxidant system of the body is represented by a number of enzymes called to negate pathogenic effect of active forms of oxygen. In pathological states balance between factors of aggression and defense is disturbed. This leads to even more deep damage of tissues. Taking into consideration important role of immunological shifts in development of atherosclerosis one can suggest that autoantibodies to enzymes represent one of mechanisms of derangement of the work of enzymatic systems. Significantly higher levels of antibodies were detected in patients with symptoms of involvement of the cardiovascular system. We measured blood serum antibodies according to elaborated by us method of indirect immuno enzyme analysis with the use of immobilized antigenic forms of enzymes.

[Antibodies to enzymes of purine metabolism as a factor of gastrointestinal tract lesions in systemic scleroderma].

Antibodies to Adenosine Deaminase (AD) and Guanin Deaminase (GDA) were founded in sera of systemic sclerosis (SS) patients, their concentration positive correlated with activity of disease. Increased levels of antibodies to AD and GDA correlated with the severity of gastrointestinal tract injury (especially pancreatitis and hepatitis). It is shown that level of antibodies to AD and GDA differs reliable from contents of these antibodies, in sera of healthy persons. It is shown that AD enzymatic activity was decreased, while activity of GDA was increased. The antibodies to enzymes may be one of the possible causes of change of enzymes activity in sera of SS patients. The method of immunoenzyme determination of level of antibodies to AD, G on the basis of immobilized form of magnet sorbent was developed for immune diagnostics of SS.

[Thyroid hormone antibodies in rheumatoid arthritis].

The goal of the study was to investigate thyroid functional activity in rheumatoid arthritis (RA) and to identify the specific features of the generation of thyroid hormone antibodies in RA patients depending on the activity of the disease. Seventy-five patients with RA (61 (81.4%) females and 14(18.6%) males; mean age 54.1±11.6years) were examined. Physical examination and measurements of the level of thyroid hormones (free T4 and free TJ and the number of their antibodies were made. Thirty healthy individuals were examined as controls. The sera from RA patients showed antibodies to T4 and T3 in 45 and 39% of cases. There was a rise in thyroxine levels and T4 antibody concentrations with the higher activity of RA. Along with the routine clinical and laboratory parameters, the values of T3 and T4 antibodies may be used as an additional tool to characterize the activity of RA genesis.

[Prospects for using antigenic nanosystems in the diagnosis and treatment of inflammatory rheumatic diseases].

AIM: To study whether immobilized antigenic nanosystems (ANS) may be designed on the basis of antigens of varying chemical nature to identify and to remove specific antibodies (Ab) from the blood of patients with systemic lupus erythematosus (SLE). SUBJECTS AND METHODS: Sixty patients with the diagnosis of SLE verified by the 1997 American College of Rheumatology criteria and 30 apparently healthy individuals were followed up. The levels of Ab to catalase (Cat), xanthine oxidase (XO), and cardiolipin (CL) were measured by enzyme immunoassay, by applying the respective ANS as an antigenic matrix. RESULTS: There was a significant relationship of the levels of Ab to Cat and XO to the activity of SLE. It was shown that Ab to Cat and XO could affect the functional activity of serum enzymes. The level of Ab to CL in patients with SLE was found to depend on two parameters - the intensity of the disease and the presence of antiphospholipid syndrome; acute cerebral circulatory disorder and thrombocytopenia were observed to have a significant unidirectional impact on the level of Ab to CL. Immobilized CL-based ANSs were effective in eliminating Ab to CL from the whole blood of patients with SLE, without resulting in a significant hemolysis of blood corpuscles and in a reduction of total protein concentrations. CONCLUSION: The development and introduction of preventive methods for the early diagnosis of SLE may be extended, by using ANS based on Cat, XO, and CL antigen. The designing and putting into practice novel ANS-based hemosorbents may allow immunosorption to occupy a prominent place in the pathogenetic therapy of inflammatory autoimmune diseases.

[Detection of serum catalase antibodies in patients with inflammatory rheumatic diseases by immobilized magnetic sorbents].

Sixty patients with systemic lupus erythematosus, 48 patients with rheumatoid arthritis, and 30 healthy individuals (a control group) were examined. Modified procedures for enzyme immunoassay (EIA) and immunofluorescence (IF) assays with an immobilized magnetic sorbent (MS) based on catalase as an antigenic matrix were used to detect catalase antibodies (Ab). The application of immobilized MSs and a photoelectric digital display attachment to a fluorescence microscope permits measurements of Ab to soluble antigens (catalase) in the IF assay. Nevertheless, for detection of catalase Ab in laboratory practice, preference should be given to EIA using a MS as a more sensitive procedure.

[Use of affinity interaction of biologically active substances in practice of public health system]. / Vozmozhnosti primeneniia affinnogo vzaimodeistviia biologicheski activnykh veshchestv v praktike zdravookhraneniia.

AIM: To determine the potentialities of use of affinity interaction of immobilized biologically active substances (bacterial cells or their fragments, toxins, antigens of various chemical nature, immunoglobulins, enzymes, gangliosides, etc.) for medical practice. MATERIALS AND METHODS: Emulsion polymerization of acrylamide monomers in the gaseous nitrogen current was used as a basic method for preparation of solid-phase magnetic immunosorbents (MIC). A procedure for preparation of siliceous MIC was also applied. The prepared MICs were used a solid phase in enzyme immunoassay and immunofluorescence assay and the recorded data were compared with those of studied conventionally used in practical medicine. RESULTS: The use of MIC made it possible to detect pathogens of particularly dangerous infections in large volumes of the samples contaminated with another microflora. With the proposed MIC, one can stand a good chance of surveying large contingents of the population, of obtaining the quantitative results in shorter periods to establish a diagnosis. With this, the sensitivity and specificity of immunoassays substantially increase. Whether MIC may be used as selective hemosorbents to remove specific antibodies from the blood of patients with rheumatic diseases for therapeutic purposes was studied. CONCLUSION: The findings are indicative of wide potentialities of use of affinity interaction of biologically active substances immobilized on inert carriers with the inserted magnetic material in the laboratory diagnosis of diseases of both infectious and autoimmune nature, which may be widely used in the in- and outpatient settings.

[Affinity sorbents having magnetic properties in the clinical picture and diagnosis of communicable and non-communicable diseases]. / Affinnye sorbenty s magnitnymi svoistvami v klinike i diagnostike infektsionnykh i neinfektsionnykh zabolevanii.

Affine magnetic sorbents which have no analogs in the practice of our country have been for the first time developed for the rapid diagnosis of various life-threatening diseases (plague, cholera, anthrax, glanders, meliodosis, tularemia, leptospirosis, dysentery, viral hepatitis A) and for the identification of their causative agents. The efficacy of new magnet-controlling test systems has been repeatedly confirmed by their applications in epidemiological events and emergencies: in the epidemiological surveillance of viral hepatitis A in Stavropol and in the Caucasian Mineralnye Vody towns, Stavropol Territory (1994), in the identification of cholera patients, in the detection of transmission factors, when monitoring during large epidemic out-bursts of cholera in Stavropol (1990), Daghestan (1994), as well as in the microbiological monitoring during military conflicts in the Chechen Republic (1995). The application of the sorbents has shown that their sensitivity is 4-5 times as much as that of conventional serological assays. In addition, biotechnologies for the production of polyacrylamide and composite aluminosilicate affine immunosorbents with magnetic properties have been developed. They have been used as the basis for designing immobilized granulated antigen reagents for the immunodiagnosis, differential diagnosis, evaluation of the time course and severity of a disease, the efficiency of therapy in patients with systemic scleroderma, proliferative arthritis, systemic lupus erythematosus, juvenile rheumatoid arthritis, osteochondrosis.

[Use of magnetosorbents in the immunodiagnosis and therapy of systemic lupus erythematosus and rheumatoid arthritis]. / Perspektivy primeneniia magnitosorbentov v immunodiagnostike i lechenii sistemnoi krasnoi volchanki i revmatoidnogo artrita.

The authors present new technology of producing immobilized granulated antigenic magnetosorbents (MS) using collagen type II, IgG, denaturated DNA and cardiolipin which were employed in enzyme immunoassay for detection of specific antibodies in the blood of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Due to high capacity, MS demonstrated effectiveness in early diagnosis, prognosis of RA and SLE as well as in control over the treatment effects. As shown in in vitro experiments, MS have high selective capacity and atraumaticity for blood corpuscles. MS can be sterilized, regenerated and reused which is a great advantage.

[Immobilized antigenic preparations with magnetic properties in the diagnosis and treatment of rheumatic diseases]. / Immobilizirovannye antigennye preparaty s magnitnymi svoistvami v diagnostike i lechenii revmaticheskikh zabolevanii.

A procedure was first developed to prepare the immobilized granulated antigen agents (IGAA) based on collagens of types I, II, III, IgG, native DNA, RNA, cardiolipin, superoxide dismutase, and glutathione reductase. The agents were applied to both immunofluorescence and enzyme immunoassay to identify specific antibodies. IGAA were effective in early diagnosis, prognosis, and control of therapy for systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis due to their high capacity. These antigen agents were used in vitro and in vivo in hyperimmune laboratory animals as magnetic sorbents and exhibited high capacities and low traumatic properties for blood cells. IGAA may be regenerated, sterilized, and reused.

[Antibodies to skeletal muscle myofibrillar proteins in the diagnosis of rheumatoid arthritis]. / Antitela k miofibrilliarnym belkam skeletnoi myshtsy v diagnostike revmatoidnogo artrita.

One of the mechanisms of muscular involvement in RA patients was elucidated by identification of antibodies to skeletal muscle components (actin and tropomyosin). Clinical symptoms of skeletal muscles involvement in RA proved to be associated with elevated count of tropomyosin antibodies against an insignificant informative value of actin. Enzyme immunoassay with tropomyosin has established a direct correlation between the number of the antibodies and RA activity.

[The properties and areas of use of an immobilized granulated streptolysin O]. / Svoistva i oblasti primeneniia immobilizovannogo granulirovannogo streptolizina-O.

The heat-resistant preparations of immobilized granulated streptolysin O were obtained with the use of emulsion polymerization techniques. In experiments the presence of gangliosides and cholesterol affected the hemolytic activity of immobilized streptolysine O. The preparations thus obtained were used in the enzyme-linked immunosorbent assay as antigens for the determination of specific antibodies in rheumatic fever patients and healthy persons.