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June 1, 2005
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June 1, 2005
Abstract
Tuberculosis, caused by the human pathogen Mycobacterium tuberculosis (M. tuberculosis) , affects an estimated 8 million people annually, resulting in approximately 2 million deaths. Human genetic variability is an important modulator of tuberculosis susceptibility. This review will discuss candidate susceptibility genes that have been implicated in tuberculosis susceptibility across various ethnic groups and epidemiological settings. Evaluating the genetic variants of tuberculosis susceptibility genes will provide us with a better understanding of the disease mechanisms in tuberculosis. Ultimately, such genetic studies may lead to the development of effective alternative treatments to cope with the growing problem of tuberculosis infections due to the AIDS pandemic, the emergence of multidrug resistant M. tuberculosis , and the limited efficacy of Mycobacterium bovis (BCG) vaccination.
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June 1, 2005
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Different phenotypes are displayed by Mycobacterium tuberculosis (M. tuberculosis) strains, fuelling speculation that certain strains are “hypervirulent” and able to evade host defenses better than others. Furthermore, differential antigen expression by M. tuberculosis strains may explain why certain patients are susceptible to a repeat episode of tuberculosis. The objective of this study was to compare protein expression by M. tuberculosis H37Rv and clinical isolates in order to determine whether differential protein expression contributes to the different phenotypes expressed by these strains. Expression of α-crystallin, the antigen 85 complex, PstS-1, L-alanine dehydrogenase and the 65 kDa antigen was analysed by Western blotting and enzyme-linked immunosorbent assays, using mouse monoclonal antibodies. We found no significant difference in the growth rate of the M. tuberculosis strains in vitro , and although M. tuberculosis protein expression showed phase variation during growth, expression seemed to be qualitatively, but not quantitatively, conserved in the strains investigated. These results have potentially important implications for vaccine development and serodiagnosis.
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June 1, 2005
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During the early development of drug resistance in Mycobacterium tuberculosis (M. tuberculosis) infection only a small proportion of resistant bacteria are present within a milieu of sensitive bacteria. This complicates the use of molecular methods to predict the presence of a resistant phenotype and has been largely ignored in many of the newly developed molecular methods. In this study, mixtures of DNA from M. tuberculosis strains with known wild-type and mutant sequences were used to evaluate the sensitivity of three different molecular methods for detection of drug resistance. The dot-blot and amplification refractory mutation system (ARMS) methods showed sensitivities that approach those of routine phenotypic methods and are able to detect the presence of mutant sequences at a ratio of 1 in 50 (corresponding to 2% mutant sequences). This is 10-fold more sensitive than the commercial kit. The ARMS method was also used to investigate the use of molecular methods to identify mixed infections, and both drug-resistant and susceptible strain populations were identified in a single clinical isolate. These findings highlight the applicability of molecular methods to the rapid detection of drug resistance in tuberculosis patients, particularly in those who are non-compliant and in contacts of known drug-resistant tuberculosis patients, and assistance in limiting the spread of drug-resistant strains.
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June 1, 2005
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Isolation and purification of mycolic acids from Mycobacterium tuberculosis have allowed them to be applied as antigen in an ELISA-based assay to detect specific antibodies in human sera. Tuberculosis patients have previously been shown to contain anti-mycolic acids antibodies. The aim of this study was to determine whether human immunodeficiency virus (HIV) co-infection increases false-negative testing rate and whether other random diseases for which hospitalisation is normally required will contribute to false-positive results. Sera from 118 human subjects were tested for the presence of antibodies to mycolic acids; 59 were patients with proven pulmonary tuberculosis and 59 were control hospitalised patients without evidence of tuberculosis. Each group consisted of HIV-seropositive and HIV-seronegative subjects. The endpoint was the detection of specific antibodies to mycolic acids in the sera, before and after precipitation of immune complexes. The two groups of subjects were well matched for age, gender, race and HIV status. On average, humans infected with Mycobacterium tuberculosis showed a specific antibody response to mycolic acids that was not affected by low CD4 T-lymphocyte counts in HIV-seropositive patients but was compromised by various other serious diseases.
