Two methods of isolating Fab- and Fc-fragments from mouse immunoglobulin G1 are presented. The first method involves fractionation of papain protein hydrolysate on a column with DEAE- (or DE-32)-cellulose adjusted with 0.005 M K-phosphate... more
Two methods of isolating Fab- and Fc-fragments from mouse immunoglobulin G1 are presented. The first method involves fractionation of papain protein hydrolysate on a column with DEAE- (or DE-32)-cellulose adjusted with 0.005 M K-phosphate buffer, pH 8. The Fab-fragment was eluted from the column with the starting buffer. The Fc-fragment was eluted, with the buffer ionic strength being increased to 0.4 M. Another method involves protein fractionation on an ion exchanger adjusted with 0.004 M Tris-H3PO4 buffer, pH 8.5. All the protein was column bound. The Fab-fragment was eluted with 0.04 M Tris-buffer containing a 0.004 M mixture of K-phosphates, pH 8.6. The Fc-fragment was eluted, with ionic strength being raised to 0.4 M with phosphates. As none of the methods assures isolation of absolutely pure Fab- or Fc-fragments, it is requird that cross absorption of antisera with respective immunosorbents may be carried on in order to obtain monospecific antisera to these fragments.
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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.... more
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.
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When infected with a high dose of Mycobacterium tuberculosis H37Rv intravenously, 1/St and C57BL/6 inbred mice display a similarly high degree of the severity of progressive disease characterized by short-term survival rates and a rapid... more
When infected with a high dose of Mycobacterium tuberculosis H37Rv intravenously, 1/St and C57BL/6 inbred mice display a similarly high degree of the severity of progressive disease characterized by short-term survival rates and a rapid body weight loss. Noteworthy, F1 hybrids between these two strains are exceptionally resistant to M. tuberculosis-triggered disease and survive about 4-fold longer than the either parental strain. The results of a segregation genetic analysis performed in this study suggest that mice of either strain carry a homozygous recessive allele at a single genetic locus which determines a high susceptibility to the disease regardless of a locus expressed in the other parental strain. Genetic complementation between these two apparently non-linked loci provides the expression of the resistant phenotype in F1 hybrids. Correspondence between these loci and the previously mapped QTLs in TB control remains to be established. There was a high correlation between th...
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In inbred CBA and CBA/N mice immunological memory was induced by subcutaneous injection of Mycobacterium bovis (BCG). Experiments with adoptive transfer of spleen T cells and ionomycin-resistant T cells (memory cells) between CBA and... more
In inbred CBA and CBA/N mice immunological memory was induced by subcutaneous injection of Mycobacterium bovis (BCG). Experiments with adoptive transfer of spleen T cells and ionomycin-resistant T cells (memory cells) between CBA and CBA/N mice in various combinations showed that immunological memory was not formed in CBA/N mice, but can be induced by adoptive transfer of cells from CBA mice.
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The availability and appropriate use of animal models is of significant importance for a better and more detailed understanding of the genetic, immunological and pathological mechanisms underlying the development of mycobacterial disease... more
The availability and appropriate use of animal models is of significant importance for a better and more detailed understanding of the genetic, immunological and pathological mechanisms underlying the development of mycobacterial disease in humans. To define a mouse model for tuberculosis severity that can be easily adapted to genetic and immunological analysis of host response to Mycobacterium tuberculosis infection. We describe here two inbred strains of mice, I/St and A/Sn (both Nramp1'), that differ vastly in commonly used parameters of susceptibility to infection with virulent and attenuated strains of M. tuberculosis. Following infection with a high dose of virulent H37Rv. M. tuberculosis and compared to their resistant A/Sn counterparts, I/St mice displayed more than a 2-fold shorter mean survival time and a more rapid onset and progression of severe body weight loss (cachexia). Moreover, I/St mice supported 20-100-fold higher multiplication of M. tuberculosis following c...
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I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10(3) or 10(5) CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs... more
I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10(3) or 10(5) CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44(-) CD45RB+ cells disappeared within 2 weeks postinfection, while the number of CD44(+) CD45RB-/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3(+) CD4(+) CD8(-) T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycob...
