[Serum microRNA-21 usefulness in inflammatory pathology of the colon]. / Utilidad de la determinación sérica de microRNA-21 en la enfermedad inflamatoria del colon.

BACKGROUND: MicroRNAs are 20-22 nucleotide molecular structures with post-transcriptional activity that are involved in the immune response, as well as in the inflammatory pathways of different cells and tissues. AIMS: We present herein a prospective study in which serum microRNA-21 expression was determined in patients diagnosed with acute appendicitis as a model of bowel inflammation. MATERIAL AND METHODS: A prospective cohort study of patients diagnosed with acute appendicitis was conducted. Serum microRNA-21 was analyzed through the PCR of blood samples taken from the patients prior to surgery. MicroRNA-21 values were compared with the analytic variables (leukocytes, hemoglobin, hematocrit, platelets, prothrombin activity, glucose, urea, and creatinine) and the anatomopathologic variables (normal appendix, phlegmonous, gangrenous, and perforated acute appendicitis). RESULTS: A total of 60 patients with acute appendicitis diagnosis were consecutively included in the study from June to October 2009. Sixty-six percent of the patients were men (40 men and 20 women), with a mean age of 26.2±14.8 years. The mean absolute level of microRNA-21 was 24.8±0.93, whereas the mean microRNA-21 gene expression was 1.04±0.28. No correlation between the analytic and anatomopathologic parameters evaluated was observed (P=.47). CONCLUSIONS: It is necessary to continue to search for the most appropriate microRNAs, so that their determination in serum can lead to greater precision in establishing the diagnosis and outcome of inflammatory disorders of the bowel.

An association between viral genes and human oncogenic alterations: the adenovirus E1A induces the Ewing tumor fusion transcript EWS-FLI1.

Malignant transformation of human cells requires the accumulation of multiple genetic alterations, such as the activation of oncogenes and loss of function of tumor suppressor genes or those related to genomic instability. Among the genetic alterations most frequently found in human tumors are chromosomal translocations that may result in the expression of chimeric products with transforming capability or are able to change the expression of oncogenes. We show here that the adenovirus early region 1A (E1A) gene can induce a specific human fusion transcript (EWS-FLI1) that is characteristic of Ewing tumors. This fusion transcript was detected by RT-PCR in normal human fibroblasts and keratinocytes after expression of the adenovirus E1A gene, as well as in human cell lines immortalized by adenoviruses. Cloning and sequencing of the RT-PCR product showed fusion points between EWS and FLI1 cDNA identical to those detected in Ewing tumors. In addition, we detected a chimeric protein by western blot analysis and immunoprecipitation and a t(11,22) by fluorescent in situ hybridization. This association between a single viral gene and a specific human fusion transcript indicates a direct link between viral genes and chromosome translocations, one of the hallmarks of many human tumors.

Laparoscopic cytoreductive surgery and HIPEC is effective regarding peritoneum tissue paclitaxel distribution.

BACKGROUND: In some patients with peritoneal carcinomatosis, we could perform the cytoreductive surgery and the HIPEC procedure by a complete laparoscopic approach to avoid morbidity. We consider that using laparoscopic approach for performing peritoneal carcinomatosis cytoreductive surgery and HIPEC with closed CO2 recirculation technique is possible and safe, with equal efficacy to conventional methods and hemodynamic complications. OBJECTIVE: Monitoring the effectiveness of the drug distribution in a laparoscopic ctoreductive and HIPEC surgery group with CO2 recirculation respect to a closed and open HIPEC group METHODS: Porcine model that included fifteen mini-pigs. Five pigs were operated with laparoscopic approach performing a pelvic and retroperitoneal lymphadenectomy. They later received a total laparoscopic closed HIPEC with CO2 recirculation (G1). Group 2 (G2): five pigs operated by an open cytoreductive surgery and closed HIPEC technique. Group 3 (G3): five animals in which an open cytoreductive surgery and an open HIPEC technique was performed. Blood and peritoneal determinations were realized after recirculation of the drug, at 60 min using chromatographic analysis. RESULTS: G1-G2: phrenic right peritoneum, p: 0.46. Phrenic left peritoneum, p: 0.46. Pelvic peritoneum, p: 0.17. Serum paclitaxel: p: 0.01. G1-G3: phrenic right peritoneum, p: 0.34. Phrenic left peritoneum, p: 0.34. Pelvic peritoneum, p: 0.17. Serum paclitaxel G1-G3, p: 0.02. CONCLUSIONS: A total laparoscopic approach for ctoreductive surgery and closed HIPEC with CO2 recirculation may be safe and feasible. In our experimental model there was no significant difference in tissue drug distribution respect the conventional techniques and there was a less toxicity because the serum drug concentration was significantly lower with laparoscopic approach respect the other groups.

