Androgen receptor transcriptional activity and chromatin modifications on the ABCB1/MDR gene are critical for taxol resistance in ovarian cancer cells.

We report here that the androgen receptor (AR) and ABCB1 are upregulated in a model of acquired taxol resistance (txr) in ovarian carcinoma cells. AR silencing sensitizes txr cells to taxol threefold, whereas ectopic AR expression in AR-null HEK293 cells induces resistance to taxol by 1.7-fold. AR activation using the agonist dihydrotestosterone (DHT) or sublethal taxol treatment upregulates ABCB1 expression in both txr cells and AR-expressing HEK293 cells. In contrast, AR inactivation using the antagonist bicalutamide downregulates ABCB1 expression and enhances cytotoxicity to taxol. A functional ABCB1 promoter containing five predicted androgen-response elements (AREs) is cloned. Deletion assays reveal a taxol-responsive promoter segment which harbors ARE4. Notably, DHT- or taxol-activated AR potentiates binding of the AR to ARE4 as revealed by the chromatin immunoprecipitation. On the other hand, txr cells display an increase in chromatin remodeling. AR/H3K9ac and AR/H3K14ac complexes bind specifically to ARE4 in response to taxol. Furthermore, acetyltransferase protein levels (p300 and GCN5) are upregulated in txr cells. Silencing of p300 or GCN5 reduces chromatin modification and enhances cytotoxicity in both parental and txr SKOV3 cells. While the phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (AKT) pathway is significantly activated by taxol, taxol-induced ABCB1 expression, histone posttranslational modifications, and p300 binding to ARE4 are suppressed following inhibition of the PI3K/AKT cellular pathway. These results demonstrate that the AKT/p300/AR axis can be activated to target ABCB1 gene expression in response to taxol, thus revealing a new treatment target to counter taxol resistance.

Gene regulatory network inference and analysis of multidrug-resistant Pseudomonas aeruginosa

BACKGROUND Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.

Genetic variability in the regulation of the expression cluster of MDR genes in patients with breast cancer.

PURPOSE: We aimed to investigate the association between the polymorphism and expression patterns of multiple drug resistance genes (MDR) in breast cancer (BC). MATERIALS AND METHODS: The MDR gene expression levels were measured in tumor tissues of 106 breast cancer patients using quantitative real-time PCR. Affymetrix CytoScan™ HD Array chips were used to assess genotypes. Pairwise correlation analysis for ABCB1, ABCC1, ABCC2 and ABCG2 gene expression levels was carried out to reveal co-expression clusters. Associations between SNPs of MDR genes and their preoperative expression levels were assessed using analysis of covariance adjusting for covariates. RESULTS: The SNPs associated with the expression of the ABCB1, ABCC1, ABCC2 and ABCG2 genes before NAC were detected. In addition, 21 SNPs associated with the expression of four ABC-transporter genes and involved in the expression regulation were identified. Validation in an independent sample confirmed the association between the MDR cluster genes and 11 SNPs. CONCLUSIONS: Four MDR genes: ABCB1, ABCC1, ABCC2 and ABCG2 were shown to form the functional expression cluster in breast tumor. Further studies are required to discover precise mechanisms of the cluster regulation, thereby providing new approaches and targets to combat the development of the MDR phenotype during chemotherapy.

Distribution and characterization of Stenotrophomonas maltophilia isolates from environmental and clinical samples in Thailand.

BACKGROUND: Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, especially in patients who are immunocompromised, suffering from malignancy or have been hospitalized for a prolonged period. Information of this bacterium in Thailand has not been elucidated. AIMS: To investigate the phenotype and genotype of environmental and clinical isolates of S. maltophilia in Songklanagarind Hospital, southern Thailand. METHODS: Isolates of S. maltophilia were collected from various environmental sources on three hospital wards and clinical samples from seven wards. Antibiotic susceptibility and minimum inhibitory concentration (MIC) testing were performed using disk diffusion and E-test, respectively. Isolates were genotyped by pulsed-field gel electrophoresis. FINDINGS: The majority of S. maltophilia environmental isolates were from sink drains (67.5%), followed by drinking water (18.7%) and tap water (7.5%). Clinical isolates of the bacterium mainly originated from sputum samples (56.2% of all isolates). Antibiotic resistance was more common in clinical isolates than in environmental isolates; resistance to co-trimoxazole was associated with the presence of the sul1 gene. The MIC values for ciprofloxacin and co-trimoxazole correlated closely with the results obtained from disk diffusion assay. DNA profile analysis revealed seven clusters with high diversity among the isolates. CONCLUSION: No genotypic relationship was detected between isolates from environmental and clinical samples. As such, acquisition of this bacterium may occur outside the hospital.

