Cytokines are early diagnostic biomarkers of graft-versus-host disease in liver recipients.

BACKGROUND: Graft-versus-host disease (GVHD) is associated with high mortality. Early diagnosis is essential to start treatment and to improve outcomes. Because of the inflammatory nature, we hypothesis that cytokine profile of patients with GVHD may serve as diagnostic markers. The present study was to evaluate the role of cytokine profile in the diagnosis of GVHD. METHODS: An immunoassay was used to detect 29 cytokines simultaneously in the serum; the measuring sensitivity of all cytokines was pg/mL. Healthy subjects undergoing annual routine physical examinations served as negative controls; 23 patients with hepatocellular carcinoma (HCC) who had undergone liver transplantation (the LT group) comprised the test subjects. A total of 22 kidney recipients with biopsy-confirmed GVHD (the RT group) were included for comparison. HCC patients with radical surgery (the HCC group, n=22) served as positive control. The liver contents of the three cytokines, IL-2, IL-18, and IFN-gamma, were detected with immunohistochemistry. Serum granzyme B and perforin were measured by flow cytometry. RESULTS: Of the 29 cytokines, the levels of IL-2 and IL-18 were increased significantly in liver recipients with GVHD compared with healthy controls (P<0.05). The serum levels of these three cytokines in the healthy, HCC, LT, and RT groups were IL-2: 0.90+/-0.02, 4.14+/-0.61, 5.10+/-0.89, and 1.48+/-0.09 pg/mL; IL-18: 80.61+/-9.35, 109.51+/-10.93, 230.11+/-12.92, and 61.98+/-7.88 pg/mL; IFN-gamma: 24.06+/-3.88, 24.84+/-3.21, 40.37+/-5.88, and 15.33+/-4.72 pg/mL, respectively. Immunohistochemistry showed that these 3 cytokines expressions in the liver were parallel to the serum cytokine. After standard anti-GVHD treatment, the expressions of IL-2, IL-18, and IFN-gamma were decreased in the liver (P<0.05). Serum granzyme B and perforin were significantly increased in GVHD patients (P<0.05). CONCLUSIONS: IL-2, IL-18 and IFN-gamma were from liver and might serve as biomarkers for monitoring GVHD development and the effects of anti-GVHD treatment. Granzyme B and perforin may play a role in increasing IL-2, IL-18, and IFN-gamma levels in GVHD patients.

A nomogram for predicting post-hepatectomy liver failure in patients with hepatocellular carcinoma based on spleen-volume-to-platelet ratio.

BACKGROUND AND OBJECTIVES: Post-hepatectomy liver failure (PHLF) is one of the most serious complications after hepatectomy in patients with hepatocellular carcinoma (HCC), and has an association with high morbidity and mortality. This study aimed to explore the risk factors of PHLF and to establish and validate a nomogram to predict PHLF. METHODS: We retrospectively analyzed 971 HCC patients undergoing major liver resection at the Eastern Hepatobiliary Surgery Hospital between 2011 and 2016, and established a nomogram based on multivariate analyses for predicting PHLF. The predictive accuracy and discriminative ability of the nomogram were evaluated by concordance index (C-index) and calibration curve. The predictive ability of PHLF of this nomogram was compared with conventional models using receiver operating characteristic (ROC) curves. RESULTS: The incidence of PHLF was 18.8%. Multivariate analysis identified age, BMI, preoperative ascites, preoperative prealbumin, spleen volume-to-platelet ratio, total bilirubin, and intraoperative blood loss as independent predictors of PHLF. The area under ROC curve (AUROC) of the predictive model was 0.668 and was higher than that of the albumin-bilirubin score (ALBI). The optimal cut-off value of the model was 124. CONCLUSIONS: We constructed a nomogram for predicting PHLF based on risk factors. The nomogram can assist clinicians in identifying patients with high-risk PHLF, eventually facilitating earlier interventions and improving clinical outcomes.

Expression of HLA-G in patients with hepatocellular carcinoma.

