Laboratory characterizations on 2007 cases of monoclonal gammopathies in East China.

Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulin in patients with or without evidence of multiple myeloma (MM), macroglobulinemia, amyloidosis (AL), or a related plasma cell proliferative disorder. This study aims to evaluate laboratory diagnostic characters of monoclonal gammopathies and investigates the correlation between monoclonal gammopathies and transforming growth factor beta1 (TGFbeta1). Immunofixation electrophoresis (IFE), serum protein electrophoresis (SPE), nephelometry and urine light chain ELISA were used for laboratory identification of monoclonal immunoglobulins. Plasma TGFbeta1 was detected with double-antibodies ELISA. Lightcycler was used for single nucleotide polymorphism (SNP) analysis. Totally 2,007 cases of monoclonal immunoglobulin (M protein) were identified in 10,682 samples. The isotypes of M protein were IgG type 47.1%, IgA 23.0%, IgM 8.7%, IgD 5.3%, free light chain kappa 6.1%, lambda 9.8%. In reference to IFE, the coherency of diagnosis was serum light chain ratio (kappa/lambda ) 94.4%, quantitation of Igs 83%, light chain quantitation 80.9%, and urine light chain ratio (kappa/lambda) 58.0%. Plasma TGFbeta1 was elevated significantly compared to normal control. The allelic frequency of codon 10 (C>T) was neither associated with the existence of the M protein nor with the M protein isotype. Monoclonal gammopathies can be identified with the combination of IFE, SPE, Igs quantitation and urine light chain determination. Although TGFbeta1, an important cytokine in immune regulation, was elevated in monoclonal gammopathies, the SNPs in coding region of TGFbeta1 gene did not confer susceptibility to the development of monoclonal gammopathies in this study.

Correlation of the expression levels of BLyS and its receptors mRNA in patients with systemic lupus erythematosus.

OBJECTIVES: To measure the levels of B-lymphocyte stimulator (BLyS) and its receptors mRNA expression in peripheral blood mononuclear cells (PBMCs) using quantitative real-time polymerase chain reaction (PCR) method and to investigate the relationship between BLyS and its receptors mRNA expression and systemic lupus erythematosus (SLE). DESIGN AND METHODS: Specific primers and TaqMan probe were designed, and real-time PCR was performed. According to the standard curve of plasmids DNA, the levels of BLyS and its receptors mRNA expression in 37 patients with SLE and 30 healthy subjects were determined. The ratio of the expression levels of BLyS mRNA to that of beta2-microglobulin (beta2M) mRNA and the ratio of the expression levels of BLyS receptors mRNA to that of beta2M mRNA were regarded as indicator for the levels of BLyS and its receptors mRNA expression. RESULTS: The expression of BLyS, TACI and BAFF-R mRNA in PBMCs from patients with SLE was significantly elevated compared to healthy controls (P<0.001 for each), and active patients with SLE group had higher mRNA expression than patients with SLE inactive group (P<0.001 for each). The patients with elevated anti-double-stranded DNA (anti-dsDNA) antibody titers had enhanced BLyS, TACI and BAFF-R mRNA expression (P<0.05 for BLyS; and P<0.01 for TACI and BAFF-R). CONCLUSION: The BLyS, TACI and BAFF-R mRNA expression levels were significantly elevated in patients with SLE, which suggests that BLyS, TACI and BAFF-R might be involved in the pathogenesis, and that mRNA expression levels might serve as a biomarker of disease activity.

[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.

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.

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.

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.

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.

Construction and expression of a humanized M2 autoantigen trimer and its application in the diagnosis of primary biliary cirrhosis.

AIM: To construct and express a humanized M(2) autoantigen trimer designated as BPO and to apply it in the diagnosis of primary biliary cirrhosis (PBC). METHODS: cDNA fragments encoding M(2)-reactive epitopes of pyruvate dehydrogenase complex E(2) (PDC-E(2)), branched chain 2-oxo-acid dehydrogenase complex E(2) (BCOADC-E(2)) and 2-oxo-glutarate dehydrogenase complex E(2) (OGDC-E(2)) were amplified with PCR using total RNA extracted from human peripheral mononuclear blood cells. The fragments were cloned into the plasmid vector pQE-30 and then transferred into E. coli M15 (pREP4) for expression, which was induced by isopropylthio-beta-D-galactoside. The expressed recombinant BPO protein was demonstrated by SDS-PAGE, Western-blotting and Immunoabsorption test, its antigenic reactivity and specificity were identified with seven M(2)-positive sera confirmed at Euroimmun Research Center (Germany). Using the purified BPO, M(2) antibodies in sera from patients with PBC and other liver related diseases were detected with ELISA. RESULTS: The expressed BPO was observed with both antigenic reactivity and specificity of M(2) autoantigens. The determination of M(2) antibodies by BPO with ELISA was more sensitive than using the Euroimmun's kit with the coefficients of variation less than 10 % in both interassay and intraassay. With the newly established method, M(2) antibodies were found in 100 % (20/20) of patients with PBC. Six cases of liver disease with unknown etiology and 1 patient with drug induced liver injury had detectable levels of serum M(2) antibodies. There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M(2)-antibody positive. CONCLUSION: Compared with the routine immunofluorescence assay and commercially available assay kit using porcine heart mitochondrial protein as the antigen, the detection system established in the present study shows higher sensitivity and specificity and may be used as a powerful tool for the diagnosis of PBC.

