Intensive Surveillance of Porcine-Rhesus Kidney Xenotransplant Using Different Ultrasound Techniques.

BACKGROUND: Significant progress has been made in kidney xenotransplantation in the past few years, and this field is accelerating towards clinical translation. Therefore, surveillance of the xenograft with appropriate tools is of great importance. Ultrasonography has been widely used in kidney allotransplantation and served as an economical and non-invasive method to monitor the allograft. However, questions remain whether the ultrasonographic criteria established for human kidney allograft could also be applied in xenotransplantation. METHODS: In the current study, we established a porcine-rhesus life sustaining kidney xenotransplantation model. The xenograft underwent intensive surveillance using gray-scale, colorful Doppler ultrasound as well as 2D shear wave elastography. The kidney growth, blood perfusion, and cortical stiffness were measured twice a day. These parameters were compared with the clinical data including urine output, chemistry, and pathological findings. RESULTS: The observation continued for 16 days after transplantation. Decline of urine output and elevated serum creatinine were observed on POD9 and biopsy proven antibody-mediated rejection was seen on the same day. The xenograft underwent substantial growth, with the long axis length increased by 32% and the volume increased by threefold at the end of observation. The resistive index of the xenograft arteries elevated in response to rejection, together with impaired cortical perfusion, while the peak systolic velocity (PSV) was not compromised. The cortical stiffness also increased along with rejection. CONCLUSION: In summary, the ultrasound findings of kidney xenograft shared similarities with those in allograft but possessed some unique features. A modified criteria needs to be established for further application of ultrasound in kidney xenotransplantation.

Prognostic value of preoperative inflammatory markers in patients with hepatocellular carcinoma who underwent curative resection.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is not optimistic. Our study focused on present inflammatory markers, including the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), gamma-glutamyl transpeptidase-to-platelet ratio (GPR), aspartate aminotransferase-to-lymphocyte ratio (ALR) and fibrinogen-to-albumin ratio (FAR), and explored their optimal combination for the prognosis of HCC after resection. METHODS: A total of 347 HCC patients who underwent curative resection were enrolled. The optimal cutoff values of the inflammatory markers were calculated using receiver operating characteristic (ROC) curve analysis, and used to divide patients into two groups whose differences were compared by Kaplan-Meier analysis. Cox univariate and multivariate analyses were used to analyze the independent prognostic inflammatory markers. The χ2 test was chosen to determine the relationship between independent prognostic inflammatory markers and clinicopathological features. We created combined scoring models and evaluated them by Cox univariate and multivariate methods. The concordance index (C-index), Akaike information criterion (AIC) and likelihood ratio were calculated to compare the models. The selected optimal inflammatory markers and their combinations were tested in different stages of HCC by Kaplan-Meier analysis. RESULTS: The ALR and GPR were independent prognostic factors for disease-free survival (DFS); the ALR, PLR, and GPR were independent prognostic factors for overall survival (OS). The proposed GPR and ALR-GPR-PLR score models were independent predictors for DFS and OS, respectively. CONCLUSION: The preoperative GPR and ALR-GPR-PLR score models were independent predictors for DFS and OS, respectively, and performed well in stratifying patients with HCC. The higher the score in the model was, the worse the prognosis.

Immortalization of porcine hepatocytes with a α-1,3-galactosyltransferase knockout background.

BACKGROUND: In vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in "bridging" liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT-KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild-type and GalT-KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation. METHODS: Primary hepatocytes extracted from GalT-KO and wild-type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT-KO porcine hepatocytes (GalT-KO-hep) and wild-type porcine hepatocytes (WT). Hepatocyte biomarkers and function-related genes were assessed by immunofluorescence, periodic acid-Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT-PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement-dependent cytotoxicity (CDC) assay was performed with GalT-KO-hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK-8 assay. RESULTS: GalT-KO and wild-type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT-KO-hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT-KO-hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum. CONCLUSIONS: Two types of immortalized cell lines of porcine hepatocytes with GalT-KO and wild-type backgrounds were successfully established. GalT-KO-hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.

Translationally controlled tumor protein exerts a proinflammatory role in acute rejection after liver transplantation.