Fast and simple detection methods for the 4-base pair deletion of canine MDR1/ ABCB1 gene by PCR and isothermal amplification.

Dogs with a 4-bp deletion in the MDR1 (or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice. Two allele-specific methods are presented, which allow discrimination of all genotypes, require little hands-on time, and show the results within ~1 h after DNA sampling. DNA from buccal swabs of 115 dogs with known genotype (no mutation, n = 54; heterozygous for the mutation, n = 37; homozygous for the mutation, n = 24) was extracted either by using a column-based extraction kit or by heating swabs in a simple NaOH-Tris buffer. Amplification was performed either by allele-specific fast polymerase chain reaction or by allele-specific loop-mediated isothermal amplification (LAMP). Analysis was done either on agarose gels, by simple endpoint visualization using ultraviolet light, or by measuring the increase of fluorescence and time to threshold crossing. Commercial master mixes reduced the preparation time and minimized sources of error in both methods. Both methods allowed the discrimination of all 3 genotypes, and the results of the new methods matched the results of the previous genotyping. The presented methods could be used for fast individual MDR1/ ABCB1 genotyping with less equipment than existing methods.

Malignancy of Cancers and Synthetic Lethal Interactions Associated With Mutations of Cancer Driver Genes.

The mutation status of cancer driver genes may correlate with different degrees of malignancy of cancers. The doubling time and multidrug resistance are 2 phenotypes that reflect the degree of malignancy of cancer cells. Because most of cancer driver genes are hard to target, identification of their synthetic lethal partners might be a viable approach to treatment of the cancers with the relevant mutations.The genome-wide screening for synthetic lethal partners is costly and labor intensive. Thus, a computational approach facilitating identification of candidate genes for a focus synthetic lethal RNAi screening will accelerate novel anticancer drug discovery.We used several publicly available cancer cell lines and tumor tissue genomic data in this study.We compared the doubling time and multidrug resistance between the NCI-60 cell lines with mutations in some cancer driver genes and those without the mutations. We identified some candidate synthetic lethal genes to the cancer driver genes APC, KRAS, BRAF, PIK3CA, and TP53 by comparison of their gene phenotype values in cancer cell lines with the relevant mutations and wild-type background. Further, we experimentally validated some of the synthetic lethal relationships we predicted.We reported that mutations in some cancer driver genes mutations in some cancer driver genes such as APC, KRAS, or PIK3CA might correlate with cancer proliferation or drug resistance. We identified 40, 21, 5, 43, and 18 potential synthetic lethal genes to APC, KRAS, BRAF, PIK3CA, and TP53, respectively. We found that some of the potential synthetic lethal genes show significantly higher expression in the cancers with mutations of their synthetic lethal partners and the wild-type counterparts. Further, our experiments confirmed several synthetic lethal relationships that are novel findings by our methods.We experimentally validated a part of the synthetic lethal relationships we predicted. We plan to perform further experiments to validate the other synthetic lethal relationships predicted by this study.Our computational methods achieve to identify candidate synthetic lethal partners to cancer driver genes for further experimental screening with multiple lines of evidences, and therefore contribute to development of anticancer drugs.

Deletions of multidrug resistance gene loci in breast cancer leads to the down-regulation of its expression and predict tumor response to neoadjuvant chemotherapy.