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Research Interests: Spleen, Tuberculosis, Mice, Female, Animals, and 5 moreVaccination, Phenotype, T lymphocytes, Isoniazid, and Mycobacterium bovis
The level and spectrum of antituberculous antibodies (AA) were assessed in mice and their hybrids sensitive or resistant to tuberculosis. The AA level in resistant animals (CBA) and hybrids (CBA x C57Bl/6) F1 surpassed that in the... more
The level and spectrum of antituberculous antibodies (AA) were assessed in mice and their hybrids sensitive or resistant to tuberculosis. The AA level in resistant animals (CBA) and hybrids (CBA x C57Bl/6) F1 surpassed that in the sensitive mice (C57Bl/6). The spectrum in resistant and hybrid mice appeared more varied than that in the sensitive animals. Resistant mice had bands in the range 15-20 kD.
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The paper discusses the genetic control of and the mechanisms of protective antituberculous immunity following BCG vaccination in inbred mice. The following genetic systems H-2 (B10.M)(H-2f) and B10.SM (H-2v) congenics were not protected... more
The paper discusses the genetic control of and the mechanisms of protective antituberculous immunity following BCG vaccination in inbred mice. The following genetic systems H-2 (B10.M)(H-2f) and B10.SM (H-2v) congenics were not protected with a high dose of BCG vaccine); Xid (X-linked immunodeficiency) CBA/N mice were not protected with regularly protective doses of BCG vaccine; Tbc-1 (I/St mice (Tbc-1s) were hypersensitive to tuberculosis. In two genetic systems (H-2 and xid), a distinct genetic control of susceptibility to primary tuberculosis and efficacy of BCG vaccination was demonstrated.
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Attempts have been made to find out 1/whether there is an interaction between normal killers (NK) and tumor cells transplanted in vivo and how both effectors' cytotoxicity against the third participant - NK-sensitive target cells, is... more
Attempts have been made to find out 1/whether there is an interaction between normal killers (NK) and tumor cells transplanted in vivo and how both effectors' cytotoxicity against the third participant - NK-sensitive target cells, is affected by the hypothetical interaction; and 2/the implication of H2-complex in the interaction of both the effectors. Use was made of an experimental three-component model in vitro. It included the effector cells of two types. As NK use was made of splenocytes from C57BL/6; CBA; B10; SM; B10.D2; B10.A(3R); B10.A(5R); BALB/C; A/Sn; B10.D2(R107); B.10.D2(R101) mice. Tumor effectors were EL-4 MX-11 (H2b), L-1210 (H2b) and SA-1(H2b) cells transplanted on syngeneic mice. EL-4 cells adapted in culture in vitro were used as standard target cells. Two types of the effector interaction are described. The homology of NK and tumor effector cells in the D-end of H2-complex in the I-C subregion was found to lead to a marked mutual suppression of both the parti...
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The object of the research was to examine whether tumor cell membranotoxicity as regards splenocytes depends on the protein synthesis in the latter ones and whether it depends on the similarity or differences in the subregions of... more
The object of the research was to examine whether tumor cell membranotoxicity as regards splenocytes depends on the protein synthesis in the latter ones and whether it depends on the similarity or differences in the subregions of H2-complex. As effectors the following tumor cells transplanted in syngeneic mice were used: EL-4 (H-2b), MX-II (H2b), L-1210 (H2d), SA-1 (H2a). As target cells, the splenocytes from the following mice were used: B10/Sn, C57BL/6, B10.A (3R), B10.A (5R), B10.D2, B10.SM, CBA, B10. D2 (R 101), B10.D2 (R 107). It was shown that noncoincidence of the effectors and targets as regards the genetic basis, K, IA, IB, IJ, IE and D-subregions does not lower the maximal membranotoxicity inherent in the entire syngeneic system. Noncoincidence of the effectors and targets as regards the D-terminal of H-2 complex reduces more than 2-fold the effect of tumor cells on splenocytes. Thus, the coincidence of the effectors and targets only as regards the IC-subregion of H-2 comp...
