Epigenetic silencing of the dual-role signal mediator, ANGPTL4 in tumor tissues and its overexpression in the urothelial carcinoma microenvironment.

Urothelial carcinoma (UC) carcinogenesis has been hypothesized to occur through epigenetic repression of tumor-suppressor genes (TSGs). By quantitative real-time polymerase chain reaction array, we found that one potential TSG, angiopoietin-like 4 (ANGPTL4), was expressed at very low levels in all bladder cancer cell lines we examined. Previous studies had demonstrated that ANGPTL4 is highly expressed in some cancers, but downregulated, by DNA methylation, in others. Consequently, owing to these seemingly conflicting functions in distinct cancers, the precise role of ANGPTL4 in the etiology of UC remains unclear. In this study, using methylation-specific PCR and bisulfite pyrosequencing, we show that ANGPTL4 is transcriptionally repressed by DNA methylation in UC cell lines and primary tumor samples, as compared with adjacent noncancerous bladder epithelium. Functional studies further demonstrated that ectopic expression of ANGPTL4 potently suppressed UC cell proliferation, monolayer colony formation in vitro, and invasion, migration, and xenograft formation in vivo. Surprisingly, circulating ANGPTL4 was significantly higher in plasma samples from UC patients than normal control, suggesting it might be secreted from other cell types. Interestingly, our data also indicated that exogenous cANGPTL4 could promote cell proliferation and cell migration via activation of signaling through the Erk/focal adhesion kinase axis. We further confirmed that mouse xenograft tumor growth could be promoted by administration of exogenous cANGPTL4. Finally, immunohistochemistry demonstrated that ANGPTL4 was downregulated in tumor cells but overexpressed in tumor adjacent stromal tissues of muscle-invasive UC tissue samples. In conclusion, our data support dual roles for ANGPTL4 in UC progression, either as a tumor suppressor or oncogene, in response to microenvironmental context.

Phosphorylation of glycogen synthase kinase-3ß in metabolically abnormal obesity affects immune stimulation-induced cytokine production.

BACKGROUND: Obesity is a severe health problem worldwide, which leads to multiple comorbidities including type 2 diabetes mellitus and cardiovascular diseases. Inflammation has been found to be an important characteristic of adipose tissue in obese subjects. However, obesity is also associated with compromised immune responses to infections and the impact of obesity on immune function has not been fully understood. SUBJECTS/METHODS: To clarify the role of obesity in the immune responses, we investigated the Toll-like receptor (TLR)-induced cytokine secretion by leukocytes from obese and lean subjects. We also investigated the relationship between insulin-induced intracellular signaling and cytokine production using peripheral blood mononuclear cells (PBMCs) and a monocytic cell line THP-1. RESULTS: We found decreased TLR-induced interferon-γ, interleukin-6 (IL-6) and tumor necrosis factor-α secretions and elevated IL-10 secretion by leukocytes from obese subjects when compared with lean controls. PBMCs from obese subjects showed enhanced basal Akt and glycogen synthase kinase-3ß (GSK-3ß) phosphorylation, which did not further increase with insulin and lipopolysaccharide (LPS) stimulation. We also found that LPS-induced IκBα degradation was inhibited in PBMCs from obese subjects. By using THP-1 cells with GSK-3ß knockdown or cells treated with hyperinsulinemic and high-fatty acid conditions, we found that LPS-induced nuclear factor κB (NF-κB) activation was inhibited and cyclic adenosine monophosphate response element-binding protein (CREB) activation was enhanced. CONCLUSIONS: These findings indicate that GSK-3ß is important in the regulation of NF-κB and CREB activation in leukocytes under the metabolic condition of obesity. Our study revealed a key mechanism through which metabolic abnormalities compromise leukocyte functions in people with obesity.

Comparative study of percutaneous nephrostomy using catheters with and without locking strings.

BACKGROUND: Although it is generally felt that a catheter with a locking string can achieve better fixation and thus prevent catheter displacement, no formal study has ever substantiated this. METHODS: We retrospectively reviewed the charts from 80 patients (mean age of 64.6 ± 14.76 y) who underwent percutaneous nephrostomy (PCN) over a 1-year period. RESULTS: Most patients had catheters without locking strings and only 17 patients (21.3%) had catheters with locking strings. The median duration of catheter placement was 29 days (interquartile range 14 - 57 d). There were no significant differences in patients' characteristics or catheter outcomes between catheters with and catheters without locking strings (p > 0.05). In addition, no significant difference in the catheter 90-day survival between catheter types was found (log rank test, p = 0.638). On univariate analysis, tumor as an indication for PCN (p = 0.018), obstruction (p = 0.021) and displacement (p = 0.007) were associated with reduced catheter survival. The multivariate analysis indicated that tumor as an indication for PCN (HR: 0.28, 95% CI: 0.13 - 0.63, p = 0.002), obstruction (HR: 0.25, 95% CI: 0.08 - 0.77, p = 0.015) and catheter displacement (HR: 0.09, 95% CI: 0.03 - 0.31, p < 0.001) were independent hazard factors for reduced catheter 90-day survival. CONCLUSION: No significant difference in either complication rate or 90-day survival was found between catheters with or without locking strings. These findings may prove helpful to the clinician in deciding the type of catheter to use during PCN.

