ABSTRACT
Rabbit anti-asialo-GM1 (ASGM1) serum or polyclonal antibodies can eliminate mouse splenic natural killer (NK) cell activity in vitro and in vivo. We developed rabbit monoclonal antibodies (mAbs) against ASGM1 using a single-cell analysis and isolation system. Five mAbs (GA109, GA115, GA116, GA131, and GA134) that were reactive to ASGM1 were isolated from the spleen lymphocytes of rabbits immunized with ASGM1. Enzyme-linked immunosorbent assay and thin-layer chromatography immunostaining results showed that the mAbs strongly reacted with ASGM1. Two mAbs (GA116 and GA134) reacted exclusively with ASGM1, whereas three mAbs (GA109, GA115, and GA131) showed slight or considerable cross-reactivity with GM1. The administration of the mAbs (4-20 µg) to BALB/c mice completely abolished NK cell activity in vivo. The anti-ASGM1 rabbit mAbs obtained in this study may provide a useful and reproducible tool for various future studies, such as depleting NK cell activity to enhance xenograft engraftment in mouse models.
Subject(s)
G(M1) Ganglioside , Killer Cells, Natural , Humans , Mice , Animals , Antibodies, Monoclonal , Mice, Inbred BALB C , Enzyme-Linked Immunosorbent Assay , Glycosphingolipids , Mice, Inbred C57BLABSTRACT
BACKGROUND: Hepatitis C virus (HCV) is one of the major causes of liver cancer. The single nucleotide polymorphisms within the IFNL3 gene, which encodes interferon (IFN)-λ(3), are strongly associated with the response to pegylated IFN-α (PEG-IFN-α) plus ribavirin (RBV) therapy in chronic hepatitis C (C-CH) patients. However, the roles of IFN-λ(3) in chronic HCV infection are still elusive. In this study, we aimed to identify clinical and immunological factors influencing IFN-λ(3) and evaluated whether serum IFN-λ(3) levels are involved or not involved in the response to PEG-IFN-α plus RBV therapy. METHODS: We enrolled 119 C-CH patients with HCV genotype 1 infection who underwent 48 weeks of PEG-IFN-α plus RBV therapy. As controls, 23 healthy subjects and 56 patients with non-HCV viral hepatitis were examined. Serum IFN-λ(3) was quantified by chemiluminescence enzyme immunoassay, and 27 cytokines or chemokines were assayed by the multiplexed BioPlex system. RESULTS: Serum IFN-λ(3) levels were higher in C-CH patients or acute hepatitis E patients than in healthy volunteers. Such levels did not differ between the IFNL3 genotypes. In C-CH patients, serum IFN-λ(3) was positively correlated with aspartate aminotransferase, alanine aminotransferase, α-fetoprotein, histological activity, fibrosis index, IFN-γ-inducible protein 10, and platelet-derived growth factor. Multivariate analysis showed that IFNL3 single nucleotide polymorphisms, fibrosis score, and macrophage inflammatory protein 1α were involved in the sustained viral clearance in PEG-IFN-α plus RBV therapy; however, serum IFN-λ(3) levels were not involved. CONCLUSION: Serum IFN-λ(3) levels are increased in C-CH patients regardless of the IFNL3 genotype. IFN-λ(3) is a biomarker reflecting the activity and fibrosis of liver disease, but is not correlated with the responsiveness to PEG-IFN-α plus RBV therapy.
