Objective This study aimed to analyze the characteristics and clinical significance of peripheral lymphocytic subsets and cytokine levels, including interleukin 1β(IL-1β), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17A, tumor necrosis factor α(TNF-α), interferon γ(IFN-γ) and IFN-α, in patients with primary biliary cholangitis (PBC), to provide some novel insights into the pathogenesis of PBC. Methods We retrospectively collected clinical features and laboratory data from hospitalized patients who were primarily diagnosed with PBC and from healthy physical examinees at the Third People's Hospital of Changzhou between January 1, 2023, and June 30, 2024. Results A total of 152 PBC patients and 96 healthy controls who met the inclusion and exclusion criteria were enrolled. Significant differences were observed in baseline characteristics and laboratory data between the two groups. After the propensity score matching (PSM) analysis, 61 PBC patients and 61 healthy controls were successfully matched, ensuring that the general characteristics (age and gender) of the two groups were balanced and comparable. Compared to the control group, the proportion of peripheral lymphocytes was significantly higher in the PBC group (31.9% vs. 17.8%), primarily due to an increase in CD4⁺ T cells (46.77% vs. 41.19%), while CD8⁺T cells were significantly decreased (19.73% vs. 22.07%). Notably, the proportions of CD4⁺ programmed cell death 1 (PD-1)⁺ T and CD8⁺PD-1⁺ T cells were elevated, with CD8⁺PD-1⁺ T cells showing a significant positive correlation with the severity of liver inflammation (r=0.41). Furthermore, the mitochondrial mass (MM) of CD4⁺ T cells was significantly increased in PBC patients, whereas no significant changes were observed in the MM of CD8⁺ T cells or the mitochondrial membrane potential (MMP) of CD3⁺ T cells. Additionally, the plasma levels of cytokines, such as IL-4, IL-8, IL-10 and IFN-α, were abnormally elevated. The plasma levels of IL-5 and IL-1β were negatively correlated with the stage of liver fibrosis in patients with PBC (r=-0.52). Conclusion The overactivation and proliferation of CD4⁺ T cells, along with the suppression of CD8⁺ T cell function and increased PD-1 expression leads to T cell exhaustion, indicating significant immunological alterations in PBC patients. These changes are closely associated with the disease progression. Additionally, cytokines are likely involved in the immune regulation process of PBC and may influence the pathogenic mechanisms of the disease. Regular monitoring of lymphocyte subsets and cytokine levels can help assess the immune status and disease activity in patients with PBC, thereby guiding the individualized treatment strategies.
Humans ; Male ; Female ; Middle Aged ; Liver Cirrhosis, Biliary/blood* ; Retrospective Studies ; T-Lymphocyte Subsets/immunology* ; Aged ; Cytokines/blood* ; Adult ; CD8-Positive T-Lymphocytes/immunology*

OBJECTIVETo investigate the characteristics and clinical significance of changes in gamma delta T cells in patients with chronic hepatitis B (CHB), including during the immune tolerant (IT) phase and the immune activated (IA) phase.

METHODSFlow cytometry was used to analyze the frequencies and absolute numbers of γδT cells and their subsets in peripheral blood and in liver from 80 CHB cases, including 20 IT carriers and 60 IA patients. Blood samples were obtained from all CHB cases and 5 healthy controls (HCs). Liver biopsies were collected from 22 IA patients and 5 IT carriers undergoing diagnosis, and from the 5 HCs.

RESULTSCompared to HCs and IT carriers, the IA patients displayed significantly lower levels of peripheral and intrahepatic γδT cells as well as the Vδ2 subsets. The levels of peripheral and intrahepatic VγδT cells were closely associated with the liver histological activity index and serum alanine aminotransferase levels.

CONCLUSIONγδT cells, especially the Vδ2 subsets, may play a protective role in decreasing liver damage in CHB patients.

Alanine Transaminase ; blood ; Case-Control Studies ; Flow Cytometry ; Hepatitis B, Chronic ; immunology ; pathology ; Humans ; Liver Cirrhosis ; immunology ; T-Lymphocyte Subsets ; cytology

OBJECTIVETo characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis.

METHODSThe full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein.

RESULTSThe recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.

CONCLUSIONThe pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.

Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; immunology ; Calmodulin ; immunology ; Clonorchiasis ; immunology ; Clonorchis sinensis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Library ; Immunoglobulin G ; blood ; Inflammation ; Liver Cirrhosis ; parasitology ; Male ; Mice ; Rats ; Recombinant Proteins ; immunology

PURPOSE: The role of IL28B gene variants and expression in hepatitis B virus (HBV) infections are not well understood. Here, we evaluated whether IL28B gene expression and rs12979860 variations are associated with HBV outcomes. MATERIALS AND METHODS: IL28B genetic variations (rs12979860) were genotyped by pyrosequencing of DNA samples from 137 individuals with chronic HBV infection [50 inactive carriers (IC), 34 chronic hepatitis B (CHB), 27 cirrhosis, 26 hepatocellular carcinoma (HCC)], and 19 healthy controls. IL28A/B mRNA expression in peripheral blood mononuclear cells was determined by qRT-PCR, and serum IL28B protein was measured by ELISA. RESULTS: Patients with IL28B C/C genotype had greater IL28A/B mRNA expression and higher IL28B protein levels than C/T patients. Within the various disease stages, compared to IC and healthy controls, IL28B expression was reduced in the CHB, cirrhosis, and HCC cohorts (CHB vs. IC, p=0.02; cirrhosis vs. IC, p=0.01; HCC vs. IC, p=0.001; CHB vs. controls, p<0.01; cirrhosis vs. controls, p<0.01; HCC vs. controls, p<0.01). When stratified with respect to serum HBV markers in the IC and CHB cohorts, IL28B mRNA and protein levels were higher in HBeAg-positive than negative individuals (p=0.01). Logistic regression analysis revealed that factors associated with high IL28B protein levels were C/C versus C/T genotype [p=0.016, odds ratio (OR)=0.25, 95% confidence interval (CI)=0.08-0.78], high alanine aminotransferase values (p<0.001, OR=8.02, 95% CI=2.64-24.4), and the IC stage of HBV infection (p<0.001). CONCLUSION: Our data suggest that IL28B genetic variations may play an important role in long-term development of disease in chronic HBV infections.
Adult ; Aged ; Alanine Transaminase/blood ; Asian Continental Ancestry Group/*genetics ; Biological Markers/blood ; Carcinoma, Hepatocellular/genetics ; Case-Control Studies ; China ; DNA, Viral/blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Genotype ; Hepatitis B virus/genetics ; Hepatitis B, Chronic/ethnology/*genetics/immunology/*virology ; Humans ; Interleukins/blood/*genetics/metabolism ; Leukocytes, Mononuclear ; Liver Cirrhosis/blood ; Liver Neoplasms/genetics ; Male ; Middle Aged ; RNA, Messenger/*genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the clinical value of serum anti-Ku86 in early detection of hepatocellular carcinoma (HCC).

METHODSExpression levels of Ku86 protein in HCC and adjacent normal liver tissues were detected by Western blotting. Serum anti-Ku86 level in 83 patients with early HCC and 124 patients with liver cirrhosis were detected by enzyme-linked immunosorbent assay (ELISA). Chemiluminescence was used to measure the serum level of α-fetoprotein (AFP).

RESULTSExpression of Ku86 protein in HCC was increased when compared with the adjacent normal liver tissues (0.21 ± 0.05 vs. 0.08 ± 0.02, P < 0.01). Serum anti-Ku86 level was significantly elevated in HCC patients compared with that in liver cirrhosis patients (0.47 ± 0.22 vs. 0.22 ± 0.06 Abs at 450 nm, P < 0.01), but there was no significant difference between HBV infection and HCV infection in HCC patients (0.51 ± 0.19 vs. 0.47 ± 0.24, P = 0.267). Of note, serum anti-Ku86 level was significantly decreased after surgical resection of the tumors in the 30 HCC cases tested (P < 0.01). The results of ROC analysis indicated a better performance of anti-Ku86 (0.857) than AFP (0.739) for early detection of HCC. In 83 HCC patients, the positive rate of anti-Ku86 was 61.4% (51/83), significantly higher than that of the AFP positive rate (27.7%, 23/83). The anti-Ku86 level was positive in 37 of 60 HCC cases with negative AFP. Combination assay of AFP and anti-Ku86 could detect 60 of 83 HCC cases (72.3%, 60/83). There was no significant correlation of anti-Ku86 and AFP (r = 0.156, P = 0.161).

CONCLUSIONSSerum anti-Ku86 level is significantly elevated and is not related to HBV and HCV infection in HCC patients. Serum anti-Ku86 antibody may be a potential biomarker for early detection of HCC, and can be used in combination with AFP in clinics.

Adult ; Aged ; Antigens, Nuclear ; immunology ; Autoantibodies ; blood ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; diagnosis ; virology ; DNA-Binding Proteins ; immunology ; Early Detection of Cancer ; Female ; Hepatitis B ; blood ; Hepatitis C ; blood ; Humans ; Ku Autoantigen ; Liver Cirrhosis ; blood ; Liver Neoplasms ; blood ; diagnosis ; virology ; Male ; Middle Aged ; ROC Curve ; alpha-Fetoproteins ; metabolism

OBJECTIVETo evaluate the clinical features of patients with primary biliary cirrhosis (PBC) and positive expression of sp100 autoantibody in order to generate a clinical screening profile that may help to increase early diagnosis and timely initiation of therapy.