Clinical implications of increased expression of CD40L in patients with acute coronary syndromes.

OBJECTIVE: To investigate clinical implications of expression of CD40L in monocytes and changes in serum soluble CD40L in patients with acute coronary syndromes (ACS). METHODS: Sixteen control and 56 patients, including 24 with stable angina (SA), 20 with unstable angina (UA) and 12 with acute myocardial infarction (AMI) enrolled in this study. Expression of CD40L in monocytes was analyzed by flow cytometry and sCD40L levels were measured by ELISA. RESULTS: Expression of CD40L in monocytes and serum levels of sCD40L in UA and AMI patients were higher than in SA patients and controls. In patients with AMI, sCD40L levels showed no significant increase when compared to patients with UA, while AMI patients had a peak level of sCD40L at 24 hours after AMI. PTCA induced a marked rise in sCD40L levels in all patients, while CD40L expression in monocytes showed no difference between patients with PTCA, before and after. CONCLUSION: Enhanced level of serum sCD40L may be a reliable prognostic indicator for ACS and may represent a marker of coronary disease activity.

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.

[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.

[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.

[Clone of M2 autoantigens from human sources and evaluation of measurement of M2 autoantibodies in diagnosis of primary biliary cirrhosis].

OBJECTIVE: To evaluate the value of measurement of M2 autoantibodies in diagnosis of primary biliary cirrhosis (PBS). METHODS: M2 autoantibodies were detected by ELISA combined with western-blot. RESULTS: Thirty-two cases presented positive M2 autoantibodies, including 20 cases of PBC, 9 liver diseases of unknown etiology, 1 autoimmune hepatitis and 1 drug-induced liver injury. CONCLUSIONS: M2 autoantibodies show good sensitivity and specificity to diagnosis of PBC. It should be used as a powerful and specific maker for the diagnosis of PBC

[Dynamic expression of tenascin in rat liver during liver fibrogenesis induced by CCl(4)].

OBJECTIVE: To study the expression of tenascin in normal and fibrotic rat liver. METHODS: Liver fibrosis induced in rat with CCl(4) were divided into three stages: the stage of hepatic injury (4 weeks), early stage of hepatic fibrosis (8 weeks) and later stage of hepatic fibrosis (12 weeks). Tenascin expression in liver tissue was observed by immunohistochemical method and in situ hybridization using digoxigenin-labelled DNA probe. RESULTS: In normal rat liver there was a weak staining for tenascin. In both liver injury stage and early stage of hepatic fibrosis, both mRNA signal and immunostaining for tenascin were significantly increased as compared to that in normal liver. In later stage of hepatic fibrosis, mRNA signal and immunostaining for tenascin were decreased compared with that in early stage of hepatic fibrosis. The cellular source of tenascin in liver mainly restricted in mesenchymal cells. CONCLUSIONS: Tenascin is a component of the extracellular matrix of liver tissue. Plays a role in early matrix organization during liver fibrogenesis.

Correlation of expression levels of BLyS and its receptors with multiple myeloma.

OBJECTIVES: To measure the expression levels of B-lymphocyte stimulator (BLyS) and its receptors in multiple myeloma (MM), and to investigate the relationship between them. DESIGN AND METHODS: BLyS and its receptors mRNA levels from peripheral blood mononuclear cells (PBMCs) of 31 MM patients and 30 healthy controls were determined by real-time PCR. The results were presented as the ratios of target genes, to beta(2)-microglobulin (beta(2)M), mRNA. BLyS serum level was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The circulating levels of BLyS in 31 MM patients were significantly elevated and correlated with the levels of Lactate dehydrogenase (LDH). More importantly, 93.55% (29/31) and 90.32% (28/31) of these MM patients had significantly higher expression levels of BLyS and BCMA mRNA respectively, which are correlated with the levels of LDH, while 64.52% (20/31) of these MM patients had significantly lower expression levels of TACI mRNA. CONCLUSION: BLyS protein concentration and BLyS, TACI and BAFF-R mRNA expression levels were significantly elevated in patients with MM, suggesting that BLyS, TACI and BAFF-R may be involved in the pathogenesis of MM, and that expression levels could serve as a biomarker of prognosis.