ABSTRACT
Mutations at codons 526 and 531 in the rpoB gene and at 315 in the katG gene are considered diagnostic markers for resistance to rifampin and isoniazid in tuberculosis. The aim of this study was to design and evaluate three TaqMan probes for the identification of these mutations in 138 respiratory samples positive for acid-fast bacilli, and 32 clinical isolates from a region with considerable levels of drug resistance. The specificities of the probes for the diagnosis of resistance to both drugs were 100%; however, the sensitivities were calculated to be 50% for isoniazid and 56% for rifampin. DNA sequencing of rpoB and katG; and the spoligotyping assay of the clinical isolates, confirmed the diversity of the mutations and the presence of 11 spoligotypes with a shared international type and eight unique spoligotypes. Analysis of the respiratory samples identified 22 (16%) as drug-resistant and 4 (3%) as multidrug-resistant tuberculosis. The diagnostic value of the TaqMan probes was compromised by the diversity of mutations found in the clinical isolates. This highlights the need for better understanding of the molecular mechanisms responsible for drug resistance prior to the use of molecular probes, especially in regions with significant levels of drug-resistant tuberculosis.
Subject(s)
Isoniazid/pharmacology , Molecular Diagnostic Techniques , Mycobacterium tuberculosis/drug effects , Rifampin/pharmacology , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis/diagnosis , Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Catalase/genetics , DNA-Directed RNA Polymerases , Genetic Variation , Humans , Mexico , Molecular Diagnostic Techniques/standards , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Sensitivity and Specificity , Sequence Analysis, DNA , Tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/geneticsABSTRACT
Previous in vitro studies have demonstrated that mast cells (MC) can be directly activated by Trichinella spiralis larvae 1 (TSL-1) antigens. To characterize even more this activation of MC and their possible role on induction and regulation of the Type 2 response generated against T. spiralis infection, we studied the interaction between a hybrid rat MC line (HRMC), murine bone marrow MC (mBMMC), and TSL-1 antigens. Immunofluorescence staining and flow cytometry analysis showed that TSL-1 antigens bound to the surface of HRMC cells, resulting in the transcriptional induction and in the release of TNF and IL-4. Besides, an increase of IL-4 intracellular expression was also observed in mBMMC. This suggests that MC may play an important role in the early immune response against T. spiralis and may be a source of cytokines, that regulate the final onset of the immune mechanisms which determine the course of the infection.
Subject(s)
Antigens, Helminth/immunology , Interleukin-4/metabolism , Mast Cells/immunology , Trichinella spiralis/immunology , Tumor Necrosis Factor-alpha/metabolism , Animals , Cell Line , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation , Interleukin-4/genetics , Larva/immunology , Mast Cells/parasitology , Mice , Mice, Inbred BALB C , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with anti-inflammatory properties that, among others, inhibits the in vitro and in vivo locomotion of human monocytes, sparing polymorphonuclear leucocytes from this effect [hence the name originally given. Monocyte Locomotion Inhibitory Factor (MLIF)]. A synthetic construct of this peptide displays the same effects as the native material. We now added MLIF to resting and PMA-stimulated cells of a human monocyte cell line and measured the effect upon mRNA and protein expression of pro-inflammatory chemokines (RANTES, IP-10, MIP-1alpha, MIP-1beta, MCP-1, IL-8, I-309 and lymphotactin) and the shared CC receptor repertoire. The constitutive expression of these chemokines and the CC receptors was unaffected, whereas induced expression of MIP-1alpha, MIP-1beta, and I-309, and that of the CCR1 receptor--all involved in monocyte chemotaxis--was significantly inhibited by MLIF. This suggests that the inhibition of monocyte functions by MLIF may not only be exerted directly on these cells, but also--and perhaps foremost--through a conglomerate down-regulation of endogenous pro-inflammatory chemokines.
Subject(s)
Cytokines/biosynthesis , Entamoeba histolytica/immunology , Oligopeptides/immunology , Animals , Chemokine CCL1 , Chemokine CCL3 , Chemokine CCL4 , Chemokines, CC/immunology , Chemotactic Factors/immunology , Cytokines/genetics , Cytokines/immunology , Down-Regulation/immunology , Entamoeba histolytica/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Lymphocyte Activation/immunology , Macrophage Inflammatory Proteins/immunology , Oligopeptides/metabolism , RNA/chemistry , RNA/genetics , Receptors, Chemokine/immunology , Reverse Transcriptase Polymerase Chain Reaction , Tetradecanoylphorbol Acetate/immunology , U937 CellsABSTRACT
Neurocysticercosis is a common problem in developing countries, and it causes neurologic disorders in children. Immunodiagnosis with Taenia solium glycoproteins as an antigen has been validated in adults but not in children. The aim of this work was to evaluate a Taenia solium glycoproteins-based enzyme-linked immunoelectrotransfer blot assay in children with neurocysticercosis. Twenty-five confirmed cases of neurocysticercosis and 50 healthy children from the same community were included. The test had a sensitivity of 72% and a specificity of 96%. Sensitivity was higher (100%) in cases with multiple cysts and in multiple sites. Sensitivity was higher when cysts were in parenchyma (86%) than when they were in the subarachnoid space. The most frequently recognized proteins were 24, 39 to 42, and 50 kDa. Diagnosis was more efficient in serum than in cerebrospinal fluid. Western blot is a reliable method for serologic diagnosis of neurocysticercosis in children. Multiple cysts and infections in multiple sites elicited a stronger immune response.
