[Use of immunological and molecular biological methods to diagnose cerebral toxoplasmosis in HIV infection].

Cerebral toxoplasmosis is one of the leading causes of neurologic diseases with high mortality rates in patients with HIV infection. Invasion was difficult to diagnose for a number of objective reasons. The objective of the investigation was to determine the clinical sensitivity of different laboratory techniques as both a single study and their various combinations to verify the diagnosis of cerebral toxoplasmosis in HIV-infected patients. Blood and cerebrospinal fluid were tested in 51 patients with Stage 4B HIV infection (AIDS) with the verified diagnosis of cerebral toxoplasmosis. Separate determination of specific antibodies of IgG, IgM, IgA and toxoplasma DNA in the blood and cerebrospinal fluid was shown to have an insufficient clinical sensitivity (37.3-68.6%). The benefits of various combinations of immunological and molecular biological assays enhancing the diagnostic efficiency up to 76.5-96.1% are demonstrated.

[Model of chronic salmonellosis: parameters of infection and immune response in inbred mice genetically variable in susceptibility to salmonellosis].

AIM: Study parameters of chronic infection and immune response in I/St and A/Sn line mice in the model of per oral infection of mice with Salmonella enterica serovar Typhimurium. MATERIALS AND METHODS: Studies were carried out in I/StSnEgYCit (I/St), A/JsnYCit (A/Sn) inbred line mice as well as their back crossing hybrids [I/StrxF1(I/StxA/Sn)]BC. Mice were infected per os by S. enterica serovar Typhimurium strain IE147 at a dose of 2 x 10(5) PFU per mice. The number of salmonellae was determined at days 3, 5 and 7, weeks 3 and 4 after the infection in various organs, the number of antibody producers--by cell EIA. Pathomorphologic changes in mice spleens were studied histologically by using hematoxylin and eosin staining. In offspring of back crossing [I/St x F1(I/St x A/Sn)]BCl segregation genetic analysis of sensitivity to salmonella infection trait and mapping of loci taking part in salmonella infection were carried out. RESULTS: The course of chronic salmonellosis in susceptible I/St line was characterized by the presence of more pronounced pathomorphologic changes in spleen and significantly higher microbial load in organs (approximately by 1000 times) when compared with A/Sn mice. Interlinear differences in susceptibility to infection correlated with differences in the type of early local and systemic immune response. In I/St mice a higher level of salmonella specific IgG2a-, IgG1- and IgA forming cells in spleen compared with A/Sn mice was detected which correlates with a pronounced splenomegaly and high concentration of salmonellae. On the contrary A/Sn mice demonstrated a higher level of salmonella specific IgA forming cells in Peyer patches that probably leads to protection of A/Sn line during per oral infection. Genetic analysis of susceptibility to salmonellosis trait inheritance showed the presence of its coupling with D9Mit89 locus of chromosome 9 on which previously Tbs2 locus was mapped that plays a role in the control of tuberculosis infection. CONCLUSION: There is a probability of the presence of general mechanisms of genetic control of tuberculosis and salmonella infections in A/Sn and I/St mice.

[Characteristic of early mucosal immune response in mice during intravaginal infection caused by Chlamydia muridarum].

AIM: Comparison of features of recruitment to infection focus of cells mediating early immune reactions in intravaginally infected mice that had previously received or not received covinan (progesterone analogue). MATERIALS AND METHODS: A/Sn and BALB/c line mice were used in the study. C. muridarum strain Nigg infection was carried out intravaginally or intraperitoneally. For synchronization of sexual cycle a group of mice received subcutaneously a synthetic analogue of progesterone--proligeston (covinan) at a single dose of 33 mg/kg. Acute urogenital infection was evaluated by culture method. Quantitative determination of C. muridarum DNA (including study of persistence) was carried out by real time PCR. Subpopulation structure of cell population of peritoneal and vaginal lavage was evaluated by flow cytofluorometry. RESULTS: Intravaginal infection of mice that had not received covinan resulted in a pronounced recruitment of cells into vaginal cavity at 24 hours after the infection. Influx of neutrophils, dendritic cells and T-lymphocytes was especially pronounced. Prior administration of covinan practically nullified cell recruitment to infection focus though partial preservation of subpopulations of activated dendritic cells and CD8+ T-cells was observed. CONCLUSION: In mice with artificially induced by progesterone sensitivity to chlamydias the ability of recruitment to the infection focus of cells that mediate early immune reactions is reduced, that gives evidence on the importance of these reactions for infection outcome.

[Toxoplasmosis in HIV infection: invasion reactivation criteria].