METHODSThe clinical data of 70 patients who were diagnosed with PBC by liver biopsy between January 2006 to December 2009 at the Second Affiliated Hospital of Kunming Medical University of Hepatobiliary and Pancreatic Medicine were retrospectively collected for analysis. The patients were divided according to expression of anti-sp100: positive patients, n = 12; negative patients, n = 58. The groups were comparatively analyzed for differences in clinical, biochemical, immunological, and histopathological parameters. Normally distributed data was compared by t-test, and non-normally data was compared by rank-sum test.

RESULTSThere was no significant difference in age among the sp100-positive and sp100-negative patients (51.6 +/- 9.5 vs. 50.0 +/- 14.7 years, P more than 0.05). The sp100-positive group had significantly more women (80.0% vs. 61.9%, X2 = 0.32, P more than 0.05) and more patients with atypical symptoms (18.2% vs. 13.8%) but the difference of the latter did not reach statistical significance. The sp100-positive group had significantly higher levels of alkaline phosphatase (ALP; 466 vs. 163 U/L, Z = 3.71), gamma-glutamyl-transpeptidase (GGT; 728 vs. 154 U/L, Z = 3.38), and immunoglobulin M (IgM; 4.25 +/- 2.86 vs. 2.81 +/- 2.15, t = 2.06, P less than 0.05). Forty of the total patients tested negative for antimitochondrial (AMA)-M2 antibodies, and eight of those were sp100-positive (20.0%) while 18 were antinuclear (ANA) antibody-positive (45.0%). There were significantly more AMA-M2-negative/ANA-positive patients than sp100-positive patients (P = 0.021). Anti-sp100 expression was not associated with the pathological stage of PBC (R1 = 5.500, P more than 0.05).

CONCLUSIONSP100-positive PBC may show a bias towards the female sex, and may be characterized by enhanced serum levels of ALP, GGT, and IgM. Further clinical differences may manifest as the disease progresses, and changes in autoantibodies' expression and liver function markers should be carefully monitored in follow-up.

Adult ; Aged ; Antibodies, Antinuclear ; blood ; Antigens, Nuclear ; immunology ; Autoantibodies ; blood ; Autoantigens ; immunology ; Female ; Humans ; Liver ; pathology ; Liver Cirrhosis, Biliary ; immunology ; pathology ; Male ; Middle Aged ; Retrospective Studies ; Young Adult

OBJECTIVETo investigate whether the level of hepatitis B surface antigen (HBsAg) represents the status of inflammation and stages of fibrosis in livers of patients with chronic hepatitis B (CHB) during the immune clearance phase (IC).

METHODSLiver biopsy samples and sera were collected from 165 consecutive patients (136 males; 29 females) with CHB in IC who were treated in our hospital between March 2009 and June 2011. Routine biochemical tests were carried out to measure indicators of liver function. The relation between HBsAg level and liver pathological stages were determined by Spearman's rank correlation analysis. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of HBsAg level for liver pathological stages. Binary logistic regression was used to analyze potentially relevant indicators, and liver pathology-predicting models were built and analyzed by the ROC method.

RESULTSThe mean values of HBsAg (IU/mL) were significantly different at the different liver inflammation stages: G1, 27 716.07+/-32 870.69; G2, 34 478.75+/-40 899.55; G3, 19 408.09+/-24 881.07; G4, 14 286.31+/-28 610.14. Likewise, the mean values of HBsAg (IU/mL) were significantly different at the different liver fibrosis stages: S1, 41 337.23+/-43 236.39; S2, 27 264.32+/-32 517.29; S3, 111 541.77+/-11 538.93; S4, 11 447.37+/-22215.44. Spearman's rank correlation analysis indicated a significant correlation between HBsAg level and liver inflammation stage (rs = -0.244) and fibrosis stage (rs = -0.365). ROC curve analysis of the diagnostic value of HBsAg for inflammation stages S more than or equal to 4 revealed that the area under the curve (AUC) was 0.70. The specificity of diagnosing S more than or equal to 4 was > 95.16% when HBsAg was less than or equal to 32995 IU/mL. Binary logistic regression analysis identified age, serum albumin, cholinesterase, and HBsAg as independent predictors of liver fibrosis.

CONCLUSIONHBsAg level is negatively correlated with liver inflammation and fibrosis stages for patients with CHB in the IC phase, and might represent a useful noninvasive marker of the degree of hepatic fibrosis.

