Association of drug metabolism gene polymorphisms with toxicities, graft-versus-host disease and survival after HLA-identical sibling hematopoietic stem cell transplantation for patients with leukemia

Individual differences in drug efficacy or toxicity can be influenced by genetic factors. We investigated whether polymorphisms of pharmacogenes that interfere with metabolism of drugs used in conditioning regimen and graft-versus-host disease (GvHD) prophylaxis could be associated with outcomes after HLA-identical hematopoietic stem cell transplantation (HSCT). Pharmacogenes and their polymorphisms were studied in 107 donors and patients with leukemia receiving HSCT. Candidate genes were: P450 cytochrome family (CYP2B6), glutathione-S-transferase family (GST), multidrug-resistance gene, methylenetetrahydrofolate reductase (MTHFR) and vitamin D receptor (VDR). The end points studied were oral mucositis (OM), hemorrhagic cystitis (HC), toxicity and venoocclusive disease of the liver (VOD), GvHD, transplantation-related mortality (TRM) and survival. Multivariate analyses, using death as a competing event, were performed adjusting for clinical factors. Among other clinical and genetic factors, polymorphisms of CYP2B6 genes that interfere with cyclophosphamide metabolism were associated with OM (recipient CYP2B6*4; P=0.0067), HC (recipient CYP2B6*2; P=0.03) and VOD (donor CYP2B6*6; P=0.03). Recipient MTHFR polymorphisms (C677T) were associated with acute GvHD (P=0.03), and recipient VDR TaqI with TRM and overall survival (P=0.006 and P=0.04, respectively). Genetic factors that interfere with drug metabolisms are associated with treatment-related toxicities, GvHD and survival after HLA-identical HSCT in patients with leukemia and should be investigated prospectively.

Polymorphisms at GLUT1 Gene Are Not Associated With the Development of TSP/HAM in Brazilian HTLV-1 Infected Individuals and the Discovery of a New Polymorphism at GLUT1 Gene

The development of HTLV-1 associated clinical manifestations, such as TSP/HAM and ATLL, occur in 2-4% of the infected population and it is still unclear why this infection remains asymptomatic in most infected carriers. Recently, it has been demonstrated that HTLV uses the Glucose transporter type 1 (GLUT1) to infect T-CD4(+) lymphocytes and that single nucleotide polymorphisms (SNP) in the GLUT1 gene are associated with diabetic nephropathy in patients with diabetes mellitus in different populations. These polymorphisms could contribute to a higher GLUT1 protein expression on cellular membrane, facilitating the entry of HTLV and its transmission cell by cell. This could result in a higher provirus load and consequently in the development of TSP/HAM. To evaluate the role of GLUT1 gene polymorphisms in the development of TSP/HAM in HTLV-1 infected individuals, the g.22999G > T, g.15339T > C and c.-2841A > T sites were analyzed by PCR/RFLP or sequencing in 244 infected individuals and 102 normal controls. The proviral load of the HTLV-1 infected patients was also analyzed using Real Time Quantitative PCR. Genotypic and allelic frequencies of the three sites did not differ significantly between controls and HTLV-1 infected individuals. There was no difference in genotypic and allelic distributions among patients as to the presence or absence of HTLV-1 associated clinic manifestations. As regards the quantification of the provirus load, we observed a significant reduction in the asymptomatic individuals compared with the oligosymptomatic and TSP/HAM individuals. These results suggest that g.22999G > T, g.15339T > C, and c.-2841A > T SNP do not contribute to HTLV-1 infection nor to the genetic susceptibility of TSP/HAM in Brazilian HTLV-1 infected individuals. J. Med. Virol. 81:552557, 2009. (C) 2009 Wiley-Liss, Inc.

Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc

Impact of thymidylate synthase promoter and DNA repair gene polymorphisms on susceptibility to childhood acute lymphoblastic leukemia

The aim of this study was to evaluate the frequency of polymorphisms in the TYMS, XRCC1, and ERCC2 DNA repair genes in pediatric patients with acute lymphoblastic leukemia using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) approaches. The study was conducted in 206 patients and 364 controls from a Brazilian population. No significant differences were observed among the analyzed groups regarding XRCC1 codon 399 and codon 194 and ERCC2 codon 751 and codon 312 polymorphisms. The TYMS 3R variant allele was significantly associated with a reduced risk of childhood ALL, represented by the sum of heterozygous and polymorphic homozygous genotypes (odds ratio 0.60; 95% confidence interval 0.37-0.99). The results suggest that polymorphism in TYMS may play a protective role against the development of childhood ALL.

Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process.

