[Production of mycobacterial antigenes merged with cellulose binding protein domain in order to produce subunit vaccines against tuberculosis].

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

[Study of a role of B cells in the formation of antituberculosis immunity].

The authors have earlier demonstrated that CBA/N mice bearing nonsense mutation in the btk gene encoding B cell Bruton's thyrosine kinase after CG vaccination develop no protective response against subsequent M. tuberculosis H37Rv challenge. Adoptive transfer approaches are applied to show that this defect is determined by lymphoid cells and B lymphocytes play an essential role in its expression. In addition, experiments on CBA/N-xid mice with crude mixtures of soluble mycobacterial antigens suggest that the processing of live BCG could be impaired in the antigen-presenting cells of these mice.

[Tuberculosis: pathogenesis, immune responses and genetics of the host].

Tuberculosis is a chronic infectious disease predominantly affecting the lung. The hallmark of tuberculosis infection is the formation of granulomata in the vicinity of infectious foci. The tuberculous granuloma is a complex, cellulary and biochemically well-orchestrated structure, which development plays a dual role. Restricting dissemination of infection and forming a battlefield for protective immunity, granulomatous process may compromise lung function, threatinig the host health. Both the susceptibility to infection per se and the degree of lung failure and disease severity are under genetic control. Tuberculosis genetics is complex and far from being resolved, but the information available clearly indicates that the control of intracellular infections depends upon biochemical networks, which have not been appreciated with this regard until recently.

[Efficiency of anti-inflammatory therapy for experimental tuberculosis in genetically tuberculosis-sensitive mice].

The study assessed trends in death rates in genetically tuberculosis-sensitive I/St mice, mycobacterial seeding from their organs, and the magnitude of abnormal changes in the lung after intratracheal Mycobacterium tuberculosis infection of recipients. The experimental animals received two anti-inflammatory regimens: 1) a very small-dose intravenous injection of immature antigen-loaded bone marrow-derived syngeneic dendritic cells; 2) the nonsteroidal anti-inflammatory drug diclofenac injected intramuscularly in the course of disease development. Both anti-inflammatory regimens caused a significant increase in the animals' survival, a reduction in the amount of mycobacteria in the lung and the pulmonary infiltration with lymphoid cells as compared with untreated infected control animals. These findings indicate that suppression of an excessive inflammatory process in the lung may have a beneficial antituberculous effect in tuberculosis-sensitive animals.

[Induction of an immune response to mycobacterial antigens by means of dendritic cells]. / Induktsiia immunnogo otveta k mikobakterial'nym antigenam s pomoshch'iu dendritnykh kletok.

The study evaluated whether a vaccine can be designed by using dendritic cells loaded by mycobacterial antigens. A method for preparing murine dendritic cells from the bone-marrow precursors was modified. The dendritic cells were characterized by the expression of surface molecules by flow cytofluorometry. The study has shown that T lymphocytes can be primed in vivo by administering the dendritic cells expressing the protective Mycobacterium tuberculosis antigens H37Rv.

[Pneumocystosis in mice under the conditions of a secondary immunodeficiency and with specific immunological tolerance for Pneumocystis carinii]. / Pnevmotsitoz u myshei v usloviiakh vtorichnogo immunodefitsita i pri spetsificheskoi immunologicheskoi tolerantnosti k Pneumocystis carinii.

To study pneumocystosis and to develop the method of its reliable diagnosis, two mouse models of this disease were created. In model 1 mice with T helpers eliminated by means of monoclonal antibodies (McAb) to T-cell marker L3T4 were used. For this purpose mice were injected intraperitoneally with McAb and then infected with P. carinii. In model 2 tolerance to P. carinii was induced in mice by the injection of cyclophosphamide and the animal were then also infected with P. carinii. In both cases the number of cysts in the lungs of the test animals significantly increased in comparison with that in the lungs of the controls. The data obtained in this study indicate that in animals treated in both ways an increase in the sensitivity of the host to this infective agent occurred.

