Capsaicin receptor TRPV1 maintains quiescence of hepatic stellate cells in the liver via recruitment of SARM1.

BACKGROUND & AIMS: Capsaicin receptor, also known as transient receptor potential vanilloid 1 (TRPV1), is involved in pain physiology and neurogenic inflammation. Herein, we discovered the presence of TRPV1 in hepatic stellate cells (HSCs) and aimed to delineate its function in this cell type and liver fibrosis. METHODS: TRPV1 expression was examined in liver biopsies from patients with liver fibrosis using quantitative real-time PCR and immunostaining. Its contribution to liver fibrosis was examined in Trpv1-/- mice, upon lentiviral delivery of the TRPV1 gene, and in human and mouse primary HSCs, using patch clamp, intracellular Ca2+ mobilization determination, FACS analyses and gain/loss of function experiments. Binding of sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) to TRPV1 was determined using mass spectrometry, co-immunoprecipitation, surface plasmon resonance, bioluminescence resonance energy transfer, and NanoBiT. RESULTS: TRPV1 mRNA levels are significantly downregulated in patients with liver fibrosis and mouse models, showing a negative correlation with F stage and α-smooth muscle actin expression, a marker of HSC activation. TRPV1 expression and function decrease during HSC activation in fibrotic livers in vivo or during culture. Genetic and pharmacological inhibition of TRPV1 in quiescent HSCs leads to NF-κB activation and pro-inflammatory cytokine production. TRPV1 requires binding of its N-terminal ankyrin repeat domain to the TIR-His583 (Toll/interleukin-1 receptor) domain of SARM1 to prevent HSCs from pro-inflammatory activation. Trpv1-/- mice display increased HSC activation and more severe liver fibrosis, whereas TRPV1 overexpression is antifibrotic in various disease models. CONCLUSION: The antifibrotic properties of TRPV1 are attributed to the prevention of HSC activation via the recruitment of SARM1, which could be an attractive therapeutic strategy against liver fibrosis. IMPACT AND IMPLICATIONS: We identified the neuronal channel protein TRPV1 as a gatekeeper of quiescence in hepatic stellate cells, a key driver of liver fibrogenesis and chronic liver disease. Physiologically expressed in healthy liver and consistently downregulated during liver fibrosis development, its therapeutic re-expression is expected to have few side effects, making it an attractive target diagnostic tool and drug candidate for industry and clinicians.

FBXO22 Accelerates Pancreatic Cancer Growth by Deactivation of the Hippo Pathway via Destabilizing LATS2.

BACKGROUND: Dysregulation of ubiquitin ligases plays a crucial role in the development and progression of various human tumors. F-box only protein 22 (FBXO22), an F-box E3 ubiquitin ligase, has been reported to participate in diverse aspects of cancer progression. However, the clinical significance and biological function of FBXO22 in pancreatic cancer remain poorly understood. AIMS: This study aimed to investigate the role of FBXO22 in promoting pancreatic cancer growth. METHODS: FBXO22 expression was detected in pancreatic cancer and adjacent normal tissues using qRT-PCR, western blotting, and immunohistochemistry. Ectopic expression and knockdown of FBXO22 were performed to measure the impact on pancreatic cancer cells growth by CCK-8, colony formation, and tumorigenicity assay. Bioinformatics analysis uncovered the potential correlation between FBXO22 and various signaling pathways. Western blotting and immunoprecipitation were performed to identify FBXO22-interacting proteins. RESULTS: We observed that FBXO22 was upregulated in samples obtained from patients with pancreatic cancer compared with its levels in the adjacent normal tissues, and an elevated FBXO22 level was obviously associated with poor prognosis among patients with pancreatic cancer. FBXO22 knockdown impaired pancreatic cancer cell growth both in vitro and in vivo, whereas FBXO22 overexpression accelerated pancreatic cancer cell growth. Furthermore, we found that FBXO22 contributed to pancreatic cancer cell growth by deactivating the Hippo pathway. Mechanistically, FBXO22 directly interacts with and destabilizes the large tumor suppressor 2 (LATS2), which is a critical regulator of the Hippo pathway. Blocking LATS2 leads to the loss of FBXO22-mediated oncogenic effect in pancreatic cancer. CONCLUSIONS: These findings provide new insights into the upstream regulation of the Hippo pathway inactivation in pancreatic cancer growth and identify FBXO22 as a potential therapeutic target for this lethal malignant tumor.

