[Comparative characteristics of the complement system in patients with C3-glomerulopathy and atypical hemolytic uremic syndrome of chronic course who suffered an acute episode of thrombotic microangiopathy].

AIM: To compare changes in the complement system in C3-glomerulopathy (C3-GP) and atypical hemolytic uremic syndrome (aHUS) after the relief of an acute episode of thrombotic microangiopathy. MATERIALS AND METHODS: The study included 8 patients diagnosed with C3-GP and 8 with aHUS in remission. The blood levels of the complement system components were determined: C3, C4, C3a, C5a, factor H (CFH), factor B (CFB), membrane-attacking complex (MAC), antibodies to C3b (anti-C3b-AT), the level of hemolytic activity (CH50), the content of factor D (CFD) in the urine. RESULTS: C3 and CH50 levels were within the reference range in both groups, however, in the C3-GP group they were at the lower limit, and C3 level was significantly lower than in the aHUS group: 0.56 [0.44; 0.96] vs 1.37 [1.16; 2.52] (p=0.003). CFB increased level was detected in both groups, but in the C3-GP group it was significantly lower than in the aHUS group - 275.1 [222.1; 356.6] vs 438.7 [323.3; 449.3] (p=0.010). C3a, C5a and MAC levels were increased in both groups, but the maximum was in the C3-GP group, and the MAC level in the C3-GP group was 2 times higher than that in aHUS, and these differences reached statistical significance - 123 555±6686 vs 5603±1294 (p=0.036). CFH and CFD levels was increased in both groups, but their highest values was in the aHUS group. CONCLUSION: Alternative complement pathway activation signs were present in both groups of patients with complement-mediated nephropathies, regardless the stage of the disease. In C3-GP, alternative complement pathway activation was more pronounced than in aHUS after the relief of an acute episode of thrombotic microangiopathy.

[Changes in the complement system in membranoproliferative glomerulonephritis]. / Izmeneniia v sisteme komplementa pri membranoproliferativnom glomerulonefrite.

AIM: To compare the clinical manifestations membranoproliferative glomerulonephritis (MPGN) in its idiopathic variant, lupus nephritis (LN), and C3 glomerulopathy (C3-GP), by comparing them with changes in the complement system. SUBJECTS AND METHODS: The clinic of nephrology followed up 42 patients with different types of MPGN in 2013 to 2015. The study included 35 patients divided into 3 groups: 1) 8 patients with C3-GP, 2) 13 with idiopathic MPGN; 3) 14 with Class IV LN. The investigators studied the blood and urine levels of components and markers for activation of the classical and alternative pathways (C3 and C4, С3а, C5a, CFH, CFB, and CFD) of the terminal complement complex (TCC). RESULTS: The detection rate of C3-GP was 19%. The patients with C3-GP were noted to have the lowest blood concentration of S3 and the highest urinary level of С3а, C5a, TCC, CFH, CFB, and CFD. C3 nephritic factor was detected in 2 patients from the C3-GP (dense deposit disease) group. CONCLUSION: Alternative complement pathway dysregulation caused by genetic or autoimmune factors plays a leading role in the pathogenesis of C3-GP.

[Complement System Abnormalities in Patients with Atypical Hemolytic Uremic Syndrome and Catastrophic Antiphospholipid Syndrome].

Background: The role of the alternative complement pathway (AP) abnormalities in the pathogenesis of aHUS is well studied. Clinical and morphological manifestations of atypical HUS and catastrophic APS are often similar. However, studies on the state of AP in patients with CAPS are virtually absent. Aims: The aim of our study was to assess the state of AP in patients with CAPS and aHUS. Patients and methods: The study enrolled 67 patients (pts) with a diagnosis of CAPS (28 pts) and aHUS (39 pts). Studies of the complement system are made of 10 pts with CAPS and 20 aHUS. Factor H, I, B, D content, functional activity of factor H, and complement components C3, C4 was determined in serum by ELISA kit. Results: Patients with CAPS and aHUS showed similar changes in complement biomarkers. The factor H level in the serum was significantly higher than the standard value. However, the specific activity of factor H reduced, mean rate 59% for aHUS and 26% for CAPS. The median value of factor D was twice higher than the normal range in both groups, indicating the activation of the AP. Conclusions: There are indications of an AP activation not only in pts with aHUS but in CAPS pts too. We suppose that the activity of factor H is a more sensitive indicator of complement system changes than factor H level. Patients with CAPS and aHUS have similar clinical and laboratory characteristics. However, CAPS is more severe, with the involvement of a larger number of vascular beds. Perhaps this is due to the double damaging effects on the endothelium ­ of antiphospholipid antibodies (aPL) and activated complement. So we hypothesize that CAPS can be called aPL-mediated TMA in pts with a complement system defect.

