Chromosome analyses in man in the course of chemoprophylaxis against tuberculosis and of antituberculosis chemotherapy with isoniazid

Summary Cytogenetic analyses were carried out in lymphocytes of 37 persons before and during a primary chemoprophylaxis or a preventive chemotherapy with isoniazid as weil as of 30 patients suffering from active pulmonary tuberculosis before and during a chemotherapy with isoniazid in combination with two other antimycobacterial drugs. The results of the four participating research groups consistently reveal no indication for a chromosome damaging activity of isoniazid, of its metabolites in the human organism and of the administered drug regimens.     

Decreased resistance of multiresistant mycobacteria to isoniazid during the treatment of experimental tuberculosis with ozone and isoniazid

Mycobacteria (MB) of the clinical strain resistant to streptomycin, isoniazid (IN), rifampicin and kanamycin were injected intravenously into 68 BALB/c mice. The animals were divided into 5 groups: two control groups 0 and 1 (intact and infected without subsequent treatment), group 2 (treated with IN), group 3 (treated with IN and injected intraperitoneally with dissolved ozone, or dO3), group 4 (injected with dO3). The animals started to die by month 4 after the infection. By month 5 all mice died with the exception of intact mice and those treated with dO3). By month 4 the study of MB cultures isolated from the lungs revealed a decrease in their resistance to IN in the groups undergoing treatment with dO3. Hepatic and splenic lesions were observed after treatment with IN only were greater than in the absence of treatment. After the use of IN + dO3 such lesions were the least. The mechanism of a decrease in the medicinal resistance of MB under the action of dO3 and the expediency of the simultaneous use IN and dO3 in cases of the unknown medicinal resistance of MB are discussed.     

结核分枝杆菌对异烟肼耐药的分子机制

抗结核一线药物异烟肼是应用最广泛的抗结核药物之一,自1952年应用于临床以来,异烟肼就成了治疗结核和潜在感染的基础药物.有报道,我国异烟肼耐药已排在首位.结核分枝杆菌对异烟肼耐药的分子机制十分复杂,涉及katG、inhA、kasA、ndh、axyR等多种基因,本研究仅就此方面的研究作一综述.     

Binding of activated isoniazid with acetyl-CoA carboxylase from Mycobacterium tuberculosis

AccD6 (acetyl coenzyme A (CoA) carboxylase), plays an important role in mycolic acid synthesis of Mycobacterium tuberculosis (Mtb). Induced gene expression by isoniazid (isonicotinylhydrazine - INH), anti-tuberculosis drug) shows the expression of accD6. It is our interest to study the binding of activated INH with the AccD6 model using molecular docking procedures. The study predicts a primary binding site for activated INH (isonicotinyl acyl radical) in AccD6 as a potential target.     

Proteome-wide profiling of isoniazid targets in Mycobacterium tuberculosis

Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts [INH-NAD(P)] of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the Mycobacterium tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, 16 other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes, including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.     

Downregulation of katG expression is associated with isoniazid resistance in Mycobacterium tuberculosis

Isoniazid (INH) is a key agent in the treatment of tuberculosis. In Mycobacterium tuberculosis, INH is converted to its active form by KatG, a catalase-peroxidase, and attacks InhA, which is essential for the synthesis of mycolic acids. We sequenced furA-katG and fabG1-inhA in 108 INH-resistant (INH(r) ) and 51 INH-susceptible (INH(s) ) isolates, and found three mutations in the furA-katG intergenic region (Int(g-7a) , Int(a-10c) and Int(g-12a) ) in four of 108 INH(r) isolates (4%), and the furA(c41t) mutation with an amino acid substitution in 18 INH(r) isolates (17%). These mutations were not found in any of 51 INH(s) isolates tested. We reconstructed these mutations in isogenic strains to determine whether they conferred INH resistance. We found that the Int(g-7a) , Int(a-10c) and Int(g-12a) single mutations in the furA-katG intergenic region decreased katG expression and conferred INH resistance. In contrast, the furA(c41t) mutation was not sufficient to confer INH resistance. These results suggested that downregulation of katG is a mechanism of INH resistance in M. tuberculosis and that mutations in the furA-katG intergenic region play a role in this resistance mechanism.     

Lack of liver injury in Wistar rats treated with the combination of isoniazid and rifampicin

Isoniazid (INH) can cause serious idiosyncratic liver injury. An animal model would greatly facilitate mechanistic studies, but it is essential that the mechanism in the model be similar to the liver injury that can occur in humans. We attempted to replicate a previous study in which Wistar rats treated with INH and rifampicin (RMP) developed liver injury, which was promising because of its delayed onset similar to the liver injury that can occur in humans. Wistar rats were treated with either a high dose of INH (150 mg/kg/day) or a combination of INH and RMP (75 mg/kg/day and 50 mg/kg/day, respectively) for up to 4 weeks. However, we did not observe any liver injury or evidence of an inflammatory infiltrate as had been reported; rather, we observed an increase in CTLA4-positive cells in the cervical lymph nodes as well as a decrease in serum CXCL1 and MCP-1. In short, we were unable to reproduce a previously reported model of delayed onset INH-induced liver injury in Wistar rats.     

