[Establishment of a mouse model of primary biliary cirrhosis by AMA M2 autoantigen injection].

OBJECTIVES: To establish a primary biliary cirrhosis (PBC) model by AMAM2 autoantigen injection into C57BL/6 mice. METHODS: Mice of the model group were immunized intraperitonealy with 200 microl of purified recombinant AMAM2 autoantigen in complete Freund's adjuvant (CFA). Mice immunized with bovine serum albumin and CFA in the same way were used as negative controls. Sixty-six weeks later, mice were sacrificed and their sera were collected. Sera samples were assayed for AMAM2 autoantibody, alkaline phosphatase (ALP), ALT and total bilirubin (TBil). Their liver, stomach, muscle and kidney tissues were sectioned and stained using HE to observe the pathological changes. RESULTS: Antibodies to AMAM2 autoantigen were readily induced in the model group. The mice in the model group had no significant changes in the level of serum ALT and TBil but had an obvious increase of ALP (P<0.05). The stomach, muscle and kidney tissues showed no evident damage while the livers had obvious pathological changes, including bile duct degeneration or proliferation, and mononuclear cell infiltration. CONCLUSION: The AMAM2 autoantigen-induced PBC animal model was successfully established in C57BL/6 mice in our experiment and its characteristic biochemical and pathology are quite similar to that in the early stage of human PBC. This model may provide a useful experimental approach for further study of the pathogenesis and clinical treatment of human PBC.

Increased levels of CD40-CD40 ligand system in patients with essential hypertension.

BACKGROUND: Although it has been hypothesized that hypertension is in part an inflammatory disorder, clinical data linking inflammation with incident hypertension are scarce. There is evidence that have shown that CD40-CD40L interaction plays a pathogenic role in inflammatory disorders. We assessed whether CD40 system expressions were altered in patients including 30 with hypertension grade 1, 80 with hypertension grade 2 and 40 with hypertension grade 3. METHODS: Twenty normal controls and 150 patients with essential hypertension were investigated. The expression of CD40 and CD40L on platelet was analyzed by indirect-immunofluorescence flow cytometry and soluble CD40L level was determined by a commercially available ELISA. C-reactive protein was also measured by ELISA. RESULTS: All patients with hypertension showed a significant increase of CD40 (67.1+/-9.6 Mean Fluorescence Intensity, MFI) and CD40L (15.3+/-5.0 MFI) coexpression on platelets as well as sCD40L levels (12.8+/-3.9 ng/ml ) compared with controls (p<0.0001). We found that CRP levels related to CD40-CD40L system. We also observed a slight correlation between sCD40L level and blood pressure. During 3 months follow-up, patients with enhanced levels of sCD40L (>15 ng/ml) indicated a tough control of blood pressure. CONCLUSION: Patients with essential hypertension show increased coexpression of CD40 system, which suggests that hypertension is in part an inflammatory disorder.

Proteomics-based identification of DEAD-box protein 48 as a novel autoantigen, a prospective serum marker for pancreatic cancer.

Patients with cancer frequently develop autoantibodies, and the identification of panels of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. This study aims to exploit the autoantibody repertoire in pancreatic cancer and identify the possible serum marker for pancreatic cancer. Sera from 55 newly diagnosed patients with pancreatic cancer and 52 healthy controls were analyzed for antibody-based reactivity against Hep-2, a human larynx epithelioma cancer cell line, with one-dimensional immunoblot assay. From this analysis, we observed a prominent band with a molecular weight of 47 kDa in 63.64% (35/55) patients, while in only 1.9% normal group (1/52). Using immunoblot analysis after two-dimensional electrophoresis combined with liquid chromatography-electrospray ionization tandem mass spectrometry, this target antigen was identified as DEAD-box protein 48 (DDX48). BLAST analysis showed that it was highly similar to eukaryotic initiation factor 4A and might play a role in pre-mRNA processing. An enzyme-linked immunosorbent assay was performed using recombinant, purified DDX48 as an antigen to detect anti-DDX48 autoantibodies in sera. Reactivity was observed in 20 of 60 (33.33%) pancreatic cancer patients, 3 of 30 (10.00%) colorectal cancer patients, 2 of 30 (6.67%) gastric cancer patients, 2 of 30 (6.67%) hepatocellular cancer patients, while none of the 20 chronic pancreatitis patients, 30 lung cancer patients, and 60 normal individuals. Together, these results demonstrate that the detection of autoantibodies to DDX48 may have clinical utility for the improved diagnosis of pancreatic cancer.