[The detection of activity of components and factors of complement according its effect on infusorians].

The article considers the developed techniques of detection of functional activity of components C1, C2, C3, C4 and membrane attacking complex of classical pathway and also factors B and D of alternative pathway of human complement using automated device measurement of immobilizing effect on infusorians Tetrahymena pyriformis. The techniques are based on application of artificially produced reagents containing all necessary components of compliment besides testing one. The mathematical mode of activities calculation is developed. The linear dependency of velocity of infusorians immobilizing from functional activities of testing component. The relevance of techniques is demonstrated by correlation of results derived using the proposed mode with the developed earlier hemolytic mode.

[Activity of B and D factors of complement alternative pathway in children with atopic dermatitis].

AIM: Development of enzyme immunoassay detection of B and D factors of complement alternative pathway functional activity for solving diagnostic and prognostic problems of patient therapy. Study activity of these factors in blood sera of children with atopic dermatitis before and after therapy for elucidation of the role of complement alternative pathway in pathogenesis of this disease. MATERIALS AND METHODS: Children aged 6 months to 18 years with atopic dermatitis were examined for functional activity of B and D factors in blood sera before and after therapy by the developed methods. RESULTS: The developed enzyme immunoassay methods for determination of functional activity of B and D complement alternative pathway showed high sensitivity and reliability. In children with atopic dermatitis factor B and D activity was significantly lower than normal before treatment. After treatment these activity increased significantly (p < 0.004) and in the case of D factor--up to normal. CONCLUSION: The data obtained in the study indicates the presence of complement alternative pathway activation in atopic dermatitis in children and the possibility of use of factor B and D functional activity analysis for diagnostic and prognostic purposes.

[Alterations of the complement system in patients with catastrophic antiphospholipid syndrome].

AIM: To investigate alterations of the complement system in patients with catastrophic antiphospholipid syndrome (CAPS). SUBJECTS AND METHODS: Four patients (2 men aged 23 and 40 years and 2 women aged 39 and 58 years) diagnosed as having CAPS, including 3 patients with systemic lupus erythematosus and secondary antiphospholipid syndrome (APS) and 1 patient with primary APS, were examined. The activity of the complement components C1-C5 and total hemolytic activity were determined in all the patients at the moment of an acute episode and in 1 patient after treatment. RESULTS: The activity of the studied complement components and total hemolytic complement activity proved to be significantly decreased in all the patients. That of complement components recovered after treatment using fresh frozen plasma. The possibility and mechanisms of complement system activation in the patients with CAPS are discussed. CONCLUSION: The preliminary results obtained by the examination of few cases may lead to the conclusion that the complement system may be involved in the development of CAPS.

A universal method for measuring functional activity of complement in humans, laboratory, domestic, and agricultural animals, amphibians, and birds.

A new universal method for measuring activity of the serum complement system in humans, laboratory, domestic, agricultural animals, birds and amphibians is based on automated evaluation of the mortality of ciliate Tetrahymena pyriformis under the effect of the complement system. In contrast to the hemolytic method, measured activity of the complement shows no erroneously high results caused by reactive lysis in febrile patients. The method can be used for studies of the complement system in humans and animals without species-specific adaptation.

[Anticoagulative and anticomplementary activity of endogenous inhibitor preparation from hepatopancreas of red king crab (Paralithosed camtschaticus) towards human blood].

Serpins (SERine Protease INhibitors)--is large and diverse group of proteins with similar structures, which can inhibit both serine and cysteine proteases by an irreversible suicide mechanism. A novel serpin from hepatopancreas of Red King Crab (Paralithosed camtschaticus) was obtained and was studied its effect on the process of human blood plasma clotting. The investigated serpin shows a noticeable anticoagulative activity, which increases dramatically in the combined action with heparine. Though the inhibitor has almost no effect on thrombin, it inhibits C1s (C1-esterase). We studied the action of the serpin from P. camtschaticus on C1s via its competitive inhibition by C1 inhibitor and the novel enzyme. The calculated inhibition constant of the serpin from P. camtschaticus towards C1s is 2.02 +/- 0.71 M. Unlike C1 inhibitor, the novel serpin from P. camtschaticus doesn't suppress fibrinolysis and at the same time prevents blood clotting. These features may be of interest for medical purposes.

[Coronarography in coronary heart disease in elderly patients of behavioral type A].

177 patients aged 32 to 74 years with CHD were examined. The data on angiographic situation and connection between the condition of coronary arteries and behavioral singularity of old-aged CHD patients of different behavioral types are revealed. The results of angiographic examination of heart vessels in old-aged CHD patients of behavioral type A are shown.