BACKGROUND: Translationally controlled tumor protein (TCTP), which has been verified to have a proinflammatory activity, plays an important role in allergy. However, it remains unclear whether TCTP has an impact on the acute rejection (AR) after liver transplantation. METHODS: Three protocols were used to delineate the role of TCTP in AR after liver transplantation. First, in rat orthotopic liver transplantation (OLT), the expression of TCTP was measured by enzyme-linked immunosorbent assay (ELISA), real-time PCR, Western blot and immunofluorescence assays. Second, in mixed lymphocyte reaction (MLR), the role of TCTP in lymphocyte proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) labeling and the impact of TCTP on inflammatory factor release was detected by cytokine arrays. Third, in human OLT, the level of serum TCTP was detected by ELISA, and the relationship between TCTP and model for early allograft function (MEAF) score was assessed by Spearman's correlation. RESULTS: In rat OLT, AR resulted in great harm to allografts, manifesting as deterioration of liver function, increasing inflammatory factors and infiltrating lymphocytes. Meanwhile, TCTP was overexpressed in serum and allografts. Higher level of TCTP was associated with higher rejection activity index (RAI). In an MLR protocol, TCTP knockdown inhibited the proliferation of mixed inflammatory cells and significantly suppressed the release of 15 cytokines and chemokines. In human OLT, the serum TCTP was up-regulated within a week after operation. Additionally, the increasing speed of serum TCTP positively correlated with MEAF scores (r = 0.449; P = 0.0088). CONCLUSIONS: Up-regulated TCTP positively affects AR after liver transplantation.

Endothelial Notch activation reshapes the angiocrine of sinusoidal endothelia to aggravate liver fibrosis and blunt regeneration in mice.

Liver sinusoidal endothelial cells (LSECs) critically regulate liver homeostasis and diseases through angiocrine factors. Notch is critical in endothelial cells (ECs). In the current study, Notch signaling was activated by inducible EC-specific expression of the Notch intracellular domain (NIC). We found that endothelial Notch activation damaged liver homeostasis. Notch activation resulted in decreased fenestration and increased basement membrane, and a gene expression profile with decreased LSEC-associated genes and increased continuous EC-associated genes, suggesting LSEC dedifferentiation. Consistently, endothelial Notch activation enhanced hepatic fibrosis (HF) induced by CCl4 . Notch activation attenuated endothelial nitric oxide synthase (eNOS)/soluble guanylate cyclase (sGC) signaling, and activation of sGC by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) reversed the dedifferentiation phenotype. In addition, Notch activation subverted the hepatocyte-supporting angiocrine profile of LSECs by down-regulating critical hepatocyte mitogens, including Wnt2a, Wnt9b, and hepatocyte growth factor (HGF). This led to compromised hepatocyte proliferation under both quiescent and regenerating conditions. Whereas expression of Wnt2a and Wnt9b was dependent on eNOS-sGC signaling, HGF expression was not rescued by the sGC activator, suggesting heterogeneous mechanisms of LSECs to maintain hepatocyte homeostasis. CONCLUSION: Endothelial Notch activation results in LSEC dedifferentiation and accelerated liver fibrogenesis through eNOS-sGC signaling, and alters the angiocrine profile of LSECs to compromise hepatocyte proliferation and liver regeneration (LR). (Hepatology 2018).

A review of pig liver xenotransplantation: Current problems and recent progress.

Pig liver xenotransplantation appears to be more perplexing when compared to heart or kidney xenotransplantation, even though great progress has been achieved. The relevant molecular mechanisms involved in xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia, are complex, and need to be systematically investigated. The deletion of expression of Gal antigens in the liver graft highlights the injurious impact of nonGal antigens, which continue to induce humoral rejection. Innate immunity, particularly mediated by macrophages and natural killer cells, interplays with inflammation and coagulation disorders. Kupffer cells and liver sinusoidal endothelial cells (LSECs) together mediate leukocyte, erythrocyte, and platelet sequestration and phagocytosis, which can be exacerbated by increased cytokine production, cell desialylation, and interspecies incompatibilities. The coagulation cascade is activated by release of tissue factor which can be dependent or independent of the xenoreactive immune response. Depletion of endothelial anticoagulants and anti-platelet capacity amplify coagulation activation, and interspecies incompatibilities of coagulation-regulatory proteins facilitate dysregulation. LSECs involved in platelet phagocytosis and transcytosis, coupled with hepatocyte-mediated degradation, are responsible for thrombocytopenia. Adaptive immunity could also be problematic in long-term liver graft survival. Currently, relevant evidence and study results of various genetic modifications to the pig donor need to be fully determined, with the aim of identifying the ideal transgene combination for pig liver xenotransplantation. We believe that clinical trials of pig liver xenotransplantation should initially be considered as a bridge to allotransplantation.