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Recombinant H-2 haplotype of mouse strain B10.D1(R108)/Y (symbol R108) obtained in experiments with skin grafting in the course of developing the CR B10.D1/Y strain (strain DBA/LacY--the donor of H-2q) was studied. Strains with... more
Recombinant H-2 haplotype of mouse strain B10.D1(R108)/Y (symbol R108) obtained in experiments with skin grafting in the course of developing the CR B10.D1/Y strain (strain DBA/LacY--the donor of H-2q) was studied. Strains with recombinant H-2 haplotypes a, h2, g1, i3, i5, i7, m, y1 were used. Alleles of different H-2 (K, I, D) regions were determined according to the presence or absence of genetic complementation in the F1 test with skin grafts. R108 recombinant was studied by serological methods with panel of anti-H-2 sera. Anti-H-2Kb (H-2.33) and anti-H-2Dq (H-2.30) monospecific antisera were used in microcytotoxicity test and in absorption experiments in vitro. It was concluded that crossing over between H-2b and H-2q chromosomes, which led to formation of recombinant H-2 haplotype of R108 mice, occurred at I region, between IA and IC subregions. The H-2 complex of R108 line has KbIAbIJ?IE?ICqSqDq alleles. bq1 symbol was proposed for the H-2 haplotype of B10.D1(R108)/Y strain.
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Genetic factors play a role in host response to infection with Mycobacterium tuberculosis, the number one infectious killer worldwide. Mice of the inbred strains I/St and A/Sn show significant differences in disease severity after... more
Genetic factors play a role in host response to infection with Mycobacterium tuberculosis, the number one infectious killer worldwide. Mice of the inbred strains I/St and A/Sn show significant differences in disease severity after intravenous injection of a lethal dose of the virulent human isolate M. tuberculosis H37Rv. Following challenge with H37Rv, only I/St mice have rapid body weight loss and short survival times. A genome wide analysis for linkage with body weight after M. tuberculosis H37Rv infection was done in (A/SnxI/St)F1xI/St mice. Among females, quantitative trait loci (QTLs) on chromosomes 9 and 3 were significantly linked to postinfection body weight (logarithm of the odds ratio [LOD] scores of 6.68 and 3.92, respectively). Suggestive linkages were found for QTLs on chromosomes 8 and 17 (LOD scores of 3.01 and 2.95, respectively). For males, QTLs on chromosomes 5 and 10 showed suggestive linkages (LOD scores of 3.03 and 2.31, respectively). These linkages can be used to identify candidate regions for tuberculosis susceptibility loci in the human genome.
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Research Interests: Survival Analysis, Biological Sciences, Infection and immunity, Virulence, Tuberculosis, and 14 moreMycobacterium tuberculosis, Mice, Female, Animals, Infection, Male, Disease Severity, Genetic linkage analysis, Quantitative Trait Loci, Body Weight, Pulmonary Tuberculosis, Chromosomes, Survival Time, and Region of Interest
We have studied the role of three mouse distinct non-H-2 genes (Bcg, Tbc-1, xid) in several phenomena of antituberculosis immunity and resistance. On the basis of median survival time (MST) of mice following infection with virulent... more
We have studied the role of three mouse distinct non-H-2 genes (Bcg, Tbc-1, xid) in several phenomena of antituberculosis immunity and resistance. On the basis of median survival time (MST) of mice following infection with virulent Mycobacterium tuberculosis H37Rv, Bcg gene did not control resistance to the lethal dose of H37Rv infection in non-vaccinated and Myco. bovis (BCG)-vaccinated mice. However, Bcgr allele, in comparison with Bcgs allele, determined more effective suppression of an early multiplication in spleens of H37Rv mycobacteria after a low dose (5x10(4) colony-forming units (CFU)) injection. CBA/N mice, which are not protected efficiently against tuberculous challenge by BCG vaccination, were characterized by a decreased in vitro proliferation of immune lymph node cells, both spontaneous and stimulated with mycobacterial antigens. The decreased proliferation was due to immunosuppression caused by interactions between responding T cells and CBA/N antigen-presenting cells (APC). We have confirmed that the defective response to BCG-vaccination in CBA/N mice is linked with the X-chromosome and thus is presumably determined by the xid gene itself. I/St mice (Tbc-1s), supersusceptible to H37Rv infection, were not able to restrict the growth of H37Rv mycobacteria in spleens, even following infection with a low dose (5x10(4)), but restricted the growth of Myco.bovis BCG more effectively than Bcgs mice.
