Evaluation of Xpert MTB/RIF for Detection of Mycobacterium tuberculosis in Cerebrospinal Fluid.

Studies investigating Xpert MTB/RIF diagnostic performance on cerebrospinal fluid (CSF) samples are lacking in resource-rich settings. Xpert MTB/RIF results for 740 CSF samples from 698 patients across England were retrospectively compared with the results of culture of the same and contemporary samples. The overall sensitivity was calculated at 55%.

Rapid molecular detection of tuberculosis and rifampicin drug resistance: retrospective analysis of a national U.K. molecular service over the last decade.

BACKGROUND: Fast and reliable detection of Mycobacterium tuberculosis complex (MTBC) and drug resistance is crucial in establishing effective treatment and enforcing timely public health measures. METHODS: The authors analysed the performance of a national U.K. molecular diagnostic service over a decade, based on the use of a line probe assay (Innolipa, LiPA) compared with conventional liquid and solid cultures with rapid molecular identification and culture-based drug resistance testing. FINDINGS: Data were available for 7836 consecutive patient samples using LiPA and the reference microbiological technique (conventional liquid and solid cultures with rapid molecular identification and culture-based drug resistance testing). For all sputum specimens (n=3382) the sensitivity, specificity, positive predictive value, negative predictive value and accuracy for MTBC detection were 93.4%, 85.6%, 92.7%, 86.9% and 90.7%; the equivalent values for smear-positive sputum specimens (n=2606) were 94.7%, 80.9%, 93.9%, 83.3% and 91.3%. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for detection of rifampicin resistance in all sputum samples (n=1667) were 92.1%, 99.3%, 89.4%, 99.5% and 98.9%, respectively; the equivalent values for smear-positive sputum specimens (n=1477) were 93.3%, 99.3%, 87.5%, 99.6% and 99%. Between January 2006 and December 2008, LiPA saved 25.3 and 32.2 days for TB diagnosis and rifampicin resistance of smear-positive samples, respectively. INTERPRETATION: A molecular diagnostic service, using a non-automated line probe assay approach, provides a rapid and reliable national service for diagnosing MTBC and rifampicin resistance.

Human Mycobacterium bovis infections in London and Southeast England.

Variable-number tandem repeat (VNTR) and spoligotyping analyses were used to assess transmission of Mycobacterium bovis between humans. VNTR was more discriminatory than spoligotyping. Low case numbers, despite a substantial animal reservoir, and resolution of all isolates provided no evidence of recent human-to-human transmission or recent significant infection from animals.

Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene.

Glucagon-like peptide 1 (GLP1) is postulated to regulate blood glucose and satiety, but the biological importance of GLP1 as an incretin and neuropeptide remains controversal. The regulation of nutrient-induced insulin secretion is dependent on the secretion of incretins, gut-derived peptides that potentiate insulin secretion from the pancreatic islets. To ascertain the relative physiological importance of GLP1 as a regulator of feeding behavior and insulin secretion, we have generated mice with a targeted disruption of the GLP1 receptor gene (GLP1R). These GLP1R-/- mice are viable, develop normally but exhibit increased levels of blood glucose following oral glucose challenge in association with diminished levels of circulating insulin. It is surprising that they also exhibit abnormal levels of blood glucose following intraperitoneal glucose challenge. Intracerebroventricular administration of GLP1 inhibited feeding in wild-type mice but not in GLP1R-/- mice; however, no evidence for abnormal body weight or feeding behavior was observed in GLP1R-/- mice. These observations demonstrate that GLP1 plays a central role in the regulation of glycemia; however, disruption of GLP1/GLP1R signaling in the central nervous system is not associated with perturbation of feeding behavior or obesity in vivo.

Long-term outcome of the use of botulinum toxin injection for functional anal pain.