Neoadjuvant chemotherapy (NAC) is intensively used for the treatment of primary breast cancer. In our previous studies, we reported that clinical tumor response to NAC is associated with the change of multidrug resistance (MDR) gene expression in tumors after chemotherapy. In this study we performed a combined analysis of MDR gene locus deletions in tumor DNA, MDR gene expression and clinical response to NAC in 73 BC patients. Copy number variations (CNVs) in biopsy specimens were tested using high-density microarray platform CytoScanTM HD Array (Affymetrix, USA). 75%-100% persons having deletions of MDR gene loci demonstrated the down-regulation of MDR gene expression. Expression of MDR genes was 2-8 times lower in patients with deletion than in patients having no deletion only in post-NAC tumors samples but not in tumor tissue before chemotherapy. All patients with deletions of ABCB1 ABCB 3 ABCC5 gene loci--7q21.1, 6p21.32, 3q27 correspondingly, and most patients having deletions in ABCC1 (16p13.1), ABCC2 (10q24), ABCG1 (21q22.3), ABCG2 (4q22.1), responded favorably to NAC. The analysis of all CNVs, including both amplification and deletion showed that the frequency of 13q14.2 deletion was 85% among patients bearing tumor with the deletion at least in one MDR gene locus versus 9% in patients with no deletions. Differences in the frequency of 13q14.2 deletions between the two groups were statistically significant (p = 2.03 × 10(-11), Fisher test, Bonferroni-adjusted p = 1.73 × 10(-8)). In conclusion, our study for the first time demonstrates that deletion MDR gene loci can be used as predictive marker for tumor response to NAC.

Evaluation of multidrug resistance-1 gene C>T polymorphism frequency in patients with asthma

OBJECTIVES:Asthma is a chronic inflammatory lung disease characterized by bronchial hyperresponsiveness and airflow obstruction. Genetic and oxidative stress factors, in addition to pulmonary and systemic inflammatory processes, play a pivotal role in the pathogenesis of asthma. The products of the multidrug resistance-1 gene protect lung tissue from oxidative stress. Here, we aimed to evaluate the association between the multidrug resistance-1 gene C>T polymorphism and asthma with regard to oxidative stress-related parameters of asthmatic patients.METHODS:Forty-five patients with asthma and 27 healthy age-matched controls were included in this study. Blood samples were collected in tubes with ethylenediaminetetraacetic acid. DNA was extracted from the blood samples. The multidrug resistance-1 gene polymorphism was detected by polymerase chain reaction and a subsequent enzyme digestion technique. The serum levels of total oxidant status and total antioxidant status were determined by the colorimetric measurement method.RESULTS:The heterozygous polymorphic genotype was the most frequent in both groups. A significant difference in the multidrug resistance-1 genotype frequencies between groups indicated an association of asthma with the TT genotype. A significant difference between groups was found for wild type homozygous participants and carriers of polymorphic allele participants. The frequency of the T allele was significantly higher in asthmatic patients. The increase in the oxidative stress index parameter was significant in the asthma group compared with the control group.CONCLUSIONS:The multidrug resistance-1 gene C/T polymorphism may be an underlying genetic risk factor for the development of asthma via oxidant-antioxidant imbalance, leading to increased oxidative stress.

HIF-1α Induces Multidrug Resistance in Gastric Cancer Cells by Inducing MiR-27a.

This study aimed to determine the correlation between HIF-1α and miR-27a expression and to evaluate the effect of inhibition of HIF-1α expression on miR-27a expression and drug resistance in gastric cancer (GC). In the present study, real time-PCR and Western blot were performed to detect the expression of HIF-1α in GC tissues and cell lines. Then, OCUM-2MD3/L-OHP cells were transfected with HIF-1α-siRNA, a miR-27a mimic or pcDNA-HIF-1α, and cell survival was determined via the MTT assay. The expression of HIF-1α, miR-27a, and MDR-related genes was measured via real time-PCR and Western blot. ChIP and dual luciferase activity assays were performed to assess the transcriptional regulation of HIF-1α and miR-27a. The results revealed that transfection with HIF-1α-siRNA markedly decreased the levels of miR-27a, resulting in dramatically enhanced inhibition of the proliferation rate of OCUM-2MD3/L-OHP cells. Compared to non-transfected cells, the survival rate was significantly reduced in the cells transfected with HIF-1α-siRNA after treatment with L-OHP. The cell survival rate was significantly increased in OCUM-2MD3/L-OHP cells transfected with the miR-27a mimic, whereas HIF-1α overexpression did not result in any clear change in cell survival. The results of the dual luciferase activity assay demonstrated that HIF-1α enhances the transcriptional activity of the miR27a promoter in cells transfected with a reporter plasmid containing the upstream promoter region of miR27a together with pcDNA-HIF-1α. ChIP analysis suggested that HIF-1α directly binds to the promoter region of miR27a. Inhibition of HIF-1α or miR27a expression decreased MDR1/P-gp, LRP, and Bcl-2 expression in OCUM-2MD3/L-OHP cells. Thus, we found that HIF-1α is closely associated with MDR in GC and that HIF-1α may suppress MDR1/P-gp, LRP and Bcl-2 expression by inhibiting miR-27a expression.