Preoperative Alfa-Fetoprotein and Fibrinogen Predict Hepatocellular Carcinoma Recurrence After Liver Transplantation Regardless of the Milan Criteria: Model Development with External Validation.

BACKGROUND/AIMS: Patient selection is critically important in improving the outcomes of liver transplantation for hepatocellular carcinoma. The aim of the current study was to identify biochemical measures that could affect patient prognosis after liver transplantation. METHODS: A total of 119 patients receiving liver transplantation for hepatocellular carcinoma were used to construct a model for predicting recurrence. The results were validated using an independent sample of 109 patients from independent hospitals. All subjects in both cohorts met the Hangzhou criteria. RESULTS: Analysis of the discovery cohort revealed an association of recurrence with preoperative fibrinogen and AFP levels. A mathematical model was developed for predicting probability of recurrence within 5 years: Y = logit(P) = -4.595 + 0.824 ×fibrinogen concentration (g/L) + 0.641 × AFP score (1 for AFP<=20ng/ml, 2 for 20 400ng/ml). At a cutoff score of -0.85, the area under the curve (AUC) was 0.819 in predicting recurrence (vs. 0.655 when using the Milan criteria). In the validation cohort, this model had reasonable performance in predicting 5-year overall survival (68.8% vs. 28.1% in using the -0.85 cutoff, p< 0.001) and disease-free survival (65.7% vs. 25.9%, p< 0.001). The sensitivity and specificity were 77.0% and 62.5%, respectively. The AUC of this newly developed model was similar to that with the Milan criteria (0.698 vs. 0.678). Surprisingly, the DFS in patients with score <= -0.85 under this model but not meeting the Milan criteria was similar to that in patients meeting the Milan criteria (53.8% vs. 60.0%, p=0.380). CONCLUSIONS: Preoperative AFP and fibrinogen are useful in predicting recurrence of hepatocellular carcinoma after liver transplantation.

Safety assessment of sorafenib in Chinese patients with unresectable hepatocellular carcinoma: subgroup analysis of the GIDEON study.

BACKGROUND: This study aimed to investigate the safety of sorafenib for the treatment of unresectable hepatocellular carcinoma in Chinese patients. METHODS: A subgroup of 345 Chinese patients from the international database of the Global Investigation of therapeutic DEcisions in hepatocellular carcinoma and Of its treatment with sorafeNib (GIDEON) study was included in this analysis. Safety assessment measures were adverse events (AEs) and serious adverse events (SAEs) graded using the National Cancer Institute Common Terminology Criteria version 3.0. RESULTS: Of 331 evaluable patients, 98% started sorafenib at 800 mg/day. The median treatment duration was 22 weeks (range, 0.1-116 weeks), and median overall survival (OS) was 322 days (10.7 months). Approximately 50% of patients had at least one adverse event, and 6% had grade 3-4 adverse events. Drug-related adverse events were experienced by 29% of patients, and 3.6% had grade 3-4 drug-related adverse events. Overall, 23% of patients (n = 77) experienced serious adverse events, among which only 1 event was drug-related (0.3%). No differences in overall adverse events, serious adverse events, and deaths were observed between Child-Pugh A and Child-Pugh B patients. The most frequent drug-related adverse events were dermatological/skin (24%), hand-foot skin reaction (20%), gastrointestinal (11%), and diarrhea (11%). The majority of adverse events occurred within 30 days of beginning sorafenib. CONCLUSION: Sorafenib has satisfactory efficacy and safety in Chinese Child-Pugh A and B patients with unresectable HCC using the recommended dosage of 800 mg/day, and the safety of sorafenib is not affected by liver function. Prophylaxis for gastrointestinal adverse events may help to decrease dose interruptions or discontinuation. TRIAL REGISTRATION: ClinicalTrials.gov ; Identifier: NCT00812175. Date of registration: December 19, 2008.