AIM: Anal pain may occur in the absence of demonstrable anal pathology. Spasm of the sphincter muscles has been suggested as a cause in some patients. We aimed to assess the effectiveness of injection of botulinum toxin in treating this condition. METHOD: Patients who had injection of botulinum toxin over a 3-year period were identified retrospectively. Patients were excluded if anal fissure or other organic pathology was found to account for their symptoms on examination under anaesthetic. Long-term outcome was assessed at a minimum 3-year post-procedure telephone follow up. RESULTS: Fourteen (eight male) patients were identified, of median age 50 years. Botulinum toxin (20-200 u) was injected into the internal sphincter. Seven of the 14 patients reported significant improvement in symptoms at 3 months. Seven were available for a structured telephone review at a median of 59 (42-68) months. The four patients who had benefited from the injection had remained asymptomatic. CONCLUSION: Injection of botulinum toxin into the internal anal sphincter has a role in alleviating symptoms in a small proportion of patients with functional anal pain.

4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses.

The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-gamma production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

Detritus-Based Food Webs: Exploitation by Juvenile Chum Salmon (Oncorhynchus keta).

Harpacticoid copepods are the principal food of chum salmon during the first critical weeks of estuarine life. Heterotrophic food sources are preferentially ingested by harpacticoids. A commercially valuable fisheries resource, usually considered to be planktivorous, is related to a detritus-based, benthically derived food web.

Differentially androgen-modulated genes in ovarian epithelial cells from BRCA mutation carriers and control patients predict ovarian cancer survival and disease progression.

Epidemiological studies have implicated androgens in the etiology and progression of epithelial ovarian cancer. We previously reported that some androgen responses were dysregulated in malignant ovarian epithelial cells relative to control, non-malignant ovarian surface epithelial (OSE) cells. Moreover, dysregulated androgen responses were observed in OSE cells derived from patients with germline BRCA-1 or -2 mutations (OSEb), which account for the majority of familial ovarian cancer predisposition, and such altered responses may be involved in ovarian carcinogenesis or progression. In the present study, gene expression profiling using cDNA microarrays identified 17 genes differentially expressed in response to continuous androgen exposure in OSEb cells and ovarian cancer cells as compared to OSE cells derived from control patients. A subset of these differentially affected genes was selected and verified by quantitative real-time reverse transcription-polymerase chain reaction. Six of the gene products mapped to the OPHID protein-protein interaction database, and five were networked within two interacting partners. Basic leucine zipper transcription factor 2 (BACH2) and acetylcholinesterase (ACHE), which were upregulated by androgen in OSEb cells relative to OSE cells, were further investigated using an ovarian cancer tissue microarray from a separate set of 149 clinical samples. Both cytoplasmic ACHE and BACH2 immunostaining were significantly increased in ovarian cancer relative to benign cases. High levels of cytoplasmic ACHE staining correlated with decreased survival, whereas nuclear BACH2 staining correlated with decreased time to disease recurrence. The finding that products of genes differentially responsive to androgen in OSEb cells may predict survival and disease progression supports a role for altered androgen effects in ovarian cancer. In addition to BACH2 and ACHE, this study highlights a set of potentially functionally related genes for further investigation in ovarian cancer.

Trends in colorectal day case surgery in NHS Trusts between 1998 and 2005.

OBJECTIVE: Day case surgery is safe and offers potential benefits to both patients and healthcare providers. This study aimed to describe national changes in colorectal day case workload between 1998 and 2005. METHODS: Admission data relating to Office of Population Censuses and Surveys Classification of Surgical Operations and Procedures (4th revision) (OPCS-4) coloproctology operation codes were analysed using the Hospital Episode Statistics (HES) database. Day case rates (DCRs) were calculated as the proportion of elective cases performed on an ambulatory basis. RESULTS: In total, 3 119 058 colorectal admissions were recorded on the HES database between 1998 and 2005; 1 891 474 (61%) of these were for lower gastrointestinal endoscopies. Emergency cases accounted for 527 665 (17%), elective inpatient cases for 406 368 (13%) and elective day cases for 293 551 (9%) admissions. Throughout the study period the DCRs for five commonly performed elective colorectal procedures were: 0.70 for anal lesion excisions (OPCS-4 codes: H48.1, H48.2 and H48.3); 0.16 for haemorrhoidectomy (OPCS-4 code: H51.1); 0.63 for anal fissure procedures (OPCS-4 codes: H56.2 and H56.4); 0.39 for elective procedures for anal fistula (OPCS-4 codes: H55.1, H55.2, H55.3 and H55.4); 0.37 for elective pilonidal surgery (OPCS-4 codes: H59 and H60.2). Two emergency operations, drainage of perianal and pilonidal abscesses (OPCS-4 codes: H58.2 and H60.3 respectively), were identified as operations potentially amenable to day surgery. Over the seven study years, an annual average of 8559 (+/-SD 307) admissions were coded to drainage of a perianal abscess and 4676 (+/-SD 478) admissions to drainage of pilonidal abscess. The average annual bed usage associated with these procedures was 18 831 (+/-SD 718) and 7623 (+/-SD 436) bed days respectively. CONCLUSIONS: Colorectal day case surgery is currently under-exploited in the NHS. By lifting some of the barriers to day case surgery significant resource savings may be possible.