Mutaciones asociadas a resistencia a rifampicina e isoniacida en aislamientos clínicos del complejo Mycobacterium tuberculosis de pacientes del Hospital Roosevelt / Mutations associated with resistance to rifampin and isoniazid in clinical isolates of the Mycobacterium tuberculosis complex from patients at Roosevelt Hospital

El aumento de tuberculosis y la multidrogo resistencia de cepas de micobacterias es un problema de los sistemas de salud, en 2009, en el Hospital Roosevelt Gordillo y cols, determinaron la TB-MDR en pacientes con tuberculosis diagnosticada microbiológicamente, la tasa de resistencia fue de 4.3%. Objetivo: Determinar los patrones de resistencia y perfiles genéticos de cepas con monoresistencia y cepas TB-MDR del Complejo M. tuberculosis. Métodos: Se utilizaron dos métodos para evaluar las cepas de M. tuberculosis, un método fenotípico, MGIT, y un método Genotípico, Genotype HAIN LifeScience para determinar el perfil genético de las cepas. Resultados: Se evaluaron 846 cepas de micobacterias de los años 2008 al 2013, encontrándose un 2.2% de TB-MDR. Las cepas evaluadas genotípicamente fueron 761, a las cuales se determinó los genes de resistencia, encontrándose monoresistencia a Isoniacida en 58 cepas, 7.6%, monoresistencia a Rifampicina en 18 cepas, 2.4% y 15 cepas MDR, 2.0%. Las mutaciones más frecuentes en monoresistencia fueron inhA MUT1 y katG MUT1 y la combinación de ambos genes 3.2%, 3.0% y 1.3%, para cepas TB-MDR la combinación rpoB Mutación silenciosa + katG MUT1 + inhA MUT1. Se encontró que en pacientes con cepas MDR el 3.1% son HIV+ y el 1.5% son HIV-...(AU)

Introduction: The increase of tuberculosis and multidrug resistance in mycobacteria strains is a problem for health systems, in 2009, in Hospital Roosevelt, Gordillo and cols, determined the TB-MDR in patients diagnosed with tuberculosis microbiologically, the resistance rate was 4.3%. Objective: To determine the resistance patterns and genetic profiles of monoresistant strains and MDR-TB strains of M. tuberculosis complex. Methods: Two methods for evaluating M. tuberculosis strains were used, a phenotypic method, MGIT, and a genotypic method, Genotype HAIN LifeScience to determine the genetic profile of the strains. Results: 846 strains of mycobacteria of the years 2008 to 2013 were evaluated, finding 2.2% of MDR-TB. The strains genotypically evaluated were 761, of wich, resistance genes were determined, finding isoniazid monoresistance in 58 strains, 7.6%, Rifampicin monoresistance in 18 strains, 2.4% and 15 MDR strains, 2.0%. The most frequent mutations for monoresistant strains were inhA MUT1 and katG MUT1 and the combination of both genes 3.2%, 3.0% and 1.3%, respectively, and the most frequent mutations for TB-MDR strains was the combination rpoB silent mutation + katG MUT1 + inhA MUT1. There was found that in patients with MDR strains 3.1% are HIV+ and 1.5% are HIV-. Conclusions: The percentage of TB-MDR strains was 2.3%, and the most common genes were rpoB silent mutation, inhA MUT1 y katG MUT1. There was found a higher percentage of monoresistance in isoniazid than rifampicin, being the HIV+ patient population the one that presented higher percentages in both monoresistance to RIF and INH and TB-MDR strains.

Effects of the pharmaceuticals diclofenac and metoprolol on gene expression levels of enzymes of biotransformation, excretion pathways and estrogenicity in primary hepatocytes of Nile tilapia (Oreochromis niloticus).