Blockade of ARHGAP11A reverses malignant progress via inactivating Rac1B in hepatocellular carcinoma.

BACKGROUND: The molecular signaling events involving in high malignancy and poor prognosis of hepatocellular carcinoma (HCC) are extremely complicated. Blockade of currently known targets has not yet led to successful clinical outcome. More understanding about the regulatory mechanisms in HCC is necessary for developing new effective therapeutic strategies for HCC patients. METHODS: The expression of Rho GTPase-activating protein 11A (ARHGAP11A) was examined in human normal liver and HCC tissues. The correlations between ARHGAP11A expression and clinicopathological stage or prognosis in HCC patients were analyzed. ARHGAP11A was downregulated to determine its role in the proliferation, invasion, migration, epithelial-to-mesenchymal transition (EMT) development, and regulatory signaling of HCC cells in vitro and in vivo. RESULTS: ARHGAP11A exhibited high expression in HCC, and was significantly correlated with clinicopathological stage and prognosis in HCC patients. Moreover, ARHGAP11A facilitated Hep3B and MHCC97-H cell proliferation, invasion, migration and EMT development in vitro. ARHGAP11A knockdown significantly inhibited the in vivo growth and metastasis of HCC cells. Furthermore, ARHGAP11A directly interacted with Rac1B independent of Rho GTPase- activating activity. Rac1B blockade effectively interrupted ARHGAP11A-elicited HCC malignant phenotype. Meanwhile, upregulation of Rac1B reversed ARHGAP11A knockdown mediated mesenchymal-to-epithelial transition (MET) development in HCC cells. CONCLUSION: ARHGAP11A facilitates malignant progression in HCC patients via ARHGAP11A-Rac1B interaction. The ARHGAP11A/Rac1B signaling could be a potential therapeutic target in the clinical treatment of HCC.

Cytotherapy with M1-polarized macrophages ameliorates liver fibrosis by modulating immune microenvironment in mice.

BACKGROUND & AIMS: Macrophages play vital roles in chronic liver injury, and have been tested as a tool for cytotherapy in liver fibrosis. However, macrophages possess ontogenic and functional heterogeneities. Some subsets are pro-fibrotic, whereas others are anti-fibrotic. This study aimed to clarify which macrophage subset is efficient for cytotherapy in liver fibrosis and to elucidate the underlying mechanisms. METHODS: Liver fibrosis was induced in mice by carbon tetrachloride injection or bile duct ligation. Bone-marrow-derived macrophages (BMDMs) were polarized into M0, M1, or M2 macrophages, respectively. BMDMs were infused into mice through the tail vein at different stages of fibrogenesis. Fibrosis progression, hepatic cell populations, and related molecular changes were evaluated. RESULTS: Both M0 and M1 BMDMs significantly ameliorated liver fibrosis, but M1 exhibited stronger therapeutic effects than M0. M2 macrophages were not effective on liver fibrosis. M1 macrophages reduced the number and activation of hepatic stellate cells (HSCs), which could be attributed at least partly to increased HSC apoptosis. M1 macrophages enhanced the recruitment of endogenous macrophages into fibrotic liver, which displayed the phenotype of Ly6Clo restorative macrophages and produced matrix metalloproteinases (MMPs) and hepatic growth factor (HGF) to enhance collagen degradation and hepatocyte proliferation, respectively. M1 macrophages also increased the number of total and activated natural killer (NK) cells in the fibrotic liver, which released TNF-related apoptosis-inducing ligand (TRAIL), inducing HSC apoptosis. CONCLUSIONS: M1 macrophages, which modulate the immune microenvironment to recruit and modify the activation of endogenous macrophages and NK cells, are effective for cytotherapy in experimental liver fibrosis. Lay summary: M1 Bone marrow-derived macrophages (BMDMs) exhibit a stronger therapeutic effect by modulating the hepatic microenvironment to recruit and modify the activation of endogenous macrophages and natural killer (NK) cells, which likely lead to hepatic stellate cells (HSCs) apoptosis and hampered fibrogenesis.

