PDF file - 639K, Supplementary Figure 1. CD133 is CSCs surface marker in PLC/PRF/5 cell line. Supplementary Figure 2. BMP4 showed pro-differentiation effect on HCC cell lines. Supplementary Figure 3. Tumorigenicity of CD133+/- HCC cells co-injected with BMP4 or vehicle embedding beads. Supplementary Figure 4. Canonical BMP4 signaling pathway switches on after exogenous BMP4 treatment. Supplementary Figure 5. BMP4 and SMAD6 expression in HCC cell lines. Supplementary Figure 6. Differentiated CSCs displayed enhanced Erk1/2 phosphorylation.

PDF file - 82K, Antibodies used in this study

IntroductionThe research demonstrated the way to compare performances of five capillary blood collection tubes for capillary blood collections among patients with pediatrics utilizing the Clinical and Laboratory Standards Institute (CLSI) document EP9-A2.MethodsA questionnaire survey was distributed to evaluate the accessibility and reliability of capillary blood collection tubes from different sources. Visual examination, microscopic examination of blood smears, and instrumental analysis were performed to evaluate the blood sample quality in different tubes. The background test, comparison test, and reliability test were used to analyze the engineering quality and the performance of additives in the tubes, and to determine the reliability of the tubes in routine blood tests.ResultsThe tubes of brand A were found to be easy to access, with a shorter blood collection time and greater acceptability by users, and the quality of blood samples in those tubes was superior, with no coagulation and less blood cell disruption.ConclusionCommercially available capillary blood collection tubes were superior to in-house' tubes. In clinical practice, in-house' capillary blood collection tubes are not recommended. The guideline of method comparison and bias estimation using patient samples' from CLSI could also be used in comparing the performances of capillary blood collections.

Primary liver carcinoma is the most important malignant disease. The nodular metastatic foci of liver carcinoma are usually found in the lung, adrenal gland or abdomen after resection or transplantation. Pulmonary lymphangitic carcinomatosis (PLC) accounts for approximately 6% to 8% of metastatic cancer in the lung. The occurrence of PLC is extremely rare in liver carcinoma. Herein we report the case of a patient with PLC after liver transplantation due to liver carcinoma. PLC was confirmed by clinical manifestations, imaging studies and cytologic examination of exfoliated cells in the pleural effusion.

Liver allografts are spontaneously accepted across MHC barriers in mice. The mechanisms underlying this phenomenon remain poorly understood. Galectin-1, an endogenous lectin expressed in lymphoid organs, plays a vital role in maintaining central and peripheral tolerance. This study was to investigate the role of galectin-1 in spontaneous tolerance of liver allografts in mice, and to evaluate the therapeutic effects of galectin-1 on liver allograft rejection induced by donor Flt3L pretreatment. Blockade of the galectin-1 pathway via neutralizing antigalectin-1 mAb did not affect survival of the liver allografts from B6 donors into C3H recipients. Administration of rGal-1 significantly prolonged survival of liver allografts from Flt3L-pretreated donors and ameliorated Flt3L-triggered liver allograft rejection. This effect was associated with increased apoptosis of T cells in both allografts and spleens, decreased frequencies of Th1 and Th17 cells, decreased expression of Th1-associated cytokines (IL-12, IL-2 and IFN-γ), Th17-associated cytokines (IL-23 and IL-17) and granzyme B, in parallel with selectively increased IL-10 expression in liver allografts. In vitro, galectin-1 inhibited Flt3L-differentiated DC-mediated proliferation of allo-CD4(+) T cells and production of IFN-γ and IL-17. These data provide new evidence of the potential regulatory effects of galectin-1 in alloimmune responses in a murine model of liver transplantation.

