[SYNTHETIC CONJUGATED ANALOGUES OF CAPSULE POLYSACCHARIDES OF PNEUMOCOCCUS - AN INSTRUMENT FOR DETECTION OF POST-VACCINATION ANTIBODIES].

AIM: Evaluation ofthe ability of capsule polysaccharides (CP) of Streptococcus pneumoniae serotype 3 and 14 and their synthetic structure analogues, conjugated with bovine serum albumin (BSA), to detect antibodies in post-vaccination sera of mice. Materials andmethods. Oligosaccharides correspond- ing to one, one and a half and two repeating links of serotype 3 and 14 S. pneumoniae CP were synthe- sized, their conjugates with BSA were produced by squarate method as well. Ligand content-per BSA molecule was controlled by MALDI-TOF spectrometry. Immune sera were obtained after 2 intraperi- toneal administrations to mice of glucoconjugates adsorbed on aluminum hydroxide or 13-valent pneumococcal conjugated vaccine. Determination of levels of post-vaccination class G antibodies and their sub-isotypes was carried out in EIA. RESULTS: Immunization of mice with neoglucoconjugates resulted in formation of predominantly IgGl recognizing serotype 3 and 14 S. pneumoniae C. IgG1 in mice immunized with a 13-valent conjugated vaccine recognized serotype 3 S. pneumoniae CP, but detected serotype 14 S. pneumoniae CP weakly. All the conjugated synthetic oligosaccharides were characterized by a high ability to bind antibodies in blood of mice immunized with the polysaccharide conjugated vaccine. BSA-tetrasaccharide of serotype 3 S. pneumoniae and BSA-tetrasaccharide of serotype 14 S.pneumoniae were characterized by the highest ability to detect IgG1 against C. CONCLUSION: Synthetic oligosaccharides, conjugated with BSA protein-carrier, may be used to develop diagnostic test-systems for determination of antibodies in post-vaccination sera.

[EPITOPIC SPECIFICITY OF A SYNTHETIC DISACCHARIDE, RECURRING LINK OF CAPSULE POLYSACCHARIDE CHAIN OF SEROTYPE 3 STREPTOCCUS PNEUMONIAE].

AIM: Study epitopic specificity of synthetic disaccharide, recurring link of serotype 3 S. pneumoniae, conjugated with bovine serum albumin (BSA). MATERIALS AND METHODS: Conjugate of the synthetic disaccharide with BSA was obtained by squarate method. Antigenic activity of the conjugate was studied in competitive EIA. Titers of IgG against capsule polysaccharide of serotype 3 S. pneumoniae were determined in EIA by using sera of mice immunized twice with disaccharide conjugate sorbed onto aluminum hydroxide. RESULTS: Disaccharide conjugate used as a well-covering antigen (4 µg/well) in EIA was characterized by a high degree of specificity and interacted only with IgG against serotype 3 S. pneumoniae in antimicrobial sera of animals without reacting with antibodies (ABs) against other pneumococcus serotypes (6B, 10A, 19A, 19F, 23F). Disaccharide conjugated with BSA was determined in competitive EIA to inhibit bonding of ABs to disaccharide by 78.8%, bacterial capsule polysaccharide by 56.9%, BSA did not inhibit the sera activity. The study of sera of mice immunized by serotype 3 S. pneumoniae disaccharide conjugate in EIA, where capsule polysaccharide was used as a plate-sorbed antigen, has established the presence of IgG against capsule polysaccharide at a titer of 1:1600. CONCLUSION: The disaccharide that is a single recurring link of serotype 3 S. pneumoniae contains a key epitope of capsule polysaccharide. The synthetic disaccharide could be used as a component of multivalent conjugated pneumococcal vaccines and for development of diagnostic test-systems.

Protective activity of a glycoconjugate based on a synthetic hexasaccharide related to a fragment of capsular Streptococcus pneumoniae serotype 14 polysaccharide chain.