Subject(s)
Hepatitis C, Chronic/blood , Inflammation Mediators/blood , Interleukins/blood , Liver Cirrhosis/blood , Adult , Aged , Antiviral Agents/therapeutic use , Biomarkers/blood , Case-Control Studies , Drug Therapy, Combination , Female , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/drug therapy , Humans , Interferon-alpha/therapeutic use , Interferons , Interleukins/genetics , Liver Cirrhosis/virology , Male , Middle Aged , Polyethylene Glycols/therapeutic use , Polymorphism, Single Nucleotide , Recombinant Proteins/therapeutic use , Ribavirin/therapeutic useABSTRACT
BACKGROUND: Genetic variation around interleukin-28B (IL28B), encoding IFN-λ3, predict non-responders to pegylated interferon-α/ribavirin (Peg-IFN/RBV) therapy in chronic hepatitis C (CHC). However, it remains unclear the expression and the role of IL28B itself. The aim of this study is to develop easy and useful methods for the prediction of treatment outcomes. METHODS: The mRNA and protein levels of IFN-λ3 induced by ex vivo stimulation of peripheral blood mononuclear cells (PBMC) or magnetically selected dendritic cells (DCs) with toll-like receptor agonists (TLR3; poly I:C, TLR7; R-837) were measured by the quantitative real-time polymerase chain reaction and our newly developed chemiluminescence enzyme immunoassays, respectively, and compared with the clinical data. RESULTS: We found that BDCA-4(+) plasmacytoid and BDCA-3(+) myeloid DCs were the main producers of IFN-λs when stimulated with R-837 and poly I:C, respectively. Detectable levels of IFN-λs were inducible even in a small amount of PBMC, and IFN-λ3 was more robustly up-regulated by R-837 in PBMC of CHC patients with favorable genotype for the response to Peg-IFN/RBV (TT in rs8099917) than those with TG/GG. Importantly, the protein levels of IFN-λ3 induced by R-837 clearly differentiated the response to Peg-IFN/RBV treatment (p = 1.0 × 10(-10)), including cases that IL28B genotyping failed to predict the treatment response. The measurement of IFN-λ3 protein more accurately predicted treatment efficacies (95.7 %) than that of IL28B genotyping (65.2 %). CONCLUSIONS: Genetic variations around IL28B basically affect IFN-λ3 production, but different amounts of IFN-λ3 protein determines the outcomes of Peg-IFN/RBV treatment. This study, for the first time, presents compelling evidence that IL28B confer a functional phenotype.
Subject(s)
Hepatitis C, Chronic/drug therapy , Interferon-alpha/therapeutic use , Interleukins/biosynthesis , Ribavirin/therapeutic use , Toll-Like Receptor 7/agonists , Aged , Aminoquinolines/pharmacology , Antiviral Agents/therapeutic use , Cells, Cultured , Dendritic Cells/metabolism , Drug Therapy, Combination , Female , Genotype , Hepatitis C, Chronic/blood , Hepatitis C, Chronic/genetics , Humans , Imiquimod , Interferons , Interleukins/blood , Interleukins/genetics , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Polymorphism, Single Nucleotide , Prognosis , RNA, Messenger/genetics , Treatment OutcomeABSTRACT
AIM: Single nucleotide polymorphisms (SNP) around interferon (IFN)-λ3 have been associated with the response to pegylated IFN-α treatment for chronic hepatitis C. Specific quantification methods for IFN-λ3 are required to facilitate clinical and basic study. METHODS: Gene-specific primers and probes for IFN-λ1, 2 and 3 were designed for real-time detection PCR (RTD-PCR). Dynamic range and specificity were examined using specific cDNA clones. Total RNA from hematopoietic and hepatocellular carcinoma cell lines was prepared for RTD-PCR. Monoclonal antibodies were developed for the IFN-λ3-specific immunoassays. The immunoassays were assessed by measuring IFN-λ3 in serum and plasma. RESULTS: The RTD-PCR had a broad detection range (10-10(7) copies/assay) with high specificity (â¼10(7) -fold specificity). Distinct expression profiles were observed in several cell lines. Hematopoietic cell lines expressed high levels of IFN-λ compared with hepatocellular carcinoma cells, and Sendai virus infection induced strong expression of IFN-λ. The developed chemiluminescence enzyme immunoassays (CLEIA) detected 0.1 pg/mL of IFN-λ3 and showed a wide detection range of 0.1-10 000 pg/mL with little or no cross-reactivity to IFN-λ1 or IFN-λ2. IFN-λ3 could be detected in all the serum and plasma samples by CLEIA, with median concentrations of 0.92 and 0.86 pg/mL, respectively. CONCLUSION: Our newly developed RTD-PCR and CLEIA assays will be valuable tools for investigating the distribution and functions of IFN-λ3, which is predicted to be a marker for predicting outcome of therapy for hepatitis C or other virus diseases.
ABSTRACT
Beer foam stability is an important characteristic. It has been suggested that isoforms of protein Z, that is, protein Z4 and protein Z7, contribute to beer foam stability. We investigated the relationship between beer foam stability and protein Z4 and protein Z7 using their deficient mutants. As a protein Z4-deficient mutant, cv. Pirkka was used. Protein Z7 deficiency was screened in 1564 barley accessions in the world collection of Okayama University, Japan. The barley samples from normal, protein Z4-deficient, protein Z7-deficient, and double-deficient were genotyped in F(2) populations and then pooled based on the DNA marker genotypes of protein Z4 and protein Z7. For a brewing trial, F(5) pooled subpopulations were used. After malting and brewing, the foam stability was determined, and the results showed that the levels of foam stability in the four samples were comparable. Two-dimensional gel electrophoresis was used to investigate the proteome in these beer samples. The results showed that low molecular weight proteins, including lipid transfer protein (LTP2), in the deficient mutants were higher than those in the normal sample. Our results suggest that the contribution of protein Z4 and protein Z7 to beer foam stability was not greater than that of other beer proteins.
Subject(s)
Beer/analysis , Hordeum/chemistry , Mutation , Plant Proteins/genetics , Serine Proteinase Inhibitors/genetics , Food Handling/methods , Plant Proteins/physiology , Seeds/chemistry , Serine Proteinase Inhibitors/physiologyABSTRACT
BACKGROUND: S100A12, also known as EN-RAGE (extracellular newly identified receptor for advanced glycation end products binding protein) is a ligand for RAGE, and has been proposed to contribute to the development of atherosclerosis. In this study, we examined the plasma S100A12 concentration in patients with ESRD and undergoing hemodialysis (HD) and evaluated the relation between S100A12 level and carotid intimal media thickness (IMT) by ultrasound. METHODS: We measured plasma S100A12 concentration in 72 HD patients and 42 control subjects. IMT of the carotid artery was measured by high-resolution B-mode ultrasonography in 46 HD patients. RESULTS: The mean plasma S100A12 level was 2.3-fold higher in HD patients than in control subjects (25.0 +/- 2.32 vs. 10.7 +/- 0.97 ng/ml, p < 0.001). Stepwise multiple regression analysis identified circulating white blood cell count as a positive independent determinant and total cholesterol and serum albumin levels as negative independent determinants of plasma S100A12 concentration. The maximum IMT was positively correlated with plasma S100A12 level. Stepwise multiple regression analysis also identified plasma S100A12 as a significant independent determinant of the maximum IMT. CONCLUSION: These findings suggest that S100A12 protein is involved in the acceleration of atherosclerosis in HD patients.
Subject(s)
Atherosclerosis/blood , Atherosclerosis/therapy , Renal Dialysis , S100 Proteins/blood , Aged , Carotid Arteries/diagnostic imaging , Case-Control Studies , Cholesterol/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Female , Glycation End Products, Advanced/metabolism , Humans , Ligands , Male , Middle Aged , S100A12 Protein , Ultrasonography/methodsABSTRACT
S100A12 is a ligand for the receptor for advanced glycation end products. It has been shown that S100A12 induces expression of adhesion molecules, and mediates activation and migration of monocytes/macrophages. Circulating S100A12 may be involved in chronic inflammation. We previously reported increased S100A12 levels in patients with non-insulin-dependent diabetes mellitus and hemodialysis. A high peritoneal solute transport rate may be associated with encapsulating peritoneal sclerosis and mortality. We measured plasma S100A12 levels in peritoneal dialysis patients and evaluated a possible relation between the increased plasma S100A12 levels in peritoneal dialysis patients and the high peritoneal solute transport rate. Subjects included 36 patients (mean age +/- SE, 46.0 +/- 12.0 years) with no apparent infection and no malignancy who had been undergoing peritoneal dialysis for 36.5 +/- 3.9 months. We developed an enzyme-linked immunosorbent assay system to measure plasma S100A12 levels. A peritoneal equilibrium test was performed and subjects were categorized as high and high-average (H) (n = 14) or low and low-average (L) (n = 22) transporters. Plasma S100A12 concentrations were significantly higher in peritoneal dialysis patients (21.6 +/- 3.0 ng/mL) than in control subjects (n = 42; 10.8 +/- 1.0 ng/mL). Plasma S100A12 concentrations were also higher in the H group (28.2 +/- 6.1 ng/mL) than in the L group (14.2 +/- 2.0 ng/mL). These results suggest that S100A12 may be a sensitive marker of subclinical inflammation and that an increased S100A12 level may be related to the high peritoneal solute transport rate.