Adult ; Female ; Hepatitis B Surface Antigens ; blood ; Hepatitis B, Chronic ; blood ; immunology ; pathology ; Humans ; Inflammation ; Liver ; immunology ; pathology ; Liver Cirrhosis ; immunology ; pathology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo observe the efficacy of ursodeoxycholic acid (UDCA) combined with Tongdan: Decoction () on immunological indices and histopathological changes in patients with primary biliary cirrhosis (PBC) of IIor III histological stage.

METHODSSixty PBC patients were assigned randomly and equally: to the control group treated with UDCA alone and the treatment group treated with UDCA combined with Tongdan Decoction. The immunological indices and histopathological changes were detected before and after 24-week treatment, and the follow-up lasted for 1-3 years.

RESULTSAfter 24-week treatment, CD4(+)CD28(-) in the peripheral blood was lowered and CD4(+)CD25(+) was increased in both groups, and better effect was shown in the treatment group (P<0.01). The levels of IgM, IgG, and IgA decreased markedly after 96-week treatment in the treatment group (P< 0.05, P< 0.01), while in the control group, only the latter two showed significant decrease after 148 week (all P<0.05). At the end of the 3-year follow-up, the medians of histopathological

CONCLUSIONCombined therapy of Tongdan Decoction and UDCA showed a better therapeutic effect: than UDCA monotherapy on PBC, especially in improving immunological indices and histopathological hepatic changes.

Antigens, CD ; blood ; Biomarkers ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Immunoglobulin G ; blood ; Inflammation ; blood ; complications ; Liver Cirrhosis, Biliary ; blood ; drug therapy ; immunology ; pathology ; Male ; Middle Aged ; Ursodeoxycholic Acid ; therapeutic use

OBJECTIVETo investigate the correlation between ER-a in the liver and cytokines of T lymphocytes subsets and serum signatures in PBC patients.

METHODSThe research is performed with cross-sectional study. 80 PBC women patients without treatment were enrolled in PBC group, 10 healthy women as baseline-matched in healthy-control group, and 20 patients with non-autoimmune liver disease in non-PBC control group. The expression of IL-6, IL-8, IL-22, TNFa, IFNgamma, AMA-M2, Sp100 and gp210 were analyzed in Peripheral Blood using ELISA in all groups, and ER-a of patients were performed on tissues from liver biopsies in PBC group and non-PBC control group with immunohistochemistry. Spearman correlation test were performed on the indices to identified the association of all Parameters. numerical data were compared with Wilcoxon rank-sum test.

RESULTSCompared with healthy-control group, expression of serum cytokines are significantly higher in PBC and non-PBC groups (P less than 0.01), while no significant difference were observed between PBC and non-PBC groups. The positive rate of ER-a in PBC patients liver tissues in PBC group is higher than that in non-PBC group (Z=4.82, P less than 0.01). Expression of ER-a is positively correlated with positive rates of AMA-M2 antibody, Sp100 and gP210 of tissues of PBC patients ( r=0.898, 0.819, 0.814, P less than 0.01). ER-a is positive correlated with the expression of cytokines, among which the coefficient of correlation of IL-22, TNFa, IFNgamma is more than 0.7 (r=0.71, 0.89, 0.82, P less than 0.01), AMA-M2, Sp100, gp210 is negative in serum of non-PBC control group. No obviously correlations were indicated between the expression of ER-a and cytokines.

CONCLUSIONA high level of expression of cytokines in the serum might be one of the factors of etiopathogenesis of PBC.

Autoantibodies ; blood ; Biomarkers ; blood ; Case-Control Studies ; Estrogen Receptor alpha ; metabolism ; Female ; Humans ; Interleukin-6 ; blood ; Interleukin-8 ; blood ; Interleukins ; blood ; Liver ; metabolism ; Liver Cirrhosis, Biliary ; blood ; immunology ; Middle Aged

Primary biliary cirrhosis (PBC) is a slowly progressive cholestatic liver disease of autoimmune etiology. The initial presentation of PBC is various from asymptomatic, abnormal liver biochemical tests to overt cirrhosis. The diagnosis of PBC is based on cholestatic biochemical liver tests, presence of antimitochondrial antibody and histologic findings of nonsuppurative destructive cholangitis. Although the diagnosis is straightforward, it could be underdiagnosed because of its asymptomatic presentation, or underrecognition of the disease. UDCA in a dose of 13-15 mg/kg is the widely approved therapy which can improve the prognosis of patients with PBC. However, one-third of patients does not respond to UDCA therapy and may require liver transplantation. Every effort to diagnose PBC in earlier stage and to develop new therapeutic drugs and clinical trials should be made.
Autoantibodies/blood ; Autoimmunity/immunology ; Cholagogues and Choleretics/therapeutic use ; Humans ; Liver Cirrhosis, Biliary/*diagnosis/pathology/*therapy ; Liver Transplantation ; Ursodeoxycholic Acid/therapeutic use