Sirolimus increases the anti-cancer effect of Huai Er by regulating hypoxia inducible factor-1α-mediated glycolysis in hepatocellular carcinoma.

BACKGROUND: Glycolysis caused by hypoxia-induced abnormal activation of hypoxia inducible factor-1α (HIF-1α) in the immune microenvironment promotes the progression of hepatocellular carcinoma (HCC), leading to enhanced drug resistance in cancer cells. Therefore, altering the immunosuppressive microenvironment by imp-roving the hypoxic state is a new goal in improving cancer treatment. AIM: To analyse the role of HIF-1α, which is closely related to tumour proliferation, invasion, metastasis, and angiogenesis, in the proliferation and invasion of liver cancer, and to explore the HIF-1α pathway-mediated anti-cancer mechanism of sirolimus (SRL) combined with Huai Er. METHODS: Previous studies on HCC tissues identified the importance of HIF-1α, glucose transporter 1 (GLUT1), and lactate dehydrogenase A (LDHA) expression. In this study, HepG2 and Huh7 cell lines were treated, under hypoxic and normoxic conditions, with a combination of SRL and Huai Er. The effects on proliferation, invasion, cell cycle, and apoptosis were analysed. Proteomics and genomics techniques were used to analyze the HIF-1α-related signalling pathway during SRL combined with Huai Er treatment and its inhibition of the proliferation of HCC cells. RESULTS: High levels of HIF-1α, LDHA, and GLUT-1 were found in poorly differentiated HCC, with lower patient survival rates. Hypoxia promoted the proliferation of HepG2 and Huh7 cells and weakened the apoptosis and cell cycle blocking effects of the SRL/Huai Er treatment. This was achieved by activation of HIF-1α and glycolysis in HCC, leading to the upregulation of LDHA, GLUT-1, Akt/mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), and Forkhead box P3 and downregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and p27. The hypoxia-induced activation of HIF-1α showed the greatest attenuation in the SRL/Huai Er (S50 + H8) group compared to the drug treatments alone (P < 0.001). The S50 + H8 treatment significantly downregulated the expression of mTOR and HIF-1α, and significantly reduced the expression of VEGF mRNA. Meanwhile, the combined blocking of mTOR and HIF-1α enhanced the downregulation of Akt/mTOR, HIF-1α, LDHA, and GLUT-1 mRNA and resulted in the downregulation of PTEN, p27, and VEGF mRNA (P < 0.001). CONCLUSION: SRL increases the anti-cancer effect of Huai Er, which reduces the promotion of hypoxia-induced HIF-1α on the Warburg effect by inhibition of the PI3K/Akt/mTOR-HIF-1α and HIF-1α-PTEN signalling pathways in HCC.

Trends of rapamycin in survival benefits of liver transplantation for hepatocellular carcinoma.

The proportion of liver transplantation (LT) for hepatocellular carcinoma (HCC) has kept on increasing over the past years and account for 20%-40% of all LT. Post-transplant HCC recurrence is considered the most important factor affecting the long-term survival of patients. The use of different types of immunosuppressive agents after LT is closely associated with an increased risk for HCC recurrence. The most commonly used conventional immunosuppressive drugs include the calcineurin inhibitors tacrolimus (FK506) and mammalian target of rapamycin inhibitor rapamycin (RAPA). Compared with tacrolimus, RAPA may carry an advantage in survival benefit because of its anti-tumor effects. However, no sufficient evidence to date has proven that RAPA could increase long-term recurrence-free survival and its anti-tumor mechanism of combined therapy remains incompletely clear. In this review, we will focus on recent advances in clinical application experience and basic research results of RAPA in patients undergoing LT for HCC to further guide the clinical practice.

Prognostic Value of Preoperative NLR and Vascular Reconstructive Technology in Patients With Pancreatic Cancer of Portal System Invasion: A Real World Study.