Detection of pancreatic islet 64,000 M(r) autoantigens in insulin-dependent diabetes distinct from glutamate decarboxylase.

Patients with insulin-dependent diabetes (IDDM) possess antibodies to islet proteins of M(r)-64,000. Potential autoantigens of this M(r) include glutamate decarboxylase (GAD) and 65 kD heat shock protein. We have detected two distinct antibody specificities in IDDM that bind 50,000 M(r) or 37,000/40,000 M(r) proteolytic fragments of 64,000 M(r) proteins. In this study, we investigated relationships of these proteolytic fragments to GAD and heat shock proteins. Polyclonal antibodies to GAD bound 50,000 M(r) fragments of islet antigen. Recombinant GAD65, but not GAD67, blocked binding to this antigen, suggesting that 50,000 M(r) fragments are derived from islet GAD65. In contrast, GAD antibodies did not recognize 37,000/40,000 M(r) fragments, and neither GAD isoforms blocked autoantibody binding to precursors of these fragments. The 37,000/40,000 M(r) fragments, but not the 50,000 M(r) fragments, were detected after trypsin treatment of immunoprecipitates from insulinoma cells that lacked expression of major GAD isoforms. Antibodies in IDDM did not bind native or trypsinized islet heat shock proteins. Thus, IDDM patients possess antibodies to GAD, but also distinct antibodies to a 64,000 M(r) protein that is not related to known GAD isoforms or heat shock proteins.

Molecular epidemiology and prevalence of mutations conferring rifampicin and isoniazid resistance in Mycobacterium tuberculosis strains from the southern Ukraine.

Understanding the molecular epidemiology of tuberculosis (TB) and mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. A structured questionnaire was used to collect basic epidemiological data from 589 patients with radiologically confirmed TB in the Odessa and Nikolaev regions of the Ukraine in 2003-2004. A non-commercial reverse hybridisation assay and DNA sequencing were used to detect mutations associated with rifampicin and isoniazid resistance. Genotyping was performed using multilocus variable number tandem repeat (VNTR) typing and spoligotyping. Mutations conferring rifampicin and isoniazid resistance were detected in 32.9% and 44.0%, respectively, of 225 Mycobacterium tuberculosis isolates from individual consecutive patients. Mutations in codon 531 and codon 315 of the rpoB and katG genes, respectively, were predominant among drug-resistant isolates. Multidrug (MDR) resistance rates were significantly higher among former prison inmates compared with non-prisoners (54.8% vs. 27.3%; RR 2.01; 95% CI 1.35-2.97) and the prevalence of mutations was higher in Beijing strains sharing the VNTR signature 223325173533424 than in other Beijing strains (71.4% vs. 45.7%; RR 1.74; 95% CI 1.17-2.57), suggesting that this group may be responsible for rapid transmission of MDR TB in the southern Ukraine.

Exploring the evidence base for Tier 3 weight management interventions for adults: a systematic review.

