PRODUCTION OF SINGLE CHAIN FRAGMENT VARIABLE (scFv) ANTIBODY AGAINST PARANOSEMA LOCUSTAE ALPHA/BETA-HYDROLASE AND IMMUNOLOCALIZATION OF MICROSPORIDIAN ENZYME IN THE INFECTED HOST CELL.

Microsporidia is a widespread group of fungi-related intracellular parasites. Direct contact of the most microsporidia species with host cytoplasm suggests that these parasites may control physiological processes of infected cells by secretion of various proteins. In previous experiments, secretion of significant amounts of microsporidia Paranosema locustae alpha/beta-hydrolase into infected cells of Locusta migratoria fat bodies was demonstrated using polyclonal antibodies against the enzyme. However, heterologous expression of microsporidian hydrolase in yeast Pichia pastoris cells was not accompanied by its secretion. In this study, we have constructed library of recombinant single chain antibodies (scFv-fragments) against proteins of fat bodies of infected locusts and isolated mini-antibody specifically recognizing the studied enzyme using phage display technology. Immunoblotting and immunofluorescent microscopy with selected scFv-fragment confirmed secretion of two different in size forms of P. locustae alpha/beta-hydrolase into infected host cell. Prospects of scFv-fragment use to explore the role of microsporidian hydrolase in host-parasite relations and mechanism of its secretion are discussed in the paper.

INTERACTIONS OF MICROSPORIDIA WITH INFECTED HOST CELL.

Microsporidia comprise a group of fungi-related obligate intracellular eukaryotic pathogens with extremely wide host range: from protists to mammals. Adaptation to intracellular parasitism drives these parasites towards significant reduction and modification of the genome and functional apparatus, which causes extreme dependence on the host cell, as well as sophisticated host-parasite relationships. In this review we summarize our results and recent literature data about microsporidian interactions with the host at the cellular level. The impacts of these pathogens to infected cells include induction of hypertrophy, restructuring and modification of the cytoskeleton and the vesicular transport system of the host cells. Microsporidians also able to stimulate the metabolic processes in the infected cells and inhibit their defensive reactions. The main tool of the direct regulatory impact of microsporidia on the host cell apparently is the secretion of the special protein effectors capable to interfere to regulatory and signaling pathways of the host cell.

Association of Apolipoprotein A1 Gene Polymorphisms with Serum Lipid Spectrum in Adolescents in East Siberia.

We studied the incidence of genotypes of polymorphic alleles (-75)G>A and (+83)C>T of apolipoprotein A1 gene in healthy Russian adolescents, residents of East Siberia. Genotyping was carried out by PCR with subsequent restriction fragment length polymorphism analysis. The incidence of allele (-75)A was 22.5%, of allele (+83)T - 7.3%. Association of allele (-75) A with high blood cholesterol level was revealed.

[Child and Adolescent Interepydemic Immunity to Influenza Viruses].

OBJECTIVE: Our aim was to evaluate the child and adolescent population immunity to influenza A viruses (IAV) and influenza B virus (IBV). METHODS: The concentration and specificity of antiviral antibodies was evaluated by hemagglutination inhibition assay (HAI) that was performed using commercial HAI diagnostic kits. RESULTS: The serum samples of 254 clinically healthy children and adolescents were examined in this study. 245 participants had the antibodies to IAV, 199--to IAV and IBV and only 4 children aged between 1 and 4 years and a 12-year-old boy had no immunity to IAVor IBV. The number of children with specific immunity increased in elder groups by 43% (from 81 to 116) for N0N1, and H3N2 subtypes and by 110% (from 38 to 80) for H1N1 subtype of IAV. In children younger than 4 years the titer of specific antibodies against the H1N1pdm09 was 1:210 and against the H3N2--1:270, whereas in adolescents of 10-14 years these figures were by 1.6 and 2.4 times lower (1:130 and 1:120) respectively. Antibodies to the subtypes H2N2 and H5N1 were not detected. CONCLUSION: The results indicate that 98.4% of child and adolescent population in interepidemic influenza season are immune to the various IAV (H3N2, H1N1, H0N1) as well as to the IBV. More than half of children and adolescents (57.4%) are immune to H1N1pdm09 subtype of IAV. The strength of immune response to the recent pathogens (H3N2 u H1N1pdm09) is higher in infants than in teenagers.