Contemporary representation of toxoplasmosis reactivation criteria in HIV infection is generalized. Significance of the issue is justified: toxoplasmosis is a leading neurological pathology in AIDS with a high lethality percentage due to complexity of clinical confirmation and difficulties of laboratory confirmation of the start of reactivation. Clinical, instrumental, immunologic, molecular genetic invasion reactivation criteria are discussed in the article and analysis of their effectiveness is performed; their most feasible combinations are justified. Further system analysis of the cerebral toxoplasmosis reactivation criteria specified in the article in combination with search of new pathogen dissemination markers will allow to obtain important information that has both fundamental interest and important practical significance.

[Features of epidemiology and diagnostics of toxoplasmosis during HIV-infection].

AIM: Comparative assessment of effectiveness of serologic methods for toxoplasmosis diagnostics in patients with HIV-infection. MATERIALS AND METHODS: Sera and CSF samples from 166 patients with AIDS stage IIIB were tested for antibodies to Toxoplasma gondii by indirect immunofluorescence, ELISA and immunoblotting. Results of serological tests were compared with clinical, pathological data as well as with results of MRI and PCR. RESULTS: Clinical value of IgG detection in blood and CSF by ELISA was shown--high level of antibodies marked reactivation of the invasion. IgG antibodies in CSF were detected only if high levels of IgG were present in the blood. Detection of antigenic determinants with molecular mass 18 - 20 and 65 - 70 kDa in immunoblotting was proposed as a criterion of cerebral toxoplasmosis reactivation. CONCLUSION: Complex laboratory serologic tests along with data obtained by MRI, PCR and microscopy of T. gondii cysts enhances the effectiveness of toxoplasmosis diagnostics. Knowledge of toxoplasmosis reactivation criteria in patients with AIDS will allow to develop the optimal protocol of toxoplasmosis diagnostics and substantiate measures for its prevention.

[Study of persistence of low virulent strain of Toxoplasma gondii on in vivo model].

AIM: To study virulence of Toxoplasma gondii strain isolated from pathologicoanatomic brain tissue of HIV-infected patients on in vivo model as well as immune response to the pathogen in immunocompetent animals and in animals with cyclophosphamide-induced areactivity. MATERIALS AND METHODS: Groups of immunocompetent and immunodeficient mice were inoculated with cysts of T. gondii obtained from brain tissues of deceased HIV-infected patients. Parasites were detected in different organs and tissues by PCR as well as by parasitological and histological methods. Antibodies to T. gondii belonging to isotypes IgG, IgG1, IgG2, IgA were detected by immunofluorescence assay. RESULTS: Cysts of the pathogen located predominantly in animals' brain, and more rarely - in spleen and liver. Using detection of antibodies of different isotypes, which marked stage of invasion, dynamics of humoral response during persistence of toxoplasms in organism was determined. Analysis of disease pathogenesis as well as peculiarities of immune response to the pathogen in immunocompetent animals and in animals with cyclophosphamide-induced areactivity was performed. CONCLUSION: Characteristics of strains of toxoplasms isolated from material of brain section of patients with HIV infection were studied. Their low virulence and ability to prolonged persistence in organism of mice were demonstrated on in vivo model. Possible association of T. gondii strains' virulence and clinical symptoms with pathogen's genetic polymorphism and its clonal population structure was discussed.

Functional activity of peritoneal macrophages from sensitive and resistant mouse strains during intravaginal infection with herpes simplex virus type 2 and mucosal vaccination.

In the early period after intravaginal infection with herpes simplex virus type 2 (2 h), macrophages from sensitive DBA/2 mice were characterized by higher capacity to engulf the antigen, decreased function of the lysosomal apparatus, lower activity of cathepsin D, and reduced oxygen metabolism compared to cells from resistant BALB/c mice. Mucosal vaccination with herpes vaccine and hyaluronic acid promoted the increase in functional activity of macrophages and improved survival of sensitive mice (by 60%).

Comparative study of macrophage response in mice after DNA immunization and infection with herpes simplex virus type 1.

Functional activity of macrophages and intensity of T cell immune response in mice were studied after intravaginal and intraperitoneal infection with herpes simplex virus type 1 and DNA vaccination in combination with adjuvant treatment (recombinant granulocyte-macrophage colony-stimulating factor and glucosaminylmuramyl dipeptide). DNA vaccination induced a virus-specific T cell immune response with no macrophagic inflammatory reaction. Infection with herpes simplex virus type 1 was accompanied by sustained inflammation, but not by the T cell immune response.

Characterization of T cell clones derived from lymph nodes and lungs of Pseudomonas aeruginosa-susceptible and resistant mice following immunization with heat-killed bacteria.

