Hematopoietic chimerism in liver transplantation patients and hematopoietic stem/progenitor cells in adult human liver.

UNLABELLED: Liver transplantation (LT) is a cure for many liver diseases. Blood chimerism of donor origin can develop after LT, which raises the possibility of the existence of hematopoietic stem/progenitor cells (HSPCs) in the liver. We characterized the blood chimerism in a large cohort of 249 LT patients and analyzed putative HSPCs in adult human livers. The overall incidence of chimerism was 6.43%, of which 11.11% was among short-term (1 day to 6 months) and 3.77% was among long-term (6 months to 8 years) LT patients. Hematopoietic Lin(-) CD34(+) CD38(-) CD90(+) populations have been demonstrated to generate long-term lymphomyeloid grafts in transplantations. In human adult livers, we detected Lin(-) CD34(+) CD38(-) CD90(+) populations accounting for 0.03% ± 0.017% of the total single liver cells and for 0.05% ± 0.012% of CD45(+) liver cells. Both Lin(-) CD34(+) and Lin(-) CD45(+) liver cells, from extensively perfused human liver grafts, were capable of forming hematopoietic myeloid-lineage and erythroid-lineage methylcellulose colonies. More importantly, Lin(-) CD45(+) or CD45(+) liver cells could be engrafted into hematopoietic cells in an immunodeficient mouse model. These results are the first evidence of the presence of putative HSPC populations in the adult human liver, where the liver is a good ectopic niche. The discovery of the existence of HSPCs in the adult liver have implications for the understanding of extramarrow hematopoiesis, liver regeneration, mechanisms of tolerance in organ transplantation, and de novo cancer recurrence in LT patients. CONCLUSION: The human adult liver contains a small population of HSPCs. In LT patients, there are two types of chimerisms: transient chimerism, resulting from mature leucocytes, and long-term chimerism, derived from putative HSPCs in the liver graft.

Activation of interleukin-6-induced glycoprotein 130/signal transducer and activator of transcription 3 pathway in mesenchymal stem cells enhances hepatic differentiation, proliferation, and liver regeneration.

Adult bone marrow-derived mesenchymal stem cells (MSCs) exist in all living species and are capable of differentiating into different types of specific cells. In this study, we demonstrate the therapeutic effectiveness of rat MSC transplantation in D-galactosamine (GalN)-induced acute liver injury and identified the novel pathways which are involved in hepatic differentiation of MSCs. In vivo, intraportal transplantation with 5 × 10(6) MSCs at 24 hours after GalN administration resulted in significant reduction in serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin compared to the control group. Engrafted MSCs actively proliferated, differentiated, and further enhanced hepatocyte proliferation activity. In vitro, coculture of MSCs with GalN-induced injured hepatocytes showed efficient differentiation and was evidenced by progressive increase in messenger RNA levels of hepatic markers, including albumin, α-fetoprotein, CCAAT-enhancer binding protein α, α-1-antitryspin, and hepatocyte nuclear factor-3ß. Immunofluorescent staining revealed that these cells were positive for albumin, α-fetoprotein, and cytokeratin 18, but not clusters of differentiation 34, cytokeratin 19, or OV6. During hepatic differentiation, signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling were constantly activated, and a gradual down-regulation of ß-catenin expression in messenger RNA and protein levels was detected. Hyper-interleukin-6 fusion protein but not interleukin-6 (IL-6) alone caused reduction in ß-catenin expression associated with the up-regulation of Wnt-5a in MSCs via activating the glycoprotein 130 (gp130)-mediated STAT3 signaling pathway, which indicates the operation of the trans-signaling mechanism. Activation of IL-6/gp130-mediated STAT3 signaling pathway in MSCs triggered wound healing, cell migration, and proliferation. In conclusion, transplantation of MSCs promotes cell proliferation and organ repair, and activation of IL-6/gp130-mediated STAT3 signaling pathway via soluble IL-6 receptor is crucial in hepatic differentiation of MSCs.

Hepatitis B virus-specific CD4 T cell immunity after liver transplantation for chronic hepatitis B.

Cellular immunity plays an important role in the long-term control of hepatitis B virus (HBV) infection. We studied the changes in HBV-specific CD4 T cell immunity after orthotopic liver transplantation (OLT) for chronic hepatitis B under antiviral prophylaxis. T cell proliferation and interferon-gamma production in response to in vitro challenge with HBV-encoded antigens were tested in 40 OLT recipients without HBV recurrence and in 12 OLT recipients with HBV recurrence more than 1 year after transplantation, and they were compared to 40 subjects with chronic HBV infection and to 23 subjects with self-limited HBV infection. The frequency and magnitude of the HBV-specific CD4 T cell response were significantly lower in 40 OLT recipients with HBV clearance, but the T cell reactivity to mitogen (phytohemagglutinin) and recall antigen (tetanus toxoid) was maintained. In the 12 OLT recipients with HBV recurrence, however, the HBV-specific T cell immunity was enhanced to a level comparable to that of patients with chronic hepatitis B, and the level was dependent on the serum viral load. In conclusion, HBV-specific CD4 T cell immunity is antigen-driven and evanesces with viral clearance, hence providing a favorable milieu for reactivation once prophylaxis is withdrawn. The cellular immunity in recipients with recurrence is not significantly different from that of individuals with chronic hepatitis B.