[Genetic association between interleukins gene polymorphisms with primary biliary cirrhosis in Chinese population].

OBJECTIVE: To determine the relationship between polymorphisms in the genes encoding IL-1, IL-6, and IL-10 with primary biliary cirrhosis (PBC) in Chinese population. METHODS: Whole-blood samples were taken from 77 patients with PBC and 160 healthy controls. DNA was extracted and the polymorphisms at positions IL-1 +3953, IL-1RN intron 2, IL-6 -174, and IL-10 -1082, -819, and -592 were determined by using sequence-specific polymerase chain reaction (SSP) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequency of IL-1RN1,1 allele in PBC group was significantly higher than in control group (90.9% vs 79.4%, P=0.026), and the frequency of IL-1RN1,2 in PBC group was significantly lower than in control group (6.5% vs 18.8%, P=0.013). There was no significant difference in the frequence of IL-1RN*2 allele between PBC group and control group (P=0.06). Of the 77 patients with PBC, 4 patients were IL-6 -174GC, 73 were IL-6 174GG. All the 160 health controls are IL-6 -174GG (P=0.0036). The frequence of IL-6 -174C allele in PBC group was significantly higher than that in control group (P=0.0038). No significant differences of polymorphisms for IL-1 +3953 and IL-10 (-1082, -819 and -592) were found between PBC group and control group. CONCLUSION: The polymorphisms of IL-1RN and IL-6 -174G/C appear to be associated with PBC, and the polymorphisms of IL-1 +3953 and IL-10 promoter gene are not associated with PBC in a Chinese population.

[Study on the relationship of CTLA-4 -318, +49 polymorphisms with autoimmune hepatitis and primary biliary cirrhosis in a Chinese population].

OBJECTIVE: To investigate the association between Chinese patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and the polymorphisms of cytotoxic T lymphocyte -associated antigen-4 (CTLA-4) gene promoter (-318) and exon 1 (+49). METHODS: The CTLA-4 promoter (-318 T/C) and exon 1 (+49A/G) polymorphisms were genotyped via restriction fragment length polymorphism methods in 62 Chinese AIH patients, 77 Chinese PBC patients and 160 healthy controls. RESULTS: There was no difference in the distribution of CTLA-4 promoter -318 T/C polymorphisms between AIH patients and controls, but the C allele frequency was significantly increased in patients with AIH, compared to controls (P=0.02, OR=2.43). The distribution of CTLA-4 gene exon 1 49 A/G genotypes exhibited significant difference between PBC patients and controls (P=0.006), and the frequency of G allele showed a significant increase in PBC group as compared with controls (P=0.0046, OR=1.8). Although the genotype distribution of the CTLA-4 exon 1-promoter gene displayed no significant difference between AIH and PBC patients and controls, the occurrence of GG-CC was increased in the patients of the two groups (AIH: 32.3%, PBC: 37.7%; control: 22.5%). CONCLUSION: The above findings suggest that the polymorphisms of CTLA-4 gene probably confer susceptibility to AIH and PBC in the Chinese population.

Diagnostic and epidemiological implications of regional differences in serum concentrations of proteins observed in six Asian cities.

