Resveratrol attenuates early diabetic nephropathy by down-regulating glutathione s-transferases Mu in diabetic rats.

Diabetic nephropathy (DN) is the major cause of end-stage renal disease. Resveratrol has been shown to ameliorate hyperglycemia in diabetic rats. However, the effects of resveratrol on DN remain unknown. The aim of the present study is to investigate the effects of resveratrol on early-stage DN. Diabetes was induced by streptozotocin injection in male Wistar rats. The diabetic rats were treated with resveratrol at a dose of 20 mg/kg body weight for 8 weeks. Plasma glucose, creatinine, kidney/body weight ratio, and 24-h urinary protein were determined. The renal pathological changes were examined with periodic acid Schiff staining, and renal mesangial cells were cultured in high glucose concentrations with indicated concentrations of resveratrol (2.5, 5.0, and 10.0 µmol/L). The proliferation of mesangial cells was evaluated by methylthiazoletetrazolium assay. Expressions of glutathione S-transferases Mu (GSTM) and nuclear factor erythroid 2-related factor 2 (Nrf2) were detected by western blot, and apoptosis was analyzed using a flow cytometer. Resveratrol reduced plasma glucose, creatinine, and urinary protein excretion, and attenuated renal hypertrophy. Moreover, resveratrol also reduced the expression of GSTM in diabetic rats. In vitro, resveratrol inhibited the proliferation of mesangial cells caused by high glucose and down-regulated GSTM and Nrf2 expressions in a dose-dependent manner. These findings suggest that resveratrol help prevent the progression of DN. The renoprotection by resveratrol is in part mediated through the inhibition of high glucose-induced rat mesangial cell proliferation and downregulation of GSTM expression.

Expression of interleukin-17 associated with disease progression and liver fibrosis with hepatitis B virus infection: IL-17 in HBV infection.

BACKGROUND/OBJECTIVES: As a proinflammatory cytokine, interleukin-17 (IL-17) contributes to the inflammation of many autoimmune diseases. We examined IL-17 levels in serum and tissues from patients with chronic hepatitis B virus infection (HBV), and especially evaluated the role of IL-17 in the pathogenesis and progression of liver fibrosis. MATERIALS AND METHODS: Whole venous blood was obtained from four patient groups: chronic hepatitis B (CHB, n = 47), liver cirrhosis (LC, n = 49), primary hepatocellular carcinoma (PHC, n = 44), chronic liver failure (CLF, n = 33), and a normal control group (n = 20). HBsAg was positive in all patients. Liver biopsy samples were acquired from asymptomatic HBsAg carriers (ASC, n = 35), CHB (n = 57), and LC (n = 31) patients. We performed ELISA to measure IL-17 levels in serum samples, and used reverse RT-PCR to measure IL-17 mRNA levels in peripheral blood mononuclear cells (PBMC). IL-17 protein expression was detected in liver biopsy tissues by immunohistochemistry. RESULTS: Compared to normal controls, serum IL-17 protein and mRNA levels were significantly higher in the four infection groups. LC patients exhibited the highest serum IL-17 and PBMC mRNA levels. No significant differences were found between the other three groups. High levels of IL-17 were also observed in tissues from CHB and LC patients, compared to ASC. IL-17 expression was mainly located in the portal area and was positively correlated with inflammation grade and fibrosis stage. CONCLUSIONS: IL-17 expression was found to be increased with increasing degrees of liver fibrosis. This suggests that IL-17 may not only induce the inflammation, but also contribute to disease progression and chronicity. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5306959258322482.

[Capability of oocyte maturation in human cryopreserved ovarian tissue following xenografting].

