[A locus involved in tuberculosis infection control in mice locates in the proximal part of the H2 complex].

One of genetic loci involved in tuberculosis (TB) infection control in mice is located within the segment of Chr. 17 occupied by the H2 complex, the mouse MHC. As far as this region includes approximately 40 Mb and contains hundreds of genes affecting immune responses and host-parasite interactions, narrowing the interval by genetic recombination is pre-requisite for identification of particular gene(s). We have developed a panel of recombinant congenic strains bearing different parts of the H2 complex from TB-susceptible I/St mice on the genetic background of TB-resistant C57BL/6 mice. By superposing the phenotype "severe vs. mild infectious course" against the chart of alleles inherited by these new strains from the two parental strains, we have mapped a locus involved in TB control within the segment 33.305-34.479 Mb (-1.1 Mb) of the Chr. 17. Such a location indicates that allelic variants of the prominent pro-inflammatory factor TNF do not affect TB course in our experimental system. This result was confirmed by the assessment of the TNF level in the lung tissue of infected mice of different strains. The QTL (quantitative trait locus) mapped in our study influences several important parameters of TB infection: multiplication of mycobacteria in the lungs, severity of lung pathology and regulation of the early inflammatory response.

[Development of pulmonary chlamydia infection in inbred mice lines differentiated by genetically determinated sensitivity to tuberculosis infection].

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. The susceptibility does not depend on the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by little interacting QTL mapped to chromosomes 3, 9, 17. To find out whether the tuberculosis-susceptible I/St mice are susceptible to other intracellular bacteria taxonomically distant pathogen of Chlamydia pneumoniae was studied. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former were more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St, 9.2 +/- 1.2 days; A/Sn, 22.0 +/- 0 days (p < 0.001)). To estimate the degree of chlamydial multiplication in the lungs, we suggested a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite's genome equivalents in infected tissue from 1 to 16 days after challenge. The interstrain difference of chlamydia burden in lungs was observed only after 24 hours after infection. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection. The numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p < 0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-? and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p < 0.05). Because the prominent difference in survival time did not correlate with permanent difference in bacterial multiplication, we suggested that both infections trigger fatal pathological processes whose dynamics depends strongly upon the host genetics.

Mycobacterium tuberculosis-susceptible I/St mice develop severe disease following infection with taxonomically distant bacteria, Salmonella enterica and Chlamydia pneumoniae.

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. To find out whether tuberculosis (TB)-susceptible I/St mice are susceptible to other intracellular bacteria, we investigated two different taxonomically distant pathogens, Chlamydia pneumoniae and Salmonella enterica serovar Typhimurium. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1(r)) demonstrated that the former are more susceptible to both salmonella and chlamydia, displaying a significantly shortened survival time following challenge. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia, despite their similar ability to control bacterial multiplication. Following infection with salmonella, substantial ( approximately 3 log) but very short (second day post-infection) interstrain differences in bacterial loads were observed, accompanied by higher levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in the peritoneal cavities of I/St mice. I/St macrophages were more permissive for salmonella growth during the first 24 h following infection in vitro. Because the prominent differences in survival time did not correlate with permanent differences in bacterial multiplication, we suggest that both infections trigger fatal pathological processes whose dynamics depend strongly upon the host genetics.

[Experimental approaches to designing vaccines against tuberculous infection reactivation].

Pulmonary tuberculosis (TB) in adults is considered to be a reactivation disease that develops after a long period of latent infection. The need for vaccines against TB reactivation is urgent; however, such vaccines are not available yet. The authors have developed an experimental model of TB reactivation in inbred mice of TB-susceptible strain I/St and attempted to vaccinate these animals in the preinfection (prophylactic) and postinfection (therapeutic) manner against the reactivation. Therapeutic vaccination with BCG resulted in an exacerbation of the disease, presumably, due to Koch's phenomenon. Prophylactic vaccination with proteins of the Rpf family induced IFN-gamma production by CD4+ T cells and slightly decreased mycobacterial multiplication in the organs. However, neither the vaccination protocol prevented infection reactivation, suggesting that heterologous prime-boost approaches should be further investigated in our model.

CD4 T cells producing IFN-gamma in the lungs of mice challenged with mycobacteria express a CD27-negative phenotype.