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Inflammation/physiopathology , Peritoneal Dialysis, Continuous Ambulatory , S100 Proteins/blood , Adult , Biological Transport , Chronic Disease , Female , Humans , Male , Middle Aged , Peritoneal Diseases/physiopathology , S100A12 Protein , Sclerosis/physiopathologyABSTRACT
BACKGROUND: Changes in the serum hepatitis B virus (HBV) RNA level during lamivudine therapy were compared to those in the serum HBV DNA and HBV core-related antigen (HBVcrAg) levels in 24 patients with chronic hepatitis B. METHODS: For measurement of HBV RNA, total nucleic acid was extracted from serum samples and treated with RNase-free DNase I. After cDNA synthesis from extracted RNA, HBV RNA was measured by real-time detection polymerase chain reaction. RESULTS: The peak fraction of HBV RNA in serum samples was consistent with peak fractions of HBV DNA and HBV core protein in a sucrose gradient analysis, indicating that HBV RNA was incorporated into virus particles. All levels of HBV DNA, HBV RNA, and HBVcrAg decreased gradually during lamivudine therapy (P < 0.001 for all). The amount of decrease from the start of lamivudine therapy was significantly higher for HBV DNA than for HBV RNA or HBVcrAg during 6 months of lamivudine therapy (P < 0.001 for all). However, a similar difference was not seen between HBV RNA and HBVcrAg levels during that period. The HBV RNA level was significantly correlated (P < 0.001 for all) with levels of HBV DNA and HBVcrAg both at the beginning and 2 months after the start of lamivudine therapy. CONCLUSIONS: HBV RNA is detectable in serum in a form indicating incorporation into virus particles, and its serum level might serve as a new viral marker with a significance different from that of HBV DNA in lamivudine therapy.
Subject(s)
Hepatitis B virus/isolation & purification , Hepatitis B, Chronic/blood , Hepatitis B, Chronic/drug therapy , Lamivudine/therapeutic use , RNA, Viral/blood , Reverse Transcriptase Inhibitors/therapeutic use , Adult , Aged , Centrifugation, Density Gradient , DNA, Viral/blood , Female , Hepatitis B Core Antigens/blood , Humans , Male , Middle Aged , Viral LoadABSTRACT
OBJECTIVE: The clinical usefulness of hepatitis B virus core-related antigen (HBVcrAg) assay was compared with that of HBV DNA assay in predicting the occurrence of lamivudine resistance in patients with chronic hepatitis B. PATIENTS: Of a total of 81 patients who were treated with lamivudine, 25 (31%) developed lamivudine resistance during a median follow-up period of 19.3 months. RESULTS: The pretreatment positive rate of HBe antigen, or pretreatment levels of HBVcrAg or HBV DNA did not differ between patients with and without lamivudine resistance. Levels of both HBVcrAg and HBV DNA decreased after the initiation of lamivudine administration; however, the level of HBVcrAg decreased significantly more slowly than that of HBV DNA. The occurrence of lamivudine resistance was significantly less frequent in the 56 patients whose HBV DNA level was less than 2.6 log copy/ml at 6 months of treatment than in the remaining 25 patients. The cumulative rate of lamivudine resistance was as high as 70% within 2 years in the latter group, while it was only 28% in the former group. Lamivudine resistance did not occur during the follow-up period in the 19 patients whose HBVcrAg level was less than 4.6 log U/ml at 6 months of treatment, while it did occur in 50% of the remaining patients within 2 years. CONCLUSION: These results suggest that measurement of HBV DNA is valuable for identifying patients who are at high risk of developing lamivudine resistance, and that, conversely, measurement of HBVcrAg is valuable for identifying those who are at low risk of lamivudine resistance.