[Masked deficiency of C4 component of complement in patients with chronic gastrointestinal tract diseases of different etiology].

AIM: Frequency of occurrence detection of C4A and C4B complement system deficiency in patients with chronic gastrointestinal tract (GIT) diseases including gastric ulcer (GU) and duodenal ulcer (DU). MATERIALS AND METHODS: 74 patients with chronic GIT diseases were examined. Endoscopy with stomach mucosa condition evaluation based on histobacterioscopic examination of gastroduodenal biopsy samples was used. Intestine microbiocenosis evaluation was performed by using microflora degree of manifestation according to Federal Standard of Russian Ministry of Health No 231 -91500.11.0004-2003. C4A and C4B isotypes in blood sera of patients were measured by using enzyme immunoassay. RESULTS: Chronic gastroduodenitis was diagnosed in 35.1%, pangastritis B--in 41.9%, GU and DU--in 23% of patients. Histological evaluation of biopsy samples revealed marked inflammatory changes in stomach and duodenum mucosa in 77% of patients. Stomach mucosa infection rate by Helicobacter pylori reached 85%. Microbiological disorders manifestation in microflora of patients matched endoscopic and histobacteriscopic changes in it and was the highest for GU and DU. In 76.0% of cases C4A and C4B isotype deficiency in blood sera matches the development of erosive-ulcerous process in stomach and duodenum mucosa with marked background dysbiotic GIT microbiota disorders. CONCLUSION: Patients with functional deficiency of C4A and C4B isotypes have a genetic burden to susceptibility to chronic GIT diseases whereas H.pylori infection deteriorate the disease.

[Enzyme immunoassay of masked complement component C4 deficiency in patients with urogenital Chlamydia infection].

AIM: Development of new method of C4B isotype functional activity evaluation in enzyme immunoassay by using pharmaceutical preparation derinat as a classical pathway complement activator and its use for blood sera isotyping in confirmed urogenital tract chlamydia infection. MATERIALS AND METHODS: Enzyme immunoassay was used to detect C4A and C4B isotype functional deficiency in blood sera of patients. Chlamydia etiology urogenital infection diagnosis was based on results of standard clinical-instrumental examination methods: vaginal clinical smear analysis, scrape sample light microscopy with consequent treatment by fluorescent monoclonal antibodies against Chlamydia trachomatis and PCR. RESULTS: In acute form of the disease C4A deficiency frequency of occurrence was 0.36, and C4B deficiency - 0.55. In chronic form of the disease deficiency frequency of occurrence was 0.38 for both isotypes. In the group of healthy people isotype deficiency was 0.08 and 0.25, respectively. CONCLUSION: Innate masked C4 deficiency interfere with the normal immune defense of organism against chlamydia infection, and antigen carbohydrate pathogenicity may possibly be more significant for the development of immune response to which C4B isotype activity is necessary.

Urgent reaction of the complement system to hypoxic exposure in rats sensitive to hypoxia.

Different modes of hypoxic exposure led to phasic changes in activities of the complement system components in rats sensitive to hypoxia starting from the first minutes of the posthypoxic period and persisting for 24 h and longer. The direction of shifts in the complement system depended on the duration and intensity of oxygen deficiency. Single one-hour interval hypoxia led to a moderate elevation of activities of virtually all the studied components. A more intense hypoxic exposure (1-h hypobaric hypoxia at a height of 5000 m) induced a biphasic response: reduction of activities of the majority of complement system components during the first hour of posthypoxic period and subsequent elevation of these activities above the normal. Exposure to severe hypobaric hypoxia (7000 m) led to a longer and more pronounced primary reduction of complement components activities, while the phase of their activity increase was blurred. Animal capacity to the formation of urgent tolerance of hypoxia was retained and increased with increasing the severity of hypoxic exposure. The complement consumption during the posthypoxic period was presumably a programmed reaction preventing hyperactivation of complement system components and essential for tolerance formation.

[Isolation and determination of activity of IgA1 protease from Neisseria meningitidis].

A method of the isolation and purification of IgAl protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and sediment obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been devised. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 mug. It was shown that IgAl protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

[Life expectancy at older ages and alternative approach to aging measurement (the case of St. Petersburg)].

St. Petersburg Institute of Bioregulation and Gerontology, NWB of RAMS, 3 pr. Dinamo, St. Petersburg 197110; For St. Petersburg, aging issues are of great importance as values of many aging indicators for St. Petersburg are higher than for Russia as a whole. Taper aims at analyzing the dynamics of life expectancy at older ages and comparing traditional (proportion of the elderly, average age, median age) and new (proportion of population with a remaining life expectancy 15 years or less, population average remaining years of life) aging indicators for St. Petersburg in 1990-2006.