Background. Th17, a newly identified CD4+ T-cell subset, has been implicated in transplant rejection. Differentiation of Th17 cells is associated with transforming growth factor-beta (TGF-beta) and interleukin-6 (IL-6), which are the main products of Kupffer cells.Objective. To determine whether Kupffer cells promote acute liver allograft rejection by inducing Th17 cell differentiation.Methods. A rat model of allogeneic liver transplantation using Dark Agouti (DA) to Brown Norway (BN) rats was established with or without gadolinium chloride (GdCl3) pretreatment. Isogeneic liver transplantation (BN to BN) was performed as a control. Concentrations of cytokines secreted by Kupffer cells or Th17-related cytokines detected in the liver and peripheral blood were analyzed using immunohistochemistry assays, flow cytometry, and enzyme-linked immunosorbent assays. Survival differences were compared between treatment groups. In vitro, Kupffer cells from liver grafts were isolated and co-cultured with naive CD4 T cells.Results. Both Kupffer cells and Th17 cells infiltrated liver allografts, accompanied by an increase in concentrations of IL-6 and TGF-beta. Pretreatment with GdCl3 attenuated intragraft infiltration of Kupffer cells and Th17 cells, and decreased IL-6 and TGF-beta concentrations. Liver function improved after pretreatment, and mean (SD) survival time was prolonged, compared with the control group (16.33 [0.96] days vs 11.50 [0.99] days, respectively; P < .01). In vitro, Kupffer cells from livers with allografts secreted significantly higher concentrations of IL-6 and TGF-beta and induced Th17 differentiation more effectively compared with livers with isografts (30.8% vs 8.1%, respectively).Conclusion. Kupffer cells have the potential to induce Th17 cells by secreting IL-6 and TGF-beta, and as a result, promote acute liver allograft rejection.

Evidence has suggested that immunosuppressive drugs impact ischemia–reperfusion injury.

Mycophenolate mofetil (MMF) has been gradually introduced into clinical liver transplantation in recent years. However, the effects of MMF on hepatic ischemia/reperfusion (I/R) injury and the potential mechanisms involved are not totally understood. We aimed to evaluate whether MMF could attenuate hepatic I/R injury. MMF (20 mg/kg) or vehicle was administered to Wistar rats by gavage. The rats were then subjected to hepatic ischemia. Liver cell apoptosis and the levels of aspartate aminotransferase, myeloperoxidase (MPO), xanthine oxidase (XOD) and malondialdehyde (MDA) were determined. Expression of vascular cell adhesion molecule-1 (VCAM-1) and activation of mitogen-activated protein kinases (MAPKs) were also investigated. Furthermore, the hepatic microcirculation was observed by intravital fluorescence microscopy. Rats pretreated with MMF exhibited significant alleviation of their postischemic liver function. Liver cell apoptosis and the tissue MPO, XOD and MDA levels were decreased by MMF pretreatment. MMF also improved I/R-induced hemodynamic turbulence, as evidenced by reduced hepatic perfusion failure and decreased numbers of rolling and adherent leukocytes. I/R injury induced activation of the MAPKs pathway while expression of VCAM-1 was downregulated by MMF pretreatment. In summary, MMF attenuates hepatic I/R injury through suppression of the production of reactive oxygen species and amelioration of postischemic microcirculatory disturbances.

BACKGROUND:Recipients of liver transplantation could have postoperative structural injury and declined absorptive function in the gastrointestinal tract. Glutamine (Gln) is a special nutrient of small intestinal mucosa and of various kinds of cells proliferating rapidly. But Gln could form a kind of poisonous pyroglutamic acid in water solution, which is the limitation of Gln in clinical practice. Glycyl-glutamine (Gly-Gln) is highly soluble and can be hydrolyzed to release glutamine. This study was undertaken to observe the effect of Gly-Gln dipeptide by enteral feeding on the intestinal structure and absorptive function after allogenetic liver transplantation in rats.METHODS:Twelve male inbred Lewis rats were selected randomly as donors, and 24 male inbred BN rats as recipients of allogenetic liver transplantation. The recipients were also randomly divided into two groups: control group (ALA group, n=12) and experimental group (GLN group, n=12). In each group, 6 normal BN rats were sampled as the normal parameter on the 3rd preoperative day. The 6 recipients in the control group received alanine 0.6 g/kg daily for 3 days before operation and 7 days after operation by gastric perfusion, and the 6 recipients in the experimental group were given Gly-Gln 0.6 g/kg daily the same way. The 12 BN recipients underwent 3-day fasting (free access to water with 0.23% sodium chloride) and orthotopic liver transplantation in aseptic conditions and were given subcutaneous injection of CsA 2 mg/kg daily after the operation. The 12 BN recipients were sampled on the 8th postoperative day. All of the 24 BN rats were subjected to examination of mucosal structure, activities of Na+-K+-ATP and disaccharidase, and D-xylose absorption test.RESULTS:The 12 BN recipients were alive after liver transplantation. On the 3rd preoperative day, mucosal structure, activities of Na+-K+-ATP and disaccharidase and D-xylose absorption in the two groups were not significantly different. On the 8th postoperative day, the parameters of the two groups were markedly changed compared with those on the 3rd preoperative day. However, the parameters of GLN group were remarkably higher than those of ALA group.CONCLUSION:Enteral feeding of Gly-Gln could improve the structure and absorptive function of the small intestine after liver transplantation in rats.