Evaluation of sorafenib in Chinese unresectable hepatocellular carcinoma patients with prior surgery and portal vein tumor thrombosis: A subset analysis of GIDEON study data.

The purpose of this study was to examine the safety and efficacy of sorafenib in Chinese patients with unresectable hepatocellular carcinoma. Data of 338 Chinese patients from the Global Investigation of therapeutic DEcisions in hepatocellular carcinoma and Of its treatment with sorafeNib study database were included. Patients were divided into those who received and did not receive sorafenib prior to surgical resection and those with and without portal vein tumor thrombosis. In the non-surgery group, the median survival was 302 days (95% confidence interval: 244-371), and the median time from diagnosis to death was 428 days (95% confidence interval: 352-556); in the surgery group, half of the patients survived for 345 days and the median time from diagnosis to death was 1000 days (95% confidence interval: 750-2816). Median progression-free survival and median time to progression were not different between the two groups. Median overall survival was 360 days (95% confidence interval: 309-435) in the non-portal vein tumor thrombosis group and 240 days (95% confidence interval: 181-296) in the portal vein tumor thrombosis group; median time between hepatocellular carcinoma diagnosis and death was 750 days (95% confidence interval: 472-1000) and 420 days (95% confidence interval: 252-567), respectively, in the two groups. Median progression-free survival was 209 days (95% confidence interval: 166-264) for patients without portal vein tumor thrombosis and 154 days (95% confidence interval: 112-202) for patients with portal vein tumor thrombosis; median time to progression was 295 days (95% confidence interval: 209-463) and 221 days, respectively. Adverse events were generally comparable regardless of prior surgery and portal vein tumor thrombosis status. We thus conclude that earlier administration of sorafenib may result in improved outcomes in patients with unresectable hepatocellular carcinoma and portal vein tumor thrombosis.

Cytokine profiles in Tibetan macaques following α-1,3-galactosyltransferase-knockout pig liver xenotransplantation.

BACKGROUND: Pig-to-nonhuman primate orthotopic liver xenotransplantation is often accompanied by thrombocytopenia and coagulation disorders. Furthermore, the release of cytokines can trigger cascade reactions of coagulation and immune attacks within transplant recipients. To better elucidate the process of inflammation in liver xenograft recipients, we utilized a modified heterotopic auxiliary liver xenotransplantation model for xeno-immunological research. We studied the cytokine profiles and the relationship between cytokine levels and xenograft function after liver xenotransplantation. METHODS: Appropriate donor and recipient matches were screened using complement-dependent cytotoxicity assays. Donor liver grafts from α1,3-galactosyltransferase gene-knockout (GTKO) pigs or GTKO pigs additionally transgenic for human CD47 (GTKO/CD47) were transplanted into Tibetan macaques via two different heterotrophic auxiliary liver xenotransplantation procedures. The cytokine profiles, hepatic function, and coagulation parameters were monitored during the clinical course of xenotransplantation. RESULTS: Xenograft blood flow was stable in recipients after heterotopic auxiliary transplantation. A Doppler examination indicated that the blood flow speed was faster in the hepatic artery (HA) and hepatic vein (HV) of xenografts subjected to the modified Sur II (HA-abdominal aorta+HV-inferior vena cava) procedure than in those subjected to our previously reported Sur I (HA-splenic artery+HV-left renal vein) procedure. Tibetan macaques receiving liver xenografts did not exhibit severe coagulation disorders or immune rejection. Although the recipients did suffer from a rapid loss of platelets, this loss was mild. In blood samples dynamically collected after xenotransplantation (post-Tx), dramatic increases in the levels of monocyte chemoattractant protein 1, interleukin (IL)-8, granulocyte-macrophage colony-stimulating factor, IL-6, and interferon gamma-induced protein 10 were observed at 1 hour post-Tx, even under immunosuppression. We further confirmed that the elevation in individual cytokine levels was correlated with the onset of graft damage. Finally, the release of cytokines might contribute to leukocyte infiltration in the xenografts. CONCLUSION: Here, we established a modified auxiliary liver xenotransplantation model resulting in near-normal hepatic function. Inflammatory cytokines might contribute to early damage in liver xenografts. Controlling the systemic inflammatory response of recipients might prevent early post-Tx graft dysfunction.

