Ismael D.C.G. Silva

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential

BackgroundThe metastatic disease rather than the primary tumor itself is responsible for death in most solid tumors, including breast cancer. The role of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in the metastatic process has previously been established. However, in all published studies only a limited number of MMPs/MMP inhibitors was analyzed in a limited number of cell lines. Here, we propose a more comprehensive approach by analyzing the expression levels of several MMPs (MMP-2, MMP-9 and MMP-14) and MMP inhibitors (TIMP-1, TIMP-2 and RECK) in different models (five human breast cancer cell lines, 72 primary breast tumors and 30 adjacent normal tissues).MethodsWe analyzed the expression levels of MMP-2, MMP-9 and MMP-14 and their inhibitors (TIMP-1, TIMP-2 and RECK) by quantitative RT-PCR (qRT-PCR) in five human breast cancer cell lines presenting increased invasiveness and metastatic potential, 72 primary breast tumors and 30 adjacent normal tissues. Moreover, the role of cell-extracellular matrix elements interactions in the regulation of expression and activity of MMPs and their inhibitors was analyzed by culturing these cell lines on plastic or on artificial ECM (Matrigel).ResultsThe results demonstrated that MMPs mRNA expression levels displayed a positive and statistically significant correlation with the transcriptional expression levels of their inhibitors both in the cell line models and in the tumor tissue samples. Furthermore, the expression of all MMP inhibitors was modulated by cell-Matrigel contact only in highly invasive and metastatic cell lines. The enzyme/inhibitor balance at the transcriptional level significantly favors the enzyme which is more evident in tumor than in adjacent non-tumor tissue samples.ConclusionOur results suggest that the expression of MMPs and their inhibitors, at least at the transcriptional level, might be regulated by common factors and signaling pathways. Therefore, the multi-factorial analysis of these molecules could provide new and independent prognostic information contributing to the determination of more adequate therapy strategies for each patient.

Gene network reconstruction reveals cell cycle and antiviral genes as major drivers of cervical cancer

Although human papillomavirus (HPV) was identified as an etiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major sub-networks up-regulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal HPV decline in women with invasive cancer who were previously unable to clear the virus.

Microarray cDNA to Identify Inflammatory Genes in Nasal Polyposis

Background The objective of this study was to investigate the spectrum of inflammatory gene expression in patients with nasal polyposis. Methods The cDNA microarray technique was used to identify gene expression in tissue samples from nasal polyps and adjacent inflammatory nasal mucosa of 21 patients with nonallergic nasal polyposis. To validate the microarray analysis, we compared the expression of five genes by reverse transcription-polymerase chain reaction (RT-PCR): tumor necrosis factor, IL-5, IL-9, fibroblast growth factor, and transforming growth factor (TGF)-β1. Results We tested 96 different inflammatory genes in our samples. Thirty-six genes exhibited differences in expression between the two tissue types. In all 36 genes the level of expression was greater in the inflammatory mucosa than the polyps. The RT-PCR confirmed the cDNA results. Conclusion We believe that the high expression of TGF-β1 in inflammatory mucosa compared with the low expression in polyps may reflect an important role in the inhibitory mechanisms of nasal polyposis.

Toll-like receptor pathway signaling is differently regulated in neutrophils and peripheral mononuclear cells of patients with sepsis, severe sepsis, and septic shock.

Objectives:Up- and down-regulation of inflammatory response was described in blood cells from septic patients, according to the stage of sepsis and the cells evaluated. This study aimed to evaluate the Toll-like receptor (TLR) signaling pathway gene expression in peripheral blood mononuclear cells (PBMC) and neutrophils in patients throughout the different stages of sepsis. Design:Prospective, observational study. Settings:Two emergency rooms and two intensive care units in one university and one teaching hospital. Patients and Controls:A total of 15 septic patients, five with sepsis, five with severe sepsis, and five with septic shock, in addition to five healthy volunteers were enrolled. Interventions:None. Measurements and Main Results:The Human-TLR Signaling Pathway, which comprises 84 genes related to TLR-mediated signal transduction, was evaluated by real time polymerase chain reaction in PBMC and neutrophils obtained from patients and controls. The fold change for each gene (2(−&Dgr;&Dgr;Ct)) was compared between the groups. Genes with fold changes greater than 2 and significant changes in &Dgr;CT are reported as differently expressed. The fold change ratios in PBMC gene expression between septic patients and healthy controls revealed a dynamic process according to the stage of sepsis, tending toward down-regulation of the TLR signaling pathway in PBMC in the more severe forms of the disease. However, the differential gene expression was restricted to five down-regulated genes in septic shock patients, which are found in the effector and downstream pathways. Neutrophils showed a different pattern of adaptation. Patients with sepsis, severe sepsis, and septic shock presented a broad gene up-regulation, which included all functional groups evaluated and persisted throughout the stages of the disease. Conclusions:TLR-signaling pathway genes are differently regulated in PBMC and neutrophils of septic patients, and are dynamically modulated throughout the different stages of sepsis.

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the β-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 μg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 μg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 μg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 μg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

Genetic polymorphisms of cytochrome P450c17α (CYP17) and progesterone receptor genes (PROGINS) in the assessment of endometriosis risk

We designed the present study in order to evaluate the eventual role of polymorphisms in the genes encoding cytochrome P450c17α (CYP17) and the progesterone receptor (PROGINS) as risk factors for endometriosis development. Eligible cases consisted of 121 women with surgically confirmed endometriosis who underwent treatment in a hospital in São Paulo, Brazil during the period from September 2003 to September 2005. The 281 controls were participants with normal gynecological as well as pelvic ultrasound evaluation, who did not have any gynecological conditions during their reproductive lives such as pelvic pain and/or dyspareunia nor infertility history. Genomic DNA was obtained from buccal cells and processed for DNA extraction using the GFX DNA extraction kit (GE Healthcare). The CYP17 (−34T→C) polymerase chain reaction–restriction fragment length polymorphism assay has been described previously, as has the progesterone receptor polymorphism (PROGINS) detection assay. PROGINS heterozygosis genotype frequencies were shown to be statistically higher in endometriosis cases compared with controls. On the other hand, differences in the CYP17 polymorphism (−34T → C) frequencies were not even close to significance (p = 0.278) according to our findings.