OBJECTIVE: To investigate the development and maturation competence of oocytes retrieved from cryopreserved and transplanted human fetal ovarian tissue by techniques of tissue culture, inducing ovary, oocyte retrieval, and in vitro maturation (IVM). METHODS: Fetal ovaries of 20 weeks were frozen-thawed and cultured for 6 days in vitro, then xenografted into kidney capsules of immunodeficient mice. All mice were stimulated with follicle stimulating hormone every second day for 23 weeks, starting 1 week after grafting. Then oocytes were retrieved from antral follicles 13 hours after human chorionic gonadotrophin injection. IVM was performed to evaluate the maturation competence of the oocytes from ovarian grafts. Human fetal ovarian tissues were examined with histological and proliferating cell nuclear antigen (PCNA) evaluation. RESULTS: There was no difference between fresh ovarian tissues and frozen-thawed ovarian tissues in the percentage of follicles at different growth stages (P > 0.05). The proportion of the primary follicles and preantral follicles in the cultured ovarian tissues was significantly larger than that of fresh ovarian tissues and frozen-thawed ovarian tissues (P < 0.05). The proportion of the primary follicles, preantral follicles, and antral follicles in the transplanted ovarian tissues was significantly higher than that of cultured ovarian tissues, fresh ovarian tissues, and frozen-thawed ovarian tissues (P < 0.05). No significant signals of PCNA in the primordial follicles in all ovarian tissues were observed. PCNA immunoreactivity first appeared in primary follicles. However, the obviously positive signals of PCNA were seen in the oocytes and/or the granular cells of cultured ovarian tissues and transplanted ovarian tissues. Oocytes from antral follicles were collected and matured in vitro, and 21.43% of the oocytes reached to MII within 48 hours IVM. CONCLUSIONS: Human ovarian follicles can survive and develop well after cryopreservation, tissue culture, and xenotransplantation. Furthermore, oocytes recovered from grafts have normal maturation competence.

Reversal of drug resistance in breast carcinoma cells by anti-mdr1 ribozyme regulated by the tumor-specific MUC-1 promoter.

Multidrug resistance (MDR) is a serious obstacle for cancer chemotherapy. The aim of this study was to reverse MDR of breast carcinoma cells specifically by degrading mdr1 mRNA with anti-mdr1 ribozyme. Our strategy was to limit the expression of ribozyme to only breast-derived cells, but not other type of cells. The results showed the recombinant ribozyme pEGFP-RZmuc was expressed in two kinds of breast carcinoma cells, but not in non-breast-derived cancer cells. Evaluation of chemosensitivity showed that a 15-fold reduction in drug resistance for Adriamycin and a 32-fold reduction in drug resistance for Vinblastine were observed in the transfected cells. Our results demonstrate the efficacy and selectivity of pEGFP-RZmuc to reverse MDR in drug resistant breast carcinoma cells in vitro.

Multivoxel 3D proton MR spectroscopy in the distinction of recurrent glioma from radiation injury.

OBJECTIVE: To explore the usefulness of multivoxel 3D proton MR spectroscopy ((1)H-MRS) in assessing the recurrent contrast-enhancing areas at the site of the previously treated gliomas. MATERIALS AND METHODS: In 28 patients who had new contrast-enhancing lesions in the vicinity of the previously resected and irradiated high-grade glioma, 3D (1)H-MRS examinations were performed on a 3.0T MR scanner. Spectral data for N-acetylaspartate (NAA), choline (Cho), and creatine (Cr) were analyzed in all patients. Receiver operating characteristic analysis was performed, and the threshold value for tumor differentiation was determined. Diagnosis of these lesions was assigned by means of histopathology and follow-up. RESULTS: Diagnostic-quality 3D (1)H-MRS with quantifiable Cho, Cr, and NAA peaks was obtained in 92.9% of the cases. The Cho/NAA and Cho/Cr ratios were significantly higher in recurrent tumor than in radiation injury (P < 0.01), whereas the NAA/Cr ratios were lower in recurrent tumor than in radiation injury (P = 0.02). The Cho/Cr and Cho/NAA ratios were significantly higher in radiation injury than in normal-appearing white matter (P < 0.01), however, the NAA/Cr ratios were lower in radiation injury than in normal-appearing white matter (P = 0.01). Using receiver operating characteristic analysis, the resulting sensitivity, specificity and diagnostic accuracy of 3D (1)H-MRS were 94.1%, 100%, and 96.2%, respectively, based on the cut-off values of 1.71 for Cho/Cr or 1.71 for Cho/NAA or both as tumor criterion. CONCLUSION: 3D (1)H-MRS could differentiate recurrent tumor from radiation injury in patients with recurrent contrast-enhancing lesions in the vicinity of the previously treated gliomas.

Distinction between recurrent glioma and radiation injury using magnetic resonance spectroscopy in combination with diffusion-weighted imaging.

PURPOSE: The aim of this study was to explore the diagnostic effectiveness of magnetic resonance (MR) spectroscopy with diffusion-weighted imaging on the evaluation of the recurrent contrast-enhancing areas at the site of treated gliomas. METHODS AND MATERIALS: In 55 patients who had new contrast-enhancing lesions in the vicinity of the previously resected and irradiated high-grade gliomas, two-dimensional MR spectroscopy and diffusion-weighted imaging were performed. Spectral data for N-acetylaspartate (NAA), choline (Cho), creatine (Cr), lipid (Lip), and lactate (Lac) were analyzed in conjunction with the apparent diffusion coefficient (ADC) in all patients. Diagnosis of these lesions was assigned by means of follow-up or histopathology. RESULTS: The Cho/NAA and Cho/Cr ratios were significantly higher in recurrent tumor than in regions of radiation injury (p < 0.01). The ADC value and ADC ratios (ADC of contrast-enhancing lesion to matching structure in the contralateral hemisphere) were significantly higher in radiation injury regions than in recurrent tumor (p < 0.01). With MR spectroscopic data, two variables (Cho/NAA and Cho/Cr ratios) were shown to differentiate recurrent glioma from radiation injury, and 85.5% of total subjects were correctly classified into groups. However, with discriminant analysis of MR spectroscopy imaging plus diffusion-weighted imaging, three variables (Cho/NAA, Cho/Cr, and ADC ratio) were identified and 96.4% of total subjects were correctly classified. There was a significant difference between the diagnostic accuracy of the two discriminant analyses (Chi-square = 3.96, p = 0.046). CONCLUSION: Using discriminant analysis, this study found that MR spectroscopy in combination with ADC ratio, rather than ADC value, can improve the ability to differentiate recurrent glioma and radiation injury.

Effect of different sites for cryopreserved ovarian tissue implantation in rabbit.

BACKGROUND: Autotransplantation of frozen-thawed ovarian tissue has proven to be an effective method to restore endocrine function and fertility. But it remains to be studied which site and which method is most effective and practical. We therefore implanted small pieces of cryopreserved ovarian tissues into different sites in rabbits to find the optimal position. METHODS: Fifteen New Zealand white female rabbits were randomly divided into three groups. In group 1, fresh ovarian tissues were implanted into the mesometrium and ovarian bursa. In group 2, cryopreserved ovarian tissues were implanted into the mesometrium and ovarian bursa. In group 3, cryopreserved ovarian tissues were implanted into the preserved ovary. RESULTS: There were no significant differences among the three groups as to the proportions of normal and morphologically changed follicles in implanted ovarian tissues. The implanted ovarian tissues in the three groups did not show any evident changes in histology and ultrastructure, and all resumed follicle development and revealed maturescent follicles. CONCLUSIONS: Cryopreservation and implantation of small pieces of ovarian tissues are feasible. Generally, the mesometrium, ovarian bursa and ovary are all available sites for implantation and have similar rates of acceptance, despite some differences in the details of implantation.

[Relationships among the expression of thymidylate synthase, thymidine phosphorylase, and dihydropyrimidine dehydrogenase and the prognosis of breast cancer].