We studied the effects of immunization with a conjugate of carrier protein and hexasaccharide ligand related to a fragment of capsular of Str. pneumoniae serotype 14 polysaccharide chain on activation of innate and adaptive immunity. It was found that two-fold immunization with the glycoconjugate adsorbed on aluminum hydroxide significantly increased the titer of IgG antibodies to capsular polysaccharide in the blood and protected 100% mice from infection with Str. pneumoniae serotype 14. Enhanced bactericidal activity of peripheral blood lymphocytes of mice was found 4 and 24 h after the first immunization with the immobilized glycoconjugate. Adsorption of the glycoconjugate on aluminum hydroxide resulted in modification of the immune processes at the stage of activation of innate immunity and subsequent strengthening of the adaptive immunity.

[Effect of aluminium hydroxide on innate immunity and immunogenicity of bacterial and synthetic antigens of Streptococcus pneumoniae].

AIM: Study the effect of aluminium hydroxide on molecular-cell mechanisms of innate immunity activation and its adjuvant effect on immunogenicity of natural bacterial and synthetic pneumococci antigens. MATERIALS AND METHODS: Surface markers of dendritic cells (DC), mononuclear leukocytes (ML) and cytokine levels were determined by flow cytometry; IgG titers--by EIA. Protective activity was evaluated in experiments with active protection of mice from infection with virulent pneumococci strains. RESULTS: Aluminium hydroxide increased the ML content of mice spleen expressing TLR2 and TLR4. Its addition into the culture of immature DC induced the appearance of a population of cells with mature DC markers--CD83, CD80, CD86, however, the level of undifferentiated cells (CD34) and cells with adhesion molecules (CD11c, CD38) did not change. DC produced IL-1ß, IL-5, IL-10, IFNγ into the cultivation medium. An increase of cytokine production took place 2 hours after the administration into mice and was retained for the observation period (24 hours). Th1 (IFNγ, TNFα) and Th2 (IL-5, IL-10, GM-CSF) cytokine production gave evidence on immune response polarization by Th1/Th2, type. After 2 administrations of aluminium hydroxide into mice the number of ML with CD19+, CD5+, NK1.1+, CD25+, MHCII+ markers increased during decrease of CD3+, CD4+ and CD8+ T-lymphocytes. Adaptive immunity activation was characterized by high IgG titers to pneumococci capsule polysaccharide and protection of 90 - 100% of the mice against infection with lethal doses of S. pneumoniae strains, was detected during 2-fold immunization of mice with conjugates of synthetic pneumococci oligosaccharides with BSA,sorbed onto aluminium hydroxide, whereas natural bacterial antigens provided 90 - 100% survival of animals during immunization without the adjuvant. CONCLUSION: Data are provided on the effect of aluminium hydroxide on key effectors of innate immunity: DC, ML, TLRs and cytokine production. A reasonable administration of this adjuvant was shown to be in association with conjugates of pneumococci synthetic oligosaccharides with a carrier protein.

Development of approaches to creation of experimental test system for evaluation of antigenic activity of synthetic oligosaccharide ligands related to fragments of the Streptococcus pneumoniae type 14 capsular polysaccharide.

A conjugate of a synthetic hexasaccharide fragment of the Streptococcus pneumoniae type 14 capsular polysaccharide with bovine serum albumin (BSA) has been prepared. The antigenic activity and specificity of this conjugate are comparable with those of natural antigens of S. pneumoniae type 14. The data suggest that the resulting synthetic conjugate can be used as a coating antigen in an experimental test system (based on enzyme immunoassay) for evaluating the antigenic activity and specificity of synthetic oligosaccharide ligands and for testing specimens of natural capsular polysaccharides and immune sera.

[Strain differences of intra-species immunogenic activity of Streptococcus pneumoniae antigen components].