The expression levels of key enzymes of the xenobiotic metabolism and excretion pathways concerning biotransformation phases I (cytochrome P4501A), II (glutathione S-transferase) and III (multidrug resistance protein) and of the estrogenic biomarker vitellogenin (vtg) were investigated in primary hepatocytes isolated from male Nile tilapia (Oreochromis niloticus) after exposure to diclofenac and metoprolol, two pharmaceuticals prevalent in the aquatic environment worldwide. The lowest test concentration (4×10(-9) M) was chosen to reflect an environmentally relevant exposure situation. Furthermore concentration dependent effects were investigated. Therefore a series of concentrations higher than the environmentally relevant range were used (10- and 100-fold). Diclofenac significantly induced all chosen biomarkers already at the environmentally relevant concentration indicating that biotransformation and elimination occur via the pathways under investigation. Estrogenic potential of this substance was demonstrated by VTG up-regulation as well. Metoprolol was either less effective than diclofenac or metabolized using different pathways. Key enzymes of the xenobiotic metabolism were less (CYP1A, GST) or not (MDRP) induced and a mild increase in vtg mRNA was detected only for 4×10(-8) M. No concentration-dependency for metoprolol was found.

Expression of multidrug resistance 1 gene in association with CXCL12 in chronic myelogenous leukaemia.

Even though the BCR-ABL tyrosine kinase inhibitor imatinib significantly improves the prognosis of chronic myelogenous leukaemia (CML) patients, drug resistance is a major obstacle to better management. We examined the interaction of recently defined bone marrow microenvironment factors CXCL12 and ATP-binding cassette (ABC) transporters in the bone marrow of CML patients in the chronic phase and blast crisis.Expression levels of mRNA extracted from frozen specimens of CML patients were measured by real-time polymerase chain reaction. The expression of the ABC transporters MDR1, ABCC1, ABCG2, and CXCL12 was significantly higher in the bone marrow samples of CML blast crisis than in those of CML chronic phase. Immunohistochemical staining for CXCL12 revealed that the proportion of CXCL12 positive reticular cell areas correlated well with the mRNA levels of CXCL12 in CML bone marrow. Finally, co-culture experiments of K562 CML cells with CXCL12 expressing mesenchymal cells (OP9 cells or human CXCL12 transfected 3T3 cells) revealed enhanced mRNA levels for MDR1 in a CXCL12 rich environment.These results suggest that imatinib treatment restores the bone marrow microenvironment in CML with the presence of CXCL12 expressing reticular cells but in turn induces the overexpression of MDR1 in haematopoietic cells due to up-regulated expression of CXCL12.

Linezolid-resistant Staphylococcus aureus strain 1128105, the first known clinical isolate possessing the cfr multidrug resistance gene.

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZD(r) clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination.

Multidrug resistance 1 gene polymorphisms may determine Crohn's disease behavior in patients from Rio de Janeiro.

OBJECTIVES: Conflicting data from studies on the potential role of multidrug resistance 1 gene polymorphisms in inflammatory bowel disease may result from the analysis of genetically and geographically distinct populations. Here, we investigated whether multidrug resistance 1 gene polymorphisms are associated with inflammatory bowel diseases in patients from Rio de Janeiro. METHODS: We analyzed 123 Crohn's disease patients and 83 ulcerative colitis patients to determine the presence of the multidrug resistance 1 gene polymorphisms C1236T, G2677T and C3435T. In particular, the genotype frequencies of Crohn's disease and ulcerative colitis patients were analyzed. Genotype-phenotype associations with major clinical characteristics were established, and estimated risks were calculated for the mutations. RESULTS: No significant difference was observed in the genotype frequencies of the multidrug resistance 1 G2677T/A and C3435T polymorphisms between Crohn's disease and ulcerative colitis patients. In contrast, the C1236T polymorphism was significantly more common in Crohn's disease than in ulcerative colitis (p = 0.047). A significant association was also found between the multidrug resistance 1 C3435T polymorphism and the stricturing form of Crohn's disease (OR: 4.13; p = 0.009), whereas no association was found with penetrating behavior (OR: 0.33; p = 0.094). In Crohn's disease, a positive association was also found between the C3435T polymorphism and corticosteroid resistance/refractoriness (OR: 4.14; p = 0.010). However, no significant association was found between multidrug resistance 1 gene polymorphisms and UC subphenotypic categories. CONCLUSION: The multidrug resistance 1 gene polymorphism C3435T is associated with the stricturing phenotype and an inappropriate response to therapy in Crohn's disease. This association with Crohn's disease may support additional pathogenic roles for the multidrug resistance 1 gene in regulating gut-microbiota interactions and in mediating fibrosis. Understanding the effects of several drugs associated with multidrug resistance 1 gene variants may aid in the selection of customized therapeutic regimens.