Comparative analysis of rabies virus isolates from Brazilian canids and bats based on the G gene and G-L intergenic region

Rabies virus (RABV) isolates from two species of canids and three species of bats were analyzed by comparing the C-terminal region of the G gene and the G-L intergenic region of the virus genome. Intercluster identities for the genetic sequences of the isolates showed both regions to be poorly conserved. Phylogenetic trees were generated by the neighbor-joining and maximum parsimony methods, and the results were found to agree between the two methods for both regions. Putative amino acid sequences obtained from the G gene were also analyzed, and genetic markers were identified. Our results suggest that different genetic lineages of RABV are adapted to different animal species in Brazil.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Detection of TGIF1 homeobox gene in oral squamous cell carcinoma according to histologic grading

Objective. TGIF1 homeobox gene involvement in oral cancer has not yet been investigated. This study analyzed the expression of TGIF1 transcripts and protein in oral squamous cell carcinoma (OSCC). Study design. Snap-frozen samples from 16 patients were taken from both OSCC and nontumoral adjacent epithelium (NT) for in situ hybridization (ISH). Forty-six paraffin-embedded samples of OSCC were submitted to immunohistochemistry (IHC). A descriptive analysis of the transcript signal detection was accomplished, and TGIF1 immunoexpression was carried out considering protein levels, localization, and cellular differentiation. Results. ISH reactions showed TGIF1 transcripts with a signal that was frequently intense in NT, and generally weak in OSCC, and that had stronger transcript signal in well-differentiated areas of OSCC when compared with poorly differentiated ones. IHC reactions had poorly differentiated cases associated with TGIF1 protein expression in both the nucleus and cytoplasm (P = .05, Fisher test). Conclusions. TGIF1 gain or loss of function might possibly play a role in oral cancer cell differentiation. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 111: 218-224)

Differential Production of Macrophage Inflammatory Protein-1 alpha, Stromal-Derived Factor-1, and IL-6 by Human Cultured Periodontal Ligament and Gingival Fibroblasts Challenged With Lipopolysaccharide From P. gingivalis

Background: Fibroblasts are considered important cells in periodontitis. When challenged by different agents, they respond through the release of cytokines that participate in the inflammatory process. The aim of this study is to evaluate and compare the expression and production of macrophage inflammatory protein (MIP)-1 alpha, stromal-derived factor (SDF)-1, and interleukin (IL)-6 by human cultured periodontal ligament and gingival fibroblasts challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Methods: Fibroblasts were cultured from biopsies of gingival tissue and periodontal ligament of the same donors and used on the fourth passage. After confluence in 24-well plates, the culture medium alone (control) or with 0.1 to 10 mu g/ml of LPS from P. gingivalis was added to the wells, and after 1, 6, and 24 hours, the supernatant and the cells were collected and analyzed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. Results: MIP-1 alpha, SDF-1, and IL-6 protein production was significantly greater in gingival fibroblasts compared to periodontal ligament fibroblasts. IL-6 was upregulated in a time-dependent manner, mainly in gingival fibroblasts (P<0.05), which secreted more MIP-1 alpha in the lowest concentration of LPS used (0.1 mu g/ml). In contrast, a basal production of SDF-1 that was inhibited with the increase of LPS concentration was detected, especially after 24 hours (P<0.05). Conclusion: The distinct ability of the gingival and periodontal ligament fibroblasts to secrete MIP-1 alpha, SDF-1, and IL-6 emphasizes that these cells may differently contribute to the balance of cytokines in the LPS-challenged periodontium. J Periodontol 2010;81:310-317.

The Effect of TAK-778 on Gene Expression of Osteoblastic Cells Is Mediated Through Estrogen Receptor

This study evaluated the effect of TAK-778 [(2R, 4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide)] on in vitro osteogenic events and on gene expression of osteoblastic cells derived from human alveolar bone and the participation of estrogen receptors (ERs) on such effect. Osteoblastic cells were subcultured, with or without TAK-778 (10(-5) M), to evaluate cell growth and viability, total protein content, and alkaline phosphatase (ALP) activity at 7, 14, and 21 days; bone-like formation at 21 days; and gene expression, using cDNA microarray, at 7 days. Also, osteoblastic cells were exposed to TAK-778 (10-5 M) combined to ICI182,780, a nonspecific ER antagonist (10(-6) M), and gene expression was evaluated by real-time polymerase chain reaction (PCR) at 7 days. TAK-778 induced a reduction in culture growth and an increase in cell synthesis, ALP activity, and bone-like formation. The cDNA microarray showed genes associated with cell adhesion and differentiation, skeletal development, ossification, and transforming growth factor-P receptor signaling pathway, with a tendency to be higher expressed in cells exposed to TAK-778. The gene expression of ALP, osteocalcin, Msh homeobox 2, receptor activator of NF-kappa B ligand, and intercellular adhesion molecule 1 was increased by TAK-778 as demonstrated by real-time PCR, and this effect was antagonized by ICI182,780. The present results demonstrated that TAK-778 acts at a transcriptional level to enhance the in vitro osteogenic process and that its effect on gene expression of osteoblastic cells is mediated, at least partially, through ERs. Based on these findings, TAK-778 could be considered in the treatment of bone metabolic disorders. Exp Biol Med 234:190-199, 2009