Protection against tuberculosis depends upon the generation of CD4(+) T cell effectors capable of producing IFN-gamma and stimulating macrophage antimycobacterial function. Effector CD4(+) T cells are known to express CD44(hi)CD62L(lo) surface phenotype. In this paper we demonstrate that a population of CD44(hi)CD62L(lo) CD4(+) effectors generated in response to Mycobacterium bovis BCG or M. tuberculosis infection in C57BL/6 mice is heterogeneous and consists of CD27(hi) and CD27(lo) T cell subsets. These subsets exhibit a similar degree of in vivo proliferation, but differ by the capacity for IFN-gamma production. Ex vivo isolated CD27(lo) T cells express higher amounts of IFN-gamma RNA and contain higher frequencies of IFN-gamma producers compared to CD27(hi) subset, as shown by real-time PCR, intracellular staining for IFN-gamma and ELISPOT assays. In addition, CD27(lo) CD4(+) T cells uniformly express CD44(hi)CD62L(lo) phenotype. We propose that CD27(lo) CD44(hi)CD62L(lo) CD4(+) T cells represent highly differentiated effector cells with a high capacity for IFN-gamma secretion and antimycobacterial protection at the site of infection.

[Significance of molecular genetic and immunological techniques in risk groups and in children with tuberculosis]. / Znachenie molekuliarno-geneticheskikh i immunologicheskikh metodov issledovaniia v gruppakh riska i u bol'nykh tuberkulezom detei.

A total of 188 children and adolescents were examined. In all the children, blood Mycobacterium tuberculosis (MBT) DNA was determined by polymerase chain reaction (PCR) and MBT antigens (AG) and antibodies (AB) were by enzyme immunoassay. The studies have shown that it is expedient to concurrently determine MBT DNA and MBT AT in order to identify local forms of tuberculosis in children from risk groups. If the tests are positive, a comprehensive examination for tuberculosis is required; the presence of the syndrome of common disturbances is generally associated with tuberculous infection. When a local form of tuberculosis is excluded, preventive chemotherapy should be performed. Further negative tests for MBT DNA and lower MBT AT may be a criterion for the efficiency of preventive treatment. In children with tuberculosis, the results of repeated blood and urine tests for MBT DNA provide a way of evaluating the course of a tuberculous process and the efficiency of chemotherapy. PCR used to determine blood and urine MBT DNA is a highly specific test as positive results were in 79% of the children with tuberculosis.

[Tuberculous IgE antibodies. Part II. Study of its concentrations in different forms of tuberculosis]. / Protivotuberkuleznye IgE-antitela. Chast' II. Issledovanie kontsentratsii pri razlichnykh formakh tuberkuleza.

Tuberculosis-afflicted lung are infiltrated by two functionally types of lymphocytes, which presumably counteract with each other by producing proinflammatory (type 1) and anti-inflammatory (type 2) cytokines. It is held that irregular sequestration of antigen into different compartments of the lung may lead to preferential activation of T-helper 1 or T-helper 2 lymphocytes. Unlike IgE antibodies, specific tuberculosis IgE antibodies are seen only in tuberculosis infection. The mean values of IgE antibodies in tuberculosis (7.661 +/- 0.849 IU/ml) are significantly greater than those in other pulmonary diseases (1.768 +/- 0.116 IU/ml). Low concentrations of tuberculosis IgE antibodies in persons with a marked hyperergic response to tuberculin (1.808 +/- 0.097 IU/ml) are of importance. Significant concentrations of mycobacterial IgE antibodies are mainly detected in fibrocavernous (14.56 +/- 1.11 IU/ml), infiltrative (10.10 +/- 1.08 IU/ml), peripheral lymph nodal (10.53 +/- 1.09 IU/ml) tuberculosis rather than intrathoracic lymph nodal tuberculosis (4.555 +/- 0.340 IU/ml). There is a particularly considerable increase in specific IgE antibodies in a phase of decay (15.98 +/- 1.64 IU/ml) and infiltration (12.66 +/- 1.08 IU/ml). These groups also show a concurrent rise in tuberculosis IgG antibodies, which nevertheless disagree with the increase of IgE (the correlation coefficient is 0.599).

[Anti-tuberculous IgE antibodies. I. Immunodominant antigens]. / Protivotuberkuleznye IgE-antitela. I chast'. Immunodominantnye antigeny.