Neurotrophic factors stimulate the activation of hepatic stellate cells in liver fibrosis.

BACKGROUND AND AIMS: Patients with liver fibrosis who have pain in the liver region may have changed nerve factors. The expression of neurokines and hepatic nerves in liver fibrosis, however, was little understood. In order to better understand how liver fibrosis develops, we plan to look into the hepatic nerve and neurokine changes and how they relate to hepatic stellate cells (HSCs). METHODS: The expression of neurokines in liver samples from 55 chronic hepatitis B patients and the carbon tetrachloride (CCl4) animal model were studied. The co-staining of Nissl and α-SMA allowed us to investigate the neurons and their interaction with α-SMA in fibrotic livers, as well as the expression of the glial cell marker glial fibrillary acidic protein (GFAP) and its relationship with α-SMA, a marker of HSCs. SH-SY5Y cells were treated with a fibrotic serum to imitate the hepatic microenvironment on neuronal cells. We also used brain-derived neurotrophic factor (BDNF) to stimulate mouse primary HSCs and LX2. RESULTS: The levels of mRNA for neurokines such as BDNF, GFAP, and growth-associated protein (GAP43) are significantly increased in both human and animal liver fibrosis. As liver fibrosis advances, we found that Nissl bodies and α-SMA may co-localize, suggesting a connection between hepatic nerves and HSCs. Human fibrotic serum may increase neurkines, notably BDNF, in SH-SY5Y cells. We also found that BDNF increased pro-inflammatory cytokines and fibrogenic markers in hHSCs. CONCLUSIONS: Patients with hepatic fibrosis had significantly higher levels of BDNF, GFAP, GAP43, and nerve fibers. HSC and nerve fibers interact, and nerves also create neurogenic substances that promote liver fibrosis and HSC activation.

Efficacy and safety of sirolimus early conversion protocol in liver transplant patients with hepatocellular carcinoma: A single-arm, multicenter, prospective study.

Mammalian target of rapamycin (mTOR) inhibitor as an attractive drug target with promising antitumor effects has been widely investigated. High quality clinical trial has been conducted in liver transplant (LT) recipients in Western countries. However, the pertinent studies in Eastern world are paucity. Therefore, we designed a clinical trial to test whether sirolimus can improve recurrence-free survival (RFS) in hepatocellular carcinoma (HCC) patients beyond the Milan criteria after LT. This is an open-labeled, single-arm, prospective, multicenter, and real-world study aiming to evaluate the clinical outcomes of early switch to sirolimus-based regimens in HCC patients after LT. Patients with a histologically proven HCC and beyond the Milan criteria will be enrolled. The initial immunosuppressant regimens are center-specific for the first 4-6 weeks. The following regimens integrated sirolimus into the regimens as a combination therapy with reduced calcineurin inhibitors based on the condition of patients and centers. The study is planned for 4 years in total with a 2-year enrollment period and a 2-year follow-up. We predict that sirolimus conversion regimen will provide survival benefits for patients particular in the key indicator RFS as well as better quality of life. If the trial is conducted successfully, we will have a continued monitoring over a longer follow-up time to estimate indicator of overall survival. We hope that the outcome will provide better evidence for clinical decision-making and revising treatment guidelines based on Chinese population data. Trial register: Trial registered at http://www.chictr.org.cn: ChiCTR2100042869.

