Indoxyl sulphate-TNFα axis mediates uremic encephalopathy in rodent acute kidney injury.

Acute kidney injury (AKI) is often accompanied by uremic encephalopathy resulting from accumulation of uremic toxins in brain possibly due to impaired blood-brain barrier (BBB) function. Anionic uremic toxins are substrates or inhibitors of organic anionic transporters (OATs). In this study we investigated the CNS behaviors and expression/function of BBB OAT3 in AKI rats and mice, which received intraperitoneal injection of cisplatin 8 and 20 mg/kg, respectively. We showed that cisplatin treatment significantly inhibited the expressions of OAT3, synaptophysin and microtubule-associated protein 2 (MAP2), impaired locomotor and exploration activities, and increased accumulation of uremic toxins in the brain of AKI rats and mice. In vitro studies showed that uremic toxins neither alter OAT3 expression in human cerebral microvascular endothelial cells, nor synaptophysin and MAP2 expressions in human neuroblastoma (SH-SY5Y) cells. In contrast, tumour necrosis factor alpha (TNFα) and the conditioned medium (CM) from RAW264.7 cells treated with indoxyl sulfate (IS) significantly impaired OAT3 expression. TNFα and CM from IS-treated BV-2 cells also inhibited synaptophysin and MAP2 expressions in SH-SY5Y cells. The alterations caused by TNFα and CMs in vitro, and by AKI and TNFα in vivo were abolished by infliximab, a monoclonal antibody designed to intercept and neutralize TNFα, suggesting that AKI impaired the expressions of OAT3, synaptophysin and MAP2 in the brain via IS-induced TNFα release from macrophages or microglia (termed as IS-TNFα axis). Treatment of mice with TNFα (0.5 mg·kg-1·d-1, i.p. for 3 days) significantly increased p-p65 expression and reduced the expressions of Nrf2 and HO-1. Inhibiting NF-κB pathway, silencing p65, or activating Nrf2 and HO-1 obviously attenuated TNFα-induced downregulation of OAT3, synaptophysin and MAP2 expressions. Significantly increased p-p65 and decreased Nrf2 and HO-1 protein levels were also detected in brain of AKI mice and rats. We conclude that AKI inhibits the expressions of OAT3, synaptophysin and MAP2 due to IS-induced TNFα release from macrophages or microglia. TNFα impairs the expressions of OAT3, synaptophysin and MAP2 partly via activating NF-κB pathway and inhibiting Nrf2-HO-1 pathway.

Mitochondrial Function in Modulating Human Granulosa Cell Steroidogenesis and Female Fertility.

Ovarian follicle steroidogenesis associated with embryo quality results in a successful pregnancy. Each follicle consists of an oocyte surrounded by granulosa cells, which secrete several steroid and peptide hormones. Follicles harvested from women who conceived after assisted reproductive therapy (ART) had significantly higher estradiol levels in follicular fluids than the follicles from women who failed to conceive after ART. The higher follicular estradiol levels correlate well with successful fertilization following ART. Mitochondria are the central sites for steroid hormone biosynthesis. The first and rate-limiting step in the biosynthesis of steroid hormones occurs in the mitochondria of granulosa cells. In the present study, we hypothesized that the mitochondria in granulosa cells are critical for maintaining oocyte quality and fertility capacity. This study aims to clarify the relationship between mitochondrial function and granulosa cell steroidogenesis, and the relationship between hormone levels and fertility capacity. Sera, follicular fluids and granulosa cells were obtained from individuals undergoing IVF-ET treatment. The oocyte numbers, oocyte quality, fertilization rate, and pregnancy rate were also recorded. The patients who provided the granulosa cells were further classified into four groups: endometriosis, ovarian endometrioma, endometriosis without ovarian endometrioma, and polycystic ovary syndrome (PCOS); patients with other female factor infertility and male factor infertility were used as controls. We measured the levels of estradiol (E2) by radioimmunoassay. Concurrently, we analyzed the mitochondrial mass and membrane potential, and apoptosis by flow cytometry using nonyl acridine orange, TMRE, Annexin V-FITC and PI. Mitochondrial morphology was visualized by transfection with pLV-mitoDsRed. In addition, we assessed the protein levels of steroidogenic enzymes, steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) by Western blot. The results showed significantly decreased serum E2 and follicular E2 levels, and decreased IVF outcomes, in the patients with endometriosis. Reduced mitochondrial mass and decreased mitochondrial membrane potential were correlated with lower E2. Furthermore, a significant decrease in StAR and 3ß-HSD was found in patients with ovarian endometrioma. The enzyme levels of StAR and 3ß-HSD were highly correlated with E2 levels. Finally, elevated cumulus cell apoptosis was found in the patient group with ovarian endometrioma and PCOS. In conclusion, mitochondrial dysfunction of human granulosa cells may contribute to the decline of steroidogenesis, decreased fertilization rate, oocyte maturation rate, and oocyte quality, and it can ultimately jeopardize fertility.

Saikosaponin-d Inhibits the Hepatoma Cells and Enhances Chemosensitivity Through SENP5-Dependent Inhibition of Gli1 SUMOylation Under Hypoxia.

Chemosensitivity is one of the key factors affecting the therapeutic effect on cancer, but the clinical application of corresponding drugs is rare. Hypoxia, a common feature of many solid tumors, including hepatocellular carcinoma (HCC), has been associated with resistance to chemotherapy in part through the activation of the Sonic Hedgehog (SHh) pathway. Hypoxia has also been associated with the increased SUMOylation of multiple proteins, including GLI family proteins, which are key mediators of SHh signaling, and has become a promising target to develop drug-resistant drugs for cancer treatment. However, there are few target drugs to abrogate chemotherapy resistance. Saikosaponin-d (Ssd), one of the main bioactive components of Radix bupleuri, has been reported to exert multiple biological effects, including anticancer activity. Here, we first found that Ssd inhibits the malignant phenotype of HCC cells while increasing their sensitivity to the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) drug system under hypoxia in vitro and in vivo. Furthermore, we had explored that GLI family activation and extensive protein SUMOylation were characteristics of HCC cells, and hypoxia could activate the SHh pathway and promote epithelial-mesenchymal transition (EMT), invasion, and chemosensitivity in HCC cells. SUMOylation is required for hypoxia-dependent activation of GLI proteins. Finally, we found that Ssd could reverse the effects promoted by hypoxia, specifically active sentrin/small ubiquitin-like modifier (SUMO)-specific protease 5 (SENP5), a SUMO-specific protease, in a time- and dose-dependent manner while inhibiting the expression of SUMO1 and GLI proteins. Together, these findings confirm the important role of Ssd in the chemoresistance of liver cancer, provide some data support for further understanding the molecular mechanisms of Ssd inhibition of malignant transformation of HCC cells, and provide a new perspective for the application of traditional Chinese medicine in the chemical resistance of liver cancer.

[Long-term follow-up and therapy of adult Wilms' tumor].

OBJECTIVE: To investigate and analyze the treatment and Long-term follow-up results of adult Wilms' tumor. METHODS: Medical records for diagnosed Wilms' tumor in patients aged more than 15 years from Jan. 1970 to Dec. 2011 were reviewed retrospectively. The clinical presentations, stages, operative details, pathologic findings, adjuvant therapies and outcomes were analyzed. All the patients received regular follow-up, and particular attention was paid to the median follow-up period and tumor specific survival time. RESULTS: The records revealed the 10 patients with a median age of 33.5 year. Male and female were each 5. Left was 4 and right was 6. In the study, 80% of the patients had clinical presentations, and 30% of the patients presented with abdominal mass, and 30% of the patients had distant metastasis at the time of diagnosis. The number of the patients with tumor stages I,II, III, and IV were 2, 1, 4, and 3. One patient just underwent fine needle biopsy due to advanced tumor stage, and the others underwent surgical operations. The range of the follow-up time was 12 to 187 months, and the median follow-up period was 20 months. One patient lost the follow up , 5 patients died, 2 patients survived with tumor recurrence, and 2 patients survived without tumor recurrence. The median survival period was 42 months, and one patient lost the follow-up. CONCLUSION: The Long-term follow-up data demonstrated the poor prognosis of adult Wilms' tumor. Early tumor stage and the combination of operation, radiotherapy and chemotherapy are key factors to improve the outcomes.

Factors associated with post-pancreaticoduodenectomy hemorrhage: 303 consecutive cases analysis.

BACKGROUND: Because of the complexity and severity of the surgery and its associated complications, pancreaticoduodenectomy (PD) is associated with significant morbidity and mortality, especially the hemorrhage post-PD. Exploring the factors associated with post-PD hemorrhage is very important for the patients' safety. METHODS: Clinical data from 303 cases of PD between January 1998 and December 2008 were analyzed retrospectively. RESULTS: The overall mortality rate was 4.95% (15/303). However, post-operative bleeding occurred in 25 patients (8.25%) with nine episodes resulting in death (36.00%). Univariate analysis was performed and identified tumor size, Child's classification, total pancreatic uncinatic process resection, and pancreatic leakage as significant risk factors for post-PD hemorrhage. In the severe hemorrhage group, incomplete resection of uncinate process of pancreas and pancreatic leakage were the main causes. The multivariate Logistic regression analysis revealed that each of these variables is an independent risk factor. CONCLUSIONS: Primary prevention of bleeding complications depends on total pancreatic uncinatic process resection and meticulous hemostatic techniques during surgery. In addition, several peri-operative factors were found to contribute to post-PD bleeding.

Extrahepatic synthesis of coagulation factor VII by colorectal cancer cells promotes tumor invasion and metastasis.

BACKGROUND: Blood coagulation factor VII (FVII) is physiologically synthesized in the liver and released into the blood. Binding of FVII to tissue factor (TF) is related to the metastatic potential of tumor cells, also a significant risk factor in the development of hepatic metastasis in patients with colorectal cancer (CRC). It has been found that some cancer cells can produce FVII extrahepatically. However, little is known about FVII and CRC. We therefore hypothesized that CRC cells may synthese FVII, leading to tumor invasion and metastasis. METHODS: We detected the expression of FVII protein in 55 CRC specimens by immunohistochemical staining. The FVII mRNA in 45 of 55 CRC cases, 6 colon cancer cell lines and one hepatoma cell line was measured by real-time reverse transcription-PCR (RT-PCR). Transwell invasion assays were performed to evaluate the changes of cell migration and invasion of LoVo cancer cells in vitro. We further observed the likely effectors regulated by the TF/FVIIa complex Western blotting assay. RESULTS: Extrahepatic synthesis of FVII was detected in the cytoplasm of 32 (58.2%) CRC specimens by immunohistochemistry, but not in normal mucosa. Liver metastasis (P = 0.003) and TNM staging (P = 0.005) were significantly correlated with FVII antigen expression. The positive ratios in stages I, II, III and IV were 33.3%, 40.0%, 52.4% and 87.5%, respectively. The expression of FVII mRNA in CRC with hepatic metastasis was significantly higher than CRC without hepatic metastasis (5.33 ± 2.88 vs. 1.47 ± 0.51, P = 0.03). Ectopic FVIIa induced a slight increase (1.34-fold) in the number of migrating cells, which was inhibited by the specific TF antibody. The formation of TF/FVIIa complex resulted in a marked increase in the expression of matrix metalloproteinases (MMP)-2 (3.5-fold) and MMP-9 (4.7-fold) in a time-dependent and dose-dependent manner. CONCLUSIONS: Extrahepatic synthesis of FVII by CRC cells may promote tumor invasion and metastasis. MMPs, as downstream effectors of TF/FVIIa signaling, facilitate the development of metastasis in colon cancer.

[Ectopic expression and clinical significance of tissue factor/coagulation factor VII complex in colorectal cancer].

OBJECTIVE: To study the expression of coagulation factor VII(FVII)/tissue factor(TF)complex in colorectal carcinoma (CRC)and its correlation with clinicopathologic factor. METHODS: The expression of coagulation factor VII protein was studied by immunohistochemistry and Western blot.The expression of tissue factor and coagulation factor VII at the mRNA levels were evaluated by quantitative realtime RT-PCR in 45 cases of CRC. RESULTS: (1) FVII overexpression was ectopicly detected in CRC specimens at protein level by immunohistochemistry and Western blot, but not in adjacent non-cancerous mucosa of colorectum;(2)FVII protein mainly localized in the cytoplasm of colon cancer cells.The positive ratios of FVII protein expression in stages I, II, III and IV by immunohistochemistry assay were 33.3%, 40.0%, 64.7% and 80.0% respectively(P=0.001); (3)The expression of FVII mRNA in CRC with hepatic metastasis was significantly higher than that in CRC without hepatic metastasis.The relative expression was 5.33+/-2.88 and 1.47+/-0.51 respectively(P=0.03). Overexpression FVII gene was unrelated with tumor size, differentiation, depth of invasion, lymph node metastasis and TNM staging.There existed some relation between the gene and protein level by Spearman correlation, r=0.58, P=0.003;(4)The expression of TF mRNA in CRC significantly correlated with lymph node metastasis, hepatic metastasis and TNM staging.The expression of tissue factor was a critical factor to predict liver metastasis by logistic regression analysis(P=0.001). CONCLUSION: Colorectal cancer can ectopicly synthesize coagulation factor VII.Tissue factor expression may play a role in the process of developing hepatic metastasis.The microenvironment of high dose FVII protein may promote tumor metastasis.

[Clinical analysis of post pancreatoduodenectomy hemorrhage].

OBJECTIVE: To explore the factors of post pancreatoduodenectomy hemorrhage. METHODS: The clinical data of 263 cases between January 1998 and April 2008 underwent pancreatoduodenectomy were analyzed prospectively. RESULTS: The overall mortality rate was 4.94% (13/263). Postoperative bleeding occurred in 23 patients (8.75%), with 8 episodes ending fatally (34.8%). The tumor size, Child classification, caput total resection and pancreatic leakage were identified as significant risk factors for post pancreatoduodenectomy hemorrhage by means of univariate analysis. The multivariate Logistic regression analysis revealed that all of the five factors turned out to be the independent risk factors. CONCLUSIONS: The prevention of these bleeding complications depends in the first place on meticulous hemostatic technique. The pancreatic leakage is also one of the most important factors due to postoperative bleeding. The prophylactic use of somatostatin is not necessary.

[The diagnosis and surgical management for patients with variants of hepatic arteries in the procedure of pancreaticoduodenectomy].

OBJECTIVE: To study the principle and surgical managements for the patients with anatomic variants of hepatic artery in the procedure of pancreaticoduodenectomy (PD). METHODS: One hundred and seventy-six patients who underwent PD between January 2000 and July 2007 were investigated retrospectively. Hepatic arterial variants were analyzed according to the intraoperative finding and CT imaging were reviewed postoperatively. RESULTS: Hepatic arterial variants were found intraoperatively in 20 cases of all 176 patients. Accessory right heptic artery, replaced right heptic artery and common heptic artery arising from the superior mesenteric artery (SMA) were present in 9 (5.1%), 5 (2.8%), 4 (2.3%) cases respectively,and replaced right heptic artery coming from the gastroduodenal artery was present in 2 cases (2.9%). All the variants of hepatic arteries arising from the superior mesenteric artery could be observed in spiral CT imaging. Most of the variant arteries were dissected intact intraoperatively except 2 cases with accessory right heptic artery arising from SMA. CONCLUSIONS: Performing CT scan preoperatively, especially CTA,is effective to diagnose these disorders. Skillful surgical techniques can manage the anatomic variants safely.

[Influencing factor analysis of delayed gastric emptying after pylorus-preserving pancreaticoduodenectomy].

OBJECTIVE: To investigate the influencing factors of delayed gastric emptying (DGE) after pylorus-preserving pancreaticoduodenectomy (PPPD) and its preventing managements. METHODS: Forty-two patients who underwent PPPD and 104 patients underwent standard Whipple procedure (SPD) between January 2000 and July 2006 were investigated retrospectively. The factors influencing the development of DGE following PPPD were analyzed statistically. RESULTS: There was no significant difference in mortality between PPPD and SPD (0/42 vs. 7/104, P = 0.193). Pancreatic fistula occurred much more frequently in SPD than in PPPD (29/104 vs. 1/42, P < 0.05). The occurrence of DGE after PPPD was higher significantly than that after SPD (35.7% vs. 18.3%, P = 0.024). More DGE occurred in patients with a operation time more than 6 hours than in patients whose operation lasted less than 6 hours (76.9% vs. 17.2%, P = 0.008). Meanwhile, DGE occurred in 20% of patients with the antero-colonic route for duodenojejunostomy and in 50% with retro-colonic route (P = 0.043). Multivariate analysis by logistic regression model showed postoperative intra-abdominal complications were not risk factors for DGE. Prophylactic use of somatostatin couldn't prevent DGE effectively. CONCLUSIONS: DGE is the most frequent postoperative complication after PPPD, it can be markedly reduced by shortening operative time and using antero-colonic duodenojejunostomy procedure. There is no medicine which could prevent DGE effectively.

