Inhibition of microRNA-449a prevents IL-1ß-induced cartilage destruction via SIRT1.

OBJECTIVE: SIRT1 has anti-inflammatory as well as protective effects in chondrocytes. The object of this study was to investigate whether microRNA-449a regulates expression of SIRT1, which inhibits expression of catabolic genes in IL-1ß-induced cartilage destruction. MATERIALS AND METHODS: MicroRNA-449a expression was determined in OA chondrocytes and IL-1ß-induced chondrocytes by real-time PCR. MicroRNA-449a binding sites on the 3'-UTR of SIRT1 mRNA and binding site conservation were examined using microRNA target prediction tools. SIRT1-overexpressing or knockdown chondrocytes were transfected with microRNA-449a or anti-microRNA-449a mimic and stimulated by IL-1ß. Expression of catabolic and anabolic genes was examined by real-time PCR and western blotting. Finally, positive effects of anti-microRNA-449a on expression of these genes were confirmed by western analysis of OA chondrocytes. RESULTS: Expression of microRNA-449a was increased in OA chondrocytes and IL-1ß-induced chondrocytes. MMP-13 expression was enhanced, whereas type II collagen and SIRT1 expression were decreased in IL-1ß-induced chondrocytes. SIRT1 overexpression resulted in decreased expression of catabolic genes such as MMPs and ADAMTSs in response to IL-1ß, but these effects were moderated by microRNA-449a. Suppression of microRNA-449a by anti-microRNA-449a inhibited expression of catabolic genes despite IL-1ß stimulation, but these effects were abolished in SIRT1 knockdown chondrocytes. Furthermore, expression of catabolic genes was decreased and expression of type II collagen as well as SIRT1 was restored by anti-microRNA-449a in OA chondrocytes as well as in IL-1ß-induced chondrocytes. CONCLUSION: Silencing of microRNA-449a had a protective effect, inhibiting catabolic gene expression and restoring anabolic gene expression, by targeting SIRT1 in IL-1ß-induced cartilage destruction.

Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53-SIRT1 axis.

NRF2 (nuclear factor erythroid-derived 2-like 2) plays an important role in defense against oxidative stress at the cellular level. Recently, the roles of NRF2 in embryonic and adult stem cells have been reported, but its role in maintaining self-renewal and differentiation potential remains unknown. We studied the mechanisms of NRF2 action in mesenchymal stem cells (MSCs) derived from human bone marrow. We found that the cellular localization of NRF2 changed during prolonged cell passage and osteogenic differentiation. Blocking the nuclear import of NRF2 using ochratoxin A (OTA) induced the loss of the self-renewal and osteogenic potential of early-passage (EP) MSCs. Conversely, reinforcing the nuclear import of NRF2 using tert-butylhydroquinone (t-BHQ) improved the self-renewal capacity and maintained the differentiation potential in the osteogenic lineage of EP MSCs. Real-time quantitative PCR and western blot analysis showed that NRF2 positively regulates sirtuin 1 (SIRT1) at the mRNA and protein levels via the negative regulation of p53. The self-renewal and osteogenic potential suppressed in OTA-treated or NRF2-targeting small hairpin RNA (shRNA)-infected EP MSCs were rescued by introducing small interfering RNA (siRNA) targeting p53. t-BHQ treatment in late-passage (LP) MSCs, which lost their self-renewal and osteogenic potential, reversed these effects. In LP MSCs treated with t-BHQ for ∼7 days, the phosphorylation and nuclear localization of NRF2 improved and SIRT1 protein level increased, whereas p53 protein levels decreased. Therefore, our results suggest that NRF2 plays an important role in regulating p53 and SIRT1 to maintain MSC stemness. This study is the first to establish a functional link between NRF2 and SIRT1 expression in the maintenance of MSC self-renewal and differentiation potential.

Difference of regeneration potential between healthy and diseased liver.