Liver transplantation for biliary atresia: A nationwide investigation from 1996 to 2013 in mainland China.

We investigated the overall situation of LT for BA in mainland China and analyzed their survival outcomes based on data from CLTR. Between January 1996 and December 2013, 509 liver transplants for BA were performed in mainland China and were included in this study. Patients' median age was 9.6 months (range: 4.8-175.2 months). KP was previously performed in 194 cases (38.1%). Grafts from living donors were used in 380 cases (74.7%). Era I (1996.1-2010.12) and era II (2011.1-2013.12) comprised 151 cases (29.7%) and 358 cases (70.3%), respectively. Twenty-five centers had performed at least one liver transplant for children with BA. Centers from Shanghai (197 cases), Tianjin (143 cases) and Beijing (81 cases) involved 82.7% of the 509 cases. One- and five-yr graft survival rates were 84.7% and 72.6%, respectively. Split grafts, center volume <20, GRWR ≥4.0%, and steroid-free immunosuppression regimen were independent risk factors for graft survival. In conclusion, the dramatic expansion of pediatric liver transplant programs in mainland China has enabled improved survival for those children affected by this devastating disease. However, screening of BA in neonates should be emphasized throughout the country to enhance early referrals for KP.

Submassive hepatic necrosis distinguishes HBV-associated acute on chronic liver failure from cirrhotic patients with acute decompensation.

BACKGROUND & AIMS: Distinguishing between acute on chronic liver failure (ACLF) and decompensated liver cirrhosis is difficult due to a lack of pathological evidence. METHODS: A prospective single-center study investigated 174 patients undergoing liver transplantation due to acute decompensation of hepatitis B virus (HBV)-associated liver cirrhosis. Two groups were distinguished by the presence or absence of submassive hepatic necrosis (SMHN, defined as necrosis of 15-90% of the entire liver on explant). Core clinical features of ACLF were compared between these groups. Disease severity scoring systems were applied to describe liver function and organ failure. Serum cytokine profile assays, gene expression microarrays and immunohistochemical analyzes were used to study systemic and local inflammatory responses. RESULTS: SMHN was identified in 69 of 174 patients proven to have cirrhosis by histological means. Characteristic features of SMHN were extensive necrosis along terminal hepatic veins and spanning multiple adjacent cirrhotic nodules accompanied by various degrees of liver progenitor cell-derived regeneration, cholestasis, and ductular bilirubinostasis. Patients with SMHN presented with more severely impaired hepatic function, a higher prevalence of multiple organ failure (as indicated by higher CLIF-SOFA and SOFA scores) and a shorter interval between acute decompensation and liver transplantation than those without SMHN (p<0.01 for all parameters). Further analyzes based on serum cytokine profile assays, gene expression microarrays and immunohistochemical analyzes revealed higher levels of anti-inflammatory cytokines in patients with SMHN. CONCLUSIONS: SMHN is a critical histological feature of HBV-associated ACLF. Identification of a characteristic pathological feature strongly supports that ACLF is a separate entity in end-stage liver disease.

Right lobe split liver transplantation versus whole liver transplantation in adult recipients: A systematic review and meta-analysis.

Split liver transplantation (SLT) has proven to be an effective technique to reduce the mortality of children on the waiting list, but whether creating 2 split grafts from 1 standard-criteria whole liver would compromise outcomes of adult recipients remains uncertain. We conducted this meta-analysis to compare outcomes of right lobe SLT and whole liver transplantation (WLT) in adult patients. PubMed, Embase, and the Cochrane Library were searched for relevant articles published before December 2014. Outcomes assessed were patient survival (PS), graft survival (GS), and major surgical complications after transplantation. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to synthesize the results. Seventeen studies with a total of 48,457 patients met the full inclusion criteria. PS and GS rates were all found to be equivalent between SLT and WLT recipients. However, SLT was associated with higher rates of overall biliary complications (OR = 1.66; 95% CI = 1.29-2.15; P < 0.001), bile leaks (OR = 4.30; 95% CI = 2.97-6.23; P < 0.001), overall vascular complications (OR = 1.81; 95% CI = 1.29-2.53; P < 0.001), hepatic artery thromboses (OR = 1.71; 95% CI = 1.17-2.50; P = 0.005), and outflow tract obstructions (OR = 4.17; 95% CI = 1.75-9.94; P = 0.001). No significant difference was observed in incidences of biliary stricture, portal vein complications, postoperative bleeding requiring surgical treatments, primary nonfunction, and retransplantations. In subgroup analyses, biliary and vascular complications only increased after ex vivo SLT rather than in situ SLT, and SLT recipients had more retransplantations if they matched with WLT recipients in terms of urgent status. In conclusion, adult right lobe SLT was associated with increased biliary and vascular complications compared with WLT, but it did not show significant inferiority in PSs and GSs.

Exome sequencing of hepatoblastoma reveals novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex.

UNLABELLED: Hepatoblastoma (HB) is the most common primary liver tumor in children. Mutations in the ß-catenin gene that lead to constitutive activation of the Wnt pathway have been detected in a large proportion of HB tumors. To identify novel mutations in HB, we performed whole-exome sequencing of six paired HB tumors and their corresponding lymphocytes. This identified 24 somatic nonsynonymous mutations in 21 genes, many of which were novel, including three novel mutations targeting the CTNNB1 (G512V) and CAPRIN2 (R968H/S969C) genes in the Wnt pathway, and genes previously shown to be involved in the ubiquitin ligase complex (SPOP, KLHL22, TRPC4AP, and RNF169). Functionally, both the CTNNB1 (G512V) and CAPRIN2 (R968H/S969C) were observed to be gain-of-functional mutations, and the CAPRIN2 (R968H/S969C) was also shown to activate the Wnt pathway in HB cells. These findings suggested the activation of the Wnt pathway in HB, which was confirmed by immunohistochemical staining of the ß-catenin in 42 HB tumors. We further used short hairpin RNA (shRNA)-mediated interference to assess the effect of 21 mutated genes on HB cell survival. The results suggested that one novel oncogene (CAPRIN2) and three tumor suppressors (SPOP, OR5I1, and CDC20B) influence HB cell growth. Moreover, we found that SPOP S119N is a loss-of-function mutation in HB cells. We finally demonstrated that one of the mechanisms by which SPOP inhibits HB cell proliferation is through regulating CDKN2B expression. CONCLUSION: These results extend the landscape of genetic alterations in HB and highlight the dysregulation of Wnt and ubiquitin pathways in HB tumorigenesis.

Influence of graft size matching on outcomes of infantile living donor liver transplantation.

We aimed to assess the impact of size mismatching between grafts and recipients on outcomes of infants or small children after LDLT. Between October 2006 and December 2014, 129 LDLT recipients weighing no more than 8 kg were retrospectively analyzed. The entire cohort was categorized into three groups by GRWR: GRWR < 3.0% (group A, n = 38), 3.0% ≤ GRWR < 4.0% (group B, n = 61), and GRWR ≥ 4.0% (group C, n = 30). Baseline characteristics were similar among groups A, B, and C. Compared with groups A and B, post-transplant alanine aminotransferase and aspartate aminotransferase within seven days were significantly higher in group C; however, differences between total bilirubin and albumin after transplantation were not prominent. Moreover, incidences of surgical complications, perioperative deaths, infections, and acute rejections were all comparable among the three groups. Five-yr patient survival rates for groups A, B, and C were 89.5%, 88.9%, and 81.6%, respectively (p = 0.872), and the graft survival rates were 89.5%, 86.6%, and 81.6%, respectively (p = 0.846). In conclusion, GRWR between 1.9% and 5.8% would not cause noticeable adverse events for infantile LDLT recipients ≤ 8 kg. However, there is still a role for considering reduction in the graft mass as an applicable strategy in selected cases.

Preoperative hepatic hemodynamics in the prediction of early portal vein thrombosis after liver transplantation in pediatric patients with biliary atresia.

BACKGROUND: Portal vein thrombosis (PVT) is one of the main vascular complications after liver transplantation (LT), especially in pediatric patients with biliary atresia (BA). This study aimed to assess the preoperative hepatic hemodynamics in pediatric patients with BA using Doppler ultrasound and determine whether ultrasonographic parameters may predict early PVT after LT. METHODS: One hundred and twenty-eight pediatric patients with BA younger than 3 years of age underwent Doppler ultrasound within seven days before LT, between October 2006 and June 2013. The preoperative hepatic hemodynamic parameters were then compared between patients with early PVT (within 1 month following LT) and those without PVT. Receiver operating characteristic analysis was performed to determine the optimal cutoff value for predicting early PVT. RESULTS: Of the 128 transplant recipients, 41 (32.03%) had a hypoplastic portal vein (PV), 52 (40.63%) had hepatofugal PV flow and 40 (31.25%) had a high hepatic artery resistance index (HARI) of ≥1. Nine cases (7.03%) experienced early PVT. A PV diameter ≤4 mm (sensitivity 88.89%, specificity 72.27%), and a hepatofugal PV flow (sensitivity 77.78%, specificity 62.18%) with a high HARI ≥1 (sensitivity 77.78%, specificity 72.27%) were hepatic hemodynamic risk factors for early PVT. CONCLUSIONS: Hepatic hemodynamic disturbances in pediatric recipients with BA were more common. Small PV diameter (≤4 mm) and hepatofugal PV flow combined with high HARI (≥1) are strong warning signs of early PVT after LT in pediatric patients with BA. Intense monitoring of vascular patency and prophylactic thrombolytic therapy should be considered in pediatric patients undergoing LT for BA.

CYP3A5 genotypes affect tacrolimus pharmacokinetics and infectious complications in Chinese pediatric liver transplant patients.

Little information is available regarding the impact of cytochrome P450 (CYP) 3A5 on the metabolism of TAC in infant LTx. Therefore, the CYP3A5 genotype of Chinese pediatric recipients (intestine) as well as donors (graft liver) was performed for the purpose of establishing an optimal dosage regimen in children. Sixty-four patients were divided according to CYP3A5 genotype (expression of *1 allele: EX and NEX) for each recipient (R) and donor (D), EX-R/EX-D (n = 21), EX-R/NEX-D (n = 8), NEX-R/EX-D (n = 8) and NEX-R/NEX-D (n = 27). Results indicated that initial TAC daily dose requirement was higher among EX-R/EX-D children compared with those who did not express CYP3A5 (0.28 ± 0.10 vs. 0.19 ± 0.08 mg/kg/day, p < 0.01). CYP3A5 expression contributed an overall of 38.35% to its C/D ratios, and graft liver was a key determinant. Additionally, the EX-R/EX-D group showed significantly higher incidence of infectious complications, lower immune response and was an independent risk factor for the development of infections (odds ratio 3.86, p = 0.025). Donor CYP3A5 expression partially explains TAC dose requirement, the effect of CYP3A5 variation may influence clinical outcomes; therefore, monitoring immune response may be important for preventing risks associated with under- and over-immunosuppression.

Living Donor Auxiliary Partial Orthotopic Liver Transplantation for Acute Liver Failure: A Case Report.