BACKGROUND: Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility complex class I molecule that has multiple immune regulatory functions including the induction of immune tolerance. The detection of HLA-G expression might serve as a clinical marker in the prediction of clinical outcomes for certain types of carcinoma. Currently, we investigated whether or not HLA-G is also expressed in patients with hepatocellular carcinoma (HCC), and whether the expression has clinical value. METHODS: Serum levels of secreted HLA-G (sHLA-G) were measured by ELISA in 36 patients with HCC, 25 patients with liver cirrhosis (LC) and 25 healthy individuals. The expression of HLA-G in liver tissue was further studied using Western blotting in 36 patients with HCC and 25 with LC. The correlations between HLA-G status and various clinicopathological parameters including survival were analyzed. RESULTS: The ELISA assay showed that the serum levels of sHLA-G in the HCC, LC and healthy groups were 132.6+/-31.4, 63.5+/-22.1, and 47.0+/-15.5 U/ml, respectively. Analysis of variance was used for inter-group comparison and differences were found between the HCC group and the other two groups (both P<0.01), while no difference was found between the LC group and the healthy group (P=0.112). HLA-G protein expression in liver tissue was found in 66.7% (24/36) of the primary sites of HCC, but not in benign lesions (LC). Further, the HLA-G expression in tumors had no significant correlation with the parameters of age, gender, histological grade and alpha-fetoprotein level. However, patients with HLA-G-positive tumors had a shorter postoperative survival time than those with HLA-G-negative tumors (P=0.014). Also, univariate analysis showed that HLA-G was an independent prognostic factor. CONCLUSION: Our results indicated that the expression of HLA-G was a characteristic feature of HCC and patients with positive expression of HLA-G in malignant liver tissue had a poor prognosis.

Impact of human leukocyte antigen matching on hepatitis B virus recurrence after liver transplantation.

BACKGROUND: Liver transplantation (LT) is an effective therapy for end-stage hepatitis B virus (HBV) infection. Recurrence of HBV is one of the frequent complications. In the present study, we investigated whether human leukocyte antigen (HLA) matching influences the incidence of HBV recurrence, and the time point of HBV recurrence after LT. METHODS: One hundred and two recipients of LT with end-stage chronic HBV infection were reviewed. The triple-drug immunosuppression regimen consisted of tacrolimus, mycophenolate, and prednisone. All patients were subjected to prophylaxis with hepatitis B immunoglobulin and lamivudine. HLA typing was performed using a sequence-specific primer-polymerase chain reaction kit. Serology for hepatitis B and HBV DNA was examined using a commercial kit. RESULTS: The incidence of recurrent HBV infection post-LT was 6.86%. The recurrent infection of HBV was independent of the degree of HLA matching (P>0.05). The time point of HBV recurrence, however, was prolonged in HLA-A matched patients compared with matchless patients (P=0.049). The recurrence of HBV infection was independent of HLA compatibility. CONCLUSIONS: This retrospective analysis showed that more HLA-A locus compatibility is associated with a prolonged time of recurrence of HBV in patients after LT for end-stage HBV infection. The incidence of HBV recurrence is independent of HLA compatibility.

Detection of human leukocyte antigen compatibility and antibodies in liver transplantation in China.

BACKGROUND: The exact roles of human leukocyte antigen (HLA) compatibility, HLA antibodies and underlying diseases in acute rejection of liver transplants are not clear. Moreover, cytomegalovirus (CMV) infection, one of the most common infections after transplantation, is related to HLA genotype and the incidence of acute rejection. METHODS: Since there are controversial reports, we analyzed the impact of HLA matching, HLA antibodies and underlying diseases in 38 liver transplant recipients in China, and assessed the association of CMV infection and HLA compatibility. RESULTS: The frequency of no HLA compatibility was high in patients without antigenemia (P=0.019). All 17 patients with HLA-A matching developed antigenemia (P<0.05). Patients with three HLA locus matches were not found in patients with acute rejection (P<0.05), and no relationship between HLA antibodies and acute rejection was found (P>0.05). In patients with acute rejection, no differences were found in the incidence of acute rejection in transplants for hepatitis B, tumors, or combined hepatitis B and tumors (P>0.05). CONCLUSIONS: There are fewer acute rejections in transplants with more HLA compatibilities. Specific investigations of underlying diseases and HLA typing may be necessary in liver transplantation. The mechanisms of CMV infection and HLA matching should be further studied. HLA before transplantation should be examined for the prevention of acute rejection and CMV infection.