Pseudomonas aeruginosa-resistant BALB/c and susceptible C57Bl/6 (B6) mice were immunized with heat-killed Pseudomonas either in the foot pad or via the trachea, and panels of Pseudomonas-specific T cell clones were developed from lymph nodes and lungs. All clones from either strain, whether of lymph node or lung origin, were CD3+CD4+CD8-TCRalphabeta+. The efficacy of cloning from lymph node cells was comparable between BALB/c and B6 mice. All lymph node BALB/c clones proliferated in response to Pseudomonas antigen in a dose-dependent manner, and this response was MHC class II-restricted. Vigorous proliferation by a considerable proportion of B6 T cell clones occurred in the absence of specific antigen. Lymph node clones from either strain could be categorized as either Th1 or Th0 on the basis of interferon-gamma (IFN-gamma)/IL-4 production. In either mouse strain the efficacy of cloning from lung tissue was substantially lower than from lymph nodes, but the efficacy of cloning from BALB/c compared with B6 lungs was higher. Four lung T cell clones from BALB/c and two from B6 mice were expanded for further analyses, and an interstrain difference was observed in cytokine production. Both B6 lung T cell clones were Th1-like and produced IFN-gamma but not IL-4 and IL-10, whereas four BALB/c lung T cell clones were Th2-like and produced IL-4 and IL-10 but not IFN-gamma. These observations suggest that differences in the CD4+ Th response in the lung may contribute to differences among inbred mouse strains in the level of resistance to bronchopulmonary Pseudomonas infection.

The Possible Immunological Mechanisms of Pathology in Mice with Congenitally Acquired Influenza Infection.

The fact that congenitally acquired viral infection often strongly influences specific and non-specific immunoreactivity is well documented. Viral infection of pregnant female may lead to serious of pathological consequences for the offspring, namely, to mortality, developmental disorders and in less severe cases to body growth retardation, wasting syndrome and immunodeficiency. In this connection, we have studied congenitally acquired influenza infection in CII mice. The progeny of C57BL/6 female mice, which were infected with influenza virus (A/WSN) by the 3rd week of pregnancy, exhibited a profound growth retardation and major morphological lesions of central nervous system, lymphoid and other organs. We have found out that mice with congenitally acquired influenza infection had autoreactive killer T cells in their lymphoid organs. CII mice exhibited some features of chronic immune activation, namely elevated spontaneous proliferation, spontaneous development of plaque forming cells, and spontaneous inhibition of migration activity. Lymphoid cells from mice with congenitally acquired influenza infection induced an enlargement of regional lymph nodes after they had been injected into syngeneic non-infected recipient in popleteal node assay. The level of this reaction depended on the level of virus-bearing cells in donor cell population and correlated with the increase of gammadelta and CD4(+) T cells. The role of these interactions in pathology is discussed herein.

Peculiarities of virus-specific immune response in mice with congenitally acquired influenza infection.

In this paper we show that progeny of C57BL/6 mice infected during pregnancy with influenza virus display a profound specific areactivity in situ according to their decreased capacity to give plaque-forming cell and delayed-type hypersensitivity responses to influenza virus. In spite of this specific areactivity in situ, spleen cells of mice with congenitally acquired influenza infection can easily transfer virus-specific delayed-type hypersensitivity and plaque-forming cell responses to syngeneic recipients. These results suggest that specific areactivity of mice with congenitally acquired influenza infection observed in situ is not connected with deletion of virus-specific immunocompetent cells after vertical virus transmission. It is also shown in this paper that spleen cells of mice with congenitally acquired influenza infection can specifically suppress local transfer of delayed-type hypersensitivity response to influenza virus in syngeneic mice. This suppression may be one of the possible causes of specific areactivity of mice with congenitally acquired influenza infection observed in situ. The presence of influenza virus antigens in lymphoid cells of mice with congenitally acquired influenza infection is also shown in this paper. The immunopathological consequences of viral persistence in lymphoid cells require further study.

[Characteristics of immune reactions to the influenza virus in mice with a slow influenzal infection]. / Osobennosti immunnykh reaktsii na virus grippa u myshei pri medlennoi grippoznoi infektsii.

It had previously been shown that intrauterine infection of mice with influenza virus resulted in growth retardation and significant immunosuppression to various nonspecific agents and influenza virus. The present study demonstrated that such mice also had lower production of specific antibody and reduced capacity to form the delayed hypersensitivity to influenza virus. Despite the lack of specific immune response, such mice had high levels of response to influenza virus in adoptive transfer. The reasons for which lymphocytes of mice with slow influenza infection fail to manifest their immunological potentials in situ require further study.