AIM: Study intra-species immunogenic activity of antigenic protein-polysaccharide components of S. pneumoniae. MATERIALS AND METHODS: Antigenic components of serotype 3, 6A, 6B, 14, 10A, 18A, 19A, 19F, 23F and unencapsulated S. pneumoniae strains were obtained by water extraction method. Synthetic hexasaccharide--corresponding to the structure of S. pneumoniae serotype 14 capsule polysaccharide repeated unit chain fragment was used as a reference preparation. Molecular mass of antigenic components was determined in SDS-electrophoresis. Antibody titers in blood sera of immunized mice were evaluated by solid-phase EIA method. Protective activity of preparations was studied in mice after 2 immunizations with consequent infection by virulent S. pneumoniae serotype 3 and 6B strains. RESULTS: Preparations from serotype 6A, 6B, 14, 19A, 19F, 23F strains in reaction with anti-microbial sera were characterized by cross serologic activity (IgG titers of 1200 - 12 800). The lowest serologic activity was detected in S. pneumoniae serotype 3 and unencapsulated strain preparations. Conjugate of synthetic hexasaccharide and bovine serum albumin interacted only with homologous antimicrobial sera up to titers of 600 +/- 89.4 and did not react with sera against serotypes 19A and 19E Cross serologic activity of preparations is probably determined by the presence of protein fractions that were detected in SDS-electrophoresis. This is confirmed by high intra-species cross protective activity of preparations from serotype 6B and 10 A strains that protect 90 - 100% of mice from infection by heterologous S. pneumoniae strains. CONCLUSION: Use of strains with cross antigenic and protective activity for production of immunogenic protein-containing fractions with the aim of enchanting and broadening specter of protective activity of vaccine preparations that are constructed based on capsule polysaccharides of S. pneumoniae is appropriate.

[Immunogenic activity of a conjugate of synthetic hexasaccharide related to a streptococcus pneumoniae serotype 14 capsule polysaccharide chain fragment].

AIM: Study antigenic and immunogenic activity of a conjugate of synthetic hexasaccharide related to a S. pneumoniae serotype 14 capsule polysaccharide chain fragment with bovine serum albumin (BSA). MATERIALS AND METHODS: Synthetic glucoconjugate based on BSA protein carrier and hexasaccharide ligand reflecting capsule polysaccharide chain fragment was obtained by using squarate method. Natural polysaccharide-protein complex from S. pneumoniae serotype 14 strain was obtained from cultural fluid supernatant by acetone precipitation. IgG titer against hexasaccharide/capsule polysaccharide was determined in antimicrobial sera and sera of mice immunized with glucoconjugate by EIA method. RESULTS: Immunogenic activity ofglucoconjugate based on BSA protein carriers and synthetic hexasaccharide reflecting S. pneumoniae serotype 14 capsule protein chain fragment was established. After 2 immunizations antibodies against hexasaccharide ligand and BSA were determined in blood sera of mice. Antibody titers against hexasaccharide exceeded the level in intact mice by 4.2 times. BSA in the conjugate did not have effect on production of antibodies against hexasaccharide. CONCLUSION: The developed experimental test-system based on synthetic glucoconjugate is useful for evaluation of level of antibodies against S. pneumoniae serotype 14 in infected and, probably, carriers of bacteria.

[Diagnosis simulation in urinary tract tuberculosis].

The paper presents the results of the statistical processing that comprises the calculation of the basic numerical statistics, the analysis of an association of signs, the estimation of Pearson's paired correlation coefficient, and the testing of its statistical significance, the construction of logit models, and the regression analysis of 350 case histories of inpatients with nephrotuberculosis treated at Samara Regional Tuberculosis Hospital One in 2002-2006. The results of the study proved it possible to simulate a clinical diagnosis for urinary tract tuberculosis. It can be simulated using the following indices: the presence of chronic renal failure, urinary leukocytes, blood urea, excretory urographic data, the electrocardiographic signs of essential hypertension, and the presence of non-tuberculous comorbidity (type 2 diabetes mellitus, gastric peptic ulcer, chronic hepatitis).

[Simulation of the quality of life in patients with chronic obstructive pulmonary disease].