Specialist weight management services provide a treatment option for severe obesity. The objective of the study is to review the characteristics, impact and practice implications of specialist weight management services for adults in the UK. Systematic review: EMBASE, MEDLINE and PsycINFO were searched from January 2005 to March 2016 with supplementary searches. Adults with a body mass index of ≥40 kg m-2 , or ≥35 kg m-2 with comorbidity or ≥30 kg m-2 with type 2 diabetes and any study of multicomponent interventions, in any UK or Ireland setting, delivered by a specialist multidisciplinary team are the inclusion criteria. Fourteen studies in a variety of settings were included: 1 randomized controlled trial, 3 controlled and 10 observational studies. Mean baseline body mass index and age ranged from 40 to 54 kg m-2 and from 40 to 58 years. The studies were heterogeneous making comparisons of service characteristics difficult. Multidisciplinary team composition and eligibility criteria varied; dropout rates were high (43-62%). Statistically significant reduction in mean body mass index over time ranged from -1.4 to -3.1 kg m-2 and mean weight changes ranged from -2.2 to -12.4 kg. Completers achieving at least 5% reduction of initial body weight ranged from 32 to 51%. There was evidence for improved outcomes in diabetics. Specialist weight management services can demonstrate clinically significant weight loss and have an important role in supporting adults to manage severe and often complex forms of obesity. This review highlights important variations in provision and strongly indicates the need for further research into effective approaches to support severely obese adults.

The impact of interventions to promote healthier ready-to-eat meals (to eat in, to take away or to be delivered) sold by specific food outlets open to the general public: a systematic review.

INTRODUCTION: Ready-to-eat meals sold by food outlets that are accessible to the general public are an important target for public health intervention. We conducted a systematic review to assess the impact of such interventions. METHODS: Studies of any design and duration that included any consumer-level or food-outlet-level before-and-after data were included. RESULTS: Thirty studies describing 34 interventions were categorized by type and coded against the Nuffield intervention ladder: restrict choice = trans fat law (n = 1), changing pre-packed children's meal content (n = 1) and food outlet award schemes (n = 2); guide choice = price increases for unhealthier choices (n = 1), incentive (contingent reward) (n = 1) and price decreases for healthier choices (n = 2); enable choice = signposting (highlighting healthier/unhealthier options) (n = 10) and telemarketing (offering support for the provision of healthier options to businesses via telephone) (n = 2); and provide information = calorie labelling law (n = 12), voluntary nutrient labelling (n = 1) and personalized receipts (n = 1). Most interventions were aimed at adults in US fast food chains and assessed customer-level outcomes. More 'intrusive' interventions that restricted or guided choice generally showed a positive impact on food-outlet-level and customer-level outcomes. However, interventions that simply provided information or enabled choice had a negligible impact. CONCLUSION: Interventions to promote healthier ready-to-eat meals sold by food outlets should restrict choice or guide choice through incentives/disincentives. Public health policies and practice that simply involve providing information are unlikely to be effective.

Cost-effectiveness of cell salvage and alternative methods of minimising perioperative allogeneic blood transfusion: a systematic review and economic model.