Lower ataxia telangiectasia mutated (ATM) mRNA expression is correlated with poor outcome of laryngeal and pharyngeal cancer patients.

BACKGROUND: Ataxia telangiectasia mutated (ATM) kinase is a critical regulator in initiating DNA damage response and activating DNA repair. However, the correlation between ATM expression and the outcome of laryngopharyngeal cancer patients is unknown. We hypothesize that ATM expression is correlated with a worse outcome in laryngopharyngeal cancer patients. PATIENTS AND METHODS: The ATM messenger RNA (mRNA) expression of 80 tumors of laryngeal and pharyngeal cancer was examined by real-time quantitative RT-PCR. Overall survival rates were measured using Kaplan-Meier estimates and the log-rank tests. The adjusted hazard rate ratios (HRRs) were computed by multivariate Cox regressions. RESULTS: Reduced ATM mRNA was found in 65 of 80 studied cases. Lower ATM expression [tumor/normal <0.3, HRR = 2.49; 95% confidence interval (CI) 1.27-4.88], younger age (<55 years, HRR = 2.71; 95% CI 1.16-6.32), and larger tumor (T(3)/T(4), HRR = 2.21; 95% CI 1.10-4.44) were independent risk factors for survival. Patients with lower ATM and younger age (HRR = 6.51; 95% CI 2.05-20.66) or with lower ATM and T(3)/T(4) tumor (HRR = 5.23; 95% CI 2.04-13.40) exhibited the poorest outcome. CONCLUSION: The expression of ATM mRNA, which is frequently downregulated in laryngeal and pharyngeal cancers, could be a valuable prognostic marker.

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

Visualization of Rab3A dissociation during exocytosis: a study by total internal reflection microscopy.

Rab3A is a small G protein in the Rab3 subfamily, and is thought to act at late stage of exocytosis. However, the detailed mechanism of its action is not completely understood. To study the role of Rab3A in exocytosis, we used a total internal reflection fluorescence microscope to examine the fluorescence changes of EGFP-Rab3A-labeled and NPY-EGFP-labeled vesicles in PC12 cells upon stimulation. The fluorescence of EGFP-Rab3A-labeled and NPY-EGFP-labeled vesicles decreased while showing different patterns. The NPY-EGFP-labeled vesicles that exocytosed showed a transient fluorescence increase before NPY-EGFP fluorescence disappearance, which represents fusion and NPY release. This transient increase was diminished in cells that co-expressed the GDP-bound Rab3A mutant. The fluorescence of EGFP-Rab3A-labeled vesicles dispersed before disappearance, which represents the dissociation of Rab3A from the vesicles. The dispersion was not found in GTP-bound Rab3A mutant-labeled vesicles. Interestingly, EGFP-Rab3A F59S, a mutant unable to bind rabphilin, dissociates slower from the vesicles than wild type Rab3A and caused a slower release of NPY-EGFP. The results provide direct evidence to support the hypothesis that GTP hydrolysis and rabphilin are involved in Rab3A dissociation from the vesicles and the occurrence of exocytosis.

Transurethral microwave thermotherapy for symptomatic benign prostatic hyperplasia: long-term durability with Prostcare.

OBJECTIVE: To evaluate the long-term durability of transurethral microwave thermotherapy (TUMT) with Prostcare for symptomatic benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: From August 1993 to July 1994, a total of 65 patients with symptomatic BPH who underwent TUMT using the Prostcare apparatus (Bruker Spectospin, Wissembourg, France) with low-energy protocol (maximal power 52 W) were enrolled into a short-term evaluation. Subsequent follow-up information was collected in July 1999. If patients had had any further therapy for BPH, the date of retreatment was considered as an endpoint of TUMT efficacy. If no further therapy for BPH had been needed, they were re-assessed for overall satisfaction. RESULTS: The median follow-up period was 49 months. Twenty patients were excluded for various reasons, including 17 with loss of follow-up and 3 with new diseases that could affect the voiding status. Thirty-eight (84.4%) of 45 valuable patients had received further therapy for BPH, including medication (n = 21, 46.7%), and endoscopic surgery (n = 17, 37.7%). The times to pharmacologic or endoscopic retreatment after TUMT were 8.9+/-11.1 and 23.0+/-14.4 months, respectively (p = 0.0003, log rank test). Only 7 (15.5%) patients had no further treatment, with 3 having satisfactory improvements, but 4 feel dissatisfied yet not needing any further therapy. In addition, 2 patients complained of erectile dysfunction after TUMT and 1 was diagnosed with prostate cancer 50 months after TUMT. In addition, there was no significant difference for all baseline values among three groups with no retreatment or retreatment with medication or endoscopic surgery. CONCLUSION: At the 5-year follow-up, the long-term durability of low-energy TUMT with Prostcare is only exhibited in a few patients and the overall retreatment rate was 84.4%. Thus, patient should be informed of the high probability of supplementary treatment after TUMT.