Potential value of autoantibodies as biomarkers of chronic graft-versus-host disease after allogeneic stem cell transplantation.

We investigated the value of autoantibodies as biomarkers of chronic graft-versus-host disease (cGVHD) by analyzing the autoantibody profiles of 65 patients (34 cGVHD and 31 non-cGVHD) surviving longer than three months after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Autoantibodies to at least one antigen were detected in 45 patients (70.8%), with multiple autoantibodies detected in 30 patients (46.2%). Antinuclear antibodies (ANAs) were the most frequently detected autoantibodies, with a significantly higher prevalence in non-cGVHD patients and cGVHD patients than that in healthy controls (HCs). ANA-nucleolar (ANA-N) was the main immunofluorescence pattern of ANA-positivity in both the non-cGVHD and cGVHD groups. There was a higher prevalence of anti-Ro52-positivity in non-cGVHD and cGVHD patients than in HC. Liver cGVHD was significantly associated with anti-Ro52-positivity. However, cGVHD activity and severity were not associated with the presence of autoantibodies. Similarly, there were no significant differences in overall survival or relapse among the four groups of patients expressing autoantibodies. Our results suggest that autoantibodies have limited value in predicting cGVHD.

Association of cytomegalovirus infection with human leukocyte antigen genotypes in recipients after allogeneic liver transplantation.

BACKGROUND: Cytomegalovirus (CMV) infection is the important cause affecting the survival rate and function of the transplanted organ after transplantation. The occurrence of CMV infection after liver transplantation (LT) is associated with many factors. Lots of studies suggest that genetic mutation between hosts and CMV may play a role in the occurrence and development of CMV infection. CMV exists in an incubative state, affect or destroy the expression of human leukocyte antigen (HLA) molecules in the host cell surface, and interfere antigen's submission. This mechanism is the key of CMV to avoid immune defense mechanism of the host. To detect HLA and CMV antibody (CMV-Ab), CMV antigen (CMV-Ag) of transplantation recipients, we evaluated the association of CMV infection and the particular HLA genotypes in recipients after LT. METHODS: 277 blood samples were collected from 39 LT recipients. CMV antibody and antigen were detected by ELISA or immunohistochemical methods. The HLA types of the recipients were determined by PCR. To analyze the association of HLA alleles and the occurrence of CMV antigenemia in the patients, relative risk degree (RR) was used as the parameter for the Chi-square test. RESULTS: The LT recipients were serum CMV IgG positive (100%), but none of them was CMV IgM positive (0%). Thirty-three LT recipients (84.6%) were CMV antigenic positive with 1-50 positive leukocytes per 50,000 leukocytes in extent and 7.2+/-4.2 positive leukocytes per 50,000 leukocytes on average. Thirteen patients developed CMV pneumonia, with CMV antigenic positive (100%) and 17.7+/-5.5 positive leukocytes per 50,000 leukocytes on average. Some HLA alleles were associated with the occurrence and extent of CMV antigenemia. HLA-A2 was the higher frequency allele for patients with antigenemia (P<0.05), and 7 patients carrying HLA-DR11 allele developed antigenemia (P<0.05). In the lower antigenemia group, HLA-A11 was higher in frequency than others (P<0.05). Besides, none of the patients carrying HLA-B16 allele developed clinical symptoms of CMV infection (P<0.05). CONCLUSIONS: The variability of HLA alleles might modulate immune response to CMV infection. HLA examination before transplantation should be made for prevention and treatment of CMV infection after operation.

Solanine-induced reactive oxygen species inhibit the growth of human hepatocellular carcinoma HepG2 cells.

The aim of the present study was to investigate the effect of solanine on promoting human hepatocellular carcinoma HepG2 cells to produce reactive oxygen species (ROS), and the molecular mechanisms leading to tumor cell apoptosis. Solanine was administered to HepG2 cells in vitro. A selection of probes targeting various cellular localizations of ROS were used to detect ROS expression using flow cytometry. The expression levels of apoptosis-associated proteins, including apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin binding protein 2 (TBP-2), and proliferation-associated proteins, including histone deacetylase 1 (HDAC1), were detected using western blotting. The percentage of cells undergoing apoptosis was measured using an Annexin V-fluorescein isothiocyanate/propidium iodide assay, and cell morphology was examined using Wright's stain followed by inverted microscopy analysis. ROS detection probes 2',7'-dichlorofluorescin diacetate and dihydrorhodamine 123 identified that abundant ROS, including hydroxyl radical (OH-) and hydrogen peroxide (H2O2), were produced in the cytoplasm and mitochondria of the solanine-treated HepG2 cells compared with the control cells (P<0.05). Superoxide anion specific probes dihydroethidium and MitoSOX™ demonstrated that there were no significant alterations in the HepG2 cells following solanine treatment compared with the control cells (P>0.05). Western blotting results revealed that solanine upregulated the expression levels of ASK1 and TBP-2 and enhanced their kinase activities, whereas solanine decreased the expression level of the proliferation-associated protein, HDAC1. The cell apoptotic rate was significantly increased (P<0.0001) in the solanine-treated HepG2 cells compared with the control cells. (P<0.05). Overall, the study indicated that solanine induces HepG2 cells to produce ROS, mainly OH- and H2O2, in a mitochondria-dependent and -independent manner. In addition, solanine stimulates the expression of ASK1 and TBP-2, and their kinase activities, but inhibits the expression of proliferation-associated proteins, such as HDAC1, thus contributing to HepG2 cell apoptosis.

In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats.

Organ decellularization is emerging as a promising regenerative medicine approach as it is able to provide an acellular, three-dimensional biological scaffold material that can be seeded with living cells for organ reengineering. However this application is currently limited to donor-derived decellularized organs for reengineering in vitro and no study has been conducted for re-engineering the decellularized organ in vivo. We developed a novel technique of a single liver lobe decellularization in vivo in live animals. Using a surgical method to generate a by-pass circulation through the portal vein and infra-hepatic vena cava with a perfusion chamber system, we decellularized the single liver lobe and recellularized it with allogenic primary hepatocytes. Our results showed that the decellularization process in vivo can preserve the vascular structural network and functional characteristics of the native liver lobe. It allows for efficient recellularization of the decellularized liver lobe matrix with allogenic primary hepatocytes. Upon the re-establishment of blood circulation, the recellularized liver lobe is able to gain the function and the allogenic hepatocytes are able to secret albumin. Our findings provide a proof of principle for the in vivo reengineering of liver.

Influence of HLA class II molecules on the outcome of hepatitis B virus infection in population of Zhejiang Province in China.

OBJECTIVE: To investigate the correlation between HLA class II molecules and the different outcome of viral hepatitis B. METHODS: Thirty patients with chronic hepatitis B and 56 subjects who had spontaneously recovered from HBV infection in Zhejiang were enrolled in this investigation. HLA class II molecules types and alleles were determined by PCR-ssp. RESULTS: HLA-DR12 was found in 21 of the 56 subjects (38%) recovered from hepatitis B, compared to 3 of the 30 patients with chronic hepatitis B [10%, relative risk (rr), 0.19; Pcorr<0.025]. The frequency of the allele of HLA-DR12 DRB1*1201 was higher in the subjects recovered from hepatitis B infection (32%) than in the patients with chronic hepatitis B (3%; rr, 0.07; Pcorr<0.005). On the contrary, more HLA-DR9 was detected in the patients with chronic hepatitis B (43%) than in the subjects who recovered from hepatitis B infection (18%; rr, 3.52; Pcorr<0.025). HLA-DQ9 was also detected with a higher frequency in the patients with chronic hepatitis B (43%) than in the subjects who recovered from hepatitis B (20%; rr, 3.13; Pcorr<0.05). CONCLUSIONS: The HLA class II molecule DR12 and its allele DRB1*1201 are associated with protection against chronic hepatitis B in Zhejiang Province, China. HLA-DR9 and DQ9 are associated with chronicity of HBV infection.