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June 1, 2005
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Our laboratory, engaged in a prospective study of adult pulmonary tuberculosis, processed on average 1186 sputum samples per year for the detection of Mycobacterium tuberculosis (M. tuberculosis) . Approximately 55% of all sputum samples were culture-positive. The study protocol required that all patients had their M. tuberculosis isolates DNA fingerprinted at diagnosis, and at subsequent time points if the patients either failed treatment or presented again with tuberculosis. Over a 22-month period, there were 14 apparent treatment failures from 109 patients who had completed 6 months of therapy. Only two of these were true treatment failures, while the other 12 had DNA fingerprints that were different from those obtained at diagnosis. It was concluded that these 12 cultures represented episodes of laboratory cross-contamination. Retrospective DNA fingerprinting of patient isolates was done so that each patient had at least two independent isolates fingerprinted. This survey revealed that 7.3% of DNA fingerprints were discordant. False-positive cultures with discordant DNA fingerprints generally arose late in chemotherapy and the isolates were usually co-processed with other strongly smear-positive sputum samples. Simple modifications of laboratory procedures were made, and over a following 10.5-month period the false-positive rate was reduced to 2.1%. These modifications did not increase the workload or the cost of processing samples and can thus be used successfully by any laboratory, and particularly by those in resource-poor settings.
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June 1, 2005
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The role of the endogenous interleukin-10 (IL-10) in the control of Mycobacterium bovis Bacille Calmette Guerin (BCG) infection was assessed using IL-10-deficient (IL-10 -/- )mice. Similar to wild-type (WT) mice, IL-10 -/- mice were resistant to intravenous challenge with Mycobacterium bovis BCG. Significantly higher plasma concentrations of IL-12 and tumour necrosis factor (TNF) indicated an elevated protective immune response of IL-10 -/- mice. Determination of bacilli burden in IL-10 -/- mice showed accelerated clearance in the lungs, spleen and the liver in comparison to WT mice. Enhanced inflammation and a vigorous granulomatous response accompanied accelerated mycobacterial clearance. Immunohistochemical analysis of hepatic granulomas from IL-10 -/- mice revealed augmented lymphocyte recruitment and macrophage activation, such as increased major histocompatibility complex (MHC) class II and inducible nitric oxide synthase (iNOS) expression. Further, it was found that enlarged granulomas persisted subsequent to mycobacterial clearance and failed to resolve in the absence of IL-10. In conclusion, endogenous IL-10 dampens the cell-mediated immune response to mycobacterial infection.
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June 1, 2005
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Characterization of human leukocyte antigen (HLA) class I restricted epitopes derived from viral pathogens is imperative for formulating therapeutic interventions, as well as for vaccine design and monitoring. Sensitive, easy and cost-effective assays that measure the frequency of antigen-specific T lymphocytes are crucial for evaluating and improving vaccines and therapies. This paper reviews the ELISPOT technique that allows for quantifying HIV-specific T lymphocytes at the single cell level from peripheral blood by detection of antigen-induced cytokine secretion. The assay can be used successfully to quantify T cell immune responses in humans infected with different pathogens and to assess T cell immunogenicity of vaccines in phase I/II and III clinical trials. This review focuses on the ELISPOT methodology and discusses how it can be standardized and potentially used by multiple international laboratories attached to clinical trial sites.
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June 1, 2005
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The discovery of chemokine receptors as HIV-1 entry molecules or “coreceptors” has lead to a greater understanding of how HIV-1 infects human cells. This has provided insight into the biological properties of HIV-1 isolates and unravelled the meaning of the syncytium-inducing and non-syncytium-inducing phenotypes. Understanding how HIV-1 exploits these coreceptors has given way to novel approaches to controlling HIV. As a result a new class of drugs has emerged that are being tested to prevent virus infection and to act as an alternative, or adjunct, to existing anti-retroviral drugs for HIV-infected individuals.