[Multicentric carpotarsal osteolysis in a rheumatologist's practice].

Multicentric carpotarsal osteolysis (MCTO) syndrome is a rare skeletal dysplasia associated with missense mutation in the MAFB gene, usually manifesting in young childhood, and showing variative phenotypic signs and course. The clinical manifestations of the syndrome include aggressive osteolysis predominantly of carpal and tarsal bones, progressive nephropathy, and mild craniofacial anomalies. The similarity between the initial clinical manifestations of MCTO and the symptoms of childhood inflammatory joint diseases makes a diagnosis very difficult, in the early stages of the disease in particular, and frequently leads to the ungrounded use of long-term immunosuppressive therapy. The paper describes a familial case of MCTO without affecting the kidneys in the mother and daughter.

[The detection of occurrence rate of genes coding capability to form pili binding in auto-strains of Escherichia coli].

E. coli is a commensal of intestine of the vertebrata. The exchange of genetic material of different types of bacteria between themselves and with other representatives of family of Enterobacteriaceae in intestinal ecosystem results in development of types of normal colibacillus with genetic characteristics of pathogenicity that can serve as a theoretical substantiation to attribute such strains to pathobionts. The entero-pathogenic colibacillus continues be an important cause of diarrhea in children in developing countries. The gene responsible for formation of pili binding is a necessary condition for virulence of entero-pathogenic colibacillus. The polymerase chain reaction was applied to examine 316 strains of different types of E. coli (normal, with weak enzyme activity and hemolytic activity) isolated from healthy children and children with functional disorders of gastro-intestinal tract for presence of genes coding capability to form pill binding. The presence of this gene in different biochemical types of E. coli permits to establish the fact of formation of reservoir of pathogenicity in indigent microbiota of intestinal biocenosis.

[Early Stages of Parkinson's Disease: Comparative Characteristics of Sleep-Wakefulness Cycle in Patients and Model Animals].

The results of study of sleep-wakefulness cycle in experimental models of pre-clinical and early clinical stages of Parkinson's disease present and compared to some clinical examples. The conclusion is, the increase in activity level and decrease in total amount of slow wave and paradoxical sleep in model animals are taking place at the same circadian period of the secretion of pineal melatonin as sleep disorders in patients.

[The State of Coagulation Hemostasis and Fibrinolysis in Children With Essential Arterial Hypertension].

Children with essential hypertension and increased risk of thrombotic complications are characterized by history of familial thrombosis, increased levels of fibrinogen and soluble fibrin-monomer complexes, activity and content of von Willebrand factor, activity of plasminogen.

[THE SPECIES CHARACTERISTIC OF BIFIDOBACTERIA IN INTESTINAL BIOTOPE OF CHILDREN WITH FUNCTIONAL DYSPEPSIA].

The study was carried out to investigate species specter and combinational variety of bifidobacteria in children with functional dyspepsia. The examined sampling included 73 children with functional dyspepsia aged from 4 to 6 years. The comparison group according common characteristic of microbiota of large intestine included 100 healthy children comparative by age. In 32 out of all examined children species specter of bifidobacteria in intestine was analyzed. The species diagnostic of bifidobacteria was implemented by using technique of polymerase chain reaction. In children with functional dyspepsia the analysis established dysbiosis alterations in intestinal biotope, characterizing by deficiency of bifidobacteria and selection of opportunistic microorganisms. The alteration of species structure of bifidobacteria in children with functional dyspepsia is demonstrated: decreasing of prevalence of populations of Bifidobacterium catenulatum and appearance of rare species of bifidobacteria B. dentium and B. angulatum. The differences are established concerning formation of species preference of bifidobacteria in conditions of conjoint vegetation in intestinal biotope of children with functional dyspepsia and in healthy children. The comprehensive analysis of micro-ecological status of intestine of children with functional dyspepsia using non-invasive techniques and including analysis of species profile, associative potential, communicative capacities of bifidobacteria complements complex of diagnostic criteria, permits to develop prognostic markers and to determine parameters of following therapy of functional dyspepsia.

[Molecular genetic markers of predisposition to thrombosis in adolescents with essential hypertension ].