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferred from the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayestan phylogenetic methods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis, Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyletic at the combined genes Within the Albimanus Section the monophyly of the Stroder Subgroup was strongly supported and within the Myzorhynchella Section Anopheles anrunesi and An lutzu formed a strongly supported monophyletic group The epidemiologically significant Albitarsis Complex showed evidence of paraphyly (relative to An lanet-Myzorhynchella) and discordance across gene trees, and the previously synonomized species of An. dunhami and An goeldii were recovered as sister species Finally, there was evidence of complexes in several species, including An antunesi, An deaneorum, and An. strodei (c) 2010 Elsevier B.V. All rights reserved

Association of genetic variants in the adiponectin encoding gene (ADIPOQ) with type 2 diabetes in Japanese Brazilians

Aim: The objective of this study is to assess the contribution of ADIPOQ variants to type 2 diabetes in Japanese Brazilians. Methods: We genotyped 200 patients with diabetes mellitus (100 male and 100 female, aged 55.0 years [47.5-64.0 years]) and 200 control subjects with normal glucose tolerant (NGT) (72 male and 128 female, aged 52.0 years [43.5-64.5 years]). Results: Whereas each polymorphism studied (T45G, G276T, and A349G) was not significantly associated with type 2 diabetes mellitus, the haplotype GGA was overrepresented in our diabetic population (9.3% against 3.1% in NGT individuals, P=.0003). Also, this haplotype was associated with decreased levels of adiponectin. We also identified three mutations in exon 3: I164T, R221S, and H241P, but, owing to the low frequencies of them, associations with type 2 diabetes could not be evaluated. The subjects carrying the R221S mutation had plasma adiponectin levels lower than those without the mutation (2.10 mu g/ml [1.35-2.55 mu g/ml] vs. 6.68 mu g/ml [3.90-11.23 mu g/ml], P=.015). Similarly, the I164T mutation carriers had mean plasma adiponectin levels lower than those noncarriers (3.73 mu g/ml [3.10-4.35 mu g/ml] vs. 6.68 mu g/ml [3.90-11.23 mu g/ml]), but this difference was not significant (P=.17). Conclusions: We identified in the ADIPOQ gene a risk haplotype for type 2 diabetes in the Japanese Brazilian population. (C) 2010 Elsevier Inc. All rights reserved.

Yerba Mate (Ilex paraguariensis) Aqueous Extract Decreases Intestinal SGLT1 Gene Expression but Does Not Affect Other Biochemical Parameters in Alloxan-Diabetic Wistar Rats

Yerba mate (Ilex paraguariensis) is rich in polyphenols, especially chlorogenic acids. Evidence suggests that dietary polyphenols could play a role in glucose absorption and metabolism. The aim of this study was to evaluate the antidiabetic properties of yerba mate extract in alloxan-induced diabetic Wistar rats. Animals (n = 41) were divided in four groups: nondiabetic control (NDC, n = 10), nondiabetic yerba mate (NDY, n = 10), diabetic control (DC, n = 11), and diabetic yerba mate (NDY, n = 10). The intervention consisted in the administration of yerba mate extract in a 1 g extract/kg body weight dose for 28 days; controls received saline solution only. There were no significant differences in serum glucose, insulin, and hepatic glucose-6-phosphatase activity between the groups that ingested yerba mate extract (NDY and DY) and the controls (NDC and DC). However, the intestinal SGLT1 gene expression was significantly lower in animals that received yerba mate both in upper (p = 0.007) and middle (p < 0.001) small intestine. These results indicate that bioactive compounds present in yerba mate might be capable of interfering in glucose absorption, by decreasing SGLT1 expression.

Emergence of Klebsiella pneumoniae carrying the novel extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and the class 1 integron-associated bla(GES-7) in Brazil

A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and bla(GES-7) was recovered. Cefoxitin and ceftazidime activity was most affected by the presence of these genes and an additional resistance to trimethoprim-sulphamethoxazole was observed. The bla(GES-7) gene was found to be inserted into a class 1 integron. These results show the emergence of novel bla(TEM) and bla(SHV) genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of bla(GES) genes into diverse nosocomial pathogens. Indeed, the bla(GES-7) gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere.