High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein-Barr virus transformed human lymphoblastoid cell lines.

Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.

Oxidative stress in nonallergic nasal polyps associated with bronchial hyperresponsiveness.

BACKGROUND: Nasal polyposis (NP) is a chronic inflammatory disease of upper airway with unknown etiology. NP is frequently associated with asthma; the interaction between these comorbidities remains interesting. Oxidative stress has been implicated in the pathophysiology of NP and asthma. The aim of this study is to investigate the significance of oxidative stress in sinonasal microenvironments by evaluating its association with clinopathological parameters and its impacts on the pathogenesis of bronchial hyperresponsiveness (BHR) in NP. METHODS: Polyp biopsy specimens were obtained from 20 nonallergic patients; control mucosas were obtained from 20 volunteers. The levels of free radicals in the tissues and in blood were determined by a sensitive chemiluminescence (CL) method. NP patients were substratified into three subgroups, NP without BHR, NP with asymptomatic BHR, and NP with BHR and asthma by the results of provocative testing. Four histological characteristics of NP, inflammatory cells, eosinophil infiltration, edema and fibrosis were estimated and applied to correlate with the tissue-CL. RESULTS: The mean CL level in polyp-tissues, but not in blood, was higher than in the control specimens. In NP patients, tissue-CL was associated with endoscopy score; high tissue-CL levels were positively correlated with the abundance of inflammatory cells and eosinophils. Tissue-CL and endoscopy score were associated with BHR/asthma phenotype. CONCLUSION: These results suggest an important role for oxidative stress in the pathophysiology of NP and a causal relation between oxidative stress and inflammatory cells, especially the eosinophils. Free radical levels in polyp-tissues associated with NP severity and with BHR/asthma phenotype in nonallergic NP patients.

Factors that affect recurrence after anterior colporrhaphy procedure reinforced with four-corner anchored polypropylene mesh.

The purpose of this study was to evaluate the effectiveness of the anterior colporrhaphy procedure reinforced with four-corner anchored polypropylene mesh in patients with severe (stage III or IV) anterior vaginal prolapse. Thirty-eight consecutive women were enlisted for this prospective study. The procedure consisted of an extensive vaginal dissection to join the vesicovaginal and retropubic space and an anchoring of a polypropylene mesh patch between the two Arcus Tendineus Fasciae Pelvis in a tension-free manner. The mean age of the study group was 63 (33-80) years. The success rate was 87% (33/38) at a mean follow-up interval of 21 (12-29) months. A total of eight (100%) patients were also cured of concomitant stress incontinence (five overt and three occult type) with an additional tension-free vaginal tape (TVT) operation. During follow-up, there were five de-novo stress incontinence cases (16.7%) and four vaginal erosions of mesh (10.5%). Four clinical variables--diabetes mellitus, recurrent anterior vaginal prolapse, chronic cough and vaginal erosions of mesh--were found to have a significant correlation with an unsatisfactory surgical result with large values of hazard ratios found by survival analysis. We concluded that the anterior colporrhaphy procedure reinforced with four-corner anchored polypropylene mesh was effective for most, but failed in some patients who had specific risk factors within short convalescence periods. Concomitant stress incontinence can be successfully treated by a TVT operation in combination with the anterior colporrhaphy procedure reinforced with four-corner anchored polypropylene mesh. However, the anterior colporrhaphy procedure may itself have adverse effects on urethral sphincter function.

Analysis of two sling procedures using polypropylene mesh for treatment of stress urinary incontinence.

OBJECTIVES: To evaluate and compare the surgical outcome between the innovative tension-free vaginal tape (TVT) and conventional pubovaginal sling (PVS) procedures using polypropylene mesh. METHODS: Eighty consecutive women with urodynamic stress urinary incontinence (SUI), who chose to undergo either a TVT (n=23) or a PVS (n=57) procedure using polypropylene mesh based on financial consideration, were recruited for this study. The surgical results were analyzed and compared subjectively and objectively. RESULTS: The mean follow-up interval was 23 months for the TVT and 20 months for the PVS procedure (P=0.062). Postoperatively, SUI (91.3% vs. 93.0%), concomitant urge symptoms (85.0% vs. 85.3%) and the negative impact of incontinence and urogenital distress on patients' quality of life (79.8% vs. 77.8%) (77.4% vs. 68.8%) had improved markedly. After a multivariable logistic regression analysis, the treatment outcome of SUI was found to be independent of the main effects of patient age, parity, concurrent gynecological surgeries, intrinsic sphincter deficiency, previous failed incontinence surgeries, and concomitant urge symptoms. However, it was significantly related to treatment procedures (TVT vs. PVS) and their interaction with patient body mass index (BMI). Based on the fitted logistic model, we see that TVT performs better than PVS when BMI is less than 27.27 kg/m2, and the advantage of TVT decreases as BMI increases. CONCLUSION: Both TVT and PVS procedures using polypropylene mesh are effective treatment modalities for female SUI. However, TVT was not as effective in treating overweight or obese women as PVS.

Molecular cloning of ras and rap genes from Entamoeba histolytica.

To better understand growth regulation in the protozoan parasite Entamoeba histolytica, ameba genes homologous to the ras oncogene and rap (Krev-1) anti-oncogene were cloned. Two putative ameba ras genes (Ehras1 and Ehras2) were identified, which contain 205 and 203 amino acid (aa) open reading frames (ORFs), respectively. The Ehras1 ORF shows an 91% positional identity with that of Ehras2, a 55% identity with Dictyostelium discoideum (Dd) ras, and a 47% identity with human (Hs) ras. Two ameba rap genes (Ehrap1 and Ehrap2) were identified, both of which contain 184-aa ORFs. The Ehrap1 ORF shows a 93% positional identity with that of Ehrap2, a 60% identity with Dd rap, a 61% identity with Hs Krev-1, and a 45% identity with that of Ehras1. Conserved aa in each ameba ras and rap ORF include GTP-binding sites, effector site, site of ADP-ribosylation by Pseudomonas exoenzyme S, and COOH-terminus CAAX. As all Xs = Leu or Phe, ameba ras and rap proteins may be gerenylgerenylated and not farnesylated. Both ras and rap genes are transcribed by trophozoites. A single 21-kDa ameba ras protein reacts with the rat Y13-259 anti-ras monoclonal antibody, which is located on the cytosolic side of the plasma membrane. These are the first ras and rap genes identified from a protozoan parasite.

Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entamoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoelectric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP(+)-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADH1 clone had an open reading frame that was 360 amino acids long and encoded a protein of approximately 39 kDa (calculated size). EhADH1 showed a 62% amino acid identity with the tetrameric NADP(+)-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADH1 contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADH1 lacked a 23-amino acid stretch present in other alcohol dehydrogenases that includes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methylpyrazole.