OBJECTIVE: To investigate the relationships among the expression of thymidylate synthase (TS), thymidine phosphorylase (TP), and dihydropyrimidine dehydrogenase (DPD) and the prognosis of breast cancer. METHODS: Immunochemistry (ABC method) was used to detect the expression of TS, TP, and DPD in the samples of breast cancer resected during operation from 28 female patients. The microvessel density (MVD) in the cancer tissue was measured by immunochemistry (LSAB method). RESULTS: The TS positive rate was 30.26% and the TP positive rate was 21%, and the DPD positive rate was 30.03%. The expression levels of TS and TP were both correlated with the tumor size, lymph node status, histological grading of tumor, and microvessel count (MVC) (all P < 0.01), and was not correlated with age, status of estrogen receptors, status of prasterone receptors (all P > 0.05). The DPD expression was not correlated with the age, tumor size, lymph node status, histological grading of tumor, status of estrogen receptors, status of prasterone receptors, and MVC. The ten-year disease-free survival rate of the TS-positive patients was 0, significantly lower than that of the TS-negative patients (25%, P < 0.01). The ten-year overall survival rate of the TS-positive patients was 3.9%, significantly lower than that of the TS-negative patients (58.8%, P < 0.01). The ten-year disease-free survival rate of the TP-positive patients was 0, significantly lower than the TP-negative patients (25.4%, P < 0.01). The ten-year overall survival rate of the TP-positive patients was 0.9%, significantly lower than that of the TP-negative patients (61.8%, P < 0.01). The ten-year disease-free survival rate and ten-year overall survival rate of the DPD positive patients were not significantly different from those of the DPD negative patients (both P > 0.05). MCV and TS expression were strong protective factors of disease-free survival rate and overall survival rate. CONCLUSION: The levels of TS and TP are both prognostic indicis of breast cancer.

Alteration of cyclin D1 in gastric carcinoma and its clinicopathologic significance.

AIM: To detect the genetic alteration and abnormal expression of cyclin D1 in gastric carcinoma and investigate its clinicopathologic significance in advanced gastric carcinoma. METHODS: Proteins of cyclin D1 were detected by immunohistochemistry in 42 cases of advanced gastric carcinoma with their follow-up data available, 27 cases of early stage carcinoma, 21 cases of gastric adenoma, 22 cases of hyperplastic polyp and 20 cases of normal mucosa adjacent to adenocarcinomas. Genetic alteration of cyclin D1 was detected by Southern blot and expression of cyclin D1 mRNA was detected by PT-PCR in 42 cases of advanced gastric carcinoma. RESULTS: Cyclin D1 protein was not expressed in normal mucosa, hyperplastic polyp and gastric adenoma, while it was only positively expressed in gastric carcinoma. The expression rate of cyclin D1 protein in early stage gastric carcinoma, advanced gastric carcinoma and lymph node metastasis was 48.1%, 47.4% and 50.0%, respectively. The amplification of cyclin D1 gene was detected in 16.6% of advanced gastric carcinomas. The overexpression of cyclin D1 mRNA was detected in 40.5% of the samples. There was no significant correlation between cyclin D1 protein expression and age, lymph-node metastasis and histological grading in patients with advanced gastric carcinoma (chi2 = 0.038, 0.059, 0.241, P>0.05). Significant correlation was observed between the expression of cyclin D1 protein and the 5-year survival rate (chi2 = 3.92, P<0.05). CONCLUSION: Detection of cyclin D1 protein by immunohistochemistry may be useful in the diagnosis of early gastric carcinomas. Patients with positive expression of cyclin D1 protein tend to have a worse prognosis.

[Reversal of multidrug resistance of tumor cells by anti-mdr1 ribozyme].

OBJECTIVE: To stably reverse the multidrug resistance (MDR) of breast carcinoma cells in vitro. METHODS: Two anti-mdr-1 ribozyme plasmids, RZ196 and RZ179, were constructed with EGFP as reporter gene and transfected into drug-resistant breast carcinoma cells in vitro. The expression of EGFP was observed by laser confocal microscopy. Flow cytometry, RT-PCR and Rhodamine123 efflux assay were used to detect P-glyco protein (p-gp) and mdr-1 mRNA. RESULTS: After transfection with RZ196 and RZ179, the mdr-1 indices were reduced from 2.20 to 0.76 and 1.40, the expression rates of p-gp were reduced from 55.0% to 4.6% and 18.2%, the fluorescence intensity increased from 22.0% to 46.2% and 70.1%, TCL reduced from 75% to 28% and 43% respectively. In addition, the expression of ribozyme plasmid in tumor cells was stable under G418 selection. After two months, the mdr-1 indices remained at 0.81 and 1.47 in the cells transfected RZ196 and RZ179 respectively. The expression rates of p-gp were 5.2% and 19.5% and the Rh123 fluorescence intensity was 51.4% and 71.6% respectively. CONCLUSIONS: Both anti-mdr-1 ribozyme RZ196 and RZ179 can stably reverse MDR phenotype of breast carcinoma cells in vitro. RZ196 construct appears to be more effective.