Multidrug resistance 1 gene polymorphisms may determine Crohn's disease behavior in patients from Rio de Janeiro

OBJECTIVES: Conflicting data from studies on the potential role of multidrug resistance 1 gene polymorphisms in inflammatory bowel disease may result from the analysis of genetically and geographically distinct populations. Here, we investigated whether multidrug resistance 1 gene polymorphisms are associated with inflammatory bowel diseases in patients from Rio de Janeiro. METHODS: We analyzed 123 Crohn's disease patients and 83 ulcerative colitis patients to determine the presence of the multidrug resistance 1 gene polymorphisms C1236T, G2677T and C3435T. In particular, the genotype frequencies of Crohn's disease and ulcerative colitis patients were analyzed. Genotype-phenotype associations with major clinical characteristics were established, and estimated risks were calculated for the mutations. RESULTS: No significant difference was observed in the genotype frequencies of the multidrug resistance 1 G2677T/A and C3435T polymorphisms between Crohn's disease and ulcerative colitis patients. In contrast, the C1236T polymorphism was significantly more common in Crohn's disease than in ulcerative colitis (p = 0.047). A significant association was also found between the multidrug resistance 1 C3435T polymorphism and the stricturing form of Crohn's disease (OR: 4.13; p = 0.009), whereas no association was found with penetrating behavior (OR: 0.33; p = 0.094). In Crohn's disease, a positive association was also found between the C3435T polymorphism and corticosteroid resistance/refractoriness (OR: 4.14; p = 0.010). However, no significant association was found between multidrug resistance 1 gene polymorphisms and UC subphenotypic categories. CONCLUSION: The multidrug resistance 1 gene polymorphism C3435T is associated with the stricturing phenotype and an inappropriate response to therapy in Crohn's disease. This association with Crohn's disease may support additional pathogenic roles ...

Pharmacogenetics of mood disorders: what clinicians need to know.

Pharmacogenetics brought the promise of matching individuals with treatments that would be efficacious while minimizing adverse events. This has been long needed in psychiatry, where treatment options have been empirical and treatment choices have been made largely based on clinical judgment. The efficacy and tolerability of antidepressants, the most common drugs used in mood disorders, have been widely studied in pharmacogenetics. Genetic association studies have been reported for pharmacokinetic genes such as the CYP450 isoenzymes or MDR1, and pharmacodynamic genes such as the serotonin transporter (SLC6A4) or the serotonin 2A receptor (HTR2A). However, despite the large number of reports, clinically useful predictors are still scarce for antidepressant monotherapy. Pharmacogenetic predictors of efficacy for mood stabilizers such as lithium and anticonvulsants have not had a dissimilar fate, and clinically meaningful markers are yet to emerge. The lack of consistent results may be in part due to small samples of heterogeneous populations and lack of consensus on phenotype definitions. Current pharmacogenetic recommendations include testing for HLA-B*1502 when using carbamazepine in Asian ancestry populations to prevent Stevens­Johnson syndrome, CYP2D6 genotypes when using pimozide, and CYP2D6 in polypharmacy to minimize drug interactions. This review, which is aimed at clinicians, lays the basis for understanding strengths and weaknesses of pharmacogenetic studies and outlines current clinical uses of these biomarkers.

Changing the expression vector of multidrug resistance genes is related to neoadjuvant chemotherapy response.