Myeloid-specific disruption of recombination signal binding protein Jκ ameliorates hepatic fibrosis by attenuating inflammation through cylindromatosis in mice.

UNLABELLED: Macrophages play multidimensional roles in hepatic fibrosis, but their control has not been fully understood. The Notch pathway mediated by recombination signal binding protein Jκ (RBP-J), the transcription factor transactivated by signals from four mammalian Notch receptors, is implicated in macrophage activation and plasticity. In this study, by using mouse hepatic fibrosis models, we show that myeloid-specific disruption of RBP-J resulted in attenuated fibrosis. The activation of hepatic stellate cells and production of profibrotic factors including platelet-derived growth factor (PDGF)-B and transforming growth factor beta1 (TGF-ß1) reduced significantly in myeloid-specific RBP-J deficient mice. The infiltration of inflammatory cells and production of proinflammatory factors were reduced in liver of myeloid-specific RBP-J-deficient mice during fibrosis. In RBP-J-deficient macrophages, the nuclear factor kappa B (NF-κB) activation was remarkably attenuated as compared with the control. This could be attributed to the up-regulation of cylindromatosis (CYLD), a negative regulator of NF-κB, in Notch signal-compromised macrophages, because the knockdown of CYLD in RBP-J-deficient macrophages or overexpression of p65 in RBP-J knockdown cells both restored NF-κB activation and the production of proinflammatory and/or profibrotic factors by macrophages. In human hepatic fibrosis biopsies, stronger Notch activation is correlated with more severe fibrosis, which is accompanied by a lower level of CYLD but irrespective of etiological reasons. CONCLUSION: RBP-J-mediated Notch signaling is required for macrophages to promote hepatic fibrosis by up-regulation of NF-κB activation through CYLD.

RRAD inhibits aerobic glycolysis, invasion, and migration and is associated with poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer worldwide. However, the mechanism underlying the HCC development remains unclear. Ras-related associated with diabetes (RRAD) is a small Ras-related GTPase which has been implicated in metabolic disease and several types of cancer, yet its functions in HCC remain unknown. A tissue microarray constructed by 90 paired HCC tissues and adjacent non-cancerous liver tissues was used to examine the protein levels of RRAD, and the messenger RNA (mRNA) expression of RRAD was also detected in a subset of this cohort. The prognostic significance of RRAD was estimated by the Kaplan-Meier analysis and Cox regression. The glucose utilization assay and lactate production assay were performed to measure the role of RRAD in HCC glycolysis. The effect of RRAD in HCC invasion and metastasis was analyzed by transwell assays. Our results suggested that the expression of RRAD was downregulated in HCC tissues compared to the adjacent non-tumorous liver tissues both in mRNA and protein levels and lower RRAD expression served as an independent prognostic indicator for the survival of HCC patients. Moreover, RRAD inhibited hepatoma cell aerobic glycolysis by negatively regulating the expression of glucose transporter 1 (GLUT1) and hexokinase II (HK-II). In addition, RRAD inhibition dramatically increased hepatoma cell invasion and metastasis. In conclusion, our study revealed that RRAD expression was decreased in HCC tumor tissues and predicted poor clinical outcome for HCC patients and played an important role in regulating aerobic glycolysis and cell invasion and metastasis and may represent potential targets for improving the treatment of HCC.

Pig BMSCs Transfected with Human TFPI Combat Species Incompatibility and Regulate the Human TF Pathway in Vitro and in a Rodent Model.