OBJECTIVES: To compare patient outcomes, resource use and costs to the NHS and NHS Blood Transfusion Authority (BTA) associated with cell salvage and alternative methods of minimising perioperative allogeneic blood transfusion. DATA SOURCES: Electronic databases covering the period 1996-2004 for systematic reviews and 1994-2004 for economic evidence. REVIEW METHODS: Existing systematic reviews were updated with data from selected randomised controlled trials (RCTs) that involved adults scheduled for elective non-urgent surgery. Any resource use or cost data were extracted for potential use in populating an economic model. Relative risks or weighted mean difference of each outcome for each intervention were assessed, taking into account the number of RCTs included in each outcome and intervention and the presence of any heterogeneity. This allowed indirect comparison of the relative effectiveness of each intervention when the intervention is compared with allogeneic blood transfusion. A decision analytic model synthesised clinical and economic data from several sources, to estimate the relative cost-effectiveness of cell salvage for people undergoing elective surgery with moderate to major expected blood loss. The perspective of the NHS and patients and a time horizon of 1 month were used. The economic model was developed from reviews of effectiveness and cost-effectiveness and clinical experts. Secondary analysis explored the robustness of the results to changes in the timing and costs of cell salvage equipment, surgical procedure, use of transfusion protocols and time horizon of analysis. RESULTS: Overall, 668 studies were identified electronically for the update of the two systematic reviews. This included five RCTs, of which two were cell salvage and three preoperative autologous donation (PAD). Five published systematic reviews were identified for antifibrinolytics, fibrin sealants and restrictive transfusion triggers, PAD plus erythropoietin, erythropoietin alone and acute normovolaemic haemodilution (ANH). Twelve published studies reported full economic evaluations. All but two of the transfusion strategies significantly reduced exposure to allogeneic blood. The relative risk of exposure to allogeneic blood was 0.59 for the pooled trials of cell salvage (95% confidence interval: 0.48 to 0.73). This varied by the type and timing of cell salvage and type of surgical procedure. For cell salvage, the relative risk of allogeneic blood transfusion was higher in cardiac surgery than in orthopaedic surgery. Cell salvage had lower costs and slightly higher quality-adjusted life years compared with all of the alternative transfusion strategies except ANH. The likelihood that cell salvage is cost-effective compared with strategies other than ANH is over 50%. Most of the secondary analyses indicated similar results to the primary analysis. However, the primary and secondary analyses indicated that ANH may be more cost-effective than cell salvage. CONCLUSIONS: The available evidence indicates that cell salvage may be a cost-effective method to reduce exposure to allogeneic blood transfusion. However, ANH may be more cost-effective than cell salvage. The results of this analysis are subject to the low quality and reliability of the data used and the use of indirect comparisons. This may affect the reliability and robustness of the clinical and economic results. There is a need for further research that includes adequately powered high-quality RCTs to compare directly various blood transfusion strategies. These should include measures of health status, health-related quality of life and patient preferences for alternative transfusion strategies. Observational and tracking studies are needed to estimate reliably the incidence of adverse events and infections transmitted during blood transfusion and to identify the lifetime consequences of the serious hazards of transfusion on mortality, health status and health-related quality of life.

A comparison of the cost-effectiveness of five strategies for the prevention of non-steroidal anti-inflammatory drug-induced gastrointestinal toxicity: a systematic review with economic modelling.