The IFCC Committee on Plasma Proteins has been investigating regional differences for commonly assayed plasma proteins to determine whether universal reference intervals can be applied. As a part of this study, we launched an Asian project analyzing the concentrations of 13 serum proteins whose values are standardized to CRM470, and five newer analytes: retinol-binding protein (RBP), cystatin C (CysC), light-chain-kappa (L-kappa), and light-chain-lambda (L-lambda). In Tokyo, Seoul, Kuala Lumpur, Hong Kong, Taipei and Shanghai, serum samples were collected from 146 to 415 apparently healthy individuals with nearly equal gender ratios. All assays were performed in Tokyo on a Behring Nephelometer II (BN II). Seven chemical analytes (aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (gammaGT), creatinine, total cholesterol (TC), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C)) were also measured. These results were used for excluding individuals with possible latent clinical disorders. Positive acute phase reactants were consistently lower, and negative ones were higher, in Tokyo than those in other cities. The most conspicuous difference was observed in C-reactive protein (CRP). There were no regional differences in transferrin, albumin, or CysC. Creatinine was much lower in Tokyo despite comparable CysC levels. ALT and gammaGT were higher in Shanghai, Taipei and Seoul; gammaGT and TG were higher in Shanghai; and HDL-C was higher in Tokyo. Gender-related differences in reference intervals were observed for immunoglobulin (Ig)M, haptoglobin, RBP, transferrin, alpha2-macroglobulin (A2M), transthyretin, alpha1-acid glycoprotein, CysC, and C4 in all cities. Slight age-related differences were observed, irrespective of the region, in IgA and ceruloplasmin (increase) and A2M (decrease). Environmental factors and lifestyle seem to have a great influence on many commonly measured analytes.

[Defect of TCR-mediated signal pathway of T cells in IDDM patients].

AIM: To study the mechanism responsible for alteration of T cell response in IDDM patients. METHODS: T cells from peripheral blood of IDDM patients were activated by anti-TCR antibodies. The level of TCR-mediated signaling pathway was analyzed. RESULTS: T cells from IDDM patients responded weakly to anti-TCR antibody-induced proliferation, as compared with T cells from normal subjects (P < 0.05). The defect could be partially remedied by the addition of rIL-2, while the anti-CD28 antibody stimulation did not restore the proliferative response of anti-TCR-induced cells from IDDM patients (P = 0.03). CONCLUSION: Unresponsiveness of the T cells from IDDM patients to anti-TCR antibody may result from a defect in the signaling pathway, the CD28 co-stimulation-signaling pathway is normal. Defect in the TCR signaling pathway increases the sensitivity of T cells from IDDM patients to apoptosis or anergy.

Transcriptional regulation of human alpha1(I) procollagen gene in dermal fibroblasts.

AIM: To clarify the fractional activity of promoters from human alpha1(I) procollagen gene, the interaction between cis-elements and consensus DNA-binding proteins responsible for high promoter activity, and the potential application of promoter competitors as well as cytokines for antifibrogenesis. METHODS: Sequence between 2483 bp upstream of the start of transcription and 42 bp downstream of this site was investigated with serial 5'-deletion. The 5'-deleted promoters recombined with chloramphenicol acetyltransferase (CAT) as reporter gene were transiently transfected to human dermal fibroblasts. Electrophoretic mobility shift assay was performed to show the DNA-protein binding capacity of the promoter sequence. Cytokines including tumor necrosis factor alpha (TNFalpha) and interferons (INFs) were added to the culture medium of transiently transfected fibroblasts. Competitor DNA for the binding sites of Sp-1, Ap-1 and NF-1 was individually cotransfected transiently in order to block the promoter-driven CAT expression. RESULTS: Sequences of -2483 to +42 bp and -268 to +42 bp of human alpha1(I) procollagen gene had high activity as promoters. Binding sites for Ap-1 and Sp-1 were among the cis-regulatory elements recognizing consensus transcription factors responsible for basal promoter activity of sequence -268 to +42 bp. TNFalpha, IFNalpha, IFNbeta showed inhibitory effects on sequence -2 483 to +42 bp as promoter with activities 43%, 62% and 60% of control respectively. Transfection of the promoter competitors could reverse the promoter activity of -268 to +42 bp 40-60%. CONCLUSION: Sequences of -2 483 to +42 bp recombined with reporter gene provide an ideal construction for transcriptional study of alpha1(I) procollagen gene. The anti-collagen capacity of TNFalpha and IFNs is associated with their transcriptional regulation. Ap-1 and Sp-1 mediate the basal transcriptional activation of human alpha1(I) procollagen gene in dermal fibroblasts. Competitors for highly active promoters might be a novel potential candidate in fibrotic blockade.

The effect of elevated serum soluble CD40 ligand on the prognostic value in patients with acute coronary syndromes.

