Twice-weekly, directly observed treatment for HIV-infected and uninfected tuberculosis patients: cohort study in rural South Africa

Objective: To determine the effectiveness of twice-weekly directly observed therapy (DOT) for tuberculosis (TB) in HIV-infected and uninfected patients, irrespective of their previous treatment history. Also to determine the predictive value of 2-3 month smears on treatment outcome. Methods: Four hundred and sixteen new and 113 previously treated adults with culture positive pulmonary TB (58% HIV infected, 9% combined drug resistance) in Hlabisa, South Africa. Daily isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E) given in hospital (median 17 days), followed by HRZE twice a week to 2 months and HR twice a week to 6 months in the community. Results: Outcomes at 6 months among the 416 new patients were: transferred out 2%; interrupted treatment 17%; completed treatment 3%; failure 2%; and cured 71%. Outcomes were similar among HIV-infected and uninfected patients except for death (6 versus 2%; P = 0.03). Cure was frequent among adherent HIV-infected (97%; 95% CI 94-99%) and uninfected (96%; 95% CI 92-99%) new patients. Outcomes were similar among previously treated and new patients, except for death (11 versus 4%; P = 0.01), and cure among adherent previously treated patients 97% (95% CI 92-99%) was high. Smear results at 2 months did not predict the final outcome. Conclusion: A twice-weekly rifampicin-containing drug regimen given under DOT cures most adherent patients irrespective of HIV status and previous treatment history. The 2 month smear may be safely omitted. Relapse rates need to be determined, and an improved system of keeping treatment interrupters on therapy is needed. Simplified TB treatment may aid implementation of the DOTS strategy in settings with high TB caseloads secondary to the HIV epidemic. (C) 1999 Lippincott Williams & Wilkins.

Emergence of multidrug-resistant tuberculosis in a community-based directly observed treatment programme in rural South Africa

OBJECTIVE: Although little studied in developing countries, multidrug-resistant tuberculosis (MDR-TB) is considered a major threat. We report the molecular epidemiology, clinical features and outcome of an emerging MDR-TB epidemic. METHODS: In 1996 all tuberculosis suspects in the rural Hlabisa district, South Africa, had sputum cultured, and drug susceptibility patterns of mycobacterial isolates were determined. Isolates with MDR-TB (resistant to both isoniazid and rifampicin) were DNA fingerprinted by restriction fragment length polymorphism (RFLP) using IS6110 and polymorphic guanine-cytosine-rich sequence-based (PGRS) probes. Patients with MDR-TB were traced to determine outcome. Data were compared with results from a survey of drug susceptibility done in 1994. RESULTS: The rate of MDR-TB among smear-positive patients increased six-fold from 0.36% (1/275) in 1994 to 2.3% (13/561) in 1996 (P = 0.04). A further eight smear-negative cases were identified in 1996 from culture, six of whom had not been diagnosed with tuberculosis. MDR disease was clinically suspected in only five of the 21 cases (24%). Prevalence of primary and acquired MDR-TB was 1.8% and 4.1%, respectively. Twelve MDR-TB cases (67%) were in five RFLP-defined clusters. Among 20 traced patients, 10 (50%) had died, five had active disease (25%) and five (25%) were apparently cured. CONCLUSIONS: The rate of MDR-TB has risen rapidly in Hlabisa, apparently due to both reactivation disease and recent transmission. Many patients were not diagnosed with tuberculosis and many were not suspected of drug-resistant disease, and outcome was poor.

Synthesis of [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)] (PF6)(2), a hydroxo-bridged copper(II) complex of N,N '-dimethyl-1-thia-4,7-diazacyclononane (Me-2[9]aneN(2)S). X-ray structural analysis, magnetic susceptibility, EPR and visible spectroscopy

The bis(mu-hydroxo) complex [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)](PF6)(2) (Me-2[9]aneN(2)S = N,N'-dimethyl-1-thia-4,7-diazacyclononane) results after reaction of [Cu(Me-2[9]aneN(2)S)(MeCN)] (PF6) with dioxygen at -78 degrees C in acetonitrile. The complex has been characterized by X-ray crystallography: orthorhombic, space group Pnma, with a 18.710(3), b 16.758(2), c 9.593(2) Angstrom, and Z = 4. The structure refined to a final R value of 0.051. The complex contains two copper(II) ions bridged by two hydroxo groups with Cu ... Cu 2.866(1) Angstrom. The solid-state magnetic susceptibility study reveals ferromagnetic coupling, the fitting parameters being J = +46+/-5 cm(-1), g = 2.01+/-0.01 and theta = -0.58+/-0.03 K. The frozen-solution e.p.r. spectrum in dimethyl sulfoxide is characteristic of a monomeric copper(II) ion (g(parallel to) 2.300, g(perpendicular to) 2.063; A(parallel to) 156.2 x 10(-4) cm(-1), A(perpendicular to) 9.0 x 10(-4) cm(-1)) with an N2O2 donor set. Thioether coordination to the copper(II) in solution is supported by the presence of an intense absorption assigned to a sigma(S)-->Cu-II LMCT transition at c. 34000 cm(-1). The single-crystal spectrum of [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)] (PF6)(2) (273 K) reveals d-->d transitions at 14500 and 18300 cm(-1) and a weak pi(S)-->Cu-II charge-transfer band at approximately 25000 cm(-1).