OBJECTIVES: To assess the relative effectiveness, patient acceptability, costs and cost-effectiveness of four strategies for the prevention of non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastrointestinal (GI) toxicity: (1) Cox-1 NSAIDs plus histamine-2 receptor antagonist (H2RA), (2) Cox-1 NSAIDs plus proton pump inhibitors (PPIs), (3) Cox-1 NSAIDs plus misoprostol, and (4) Cox-2 NSAIDs (later expanded to 4a Cox-2 coxibNSAIDs and 4b Cox-2 preferential NSAIDs). DATA SOURCES: Electronic databases up to May 2002. REVIEW METHODS: Relevant studies were selected, assessed and analysed. Pooled relative risk ratios (RR) from the systematic review were combined with up-to-date UK resource use and unit costs data in an incremental economic analysis. A probabilistic decision-analytic model was designed and populated with data to carry out incremental economic analysis. Incremental cost-effectiveness ratios (ICERs) were generated for the outcome measure, endoscopic ulcer or serious GI event averted, against total cost, and non-parametric bootstrapping was used to simulate variance of these ICERs. RESULTS: Of 118 selected trials, including 125 relevant comparisons (which included 76,322 participants) only 138 deaths and 248 serious GI events were reported. Seven comparisons were judged to be at low risk of bias. Comparing the gastroprotective strategies against placebo, there was no evidence of effectiveness of H2RAs against any primary outcomes (few events reported), PPIs may reduce the risk of symptomatic ulcers [RR 0.09, 95% confidence interval (CI) 0.02 to 0.47], misoprostol reduces the risk of serious GI complications (RR 0.57, 95% CI 0.36 to 0.91) and symptomatic ulcers (RR 0.36, 95% CI 0.20 to 0.67), Cox-2 'preferentials' reduce the risk of symptomatic ulcers (RR 0.41, 95% CI 0.26 to 0.65) and Cox-2 'coxibs' reduce the risk of symptomatic ulcers (RR 0.49, 95% CI 0.38 to 0.62) and possibly serious GI events (RR 0.55, 95% CI 0.38 to 0.80). All strategies except Cox-2 'preferentials' reduce the risk of endoscopic ulcers. There were only 12 direct comparisons between gastroprotective strategies. All they suggest is that Cox-2 preferentials are better than misoprostol for preventing GI complications. Indirect comparisons suggested that PPIs may prevent symptomatic ulcers better than Cox-2 coxibs, but this is very weak evidence. For prevention of endoscopic ulcers PPIs and misoprostol appear more successful than H2RAs and misoprostol is better than Cox-2 preferentials. There were no UK head-to-head published economic analyses with regard to the main gastroprotective strategies. There were generally insufficient data with regards to cardiac or renal outcomes, serious GI outcomes or life-years gained to populate the mode. Mean (2.5th and 97.5th percentile) costs per endoscopic ulcer averted compared with Cox-1 NSAIDs alone were as follows: Cox-1 plus H2RAs, -186 pounds (-555 to 804); Cox-1 plus PPIs, 454 pounds (251 to 877); Cox-1 plus misoprostol, 54 pounds (-112 to 238); Cox-2 selective NSAIDs, 263 pounds (-570 to 1280), or Cox-2 specific NSAIDs, 301 pounds (189 to 418). With regard to the prevention of endoscopic ulcers, Cox-1 NSAID plus H2RA is a dominant option. Cost-effectiveness acceptability analysis showed a 95% probability that this combination was less costly and more effective. Cost-effectiveness acceptability frontiers showed that if the decision-maker is willing to pay up to 750 pounds to avoid an endoscopic ulcer, then Cox-1 plus H2RA is the optimal strategy. If the decision-maker is willing to pay over 750 pounds, the optimal strategy is NSAID plus misoprostol. Between 1900 pounds and 3750 pounds, Cox-2 selective inhibitors are optimal, and over 3750 pounds, Cox-2 specific inhibitors become optimal. NSAID plus PPI is never the optimal strategy. Sensitivity and subgroup analyses suggest that Cox-1 NSAID plus H2RA and Cox-1 NSAID plus misoprostol become more cost-effective in the older age group. Some conclusions were associated with high levels of uncertainty. CONCLUSIONS: Although there is a very large body of evidence comparing Cox-2 NSAIDs with Cox-1 NSAIDs, this is not matched by studies of the other types of gastroprotectors or by studies directly comparing active gastroprotective strategies. This lack of direct comparisons led to the use of indirect comparisons to help understand the relative efficacy of these strategies. Indirect evidence in itself is weak and was also hampered by lack of evidence in the underlying studies (where the gastroprotectors were compared with placebo). Economic modelling suggests that Cox-1 NSAID plus H2RA or Cox-1 NSAID plus PPI are the most cost-effective strategies for avoiding endoscopic ulcers in patients requiring long-term NSAID therapy. All strategies other than Cox-2 selective inhibitors reduce the rate of endoscopic ulcer compared with Cox-1 alone. The economic analysis suggests that there may be a case for prescribing H2RAs in all patients requiring NSAIDs. Misoprostol is more effective, but is associated with a greater cost and GI side-effects which may be unacceptable for patients. However, when assessing serious GI events, the economic analysis is sufficiently weakened by the data available as to render clear practice recommendations impossible. Further large, independent RCTs directly comparing various gastroprotective strategies are needed. These should report items such as major outcomes, primary data, adverse events, assessment of practice and patient preference.

The use of macroarrays for the identification of MDR Mycobacterium tuberculosis.

The emergence of Mycobacterium tuberculosis (Mtb), resistant to both isoniazid (INH) and rifampicin (RIF) (MDR-TB), is an increasing threat to tuberculosis control programs. Susceptibility testing of Mtb complex isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used. Low density oligonucleotide arrays (macroarrays) have been used successfully for the detection of RIF resistance in Mtb. We describe the use of macroarray technology to identify Mtb complex isolates resistant to INH and/or RIF. The macroarray MDR-Mtb screen has been designed to detect mutations in the RIF resistance determining region (RRDR) of Mtb rpoB and loci in katG and mabA-inhA associated with INH resistance. A panel of Mtb isolates containing 38 different RRDR genotypes, 4 different genotypes within codon 315 of katG and 2 genotypes at mabA-inhA was used to validate the macroarray. The wild type (WT) genotype was correctly identified at all three loci. Of the 37 mutant rpoB genotypes, 36 were correctly detected; the single mutant not detected contained a 9 base insertion. All mutations within katG and mabA-inhA were correctly identified. We conclude that this low cost, rapid system can usefully detect the mutations associated with the vast majority of MDR-Mtb.