[Experimental influenzal and tuberculous infections under conditions of specific and nonspecific immunosuppression]. / Eksperimental'naia grippoznaia i tuberkuleznaia infektsii v usloviiakh spetsificheskoi i nespetsificheskoi immunosupressii.

The course of influenza and tuberculosis infections under the conditions of disturbances in the immune response of experimental animals has been studied. As revealed in the survival test, the induction of secondary T- and B-cell-mediated immunodeficiency in mice leads to an increase in the sensitivity of the body to influenza virus, especially in cases of T-cell-mediated immunodeficiency. The injection of BCG in combination with cyclophosphamide into mice induces tolerance to this antigen in the animals; this tolerance has a "split" character, i.e. it affects only T-cell-mediated, but not humoral immunity. The induction of T-cell-mediated immunodeficiency or tolerance to BCG in mice has been shown (in the survival test) to lead to the development of the sensitivity of the animals to experimental tuberculosis infection. B-cell-mediated immunodeficiency did not influence the animal survival rate.

[Nonspecific non-reactivity in mice induced by joint administration of Newcastle disease virus and cyclophosphamide]. / Nespetsificheskaia areaktivnost' u myshei, vyzvannaia sochetannym vvedeniem virusa bolezni N'iukasla i tsiklofosfamida.

The immunomodulating effect of Newcastle disease virus (NDV) was investigated in vitro and in vivo in mice. NDV was shown to induce a mitogenic effect in splenocytes in vitro. Combined injections of NDV and CP resulted in nonspecific suppression of immunoreactivity in mice. The antibody production and development of delayed type hypersensitivity to sheep erythrocytes were markedly reduced. Injections of NDV alone slightly increased the reactions. The NDV + CP injections led also to a reduction of immune response to thymus-independent antigen, LPS. Thus, the combined injections of NDV and CP led to nonspecific suppression of T and B cell immunity in mice. The mechanisms of this form of anergy require further study.

[Changes in antigenic properties of lipopolysaccharides of Shigella sonnei phase I having lost the virulence due to transposon integration into the invasiveness plasmid PSS120]. / Izmenenie antigennykh svoistv lipopolisakharida shigell Zonne I fazy, utrativshikh virulentnost' v sviazi s integratsiei transpozona v plazmidu invazivnosti PSS120.

A nonvirulent strain of Shigella sonnei phase I has been obtained by integration of the transposon Tn5 into the invasiveness plasmid pSS120 in the virulent strain and designated NR18. The presence of the plasmid pSS120 in both strains results in the similar morphology and bacterial ability to agglutinate in the presence of antiserum to Shigella sonnei phase I antigen. The lipopolysaccharide preparations from the virulent and nonvirulent strains give the similar reactions with the antiserum in the reaction of hemagglutination. However, in the reaction of passive local hemolysis in the gel (Jerne reaction) the significant difference is revealed in the immunogenicity of the preparations, with the preparations from the virulent strain being 4-5 fold more immunogenic. In crossreaction, the antibodies secreted by the mouse spleen cells immunized by LPS from the virulent strain show a weak reaction with the ram erythrocytes sensitized by the LPS of the nonvirulent strain. Thus, the biological changes in the LPS of the nonvirulent strains that are, evidently, the consequence of the structural changes, are identified only by the most sensitive immunological techniques.

[The nature of immunodeficiency induced by injections of lectins and cyclophosphamide]. / Priroda immunodefitsita, indutsirovannogo in''ektsiiami lektinov i tsiklofosfamida.

Consecutive injections of T-cell mitogen (LcA, Con A) and cyclophosphamide (CY) produce an inhibition of T-cell, but not B-cell functions. This phenomenon was not a result of suppressor-cell activity of the action of some suppressor serum factors. Immunoreactivity of mice, treated with Con A CY is restored by thymocytes from intact donors, but not bone marrow cells. Using, monoclonal antibodies to Thy-1.2 antigen or anti-Ig serum it has been shown that pretreatment of mice with lectin and CY resulted in a decrease in T-cell, but not B-cell number in comparison with control mice. The above facts are indicative of CY-mediated elimination of T cells involved in proliferation and differentiation by lectin.