AIM: To estimate the spread and distribution of genotypes and alleles of thrombosis predisposition gene polymorphisms in adolescents with essential hypertension (EH). SUBJECTS AND METHODS: One hundred and thirty-seven 14- to 17-year-old adolescents were examined. There were 3 examined groups: 1) 37 EH patients with prothrombotic changes (PTC); 2) 60 EH patients without PTC; 3) 40 healthy adolescents. Prothrombin time, activated partial thromboplastin time, and the levels of fibrinogen and soluble fibrin monomer complexes were determined. The adolescents were assigned to a PTC group if they had changes by one or several above indicators. Polymerase chain reaction assay was used to type coagulation factor II (FII) and factor V (FV), plasminogen activator inhibitor type 1 (PAI-7), 5,10-methylenetetrahydrofolate reductase (MTHFR), and methionine syntase reductase (MTRR) gene polymorphisms. Group differences were determined by qualitative signs, by using.the χ2test and Yates' correction for continuity and Fisher's exact test if one of the groups had at least 5 individuals. All differences were considered statistically significant at p < 0.05. RESULTS: FV R506Q, FII G20210A, PAI1-675 4G/5G, MTHFR A1298C, and MTRR A66G polymorphisms have not been found to affect the hemostatic system in adolescents with EH. MTHFR C677T may act as a risk factor of PTC in adolescents with EH, by increasing the risk of thrombotic events at an old age. CONCLUSION: It is recommended that the determination of molecular genetic markers for predisposition to thrombosis be actively used in adolescents with EA to form a risk group for thrombotic events and to implement preventive measures..

[The experience with the application of the non-medication methods for the treatment of essential arterial hypertension in the adolescents presenting with obstructive sleep apnea/hypopnea syndrome].

The present study was designed to estimate the effectiveness of non-invasive auxiliary lung ventilation under continuous positive airway pressure (CPAP therapy) and phototherapy applied for the treatment of adolescents presenting with essential arterial hypertension and obstructive sleep apnea/hypopnea. It was shown that CPAP therapy decreased the level of arterial pressure (AP) either to normal or to high normal values in accordance with the patients' gender and age. It was confirmed that phototherapy produced beneficial effect on the system of circadian rhythms in the patients with essential hypertension. These findings give reason to recommend the combined application of the above methods for the treatment of adolescents with essential hypertension and obstructive sleep apnea/hypopnea. The proposed treatment makes it possible to decrease arterial pressure due to the correction of obstructive breathing disorders in sleep and to normalize the circadian rhythm system in the adolescents suffering from essential hypertension and obstructive sleep apnea/hypopnea. The clinical application of this method would permit to reduce morbidity, the incidence of disability, and mortality from complications developing in adulthood.

[Immunolocalization of microsporidium paranosema locustae canning lSP70 family proteins in locust infected fat body].

Immunolabeling method of microsporidium Paranosema locustae proteins on cryosections of locust infected fat body was proposed. In contrast to single parasite cells and artificially infected host cell cultures, this method allows to study molecular mechanisms of host-parasite relationships and in particular the secretory microsporidial proteins either at cellular or tissue level. Immunolocalization of the EPR-specific and cytoplasmic forms of Hsp70 family of molecular chaperones on cryosections showed accumulation of these proteins in the respective compartments of intracellular developmental stages of P. locustae and their absence in host structures. This allows to use them in diagnostics of microsporidiosis lesions in infected tissues as well as in colocalization analysis with P. lociustatre secretory proteins as a marker of parasite. The cytoplasmic chaperone stains cytoplasmic compartment homogeneously, but in the infected host cell during sporogony it disappears partially from the intracellular stages of development which damaged by maturing spores. Thereby study of molecular mechanisms of host-parasite relationships is to be carried out at the earlier stages of infection before active sporogony.

[The contribution of genes polymorphism of thrombophilia in clinical variability of hemorrhagic vasculitis].