The purpose was aimed to establish a simple computational model to predict tumor prognosis by combining neutrophil to lymphocyte Ratio (NLR) and biomarkers of oncological characteristics in patients undergoing vascular reconstructive radical resection of PDAC. The enrolled patients was divided into high or low NLR group with the cutoff value determined by the receiver operator characteristic (ROC) curve. Different vascular anastomoses were selected according to the Chaoyang classification of PDAC. Survival rates were calculated using the Kaplan-Meier and evaluated with the log-rank test. Cox risk regression model was used to analyze the independent risk factors for prognostic survival. The optimal cut-off value of NRL was correlated with the differentiation, tumor size, TNM stage and distant metastasis of advanced PDAC. A curative resection with vascular reconstructive of advanced PDAC according to Chaoyang classification can obviously improve the survival benefits. Cox proportional hazards demonstrated higher evaluated NLR, incisal margin R1 and lymphatic metastasis were the independent risk predictor for prognosis with the HR > 2, meanwhile, age beyond 55, TNM stage of III-IV or Tumor size > 4cm were also the obvious independent risk predictor for prognosis with the HR ≤ 2. The advanced PADC patients marked of RS group (3 < RS ≤ 6) showed no more than 24 months of survival time according to RS model based on the six independent risk predictors. Vascular reconstruction in radical resection of advanced PDAC improved survival, higher elevated NLR (>2.90) was a negative predictor of DFS and OS in those patients accompanying portal system invasion.

PD-L1+NEUT, Foxp3+Treg, and NLR as New Prognostic Marker with Low Survival Benefits Value in Hepatocellular Carcinoma.

Background: This presented study was aimed to evaluate the diagnostic and prognostic value of PD-L1+Neutrophils (PD-L1+NEUT) and neutrophil to lymphocyte ratio (NLR) based on our previous experience of Foxp3+Treg in transplantation. Methods: the NLR cutoff value of 1.79 was used to include 136 cases from the 204 patients with hepatocellular carcinoma (HCC) confirmed by clinical pathology, which were divided into highly-moderately and poorly differentiated HCC groups. The expressions of PD-L1+NEUT and Foxp3+Treg in peripheral blood and cancer tissue were detected with flow cytometry, meanwhile, PD-L1 and Foxp3 expressed in carcinoma and para-carcinoma tissues were marked by immunohistochemistry. Survival rates, including overall survival and disease-free survival, were calculated by the Kaplan-Meier curve and evaluated with the log-rank test. Finally, Cox risk regression model was used to analyze the independent risk factors for prognostic survival. Results: The level of PD-L1+NEUT, Foxp3+Treg, and NLR in peripheral blood of patients with poorly differentiated HCC were significantly increased (all P < .001). Both PD-L1+NEUT and NLR were positively correlated with Foxp3+Treg (r = 0.479, P = .0017; r = 0.58, P < .0001). The level of PD-L1+NEUT and Foxp3+Treg as well as PD-L1 and Foxp3 in cancer tissue and patients with poorly differentiated HCC were obviously increased (all P < .01), respectively. Cox regression analysis indicated that PD-L1+NEUT, NLR, and Foxp3+Treg were independent risk factors for the prognosis (P = .000, .000, .006) with a RR and 95%CI of 2.704-(2.155-3.393), 3.139-(2.361-4.173), 1.409-(1.105-1.798), respectively. Conclusion: PD-L1+NEUT, NLR, and Foxp3+Treg are independent risk factors for prognosis which maybe new marker of lower survival benefits.

Rapamycin treated tol-dendritic cells derived from BM-MSCs reversed graft rejection in a rat liver transplantation model by inducing CD8+CD45RC-Treg.

OBJECTIVE: To investigate the influence of tolerance dendritic cells (tolDCs), generated from Bone marrow mesenchymal stem cells (BM-MSCs) treated with rapamycin (Rapa) on liver allograft survival in a rat acute liver transplantation model. METHODS: Different GM-CSF induction project was used to obtain immature DCs (imDCs), mature DCs (matDCs) or tolDCs from BM-MSCs. First, MLR was performed to analyze the activity of tolDCs on polyclonaly stimulated total T cells. Then, co-cultured imDCs, matDCs and tolDCs with CD8+T cells isolated by magnetic activated cell sorting to analyze the influence on its regulatory characteristic. Last, the established rat acute liver transplantation model were adoptive transfused with imDCs, matDCs or tolDCs isolated by anti-CD11c immunomagnetic beads. The phenotype of DC cells and level of CD8+Treg in the culture system and in vivo, the expression of CD8 and CD45RC in the tissues were analyzed by flow cytometry and immunohistochemistry, respectively. RESULTS: The loGM-CSF plus IL-4 decreased the costimulatory molecules of CD80/86 and MHC class II of DCs comparison with hiGM-CSF from BM-MSCs no matter whether stimulation by LPS (P<0.05). Rapa treated not only reduced the expression of CD80/86 and MHC class II but also down-regulated the expression of CD11c after LPS stimulation which was more obviously in tolDCs by loGM-CSF project (P<0.05). Moreover, tolDCs displayed a rather higher level of IL-10 and low level of IL-12p70 than others (P<0.01), which shown a rather lower stimulative effect on the proliferation of T cells comparison with matDCs and imDCs. Co-cultured with CD8+Treg showed an improvement on induction of CD8+TCR+CD45RC-T cells (CD8+Treg) in ex vivo. The rats transfused with tolDCs has a delayed survival benefits with high level of CD8+Tregs (P<0.01) and high expression of CD45RC in liver tissue (P<0.01) and spleen when comparison with other groups. The infused tolDCs improved a mean survival time (MST) of 32 days comparison with a MTS of 9.5 days and 15.75 days displayed by rat that per-infused with matDCs and imDCs, respectively. CONCLUSION: Rapa modified tolDCs derived from BM-MSCs reversed graft rejection by improve tolerance characteristics of CD8+CD45RC-Treg in acute liver rat transplantation.

Reduction of FoxP3+ Tregs by an immunosuppressive protocol of rapamycin plus Thymalfasin and Huaier extract predicts positive survival benefits in a rat model of hepatocellular carcinoma.

BACKGROUND: Investigate immunoregulation and anti-tumor immunity of FoxP3+Tregs after treatment with rapamycin (RAPA/SRL) plus thymalfasin (Zadaxin) and Huaier extract (PS-T) in a hepatocellular carcinoma (HCC) rat model simulating HCC relapse after liver transplant (LT). METHODS: We successfully established a rat model simulating HCC relapse after LT using an optimized chemical induction method with TACROLIMUS, methylprednisolone, and diethylnitrosamine as identified by visible liver nodules and hematoxylin-eosin staining. The model rats were then treated with RAPA, Zadaxin, and PS-T. Immune status changes were analyzed by flow cytometry, and protein expression of Akt and mTOR was determined by western blotting. Cytokines were measured by ELISAs. RESULTS: Combined therapy by RAPA plus Zadaxin and PS-T obviously alleviated hepatic pathological changes and significantly decreased the levels of FoxP3+Tregs in peripheral blood, the spleen, and the liver (P<0.05) and expression of mTOR protein (P<0.01) in the liver, obviously improved survival time (P=0.02). Moreover, the levels of CD8+T cells were increased significantly to almost normal levels (P<0.05) in comparison with no SRL monotherapy protocols. Inhibitory cytokines were also decreased in accordance with FoxP3+Tregs. Significant decreases of IL-10 and TGF-ß were observed after SRL-based therapy (P<0.01) in comparison with the other groups. Serum alpha fetoprotein (AFP) and vascular endothelial growth factor (VEGF) levels were also decreased significantly (P<0.05). FoxP3+Tregs showed a negative correlation with CD8+ and CD4+/CD8+T cells and a positive correlation with AFP, and VEGF (P<0.05). CONCLUSIONS: SRL-based therapy reduces FoxP3+Tregs to decrease secreted inhibitory cytokines which may enhancement the viability and number of CD8+T cells to exert anti-tumor effects that are mainly mediated through the AKT-mTOR signaling pathway.

The advantage of Sirolimus in amplifying regulatory B cells and regulatory T cells in liver transplant patients.

Sirolimus has been shown to ameliorate steroid-resistant rejection and induce long-term immune tolerance among liver transplant patients. However, the detailed mechanism of how Sirolimus achieve these advantages is still lacking. This study attempts to reveal some possible mechanisms by investigating regulatory B cells (Bregs), regulatory T cells (Tregs) and some cytokines in liver transplant recipients whose Tacrolimus was partially converted to Sirolimus. The results showed that CD19+CD24+CD38+Bregs and CD4+CD25+FoxP3+Tregs increased significantly during the first month after drug conversion (P < 0.01 and P < 0.05). The percentages of IL-10+Bregs and TGF-ß1+Bregs were also elevated (P < 0.05 and P < 0.01), and the same trend was observed in the levels of IL-10 and TGF-ß1 (P < 0.01 and P < 0.01). However, in the observation period, these investigated lymphocyte subsets and cytokines didn't change significantly in patients without Sirolimus usage. The incidence of biliary stenosis in the conversion group were significantly lower than that in the control group (P < 0.05). At the same time, in vitro experiments showed that Sirolimus could significantly amplify Bregs and Tregs (P < 0.01 and P < 0.01) while Tacrolimus did not show the amplifications effects. Sirolimus' function of amplifying Bregs was weakened, and its function of amplifying Tregs even disappeared after IL-10 and TGF-ß1 were neutralized. In conclusion, Sirolimus could amplify Bregs and Tregs among liver transplant recipient, which might be benefit to mitigate the immune response, decrease chances of rejection and alleviate biliary complication. IL-10 and TGF-ß1 may play important roles during this process.

Novel strategy of sirolimus plus thymalfasin and huaier granule on tumor recurrence of hepatocellular carcinoma beyond the UCSF criteria following liver transplantation: A single center experience.

Although liver transplantation (LT) lengthens the survival time of patients with hepatocellular carcinoma (HCC), LT patients exhibit a high recurrence rate; particularly those that had advanced HCC associated with the tumor biological characteristics and long-term application of immunosuppressants. A consensus on optimal prophylaxis and treatment for recurrent HCC following LT does not currently exist. The present study retrospectively analyzed data from 36 non-University of California at San Francisco criteria-eligible patients with advanced HCC who underwent LT, and then treated them with sirolimus (SRL)-based therapy with thymalfasin and huaier granules (SRL+, n=18), or with tacrolimus-based therapy (controls; n=18). The SRL+ group had significantly longer recurrence times (P=0.008) and survival times (P<0.0001) (OS, 1-year: 100%, 3-year: 94.4%, 5-year: 77.8%; DFS, 1-year: 88.9%, 3-year: 55.6%, 5-year: 50.0%). Furthermore, compared with pre-LT values and the control group, the SRL+ group had significantly lower serum α-fetoprotein (AFP) levels (both P<0.0001) and percentage of Forkhead box P3 (FoxP3)+ Treg lymphocytes (P<0.001) during the first year. In the SRL+ group, FoxP3+/cluster of differentiation (CD)8+ Treg lymphocyte percentages decreased significantly following LT (P<0.001); however, CD8+/CD3+ T-cells significantly increased (P<0.001). Levels of serum AFP and FoxP3+ Treg cells increased when tumors relapsed, and decreased to near-normal when relapse foci were cured or stabilized. SRL+ therapy may decrease AFP and Treg levels, while increasing CD8+ T cells, indicating an associated mechanism among them. In conclusion, SRL+ therapy appears to be safe and effective in preventing HCC recurrence following LT with no significant adverse events, and warrants further investigation.

[Immature dendritic cells phagocytosing human spleen cells treated by PUVA present the characteristics of regulatory dendritic cells].

Objective To investigate the effect of psoralen combined with A-band ultraviolet (PUVA)-treated human spleen lymphocytes on the phenotype and function of immature dendritic cells (imDCs). Methods Human peripheral blood mononuclear cells (PBMCs) were isolated and induced to produce DCs by interleukin-4 (IL-4) and recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF). On the sixth day, the imDCs were obtained and stimulated by lipopolysaccharide (LPS). One day later, mature DCs were harvested. Human spleen cells (SPs) were isolated and treated with PUVA to prepare apoptotic PUVA-SPs. Co-culture of imDCs with PUVA-SPs resulted in extracorporeal photochemotheraputic DCs (ecpDCs). Co-culture of imDCs with SPs resulted in SP-DCs. The expressions of CD11c, CD83 and CD86 were detected by flow cytometry. The levels of IL-10 and IL-12 in the supernatants of the above cells were determined by ELISA. Results The early apoptosis rate of PUVA-SPs was (94.21±3.75)%. There was no significant difference in the expressions of CD83 and CD86 between imDCs and ecpDCs. But the positive rates of CD83 and CD86 in ecpDCs were lower than those in DCs. However, the positive rates of CD83 and CD86 in SP-DCs were significantly higher than those of the imDCs. Conclusion The imDCs phagocytosing apoptotic human SPs present phenotype and function of regulatory DCs.

Sirolimus treatment for cirrhosis or hepatocellular carcinoma patients accompanied by psoriasis after liver transplantation: A single center experience.

There is currently no consensus on the most suitable therapeutic approach for psoriasis (PS) co-existing with posthepatic cirrhosis (PCs) and hepatocellular carcinoma (HCC) following liver transplantation (LT). The present study provides an analysis of the therapeutic experience of such patients. Five LT recipients (two with PC and three with HCC) with accompanying PS were included. The induction program consisted of methylprednisolone plus basiliximab treatment. The initial postoperative treatment scheme consisted of tacrolimus (FK506) plus mycophenolate mofetil (MMF) and hormone; the latter was withdrawn 1 week after LT. The patients with PC had been using FK506 with or without a postoperative MMF program; the patients with HCC and recurrence of PS had been switched to a sirolimus (SRL)-based replacement therapy. Furthermore, all patients received anti-hepatitis B virus (HBV) therapy. The patients were followed up after 8.3±1.5 years. There was a positive correlation between HBV-DNA copy numbers, and psoriatic area and severity index (PASI) scores (r=0.97; P=0.006). The PASI scores were decreased significantly at 6 months following surgery compared with pre-transplantation (P<0.05). The patients who had received the FK506-based treatment experienced PS recurrence two years post-transplantation. The PASI scores increased significantly (P<0.05) and then declined gradually, maintaining a stable level (P<0.05) by 1 year after switching to the SRL-based treatment. The patients who had received the SRL-based treatment exhibited no recurrence of PS. The results of the present study suggest that SRL therapy provides a promising novel treatment method for patients with PS following LT that may be superior to tacrolimus treatment. When co-existing HBV is present pre-transplantation, regular injection of human hepatitis B immunoglobulin should be used to prevent the HBV from relapsing or aggravating the PS.

[Immature dendritic cells phagocytosing apoptotic human spleen cells treated with PUVA inhibits the maturation induced by LPS].

Objective To investigate whether lipopolysaccharide (LPS) can induce the maturation of immature dendritic cells (imDCs) which phagocytose apoptotic spleen lymphocytes. Methods Human peripheral blood mononuclear cells (PBMCs) were induced to produce DCs by interleukin 4 (IL-4) and recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF). Human spleen cells (hSPs) were isolated and treated with psoralen combined with ultraviolet A(PUVA) to obtain apoptotic PUVA-hSPs. Co-culture of imDCs with PUVA-hSPs resulted in extracorporeal photochemotherapeutic dendritic cells (ecpDCs). The imDCs and ecpDCs were collected and stimulated by 10 ng/mL LPS for 1 day. The expressions of CD11c, CD83 and CD86 were detected by flow cytometry. The level of IL-10 in the supernatants of the above cells was detected by ELISA. Results There was no significant difference in the expressions of CD83 and CD86 between ImDCs and ecpDCs. However, the positive rates of CD83 and CD86 in the imDCs stimulated by LPS were significantly higher than those in the ecpDCs treated by LPS. The level of IL-10 in imDCs culture supernatant was lower than that in ecpDCs. The level of IL-10 in LPS-stimulated imDCs was lower than that in LPS-stimulated ecpDCs. Conclusion Both imDCs and ecpDCs showed immature phenotype, but ecpDCs can inhibit the maturation of DC induced by LPS.

The role of regulatory B cells (Bregs) in the Tregs-amplifying effect of Sirolimus.

Sirolimus can significantly amplify regulatory T cells (Tregs) in vivo and in vitro, but the specific mechanism of this has not been well documented. The role of regulatory B cells (Bregs) in the Tregs-amplifying effect of Sirolimus was investigated in peripheral blood mononuclear cells (PBMCs) in vitro in this study. The results showed that the percentages of both CD19+CD24+CD38+TGF-ß1+ Bregs and CD19+CD24+CD38+IL-10+ Bregs to B cells were elevated by Sirolimus in PBMCs including B cells. Sirolimus significantly enhances the cytokine production of transforming growth factor-ß1 (TGF-ß1) and interleukin-10 (IL-10) in PBMCs with B cells, and the enhancement significantly decreased in PBMCs without B cells. The percentage of CD4+CD25+Foxp3+ Tregs to T cells was also elevated by Sirolimus in PBMCs including B cells. The elevation of Tregs percentage decreased in PBMCs without B cells and recovered when additional TGF-ß1 and IL-10 were added. The amplification of Tregs by Sirolimus was partially inhibited when either TGF-ß1 or IL-10 was neutralized, and it even disappeared when these two cytokines were both neutralized. These results indicate that Sirolimus can amplify Bregs and Tregs in PBMCs in vitro, and Bregs may be the why Sirolimus amplifies Tregs.

Individualized Immunosuppressive Protocol of Liver Transplant Recipient Should be Made Based on Splenic Function Status.

BACKGROUND: Lymphocyte subsets play important roles in rejection in liver transplant recipients, and the effect of splenic function on these roles remains unknown. The aim of this study was to explore the feasibility to adjust immunosuppressive agents based on splenic function status through detecting the lymphocyte subsets in liver transplantBeijing recipients. METHODS: The lymphocyte subsets of 49 liver transplant recipients were assessed in the 309th Hospital of Chinese People's Liberation Army between June 2014 and August 2015. The patients were divided into splenectomy group (n = 9), normal splenic function group (n = 24), and hypersplenism group (n = 16). The percentages and counts of CD4+ T, CD8+ T, natural killer (NK) cell, B-cell, regulatory B-cell (Breg), and regulatory T-cell (Treg) were detected by flow cytometer. In addition, the immunosuppressive agents, histories of rejection and infection, and postoperative time of the patients were compared among the three groups. RESULTS: There was no significant difference of clinical characteristics among the three groups. The percentage of CD19+CD24+CD38+ Breg was significantly higher in hypersplenism group than normal splenic function group and splenectomy group (3.29 ± 0.97% vs. 2.12 ± 1.08% and 1.90 ± 0.99%, P = 0.001). The same result was found in CD4+CD25+FoxP3+ Treg percentage (0.97 ± 0.39% vs. 0.54 ± 0.31% and 0.56 ± 0.28%, P = 0.001). The counts of CD8+ T-cell, CD4+ T-cell, and NK cell were significantly lower in hypersplenism group than normal splenic function group (254.25 ± 149.08 vs. 476.96 ± 225.52, P= 0.002; 301.69 ± 154.39 vs. 532.50 ± 194.42, P= 0.000; and 88.56 ± 63.15 vs. 188.33 ± 134.51, P = 0.048). Moreover, the counts of CD4+ T-cell and NK cell were significantly lower in hypersplenism group than splenectomy group (301.69 ± 154.39 vs. 491.89 ± 132.31, P= 0.033; and 88.56 ± 63.15 vs. 226.00 ± 168.85, P = 0.032). CONCLUSION: Splenic function status might affect the immunity of liver transplant recipients, that should be considered when we make immunosuppressive protocols.

[Triplet anti-tumor therapy based on thymosin α-1 attenuates incidence of hepatoma and serum alpha-fetoprotein level in rat hepatoma model].

OBJECTIVE: To explore the impact of triple anti-tumor therapy based on thymosin α1 (Tα1) combined with Huaier granule(HG) and sirolimus on the level of serum alpha-fetoprotein (AFP) in rat models of liver cancer. METHODS: Ninety Sprague-Dawley rats were randomly divided into triple anti-tumor therapy group, Tα1 group, HG group, sirolimus group, positive control and blank control groups, with 15 rats in each group. Except the blank control group, the rats in the other groups were induced using diethylnitrosamine (DEN) to establish liver cancer models. After DEN treatment, the triple therapy group underwent 0.8 mg/kg Tα1 subcutaneous injection (from once a day for two weeks to twice a week since the third week), 0.35 g/kg HG gavage (three times a day) and 1 mg/kg sirolimus gavage (once a day). The dose of the rest single drug groups were the same with that of the triple therapy group. The positive control and blank control groups were not treated with the drugs. The treatment lasted 20 weeks. Then, the behavior of the rats were observed at the different time points, and the level of serum AFP in the rats were detected at 6, 16, 18, 20 weeks, respectively. RESULTS: The typical symptoms of liver cancer were seen in the DEN-induced rats at 16 weeks. Since the tenth week, 6 rats died one after another. Pathological section of rat liver tissue suggested that the rat models were established successfully. According to the incidence rate of liver cancer and the survival rate at 20 weeks, the triple anti-tumor therapy was significantly superior to the single drug treatments. In addition, the triple anti-tumor therapy significantly reduced the level of serum AFP in the rats. CONCLUSION: The triple anti-tumor therapy can significantly prolong the survival time of rats with liver cancer, decrease the cancer incidence rate and the level of serum AFP.

New strategies for prevention and treatment of splenic artery steal syndrome after liver transplantation.

AIM: To explore a prophylactic procedure to prevent splenic artery steal syndrome (SASS), as well as a therapeutic intervention to correct it. METHODS: Forty-three liver transplant patients were enrolled in a non-randomized controlled trial, with the eligible criterion that the diameter of the splenic artery is more than 5 mm and/or 1.5 times of the diameter of the hepatic artery. The procedure of splenic artery banding was performed in 28 of the 43 patients, with the other 15 patients studied as a control group. SASS and other complications were compared between these two groups. A new therapeutic intervention, temporary incomplete blockade of the splenic artery with a balloon, was performed to treat SASS in this study. RESULTS: The incidence of SASS was decreased by banding the splenic artery (0/28 vs 5/15, P = 0.006), and the same result was observed in total complications associated with prophylactic procedures (2/28 vs 6/15, P = 0.014). Five patients in the control group developed SASS within 5 d after OLT, 2 of whom were treated by coil embolization of the splenic artery, whereas the other 3 by temporary blockade of the splenic artery. Reappeared or better hepatic arteries with improved systolic amplitude and increased diastolic flow were detected by Doppler ultrasonography in all the 5 patients. Local splenic ischemic necrosis and nonanastomotic biliary stricture were diagnosed respectively in one patient treated by coil embolization, and no collateral complication was detected in patients treated by temporary blockade of the splenic artery. CONCLUSION: SASS should be avoided during the operation by banding the splenic artery. Temporary blockade of the splenic artery is a new safe and effective intervention for SASS.

[The modified extracorporeal photochemotherapy promotes apoptosis of spleen lymphocytes].

OBJECTIVE: To explore the efficacy of the modified extracorporeal photochemotherapy (ECP) in improving the apoptotic rate of lymphocytes in vitro. METHODS: The spleens which were obtained from liver transplantation donor under aseptic condition were used as experimental materials. Splenic lymphocytes (SPs) suspensions were prepared by modified and traditional ECP method, respectively. And then the isolated SPs were treated by the irradiation of 8-methoxypsoralen (8-MOP) combined with ultraviolet A (UVA) named PUVA, 8-MOP and UVA, and compared with a blank group meanwhile. The treated SPs were cultured overnight in an incubator at 37 Degrees Celsius, in a humidified atmosphere of 50 mL/L CO2 for 6-8 hours. The morphological changes of cells were observed using an inverted microscope, the apoptotic rates of SPs were detected by flow cytometry, and the difference between groups was analyzed finally. RESULTS: The apoptotic rate at early stage and the total apoptotic rate of SPs prepared by the modified ECP method were respectively (95.33±3.03)% and (97.10±2.12)% after treated by PUVA, (23.39±4.55)% and (36.32±6.63)% after treated by 8-MOP, and (66.98±3.60)% and (68.65±4.35)% by UVA. Compared with control group (12.82±1.86% and 13.4±2.65%), there were statistically significant differences (P<0.01). The apoptotic rate at early stage and the total apoptotic rate of SPs prepared by the traditional ECP method were respectively (79.73±4.21)% and (82.70±4.13)%, (61.42±2.28)% and (68.91±2.18)%, (19.30±1.78)% and (28.06±1.88)%, (10.84±0.98)% and (12.77±1.22)%, and the statistical comparisons between groups also had significant difference (P<0.01). In addition, there was a significant difference in the early and total apoptosis between the modified and traditional ECP (P<0.01), but no obvious variation in the end-stage apoptosis in the two groups (P>0.05). CONCLUSION: The modified ECP method can promote apoptosis of SPs in vitro conveniently, safely and efficiently, especially in the early stage. This can lay a foundation for the further study on dendritic cell immunomodulation induced by ECP method.

What is the work environment of orthopaedic surgeons in China?

BACKGROUND: Physicians in China face heavy demands from patients and the government for services but deal with the threat of unpredictable legal and physical conflicts with patients, some ending with the death of doctors. More than 40 doctors and nurses have been killed by patients since 2001. QUESTIONS/PURPOSES: We sought to evaluate (1) the demographics of orthopaedic practice, (2) duty periods, (3) practice support, and (4) job satisfaction among orthopaedic surgeons in China. METHODS: Questionnaires were posted online at www.OrthoChina.org for download by orthopaedic surgeons in 2006 to 2007, and sent to those attending meetings in 2013. In 2013, a total of 1350 surgeons were invited and 456 participated in the survey at meetings. In 2007, during the period of the survey, 9759 individuals were qualified orthopaedic surgeons, and 334 participated in the survey at www.OrthoChina.org . RESULTS: Ninety-one percent of orthopaedic surgeons work in public and 9% in private hospitals. Ninety-four percent work more than 8 hours per day 6 to 7 days a week. Twenty-five percent work more than 12 hours per day 6 to 7 days a week without extra compensation. The majority of orthopaedic surgeons must work on national statutory holidays. Almost none received contractually mandated income for weekends and national holidays. Approximately 80% of participants reported an attack of some kind, including physical or psychologic harm. With respect to job satisfaction, 73% stated they would not choose to be a physician again and 86% reported that they do not want their children to become a physician. CONCLUSIONS: China's rapid economic growth and resulting demands for modern health care have resulted in heavy pressure on orthopaedic surgeons, financially and personally. Chinese orthopaedic surgeons are overworked, suffer lack of respect, and face the possibility of serious personal harm. As a consequence, they are demoralized and unsatisfied. Significant reforms are needed.

Comparison of hepatitis B X gene mutation between patients with hepatocellular carcinoma and patients with chronic hepatitis B.

Hepatitis B virus (HBV), a major causative agent of hepatocellular carcinoma (HCC), encodes an oncogenic X protein (HBx) that transcriptionally activates multiple viral and cellular promoters. The present study aimed to identify the specific gene mutation related to the clinical outcome of HCC. Seventy-two HBV-infected patients (38 with chronic HBV infection and 34 with HCC) with well-characterized clinical profiles were enrolled. The HBx region was amplified from patient serum samples and analyzed by sequencing. Genotypes were determined using the National Center for Biotechnology Information genotype tool. All isolates were genotype B or C. Enhancer II nucleotide substitutions in the HCC group were significantly different from those in the chronic hepatitis B (CHB) group (Ρ < 0.05). HCC patients with genotype C had a higher risk of harboring the 1762/1764 double mutation than those with genotype B. The incidence of the 1762/1764 double mutation was higher in the high viral load group (>10(6) copies/ml) than in the low viral load group (≤10(6) copies/ml) (P = 0.03). The 1762/1764 double mutations may be related to the genotype and viral load. We found significantly more direct repeat sequence 1 (DR1) nucleotide substitutions in the HCC group (32.4%, 11/34) than in the CHB group (10.5%, 4/38) (Ρ < 0.05). Patients with higher viral load and genotype C had a higher incidence of 1762/1764 double mutations, which may not be related with development of HCC. Enhancer II and DR1 may play an important role in HCC development via nucleotide substitution.

Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4+CD25+Foxp3+ regulatory T cells and down-regulates cardiac allograft rejection.

Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient's autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-gamma by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naïve T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4(+)CD25(high)Foxp3(+) regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.

[Immune regulation effect of rat dendritic cells phagocytosing photochemotherapy-treated allogeneic cells on syngeneic T cells].

The aim of this study was to investigate the immune regulatory effect of dendritic cells phagocytosing photochemotherapy-treated allogeneic spleen lymphocytes on syngeneic T cells. DA rat spleen lymphocytes were treated with 8-methoxypsoralen plus UVA irradiation (PUVA). LEW rat bone marrow-derived DCs were co-cultured with PUVA-treated DA spleen lymphocytes (PUVA-SP), and the surface markers (MHC-II, CD86 and CD40) of treated DC were detected by flow cytometry. CFSE-labeled PUVA SP were incubated with LEW DCs and the phagocytosis of DCs on PUVA-SP was observed by using fluorescent microscope. The ability of DC phagocytosing allogeneic PUVA-SP (PUVA-SP DC) to stimulate the proliferation of LEW T cells was analyzed by mixed leukocyte reactions (MLR). The production of IL-4, IL-10, IL-2, IFN-gamma in MLR culture supernatant was determined by luminex method. The results indicated that the PUVA treatment effectively induced early apoptosis of DA rat spleen lymphocytes. After co-culture, DC efficiently phagocytosed allogeneic PUVA-SP and still maintained an immature phenotype with low levels of MHC II, CD40 and CD86. PUVA-SP DC induced LEW T cell hyporesponsiveness to DA rat antigen, and led to skewing of T cell cytokine expression toward Th2 (IL-10 and IL-4). It is concluded that the PUVA-SP DC effectively down-regulate T cell response to alloantigen and induce Th2 immune deviation in vitro.