It is widely accepted that protection against tuberculosis is provided by the formation of type 1 immune response, which is characterized by the production of IFN-gamma and IL-2. However, type 2 antimycobacterial immune response is also present: specific IgE antibodies that are IL-4 dependent, are usually found in tuberculosis patients. There is elevated production of type 2 cytokines in some cases. Thus, both types of an immune response can simultaneously develop, probably counteracting with each other. It is unknown which of mycobacterial antigens are capable of inducing a preferential type 2 response. To detect these antigens, the authors studied tuberculosis IgE antibodies in the sera of 500 tuberculosis patients by using the ELISA assay with ultrasonic disintegrated M. Tuberculosis H37Rv (sonicate). Antigens recognized by IgE antibodies were found to be localized in the cell wall of mycobacteria. The IgE-response was specific since the sera did not react with the antigens of atypical mycobacteria and other bacterial species.

[Improving the efficacy of tuberculosis immunodiagnosis using anti-human IgG monoclonal antibodies]. / Povyshenie éffektivnosti immunodiagnostiki tuberkuleza putem primeneniia monoklonal'nykh antitel protiv IgG cheloveka.

Monoclonal antibodies (MAb) F5 to human IgG were used for creating immunoperoxidase conjugate. MAb dissociation constant was 10(-9)M-1 and the number of binding sites 1. Enzyme immunoassay (EIA) and immunoblotting showed that MAb F5 specifically recognize conformation epitope on intact human IgG molecule but not on other human immunoglobulins or denatured IgG. The resultant peroxidase conjugate with MAb F5 was used for EIA titration of antibacterial antibodies in sera from 30 patients with pulmonary tuberculosis, 28 patients with nonspecific pulmonary diseases (bronchitis and/or asthma, pneumonia), and 12 donors. For comparison similar studies were carried out with reference commercial immunoperoxidase conjugate to human IgG(H + L) manufactured at N. F. Gamaleya Institute of Epidemiology and Microbiology. Mycobacterium tuberculosis monoantigen (mol. weight 15-18 kD), affinity isolated by antibacterial MAb S4C1G4 (alpha S4C1G4), and PPD (Batch RT 45, Stattens Seruminstitut, Denmark) were used. Sensitivity and specificity of serum antibacterial antibodies were compared. The specificity of conjugate based on MAb F5 with monoantigen alpha S4C1G4 was 78.21%, sensitivity 94.50%, while those of conjugate to human IgG(H + L) were 53.30 and 76.89%, respectively (p < 0.001). For PPD the specificity and sensitivity were 56.75 and 72.33%, respectively (conjugate with MAb F5) versus 47.67 and 62.38% for conjugate against IgG(H + L), p < 0.001. Similar values were obtained in assessment of the concentrations of antibodies to alpha S4C1G4 for MAb F5 conjugate: specificity and sensitivity 47.16 and 71.56%, respectively, versus 23.67 and 95.16% for PPD (p < 0.05). No statistically significant differences between the experimental groups were detected with IgG(H + L) conjugate. We believe that specific MAb-based conjugate to human IgG will improve the efficacy of EIA as a method for the diagnosis of pulmonary tuberculosis.

[Detection of antituberculosis antibodies and antigens by enzyme immunoassay in young and preschool children suffering from tuberculosis]. / Opredelenie protivotuberkuleznykh antitel i antigenov metodom immunofermentnogo analiza u detei rannego i doshkol'nogo vozrasta, bol'nykh tuberkulezom.

Antituberculosis antibodies and mycobacterial antigens were detected in 74 young and preschool children suffering from tuberculosis by using enzyme immunoassay (EIA). They were found in 75.7% and 68.9% of children, respectively. The highest levels of antibodies were significantly greater in patients with active disseminated processes than in those with active restrictive processes. There were no great differences in the levels of antigens between the patients having different activities of a tuberculosis process. Follow-up indicated that there was a reduction in the levels of antibodies and antigens at 6-month treatment. The use of enzyme EIA in children ill with tuberculosis may serve an additional criterion for diagnosis of tuberculosis, evaluation of its activity and course.

[Serological and immunochemical properties of carbohydrate-containing fraction from mycobacterium tuberculosis H37Rv]. / Serologicheskie i immunokhimicheskie svoistva uglevodosoderzhashchei fraktsii iz mycobacterium tuberculosis H37Rv.

