Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production.

Since a dysregulated synthesis of tumor necrosis factor alpha (TNF-alpha) may be involved in the pathogenesis of autoimmune diseases, it was of interest to precisely locate the recently reported NcoI restriction fragment length polymorphism (RFLP) of the TNF-alpha region. However, by mapping of 56.8 kb of overlapping cosmid clones and direct sequencing, we could localize the polymorphic NcoI restriction site within the first intron of the TNF-beta gene and not in the TNF-alpha gene. To study whether regulatory mechanisms are affected by this polymorphism, we analyzed the TNF-alpha/TNF-beta production of phytohemagglutinin-stimulated peripheral blood mononuclear cells of individuals homozygous for the TNF-beta NcoI RFLP by ELISA and concomitant Northern blot analysis. On days 2-4 after stimulation with mitogen, the TNFB*1 allele corresponding to a 5.3-kb NcoI fragment presented with a significantly higher TNF-beta response. A mRNA analysis demonstrated that higher protein levels of TNF-beta correlate also with increased amounts of TNF-beta transcripts. No allelic association was found in respect to TNF-alpha production. To further investigate a possible allelic influence on transcription, we determined the DNA sequence of 2 kb of the 5' portion of our cloned TNFB*2 allele and compared it with the available TNF-beta sequences. By computer-aided recognition motif search of DNA binding factors, we report putative binding sites conserved between mouse and man in the 5' flanking region as well as in intron 1 of the TNF-beta gene, found also in other cytokine promoter sequences. In addition, by polymerase chain reaction amplification and sequencing of 740 bp of the 5' part of TNF-beta of individuals typed homozygously for the NcoI RFLP, we could show that amino acid position 26 is conserved as asparagine in the TNFB*1 and as threonine in the TNFB*2 sequence. A previously reported, EcoRI RFLP in the 3' untranslated region of TNF-beta does not segregate with either of the two alleles. Thus, four TNFB alleles can be defined at the DNA level.

p53 and K-RAS alterations in pancreatic epithelial cell lesions.

We have analysed the expression of p53 at the mRNA level, and extensively at the protein level by immunostaining, Western blotting, and ELISA measurements revealing a p53 increase in 8 out of 14 cell lines established from human pancreatic carcinomas. The mRNA levels closely paralleled the protein levels in most of the cell lines. Overexpression of p53 in tumor cells correlated with mutations in the p53 gene. Immunocytochemistry was also performed with tissue cryosections showing a nuclear p53 staining in 8 out of 12 exocrine, and 2 out of 2 endocrine tumors. In addition, nonmalignant peri-tumoral tissue specimens and cells derived from pancreatic juice of acute pancreatic patients were also positively stained. These findings may suggest functions of p53 in stress situations induced by acute inflammation or tissue regeneration. Genomic mutations in the tumor suppressor gene were associated with point mutations in either codon 12, 13 or 61 in the c-K-RAS oncogene in about two-thirds of cell lines. The frequent activations of a RAS oncogene in combination with mutations of a tumor suppressor gene are likely to contribute to the malignant phenotype of pancreatic adenocarcinomas.

Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow.

PURPOSE: This study was designed to evaluate the potential of reverse-transcriptase polymerase chain reaction (RT-PCR) analyses for the detection of micrometastatic carcinoma cells in bone marrow (BM). PATIENTS AND METHODS: The specificity of RT-PCR assays with primers specific for various tumor-associated and organ-specific mRNA species was examined by analysis of 53 BM aspirates from control patients with no epithelial malignancy. In addition, BM samples from 63 patients with prostate cancer (n = 53) or breast cancer (n = 10) were analyzed by RT-PCR with primers specific for prostate-specific antigen (PSA) mRNA. As a reference method, all samples were analyzed simultaneously by an established immunocytochemical assay, using monoclonal antibodies (mAbs) against cytokeratins (CK) for tumor-cell detection. RESULTS: Seven of eight marker species could be detected in a considerable number of BM samples from control patients: epithelial glycoprotein-40 (EGP-40; 53 of 53 samples), desmoplakin I (DPI I; five of five), carcinoembryonic antigen (CEA; five of 19), erb-B2 (five of seven), erb-B3 (six of seven), prostate-specific membrane antigen (PSM; four of nine), and CK18 (five of seven). Only PSA mRNA was not detected in any of the 53 control BM samples. In serial dilution experiments, the PSA RT-PCR assay was able to detect five LNCaP prostate carcinoma cells in 4 x 10(6) BM cells. CK-positive cells were found in 20 patients (37.7%) with prostate cancer, while PSA mRNA was found in only 15 (28.3%; P = .04). Moreover, despite the recent observation that PSA is also expressed in mammary carcinomas, none of the 10 CK-positive BM samples were PSA mRNA-positive. CONCLUSION: Limiting factors in the detection of micrometastatic tumor cells by RT-PCR are (1) the illegitimate transcription of tumor-associated or epithelial-specific genes in hematopoietic cells, and (2) the deficient expression of the marker gene in micrometastatic tumor cells.