Characterization of the binding of isoniazid and analogues to Mycobacterium tuberculosis catalase-peroxidase

The first-line antituberculosis drug isonicotinic hydrazide (INH) is a prodrug whose bactericidal function requires activation by Mycobacterium tuberculosis catalase-peroxidase (KatG) to produce an acyl-NAD adduct. Peroxidation of INH is considered a required catalytic process for drug action. The binding of INH and a series of hydrazide analogues to resting KatG was examined using optical and calorimetric techniques to provide thermodynamic parameters, binding stoichiometries, and kinetic constants (on and off rates). This work revealed high-affinity binding of these substrates to a small fraction of ferric enzyme in a six-coordinate heme iron form, a species most likely containing a weakly bound water molecule, which accumulates during storage of the enzyme. The binding of hydrazides is associated with a large enthalpy loss (>100 kcal/mol); dissociation constants are in the range of 0.05-1.6 microM, and optical stopped-flow measurements demonstrated kon values in the range of 0.5-27 x 10(3) M-1 s-1 with very small koff rates. Binding parameters did not depend on pH in the range 5-8. High-affinity binding of INH is disrupted in two mutant enzymes bearing replacements of key distal side residues, KatG[W107F] and KatG[Y229F]. The rates of reduction of KatG Compound I by hydrazides parallel the on rates for association with the resting enzyme. In a KatG-mediated biomimetic activation assay, only isoniazid generated in good yield the acyl-NAD adduct which is considered a key molecule in INH action, providing a better understanding of the action mechanism of INH.     

Selection Practices of Bonga Sheep Reared in Southern Ethiopia

The study was conducted to assess the traits traditionally used to select the Bonga sheep reared in Southern Ethiopia. The breed was included for improvement under the community based breeding program (CBBP) launched in the year 2009. The results are based on the data collected from the project between 2009 till 2012. The findings are based on focus group discussions with the community elders who have been rearing the Bonga sheep for more than 20 years. The present findings are based on information obtained from 50% of the total respondents who were involved in CBBP. The Bonga sheep is a mutton type breed and the ewes are moderately prolific. In the past this breed of sheep were bartered to settle legal disputes and were traded against household commodities.. However, over the years (and also after the intervention of CBBP) the sheep have been selected for their body weight as well as for their distribution in many parts of Ethiopia The respondents selected the lambs at both pre weaning and post weaning stages. Traditionally the traits of rams are selected based on their body length, canon circumference, broad face, enlarged thyroid, while for the ewes, traits prolificacy, skin thickness and pelvic width are determining characters. Fat tail and brown coat color were preferred irrespective of the sexes in trait selection. The ram lambs selected for breeding purpose are locally known as “Dookoo”. These rams are selected based on some predetermined phenotypic traits and are initially selected at preweaning stage and further, again at the post weaning stage. These rams are preferentially cared and are provided with supplementary feed and comfortable housing.     

Luteolin as a potential host-directed immunotherapy adjunct to isoniazid treatment of tuberculosis

Tuberculosis (TB) remains a major health problem throughout the world with one third of the population latently infected and ~1.74 million deaths annually. Current therapy consists of multiple antibiotics and a lengthy treatment regimen, which is associated with risk for the generation of drug-resistant Mycobacterium tuberculosis variants. Therefore, alternate host directed strategies that can shorten treatment length and enhance anti-TB immunity during the treatment phase are urgently needed. Here, we show that Luteolin, a plant-derived hepatoprotective immunomodulator, when administered along with isoniazid as potential host directed therapy promotes anti-TB immunity, reduces the length of TB treatment and prevents disease relapse. Luteolin also enhances long-term anti-TB immunity by promoting central memory T cell responses. Furthermore, we found that Luteolin enhances the activities of natural killer and natural killer T cells, both of which exhibit antitubercular attributes. Therefore, the addition of Luteolin to conventional antibiotic therapy may provide a means to avoid the development of drug-resistance and to improve disease outcome.     

Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.     

The genetics and biochemistry of isoniazid resistance in mycobacterium tuberculosis

Although the primary targets of activated isoniazid (INH) are proteins involved in the biosynthesis of cell wall mycolic acids, clinical resistance is dominated by specific point mutations in katG. Mutations associated with target mutations contribute to, but still cannot completely explain, resistance to INH. Despite the wealth of genetic information currently available, the molecular mechanism of cell death induced by INH remains elusive.     

Lymphocyte adherence in multiple sclerosis: Lack of virus specificity

The virus specificity of adherence of peripheral blood mononuclear leukocytes from patients with multiple sclerosis and from age- and sex-matched healthy volunteers to tissue culture cells infected with measles virus, Newcastle disease virus, and vesicular stomatitis virus was studied. Lymphocyte adherence to uninfected cells is uniformly low (5–15% tissue culture cells with > 3 lymphocytes adhered). Adherence to cells infected with virus is enhanced 2- to 4-fold in controls and 2- to 10-fold in patients with multiple sclerosis. Virus-specific antigen, antiserum, and receptor, at least in part, inhibited adherence to all cells tested. It is concluded from these studies that increased lymphocyte adherence in multiple sclerosis is not measles virus specific.     

Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates

Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.     

Lack of adrenarche in two children with precocious puberty secondary to hypothalamic hamartoma

Adrenarche, which occurs earlier than gonadarche in normal children, is marked by increases in plasma dehydroepiandrosterone and its sulfate (DHAS). Adrenarche and gonadarche can be dissociated in various situations, e.g. central precocious puberty, indicating that they are controlled by independent mechanisms. This report concerns 2 children with central precocious puberty secondary to hypothalamic hamartoma. Their plasma basal DHAS values, compared to other cases with central precocious puberty not secondary to hamartoma, remained low for chronological age and bone age over a follow-up of 6.3 (case 1) and 9.2 9.2 years (case 2): in case 1 (boy), DHAS was 9 micrograms/dl at chronological age 7.7 and bone age 13 years; in case 2 (girl), DHAS was 11 micrograms/dl for chronological age 10.5 and bone age 13.5 years. GH secretion was normal. Basal plasma cortisol levels as the levels during hypoglycemia and after corticotropin stimulation were all normal. These data suggest that hypothalamic hamartoma may affect the central control of adrenarche. They may also contribute to the diagnosis of hypothalamic hamartoma.