PURPOSE: We aimed to examine the association between alterations in multidrug resistance (MDR) gene expression, measured before and after neoadjuvant chemotherapy (NAC), and short-term response in a cohort of stage IIA-IIIC breast cancer patients (n = 84). METHODS: All patients were treated with two to four preoperative cycles of FAC (5-fluorouracil-adriamycin-cyclophosphamide), CAX (cyclophosphamide-adriamycin-xeloda) or taxane regimes. The expression levels of key MDR genes (ABCB1, ABCC1, ABCC2, ABCC3, ABCC5, ABCG1, ABCG2, GSTP1, and MVP) were evaluated in both tumor tissues obtained pre-therapy and in specimens removed by final surgery, using TaqMan-based quantitative reverse transcriptase PCR. RESULTS: No significant difference in the average level of MDR gene expression in paired breast tumors before and after NAC was found when analyzed in both responsive and non-responsive patients. There was no correlation between the expression levels of MDR genes in pre-NAC tumors and immediate NAC response. In the group with tumor responses, we found a statistically significant downregulation of expression of ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, ABCG2, GSTP1, and MVP genes following NAC in FAC and CAX-treated patients (67-93% of cases). In contrast, we found that expression of these genes was upregulated after NAC, mostly in non-responsive patients (55-96% of cases). Responsiveness to taxotere was related to reduced levels of ABCB1, ABCC2, ABCG1, ABCG2, and MVP mRNA in tumor samples collected after chemotherapy. CONCLUSION: Our results suggest that reductions in MDR gene expression in post-NAC samples in comparison with pre-NAC are associated with tumor response to FAC and CAX as well as taxotere-based NAC, while patients displaying MDR gene upregulation had resistance to therapy.

Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil / Genes de multirresistência, incluindo blaKPC e blaCTX-M-2, em Klebsiella pneumoniae isoladas em Recife, Brasil

INTRODUCTION: The prevalence of cephalosporins and carbapenem-resistant Klebsiella pneumoniae strains is rising in Brazil, with potential serious consequences in terms of patients' outcomes and general care. METHODS: This study characterized 24 clinical isolates of K. pneumoniae from two hospitals in Recife, Brazil, through the antimicrobial susceptibility profile, analyses of β-lactamase genes (blaTEM, blaSHV,blaCTX-MblaKPC, blaVIM, blaIMP, and blaSPM), plasmidial profile and ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTS: ERIC-PCR and plasmidial analysis grouped the isolates in 17 and 19 patterns, respectively. Six isolates from one hospital presented the same pattern by ERIC-PCR, indicating clonal dissemination. All isolates presented blaSHV, 62.5% presented blaCTX-M-2, 29% blaTEM, and 41.7% blaKPC. Metallo-β-lactamase genes blaand blawere not detected. Eleven isolates were identified carrying at least 3 β-lactamase studied genes, and 2 isolates carried blaSHVblaTEM, blaCTX-M-2 and blaKPC simultaneously. CONCLUSIONS: The accumulation of resistance genes in some strains, observed in this study, imposes limitations in the therapeutic options available for the treatment of infections caused by K. pneumoniae in Recife, Brazil. These results should alert the Brazilian medical authorities to establish rigorous methods for more efficiently control the dissemination of antimicrobial resistance genes in the hospital environment.

INTRODUÇÃO: A prevalência de cepas de Klebsiella pneumoniae resistentes a cefalosporinas e carbapenêmicos está aumentando no Brasil, com sérias consequências em termos de desfechos dos pacientes e cuidados gerais. MÉTODOS: Este estudo caracterizou 24 isolados clínicos de K. pneumoniae provenientes de dois hospitais de Recife, Brasil, através do perfil de susceptibilidade a antimicrobianos, análise de genes de β-lactamase (blaTEM,blaSHV,blaCTX-MblaKPC,blaVIM, blaIMP,and blaSPM), perfil plasmidial e ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTADOS: A análise da ERIC-PCR e do perfil plasmidial agrupou os isolados em 17 e 19 perfis, respectivamente. Seis isolados de um hospital apresentaram o mesmo padrão de ERIC-PCR, indicando disseminação clonal. Todos os isolados apresentaram blaSHV, 62,5% apresentaram blaCTX-M-2, 29% blaTEM e 41,7% blaKPC. Genes de metalo-β-lactamase blaVIM, blaIMP e blaSPM não foram detectados. Onze isolados foram identificados carreando, pelo menos, três dos genes de β-lactamase estudados, dentre estes, dois isolados continham blaSHV,blaTEM, blaCTX-M-2 e blaKPC simultaneamente. CONCLUSÕES: O acúmulo de genes de resistência em algumas cepas, observado nesse estudo, impõem limitações nas opções terapêuticas disponíveis para o tratamento de infecções causadas por K. pneumoniae em Recife, Brasil. Estes resultados devem alertar as autoridades médicas brasileiras para estabelecer rigorosos métodos para controlar eficientemente a disseminação de genes de resistência a antimicrobianos no ambiente hospitalar.