[Graft versus host disease after liver transplantation: a report of 3 cases].

OBJECTIVE: To document the clinical experience in the diagnosis and treatment of graft-versus-host disease(GVHD) after liver transplantation. METHODS: Clinical course was followed up and laboratory examinations were done in 3 patients with orthotopic liver transplantation (OLT) who developed acute GVHE. The diagnosis depended on clinical manifestations, skin biopsy, HLA typing and PCR short tandem repeat (PCR-STR). Immunosuppressive drugs were transferred and adjusted. RESULTS: Fever, shin rash, diarrhea and pancytopenia were found within 3 to 8 weeks after liver transplantation. The liver function was normal. CMV antigen (pp65) and EBV antibody (IgM) were negative. The donor's HLA was detected in the host's peripheral blood cells. One female recipient had the donor's Y chromosome microchimerism detected by PCR-STR. All 3 patients died from infection, alimentary tract bleeding, or multiple organ failure in the end. CONCLUSION: GVHD is not a rare complication easily misdiagnosed with pessimism out come after liver transplantation.

[The study on high-resolution HLA and human cytomegalovirus (HCMV) viremia in bone marrow transplantation recipients].

OBJECTIVE: To study the correlation between high-resolution HLA-A * 1101, HLA-A * 0201, HLA-A * 2402, HLA-B * 4001, HLA-DRB1 * 0901 with HCMV pp65 antigenemia after bone marrow transplantation (BMT) in China. METHODS: 48 recipients doing BMT during 2009. 2-2010. 10 were selected in my hospital; HCMV pp65 was detected by ELISA or immunohistochemical methods. The frequency of HLA-A * 1101, HLA-A * 0201, HLA-A * 2402, HLA-B * 4001, HLA-DRB1 * 0901 alleles were determined by Polymerase chain reaction-sequence based typing (PCR - SBT). RESULTS: (1) The BMT recipients were HCMV pp65 antigenic positive(100%); (2) The positive rate of HLA-A * 1101, HLA-A * 0201, HLA-A * 2402, HLA-B * 4001 showed no obvious difference between 12 lower antigenemia group and 36 higher antigenemia group, the positive rate: HLA-A * 1101 were 33.3% (8/24) and 20.8% (15/72), HLA-A * 0201 were 4.2% (1/24) and 13.9% (10/72), HLA-A * 2402 were 12.5% (3/24) and 19.4% (14/72), HLA-B* 4001 were 16.7% (4/24) and 12.5% (9/72); (3) HLA-DRB1 * 0901 positive rate in higher antigenemia group was higher than the lower (P = 0.048), the positive rate were 4.2% (1/24) and 19.4% (14/72); (4) HLA-DRB1 * 0901 recipients were higher pp65 antigenemia than HLA-A * 2402 recipients (P = 0.007) and HLA-A * 1101 recipients (P = 0.028), HLA-A * 0201 recipients were higher pp65 antigenemia than HLA-A * 2402 (P = 0.02), the pp65 antigenemia showed no obvious difference among the rest of high-resolution HLA groups (P > 0.05). CONCLUSION: HLA-DRB1 * 0901 alleles might be correlated with BMT recipients happened higher pp65 antigenemia, HLA-A * 2402 alleles might be correlated with BMT recipients happened lower pp65 antigenemia.