BACKGROUND: We sought to evaluate total and segmental liver regeneration by comparing preoperative computed tomographic (CT) volumetry and CT volumetry on postoperative day (POD) 7 after a right hepatectomy, in patients with various status and surgical indications. METHOD: We included 36 patients who underwent right lobectomy for living donor liver transplantation (healthy group), and 29 for hepatocellular carcinoma treatment (disease group). All of the disease group patients were Child-Turcotte-Pugh (CTP) class A. The regeneration of lateral, medial segment and total remnant liver volumes were assessed on POD 7 using a CT-based program. Total volumes and segmental volumes were measured for total liver, future liver remnant (FLR), and liver remnant. We calculated total and segmental early regeneration indexes, defined as [(VLR-VFLR)/VFLR]×100, where VLR is volume of the liver remnant and VFLR is volume of the FLR. RESULT: The VLR at POD 7 showed a 72.9% increase in volume among the healthy versus 55% in the disease group, (P=.012) In the disease group, segmental volume and regeneration indexes were also significantly lower than among the healthy group: 59.0% versus 46.9% in the medial and 86.8% versus 57.7% in the lateral segment (P=.023 and P<.001) respectively. CONCLUSION: The volume regeneration potential in diseased livers is significantly lower than that of a normal, healthy liver. So, we must consider a patient's liver status and volume profile before an extensive liver.

Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture.

OBJECTIVES: This study has aimed to repopulate 'primitive' cells from late-passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. MATERIALS AND METHODS: Effects of low density culture compared t high density culture on late-passage bone marrow (BM)-derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. RESULTS AND CONCLUSIONS: We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2.

Dominance specific visual extinction associated with callosal disconnection.

Callosal disconnection signs are closely related to asymmetric hemispheric specialization of cognitive functions. Although extinction is more commonly associated with the right parietotemporal lesion, it may occur following lesions of the corpus callosum. After an infarction involving the left splenium, a 58-year-old right-handed man had no disconnection symptoms that had been reported earlier, but showed visual extinction with left or right visual hemifield dominant stimuli. Our results suggest that dominance specific visual extinction might be another disconnection sign associated with splenial lesion.

Continuous removal of hydrogen peroxide with immobilised catalase for wastewater reuse.

Hydrogen peroxide was continuously removed for wastewater reuse using an immobilised biocatalyst. A commercial catalase, which is an enzyme to decompose hydrogen peroxide to water and oxygen, was entrapped in chitosan beads. Hydrogen peroxide in aqueous solutions of varying pH, temperature and concentration was continuously removed through a reactor containing the catalase-entrapped chitosan beads at high efficiency for 24 h. Additional silicate coating of the chitosan beads resulted in significant improvements in the catalase performance under harsh conditions, which are often found in peroxide-based industrial processes. We expect that immobilisation of catalases can enhance their applicability for continuous degradation of hydrogen peroxide for wastewater reuse.

Association of maspin expression with the high histological grade and lymphocyte-rich stroma in early-stage breast cancer.

AIMS: Maspin is a recently described member of the serpin family or protease inhibitors that is known to be a tumour suppressor gene product. Loss of maspin expression has been found in most breast cancer cases and is correlated with cell motility and tumour invasiveness. However, its precise role in human breast cancer remains to be discovered. We aimed to evaluate the role of maspin in early-stage breast cancer. METHODS AND RESULTS: We analysed the expression of maspin in 192 stage I and II primary breast cancers by immunohistochemistry. Of these cases, 34.4% showed maspin expression. Maspin expression was more frequently found in invasive ductal carcinoma (36.4%) than in invasive lobular carcinoma (7.1%). High maspin expression was demonstrated in breast cancers showing high histological grade or lymphocyte-rich stroma (P < 0.05). Maspin expression was not associated with overall and disease-free survival rate of breast cancer. CONCLUSIONS: The results indicate that different biological mechanisms may be responsible for maspin expression in histologically distinct types of breast cancer. Our survey suggests that maspin expression in breast cancer might have a compensatory role rather than prognostic one.

Genetic mapping and DNA sequence-based analysis of deleted regions on chromosome 16 involved in progression of bladder cancer from occult preneoplastic conditions to invasive disease.

Histologic and genetic mapping with 30 hypervariable markers mapped to chromosome 16 were performed on 234 DNA samples of five cystectomy specimens from patients with invasive bladder cancer. Allelic losses of individual markers were related to microscopically identified precursor conditions in the entire bladder mucosa and invasive cancer. Their significance for the development and progression of neoplasia from in situ preneoplastic conditions to invasive disease was analysed by the nearest neighbor algorithm and binomial maximum likelihood analysis. Using this approach we identified five distinct regions of allelic losses defined by their flanking markers and predicted size as follows. p13.3(D16S418-D16S406, 1.2 cM), p13.1(D16S748-D16S287, 12.9 cM), q12 1(D16S409-D16S514, 24.0 cM), q22.1 (D16S496-D16S515, 5.4 cM), and q24 (D16S507-D16S511, 5.9 cM and D16S402-D16S413, 17.4 cM). The regions mapping to p13.1 and q24 were involved in early intraurothelial phases of bladder neoplasia such as mild to moderate dysplasia. On the other hand the deleted region mapping to p13.3 was involved in progression of severe dysplasia/carcinoma in situ to invasive bladder cancer. Testing of markers that exhibited statistically significant LOH in relation to progression of neoplasia from precursor conditions to invasive cancer on 28 tumors and voided urine samples from 25 patients with bladder cancer revealed that q12.1 showed LOH in 46.4% of tumor and 32.0% of voided urine samples. The LOH of a single marker D16S541 could be detected in approximately 28% of tumors and 20% of voided urine samples of patients with bladder cancer. These data imply that the deleted region centered around marker D16S541 spanning approximately 10 cM and flanked by D16S409 and D16S415 contains a novel putative tumor suppressor gene or genes playing an important role in the development of human bladder cancer. To facilitate more precise positional mapping and identification of pathogenetically relevant genes, we analysed of human genome contig and sequence databases spanning the deleted regions. Multiple known candidate genes and several smaller gene-rich areas mapping to the target regions of chromosome 16 were identified.

Mapping and genome sequence analysis of chromosome 5 regions involved in bladder cancer progression.

We studied the evolution of allelic losses on chromosome 5 by whole-organ histologic and genetic mapping in 234 mucosal DNA samples of 5 cystectomy specimens with invasive bladder cancer and preneoplastic changes in adjacent urothelium. The frequency of alterations in individual loci was verified on 32 tumors and 29 voided urine samples from patients with bladder cancer. Finally, deleted regions on chromosome 5 were integrated with the human genome contigs and sequence-based databases. Deleted regions on chromosome 5 involved in intraurothelial phases of bladder neoplasia defined by their nearest flanking markers and predicted size were identified as follows: q13.3-q22 (D5S424-D5S656; 38.8 centimorgan [cM]); q22-q31.1 (D5S656-D5S808; 19.2 cM), q31.1-q32 (D5S816-SPARC; 11.5 cM), and q34 (GABRA1-D5S415; 6.4 cM). The two most frequently deleted neighbor markers (D5S2055 and D5S818) mapping to q22-q31.1 defined a 9 cM region, which may contain genes that play an important role in early phases of urinary bladder carcinogenesis. Human genome database analysis provided an accurate map of deleted regions with positions of 138 known genes and revealed several smaller gene-rich areas representing putative targets for further mapping. The strategic approach presented here, which combines whole-organ histologic and genetic mapping with analysis of the rapidly emerging human genome sequence database, facilitates identification of genes potentially involved in early phases of bladder carcinogenesis.

Composite neuroendocrine and adenocarcinoma of the common bile duct associated with Clonorchis sinensis: a case report.

The biliary tract has neuroendocrine cells of endoderm origin similar to the gastrointestinal tract, however neuroendocrine tumors of the biliary tract are rare. We report a composite glandular-endocrine cell carcinoma of the common bile duct in a 64-year-old Korean man which was associated with Clonorchis sinensis. The patient complained of right upper quadrant abdominal pain. Several parasites of Clonorchis sinensis were removed during the percutaneous transbiliary drainage. Endoscopic retrograde cholangiopancreatography revealed a polypoid mass (3 x 3 cm) with central ulceration in the common bile duct. Pancreaticoduodenectomy was performed. Microscopic examination of the tumor revealed a composite small cell neuroendocrine carcinoma and adenocarcinoma. The small cell carcinoma component showed positive reaction to chromogranin A and neuron-specific enolase and it was located mainly in the deeper portion of the mass. The well-differentiated adenocarcinoma component showed a positive reaction to carcinoembryonic antigen and it was situated in the superficial portion of the mass. Exclusively, the small cell component metastasized to the lymph node. It is suggested that this tumor could arise from a multipotential stem cell and showed neuroendocrine and glandular differentiation and that Clonorchis sinensis could be a predisposing factor, as in cholangiocarcinoma.

Exercise therapy effect on natural killer cell cytotoxic activity in stomach cancer patients after curative surgery.

OBJECTIVE: To evaluate the effect of early exercise therapy on the natural killer cell cytotoxic activity (NKCA) of patients who had undergone curative resection of stomach cancer. DESIGN: Prospective study. PATIENTS: Thirty-five stomach cancer patients who had undergone curative surgery were randomly divided into an exercise group (n = 17) and a control group (n = 18). INTERVENTION: From postoperative day 2, moderated exercise using arm and bicycle ergometers performed twice a day, 5 times a week, for 14 days. The intensity of exercise was 60% of maximal heart rate. Venous blood samples were obtained on postoperative days 1, 7, and 14. MAIN OUTCOME MEASURE: Mean sequential change of NKCA. RESULTS: The mean sequential change of NKCA decreased until postoperative day 7 and then increased. Mean NKCA of day 7 decreased in both groups, compared with that at postoperative day 1. At day 14, the mean NKCA of the exercise group demonstrated a significant increase compared with that of the control group (p < .05). CONCLUSION: This study suggests that early moderate exercise has a beneficial effect on the function of in vitro NK cells in stomach cancer patients after curative surgery.

Rupture of the rectosigmoid colon with evisceration of the small bowel through the anus.

Spontaneous rupture of the rectosigmoid colon and herniation of the small intestine through the rupture site and eventual evisceration through the anus is a very rare event. In the literature, only 42 cases have been reported. The majority of them occurred in patients with rectal prolapse and one case was reported in association with a third-degree uterine prolapse. We experienced an 81-year-old female patient with rectal prolapse and second-degree uterine prolapse complicated by spontaneous perforation of the rectosigmoid colon and anal evisceration of the small intestine. Segmental resection of the nonviable small intestine, primary repair of the ruptured rectosigmoid colon, and sigmoid loop colostomy were performed, and the patient recovered well. In our patient, both rectal and uterine prolapses cooperatively damaged the anterior wall of the rectosigmoid colon and resulted in perforation. So, rectal and uterine prolapses should be treated before the complication develops. In this patient, uterine prolapse should be treated because of the recurrence of this rare episode.

Reduced expression of the cell-cycle inhibitor p27Kip1 is associated with progression and lymph node metastasis of gastric carcinoma.

AIMS: p27Kip1 (p27), a cyclin-dependent kinase inhibitor, plays an important role as inhibiting the progression of the cell cycle. Decreased expression of p27 is associated with high histological grade and aggressiveness of several human tumours. We aimed to evaluate the role of p27 in the progression and metastasis of gastric carcinoma. METHODS AND RESULTS: We analysed the expression of p27 in 67 primary gastric carcinomas and 31 lymph node metastases by immunohistochemistry. Reduced expression of p27 was found more frequently in advanced gastric cancer (40.9%) than in early gastric cancer (15.6%) (P < 0.001). Decreased p27 expression correlated with large tumour size, high histological grade, lymphatic invasion, advanced stage, deep invasion, lymph node metastasis and recurrence. The expression of p27 showed an inverse correlation with the Ki67 labelling index. There was a significant reduction of p27 expression in metastatic tumour cells in lymph nodes (mean positive cells: 3. 7%) when compared to the corresponding primary gastric carcinomas (mean positive cells: 8.1%) (P = 0.008). CONCLUSIONS: Alterations of p27 expression may play an important role in the progression and metastasis to lymph node of tumour cells in human gastric carcinoma.