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The 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette-Guérin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinant protein in two saprophytic... more
The 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette-Guérin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinant protein in two saprophytic mycobacteria-M. vaccae and M. smegmatis-resulted in abrogation of their ability to confer protection against M. tuberculosis in a murine challenge model, and in their ability to prime a DTH response to cross-reactive mycobacterial antigens. Induction of an immune response to the 19-kD antigen by an alternative approach of DNA vaccination had no effect on subsequent M. tuberculosis challenge. These results are consistent with a model in which the presence of the 19-kD protein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targeted inactivation of genes encoding selected antigens represents a potential route towards development of improved vaccine candidates.
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Mouse alloantiserum obtained by immunization of/DBA/2X XB10. A (3R)/F1 hybrids with thymic lymphocytes stimulated with Con A of B10.A(%) mice (anti-I-Jk) was tested in the microcytotoxic assay and in functional test of the survival of... more
Mouse alloantiserum obtained by immunization of/DBA/2X XB10. A (3R)/F1 hybrids with thymic lymphocytes stimulated with Con A of B10.A(%) mice (anti-I-Jk) was tested in the microcytotoxic assay and in functional test of the survival of mice infected with tuberculosis. Serum was found to react with population of the cells from the lymph nodes of A/Sh, A2G, B10.AKM, and A.TL (I-Jk) mice, and was not active with B10.HTT (I-Js), B10 (I-Jb), B10,D2 (R107) (I-Jb), B6-H-2bm3 (I-Jb). Administration of the antiserum to mice infected with tuberculosis made the survival of the I-Jk-bearing strains significantly longer.
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... of back cross-ing (F l • BI0-fzY)BCI, (Bcgr/s and BcgS/S). Instead of mice of line BI0 (Bcg s, +) we used Bl0-fzY (Bcg s, fzY) mice which, unlike BI0, carry the recessive mutation fzY in chromosome I, leading to a readily identified... more
... of back cross-ing (F l • BI0-fzY)BCI, (Bcgr/s and BcgS/S). Instead of mice of line BI0 (Bcg s, +) we used Bl0-fzY (Bcg s, fzY) mice which, unlike BI0, carry the recessive mutation fzY in chromosome I, leading to a readily identified disturbance of the hair cover in the homozygote. ...
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A phospholipid-based nanoemulsion formulation of SQ641 (SQ641-NE) was active against intracellular Mycobacterium tuberculosis in J774A.1 mouse macrophages, although SQ641 by itself was not. Intravenous (i.v.) SQ641-NE was cleared from... more
A phospholipid-based nanoemulsion formulation of SQ641 (SQ641-NE) was active against intracellular Mycobacterium tuberculosis in J774A.1 mouse macrophages, although SQ641 by itself was not. Intravenous (i.v.) SQ641-NE was cleared from circulation and reached peak concentrations in lung and spleen in 1 h. In a murine tuberculosis (TB) model, 8 i.v. doses of SQ641-NE at 100 mg/kg of body weight over 4 weeks caused a 1.73 log10 CFU reduction of M. tuberculosis in spleen and were generally bacteriostatic in lungs.
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New delivery vehicles and routes of delivery were developed for the capuramycin analogue SQ641. While this compound has remarkable in vitro potency against Mycobacterium tuberculosis, it has low solubility in water and poor intracellular... more
New delivery vehicles and routes of delivery were developed for the capuramycin analogue SQ641. While this compound has remarkable in vitro potency against Mycobacterium tuberculosis, it has low solubility in water and poor intracellular activity. We demonstrate here that SQ641 dissolved in the water-soluble vitamin E analogue alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) or incorporated into TPGS-micelles has significant activity in a mouse model of tuberculosis.