Who fails to complete tuberculosis treatment? Temporal trends and risk factors for treatment interruption in a community-based directly observed therapy programme in a rural district of South Africa

SETTING: Hlabisa Tuberculosis Programme, Hlabisa, South Africa. OBJECTIVE: To determine trends in and risk factors for interruption of tuberculosis treatment. METHODS: Data were extracted from the control programme database starting in 1991. Temporal trends in treatment interruption are described; independent risk factors for treatment interruption were determined with a multiple logistic regression model, and Kaplan-Meier survival curves for treatment interruption were constructed for patients treated in 1994-1995. RESULTS: Overall 629 of 3610 surviving patients (17%) failed to complete treatment; this proportion increased from 11% (n = 79) in 1991/1992 to 22% (n = 201) in 1996. Independent risk factors for treatment interruption were diagnosis between 1994-1996 compared with 1991-1393 (odds ratio [OR] 1.9, 95% confidence interval [CT] 1.6-2.4); human immunodeficiency virus (HIV) positivity compared with HIV negativity (OR 1.8, 95% CI 1.4-2.4); supervised by village clinic compared with community health worker (OR 1.9, 95% CI 1.4-2.6); and male versus female sex (OR 1.3, 95% CI 1.1-1.6). Few patients interrupted treatment during the first 2 weeks, and the treatment interruption rate thereafter was constant at 1% per 14 days. CONCLUSIONS: Frequency of treatment interruption from this programme has increased recently. The strongest risk factor was year of diagnosis, perhaps reflecting the impact of an increased caseload on programme performance. Ensuring adherence to therapy in communities with a high level of migration remains a challenge even within community-based directly observed therapy programmes.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.

Glucocorticoid receptors in human preadipocytes: regional and gender differences

Glucocorticoid excess causes visceral obesity and its accompanying insulin resistance, dyslipidemia and hypertension. Glucocorticoids enhance preadipocyte (PA) differentiation and increase their aromatase activity (oestrogen production) and there is regional variability in these PA processes. Therefore, we studied human PAs for the presence of, and any regional or gender differences in, glucocorticoid receptors (GRs). Confluent subcultured human subcutaneous (Sc) and visceral (Vis) PAs from both genders contained GRs as assessed by GR gene expression and specific glucocorticoid (dexamethasone) binding. The dissociation constant was similar to that of other human cells and there was no difference between Sc and Vis sites or between males and females. There was significantly less GR mRNA in Vis PAs compared with Sc PAs in females (P=0.008) but not in males. There was less glucocorticoid binding in Vis compared with Sc PAs in females, measured by maximal binding capacity (P=0.035) or single saturating dose glucocorticoid binding (Bssd) (P=0.019). There was no regional difference in specific glucocorticoid binding in males. There was a gender difference with fewer GRs in Vis PAs in females compared with males measured by Bssd (P=0.006). In summary, GRs are present in human PAs. There is a lower GR density in Vis compared with Sc PAs in females, and females have fewer GRs in Vis PAs compared with males. These differences are likely to affect regional aromatase activity and to contribute to the smaller visceral fat mass in females compared with males.

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial polytope minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class 1 alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems. (C) 2000 Academic Press.

Seeing is believing: Recent trends in the measurement of Ca2+ in subcellular domains and intracellular organelles

The role of Ca2+ in the regulation of the cell cycle has been investigated mostly in studies assessing global cytosolic free Ca2+. Recent studies, however, have used unique techniques to assess Ca2+ in subcellular organelles, such as mitochondria, and in discrete regions of the cytoplasm. These studies have used advanced fluorescence digital imaging techniques and Ca2+-sensitive fluorescence probes, and/or targeting of Ca2+-sensitive proteins to intracellular organelles. The present review describes the results of some of these studies and the techniques used. The novel techniques used to measure Ca2+ in microdomains and intracellular organelles are likely to be of great use in future investigations assessing Ca2+ homeostasis during the cell cycle.

PMCA1 mRNA expression in rat aortic myocytes: A real-time RT-PCR study

The plasmalemmal Ca2+ adenosine triphosphatase (PMCA) is a key regulator of Ca2+ efflux in vascular smooth muscle. In these studies are developed a realtime reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay for assessing PMCA1 mRNA levels in rat primary cultured aortic myocytes. This assay detected fetal bovine serum-induced increases in PMCA1 mRNA (relative to 18S rRNA) 4, 8, and 24 h after stimulation. Early fetal bovine serum-induced increases in PMCA1 mRNA were insensitive to the Ca2+ channel blockers nifedipine, flunarizine, and SKF-96365. These studies demonstrate the feasibility of real-time RT-PCR to assess mRNA levels of PMCA1 and illustrate dynamic regulation of this Ca2+ pump isoform in rat primary cultured aortic myocytes, (C) 2000 Academic Press.

Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas

Purpose: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. Methods: Allelic loss at CyP40 was determined from patients' matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. Results: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. Conclusions: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.

Neuronal sodium-channel alpha 1-subunit mutations in generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome characterized by the presence of febrile and afebrile seizures. The first gene, GEFS1, was mapped to chromosome 19q and was identified as the sodium-channel beta1-subunit, SCN1B. A second locus on chromosome 2q, GEFS2, was recently identified as the sodium-channel alpha1-subunit, SCN1A. Single-stranded conformation analysis (SSCA) of SCN1A was performed in 53 unrelated index cases to estimate the frequency of mutations in patients with GEFS+. No mutations were found in 17 isolated cases of GEFS+. Three novel SCN1A mutations-D188V, V1353L, and I1656M-were found in 36 familial cases; of the remaining 33 families, 3 had mutations in SCN1B. On the basis of SSCA, the combined frequency of SCN1A and SCN1B mutations in familial cases of GEFS+ was found to be 17%.

Mutant GABA(A) receptor gamma 2-subunit in childhood absence epilepsy and febrile seizures

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants(1,2). We have found a mutation in a gene encoding a GABA, receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS), There is a recognized genetic: relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.