Xiao-Meng Zhang

Identification of Cerebral Infarction-Specific Antibody Markers from Autoantibodies Detected in Patients with Systemic Lupus Erythematosus

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease which may be caused by development of the autoantibodies. On the other hand, SLE is a high-risk group of atherosclerosis, so it is possible that some of autoantibodies in SLE are the result of atherosclerosis-related diseases such as cerebral infarction (CI), cardiovascular disease (CVD) and diabetes mellitus (DM). Methods: The initial screening of autoantibodies was performed using the protein array method. AlphaLISA was used to analyze the serum antibody levels using synthetic polypeptides as antigens. Results: After the initial screening using protein array, we identified 67 antigens that were recognized by IgG antibodies in sera of patients with SLE. In the second screening, 170 peptides derived from amino acid sequences of 67 antigens were synthesized and used as antigens for analysis of serum antibody levels by AlphaLISA. The antibody levels for ten peptides were significantly higher in the sera of patients with SLE than in those of healthy donors. Further AlphaLISA analysis of sera of patients with CI, CVD or DM revealed that the serum antibody levels for four peptides derived from SOSTDC1, CTNND1, CLDND1 and CCNG2 were elevated in patients as compared to those of healthy donors. Conclusions: Serum antibody levels against peptide antigens of SOSTDC1, CTNND1, CLDND1 and CCNG2 are useful markers for diagnosis of the progression of CI, CVD and/or DM.

Association of serum levels of antibodies against MMP1, CBX1, and CBX5 with transient ischemic attack and cerebral infarction

Transient ischemic attack (TIA) is a predictor for cerebral infarction (CI), and early diagnosis of TIA is extremely important for the prevention of CI. We set out to identify novel antibody biomarkers for TIA and CI, and detected matrix metalloproteinase 1 (MMP1), chromobox homolog 1 (CBX1), and chromobox homolog 5 (CBX5) as candidate antigens using serological identification of antigens by recombinant cDNA expression cloning (SEREX) and Western blotting to confirm the presence of serum antibodies against the antigens. Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) revealed that serum antibody levels were significantly higher in patients with TIA or acute-phase CI (aCI) compared with healthy donors (P < 0.01). Spearman’s correlation analysis and multivariate logistic regression analysis demonstrated that levels of anti-MMP1, anti-CBX1, and anti-CBX5 antibodies were associated with age, cigarette-smoking habits, and blood pressure. Thus, serum levels of antibodies against MMP1, CBX1, and CBX5 could potentially serve as useful tools for diagnosing TIA and predicting the onset of aCI.

Elevation of autoantibody level against PDCD11 in patients with transient ischemic attack

Background Disease specific autoantibodies have been detected in the sera of patients with atherosclerosis-related diseases, such as cerebral infarction, cardiovascular disease. In the present study, we aimed to identify novel autoantibodies responsible for transient ischemic attack (TIA), a prodromal condition for cerebral infarction. Methods To identify candidate antigens, we screened a human aortic endothelial cell cDNA library using sera from 20 patients with TIA. Serum antibody levels were measured using amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in 2 independent patient/healthy donor (HD) cohorts (n = 192 and n = 906 in the second screening and validation cohort, respectively). Results First screening identified 3 candidate antigens. Of these, programmed cell death 11 (PDCD11) was determined to be associated with stroke (p < 0.0001), as evidenced from the second screening using AlphaLISA. The validation cohort revealed significantly higher antibody levels against PDCD11 (PDCD11-Ab levels) in patients with TIA than in HDs. Multivariate logistic regression analysis indicated that the predictive value of PDCD11-Ab levels for TIA [Odds ratio (OR): 2.44, 95% confidence interval (CI): 1.33-4.57, p = 0.0039] was not inferior to other known risk factors for ischemic stroke, including age (OR: 4.97, 95% CI: 2.67–9.48, p < 0.0001); hypertension (OR: 3.21, 95% CI: 1.76–5.86, p = 0.0001); and diabetes (OR: 4.31, 95% CI: 1.74–11.2, p = 0.0015). Conclusion Serum PDCD11-Ab level may serve as a potential biomarker for TIA.

Serum SH3BP5-specific Antibody Level is a Biomarker of Atherosclerosis

Abstract Background: The discovery and development of novel biomarkers that could facilitate early diagnosis and thus prevent the progression of atherosclerosis-related diabetes mellitus (DM), cerebral infarction (CI), and cardiovascular disease (CVD) has garnered much research interest. Notably, recent reports have described a number of highly sensitive antibody markers. In this study, we aimed to identify additional antibody markers that would facilitate screening. Methods: The amplified luminescent proximity homogeneous assay (AlphaLISA) method, which incorporates glutathione- or streptavidin-donor beads and anti-human-IgG-acceptor beads, was used to evaluate serum antibody levels in serum samples. The protein array method was used for the initial screening, and peptide arrays were used to identify epitope sites. Results: The protein array identified SH3 domain-binding protein 5 (SH3BP5) as a target antigen of serum IgG antibodies in the sera of patients with atherosclerosis. We prepared recombinant glutathione S-transferase (GST)- fused SH3BP5 protein. Peptide arrays revealed that the epitope site recognized by serum antibodies is located within amino acids 161–174 of SH3BP5. AlphaLISA revealed significantly higher serum antibody levels against both the SH3BP5 protein and peptide in patients with DM, acute-phase CI, transient ischemic attack, CVD or chronic kidney disease (CKD), than in healthy donors. Furthermore, areas under the receiver operating characteristic curves of these antibodies were higher in patients with CKD and DM than in other patients. Spearman correlation analysis revealed associations between the serum antibody levels against SH3BP5 peptide and artery stenosis, hypertension, and smoking. Conclusions: The serum anti-SH3BP5 antibody marker appears to be useful for estimating the progress of atherosclerosis and may discriminate atherosclerosis associated with hypertension and/or habitual smoking.

Elevated Levels of Autoantibodies against ATP2B4 and BMP-1 in Sera of Patients with Atherosclerosis-related Diseases

Background: Atherosclerosis-related life-style diseases such as cerebral infarction (CI), cardiovascular disease (CVD), diabetes mellitus (DM), and chronic kidney disease (CKD) are a serious problem in the recently aging society. The development of novel and sensitive diagnostic markers is necessary and expected for the early treatment. Methods and Results: Through the first screening by phage expression cloning, we identified ATPase, Ca++ transporting, plasma membrane 4 (ATP2B4) and bone morphogenetic protein 1 (BMP-1) as antigens recognized by IgG antibodies in the sera of patients with atherosclerosis. The presence of autoantibodies against these antigens in serum specimens was confirmed by Western blotting. We then compared serum antibody levels against recombinant ATP2B4 and BMP-1 proteins between healthy donors (HD) and patients with atherosclerotic diseases, such as CI, transient ischemic attack (TIA), CVD, DM, or CKD, by the Alpha (amplified luminescent proximity homogeneous assay)-LISA method. The results revealed that both antibody levels were significantly higher in patients with these diseases than in HD and exhibited most prominent differences in CKD vs. HD. Correlation analysis showed that both antibody levels were well correlated with the degree of artery stenosis, such as maximum intima-media thickness with some different patterns, i.e., anti-ATP2B4 antibody levels were related to hypertension, whereas anti-BMP-1 antibodies were related to smoking habits. Conclusions: The serum antibody levels against ATP2B4 and BMP-1 can be useful diagnostic markers for atherosclerosis and its related diseases caused by hypertension and smoking habits, respectively.

Association of Serum Anti-Prolylcarboxypeptidase Antibody Marker with Atherosclerotic Diseases Accompanied by Hypertension

Background: Atherosclerosis is leading to mortal diseases such as Cerebral Infarction (CI), and Cardiovascular Disease (CVD), and closely related to Diabetes Mellitus (DM) and Chronic Kidney Disease (CKD). Biomarkers are useful but not sufficient for early detection. In the present study, we aimed to identify novel antibody markers for atherosclerosis-related diseases. Methods: The protein array method was used for the initial screening, and a peptide containing a possible epitope domain was used to evaluate serum antibody levels using the Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA) method. Results: The protein array identified prolylcarboxypeptidase (PRCP) as one of the target antigens recognized by IgG antibodies in the sera of patients with atherosclerosis. We then prepared a possible antigenic peptide of amino acids 214-227 of PRCP. Results of AlphaLISA showed significantly higher serum antibody levels against the PRCP peptide in patients with DM, CVD, acute-phase cerebral infarction, transient ischemic attack or CKD, than in healthy donors. Furthermore, areas under the receiver operating characteristic curves of these antibodies were higher in patients with DM or CKD than in other patients. Spearman correlation analysis revealed that the serum anti-PRCP antibody levels were associated with hypertension, artery stenosis, and smoking habit.. Conclusion: The serum anti-PRCP antibody may be useful for early detection of atherosclerosis-related diseases, and may have a pathogenic role in the development of atherosclerosis.

Identification of Serum Anti-GADD34 Antibody as a Common Marker ofDiabetes Mellitus and Parkinson Disease

Background: Growth arrest and DNA-damage-inducible gene 34 (GADD34) has been identified as an antigen by serological identification of antigens by cDNA expression cloning (SEREX) using the sera of patients with atherosclerosis. It is possible that GADD34 is associated with atherosclerosis-related diseases such as diabetes mellitus (DM), acute-phase cerebral infarction (aCI), cardiovascular disease (CVD), and chronic kidney disease (CKD) as well as endoplasmic reticulum stress-related Parkinson disease (PD). Methods: GADD34 protein was bacterially expressed and purified. Amplified luminescent proximity homogeneous assay (AlphaLISA) was used to evaluate serum antibody levels against GADD34 protein in serum samples. Results: AlphaLISA revealed significantly higher serum antibody levels against GADD34 protein in patients with DM, aCI and CVD than those in healthy donors (HDs). The difference in levels between DM and HD was more prominent than that between aCI or CVD and HDs. The anti-GADD34 antibody levels were also elevated in the sera of diabetic CKD patients; thus, the anti-GADD34 antibodies appeared to be the most associated with DM, which is also a risk factor of PD. Anti-GADD34 antibody levels were also higher in patients with PD but not in those with amyotrophic lateral sclerosis as compared with those in HDs. Conclusion: Anti-GADD34 antibody may be a useful diagnostic tool for DM and PD. GADD34 may account for the pathophysiological relationship between DM and PD.

Elevated Adiponectin Antibody Levels in Sera of Patients with Atherosclerosis-Related Coronary Artery Disease, Cerebral Infarction and Diabetes Mellitus

Adiponectin secreted from the adipocytes plays pleiotropic, anti-atherosclerotic roles, such as enhancement of insulin secretion and an increase in energy expenditure. The measurement of levels of circulating adiponectin is useful to evaluate the progression of atherosclerosis-related diseases, such as coronary artery disease (CAD), cerebral infarction (CI) and diabetes mellitus (DM). We examined the serum antibody levels against recombinant adiponectin protein via the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) method. The results revealed that the antibody levels were significantly higher in patients with CAD, CI and type 2 DM, than in healthy donors. Receiver operating curve analysis showed that the sensitivity was in a range of 41–48% for CAD, CI and DM. Thus, the serum anti-adiponectin antibody levels could be a common marker for atherosclerosis-related diseases.