BACKGROUND: The article is devoted to the study of clinical and laboratory characteristics of the current of hemorrhagic vasculitis in children in the Republic of Buryatia. PATIENTS AND METHODS: The study included 27 patients aged 7.6 +/- 4.02 years, who conducted clinical and laboratory tests, immunological study of antiphospholipids of antibodies, genetic testing for thrombophilia markers of candidate genes. RESULTS: The results showed that hemorrhagic vasculitis often affects children of Buryat nationality. In 96% of cases there are mixed clinical forms of the disease. 63% of children of hemorrhagic vasculitis preceded by various factors, a higher percentage of infectious diseases. The first clinical symptom in 63% of patients is a typical purpura hemorrhagic rash. Results of clinical laboratory blood tests revealed no significant deviations. Circulation of lupus anticoagulant was detected in 37% of subjects. The alphaCL IgM detected in 3 children, alphabeta2-GP-I IgA--in 4, alphabeta2-GP-I IgM--in 1 patient. Carriers of thrombophilia polymorphisms were in 95% of children. Noted that homozygous variants of genes polymorphisms of methylenetetrahydrofolatered reductase and plasminogen activator inhibitor-1 correlate with the presence of urinary symptoms and recurrence of the rash. CONCLUSION: The study shows the risk of recurrent flow of hemorrhagic vasculitis and nefritis of Henoch-Schonlein in children with thrombophilia gene polymorphism.

[Evaluation of erectile function during the night in men with obstructive sleep apnea syndrome: new possibilities of modern sleep medicine].

Currently, the relationship between the sleep and erectile function (EF) is not in doubt; therefore, evaluation of this relationship in the context of sleep medicine (somnology) is the most informative diagnostic method. The article presents the results of polysomnography with the option of simultaneous monitoring of spontaneous episodes of nocturnal erections in 51 men aged 46-55 years without complaints of violation of the EF. The main group (n = 37) consisted of patients with obstructive sleep apnea syndrome (OSAS). The violation of pattern (quantitative and qualitative characteristics) of nocturnal episodes of spontaneous erections in this group of men was revealed, which was regarded as the initial (pre-clinical) manifestation of erectile dysfunction. Thus, modern diagnostic capabilities of sleep medicine allow to effectively evaluate the EF in patients with OSAS and timely assign pathogenetically justified therapy.

[Uric acid and factors of cardiometabolic risk in adolescents with arterial hypertension].

AIM: To elucidate links of blood serum uric acid level (SUAL) with cardiometabolic risk factors (CMR) in adolescent boys with arterial hypertension (AH). MATERIAL AND METHODS: We examined 261 adolescent boys (mean age 15.8+/-1.3 years) with primary AH. Examination included 24-hour blood pressure (BP) monitoring, and measurement of office BP, BMI Z-score, levels of serum glucose, uric acid, total cholesterol (TC), high density lipoprotein cholesterol (HDL) and triglycerides (TG). Low density lipoprotein cholesterol (LDL) was calculated using Friedwald equation. RESULTS: SUAL was significantly and independently of patient age related to BMI Z-score (=0.0000001) and TG level (=0.01). There were no correlation between SUAL and glucose level. According to logistic regression analysis highest tertile of SUAL was regardless of age significantly associated with excess body mass and obesity (odds ratio [OR] 3.5 [95% confidence interval [CI] 1.9-6.7; =0.0001]), and dyslipidemia (OR 3.05, 95%CI 1.3-6.9; =0,007]). Significant relationship was also found between elevated SUAL and clasterization of 2-3 CMR (OR 3.2, CI 1.3-7.7; =0.01]). CONCLUSION: Hyperuricemia in adolescent boys with AH is a marker of high cardiometabolic risk. This justifies screening for high SUA levels among boys with elevated BP with subsequent estimation of individual risk and timely initiation of preventive measures.

Optimization of the Protocol for the Isolation and Refolding of the Extracellular Domain of HER2 Expressed in Escherichia coli.

Receptor 2 of the human epidermal growth factor (HER2/neu, c-erbB2) is a 185 kDa proto-oncogene protein characterized by an overexpression in some oncological diseases, including 30% of mammary glands cancers, as well as tumors in the ovary, stomach and other organs of the human body. Since HER2- tumor status testing is the essential part of a successful cancer treatment, the expression and purification of substantial amounts of the extracellular domain (ECD) of HER2 is an important task. The production of ECD HER2 in Escherichia coli has several advantages over the use of eukaryotic expression systems, but the bulk of the recombinant product in bacteria accumulates as insoluble protein inclusion bodies. In this study, we obtained ECD HER2 in Escherichia coli as insoluble inclusion bodies and elaborated a simple, efficient, and fast protocol for the solubilization, refolding, and isolation of the protein in soluble form.