Using affinity chromatography on concanavalin A (Con A) sepharose CL 6B, a carbohydrate-containing fraction was derived from Mycobacterium tuberculosis H37Rv sonicate. Surprisingly, the main component of Con A fraction was a protein having a molecular weight of a 30-kD range which is generally absent in the Con A-adsorbed fraction from the culture filtrate. ELISA by means of an antimycobacterial monoclonal antibody panel showed that the 30-kD range of Con A fraction contained antigen 85. It is suggested that the derived components antigen 85 are a glycosylated form of the proteins associated with the mycobacterial cell wall. The Con A fraction, antigen 85 (Department of Virology, Pasteur Institute, Brussels, Belgium), and PPD (Batch RT 45, Stattens Seruminstitute, Denmark) were used for ELISA determination of antimycobacterial antibody titers in the sera of 30 patients with pulmonary tuberculosis, 28 patients with nonspecific lung diseases (bronchitis and/or asthma, pneumonia), as well as in the sera of 12 healthy volunteers. The sensitivities were 46.42, 57.34, and 72.33% and the specificities were 36.97, 26.73, and 56.75% for Con A fraction, antigen 85, and PPD, respectively. The authors suggest that serodiagnostic properties of Con A fraction are extremely limited.

[Interaction of Mycobacteria isolated from sarcoidosis patients with antituberculosis antibodies]. / Vzaimodeístvie mikobakterií, bydelennykh ot bol'nykh sarkoidozom, s protivotuberkuleznymi antitelami.

In 80% of cases, antituberculosis antibodies from the sera of patients with tuberculosis were ascertained to react in enzyme immunoassay (EIA) with antigens (ultrasound disintegrants (USDs)) of reverse mycobacteria isolated (initially) from patients with sarcoidosis. The USDs of reverse mycobacteria isolated from patients with sarcoidosis reacted in EIA with monoclonal antibodies (MAb) against M. tuberculosis complex antigens (unique and crossover). Both common and distinctive (unique) antigenic determinants were detected via MAb against different mycobacterial types by immunoblotting in the antigenic complexes of M. tuberculosis H37Rv and reverse strains isolated from patients with sarcoidosis.

[Examining of humoral immunity on mycobacteria antigens in sarcoidosis]. / Izuchenie gumoral'nogo immuniteta na antigeny mikobakterii pri sarkoidoze.

Antibodies to Mycobacterium tuberculosis antigens H37Rv and reverse strains previously isolated from patients with sarcoidosis with granular isolates were determined in 50 patients with sarcoidosis (including 16 patients isolating granular types) and 56 patients with tuberculosis, by using ELISA and immunoblotting. Serum antibodies from patients with sarcoidosis were ascertained to more commonly react in ELISA with the antigen (ultrasound disintegrant (USDs) obtained from reverse mycobacteria isolated (initially) from patients with sarcoidosis (AGS) than with the USD of the M. tuberculosis H37Rv (AGT) and, on the contrary, serum antibodies from patients with tuberculosis more frequently reacted with the M. tuberculosis H37Rv. The spectrum of serum antibodies from patients with sarcoidosis greatly differed at immunoblotting with AGS and AGT. There was most commonly a reaction with the antigenic determinants 79, 27, 30, and 50 kDa to AGS and that of the determinants 17, 35, 32 kDa to AGT.

[The isolation and characteristics of monoclonal antibodies to Mycobacterium smegmatis and M. kansasii]. / Poluchenie i kharakteristika monoklonal'nykh antitel k mikobakteriiam smegmatis i kansasii.

Immunization of BALB/c mice with sonicates of M. smegmatis and kansasii and fusion of splenocytes of the immunized mice with cells of syngeneic myeloma P3X63Ag8653 yielded hybrid clones synthetizing antibodies to these antigens. The selection of hybrid clones and analysis of the antibodies specificity were performed at ELISA using the immunization antigens and antigens from other mycobacteria and E. coli. To define immunoglobulin class of the antibodies the authors used antiglobulin sera of class A, M, G. The monoclonal antibodies belonged to IgG. Molecular mass of polypeptides carrying the epitope against which the antibodies were directed was measured at immunoblotting. Two antibodies reacted only with M. smegmatis, 38 and 10 kD proteins. The other 2 cross-reacted with other mycobacteria and were directed against epitopes on polypeptides varying in molecular mass.

[Affinity isolation of mycobacterial antigens on monoclonal antibodies]. / Affinnoe poluchenie antigenov mikobakterii na monoklonal'nykh antitelakh.

Affinity chromatography on monoclonal antibodies specific to tuberculous mycobacteria was used to isolate mycobacterial antigens from ultrasonic disintegrate M. tuberculosis H37Rv. Disk-electrophoresis on polyacrylamide gel showed purified antigens to consist of polypeptides with molecular mass 16, 19, 32, 60, 27, 70 and 55 kD. The antigens were tested for enzyme immunoassay of antibodies in the sera from patients with infiltrative pulmonary tuberculosis.