Negative regulation of expression of the pituitary-specific transcription factor GHF-1/Pit-1 by thyroid hormones through interference with promoter enhancer elements.

Expression of the growth hormone gene is due to the presence of the pituitary-specific transcription factor GHF-1/Pit-1. The action of the thyroid hormone T3 is mediated by nuclear receptors that regulate transcription by interaction with DNA elements located near promoters of the regulated genes. In this study, we show that T3 inhibits expression of the GHF-1/Pit-1 gene in rat pituitary GH4C1 cells by a novel mechanism that involves transcriptional interference with other regulatory elements of the promoter. Sequences between bp -90 and -200 of the rat GHF-1/Pit-1 gene which do not contain a hormone response element but contain two cyclic AMP-responsive elements mediate most of the repressive effect of T3. The hormone reduces basal levels of GHF-1/Pit-1 promoter activity and antagonizes its response to cyclic AMP and the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). A similar repression is found with a heterologous promoter that contains four copies of the cyclic AMP-responsive element motif. This regulation provides a novel example of the cross-talk between the thyroid hormone receptor and the signal transduction pathways used by different hormones and growth factors. Additionally, T3 interferes with in vitro binding of GHF-1/Pit-1 to a positive autoregulatory element located at bp -45 to -63 and has a detectable inhibitory effect on the activity of a promoter construct which extends to bp -90 of 5'-flanking DNA. The regulation of the transcription factor provides a novel example of negative transcriptional regulation by thyroid hormones.

Retinoic acid induces expression of the transcription factor GHF-1/Pit-1 in pituitary prolactin- and growth hormone-producing cell lines.

PRL and GH gene expression depend on the presence of the pituitary-specific transcription factor GHF-1/Pit-1. We have examined the effects of retinoic acid (RA) on the expression of the GHF-1/Pit-1 gene. RA induced a time- and dose-dependent increase in GHF-1/Pit-1 messenger RNA in the PRL-producing cell line 235-1. A maximal effect (a 2- to 3-fold increase) was obtained after 12-24 h of incubation with 1 microM RA. The level of the transcription factor determined by both Western blotting and gel retardation analysis with a GHF-1/Pit-1-binding site was increased in RA-treated cells compared to that in control cells. Sequences located between -400 and -90 bp mediated a 2- to 3-fold activation of the GHF-1/Pit-1 promoter by RA. The retinoid also increased the response to cAMP and phorbol esters that is mediated by two cAMP-responsive elements (CREs) located in the same promoter fragment. Both CREs are required for RA induction, as deletion of either CRE abolished the response to the retinoid RA also induced GHF-1/Pit-1 gene expression in GH4C1 cells, which produce both PRL and GH. T3 did not affect expression of the GHF-1/Pit-1 gene in 235-1 cells, but decreased basal GHF-1/Pit-1 messenger RNA and promoter activity in GH4C1 cells and blocked the stimulatory effect of RA.

The transcription factor GHF-1, but not the splice variant GHF-2, cooperates with thyroid hormone and retinoic acid receptors to stimulate rat growth hormone gene expression.