[Treatment of paraneoplastic pemphigus with Castleman's disease].

OBJECTIVE: To discuss the clinical findings and treatment of paraneoplastic pemphigus (PNP) with Castleman's disease. METHODS: To investigate the clinical, histopathologic and CT findings of 8 cases paraneoplastic pemphigus with Castleman's disease. RESULTS: All of 8 patients were diagnosed PNP first and were found Castleman's tumor incidently during routine examination. All 8 cases showed severe erosion or ulcer of the oral mucosa with various skin lesions. Histopathologically, there were intraepidermal acantholytic vesicle, basal cell liquefaction, necrotic keratinocytes in the epidermis and lymphocyte infiltration in the upper dermis. CT scan appeared solitary mass in these patients. Some of them were attacked by bronchiolitis obliterans. All 8 patients were failed by use of predisone. Obvious relief of PNP and pulmonary lesion occurred after tumor was rescted. CONCLUSIONS: Paraneoplastic pemphigus with Castleman's disease is a rare disease. The key step is to find and resect the tumor in abdomen. CT scan should be used to detect the tumor in patients with PNP, especially, when predisone was failed in treatment.

First two cases of living related liver transplantation with complicated anatomy of blood vessels in Beijing.

AIM: Living related liver transplantation (LRLT) has been developed in response to the paediatric organ donor shortage. Though it has been succeeded in many centers worldwide, the safety of the donor is still a major concern, especially in donors with anatomy variation. We succeeded in performing the first two cases of living related liver transplantation with complicated anatomy of blood vessels as a way to overcome cadaveric organ shortage in Beijing. METHODS: Two patients, with congenital liver fibrosis and congenital biliary atresia were performed with living donor liver transplantation in our hospital and then followed up from November 12 to December 13, 2001. The two living donors, mother and father, were healthy aged 34 and 35 years. One right lobe (segment V, VI, VII, VIII) and one left lateral lobe (segment II and III) were used. The grafts weighed 394 g and 300 g. The ratio of graft weight to the standard liver volume (SLV) of donors was 68% and 27%. The graft weight to recipient body weight ratio was 3.2% and 4.4%. The graft weight to recipient estimated standard liver mass (ESLM) ratio was 63% and 85%. The two donors had complicated blood vessel variation. RESULTS: Two patients undergone living donor liver transplantation had good results. Abnormal liver function with high bilirubin level appeared in a few days after operation, but liver function returned to normal one month after operation with bilirubin level almost decreased to near normal. No bleeding, thrombosis, infection and bile leakage occurred. One had an acute rejection and recovered. The two donors recovered in two weeks. One had slight fever because of a little collection in abdomen and recovered after paracentesis and drainage. CONCLUSION: Living donor liver transplantation has been proved to be a good way that offers a unique opportunity of getting a timely liver graft as a response to shortage of pediatric donors, though it could be a technically difficult operation if there is anatomical variation.

Purification of p53-associated Proteins from Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a common cancer in southeast China that is closely associated with Epstein-Barr virus. We investigated the potential interaction between p53 and viral or cellular proteins in NPC tumor leading to the accumulation of p53 protein, by using immunoaffinity purification of p53 protein in NPC tumor specimens. Two immunoaffinity columns, each with monoclonal antibodies against p53, pAb1801 and pAb240 were used for the p53 protein purification, respectively. The p53 binding proteins were purified from metastasized lymph node tumors of NPC. p53 protein was highly purified with either of the two columns, as indicated by silver staining and Western blot analysis. In addition, two other protein bands could be visualized of 74 and 26 kD. The 26 kD band was detected by anti-p53 antibodies. The 74 kD protein bound to p53 protein by secondary bonds in NPC cells and weakly reacted with NPC patien's serum.