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June 1, 2005
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Paclitaxel (Taxol ® ) and docetaxel (Taxotère ® ) are currently two of the most important anticancer drugs in cancer chemotherapy. However, clinical treatment with these taxane agents often encounters undesirable side effects and multidrug resistance (MDR) caused by overexpression of P-glycoprotein (Pgp). Photoaffinity labeling of Pgp using photoreactive radiolabeled paclitaxel analogs along with molecular modeling has revealed a unique binding region for paclitaxel on the C-terminal half of Pgp. Highly efficient taxane-based MDR reversal agents (TRAs) have been developed. Extensive structure-activity relationship (SAR) studies have led to the development of new generation taxanes that possess 2–3 orders of magnitude higher potencies against human cancer cell lines expressing the MDR phenotype. One of these taxanes, SB-T-110131 (IDN5109, BAY59-8862), exhibits excellent activity against a variety of drug-sensitive and drug-resistant cancer cell lines as well as human tumor xenografts in mice. This taxane is orally active with excellent bioavailability, and is currently undergoing phase II human clinical trials. Novel taxane-antibody immunoconjugates have shown very promising results for tumor-specific delivery and release of an extremely cytotoxic taxane, wherein epidermal growth factor receptor is used as the specific antigen on the tumor surface of human squamous cancer xenograft in SCID mice.
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June 1, 2005
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Microsatellite typing is frequently used in disease diagnosis and in genetic association studies. Inter-study consistency and comparability is essential in both applications. In this study, we show that the interlaboratory comparison of microsatellite sizes is often discrepant and misleading. This is a matter of great concern in the recent literature. However, accurate allele designation is easily attainable by the simple procedures we report, which are applicable to all gel-based genotyping methods. These involve: 1) the creation of dedicated standards for a specific microsatellite by PCR-amplifying representative alleles to generate an allelic ladder with comparable electrophoretic characteristics; 2) including both internal and external standards during electrophoresis to facilitate alignment. In addition, we recommend procedures that will improve inter-study comparability of all microsatellite analyses regardless of genotyping method. These involve: 1) cloning and sequencing representative microsatellite alleles to obtain accurate size designation; 2) sharing alleles of known sequence between laboratories to use as standards. We report on the typing of natural resistance-associated macrophage protein ( NRAMP2 ) and interferon-γ ( IFNG ) gene microsatellites as examples, the latter of which is crucial to many pathogenic processes. We describe in detail the varying allele sizes obtained by different methods, which prevent meaningful inter-study comparisons.
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June 1, 2005
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South African blacks rarely form kidney stones compared with whites. This study investigated whether purified urinary prothrombin fragment 1 (UPTF1) derived from blacks is a more potent inhibitor of calcium oxalate crystallisation than that from whites. UPTF1 was purified from the urine of both population groups and their inhibitory activities were compared in a cross-over design in which each protein was tested in ultrafiltered urine from both population groups. Coulter Multisizer, [ 14 C]-oxalate deposition and scanning electron microscopy experiments were used to monitor crystallisation. The study has demonstrated for the first time that UPTF1 promotes nucleation and that inhibitory activity is synergistically dependent upon urine composition. The activity of the whites' UPTF1 was greater than that of the blacks in the whites' urine ( e.g. particle size decrease: 31.7% vs. 25.2%), while the blacks' UPTF1 was superior to that of the whites in the blacks' urine ( e.g. particle size decrease: 46.5% vs. 32.4%). In addition, when tested in their respective endogenous urines, the blacks' UPTF1 demonstrated superior inhibitory activity on an absolute scale ( e.g. particle size decrease: 46.5% vs. 31.7%). Thus, the urine composition of black South Africans may influence their UPTF1 conformation, conferring greater efficacy for inhibition of calcium oxalate crystallisation.
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June 1, 2005
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We conducted a cross-sectional study in Bolifamba village in the South West Province of Cameroon to determine antibody responses to crude Plasmodium falciparum antigens. A total of 347 subjects were examined. Parasite counts were obtained on thick blood films stained with Field's stain. Total immunoglobulin (Ig)G and IgG subclass levels were determined in serum samples from four groups comprising children 1 to 5 years old and adults ≥18 years with or without falciparum malaria parasites, using enzyme-linked immunosorbent assay with crude blood-stage antigens of Plasmodium falciparum strain F32 as target. Depending on the age group, malaria prevalence varied between 10% and 65% with a mean of 30.8%. Prevalence rate and parasite density declined with increasing age. Total IgG and IgG1-3 levels were significantly higher in adults than in children (p<0.05). Parasite-bearing individuals in both age groups had higher IgG titres than their non-infected counterparts, while subtype levels were not significantly different (p=0.05). These findings indicate that Bolifamba village could be a convenient site to study further the protective immunity to malaria.