Endometrial claudin-4 and leukemia inhibitory factor are associated with assisted reproduction outcome.

BackgroundClaudin-4 (CLDN4) is one of several proteins that act as molecular mediators of embryo implantation. Recently, we examined immunolabeling of leukemia inhibitory factor (LIF) in the endometrial tissue of 52 IVF patients, and found that LIF staining intensity was strongly correlated with successful pregnancy initiation. In the same set of patients, we have now examined endometrial CLDN4 expression, to see how expression intensity may vary with LIF. We examined CLDN4 in the luteal phase of the menstrual cycle, immediately preceding IVF treatment. Our aim was to compare expression of LIF and CLDN4 in the luteal phase, and document these patterns as putative biomarkers for pregnancy.MethodsEndometrial tissue was collected from women undergoing IVF. Endometrial biopsies were obtained during the luteal phase preceding IVF, and were then used for tissue microarray (TMA) immunolabeling of CLDN4. Previously published LIF expression data were then combined with CLDN4 expression data, to determine CLDN4/LIF expression patterns. Associations between successful pregnancy after IVF and combined CLDN4/LIF expression patterns were evaluated.ResultsFour patterns of immunolabeling were observed in the endometrial samples: 16% showed weak CLDN4 and strong LIF (CLDN4-/LIF+); 20% showed strong CLDN4 and strong LIF (LIF+/CLDN4+); 28% showed strong CLDN4 and weak LIF (CLDN4+/LIF-); and 36% showed weak CLDN4 and weak LIF (CLDN4-/LIF-). Successful implantation after IVF was associated with CLDN4-/LIF+(p = 0.003). Patients showing this endometrial CLDN4-/LIF+ immunolabeling were also 6 times more likely to achieve pregnancy than patients with endometrial CLDN4+/LIF- immunolabeling (p = 0.007).ConclusionThe combined immunolabeling expression of CLDN4-/LIF+ in endometrial tissue is a potential biomarker for predicting successful pregnancy in IVF candidates.

New approach reveals CD28 and IFNG gene interaction in the susceptibility to cervical cancer

Cervical cancer is a complex disease with multiple environmental and genetic determinants. In this study, we sought an association between polymorphisms in immune response genes and cervical cancer using both single-locus and multi-locus analysis approaches. A total of 14 single nucleotide polymorphisms (SNPs) distributed in CD28, CTLA4, ICOS, PDCD1, FAS, TNFA, IL6, IFNG, TGFB1 and IL10 genes were determined in patients and healthy individuals from three independent case/control sets. The first two sets comprised White individuals (one group with 82 cases and 85 controls, the other with 83 cases and 85 controls) and the third was constituted by non-white individuals (64 cases and 75 controls). The multi-locus analysis revealed higher frequencies in cancer patients of three three-genotype combinations [CD28+17(TT)/IFNG+874(AA)/TNFA-308(GG), CD28+17(TT)/IFN+847(AA)/PDCD1+7785(CT), and CD28 +17(TT)/IFNG+874(AA)/ICOS+1564(TT)] (P < 0.01, Monte Carlo simulation). We hypothesized that this two-genotype [CD28(TT) and IFNG(AA)] combination could have a major contribution to the observed association. To address this question, we analyzed the frequency of the CD28(TT), IFNG(AA) genotype combination in the three groups combined, and observed its increase in patients (P = 0.0011 by Fishers exact test). The contribution of a third polymorphism did not reach statistical significance (P = 0.1). Further analysis suggested that gene-gene interaction between CD28 and IFNG might contribute to susceptibility to cervical cancer. Our results showed an epistatic effect between CD28 and IFNG genes in susceptibility to cervical cancer, a finding that might be relevant for a better understanding of the disease pathogenesis. In addition, the novel analytical approach herein proposed might be useful for increasing the statistical power of future genome-wide multi-locus studies.

Estrogen receptor alpha polymorphism and susceptibility to uterine leiomyoma

Uterine leiomyoma is the most frequent pelvic tumor found in female genital tract. Some studies have suggested an association between single nucleotide polymorphisms (SNPs) in estrogen receptors genes with susceptibility in developing uterine leiomyoma. In this work, we estimated the frequency of two SNPs: one located in the intron 1 (rs9322331) and other in the exon 1 (rs17847075) of the estrogen receptor alpha (ESR1) gene in 125 women with uterine leiomyoma and 125 healthy women. To do this we used a PCR-RFLP method with MspI and HaeIII restriction enzymes to respectively detect C/T SNPs in the intron 1 and in the exon 1 of ESR1. To our knowledge this is the first study aimed to investigate the association of ESR1 SNPs with the risk of developing uterine leiomyoma in Brazilian women. Our results showed that the allele frequencies of the exon 1 and the intron 1 of the ESR1 gene did not differ between cases and controls (P = 0.325 and 0.175, respectively). Furthermore, our findings provided little support for the association of these SNPs on ESR1 with leiomyoma. However, we found that the SNP in the intron 1 of the ESR1 gene was underrepresented in the Brazilian female population.