BACKGROUND: Increasing evidence shows that high expression of CD40L plays an important role in the pathogenesis of atherosclerosis and coronary artery disease. We evaluated the clinical predictive value of increased serum soluble CD40 ligand (CD40L) in patients with acute coronary syndromes (ACS) and acute chest pain. METHODS: Serum levels of soluble CD40 ligand were measured by ELISA in 128 patients with ACS and in 68 patients with acute chest pain. Platelet activation was assessed by flow cytometry. RESULTS: The levels of soluble CD40 ligand were increased in 57.8% patients with ACS (>8.0 ng/ml) and in 35 patients with acute chest pain (>8.0 ng/ml), respectively. The level of soluble CD40 ligand was slightly correlated with measured levels of troponin T (r=0.21, p<0.05), and the increased soluble CD40L levels (>8.0 ng/ml) were associated with higher risk for AMI, sudden death and recurrent angina. Patients with elevated serum levels of sCD40L and cTnT showed a significantly increased risk of major adverse cardiovascular events (including AMI, sudden death and recurrent angina) in the two groups during 30 days and 6 months of follow-up. CONCLUSION: In patients with unstable coronary artery disease, elevation of serum soluble CD40L levels indicated an independent increased risk of major adverse cardiovascular events.

Relation between upregulation of CD40 system and complex stenosis morphology in patients with acute coronary syndrome.

AIM: To investigate whether upregulation of CD40-CD40 ligand system is related to matrix metalloproteinases level and stability of coronary atherosclerotic plaque in patients with acute coronary syndrome (ACS). METHODS: Sixteen normal controls and 56 patients including 24 with stable angina (SA), 20 with unstable angina (UA), and 12 with acute myocardial infarction (AMI) were investigated. The expression of CD40 and CD40L on platelet was analyzed by flow cytometry. Serum soluble CD40L (sCD40L), MMP-9 and MMP-3 level was determined by ELISA. All coronary stenosis with > or =30% diameter reduction were assessed by angiographic coronary stenosis morphology. RESULTS: Patients with ACS showed a significant increase of CD40 (75 +/- 12 MIF) and CD40L (13 +/- 4 MIF) coexpression on platelets compared with control and SA group (P<0.01). sCD40L also showed higher level in patients with ACS (10.2 +/- 3.5 microg/L) than in control (3.1 +/- 1.4 microg/L, P<0.01) and SA group (3.3 +/- 1.6 microg/L, P<0.01). Serum MMP-3 and MMP-9 in patients with ACS were two times greater than those in control. A positive correlation was found between MMP-9, MMP-3, and CD40L expression on platelets as well as sCD40L levels, but not for CD40 expression on platelets. An obvious correlation was also observed between sCD40L concentration and complex coronary stenoses (r=0.60, P<0.01). CONCLUSION: Patients with ACS show increased coexpression of CD40 system, especially expression of CD40L, which may create a proinflammatory and prothrombotic milieu for aggravating the development of atherosclerosis and instability of atherosclerotic plaques, and may be a valuable marker for predicting the severity of ACS.

[Mechanism of arsenic trioxide-induced cytotoxicity on multiple myeloma cells].

BACKGROUND & OBJECTIVE: Multiple myeloma (MM), a plasma cell tumor, is difficult to cure by now. Previous study showed that As2O3 could inhibit the proliferation and induce the apoptosis of myeloma cell in vitro. The aim of this study was to explore the possible mechanism of arsenic trioxide (As2O3) on multiple myeloma cells. METHODS: The cytotoxic effects of As2O3 on five myeloma cell lines U266, SKO-007, LP-1, HS-Sultan, and KM3 were examined using MTT bioassay, and the concentration of 50% growth inhibition (IC(50)) was calculated. The synergistic or antagonistic effects of menadione (VK(3)), N-acetyl-cysteine (NAC), and reduced glutathione (GSH) combined with As2O3 were also examined. The cellular GSH levels in five MM cell lines and its changes in U266 cells after treated with As2O3, VK(3), NAC, and exogenous GSH were determined by colorimetric assay, and the relationship between IC(50) and cellular GSH levels was analyzed. RESULTS: As2O3 inhibited the proliferation of all five myeloma cells, but with different sensitivity. GSH contents in five MM cells were correlated with its IC(50) significantly (r=0.87,P< 0.05). Oxidant VK(3) had significant synergistic effect with As2O3, and antioxidants NAC and GSH partly blocked the growth inhibition of As2O3. Both As2O3 and VK(3) decreased the GSH contents, NAC and GSH increased them contrarily. CONCLUSION: One of the mechanisms of effect of As2O3 on myeloma cells may be through decreasing the cellular GSH levels and inducing myeloma cell apoptosis.