[T-cell immunodeficiency in mice receiving lectin and cyclophosphamide]. / T-kletochnyi immunodefitsit u myshei, poluchivshikh lektin i tsiklofosfamid.

Cyclophosphamide injections to mice following T cell mitogen (lectins from Lens culinaris and concanavalin A) were shown to suppress (20-40-fold) thymus-dependent response to SRBC. At the same time no damage-specific and polyclonal response to thymus-independent antigen and polyclonal activator of B cells--lipopolysaccharide--has been observed. Injections of lectin and cyclophosphamide to mice prevented the onset of DTH reaction to SRBC and induction of antigen-specific DTH suppressor cells. Thus, cyclophosphamide injection after T cell mitogen leads to T-cell anergy, with B-cell activity remaining unchanged.

[Mechanisms of polyclonal tolerance induced by lipopolysaccharide and cyclophosphamide]. / Mekhanizmy poliklonal'noi tolerantnosti, indutsirovannoi lipopolisakharidom i tsiklofosfamidom.

The treatment of recipient mice with LPS from S. marcescens followed by the injection of CY 48 h later inhibited a subsequent antibody production against unrelated antigen (SRBC) and polyclonal mitogen (LPS from Br. abortus). Such a reactivity persisted for 2-3 weeks after treatment. It was shown that the number of Ig+ cells in the spleens of treated mice was decreased, while the population of spleen Thy-1.2+ cells remained unaltered. Cell-cooperative test revealed that the function of B cells, but not T cells, was inhibited by the treatment. There were no changes in DTH response to SRBC. Thus, a subsequent treatment of mice with LPS and CY led to B-cell deficiency. The nature of this phenomenon is presumably the same as the nature of CY-induced antigen-specific immunological tolerance.

[Characteristics of the reaction of immunologically tolerant mice to the polyclonal stimulant salmozan]. / Osobennosti reaktsii immunologicheski tolerantnykh myshei na poliklonal'nyi stimuliator sal'mozan.

The nonspecific stimulant of the immune response salmozan (Sal) increases the number of PFC against SRBC in intact mice, B mice (thymectomized, irradiated and reconstituted with embryonic liver cells), and in mice pretreated with cyclophosphamide (CY). This effect was decreased in mice pretreated with SRBC and CY. The subsequent injections of SRBC and Sal into tolerant mice did not increase the response under study. It is concluded that the effect observed is due to partial alteration of antigen-specific B cells of tolerant mice and this alteration cannot be explained by the lack of the regeneration of membrane immunoglobulins.

[A reactivity to polyclonal stimulation of immunogenesis with salmozan after its repeated administration]. / Areaktivnost'k poliklonal'noi stimuliatsii immunogeneza sal'mozanom posle ego povtornogo vvedeniia.

The injection of 100 micrograms of salmozan (polysaccharide isolated from Salmonella typhi somatic O-antigen) or 50 micrograms of Escherichia coli lipopolysaccharide into mice induced a considerable increase in the number of antibody-forming cells in the spleen in response to the injection of sheep red blood cells (SRBC) 2-3 days later. This polyclonal effect was essentially weaker if the animals previously received 500 micrograms of salmozan (9-10 days prior to the injection of SRBC). The absence of reactivity was not linked with antibodies to salmozan or with some other serum factor. The lymphocytes of nonreactive mice proved to be capable of polyclonal response in the adoptive system, and at the same time the polyclonal response of intact lymphocytes to salmozan in the body of nonreactive irradiated mice was essentially weakened. The features making the above phenomenon similar to, as well as different from, the so-called "endotoxin tolerance" are analyzed.

[Thymus-dependence of genetic resistance to (CBA x M523)F1 lymphocytes]. / Timuszavisimost' geneticheskoi rezistentnosti k limfotsitam (CBA x M523)F1.