[Determination of protease activity in blood and in microorganisms].

For determination of protease activity it is possible to use immunoglobulins. Since proteolytic products apparently do not retain substrate antigenic determinants, it is possible to use ELISA methodsfor monitoring for enzymatic process. ELISA determination of functional activity of specific IgA1-protease has been applied not only for detection of this enzyme, but also for measurement of its inhibition constants. Fixed on a micropanel IgG may be used for evaluation of total proteolytic activity. Depending on pH values, it is possible to measure activity of neutral, alkaline and acid proteases. This approach has allowed to estimate total proteolytic activity of neutral proteases of serum. Measurement of a total level of serum pepsinogene activity can have diagnostic importance in gastroenterology, due to decisive contribution of pepsinogen I to the measured activity.

[Protease activity of microflora in the oral cavity of patients with periodontitis].

Microbial spectrum and non-specific as well as specific IgA1 protease activity of isolated microorganisms were investigated in gingival liquid of patients with periodontitis. Microorganisms from the gingival liqud of these patients belonged to conditional-pathogenic obligate and facultatively anaerobic bacteria. 24 strains of microorganisms have been identified. Nonspecific proteolytic activity was found in the following microorganisms: Actinomyces israelii, Actinomyces naeslundii, Aerococcus viridans, Bifidobacterium longum, Neisseria subflave, Streptococcus parvulus, Eubacterium alactolyticum, Lactobaccilus catenoforme, Bacillus spp. Specific IgA1-protease activity and lack of proteolytic activity towards IgG was found in Streptococcus acidominimus, Streptococcus hansenii, Streptococcus salivarius, Leptotrychia buccalis, Staphylococcus haemolyticus and Neisseria sicca. No proteolytic activity was found in cultivation medium of Eubacterium alactolyticum (1 strain), Prevotella buccalis, Aerococcus viridans and Streptococcus sanguis.

[Artificial inhibition of the complement system].

A great number of natural substances affect the complement system in addition to its natural regulators. Among the complement effectors, the most important are inhibitors of the activation cascade. The necessity of searching for preparations capable of a purposeful effect on complement by inhibition of single stages of the activation cascade and without influence on its other functions is connected with the current importance of use in medicine of novel therapeutic regulators of the complement system. Important directions are the search for complement inhibitors that (a) interfere with the rejection of transplants; (b) can replace C1 inhibitor in hereditary angioedema, and (c) have a high anti-inflammatory activity in the therapy of rheumatic diseases, diabetes, and other autoimmune disorders. It is expedient to use the available techniques for the directed detection of the action of medicinal substances on complement, which allow the determination of their action on the complement system at various stages of the cascade of its activation.

[Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms].

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.

[Effect of medicinal preparations on the complement: inhibition of subcomponent C1q binding to a target].

A method is developed for determining the inhibition constants of substances capable of acting on the complement system at the stage of target recognition (immune complexes) by the first component of the complement (and, hence, blocking activation of the classical pathway of the complement). The ability of some drugs to inhibit the binding of subcomponent C1q to a target has been studied. It is shown that some drugs possess a pronounced ability to block the complement activation. The inhibition constants are compared to the therapeutic dozes of drugs. Some of the investigated preparations (suramin, sodium deoxiribonucleate, curcumin, heparin, sulfetron, guttalax, lysozyme) upon administration can present in the blood flow in concentrations capable of blocking the complement. The method is useful in the search for preparations capable of inhibiting the complement and in the study of side effects of medicinal preparations.

[Influence of serum IgG antibodies to Chlamydia trachomatis on the functional activity of complement components].

The immunoenzyme analysis and the method for the determination of IgG-containing immune complexes, carrying C1q component of the complement, were developed. In human blood sera the functional activity of components C3, complex C1r2s2, the content of C1 inhibitor and complement-activating immune complexes were determined. The comparative analysis of the activity of components C3 and C1r2s2, as well as between the content of C1 inhibitor and the activity of complex C1r2s2 for seropositive and seronegative sera, was made. Pronounced correlation for seropositive sera was observed. In addition, for seropositive sera correlation between an increase in IgG immune complexes and a drop in the functional activity of complex C1r2s2, as well as a drop in the functional activity of complex C1r2s2 and a growth in the titers of IgG antibodies to Chlamydia trachomatis, were established. The decreased functional activity of key complement components, simultaneously with the presence of complement-activating immune complexes and high titers of specific antibodies could be the diagnostic criteria of carrier state.