Development of an enzyme immune assay for detecting M2 autoantibodies specific for primary biliary cirrhosis.

OBJECTIVE: To develope a new enzyme immune assay (ELISA) for detection of M2 antibody specific for primary biliary cirrhosis (PBC) by using a triple hybrid clone as antigen, which coexpresses the three immunodominant lipoyl domains of PDC-E2, BCOADC-E2 and OGDC-E2 from human sources. METHODS: After expressing autoantigens of PBC in prokaryote by constructing recombinant expressive plasmid successfully, the fusion protein was purified by affinity chromatography. The sera of 17 PBC patients were examined. As controls, the sera of 167 non-PBC patients and the sera of 1225 normal controls aged under 28 were examined. RESULTS: None of the sera from the non-PBC patients or the normal controls was positive for anti-M2 shown by the new ELISA. However, the positivity rate for anti-M2 in the PBC patients was 100% (17/17), as shown by the new ELISA. CONCLUSION: The detection system with a good sensitivity and specificity may be used as a powerful method for the diagnosis of PBC.

Effect of CD40--CD40 ligand interaction on diacylglycerol-protein kinase C and inositol trisphosphate-Ca(2+) signal transduction pathway in human umbilical vein endothelial cells.

BACKGROUND: Increasing evidence shows that CD40-CD40L interaction plays a crucial role in the pathogenesis of atherosclerosis and coronary artery disease. The mechanism of CD40-CD40L interaction might be related to signal transduction via receptor. The transduction pathway of the CD40 receptor may involve the activation of phospholipase C (PLC) which induces the production of inositol trisphosphate (IP(3)) leading to the increase of the intracellular free calcium on one hand, and of diacylglycerol (DAG) which stimulates the translocation to the membrane of protein kinase C (PKC). METHODS: Endothelial cells were isolated from human umbilical vein and incubated with indicated concentrations of CD40 ligand (CD40L) for various periods. The DAG levels in HUVEC were studied with radioenzymatic assay. Quantitative measurements of 32P phosphatidic acid were performed by thin-layer chromatography and autoradiography. IP(3) was quantitatively measured by the radioreceptor binding assay. The activity of PKC and [Ca(2+)]i induced by CD40L were measured by its ability to transfer phosphate from [gamma-32P]ATP to lysine-rich histone and flow cytometric analysis loading with the Ca(2+) dye fluo3/Am, respectively. RESULTS: The DAG levels were raised by CD40L in a dose-dependent, biphasic manner. The early phase was rapid and transient, peaking at 20 s; and the late phase reached the maximal level at 10 min and then decayed slowly. CD40L increased the PKC total activity in a dose-dependent manner with phase peaking at 12 min, then decreased slowly and maintained for at least 20 min. The results also showed that CD40L induced PKC activity translocation from the cytosolic to membrane. Similarly, the CD40L-induced transient IP(3) formation was coincident with the first peak of DAG formation. Moreover, CD40L also induced biphasic [Ca(2+)]i responses including the rapid initial transient phase and the sustained phase. Anti-CD40 monoclonal antibody can significantly suppress CD40L-induced DAG-PKC and IP(3)-[Ca(2+)]i signal pathway activation in HUVEC. CONCLUSIONS: CD40-CD40 ligand interaction can induce a robust stimulation of the DAG-PKC and inositol trisphosphate-Ca(2+) signal transduction pathway in HUVEC.

Characterization of M2 antibodies in asymptomatic Chinese population.