Down-regulation of transforming growth factor beta receptors by androgen in ovarian cancer cells.

Steroid hormones have been implicated in the etiology and/or progression of epithelial ovarian cancer. As ovarian surface epithelial cells are growth inhibited by transforming growth factor beta (TGF-beta), we tested whether steroid hormones could regulate the expression of TGF-beta1 or its receptors in ovarian cancer cells, as assessed by quantitative reverse transcription-PCR. Treatment of ovarian cancer HEY cells with 500 nM 5alpha-dihydrotestosterone (DHT), but not estradiol-17beta or progesterone, for 60 h down-regulated the expression of mRNA for TGF-beta receptors I and II (TbetaR-I and TbetaR-II), betaglycan, and endoglin but had no effect on TGF-beta1 mRNA levels. Androgen receptor (AR) mRNA expression in HEY cells was compared to other ovarian cancer cell lines. OVCAR-3 cells expressed AR mRNA levels similar to that of androgen-responsive LNCaP prostate cancer cells, whereas SKOV-3 and HEY cells expressed only 3 and 0.01%, respectively. Western blot analysis and saturation binding assays confirmed the expression of AR protein in these three cell lines, but at the limit of detection in SKOV-3 and HEY cells. Treatment of SKOV-3 and HEY cells for 24 h with 1-50 nM DHT resulted in a dose-dependent down-regulation of TbetaR-II mRNA. The AR antagonist hydroxyflutamide did not reverse the effect of DHT on SKOV-3 cells but by itself down-regulated TbetaR-II mRNA. This apparent androgen-mimetic action of hydroxyflutamide and the ability of SKOV-3 and HEY cells to respond to DHT may be due to their expression of AR-associating protein 70, an AR co-activator reported to amplify AR transactivation and to result in agonist activity of AR antagonists. DHT was able to reverse TGF-beta1 growth-inhibitory action in SKOV-3 cells and in a primary culture of ovarian cancer cells derived from ascites. Thus, androgens may promote ovarian cancer progression in part by decreasing TGF-beta receptor levels, thereby allowing ovarian cancer cells to escape TGF-beta1 growth inhibition.

Androgen-induced inhibition of cell proliferation in an androgen-insensitive prostate cancer cell line (PC-3) transfected with a human androgen receptor complementary DNA.

A full length human androgen receptor complementary DNA was introduced into androgen receptor-negative PC-3 cells to determine if androgen sensitivity could be established in this cell line and to assess what influence, if any, androgen exposure would have on the growth of these cells. The androgen receptor complementary DNA was inserted into pSG5 in the region controlled by the SV40 promoter. This construct was cotransfected with pSR1neo into PC-3 cells and stably transfected cells were selected and screened for the expression of the androgen receptor. Active expression of the receptor was demonstrated by Western blotting using a rabbit anti-androgen receptor antiserum and by [3H]methyltrienolone binding to cytosol extracts. Saturation ligand-binding analysis revealed the presence of a single class, high affinity (Kd = 0.122 nM) androgen-binding site in cytosol extracts of transfected cells but not in extracts from mock-transfected cells. In cells expressing the transfected androgen receptor, androgen decreased the proliferation rate and cloning efficiency and induced a more differentiated phenotype. These results demonstrate that PC-3 cells have retained the mechanisms required to respond to the activated androgen receptor and that the loss of androgen sensitivity in these cells is due to the lack of functional androgen receptor. This also provides a technique for determining whether androgen-resistant tumor cells contain functional androgen receptors or whether androgen sensitivity is due to abnormalities in downstream signaling pathways. The apparent androgen-induced decreased malignant state of these transfected cells suggests new directions for the treatment of prostate cancer.