Transduction of mesenchymal stem cells with multidrug resistance gene provides protection for bone marrow toxicity after being transplanted into a nude mice model.

BACKGROUND: Myelosuppression is the main dose-related toxicity of many chemotherapeutic drugs. The human multidrug resistance (mdr1) gene is well-known for its ability to confering drug resistance. In this study, we meant to transplant the placenta mesenchymal stem cells (P-MSCs) moderated by mdr1 gene into a nude mice model radiated by γ-Co(60) and to explore the chemoprotection for bone marrow (BM) toxicity. METHODS: Human P-MSCs were isolated from trypsin-digested term placentas and then transduced by with reconstructed retroviral vector containing mdr1 gene and green fluorescent protein (GFP) reporter gene. The integration and expression of mdr1 gene was observed indirectedly by the expression of GFP. A nude mice model was constructed after irradiation with a sublethal dosage of γ-Co(60). These irradiated mice were transplanted with mdr1-MSCs through the caudal vein and then received paclitaxel (PAC) intraperitoneal chemotherapy. The Peripheral peripheral blood (PB) of the nude mice was collected, and the PB cells counts and values were determined using an automatic analyzer. RESULTS: After PAC treatment, mdr1-MSCs transplanted mice showed markedly improved survival upon compared to MSCs transplanted mice (85.7% vs. 57.1%). White blood cell (WBC) and red blood cell (RBC) counts as well as the hemoglobin (Hb) values were significantly increased in PAC treated mdr1-MSCs mice compared to PAC treated control mice when PAC chemotherapy had been finished (all P < 0.05), but the difference was not found in the plateltes (PLT) count (P > 0.05). CONCLUSION: Human P-MSCs moderated by mdr1 gene when transplanted into nude mice may provide chemoprotection for hematopoietic toxicity.

Gefitinib enhances cytotoxicities of antimicrotubule agents in non-small-cell lung cancer cells exhibiting no sensitizing epidermal growth factor receptor mutation.

INTRODUCTIONS: Although randomized clinical trials showed no benefit from combining epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) with standard chemotherapy for advanced non-small-cell lung cancer (NSCLC), better results might be obtained by combining EGFR-TKI with individual agents that are substrates for the adenosine triphosphate binding cassette transporters (ABCTs) because EGFR-TKIs can inhibit their efflux. The combination effects deserved to be further examined in vitro. METHODS: The combination effects of gefitinib with three antimicrotubule agents (AMTAs), paclitaxel, docetaxel or vinorelbine, or with gemcitabine were tested in 17 NSCLC cell lines using the tetrazolium colorimetric assay and classical isobole method. The effects of drug combinations, identified by the values of mean combination index (mCIs), were correlated with the expression levels of ABCTs. Dose-versus-log-response curves were analyzed to further evaluate the possible mechanisms of drug interactions. RESULTS: Synergistic gefitinib/AMTA interactions were observed in the tested cell lines. The synergism was more robust in the four lines overexpressing de novo or acquired P-glycoprotein (Pgp; individual mCIs range, 0.484-0.859; all p values were < 0.05), or in 12 cell lines exhibiting no sensitizing EGFR mutations (group mCIs for gefitinib/paclitaxel, gefitinib/docetaxel, and gefitinib/vinorelbine were 0.869, 0.82, and 0.853, respectively. All p values were < 0.02). The synergism could be observed in cells expressing nearly undetectable Pgp and other ABCTs tested in this study. The combination of gefitinib/gemcitabine was additive (mCI = 1.027). CONCLUSIONS: Combined gefitinib/AMTAs showed synergism in NSCLC cells harboring no sensitizing EGFR mutations. Gefitinib could enhance AMTA effects through mechanisms not restricted to Pgp blockage.