Transurethral microwave thermotherapy for symptomatic benign prostatic hyperplasia: short-term experience with Prostcare.

PURPOSE: To assess our short-term experience with transurethral microwave thermotherapy (TUMT) for symptomatic benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: From August 1993 through July 1994, in total 65 patients with symptomatic BPH were enrolled into this study. The patients' ages ranged from 56 to 95 years with a mean of 70 years. Under local anesthesia with intraurethral instillation of Xylocaine jelly only, all patients received one session of TUMT for up to 60 min with Prostcare equipment. Uroflowmetry was performed and international prostatic symptom score (IPSS) determined before 3 and 6 months after TUMT for assessment of efficacy. All adverse events were recorded and evaluated for clinical relevance. RESULTS: At 3 and 6 months following TUMT, the mean IPSS decreased from 19.7 +/- 6.8 (baseline) to 12.8 +/- 8.2 (-46%) and to 15.5 +/- 9.0 (-21%), respectively; the maximal urine flow rate at 3 and 6 months increased from 9.1 +/- 4.8 ml/s (baseline) to 11.0 +/- 4.9 ml/s (+21%) and to 10.9 +/- 5.6 ml/s (+19%), respectively. During TUMT, burning sensation was the most frequent complaint (38.5%), followed by urethral discomfort (29.2%) and urgency (9.2%). Two patients (3.1%) interrupted TUMT, because of intolerable pain. Following TUMT micturition pain (73.8%) and gross hematuria (45.9%) were the most adverse events. Most of these adverse events disappeared within 2 weeks. One patient suffered from skin erosion over the penoscrotal junction 1 week later. None had retrograde ejaculation; 1 patient complained of erectile dysfunction. CONCLUSION: Although the efficacy of TUMT with Prostcare became less prominent 6 months after TUMT, TUMT was still a tolerable, safe alternative treatment of BPH, especially in patients who were not suitable for transurethral resection of the prostate or anesthesia.

Postoperative administration of interleukin-12 significantly enhances the anti-tumor immune response of MBT-2 bladder cancer bearing mice.

This study, using the MBT-2 murine bladder tumor model, mainly investigated the role of interleukin-12 (IL-12) in the specific antitumor immune response of a tumor-bearing host when systemically administrated after surgery. Day 17 tumor-bearing mice (D17TBM) along with non-tumor bearing naive mice were treated with daily intraperitoneal (i.p.) injection of IL-12 (0.25 microg/mouse) from day 18 to day 24 for a total of 7 doses. Their splenocytes were obtained on Day 31 for natural killer cells (NK), lymphokine activated killer cells (LAK) and cytotoxic T lymphocyte (CTL) activity assay and lymphocyte subsets phenotypic analysis. The tumor suppression effect of systemic IL-12 administration was evaluated based on the tumor outgrowth of the higher number of tumor cells rechallenged 24 hours after resectioning of the primary tumor. After evaluation on Day 31, the result of in vitro cytotoxicity assay revealed that systemic administration of IL-12 mainly enhanced the splenic LAK and CTL activities in non-tumor-primed naive mice, and the NK activity in tumor-primed D17TBM, respectively. However, in vivo administration of IL-12 with or without IL-2 failed to upgrade the proportions of either CD4+ CD44+ or CD8+ CD44+ T cells subsets in the spleens and regional inguinal lymph nodes (LNs) of both the D17TBM and naive mice. However, the splenic CD8+ CD44+ T-cell subset in the IL-12-treated D17TBM increased prominently after further culturing in the presence of IL-2 400 units/ml plus IL-12 25 ng/ml for 4 days. The fact that systemic administration of IL-12 significantly suppressed the outgrowth of Day-18 challenged tumor cells, especially in D17TBM, clearly indicates that the established specific antitumor immunity in tumor-primed D17TBM was efficiently augmented. From the results of this study, we conclude that, after surgical resection of a primary tumor, systemic administration of IL-12 can be an effective adjuvant therapy because it demonstrates a significant augmentation effect on the tumor-specific immune response in the tumor-primed host.

