ABSTRACT
The correlation of the Beijing/W and LAM strains with multiple drug resistance was studied. The transmissibility of the multidrug-resistant strains of these families was demonstrated.
Subject(s)
Drug Resistance, Multiple, Bacterial/genetics , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/genetics , Tuberculosis, Multidrug-Resistant/transmission , Tuberculosis, Pulmonary/genetics , Tuberculosis, Pulmonary/transmission , Genotype , Humans , Mycobacterium tuberculosis/isolation & purification , Prisoners , RussiaABSTRACT
Above two million people annually die worldwide of tuberculosis. There has been a serious uptrend in the tuberculosis morbidity observed during the recent decade in Russia. The growing prevalence of drug-resistant strains of pathogens is highly dangerous. A timely diagnosis reduces significantly the risk of epidemics. The use of molecular methods cuts the time of tuberculosis diagnosis, including its resistant forms, from several weeks to several days. The presented survey contains a discussion of modem methods used for identification of Mycobacterium tuberculosis resistance to the main anti-TB drugs.
Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Alleles , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Humans , Mutation , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Hybridization , Polymerase Chain Reaction , Sequence Analysis , Tuberculosis/drug therapyABSTRACT
Efficient system for producing the recombinant Fab of DNA-hydrolyzing antibody BV04-01 in metylotrophic yeast Pichia pastoris was developed. Addition of peptides encompassing the Jun-Fos leucine zipper at the C-termini of the antibody chains facilitated the in vivo assembling of the Fab. The yield of secreted functionally active BV04-01 Fab was about 3 mg/L. Catalytic efficiency of supercoiled DNA hydrolysis by the Fab obtained was 1.8 x 10(6) M(-1) min(-1).
Subject(s)
DNA, Fungal/metabolism , Immunoglobulin Fab Fragments/immunology , Pichia/genetics , Base Sequence , DNA Primers , DNA, Fungal/immunology , Hydrolysis , Recombinant Proteins/immunologyABSTRACT
Cytotoxicity of anti-DNA autoantibodies from sera of SLE and CLL patients was assayed on permanent cell lines L929, HL-60, Raji, and K562. L929 cells appeared to be the most sensitive to antibody treatment. DNA-hydrolyzing properties of the same autoantibody preparations were analyzed in parallel. The data obtained outlined the correlation between cytotoxicity and DNA-hydrolyzing properties of these autoantibodies. It was shown that treatment of the cells with cytotoxic anti-DNA autoantibodies induced internucleosomal DNA fragmentation and Annexin V binding to the cell surface characteristic of apoptotic pathway of cell death. A time-dependent profile of antibody-mediated toxicity to L929 cells suggested recruitment of at least two distinct mechanisms of cell death. The first peak of cell death observed in 3 h of incubation was completely inhibited by preincubation of cells with caspase inhibitor YVAD-CHO, while the second increase in cell mortality (18-30 h) persisted. Possible mechanisms for anti-DNA autoantibody cytotoxicity are discussed.
Subject(s)
Antibodies, Antinuclear/immunology , Antibodies, Antinuclear/toxicity , DNA/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Lupus Erythematosus, Systemic/immunology , Caspase Inhibitors , Caspases/metabolism , Cell Death/drug effects , DNA/metabolism , DNA Fragmentation/drug effects , Flow Cytometry , Humans , Hydrolysis/drug effects , Immune Sera/immunology , Immune Sera/toxicity , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/toxicity , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Lupus Erythematosus, Systemic/genetics , Time Factors , Tumor Cells, CulturedABSTRACT
The cytotoxicity of DNA-specific autoantibodies from sera of patients with systemic lupus erythematosis (SLE) and with lymphoproliferative diseases, and from blood of healthy donors was examined on tumor-cell lines L929 and HL-60. DNA-binding IgG fractions from SLE and chronic lymphocytic leukemia (CLL) sera were cytotoxic at concentrations of up to 10(-10) M. No detectable changes in cell viability were observed after incubation with antibodies devoid of DNA-binding activity and DNA-specific antibodies isolated from blood of healthy donors and patients with T-cell lymphoma, B-cell lymphosarcoma, and acute B-cell leukemia. There was good correlation between the cytotoxic activity and DNA-hydrolyzing activity of anti-DNA antibodies. The cytotoxic effect of DNA-binding antibodies presumably was complement-independent, because it was attributed only to the Fab fragment. The cytotoxic effect was completely inhibited by preincubation with double-stranded DNA (dsDNA). Both the cytotoxic effect and the DNA-hydrolyzing activity of anti-DNA antibodies were significantly increased in the antibody fraction that displayed cross-reactivity with nuclear matrix proteins. Possible mechanisms for the formation and pathogenicity of cytotoxic anti-DNA antibodies are discussed in this article.