Subject(s)
Drug Resistance , Hepatitis B e Antigens/immunology , Hepatitis B virus/immunology , Hepatitis B/drug therapy , Lamivudine/therapeutic use , Viral Core Proteins/immunology , Adult , Aged , Cohort Studies , DNA, Viral/analysis , Female , Hepatitis B/immunology , Hepatitis B/pathology , Hepatitis B e Antigens/analysis , Humans , Liver Function Tests , Male , Middle Aged , Predictive Value of Tests , Probability , Prognosis , Retrospective Studies , Risk Assessment , Sensitivity and Specificity , Severity of Illness Index , Statistics, Nonparametric , Viral Core Proteins/drug effectsABSTRACT
The characteristic differences between patients with and without loss of hepatitis B virus (HBV) DNA after achieving hepatitis B e antigen seroconversion were analyzed by comparing changes in HBV DNA and HBV core-related antigen levels during a period from 3 years before to 3 years after the seroconversion. Of the 24 seroconverters, 6 (inactive replication group) showed continuous loss of HBV DNA in serum after the seroconversion and the remaining 18 did not lose HBV DNA (active replication group). The HBV DNA level was similar between the two groups, while the HBV core-related antigen level was significantly lower in the active replication group than in the inactive replication group before the seroconversion. The levels of both HBV DNA and HBV core-related antigen decreased remarkably around the time of seroconversion in the inactive replication group, while these levels did not change or decreased slightly in the active replication group. After the seroconversion, the HBV DNA level was significantly higher in the active replication group than in the inactive replication group, while the HBV core-related antigen level was similarly low between the two groups. Because the serum level of HBV core-related antigen mainly reflects that of HBe antigen, the low level of HBV core-related antigen seen after seroconversion in both groups might have contributed to the occurrence of seroconversion. The precore and core promoter mutations which cause diminished excretion of hepatitis B e antigen were significantly more frequent in the active replication group than in the inactive replication group. It was therefore considered that the seroconversion was caused mainly by a decrease in viral replication in the inactive replication group, and mainly by a decrease in HBe antigen production in the active replication group.
Subject(s)
DNA, Viral/blood , Hepatitis B e Antigens/blood , Hepatitis B virus/genetics , Hepatitis B/virology , Adult , Aged , Female , Hepatitis B Core Antigens/blood , Humans , Male , Middle Aged , Mutation , Promoter Regions, GeneticABSTRACT
BACKGROUND AND AIMS: Hepatitis B virus (HBV) core-related antigen (HBcrAg) and HBV core antigen (HBcAg) assays were developed for the measurement of serum HBV load. The aim of this study was to assess the clinical utility of these assays in Chinese patients with chronic genotype B and C HBV infection. METHODS: One hundred and ninety-three chronic hepatitis B patients were enrolled. Serum HBcrAg and HBcAg were measured by chemiluminescence enzyme immunoassay, and HBV-DNA was measured by using a sensitive polymerase chain reaction assay. The data were analyzed in patients with HBV genotype B (HBV/B) and genotype C (HBV/C). The HBcrAg/HBcAg ratio was calculated and compared between patients with and without hepatitis B e antigen (HBeAg). RESULTS: The concentrations of HBcrAg and HBcAg showed significant positive correlation with the HBV-DNA concentration in both HBV/B (r = 0.79, P < 0.001, and r = 0.77, P < 0.001, respectively) and HBV/C (r = 0.87, P < 0.001, and r = 0.90, P < 0.001, respectively). The cut-off for a positive HBcAg corresponded to approximately 4.5 log copies/mL, and that for a positive HBcrAg result corresponded to 3-4 log copies/mL. The HBcrAg/HBcAg ratio was higher in patients with HBeAg than in those without HBeAg. CONCLUSIONS: The HBcrAg assay and HBcAg assay are clinically useful in viral quantitation of HBV/B and HBV/C. A combination of these assays would be a valuable tool for analyzing the clinical status of HBV infection.