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June 1, 2005
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Oesophageal squamous cell cancer is the leading cause of cancer death amongst African males in South Africa. DNA was isolated from normal and tumour biopsies of the oesophagi of 33 African patients with squamous cell carcinoma of the oesophagus and was analysed with two dinucleotide repeat polymorphisms, a GT repeat sequence in the first intron, and a CA repeat in the promoter of the human α 2 (I) procollagen gene (COL1A2) , using the polymerase chain reaction (PCR). Normal and tumour DNAs from each individual were compared to identify changes present in the tumour DNA, but absent in normal DNA. Twenty two cases were informative (heterozygous) for the promoter polymorphism and 24 cases were informative for the intronic polymorphism. Loss of heterozygosity (LOH) was seen in 2/22 (9.1%) for the promoter and 3/24 (12.5%) for the intronic polymorphism. These changes involved a total of three patients: two patients displayed the lost allele incorporating both the CA repeat and GT repeat loci; the third patient revealed LOH at the intronic polymorphism, but was non-informative (homozygous) for the promoter polymorphism. Deletions within the procollagen genes may represent an as yet unrecognised but rare event in the multistep process of carcinogenesis.
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June 1, 2005
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Zearalenone (6-[10-hydroxy-6-oxo-trans-1-undecenyl]-β-resorcylic acid-μ-lactone), a mycotoxin produced in corn, is able to adopt a conformation which sufficiently resembles 17β-oestradiol to allow it to bind to the oestrogen receptor in target cells of the body and exert (agonist) oestrogenic action. We adopted an analytical approach to isolate and quantify the mycotoxin and its derivatives using high-performance liquid chromatography and gas chromatography-mass spectrometry. In this study, the quantity of zearalenone and its congeners (α-zearalenol and β-zearalenol) present in the plasma of patients with breast (n=28) and cervical carcinoma (n=54) were compared with levels in patients presenting with other diagnoses (n=26) and healthy volunteers (n=24). There were no significant differences between the groups in the levels of zearalenone and its congeners, using analysis of covariance (0.2<p<0.6). These results suggest that the presence of this mycotoxin in blood does not indicate causal relationship between exposure to this exogenous myco-oestrogen and the subsequent biological effect in our study population but may be used as an indicator of exposure. The presence of zearalenone in the study groups is, however, of growing concern, due to the possible effects of cumulative long-term exposure of oestrogenic target organs to this compound.
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June 1, 2005
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The co-ordinate expression and regulation of the drug metabolising enzymes, cytochrome P4501A1 (CYP1A1) and glutathione transferases (GSTM1, GSTT1 and GSTP1), and their metabolic balance in the cells of target organs may determine whether exposure to carcinogens results in cancer. Besides showing variability in activity due to induction and inhibition, these enzymes also exhibit genetic polymorphism that alter enzyme levels and activity. We determined frequencies of common allelic variants of CYP1A1 and glutathione ( M1 , T1 and P1) among Tanzanians, South African Venda and Zimbabweans using PCR/restriction fragment length polymorphism techniques. The CYP1A1 Val 462 mutant variant was found at a frequency of 1.3% among 114 subjects. The GSTM1*0 genotype was found at a frequency of 29% and 33% among Tanzanian psychiatric patients and healthy volunteers, respectively. Similarly, the GSTT1*0 polymorphism was present with a frequency of 25% in both the psychiatric patients and healthy controls. The frequency of GSTP1 Val 105 variant was 16%, 12% and 21% among Tanzanians, South African Venda and Zimbabweans, respectively. We conclude here that CYP1A1 Val 462 polymorphism is very rare among Africans. This is the first report of the GSTP1 Val 105 variant frequency in African populations. We show here that there are no differences in frequencies of the variant alleles for CYP1A1, GSTM1, GSTT1 and GSTP1 in the three African populations.
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July 27, 2005