Chronicity in hepatitis B virus (HBV) infection is maintained by increased type 2 T-helper cell response, possibly because of increased interleukin-10 (IL-10) productions. B7-H1 can negatively regulate T-cell responses via its receptor, programmed death 1. Ligation of B7-H1 to T-cells can result in the preferential secretion of IL-10. In this study, we investigated whether there was an upregulated expression of B7-H1 in peripheral blood mononuclear cells in patients chronically infected by HBV and further explored the correlation between B7-H1 expression and serum interleukin 2, interferon-gamma, IL-10, HBeAg, alanine aminotransferase (ALT) levels and viral load. Fifty-five patients with chronic HBV infection and 20 healthy controls (HCs) were enrolled in the present study. The results showed that in patients with chronic hepatitis B CD14+ monocytes but not CD3+ and CD19+ cells had a significantly increased expression of B7-H1 compared with HCs, which positively correlates with serum IL-10 levels and the presence of HBeAg and negatively correlates with serum ALT levels. In conclusion, chronic HBV patients harbour an increased B7-H1 expression in CD14+ monocytes compared with controls, which may be responsible for the increased serum IL-10 levels. This might be an important way by which HBV evades an adequate immune response, leading to viral persistence and disease chronicity.

In addition to its effects on lymphocytes, mycophenolic acid (MPA) may inhibit the allostimulatory capacity of dendritic cells (DC) via unknown mechanisms. B7-H1 and B7-DC surface markers on DC negatively regulate T-cell responses via the receptor PD-1. In this study, we sought to investigate whether B7-H1 and B7-DC are responsible for the inhibitory functions of MPA on DC. Mouse bone marrow-derived DC were cultured with recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 in the presence or absence of MPA (0.01 micromol and 0.1 micromol). The DC phenotype was assessed by flow cytometry, their immunostimulatory capacity measured by mixed lymphocyte reaction (MLR), and cytokine production by ELISA. The results showed that MPA not only inhibited the expression of major histocompatibility complex (MHC) class II and costimulatory molecules CD80 and CD86 but upregulated B7-DC expression on mature DC induced by LPS. These findings were associated with a reduced allostimulatory ability and an altered cytokine secretion pattern of DC. Thus, we suggest that MPA upregulates B7-DC expression during DC maturation induced by LPS in vitro, an effect that may be responsible for MPA-mediated inhibitory effects on the allostimulatory capacity of DC.

OBJECTIVE:To investigate the effect of combined CsA and FK506 with 5-FU on hepatocellular carcinoma rats.METHODS:A syngeneic rat model of hepatocellular carcinoma was used. Control group (A) underwent 4 ml 5% GS. Treatment group was divided into 3 groups namely, group B: only 5-FU and 5% GS; group C: 5-FU, CsA and 5% GS; group D: 5-FU, FK506 and 5%GS. Cell cycle, apoptosis, necrosis and mitochondrial transmembrane potential were measured by flow cytometry, laser scanning confocal microscopy, and electron transmission microscopy. Statistical analysis was performed by SPSS 10.0 for Windows software. Statistical comparisons were made with ANOVA followed by Dunnett's T3 or LSD test.RESULTS:Compared to the control group, the percentage of apoptotic cells including trifle necrotic cells was significantly higher, and among the treatment group, group D was the highest, and group C was higher than group B. In the treatment group, cell cycle of hepatoma cells was mainly arrested at S phase, but in group D, G0/G1 phase cells were significantly decreased and S phase cells significantly increased. Compared to the control group, mitochondrial transmembrane potential was significantly decreased in the treatment group, among with, group B was the lowest, group C was higher than group D. Morphological changes demonstrated by electron microscopy included dispersed nuclear chromatin, loss of nucleoli, membrane bleeding, cell shrinkage, typical apoptotic bodies and marked swelling of mitochondria in the treatment group. In the control group, however, they were characterized by normal cell ultrastructure.CONCLUSIONS:The present study reveals that 5-FU combined with CsA or FK506 demonstrated a synergistic effect on hepatocellular carcinoma rats. For FK506, the powerful mutual effect is related to the increase of tumor cell's quantity in S phase. Both CsA and FK506 can provide protection on mitochondrial transmembrane potential reduction against hepatoma cells damage from 5-FU.