The rat growth hormone (GH) promoter was significantly activated in non-pituitary cells by the expression of unliganded trioodothyronine (T3) and retinoic acid (RA) receptors. Furthermore, a strong ligand-dependent activation was found in the presence of the pituitary-specific transcription factor GHF-1. When compared with GHF-1, the splice variant GHF-2 showed a decreased ability to bind the cognate site in the GH promoter. As a consequence, expression of GHF-2 had little stimulatory effect on the GH promoter and did not show cooperation with T3 or RA receptors even in the presence of ligands. Furthermore, over-expression of GHF-2 inhibited the response to T3 and RA in pituitary cells. These results show that alternative splicing of the GHF-1 gene gives rise to two isoforms that differ in their transactivating properties and in their ability to synergize with the nuclear thyroid hormone and retinoic acid receptors on GH gene expression.

[Prognostic significance and clinic utility of serum and immunohistochemical cathepsin B levels in colorectal cancer]. / Significación pronóstica y utilidad clínica de la determinación sérica e inmunohistoquímica de catepsina B en cáncer colorrectal.

BACKGROUND: Aproximately one third of node-negative colorectal cancer recur suggesting the presence of micrometastasis not detected by conventional histopathologic methods. We think that the role of enzymes like Cathepsin B play in the process of invasion and metastasis in colorectal cancer might identify at earlier stages patients with high risk of shorter survival and who need more aggressive treatment. Our porpuse is to evaluate the prognostic significance of preoperative serum and inmunohistochemical levels of cathepsin B to identify colorectal carcinomas with worse prognostic. METHODS: Fifty five patients undergoing surgical treatment for colorectal cancer from 1998 to 2000. As a control group sera from 23 patients with acute appendicitis. Serum levels of cathepsin B were obtained preoperatively (KRKA, Novo Slovenia;ng/ml); cathepsin B inmunoreactivity was determinated after surgical treatment, (C-19, Santa Cruz Biotechnology). Serum levels of CEA (Inmulit 2000 CEA), and CA 19,9 (Inmulite Gi-Ma, Diagnostic Products Corporation, Los Angeles, CA), and p53 expression (Dako) were determinated in patients with colorectal cancer. Survival analysis was realized using Cox and Kaplan-Meier methods (SPSS 10.0 for Windows). RESULTS: The mean age of patients with colorectal cancer was 68 years (range 39-87 years). 29 males and 26 females. Tumor size was 4.6 cms., range 1-12. Rectal localization, 32.2%. Moderately differentiated, 49.1%. The median serum and inmunohistochemical levels of cathepsin B were 5.74 ng/ml and 29.56% in patients with acute appendicitis respectively. Preoperative serum levels in patients with colorectal cancer were: CEA, 46.04 ng/ml (range 0.21-7.32 ); CA 19,9, 110.52 UI/ml, (range 2.5-1920); and Cathepsin B, 6.94 ng/ml, range 3.57-11.6). Inmunohistochemical results were: p53, 44.36%, (range 0-95); Cathepsin B, 66.9% (range 10-90). Serum and inmunhistochemical values were significantly increased in patients with colorectal cancer when compared with control group, p=0,011 and p=0,000. High serum levels of cathepsib B were significantly associated wiyh shorter survival of patients with colorectal cancer in univariate and multivariate methods, p=0.041;HR 1.281 95%CI (1.043-1.716) and p= 0.022; HR 1.338.955 CI (1.043-1.716). CONCLUSIONS: Cathepsin B can be used like an independent prognostic tumoral marker in colorectal cancer. Preoperative serum levels over 6.94 ng/ml, are associated with worse prognostic and shorter survival.

Role of GHF-1 in the regulation of the rat growth hormone gene promoter by thyroid hormone and retinoic acid receptors.

