Identification of genes differencially expressed in women endometrium during the menstrual cycle

As variações fisiológicas dos hormônios sexuais ao longo do ciclo menstrual promovem alterações histomorfológicas e funcionais no endométrio. Para melhor entender as variações de expressão genética responsáveis por tais eventos, motivamo-nos a avaliar as eventuais variações na expressão gênica nas fases proliferativa e secretora do ciclo menstrual. Para tanto, estudamos o endométrio normal de 24 pacientes submetidas à histerectomia por causas benignas. Retiraram-se fragmentos do endométrio para extração de RNA, os quais foram imediatamente acondicionados em gelo seco para futura utilização. As amostras foram submetidas a ensaios do tipo cDNA microarray em lâminas contendo 10.000 genes. A análise da expressão diferencial foi realizada pelo software SAM e os resultados confirmados, individualmente, por PCR em tempo real para RNA mensageiro, além de imunohistoquímica para a correta confirmação em nível protéico. Dos 10.000 genes estudados, obtivemos um total de 1127 genes diferencialmente expressos. Dentre estes, 364 genes estavam superexpressos na fase proliferativa e 763 genes tinham seu RNA mensageiro em maior quantidade na fase secretora. A fase secretora apresentou-se, do ponto de vista de variedade de expressão gênica, como uma fase marcadamente mais rica, em especial quando analisamos os genes envolvidos na resposta imune, e os genes que respondem aos hormônios tireoideanos, bem como aqueles envolvidos no metabolismo de cálcio.In order to further comprehend the genetic events underlying the endometrial steroids response we undertook a 10.000 genes microarray analysis between human normal endometrium in the proliferative and secretory phases of the menstrual cycle. We selected 24 patients out of 72 women who were diagnosed as having uterine leiomyomata. Neither patient had received any hormonal treatment during the six months previously the diagnosis of leiomyomata, endometrial fragments were divided in proliferative and secretory after histological analysis. Total RNA was extracted from samples by using prior to reverse transcription, two pools of RNA were obtained by mixing 3 µg of total RNA from each individual case in two separated tubes representing endometrial tissues in proliferative (group 1) and secretory phase (group 2). These two pools were then hybridized two times each to four CodeLink UniSet Human I Bioarrays (Amersham Biosciences, Piscataway, NJ). After analysing the results by the SAM software 1127 genes were found differentially expressed between secretory and proliferative phases when a 2,5 difference was used as a cut-off criteria. 364 genes were up-regulated in the proliferative phase and 763 genes were up-regulated in the secretory phase. Among them, genes involved in immune response, thyroid and calcium metabolism were found differentially expressed specially in the secretory phase. It is hypothesize that overexpression of calcium related, thyroid and immune responsive genes might be one of the hallmarks of the second phase of menstrual cycle

Epstein-Barr virus detection in invasive and pre-invasive lesions of the uterine cervix

In the present study, our aim was to investigate whether EBV DNA could be found in association with invasive and pre-invasive cervical cancer lesions. We hypothesize that EBV is not merely a commensal agent when present in malignant cervical lesions. DNA was extracted from cervical scrapings followed by nested PCR-based amplification. The patients were 66 women with high grade cervical intraepithelial neoplasia and 14 women with invasive cervical cancer. The control group consisted of 89 women with a normal Pap smear and colposcopy as well as a negative HPV DNA test. Analysis of our results, in conjunction with the work of other authors, leads us to propose that EBV is not merely a commensal agent when present in malignant cervical lesions. The presence of DNA from EBV is significantly associated with cervical cancer.FAPESP/Ludwig[99/03366-6

Differential gene expression assessed by cDNA microarray analysis in breast cancer tissue under tamoxifen treatment

Our purpose was to identify tamoxifen (TAM) responsive genes after 30 days of TAM treatment in tumor tissues obtained from women with breast cancer using microarray expression analysis. In our study, we identified 12 candidates to be considered as tamoxifen-modulated genes. Among them, we selected two candidates the TEGT BI-1 (testis enhanced gene transcript Bax Inhibitor-1) and the CD63 gene in order to further confirm their differential expression under tamoxifen effects. We observed that both were down-regulated in tumor tissues of patients during TAM treatment. TEGT is able to inhibit the expression of Bax, which is known to promote apoptosis. On the other hand, CD63 encodes a cell membrane protein and it seems to be involved in mechanisms of platelet activation. cell adhesion and cell motility. We therefore hypothesize that TAM would be able to modulate tumor growth by down-regulating genes involved in mechanisms such as cell cycle control, tumor invasion and metastasis.Univ Fed Sao Paulo, Dept Gynecol, Mol Gynecol Lab, Paulista Sch Med, Sao Paulo, SP, BrazilLudwig Inst Canc Res, Sao Paulo Branch, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Gynecol, Mol Gynecol Lab, Paulista Sch Med, Sao Paulo, SP, BrazilWeb of Scienc

A Transcript Finishing Initiative for Closing Gaps in the Human Transcriptome

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used