Validez y confiabilidad de una escala de clasificación de limpieza gástrica en endoscopia digestiva alta en población chilena / Validity and reliability of a gastric cleansing scale in upper endoscopyin chilean population

The presence of foam and bubbles during upper gastrointestinal endoscopy (UGE) obscures the view of gastric lesions. Objective: To assess the confidence of a gastric cleansing scale in UGE. Methods: Prospective, multicenter study. The instrument was administered to patients undergoing a UGE examination. For the gastric visualization scale, the stomach was divided in 4 parts and a 1-4 scale was used to classify each part, with a total score of 4 (optimal view of gastric mucosa) and 16 (poor view of gastric mucosa), assessed by 2 independent endoscopists. An initial cleansing score was obtained and later, after cleansing of each studied section, and total. Inter-observer concordance was established by means of Kappa test, and the agreement on the global cleansing score was established with the Bland-Altman plot. Results: 53 patients went under UGE, with an average age of 48,7 years and 62,3 percent female subjects. The main indication for performing the UGE examination was gastroesophageal reflux disease (GERD) (32.1 percent). Average duration of the procedure was 13.6 minutes. The average total gastrointestinal view before cleansing with water was 6.26 points (scale from 4 to 16) and 5.1 points (p < 0.001) after cleansing. 37.7 percent required at least 50 cc of water for cleansing. The difference in the pre and post cleansing score inter-observers was no different of 0. Kappa value obtained in gastric fundus, upper body, lower body and antrum before cleansing was 0.81; 0.71; 0.9 and 0.8, respectively. Kappa value obtained after cleansing of gastric fundus, upper body, lower body and antrum was 0.84; 0.65; 0.81 and 0.78; respectively. The mean difference between inter-observer scores before cleansing was 0.08 (p = 0.51), and after cleansing, 0.02 (p = 0.78)...

La presencia de espuma y burbujas durante la endoscopia digestiva alta (EDA) es una limitante para la visualización de lesiones gástricas. Objetivo: Evaluar la confiabilidad de una escala de clasificación de limpieza gástrica en EDA. Métodos: Estudio prospectivo, multicéntrico. Se aplicó el instrumento a pacientes que estaban agendados para EDA. Para la clasificación de visualización gástrica, el estómago se dividió en 4 porciones y se utilizó una escala de 1 a 4 por porción, sumando un puntaje total entre 4 (óptima visualización de la mucosa) y 16 (pobre visualización de ésta), evaluada por 2 endoscopistas independientes. Se obtuvo un puntaje de limpieza inicial y luego de la limpieza con agua de cada segmento estudiado y total. La concordancia inter-observador se estableció por medio del test de Kappa y el acuerdo para el puntaje global de limpieza fue establecido mediante el gráfico de Bland-Altman. Resultados: 53 pacientes fueron sometidos a EDA, con edad promedio de 48,7 años y 62,3 por ciento de sexo femenino. La principal indicación de EDA fue enfermedad por reflujo gastroesofágico (32,1 por ciento). El tiempo promedio del procedimiento fue 13,6 min. El promedio de visualización gástrica total previo a limpieza con agua fue de 6,26 puntos (escala 4 a 16) y post limpieza 5,1 puntos (p < 0,001). Para la limpieza el 37,7 por ciento requirió al menos 50 cc de agua. La diferencia de puntaje de visualización pre y post limpieza inter observador no fue distinta de 0. En fondo gástrico, cuerpo alto, cuerpo bajo y antro se obtuvo un valor de Kappa previo a limpieza de 0,81; 0,71; 0,9 y 0,8, respectivamente. El valor de Kappa posterior a limpieza en fondo gástrico, cuerpo alto, cuerpo bajo y antro fue 0,84; 0,65; 0,81 y 0,78, respectivamente. La diferencia media de los puntajes inter-observadores previos a la limpieza gástrica fue de 0,08 (p = 0,51) y posterior a la limpieza gástrica fue de 0,02 (p = 0,78)...

Allelic variation in the TNF-beta gene does not explain the low TNF-beta response in patients with primary biliary cirrhosis.

Autoimmune disorders in humans are often associated with particular alleles of major histocompatibility genes. However, the chronic inflammatory liver disease primary biliary cirrhosis (PBC) has not been found to be correlated with certain haplotypes so far. Interestingly, an impaired production of tumour necrosis factor beta (TNF-beta) upon mitogen stimulation was observed for PBC patients, especially in the immunologically active stages of the disease. Furthermore, the identification of alleles of the TNF-beta gene which differ in one unique amino acid, and in the production of TNF-beta after phytohaemagglutinin stimulation, has prompted the idea of a possible linkage between the impaired TNF-beta response in PBC and the genetic prevalence of a certain TNF haplotype. We report here a rapid method for typing the TNFB*1 and TNFB*2 genes by a standard polymerase chain reaction. PBC patients (n = 60) as well as randomized healthy controls (n = 179) of the Munich area were studied for the occurrence of the TNF alleles. No deviation was found in the PBC collective (0.7) for the TNFB*2 distribution when compared with the control (0.67).