[Humoral immune response in humans and animals infected and immunized with Mycobacteria of the MAIS complex]. / Gumoralnyi immunnyi otvet u zarazhennykh i immunizirovannykh mikobakteriiami kompleksa MAIS liudei i zhivotnykh.

Hyperimmunization of mice and rabbits with surface (whole cells) and complex (ultrasound disintegrants) with the MAIS complex Mycobacterium antigens induced a humoral immune response which was largely aimed against cross-reacting than "unique" Mycobacterium antigens. Infection of BALB/c mice and rabbits with M. avium induced a humoral immune response which was chiefly against cross-antigenic Mycobacterium determinants. A series of consecutive immunochemical procedures were used to isolate from M. avium antiserum the antibody reacting with the antigenic determinants 18, 20, 24, 27, 39, 68, 94, kDa. Mab to the antigenic determinant 79 kDa of M. avium, which belong to the IgG class and unreacting with antigens of other Mycobacterium types were obtained. The antibodies isolated from hyperimmune serum and Mabs were used in the competitive EIA to determine antimycobacterial antibodies in man, mice, and rabbits. With Mab, positive results were obtained in 75% of M. avium-infected mice, 63.6% of rabbits and 63.6% of patients with M. avium infection (with 5.0% false positive results in humans). With antiserum, 83.3% of positive results were obtained in mice (with 10.0% of false positive results), 81.8 in rabbits (with 25% of false positive results) and 90.9% in M. avium-infected persons (with 30% of false positive results).

[Dynamics in bacterial isolation and antigenemia in patients with infiltrative pulmonary tuberculosis]. / Dinamika bakteriovydeleniia i antigenemii u bolnykh infiltrativnym tuberkulëzom lëgkikh.

A hundred patients with infiltrative pulmonary tuberculosis were studied. They were examined for bacterial isolates and mycobacterial antigens in the immune complexes. The isolates were detected in 76%. In most cases, isolation stopped 3 months following chemotherapy and in the absolute majority of cases (except one) 9 months after. Isolation of L-forms remained for a longer period of time, at month 6 of therapy there was an increase in the number of patients who were found to have L-forms of the bacteria. Most (97%) patients with infiltrative tuberculosis displayed mycobacterial antigens circulating in the immune complexes. Antigenemia retained in a considerable (40%) number of patients long (up to 9 months) and in most cases with the unfavourable course of the disease.

[A genotypic study of children ill with tuberculosis and of healthy BCG-revaccinated ones of Tuvinian nationality]. / Izuchenie genotipa u bol'nykh tuberkulezom i zdorovykh revaktsinirovannykh BTsZh detei tuvinskoi natsional'nosti.

The genotype was studied according to the 8 polymorphic genetic systems (Gm, Hp, Tf, Gc, ESD, PGM, ACP and ADA) in 73 children suffering from tuberculosis and 251 healthy children of the Tuvinian nationality. Analysis of the data obtained showed that a positive association was found in the allotype G1m (2), phenotype G1m (+1, +2, +4) and the genetic variant ESD 2-2 with tuberculosis in subjects of the Tuvinian nationality, and a negative association with the genotype ESD 1-1. It has been suggested that these genetic markers play a definite role in susceptibility to tuberculosis.

[Comparative analysis of the effects of the unpurified preparations of murine erythropoietin and human recombinant erythropoietin on erythroid and granulocyte-macrophage progenitor cells in semi-solid mouse bone marrow cultures]. / Sravnitel'nyi analiz deistviia neochishchennykh preparatov myshinogo éritropoétina i rekombinantnogo éritropoétina cheloveka na éritroidnye i granulotsitarno-makrofagal'nye kletki-predshestvenniki vpolutverdykh kul'turakh kostnogo mozga myshei.

Effects of unpurified murine erythropoietin and unpurified human recombinant erythropoietin on the growth of erythroid--BFU-E and granulocyte--macrophage progenitor cells--CFU--GM from the mouse bone marrow were compared using a methylcellulose culture system. Average erythropoietin titers for murine serum and supernatant human recombinant erythropoietin were 16 U/ml and 33 U/ml, respectively. The maximal stimulation was observed at 1-2 U/ml culture recombinant erythropoietin and 0.5 U/ml culture murine erythropoietin. Murine erythropoietin was more effective then human one. Murine and human recombinant erythropoietin had no significant effect on the number of CFU-GM colonies. But human recombinant erythropoietin could be preferentially used when studying the mechanism of erythropoiesis in man and animals because there were erythropoiesis inhibitors in mouse serum.