BACKGROUND: The activation of tissue factor (TF) is one of the major reasons for coagulation dysregulation after pig-to-primate xenotransplantation. Tissue factor pathway inhibitor (TFPI) is the most important inhibitor of TF. Studies have demonstrated species incompatibility between pig TFPI and human TF. METHODS: A pig-to-macaque heterotopic auxiliary liver transplantation model was established to determine the origin of activated TF. Chimeric proteins of human and pig TFPI were constructed to assess the role of Kunitz domains in species incompatibility. Immortalised pig bone marrow mesenchymal stem cells transfected with human TFPI were tested for their ability to inhibit clotting in vitro. RESULTS: TF from recipient was activated early after liver xenotransplantation. Pig TFPI Kunitz domain 2 bound human FXa, but Kunitz domain 1 did not effectively inhibit human TF/FVIIa. Immortalised pig bone marrow mesenchymal cells (BMSCs) transfected with human TFPI showed a prolonged recalcification time in vitro and in a rodent model. CONCLUSION: Recipient TF is relevant to dysregulated coagulation after xenotransplantation. Kunitz domain 1 plays the most important role in species incompatibility between pig TFPI and human TF, and clotting can be inhibited by human TFPI-transfected pig BMSCs. Our study shows a possible way to resolve the incompatibility of pig TFPI.

c-Myc-mediated epigenetic silencing of MicroRNA-101 contributes to dysregulation of multiple pathways in hepatocellular carcinoma.

UNLABELLED: The MYC oncogene is overexpressed in hepatocellular carcinoma (HCC) and has been associated with widespread microRNA (miRNA) repression; however, the underlying mechanisms are largely unknown. Here, we report that the c-Myc oncogenic transcription factor physically interacts with enhancer of zeste homolog 2 (EZH2), a core enzymatic unit of polycomb repressive complex 2 (PRC2). Furthermore, miR-101, an important tumor-suppressive miRNA in human hepatocarcinomas, is epigenetically repressed by PRC2 complex in a c-Myc-mediated manner. miR-101, in turn, inhibits the expression of two subunits of PRC2 (EZH2 and EED), thus creating a double-negative feedback loop that regulates the process of hepatocarcinogenesis. Restoration of miR-101 expression suppresses multiple malignant phenotypes of HCC cells by coordinate repression of a cohort of oncogenes, including STMN1, JUNB, and CXCR7, and further increases expression of endogenous miR-101 by inhibition of PRC2 activation. In addition, co-overexpression of c-Myc and EZH2 in HCC samples was closely associated with lower expression of miR-101 (P < 0.0001) and poorer prognosis of HCC patients (P < 0.01). CONCLUSIONS: c-Myc collaborates with EZH2-containing PRC2 complex in silencing tumor-suppressive miRNAs during hepatocarcinogenesis and provides promising therapeutic candidates for human HCC.

Overexpression of miR-200a suppresses epithelial-mesenchymal transition of liver cancer stem cells.

Due to high incidence of invasion and intrahepatic metastasis, hepatocellular carcinoma (HCC) is one of the most aggressive tumors in the world, which is also associated with the acquisition of epithelial-mesenchymal transition (EMT). Increasing evidence suggests that cancer cells with EMT traits share many biological characteristics with cancer stem cells. And miR-200a has been known as a powerful regulator of EMT. Here, we sought to investigate the role of miR-200a in regulation of EMT phenotype of liver cancer stem cells (LCSCs). We used side population (SP) sorting to obtain cancer stem-like cells from HCC cell lines and identified that the SP fraction could be enriched with LCSCs. Then, we detected the expression of miR-200a and EMT makers in SP and non-SP cells. Our results suggested that miR-200a was down-regulated in SP cells, along with relatively low epithelial marker and high mesenchymal marker. In order to find the role of miR-200a in the manipulation of EMT, we transfected miR-200a mimic into LCSCs and found that overexpression of miR-200a resulted in down-regulation of N-cadherin, ZEB2, and vimentin, but up-regulation of E-cadherin. Moreover, overexpression of miR-200a resulted in decreased migration and invasion ability in LCSCs. In conclusion, our study revealed that miR-200a played an important role in linking the characteristics of cancer stem cells with EMT phenotype in HCC, and targeting miR-200a might be an effective strategy to weaken the invasive behavior of LCSCs.

Decreased pain threshold and enhanced synaptic transmission in the anterior cingulate cortex of experimental hypothyroidism mice.