The statistically significant formulas calculated on the basis of statistical processing of the data of the Saint George test and physical, functional, laboratory, and radiation studies in 117 patients with chronic obstructive pulmonary disease (COPD) are given. Based on the external function test, the study has provided highly significant statistical formulas for calculating the subjective estimate of the severity of COPD, the degree of its associated psychological problems and overall distress due to COPD in the presence of moist rales and chronic cor pulmonale or in its absence.

[The specific features of an attitude of patients with pulmonary tuberculosis towards disease].

The attitude of a patient with tuberculosis to his/her disease has not been described in the literature. The purpose of the study was to explore the attitude of a tuberculosis patient towards his/her disease. A hundred Samara dwellers (62 males and 38 females) aged 22 to 56 years who had pulmonary tuberculosis and were receiving a course of inpatient therapy were examined. Of them there were 50 patients with infiltrative pulmonary tuberculosis and 50 with fibrocavernous tuberculosis. The duration of the disease was 2 to 5 years. To study the attitude of a patient with pulmonary tuberculosis towards his/her disease, the authors developed a special questionnaire comprising 23 statements, which was based on the respondent's acceptance or denial of certain statements (innovation No. 511 by the Samara Military Medical Institute). Positive and negative replies were statistically processed by the generally recognized procedures. The findings indicated that tuberculosis worsened the relations of a patient with his/her relatives and associates, actually restricted contacts, lowered the social position, living standards, made him/her need for financial support, narrowed the range of interests, and drastically impaired the plans for future life. Patients with tuberculosis commonly develop social phobia. To overcome the latter, it is necessary to make a differential social correction. More than half the patients with newly diagnosed tuberculosis and only a third of chronic tuberculosis patients make sure of that they will be able to recover; half the patients want to give up smoking; virtually all patients consider the prescribed treatment to be effective; however, it satisfies just more than the half of them. To improve patient compliance and interaction with medical staff, it is necessary to correct the emotional status and alleviate depressive reactions. The attitude of patients with tuberculosis towards their disease is destructive and it is to be corrected.

[Formation of treatment motivation in patients with pulmonary tuberculosis].

Incompliance is a reason for inefficient therapy in patients with pulmonary tuberculosis. The purpose of the investigation was to study the values in patients with pulmonary tuberculosis and, on their basis, to form treatment motivation. A special questionnaire (the authors' innovation) was drawn up and used to examine 129 tuberculosis patients, Samara residents, aged 18 to 65 years. The results were statistically processes, by using the SPSS program, version 10.0. The patients from different sociomedical groups, who received therapy of varying duration, were found to have different personality values. Therefore, different treatment motivations should be used to increase compliance. The most governing motives were identified for tuberculosis patients from different sociomedical groups.

[The satisfaction of personal needs in patients with pulmonary tuberculosis].

The founder of humanistic psychology A. Maslow divided the needs of a personality into several levels--from the lowest to the highest ones. Higher-leveled needs rise when the lower-leveled needs are satisfied. A great deal of factors affect the origination and satisfaction of needs, but they are always interrelated with social values. The extent to which personality needs are satisfied in patients with pulmonary tuberculosis has not been studied. A special questionnaire has been drawn up to study the extent to which personality needs are met. Its suitability has been determined, by using a group of patients with bronchial asthma. The extent to which personality needs are satisfied in patients with pulmonary tuberculosis was studied in 178 patients with infiltrative pulmonary tuberculosis and 253 patients with fibrocavernous pulmonary tuberculosis. The results have shown that the extent to which personality needs are satisfied in patients with tuberculosis is lower than that in apparently healthy individuals of the same social status. In females with infiltrative pulmonary tuberculosis, the needs for safety are satisfied to a lesser extent. In those with fibrocavernous pulmonary tuberculosis, the extent to which the physiological, safety, and self-realization needs is decreased. In males with infiltrative pulmonary tuberculosis, the physiological, noetic, and self-realization needs are satisfied to a lesser extent. In those with fibrocavernous pulmonary tuberculosis, the extent to which the physiological, safety, group decision-making, noetic and self-realization needs is decreased.

[Need for psychological correction in patients with pulmonary tuberculosis]. / Neobkhodimost' psikhologicheskoi korrektsii u bol'nykh tuberkulezom legkikh.