[Electric status epilepticus in sleep (case report)]. / Élektricheskii épilepticheskii status sna (klinicheskoe nabliudenie).

Electric status epilepticus in sleep (ESES) syndrome is the partially reversible, age-dependent children's epileptic encephalopathy with continuous spike-wave complexes in a slow sleep phase. It is a nonspecific electrographic phenomenon in many epileptic syndromes. It can be a single indicator of the epileptic nature of disorders and a marker of epileptic encephalopathy. A sleep EEG should be performed in children with autistic, speech, behavioral disorders to specify their etiology and prescribe pathogenetic treatment.

[Peculiarities of the expression, structure, and localization of the subtilisin-like protease in the microsporidium Paranosema locustae].

Peculiarities of the expression, localization, and structure of the subtilisin-like protease from the microsporidium Paranosema locustae, a parasite of the migratory locust and other orthopteran species, are analyzed. Heterologous expression of the microsporidian ferment in the bacterium Escherichia coli allowed obtaining antibodies to the recombinant protein and to start its examination. In spite of the presence of the N-tail signal peptide in the ferment, potentially able to secret it into the cytoplasm of the infected cell, immunoblotting with obtained antibodies had demonstrated specific accumulation of the protease in the insoluble fraction of spore homogenate. At the same time, the ferment was absent in intracellular stages.of the parasite and also in the cytoplasm of infested host cells. Accumulation of mRNA, coding the studied protein in microsporidian spores was confirmed with the use of RT-PCR method. Heterologous expression of the protease in the methylotrophic yeast Pichiapastoris demonstrated the same result. The ferment of P. locustae was not secreted into a culture medium and was absent in the cytoplasm of yeast cells, accumulating in a dissoluble (membrane) fraction of the homogenate. On the whole, the obtained data testify to the fact that the subtilisin-like protease of P. locustae plays an important role in the physiology of spores rather than participates in host-parasite relations during intra-cellular development.

[Peculiarities of functional hemispheric asymmetry formation in children and adolescents with hypertension].

OBJECTIVE: Peculiarities and age dynamics of hemispheric asymmetry investigation in children and adolescents with hypertension. METHODS: 196 children and adolescents aged 10 to 17 years (15.0-1.4), including 98 patients with arterial hypertension (AH) (15.1 ± 1.5) and 98 healthy children of the control group (14.9 ± 1.3). The number of boys in the comparison group and the main group of patients with AH was 60 (61.2%) and 59 (60.2%), girls--38 (38.8%) and 39 (39.8%), respectively. RESULTS: The majority of children and adolescents with hypertension sensorimotor asymmetry differed by low intensity, indicating a decline in the dominance of the left hemisphere. Accumulation of ambi- and left lateral features traced mainly in motor (53.6%) and auditory (60.2%) system, while asymmetry in the visual analyzer did not differ from the control group. Differences in the degree of hemispheric asymmetry increases in the ontogeny compared to control group. CONCLUSION: Identification of specificity hemispheric interaction in patients with hypertension (low asymmetry and its weak performance in ontogeny) can be considered not only as a pathological consequence of hypertension, but also as a compensatory mechanism for the redistribution of loads to the right hemisphere in terms of the functional deficit of the left hemisphere. It can be assumed that the rearparts of the brain may play a significant role in the processes of adaptation and compensation of occurring disorders in hypertension.

[Moving activity and wakefulness-sleep cycle changes in a mouse MPTP model of Parkinson's disease].

A group of mice with preliminary implanted (under general anesthesia) electrodes for cortical EEG and nuchal EMG was subjected to continuous baseline 24-hr video and digital polysomnographic recording with the 12/12 light/dark schedule, and then injected subcutaneously with 24 or 48 mg/kg of MPTP toxin or (the control group) saline. The recordings were continued for 2 weeks more. A significant increase in activity and the waking percentage as well as decrease in REM sleep and NREM sleep (tendency) during the dark period as compared to the baseline and control recordings was found. The effect was seen just on the 7th day following MPTP administration and became significant by the 14th day. The effect was more pronounced after 48 mg/kg injection than after 24. There were no changes during the light period. Morphological control revealed a 70% and 35% decreases in the amount of tyrosine hydroxylase positive neurons in substancia nigra/pars compacta after 48 and 24 mg/kg of MPTP, respectively, as compared to the saline group.