In non-pituitary HeLa cells the unliganded thyroid hormone or retinoic acid receptors cause a strong activation of the rat growth hormone promoter that is repressed by their ligands. In contrast, after expression of the pituitary-specific transcription factor GHF-1, thyroid hormone and retinoic acid produce a stimulation similar to that found in pituitary cells. Therefore, GHF-1 changes a ligand-dependent inhibition into a ligand-dependent activation. The essential role of GHF-1 on the rat growth hormone promoter was also demonstrated with AF-2-defective T3 receptor mutants that show a normal activation of this promoter in the presence of GHF-1. Furthermore, a truncated T3 receptor, which lacks the N-terminus and the DNA binding domain, was able to stimulate this promoter in the presence of GHF-1 and exogenous RXR receptors, suggesting the importance of protein to protein interactions in this regulation. This study shows that the final transcriptional effect depends not only on the type of regulatory promoter response elements but also on the presence of other transcriptional activators, in the case of the growth hormone promoter, the tissue-specific transcription factor GHF-1, which plays a coactivator-like role in this promoter.

[Abdominal cancer surgery in patients over 70]. / Onkologische abdominale Chirurgie bei über 70jährigen Patientinnen.

Between 1971 and 1986, 63 oncological patients were submitted to a major abdominal operation at our hospital. Mortality, complication rate and mean hospital stay of these patient group were comparable to those found in a control group of geriatric patients whose indication for laparotomy was a benign condition. From the presented data it is concluded that if there is an indication for surgery, in the absence of an equivalent medical alternative, the operation should be undertaken also in oncological patients, regardless of age.

Determination of the promoter elements that mediate repression of c-fos gene transcription by thyroid hormone and retinoic acid receptors.

Basal and stimulated activity of the c-fos promoter is reduced by triiodothyronine (T3) and retinoic acid (RA) in GH1 cells. We examined the influence of these ligands on the activity of reporter constructs containing the AP-1 site, the serum response element (SRE) and the cyclic AMP responsive element (CRE) of the c-fos promoter under control of an heterologous promoter. T3 and RA decreased the response of AP-1 and SRE sequences to phorbol esters, forskolin or serum but they did not reduce basal or forskolin-stimulated activity mediated by the CRE. Therefore, repression of c-fos gene expression by T3 and RA receptors appears to be exerted through transcriptional interference with the SRE and the AP-1 binding site of the promoter.

A direct protein-protein interaction is involved in the cooperation between thyroid hormone and retinoic acid receptors and the transcription factor GHF-1.

The nuclear receptors for thyroid hormone (TRs) and retinoic acid (RARs and RXRs) cooperate with the pituitary-specific transcription factor GHF-1 to activate the rat growth hormone (GH) gene. The GH promoter contains a hormone response element (HRE), which binds TR/RXR and RAR/RXR heterodimers, located close to two binding sites for GHF-1. GHF-1 inhibits binding of TR/RXR and RAR/RXR heterodimers to an isolated HRE. Similarly, the receptors inhibit binding of GHF-1 to its cognate site. These results suggest the existence of direct protein to protein interactions between the receptors and the pituitary transcription factor. This was confirmed by in vitro binding studies with GST fusion proteins, which demonstrated a strong association of GHF-1 with RXR and a weaker interaction with RAR and TR. GHF-1 and the receptor heterodimers form a ternary complex with a fragment of the rat GH promoter, which contains binding sites for both, and GHF-1 increases receptor binding to the promoter when present in limiting conditions. These results suggest that the synergistic activation of the rat GH gene involves protein-DNA interactions as well as a physical association between the nuclear receptors and the pituitary-specific transcription factor GHF-1.

Correlation between plasma estradiol and estrone-3-glucuronide in urine during the monitoring of ovarian induction therapy.

An improved radioimmunoassay was developed to determine estrone-3-glucuronide in daily urine. Resulting levels were compared with those of estradiol in plasma of 10 healthy women and 14 undergoing ovulation induction therapy with human menopausal gonadotropin and human chorionic gonadotropin. A highly significant correlation between plasma estradiol and urinary estrone-3-glucuronide in normal (r = .9209; P less than .01) and stimulated (r = .9229; P less than .01) women was demonstrated. These results proved that the pattern of excretion of estrone-3-glucuronide perfectly reflected the changes in plasmatic estradiol levels when monitoring ovarian induction and that estrone-3-glucuronide determinations can provide clinically useful information in human induction therapy.