Expression of biliary antigen and its clinical significance in hepatocellular carcinoma.

In order to classify the hepatocellular carcinomas (HCCs) which had diverse clinicopathologic characteristics, we divided HCCs into two groups according to the expression of biliary antigen on the basis of the hypothesis that the hepatocyte and biliary epithelial cell originate from the same precursor cell, and then we investigated the clinical and pathologic characteristics in the two groups. Forty HCC cases with no preoperative treatment and at least two-year follow-up data were selected among 202 cases of HCC files from 1991 to 1995. Expression of biliary antigen (AE1, cytokeratin 19), p53, AFP, and Ki-67 in the tumor tissue were assessed by immunohistochemistry. Positive cytokeratin 19 was noted in one case (2.5%); AE1 was detected in 40% of patients; p53 was overexpressed in 20% of patients; and AFP was detected in 45% of patients. No statistical difference between the biliary antigen positive group (16 cases) and the negative group (24 cases) were noted in terms of mean age, sex, presurgical serum AFP level, Child class, and tumor size. HBsAg positive rate was 66.7% for the biliary antigen (-) group and 93.8% for the biliary antigen (+) group with a statistically significant difference (p = 0.048). The number of cases for Edmonson-Steiner grade I/II and III/IV were 15 and 9 in the biliary antigen (-) group, and 4 and 12 in the biliary antigen (+) group, respectively, with a statistically significant difference (p = 0.024). The 1, 3 and 5-year disease-free survival rates were 69.7, 40.9 and 40.9% for the biliary antigen (-) group and 73.7, 39.1, 39.1% for the biliary antigen (+) group with no statistically significant difference. The 1, 3 and 5-year overall survival rates were 91.7, 73.8, 66.4% for the biliary antigen (-) group and 68.8, 34.4, 34.4% for the biliary antigen (+) group, with a significantly greater overall survival rate for the biliary antigen negative group (p = 0.045). Poor histopathological differentiation, a high HBsAg positive rate and poor overall survival rate were noted in the biliary antigen positive group and the differences were statistically significant. In conclusion, HCCs with positive biliary antigen, which originates from more primitive cells, is suggested to be more aggressive than HCCs with negative biliary antigen.

Non-alcoholic duct-destructive chronic pancreatitis: recognition before definitive treatment.

Non-alcoholic duct-destructive chronic pancreatitis is a new entity that differs morphologically and pathogenetically from alcoholic chronic pancreatitis. Some clinical and imaging features of this entity resemble those of pancreatic cancer, and hence most of the reported cases underwent pancreatic resections including an invasive pancreaticoduodenectomy. Recognition of this new entity before a definitive treatment is therefore important to avoid an unnecessary pancreatic resection. Recently, we experienced a case of non-alcoholic duct-destructive chronic pancreatitis in an 80-year-old man presenting with obstructive jaundice and whose radiologic features were characteristic as originally described. Recognition of this new entity before definitive treatment enabled us to manage this patient optimally. In addition, the relation between non-alcoholic duct-destructive chronic pancreatitis and chronic pancreatitis with diffuse irregular narrowing of the main pancreatic duct is discussed.

Enhanced solubility and dissolution rate of itraconazole by a solid dispersion technique.

The aim of the present study was to improve the solubility and dissolution rate of a poorly water-soluble drug, itraconazole, by a solid dispersion technique. Solid dispersion particles of itraconazole were prepared with various pH-independent and -dependent hydrophilic polymers and were characterized by differential scanning calorimetry, powder X-ray diffraction and scanning electron microscopy. Of the polymers tested, pH-dependent hydrophilic polymers, AEA and Eudragit E 100, resulted in highest increases in drug solubility (range, 141.4-146.9-fold increases). The shape of the solid dispersion particles was spherical, with their internal diameter ranging from 1-10 microm. The dissolution rate of itraconazole from the tablets prepared by spray drying (SD-T) was fast, with > 90% released within 5 min.SD-T prepared with AEA or Eudragit E 100 at a 1:1 drug hydrophilic polymer ratio (w/w) showed approximately 70-fold increases in the dissolution rate over a marketed product.