Subject(s)
Asian People , Hepatitis B Core Antigens/blood , Hepatitis B virus/genetics , Hepatitis B, Chronic/immunology , Hepatitis B, Chronic/virology , Adolescent , Adult , Aged , Child , DNA, Viral/blood , Female , Genotype , Hepatitis B, Chronic/ethnology , Humans , Immunoenzyme Techniques , Luminescent Measurements , Male , Middle Aged , Osmolar ConcentrationABSTRACT
Four proteomic biomarkers (human neutrophil peptide 1 [HNP1], HNP2 [defensins], calgranulin C [Cal-C], and Cal-A) characterize the fingerprint of intra-amniotic inflammation (IAI). We compared proteomic technology using surfaced-enhanced laser desorption-ionization-time of flight (SELDI-TOF) mass spectrometry to enzyme-linked immunosorbent assay (ELISA) for detection of these biomarkers. Amniocentesis was performed on 48 women enrolled in two groups: those with intact membranes (n = 27; gestational age [GA], 26.0 +/- 0.8 weeks) and those with preterm premature rupture of the membranes (PPROM; n = 21; GA, 28.4 +/- 0.9 weeks). Paired abdominal amniotic fluids (aAFs)-vaginal AFs (vAFs) were analyzed in PPROM women. Quantitative aspects of HNP1-3, Cal-C, Cal-A, and calprotectin (a complex of Cal-A with Cal-B) were assessed by ELISA. SELDI-TOF mass spectrometry tracings from 16/48 (33.3%) aAFs and 13/17 (88.2%) vAFs were consistent with IAI (three or four biomarkers present). IAI (by SELDI-TOF mass spectrometry) was associated with increased HNP1-3 and Cal-C measured by ELISA. However, immunoassays detected Cal-A in only 4 of the AFs even though its specific SELDI-TOF mass spectrometry peak was identified in 19/48 AFs. Calprotectin immunoreactivity was decreased in AFs retrieved from women with IAI (P = 0.01). In conclusion, IAI is associated with increased HNP1-3 levels. In the absence of isoform-specific ELISAs, mass spectrometry remains the only way to discriminate the HNP biomarker isoforms. Monomeric Cal-A is not reliably estimated by specific ELISA as it binds to Cal-B to form the calprotectin complex. Cal-C was reliably measured by SELDI-TOF mass spectrometry or specific ELISA.
Subject(s)
Amniotic Fluid/metabolism , Calgranulin B/analysis , Chorioamnionitis/metabolism , Proteomics , Biomarkers/analysis , Defensins/analysis , Female , Humans , Mass Spectrometry , Pregnancy , Proteomics/methods , Sensitivity and SpecificityABSTRACT
DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.