The paper presents the results of examination using a standardized multifactorial personality study schedule in patients with pulmonary tuberculosis, residents in the Samara Region, including 123 males and 55 females with first diagnosed tuberculosis, 152 males and 101 females with chronic pulmonary tuberculosis. Ninety-eight apparently healthy males and 142 females of the same age were examined as a control group. The type of a response in the psychotraumatic situation and psychological traits were identified. The leading type of a response (66%) was hyposthenic (an inhibitory response to stress) in males with first identified tuberculosis. Conflict somatization was peculiar to other males, as in 46% of the females. The behavioral type of dysadaptation and asocial and antisocial responses occur in 38.9% of the males with chronic pulmonary tuberculosis and in 25% of the females. 22.2% of the males and 50% of the females show a somatic response, which leads to the transition to the psychophysiological level, to the occurrence of psychosomatic diseases. Irrespective of the duration of the disease, all the patients, both males and females, need psychological correction.

[Tuberculosis as a component of the sub-culture]. / Tuberkulez kak élement subkul'tury.

The paper presents the results of a study of 152 males (mean age, 45.7+/-3.8 years) with fibrocavernous pulmonary tuberculosis and 123 males (mean age, 41.2+/-3.6 years) with infiltrative pulmonary tuberculosis, who reside in the Samara Region. The examination of all patients was comprehensive, which included physical examination, functional studies, psychological tests, and a special questionnaire survey. The data on 108 apparently healthy males served as a control of SMPI and Luscher tests. The social situation was analyzed. A correlation analysis was made to define a relationship of the satisfaction of main personality needs to the functional parameters of the lung. The comprehensive study has demonstrated that the disease is socially advantageous for males with infiltrative pulmonary tuberculosis; this is an original marker of group affiliation, an element of subculture, a way of enhancing a self-appraisal in the eyes of the members of a reference group. No intention to treatment accounts for poor drug regiment compliance. Males with chronic forms of pulmonary tuberculosis lose a secondary social advantage. A sensation of weakness, malaise, and a fear of violence, which are an obstacle for them to become fully-fledged members of a reference group are in the foreground.

["Phthisia-School": a complex action system for a patient with tuberculosis]. / "Ftiza-shkola" -- sistema kompleksnogo vozdeistviia na bol'nogo tuberkulezom.

To make the outcomes of treatment in patients with tuberculosis better, a complex action system has been developed, by using the data obtained from studies of the clinical, psychological, and social characteristics. Four hundred and sixty four patients (211 new cases of tuberculosis and 253 with fibrocavernous tuberculosis) were examined. The examination was comprehensive and involved a package of psychological tests and a special questionnaire survey. Analysis of the results of the study made it possible to develop a complex action system for a patient with tuberculosis, which was called the "Phthisia-School". It includes the provision of positive information, regulated drug therapy, and a corrective package aimed at altering the patient's attitude towards to his/her disease, intent-to-treat development, enhancing systemic and psychological adaptation. There was a statistically significant increase in the efficiency of treatment, a tendency towards harmonization of the type of the patient's attitude towards his/her disease, a change in this attitude, treatment, personal hygiene rules, untraditional treatments, an increase in the discipline of treatment and confidence in good prognosis.

[Some specific features of life quality in patients with pulmonary tuberculosis]. / Nekotorye osobennosti kachestva zhizni bol'nykh tuberkulezom legkikh.

The paper presents the results of examination of two groups of male patients with pulmonary tuberculosis: 152 males with fibrosocavernous pulmonary tuberculosis and 123 males with infiltrative pulmonary tuberculosis; which included physical examination, X-ray, laboratory, and functional studies, multifactorial psychological personality testing, survey using a special questionnaire. The functional parameters of health have been found to be worse in male patients with chronic pulmonary tuberculosis. Analyzing the social situation revealed no statistically significant differences in males with chronic or first diagnosed pulmonary tuberculosis. The psychological characteristics were more impaired in male patients with chronic pulmonary tuberculosis. By recognizing that life quality is an integrative indicator of the functional parameters of health and the social and psychological parameters of living standards and life way, life quality in all male patients with pulmonary tuberculosis may be considered to be low. Life quality in male patients with chronic pulmonary tuberculosis is still worse than in those with first diagnosed pulmonary tuberculosis.