The nature of cells responsible for the genetic resistance of lethally irradiated CBA mice to lymphocytes of (CBA x M523)F1 hybrids was studied. Preirradiation of the hosts was shown to abolish the resistance. The latter was generally recovered by syngeneic thymocytes or splenocytes while embryonic liver and bone marrow cells or splenocytes treated with anti-Thy-I serum plus complement before injection into host were ineffective. It is postulated that some cells with T cell characteristics are responsible for the phenomenon of parental resistance. These cells differ in several respects from T cells that mediate the transplantation immunity and from M cells that control other forms of the genetic resistance.

[Development of delayed hypersensitivity to sheep erythrocytes after separate and combined administration of the antigen and cyclophosphamide]. / Formirovanie giperchuvstvitel'nosti zamedlennogo tipa k éritrotsitam barana pri razdel'nom i sochetannom vvedenii antitela i tsiklofosfamida.

The ability to the development of delayed hypersensitivity (DHS) to the appropriate antigen was studied in mice treated with large doses of SRBC and cyclophosphamide at varying time prior to the experiment. Suppression of DHS development induced by administering either the cytostatic alone or a large dose of the antigen was examined at the same periods of time. The combined treatment was shown to induce tolerance according to diverse tests for DHS (skin and macrophage migration inhibition tests). At the basis of this tolerance lies genuine deficiency of the appropriate clone of T cells. Administration of cyclophosphamide alone leads to a slight suppression of DHS, while a large dose of the antigen induces a different form of areactivity due to the suppressor cells whose nature is not yet clear.

[Parental resistance of irradiated mice (CBA X M523)F1 to lymphocytes: fate of transplanted cells, duration of resistance and its specificity]. / Roditel'skaia rezistentnost' obluchennykh myshei k limfotsitam (CBA X M523)F1: sud'ba transplantirovannykh kletok, dlitel'nost' rezistentnosti i ee spetsifichnost'.

The immune response of (CBA X M523)F1 mouse lymphocytes to the antigen (sheep red blood cells) in CBA mice after lethal irradiation was studied. When irradiation, cell transfer and antigen test-injection were performed at the same day the graft activity was inhibited, in comparison with syngeneic system. The activity of donor cells restored on increasing the interval between these processes up to 3 days. Retransplantation of the spleen cells from recipients to irradiated CBA and F1 mice revealed viability of transplanted cells and lack of their readaptation to insyngeneic microenvironment. The recipient's resistance could be specifically overcome by the preinjection of mouse cells combined with cyclophosphamide or without it. The results obtained allow a conclusion that genetic parental resistance of CBA mice to F1 mouse cells is due to the recipient immunocompetent cells which are inactivated 3 days following irradiation. They do not produce cytotoxic effects with respect to donor cells, blocking, however, their activity for some time.

[Phenomenon of parental resistance and its genetic regulation]. / Fenimen roditel'skoi rezistentnosti i ego geneticheskaia reguliatsiia.

Lymphocytes of mice F1 (CBA X M523) and F1 (A X M523) transplanted to 1000 R irradiated CBA or A mice responded to the test antigens--SRBC or S. typhi Vi-antigen--by formation of 100--1000 times less antibody forming cells than in syngeneic recipients. An intermediate result is achieved when the lymphoid cells are transplanted to the irradiated M523 mice. Lymphocytes of mice F1 (A X CBA), F1 (CBA X C57Bl/6), or F1 (A X A.CA) developed a similar immune response in the irradiated syngeneic mice and in both parental lines. The ability of parental line M523 to respond to SRBC was the same as in the other lines studied when examined in situ or in adoptive transfer experiments. The stem hemopoietic cells of mice F1 (CBA X M523) develop in the spleen of CBA mice 2--2.5 times less hemopoietic colonies than in the spleen of syngeneic animals. A conclusion was drawn that mutation M523 in CBA mice inhibited the proliferation and differentiation of hemopoietic and lymphoid cells in the irradiated nonsyngeneic recipients.