Acute liver failure (ALF) is a life-threatening end-stage liver disease, causing high mortality all over the world. Here, we presented a 54-year-old male patient, suffering from drug-induced ALF on the basis of hepatitis B virus (HBV) infection. At that time, the only available donor we could find was his 44-year-old sister. We performed living donor auxiliary partial orthotopic liver transplantation (APOLT) on the second day of admission. The right lobe of the donor liver was implanted into the recipient orthotopically. The actual graft-to-recipient weightratio (GRWR) was 0.85% and the postoperative immunosuppression was a combination of tacrolimus, mycophenolate mofetil (MMF) and steroids. The patient recovered consciousness on the third post-operative day and was discharged with normal liver function after 43 days of transplantation. After a 48-month follow-up, the recipient has been restored to normal life and the immunosuppression has been tapered off (tacrolimus, 1 mg/d). Besides, the size of the native liver has extended to 29% of standard liver volume (SLV) and the native liver cells have returned to normal pathological condition. In conclusion, APOLT is a life-saving treatment for ALF with excellent post-operative outcomes.

Living donor liver transplantation in 43 children with biliary atresia: a single-center experience from the mainland of China.

BACKGROUND: There is no large-cohort report on living donor liver transplantation (LDLT) for biliary atresia (BA) patients from the mainland of China. This single-center study describes our initial experience with 43 LDLTs for BA patients aged two years or younger. METHODS: In this study, the eligibility criteria were BA as the primary diagnosis and two years of age or younger. From October 2006 to December 2010, the clinical data of 43 LDLTs, including pre-operative evaluations, surgical techniques, postoperative complications and outcomes of donors and recipients, were retrospectively analyzed. RESULTS: Donor graft type was the left lateral segment with compatible ABO blood groups. Forty-three recipients were selected in this study. The median patient age at operation was 9 months (range 6-24), and the median body weight was 8 kg (range 5.7-12.5). Fourteen (32.6%) recipients received Kasai operations before liver transplantation. The overall one- and two-year cumulative survival rates for grafts and recipients were 81%, 81% and 76%, 76%, respectively. No donor mortality was encountered, with a minimal morbidity and no long-term sequelae. Nine out of 43 recipients died. Postoperative complications of recipients were biliary leakage and refluxing cholangitis (11/43, 25.6%), hepatic artery thrombosis (4, 9.3%), pulmonary infections (4, 9.3%), portal vein thrombosis (3, 7.0%), wound disruption (3, 7.0%), acute rejection (3, 7.0%), cytomegalovirus infection (2, 4.7%), and intra-abdominal bleeding (1, 2.3%). CONCLUSION: Despite the relatively low survival rates due to lack of experience initially, LDLT still provides encouraging outcomes for pediatric recipients with BA, even small children under two years old.

Evaluation of Early Retinal Nerve Injury in Type 2 Diabetes Patients Without Diabetic Retinopathy.

Objectives: To investigate the damage to the retinal nerve fiber layer (RNFL) and ganglion cell complex layer (GCL+) in diabetic patients without retinal microangioma and to determine the kind of nerve damage more likely to indicate early injury. Subjects and Methods: We included 360 patients (360 eyes) with type 2 diabetes mellitus and 168 healthy volunteers (168 eyes). Patients with retinal microangioma were excluded by fundus fluorescein angiography (FFA). The parameters around the optic disc and macular area were measured by optical coherence tomography (OCT). Results: The peripapillary RNFL thickness was thinner in the temporal (72.98 ± 13.76 µm, P < 0.0001) and inferior (120.71 ± 21.43 µm, P = 0.0103) sectors in patients with no diabetic retinopathy (NDR) compared to healthy controls. The reduction of retinal thickness in the macular region was prominent in the inferior sector in patients (34.74 ± 4.92 µm, P < 0.0001) compared to normal controls. Thinning of GCL+ in the second region of the macular area was significant in patients with NDR compared to normal controls (P < 0.05). However, no difference in the GCL+ and retinal thicknesses of the central macular region was observed between the patients with NDR and healthy controls. Using the 5th percentile (P5) of normal controls as the reference value, we found that the parameters with the highest indices in patients with NDR were the inferior and temporal peripapillary RNFL thickness (13.0%), the inferior RNFL thickness in the macular area (20%), the inferior retinal thickness in the outer ring of the macular area (10.8%), and the inferior GCL+ thickness in the macular area (10.6%). The GCL+ and RNFL thicknesses in the central macular area accounted for the smallest proportion in P5 of normal controls (3%). Conclusions: Retinal nerve injury can occur in patients without retinal microangioma. The inferior RNFL in the macular area and the inferior and temporal peripapillary RNFL were most sensitive to glucose damage. These areas might be associated with early detection of diabetic retinopathy (DR) as they are more likely to indicate early damage.

Diagnostic Value of Systematic Imaging Examination in Embedded Optic Disc Drusen in Adolescents with Mild Visual Impairment.

AIM: To evaluate the diagnostic value of systematic ophthalmologic imaging examination in the diagnosis of embedded optic disc drusen (ODD) in adolescents with mild visual impairment. METHODS: Eleven patients were evaluated through optometric examination, fundus photography, visual field inspection, optical coherence tomography (OCT), ultrasonography (US), and fundus fluorescein angiography (FFA). Of the 11 patients, three also underwent cranial and orbital magnetic resonance imaging (MRI). RESULTS: All 11 patients had either no apparent abnormality or only mild refractive abnormalities. In all patients, fundus inspection revealed flushing the optic disc with varying degrees of limited boundary ambiguity and optic disc congestion with disappearance of the fovea. One patient had a visual field defect during the period of edema of ODD, but the visual field returned to normal after the optic disc edema subsided. US revealed discoid acousto-optic masses in front of the optic disc in six patients. OCT showed a slight elevation and thinning of the retinal nerve fiber layer (RNFL) of the optic disc in all patients. Quasicircular, hyperreflex signals of different sizes could be observed below the RNFL. Late-stage FFA revealed focal staining at the edge of the optic disc without fluorescence leakage in all patients. Orbital and cranial MRI findings were normal in the three patients. CONCLUSION: A systematic ophthalmologic imaging examination can not only improve the detection rate of embedded ODD but also avoid excessive examinations and treatments.

Hepatectomy combined with sorafenib in patients with intermediate-advanced hepatocellullar carcinoma.

PURPOSE: To evaluate the efficacy and tolerability of hepatectomy in combination with sorafenib in the treatment of intermediate-advanced hepatocellular carcinoma (HCC). METHODS: One hundred and eighty-sixty consecutive patients with intermediate-advanced HCC who were treated with sorafenib were enrolled in this study. They were divided into two groups: sorafenib group (39) and hepatectomy combined with sorafenib group (147). Survival rates of the patients were analyzed by the Kaplan-Meier method. Cox's proportional hazards model was used to analyze variables associated with survival. Adverse events induced by sorafenib were observed and recorded. RESULTS: The median follow-up duration was 13.0 months (range 1-41). There are 77 patients with intermediate HCC (BCLC stage B) (41.4%) and 109 patients with advanced HCC (BCLC stage C) (58.6%). The overall survival was greater in patients with intermediate HCC than in patients with advanced HCC (p=0.011). Surgery before administration of sorafenib did not contribute to overall survival of patients with intermediate HCC (p=0.312). For patients with advanced HCC, the survival of those who underwent surgery before sorafenib was significantly longer than that of patients who received sorafenib alone (15.0 months, 95% CI 12.3-17.7 vs. 8.0 months, 95% CI 5.5-10.5; p=0.024) and surgery before sorafenib was identified as the only predictor of survival for patients with advanced HCC (HR, 0.582; 95%CI, 0.353-0.932; p=0.035). CONCLUSIONS: The combination of surgery and sorafenib is safe and significantly prolongs overall survival of patients with advanced HCC.

A DMSO-free hepatocyte maturation medium accelerates hepatic differentiation of HepaRG cells in vitro.

The most essential tools for studying drug hepatotoxicity, liver diseases, and bioartificial livers have always been models that can recapitulate liver physiology in vitro. The liver progenitor cell line HepaRG represents an effective surrogate of the primary hepatocyte. However, the differentiation of HepaRG relies on long-term induction using a high concentration of dimethyl sulfoxide (DMSO), which may compromise the research of drug metabolism and restrict the applicability of this hepatic model. Here, we present a novel hepatic maturation medium (HMM) for the differentiation of HepaRG, which is based on a cocktail of soluble molecules that mimick the in vivo environment. We showed that HMM could rapidly (about nine days) induce HepaRG differentiation into polarized hepatocytes with maturely metabolic functions. In addition, under three-dimensional culture conditions, the hepatic spheroids showed multiple liver functions and toxicity profiles close to those of primary human hepatocytes (PHH). Our work demonstrates the utility of HMM as an alternative to the DMSO-dependent differentiation protocol for HepaRG; moreover, these results facilitate the application of HepaRG.

Generation of hepatic spheroids using human hepatocyte-derived liver progenitor-like cells for hepatotoxicity screening.

Rationale: The idiosyncratic drug-induced liver injury (iDILI) is a major cause of acute liver injury and a key challenge in late-stage drug development. Individual heterogeneity is considered to be an essential factor of iDILI. However, few in vitro model can predict heterogeneity in iDILI. We have previously shown that mouse and human hepatocytes can be converted to expandable liver progenitor-like cells in vitro (HepLPCs). However, the limited proliferation potential of human HepLPCs confines its industrial application. Here, we reported the generation of a novel hepatocyte model not only to provide unlimited cell sources for human hepatocytes but also to establish a tool for studying iDILI in vitro. Methods: Human primary hepatocytes were isolated by modified two-step perfusion technique. The chemical reprogramming culture condition together with gene-transfer were then used to generate the immortalized HepLPC cell lines (iHepLPCs). Growth curve, doubling time, and karyotype were analyzed to evaluate the proliferation characteristics of iHepLPCs. Modified Hepatocyte Maturation Medium and 3D spheroid culture were applied to re-differentiate iHepLPCs. Results: iHepLPCs exhibited efficient expansion for at least 40 population doublings, with a stable proliferative ability. They could easily differentiate back into metabolically functional hepatocytes in vitro within 10 days. Furthermore, under three-dimensional culture conditions, the formed hepatic spheroids showed multiple liver functions and toxicity profiles close to those of primary human hepatocytes. Importantly, we established a hepatocyte bank by generating a specific number of such cell lines. Screening for population heterogeneity allowed us to analyze the in vitro heterogeneous responses to hepatotoxicity induced by molecular targeted drugs. Conclusions: In light of the proliferative capacity and the heterogeneity they represented, these iHepLPCs cell lines may offer assistance in studying xenobiotic metabolism as well as liver diseases in vitro.

[Clinical study of diabetic retinopathy treated by compound danshen dripping pills].

OBJECTIVE: To observe the therapeutic effect of compound danshen dripping pills in visual function recovery of diabetic retinopathy. METHODS: 42 patients(78 eyes) with diabetic retinopathy of I-III phase were divided into two groups in random. 43 eyes were treated with compound danshen dripping pills and the other 35 eyes with vitamin BI and Luding C for three months. The visual acuity, fundus, visual field, fundus fluorescein angiography and visual electro-physiology were observed. RESULTS: The visual acuity in treated group were significantly improved after treatment and had significant difference with those in control group (P < 0.05). The number of micro-hemorrage, microaneurysm and the mean defect (MD) of visual field in treated group decreased obviously after treatment and had significant difference with that in control group (P < 0.05). The latent period of P100 wave and a, b wave in treated group were shorter than control group, and the wave amplitude were higher than that in control group. CONCLUSION: The compound danshen dripping pills can improve the visual acuity, control the micro-hemorrage and microaneurysm of fundus and have the effect in visual function recovery of diabetic retinopathy.