[Behavioral aggression in patients with pulmonary tuberculosis and a way of its correction]. / Povedencheskaia agressiia bol'nykh tuberkulezom legkikh i sposob ee korrektsii.

This study deals with the specific psychological and social features in patients with tuberculosis, including their attitude to others and their behavioral features. The patients underwent a comprehensive examination, including standard multifactorial personality study and Lusher tests, special questionnaire surveys. There were 253 patients with fibrocavernous pulmonary tuberculosis, including 152 males (mean age, 45.7 +/- 3.8 years) and 101 females (mean age, 44.0 +/- 3.9 years) and 178 patients with infiltrative pulmonary tuberculosis, including 123 males (mean age, 41.2 +/- 3.6 years) and 155 females (mean age, 42.0 +/- 3.5 years). The diagnosis of tuberculosis was verified by physical, laboratory, and X-ray studies, external respiratory function tests, and brush biopsy at bronchoscopy. The comprehensive study has indicated that irrespective of the duration of the disease, specific psychological peculiarities, altered behavior and attitude to themselves and others appear in both males and females, leading to the socially dangerous manifestation of behavioral aggression. The study has developed a procedure to prevent the manifestation of aggressive behavior in patients with pulmonary tuberculosis. Goal-oriented correction prevents distresses resulting in decompensation.

Preparative route to glucuronyl donors bearing temporary protecting group at O-3 via 6,3-lactonisation by Bz(2)O or Piv(2)O.

Heating of non-substituted beta-D-glucopyranuronic acids in the presence of pivalic or benzoic anhydride in DMF gives 4-O-monoacylated or 2,4-di-O-acylated 6,3-lactones that can be easily transformed into corresponding methyl uronates bearing free OH-groups at C-2 and C-3 or C-3 only, respectively. This method was used for preparation of selectively 3-O-protected glucuronyl donors of thioglycoside and trichloracetimidate types.

Study of glycosylation with N-trichloroacetyl-D-glucosamine derivatives in the syntheses of the spacer-armed pentasaccharides sialyl lacto-N-neotetraose and sialyl lacto-N-tetraose, their fragments, and analogues.

The syntheses of 2-aminoethyl glycosides of the pentasaccharides Neu5Ac-alpha(2-->3)-Gal-beta(1-->4)-GlcNAc-beta(1-->3)-Gal-beta(1-->4)-Glc and Neu5Ac-alpha(2-->3)-Gal-beta(1-->3)-GlcNAc-beta(1-->3)-Gal-beta(1-->4)-Glc, their asialo di-, tri-, and tetrasaccharide fragments, and analogues included a systematic study of glycosylation with variously protected mono- and disaccharide donors derived from N-trichloroacetyl-D-glucosamine of galactose, lactose, and lactosamine glycosyl acceptors bearing benzoyl protection around the OH group to be glycosylated. Despite the low reactivity of these acceptors, stereospecificity and good to excellent yields were obtained with NIS-TfOH-activated thioglycoside donors of such type, or with AgOTf-activated glycosyl bromides, while other promotors, as well as a trichloroacetimidate donor, were less effective, and a beta-acetate donor was inactive. In NIS-TfOH-promoted glycosylation with the thioglycosides, the use of TfOH in catalytic amount led to rapid formation of the corresponding oxazoline, but the quantity of TfOH necessary for further efficient coupling with an acceptor depended on the reactivity of the donor, varying from 0.07 equiv for a 3,6-di-O-benzylated monosaccharide derivative to 2.1 equiv for a peracetylated disaccharide one. In the glycosylation products, the N-trichloroacetyl group was easily converted into N-acetyl by alkaline hydrolysis followed by N-acetylation.