Subject(s)
Arginine/chemistry , DNA, Viral , Hepatitis B virus/genetics , Antibodies, Monoclonal/chemistry , Antigens, Viral/chemistry , Blotting, Western , Capsid , Centrifugation, Density Gradient , Chromatography, Gel , Cytoplasm/metabolism , DNA, Viral/metabolism , Dose-Response Relationship, Drug , Female , Genome, Viral , Hepatitis B/blood , Hepatitis B/virology , Humans , Immunoenzyme Techniques , Immunoprecipitation , Male , Mass Spectrometry , Microscopy, Electron , Models, Genetic , Protein Structure, Tertiary , Protein Transport , Recombinant Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sucrose/pharmacology , Viral Core Proteins/chemistryABSTRACT
S100A12, also called EN-RAGE (extracellular newly identified receptor for advanced glycation end products binding protein) or calcium-binding protein in amniotic fluid-1, is a ligand for RAGE. It has been shown that S100A12 induces adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in the vascular endothelial cell and mediates migration and activation of monocytes/macrophages through RAGE binding and that infusion of lipopolysaccharide into mice causes time-dependent increase of S100A12 in the plasma. Therefore, circulating S100A12 protein may be involved in chronic inflammation in the atherosclerotic lesion. In this study, we developed an ELISA system that uses specific monoclonal antibodies against recombinant human S100A12 to measure plasma S100A12 levels in patients with diabetes. On using our S100A12 ELISA system, the coefficients of variation of intra- and interassay were less than 4 and 9%, respectively. The analytical lower detection limit was 0.2 ng/ml. When plasma S100A12 levels were measured by this system, the concentrations were more than twice as high in the patients with diabetes, compared with those without. Using univariate analysis in all subjects, plasma S100A12 concentrations correlated with hemoglobin A1c, fasting glucose, high-sensitivity C-reactive protein and white blood cell count. Stepwise multiple regression analyses, however, revealed that only white blood cell count and hemoglobin A1c remained significant independent determinants of plasma S100A12 concentration. These results suggest that plasma S100A12 protein levels are regulated by factors related to subclinical inflammation and glucose control in patients with type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 2/blood , S100 Proteins/blood , Enzyme-Linked Immunosorbent Assay , Female , Humans , Leukocyte Count , Male , S100A12 ProteinABSTRACT
EN-RAGE is a ligand for the receptor for advanced glycation end products (RAGE) and may be involved in the development of diabetic macro- and micro-angiopathy. This study is designed to investigate the regulation of EN-RAGE gene expression in human macrophages. The amounts of EN-RAGE mRNA were measured in cultured human THP-1 macrophages after treatment with various stimuli known to modulate atherosclerosis. First, interleukin-6 (IL-6), a proinflammatory cytokine, increased the level of EN-RAGE mRNA by approximately 2-fold in a time- and a dose-dependent fashion. EN-RAGE protein was detected in the cultured medium and increased significantly by the addition of IL-6. The induction was abolished by pretreatment with the JAK kinase inhibitor and cycloheximide, but not with the MEK kinase inhibitor. Second, pioglitazone (PIO), a thiazolidinedione, decreased the level of EN-RAGE mRNA by approximately 25% of the basal in a time- and a dose-dependent fashion. Pioglitazone also inhibited the induction of EN-RAGE mRNA by IL-6. These results indicate the production of EN-RAGE is induced by IL-6 through de novo protein synthesis via the JAK-STAT kinase pathway and inhibited by the activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) in human macrophages.
Subject(s)
Gene Expression Regulation , Interleukin-6/metabolism , Macrophages/metabolism , S100 Proteins/genetics , Signal Transduction , Analysis of Variance , Base Sequence , Cell Line, Tumor , Down-Regulation/drug effects , Enzyme Activation/drug effects , Humans , Interleukin-6/pharmacology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Macrophages/cytology , Pioglitazone , RNA, Messenger/analysis , Receptors, Cytoplasmic and Nuclear/agonists , S100 Proteins/biosynthesis , S100A12 Protein , Thiazolidinediones/pharmacology , Transcription Factors/agonists , Tumor Cells, CulturedABSTRACT
A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 microg/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative, n = 160) and anti-hepatitis C virus-positive (n = 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r = 0.946, n = 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed.
Subject(s)
DNA, Viral/analysis , Hepatitis B Core Antigens/analysis , Hepatitis B virus/isolation & purification , Immunoenzyme Techniques/methods , Antibodies, Monoclonal , Antiviral Agents/therapeutic use , Centrifugation, Density Gradient , DNA, Viral/blood , Hepatitis B Antibodies , Hepatitis B, Chronic/diagnosis , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/virology , Humans , Immunoenzyme Techniques/statistics & numerical data , Lamivudine/therapeutic use , Reproducibility of ResultsABSTRACT
A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10(3) U/ml, all healthy control (HBsAg/HBV-DNA negative; n = 108) and anti-HCV antibody-positive (n = 59) sera were identified as negative. The assay showed a detection limit of 4 x 10(2) U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r = 0.91, n = 29) and in-house real-time detection-PCR (r = 0.93, n = 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.