Modulating the antitumor immunity of MBT-2 murine bladder tumor bearing mice by postoperative administration of interferon-alpha.

This study was conducted mainly to investigate the effect of interferon-alpha (IFN-alpha) on the antitumor immunity of a tumor bearing host (TBH) when postoperatively administrated with or without lethally irradiated autologous tumor cells. Using the C3H/He-MBT-2 murine bladder tumor model, a status of postoperative residual tumor was mimicked by rechallenging tumor cells 24 hours after resecting the day-17 tumor. Using immunohistochemical analysis we demonstrated that after treating with lethally irradiated MBT-2 tumor cells (IRMBT-2) + IL-2 cells of CD4+, CD8+, CD44+ and CD11b+ phenotypes prominently infiltrate the subcutaneous local injection sites. In contrast, only scanty immune responding cells could be seen locally if treated with IRMBT-2 + IFN-alpha 2b, albeit in the presence of interleukin-2 (IL-2). However, the spleens of D17TBM treated with IRMBT-2 + IFN-alpha 2b contained the highest percentage of CD44+ memory T cells and cells of the CD11b+ phenotype; moreover, their natural killer (NK), lymphokine activated killer (LAK) and cytotoxic T lymphocytes (CTL) activities were significantly augmented. The results of in vivo tumor rechallenge revealed that administration of IFN-alpha, either alone or combined with IRMBT-2, could both effectively suppress the outgrowth of perioperative rechallenged tumor cells as well as prolong the survival of TBH. We conclude that despite the presence of autologous tumor vaccine, postoperative administration of IFN-alpha can further enhance the antitumor immunity of TBH and therefore can be an effective adjuvant therapy to improve the therapeutic results of surgery on a tumor bearing host.

Unilateral pulmonary edema during general anesthesia--report of two cases.

Unilateral presentation of pulmonary edema, though well known to occur, is an uncommon entity. Previous reviews of this subject have discussed the different etiologies, which include rapid reexpansion of collapsed lung, down lung syndrome (gravitational edema), systemic-to-pulmonary arterial shunts, heart failure, compression or occlusion of pulmonary vasculatures, obstruction of a bronchus and an acute manifestation of neuropulmonary reaction (neurogenic pulmonary edema). Occurrence of this complication during surgery, however, is even rarer. We report 2 cases of unilateral pulmonary edema occurring during general anesthesia for elective surgery.

Supplementation with vitamins C and E enhances cytokine production by peripheral blood mononuclear cells in healthy adults.

The effect of supplementation with vitamins C and E on cytokine production of healthy adult volunteers was studied in a single-blind trial. Ten subjects in each group received daily vitamin C (1 g ascorbic acid), vitamin E (400 mg dl-alpha-tocopheryl acetate), or vitamins C and E for 28 d. Plasma concentrations of alpha-tocopherol, ascorbate, and lipid peroxides as well as the production of cytokines by peripheral blood mononuclear cells (PBMCs) were measured before, during, and at the end of the supplementation and 1 wk later. PBMCs were cultured in the presence of absence of lipopolysaccharide for 24 h. The interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) in the culture supernates were assayed by enzyme-linked immunosorbent assay methods. Production of IL-1 beta and TNF-alpha in the group supplemented with vitamins C and E was significantly higher (P < 0.05) than that of the groups given vitamin E or vitamin C alone. The enhancing effect of supplementation with a combination of vitamins E and C coincided with peak plasma alpha-tocopherol and ascorbate concentrations and the lowest plasma lipid peroxide concentrations (P < 0.05) on day 14. In addition, an in vitro experiment with PBMCs showed that vitamins E and C reduced lipopolysaccharide-induced prostaglandin E2 production and enhanced TNF-alpha production. These results indicate that combined supplementation with vitamins C and E is more immunopotentiating than supplementation with either vitamin alone in healthy adults.

Metabolic development in erythropoietin-dependent maturation of erythroid cells.

1. Metabolic development was investigated in splenic erythroblasts isolated from Friend virus infected mice and maintained in culture with erythropoietin. 2. We found that immature erythroid cells not only possess a highly oxidative metabolism, but also glycolytic activity, both of which decrease drastically in the course of maturation. 3. Most purine and pyrimidine compounds which are found in immature erythroid cells are still present in mature erythrocytes. 4. While total adenine nucleotide levels fall, the 2,3-DPG pool increases during erythroid development.

Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells.

A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles.

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.