Villous adenoma of the bile ducts: a case report and a review of the reported cases in Korea.

Villous adenomas are benign epithelial lesions with malignant potential which can occur at any site in the gastrointestinal tract. They are usually encountered in the rectum and colon, less frequently in the small bowel and very rarely in the biliary trees. Nine cases of bile duct villous adenomas have been reported in the literature. However, 4 cases of bile duct villous adenomas have been reported in the Korean literature. Recently, we experienced a case of villous adenoma in the common hepatic duct in a 77-year-old man presenting with obstructive jaundice in which preoperative histologic diagnosis of villous adenoma played a critical role in managing this patient. Herein, we present a case report of bile duct villous adenoma and a review of the reported cases in Korea to help define and manage this rare disease entity in the bile ducts. In addition, confusing nomenclature of bile duct adenomas is discussed.

Do dialysis patients need extra folate supplementation?

To assess folate status and to evaluate the need for conventional folate supplementation in patients on dialysis, we measured serum folate, vitamin B12, and red cell folate concentrations by radioimmunoassay. Thirty-four continuous ambulatory peritoneal dialysis (CAPD) patients and 60 hemodialysis (HD) patients who had not been supplemented with folate were enrolled. Serum folate levels (5.8 +/- 3.6 ng/mL vs 2.0 +/- 1.1 ng/mL, p < 0.001) and vitamin B12 levels (831.4 +/- 416.9 pg/mL vs 513.9 +/- 213.3 pg/mL, p < 0.001) were significantly higher in CAPD patients than HD patients. The red cell folate levels (849.7 +/- 489.4 ng/mL vs 491.0 +/- 253.2 ng/mL, p < 0.001) were also significantly higher in CAPD patients. The incidences of folate deficiency in CAPD and HD patients were overestimated using the cut-off value for serum folate concentration (3.0% vs 71.7%, respectively), but the incidence of true folate deficiency was lower using the cut-off value for red cell folate level (0.0% vs 10.0%, respectively). In conclusion, the true incidence of folate deficiency in stable CAPD and HD patients is surprisingly low, even in patients who may not be taking folate supplements. The need for conventional folate supplementation in patients with end-stage renal disease on dialysis must therefore be re-evaluated. Before the decision is made to use folate supplementation, measurement of red cell folate is essential to assess of folate reserves of the patients on dialysis.

Cell proliferation index and the expression of p53 and Bcl-2 in tumorous and non-tumorous lesions of hepatocellular carcinoma and metastatic liver cancer.

In the development of a cancer, unlimited cell proliferation has been believed to play an important role. In addition, a programmed cell death called apoptosis, which is regulated by several oncogenes and tumor suppressor genes, has been suggested to be another important different pathway of carcinogenesis. Recently, several reports on cell proliferation capacity and apoptosis in the development of human liver disease have been published, but the cell proliferation index and its relationship between the expression of the bcl-2 and p53 genes involving apoptosis has not yet been discussed in view of the clinical differences of primary and metastatic liver cancer. In this study, we investigated the cell proliferation index and expression of p53 and bcl-2 in the tumorous and non-tumorous portions of both hepatocellular carcinoma and metastatic liver cancer. The expression of p53 was observed in both hepatocellular carcinoma and metastatic liver cancer, but bcl-2 expression was observed neither in hepatocellular carcinoma nor in metastatic liver cancer. In hepatocellular carcinoma, the p53 positive group showed a higher Ki-67 score (cell proliferation index) and more tumor numbers than the p53 negative group (p < 0.05). In metastatic liver cancer, the results were the same as in hepatocellular carcinoma (p < 0.05). However, we could not correlate the p53 expression and its prognostic significance in hepatocellular carcinoma.