Modification of membrane physical properties, biological response and insulin binding in Friend cells by low serum concentration.

The effect of low serum concentration on plasma membrane fluidity and lipid composition, differentiation and insulin binding was investigated in three Friend erythroleukemia clones. Both FLC (clones No. 745) and F(+) (Ostertag F4N) Friend erythroleukemia cells can be induced to differentiate and to produce hemoglobin when exposed to DMSO. Clone R(3) (Ostertag F4-D5-1) is a DMSO-resistant clone when grown under normal conditions (15% serum) but could undergo differentiation with accumulation of protoporphyrin IX upon induction with DMSO when grown in low serum concentration (2.5% serum). Electron spin resonance measurements of the order parameters (S) and S(T parallel) demonstrate that R(3) has a more fluid plasma membrane than the FLC and F(+). The order parameters of the outer hyperfine splittings S(T parallel) at 37 degrees C are 0.60 +/- 0.009, 0.62 +/- 0.008 and 0.64 +/- 0.009 for R(3), F(+) and FLC, respectively. We have used the insulin receptors as a model for a polypeptide hormone receptor associated with the plasma membrane of the Friend clones. Insulin binding studies demonstrated that the receptor of R(3) had a decreased affinity for insulin manifest as a 10-fold increase in the amount of insulin required to compete for half of the tracer binding (18 nM for R(3) vs. 2 nM for FLC and F(+)). Computer-fit Scatchard plot analysis by the negative cooperativity model reveal that R(3) possessed a similar number of sites/cell (about 70,000) as the FLC or F(+) cells, with similar high and low affinities. Growing the DMSO-resistant clone R(3) in low serum concentration caused a decrease in receptor number by 35%, and an increase in receptor affinity to that seen with the differentiable clones. Thus, the abnormal properties of the plasma membrane and insulin receptor of the DMSO-resistant clone in our earlier report (Simon et al. (1984) Biochim. Biophys. Acta 803, 39-47) were partially reversed by growing the cells in a low serum concentration, restoring the cellular response to the differentiation agent.

Abnormal insulin binding and membrane physical properties of a Friend erythroleukemia clone resistant to dimethylsulfoxide-induced differentiation.

We have compared insulin binding, plasma membrane fluidity, and phospholipid composition of three different Friend erythroleukemia clones, a wild type (FLC) a mutant (R3) and the revertant to wild type F+. The R3 clone is a non-differentiating DMSO-resistant clone (R3) and has altered membrane fluidity and dramatically altered insulin-binding properties. The receptor of R3 bound insulin as if it possessed a single class of low affinity receptors that lacks the property of negative cooperativity. The Scatchard plot is linear and there is no ligand-induced acceleration of dissociation. The Hill coefficient for R3 is 1, implying 'no cooperativity', whereas the Hill coefficient for the two DMSO-inducible clones, (FLC and F+) is 0.3, implying 'negative cooperativity'. In addition, the insulin receptor of R3 has a decreased affinity for insulin, manifested as a 40-fold increase in the amount of insulin required to compete for half of the tracer binding (41 nM for R3 vs. 1 nM for FLC and F+). Computer-fitted Scatchard plots analyzed by the negative cooperativity model reveal that R3 has 95 000 receptor sites/cell, with a high affinity constant Ke of 0.016 nM-1, and a low affinity constant, Kf of 0.012 nM-1. Both DMSO-inducible clones have about 40 000 receptor sites/cell with Ke of 0.11 nM-1 and Kf of 0.02 nM-1. Electron spin resonance measurements with the 5-nitroxy stearate spin probe demonstrate that R3 had a more fluid plasma membrane than the FLC and F+ clones. The lipid composition of R3 is different from that of the DMSO-inducible clones. The weight ratio for unsaturated fatty acids to saturated fatty acids for R3 is 2.5, and the FLC clone has a lower ratio of 1.9. These results are consistent with our earlier findings in FLC that very high membrane fluidity is associated with alterations in the binding properties of the insulin receptor.