Liver intestine-cadherin (CDH17) haplotype is associated with increased risk of hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC), the most common form of liver cancer, is a leading cause of cancer death worldwide. We previously showed that aberrant mRNA splicing of the liver intestine-cadherin gene CDH17 in liver tissues was triggered by the specific constellation of two CDH17 single nucleotide polymorphisms (651T and IVS6+35G). CDH17 aberrant splicing was highly associated with tumor dissemination and shorter survival of HCC patients. Consequently, it is highly relevant to assess whether the presence of these single nucleotide polymorphisms in the general population represents a risk to the development of HCC. EXPERIMENTAL DESIGN: We conducted a case-control study including 164 HCC and 99 cirrhosis patients and 293 healthy controls. Genotyping was done by PCR and direct sequencing. Odds ratio (OR) and chi2 analysis were used to analyze genotypes and haplotypes. RESULTS: Genotypes 651TT [OR, 2.62; 95% confidence interval (95% CI), 1.34-5.03] and IVS6+35 GG (OR, 1.95; 95% CI, 1.04-3.62) were highly associated with HCC disease. The 651T (C>T) and IVS6+35G (A>G) alleles were also overrepresented in HCC patients and, in particular, the T-G haplotype was the most prevalent in HCC patients when compared with healthy controls (OR, 1.57; 95% CI, 1.167-2.109; P=0.004), which was in agreement with the aberrant splicing observed in tumor tissues. There was no significant difference in genotype and allele frequencies between cirrhosis patients and controls. CONCLUSION: The functional T-G haplotype of CDH17 (651 C>T and IVS6+35A>G) is a genetic susceptibility factor for the development of HCC in a Chinese population.

Identification of hepatitis B virus-specific lymphocytes in human liver grafts from HBV-immune donors.

Both animal and human studies have demonstrated the adoptive transfer of immunity against hepatitis B virus (HBV) through liver transplantation that may be attributed to the presence of HBV-specific immunocompetent cells of donor origin in liver grafts. In this study, we characterized the resident lymphocytes in 41 human liver grafts by immunohistochemical staining and flow cytometry and directly identified the intragraft HBV-specific lymphocytes in relation to the donor's and subsequent recipient's immunity using enzyme-linked immunospot assay. A significant number of HBV-specific T and B cells were detectable in 59.4% (19/32) and 28.1% (9/32), respectively, of liver grafts from HBV-immune donors. The presence of various HBV-specific lymphocytes was closely associated with each other and with a higher serum titer of antibody against hepatitis B surface antigen (anti-HBs) in donors (P < 0.05). After liver transplantation, 17 of 35 (48.6%) patients with chronic HBV infection showed a spontaneous anti-HBs production, which was significantly associated with a higher number of donor-derived T lymphocytes specific for hepatitis B surface antigen (P = 0.043). In conclusion, the presence of considerable numbers of donor-derived HBV-specific immunocompetent cells in grafts may account for the adoptive transfer of HBV immunity through liver transplantation.

Significance of hepatitis B virus genotype in liver transplantation for chronic hepatitis B.

Hepatitis B virus (HBV) genotype influences chronic hepatitis B disease profile but its relevance in liver transplantation (LTx) is not known. HBV genotype was identified by direct sequencing from pre-transplant sera of 119 patients who underwent LTx using lamivudine prophylaxis (genotype A,1; B,43; C,74; D,1). The baseline characteristics and outcome of 43 genotype B and 74 genotype C patients were compared. Genotype B patients had significantly more pre-transplant acute flare, worse liver functions and higher model for end-stage liver disease score. Fewer genotype B patients had HBeAg (13% vs. 32%; p=0.017), but HBV DNA seropositivity (by bDNA assay) was comparable (26% vs. 23%; p=0.727). The 3-year graft survival was 83% for genotype B and 89% for genotype C (p=0.2). The rate of HBsAg clearance or seroreversion was the same. The cumulative rate of viral breakthrough due to lamivudine-resistant mutants at 3 years was 4% for genotype B and 21% for genotype C (p=0.017). Liver biopsy after viral breakthrough showed recurrent hepatitis B in 7 of 10 genotype C patients, including 2 with fibrosing cholestatic hepatitis, and no histologic recurrence in 2 genotype B patients. In conclusion, HBV genotypes B and C are associated with different patterns of end-stage liver diseases that required transplantation, and genotype C may carry a greater risk and severity of recurrence due to lamivudine-resistant mutants.

Up-regulation of vascular endothelial growth factor (VEGF) in small-for-size liver grafts enhances macrophage activities through VEGF receptor 2-dependent pathway.

This study aims to investigate the potential role of vascular endothelial growth factor (VEGF) and VEGF-R2 (fetal liver kinase (Flk)-1) in mediating macrophage activities in small-for-size liver transplantation. A rat orthotopic liver transplantation model was performed using either whole, 50, or 30% liver grafts (both 50 and 30% were regarded as small-for-size) in syngeneic or allogeneic combinations, respectively. Firstly, the mRNA and protein levels of VEGF and Flk-1 in liver grafts were detected by RT-PCR and Western blot, and the number of Flk-1(+) macrophages (labeled by ED1) was determined by flow cytometry. It was found that the small-for-size isografts and allografts presented higher levels of VEGF and Flk-1 expression than the whole isograft and allograft. In addition, a higher number of Flk-1(+)ED1(+) cells were detected in the small-for-size isografts and allografts than the whole isograft and allograft. Secondly, our study revealed that macrophage cell lines did not initially express detectable Flk-1, but could be induced by VEGF, and the inducible expression of Flk-1 in macrophages was related to their migration and proliferation activities. Finally, our study demonstrated that the induction of Flk-1 expression on macrophages by VEGF was associated with the expression of NF-kappaB and heat shock protein 90. In conclusion, the present study showed that the up-regulated expression of VEGF and its interaction with Flk-1 in small-for-size liver grafts might facilitate the activities of macrophages.