BACKGROUND: Thyroid hormones are essential for the maturation and functions of the central nervous system. Pain sensitivity is related to the thyroid status. However, information on how thyroid hormones affect pain processing and synaptic transmission in the anterior cingulate cortex (ACC) is limited. Nociceptive threshold and synaptic transmission in the ACC were detected in the experimental hypothyroidism (HT) mice. RESULTS: HT was induced by methimazole and potassium perchlorate in distilled drinking water for 4 weeks. The threshold of pain perception to hot insults, but not mechanical ones, decreased in hypothyroid mice. After treatment with tri-iodothyronine (T3) or thyroxine (T4) for 2 weeks, thermal pain threshold recovered. Electrophysiological recordings revealed enhanced glutamatergic synaptic transmission and reduced GABAergic synaptic transmission in the ACC. Supplementation with T3 or T4 significantly rescued this synaptic transmission imbalance. In the same model, HT caused the up-regulation of the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and NR2B-containing N-methyl-D-aspartate receptors, but it down-regulated γ-aminobutyric acid A receptors in the ACC. Supplementation with T3 or T4 notably recovered the levels of above proteins. CONCLUSIONS: These results suggest that HT promotes hypersensitivity to noxious thermal, and that supplementation with T3 or T4 rescues the imbalance between excitatory and inhibitory transmission in the ACC.

Combination therapy with sorafenib and radiofrequency ablation for BCLC Stage 0-B1 hepatocellular carcinoma: a multicenter retrospective cohort study.

OBJECTIVES: The objective of this study was to evaluate the efficacy of combined therapy using Sorafenib and radiofrequency ablation (RFA) with curative intent for all detectable lesions in patients with Barcelona Clinic Liver Cancer (BCLC) Stage 0-B1 hepatocellular carcinoma (HCC). METHODS: One hundred and twenty-eight patients with HCC from 12 centers were enrolled in this retrospective study; 64 patients who received Sorafenib plus RFA (Sorafenib-RFA) were compared with a control group treated with RFA alone. The two patient groups were selected with a predefined criterion and matched in terms of their clinical and tumor characteristics at baseline. The primary end point of the study was the incidence of post-RFA HCC recurrence. Secondary end points were overall survival (OS) and treatment toxicity. RESULTS: During a median follow-up of 134.1 weeks, 49 patients died and 79 survived. The 1-, 2-, and 3-year cumulative incidences of post-RFA recurrence were 40.5%, 62.9%, and 74.5%, respectively, in the Sorafenib-RFA group, and 62.8%, 85.4%, and 92.7%, respectively, in the RFA group. The 1-, 2-, 3-, and 4-year OS rates were 85.6%, 64.0%, 58.7%, and 50.3%, respectively, in the Sorafenib-RFA group, and 80.7%, 47.2%, 30.9%, and 30.9%, respectively, in the RFA group. Thus, the Sorafenib-RFA group exhibited better survival than the RFA alone group. CONCLUSIONS: Combined therapy with Sorafenib-RFA was associated with a lower incidence of post-RFA recurrence and better OS than RFA alone in patients with BCLC Stage 0-B1 HCC. Although these findings suggest that Sorafenib and RFA is safe and effective for the treatment of early HCC, prospective and randomized controlled trials are needed to validate them.

PCBP1 is an important mediator of TGF-ß-induced epithelial to mesenchymal transition in gall bladder cancer cell line GBC-SD.

Gall bladder carcinoma (GBC) is the seventh most common cancer across the globe and the most common malignancy of the biliary tract. Most GBC related deaths occur due to secondary progression and metastasis to distant organs. Epithelial-mesenchymal transition (EMT) is an important pre-requisite for tumor metastasis, however its mechanism in GBC has not yet been defined. Using the GBC-SD cell line, we have uncovered an important mediator, poly r(C) binding protein-1 (PCBP1), of transforming growth factor-beta (TGF-ß)-induced EMT in GBC. Our results show that TGF-ß treatment resulted in PCBP1 phosphorylation in accordance with similar observation in other model systems. We further showed through gain- and loss-of-function assays that PCBP1 expression levels regulate the capacity of GBC-SD cells to migrate and invade in vitro. Finally, our results showed that PCBP1 expression levels also regulate generation of CD44(+)CD24(-) progenitor cell population in GBC-SD cells after TGF-ß treatment. Cumulatively, our results indicate, pending further validation, that PCBP1 might be a prognostic marker for GBC metastasis.