Mesenchymal stromal cell-dependent immunoregulation in chemically-induced acute liver failure.

Drug-induced liver injury (DILI), which refers to liver damage caused by a drug or its metabolites, has emerged as an important cause of acute liver failure (ALF) in recent years. Chemically-induced ALF in animal models mimics the pathology of DILI in humans; thus, these models are used to study the mechanism of potentially effective treatment strategies. Mesenchymal stromal cells (MSCs) possess immunomodulatory properties, and they alleviate acute liver injury and decrease the mortality of animals with chemically-induced ALF. Here, we summarize some of the existing research on the interaction between MSCs and immune cells, and discuss the possible mechanisms underlying the immuno-modulatory activity of MSCs in chemically-induced ALF. We conclude that MSCs can impact the phenotype and function of macrophages, as well as the differentiation and maturation of dendritic cells, and inhibit the proliferation and activation of T lymphocytes or B lymphocytes. MSCs also have immuno-modulatory effects on the production of cytokines, such as prostaglandin E2 and tumor necrosis factor-alpha-stimulated gene 6, in animal models. Thus, MSCs have significant benefits in the treatment of chemically-induced ALF by interacting with immune cells and they may be applied to DILI in humans in the near future.

Efficient generation of functional hepatocyte-like cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells.

BACKGROUND: Differentiation of liver progenitor cells (LPCs) to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system. METHODS: Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line (HSC-Li) we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS: Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, low-density lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity. CONCLUSIONS: Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Mesenchymal stem cells from the human umbilical cord ameliorate fulminant hepatic failure and increase survival in mice.

BACKGROUND: Cell therapy has been promising for various diseases. We investigated whether transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs) has any therapeutic effects on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced fulminant hepatic failure in mice. METHODS: hUCMSCs isolated from human umbilical cord were cultured and transplanted via the tail vein into severe combined immune deficiency mice with GalN/LPS-induced fulminant hepatic failure. After transplantation, the localization and differentiation of hUCMSCs in the injured livers were investigated by immunohistochemical and genetic analyses. The recovery of the injured livers was evaluated histologically. The survival rate of experimental animals was analyzed by the Kaplan-Meier method and log-rank test. RESULTS: hUCMSCs expressed high levels of CD29, CD73, CD13, CD105 and CD90, but did not express CD31, CD79b, CD133, CD34, and CD45. Cultured hUCMSCs displayed adipogenic and osteogenic differentiation potential. Hematoxylin and eosin staining revealed that transplantation of hUCMSCs reduced hepatic necrosis and promoted liver regeneration. Transplantation of hUCMSCs prolonged the survival rate of mice with fulminant hepatic failure. Polymerase chain reaction for human alu sequences showed the presence of human cells in mouse livers. Positive staining for human albumin, human alpha-fetoprotein and human cytokeratin 18 suggested the formation of hUCMSCs-derived hepatocyte-like cells in vivo. CONCLUSIONS: hUCMSC was a potential candidate for stem cell based therapies. After transplantation, hUCMSCs partially repaired hepatic damage induced by GalN/LPS in mice. hUCMSCs engrafted into the injured liver and differentiated into hepatocyte-like cells.

Nature and mechanisms of hepatocyte apoptosis induced by D-galactosamine/lipopolysaccharide challenge in mice.

Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6-10 h after the intraperitoneal injection of D-GalN (700 mg/kg) and LPS (10 µg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering D-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end­labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following D-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-ß1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the D-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by D-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-ß signaling pathway may also play a vital role in this process of hepatocyte apoptosis.

Detection of the JAK2 mutation in myeloproliferative neoplasms by asymmetric PCR with unlabeled probe and high-resolution melt analysis.

BACKGROUND: Several methods have been established to detect the JAK2 V617F mutation, a frequent event involved in the pathogenesis of myeloproliferative neoplasms (MPNs). High-resolution melt (HRM) analysis is a newly established technique without the requirement of any gel-based post-PCR handling. METHODS: An asymmetric PCR with unlabeled specific probe was developed and combined to HRM analysis o screen for JAK2 V617F mutation. RESULTS: Heterozygous mutation was easily distinguished from homozygous JAK2 for the obvious shape change. Homozygous JAK2 mutant can be also well separated from wild-type JAK2 in the presence of internal temperature calibrators. The easily recognizable and maximal sensitivity of HRM analysis was 5% for the detection of JAK2 V617F mutation, higher than 25% of direct sequencing. In the test of blind screening of 223 samples (111 Ph- MPNs, 60 Ph+ chronic myeloid leukemia, and 52 acute myeloid leukemia), JAK2 V617F mutations were found in 78 (70%) patients with MPNs, but in none with chronic and acute myeloid leukemia. HRM analysis of all cases was fully concordant with the results of PCR-RFLP and direct sequencing. CONCLUSIONS: The HRM method with unlabeled probe could be used as convenient, sensitive and reliable diagnostic test for detection of JAK2 V617F mutation.

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1).

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4(+) T cells and the IFN-gamma expression of CD8(+) T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-gamma expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.

Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy.

Acute liver failure and metabolic liver disorder animal models have demonstrated that hepatocytes transplanted into the liver or spleen survive and participate in the liver repopulation process, and recent studies have documented the usefulness of hepatocyte transplantation in humans. However, despite the promising cell therapy, there are still many restrictions, such as the shortage of donor human livers and the limited lifespan and the functional insufficiency of primary cultured hepatocytes. The immortalized and highly differentiated human hepatocyte could provide an unlimited supply of transplantable cells. In this study, we established an efficient and highly differentiated immortalized human hepatocyte line for bioartificial liver and hepatocyte transplantation research. Hepatocytes isolated from the liver of a 25 year old, brain dead male were transfected with pcDNA3.1 (-) recombinant plasmid containing the genes encoding simian virus 40 (SV40) large tumor antigen. One of the hepatocyte clones, HepLL, displayed highly differentiated liver functions with immortalized characteristics and was selected with a 700-300 microg/ml of G418 technique in 42 days. To characterize this immortalized cell line for cell therapy in the near future, HepLL cells were studied with immunohistochemistry, reverse transcription-polymerase chain reactions, immunoblotting, and tumorigenicity tests. The results revealed that HepLL cells displayed morphologic characteristics of liver parenchymal cells, secreted albumin, synthesized urea and glycogen, and expressed liver enriched functional markers, but there were no tumorigenic qualities after transplantation into severe combined immunodeficiency mice. Thus this immortalized human hepatocyte line is expected to be a useful tool for studying the functions of differentiated human hepatocyte and a promising strategy to resolve the shortages of donor organs and the limits of primary human hepatocyte for transplantation and bioartificial liver support systems.

Determination of platelet-activating factor by reverse phase high-performance liquid chromatography and its application in viral hepatitis.

AIM: To detect the platelet-activating factor (PAF) and the plasma or serum levels of tumor necrosis factor-alpha (TNF-alpha) malondialdehyde (MDA), endotoxin (ET) and to discuss their significance in various types of viral hepatitis. METHODS: PAF, TNF-alpha, MDA, and ET levels in 60 controls, 16 cases of acute viral hepatitis, 71 cases of chronic viral hepatitis, 19 cases of severe viral hepatitis were detected by reverse phase high-performance liquid chromatography (rHPLC), bio-assay, ELISA, thiobarbituric acid (TBA), and limulus lysate test (LLT), respectively. RESULTS: The rHPLC was more sensitive and specific than bio-assay (r = 0.912, P<0.01). The plasma levels of PAF, TNF-alpha, MDA, and ET in patients with viral hepatitis were higher than those in controls (P<0.01). CONCLUSION: rHPLC is more reliable and accurate for the detection of PAF.