AIM: To investigate the presence of M2 antibodies specific for primary biliary cirrhosis (PBC) in asymptomatic Chinese and identify patients with early PBC. METHODS: Enzyme-linked immunosorbent assay (ELISA) tests for M2 antibodies to recombinant protein were performed in 5 011 subjects (age range, 26-85 years; mean age: 45.81+/-15.02 years) who took an annual physical examination. M2-positive subjects were further analyzed for immunoglobulin (Ig) classes and subclasses of M2 antibodies. Clinical, biochemical and immunological data were obtained for M2-positive subjects. In addition, ultrasonography (US) or endoscopic retrograde cholangio-pancreatography (ERCP) was performed to exclude any disorders other than PBC. RESULTS: M2 antibodies were detected in 8 (0.16 %) of the 5 011 subjects studied. Of the 8 subjects, 7 were female and 1 was male (age range: 40-74 years). An unexplained increase of serum alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (gamma-GT) values, often to striking levels, was detected in 4 M2-positive subjects, 3 of them accorded with the diagnostic criteria recommended by the American Association for the Study of Liver Diseases, even though they had no symptoms of PBC (such as fatigue, pruritus or jaundice). Liver biopsy was performed in two M2-positive subjects and the histology was compatible with PBC in both cases. CONCLUSION: Our data, while not assessing the true prevalence of asymptomatic PBC in the general population, suggest that asymptomatic PBC is much more common in China than has been supposed.

[Effects of arsenic trioxide on cell cycle and expression of cyclin dependent kinase inhibitors of multiple myeloma cells].

OBJECTIVE: To study the effects of arsenic trioxide (As(2)O(3)) on cell cycle and expression of cyclin dependent kinase inhibitors (CDKIs) in multiple myeloma (MM) cells, and explore its pharmacological mechanism. METHODS: The DNA content of MM cells line HS-Sultan was analyzed by flow cytometry after exposure to As(2)O(3), the effects on expression of CDKI P15, P16 AND P21 were studied by reverse transcriptase PCR. RESULTS: DNA flow cytometric analysis showed that As(2)O(3) induced most of HS-Sultan cells, arrest at G(0)/G(1) phase and a small fraction at G(2)/M phase and apoptosis occurred mainly in S phase. There was no expression of P15 and P16 mRNA in untreated HS-Sultan cells and 1.0 micromol/L As(2)O(3) could make them expressed after exposed 24 or 48 hours respectively. Expression of P12 mRNA was obviously elevated by As(2)O(3) comparing with that of control. CONCLUSION: One of the pharmacological mechanisms of As(2)O(3) is to activate the expression of CDKI P15, P16 and P21, and consequently affect cell proliferation cycle.

Effect of CD40-CD40 ligand interaction on diacylglycerol-protein kinase C signal transduction pathway and intracellular calcium in cultured human monocytes.

AIM: To investigate whether CD40-CD40 ligand interaction can activate diacylglycerol (DAG)-protein kinase C (PKC) signaling pathway and intracellular free calcium ([Ca2+]i) in cultured human peripheral blood monocytes (PBMC). METHODS: The DAG levels in PBMC were studied with radio-enzymatic assay. Quantitative measurements of (32)P-phosphatidic acid were performed by thin-layer chromatography and autoradiography. The activity of PKC and [Ca2+]i induced by CD40 ligand (CD40L) in PBMC were measured by its ability to transfer phosphate from [gamma-32P]ATP to lysine-rich histone and flow cytometric analysis loading with the Ca2+ dye Fluo-3/Am, respectively. RESULTS: The DAG levels in PBMC were increased by CD40L in a dose-dependent, biphasic manner. The early phase was rapid and transient, peaking at 20 s; the late phase reached the maximal level at 10 min and then decayed slowly. CD40L increased the PKC total activity in a dose-dependent manner with phase peaking at 12 min, then decreased slowly and maintained for at least 20 min. CD40L induced PKC activity translocation from the cytosol to membrane. Moreover, CD40L also induced biphasic [Ca2+]i responses including the rapid initial transient phase and the sustained phase. Removal of extracellular Ca2+ did not inhibit the rapid phase of CD40L-induced rise in [Ca2+]i, but abolished the sustained phase of [Ca2+]i response to CD40L. Anti-CD40 monoclone antibody 10 mg/L significantly suppressed CD40L-induced DAG-PKC signal transduction pathway activation and [Ca2+]i changes in PBMC. CONCLUSION: CD40-CD40 ligand interaction induced a robust stimulation of the DAG-PKC pathway and calcium mobilization from intracellular pool in PBMC.