Background and Purpose-About one half of those who develop adult-onset moyamoya disease experience intracranial hemorrhage. Despite the extremely high frequency of rebleeding attacks and poor prognosis, measures to prevent rebleeding have not been established. The purpose of this study is to determine whether extracranial-intracranial bypass can reduce incidence of rebleeding and improve patient prognosis. Methods-This study was a multicentered, prospective, randomized, controlled trial conducted by 22 institutes in Japan.Adult patients with moyamoya disease who had experienced intracranial hemorrhage within the preceding year were given either conservative care or bilateral extracranial-intracranial direct bypass and were observed for 5 years. Primary and secondary end points were defined as all adverse events and rebleeding attacks, respectively. Results-Eighty patients were enrolled (surgical, 42; nonsurgical, 38). Adverse events causing significant morbidity were observed in 6 patients in the surgical group (14.3%) and 13 patients in the nonsurgical group (34.2%). Kaplan-Meier survival analysis revealed significant differences between the 2 groups (3.2%/y versus 8.2%/y; P=0.048). The hazard ratio of the surgical group calculated by Cox regression analysis was 0.391 (95% confidence interval, 0.148-1.029).Rebleeding attacks were observed in 5 patients in the surgical group (11.9%) and 12 in the nonsurgical group (31.6%), significantly different in the Kaplan-Meier survival analysis (2.7%/y versus 7.6%/y; P=0.042). The hazard ratio of the surgical group was 0.355 (95% confidence interval, 0.125-1.009). Conclusions-Although statistically marginal, Kaplan-Meier analysis revealed the significant difference between surgical and nonsurgical group, suggesting the preventive effect of direct bypass against rebleeding. Clinical Trial Registration

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.

BackgroundBecause circulating antibodies against a variety of antigens have been detected in patients with coronary heart disease, carotid atherosclerosis and those who have suffered a stroke, it is suspected that immune response may be one of the mechanisms of atherogenesis The objective of this study is to identify novel antibodies in ischemic stroke patients by screening the expressed recombinant proteins using a human cDNA library (SEREX).MethodsTo identify the candidate antigens, cDNA library was screened by SEREX using plasma from ten patients with ischemic stroke. Subsequently, via ELISA using recombinant proteins and synthetic peptides, the serum antibody levels were measured in two independent patient/healthy donor (HD) cohorts (142 and 78 in the 2nd screening and a validation cohort, respectively).ResultsThe initial screening resulted in the identification of six candidate antigens. Of these antigens, replication protein A2 (RPA2) was determined to be the antigen associated with stroke (P < 0.05) by ELISA with 2nd screening and validation cohort. Multifactorial logistic regression analysis showed that the increased levels of the RPA2 antibodies (RPA2-Abs) associated with stroke independent of other risk factors for stroke (P < 0.05). Receiver operating curve analysis demonstrated that the area under the curve from ELISA using GST fusion RPA2 and synthetic peptides (bRPA2-132) were 0.867 (95% CI: 0.798-0.936) and 0.971 (95% CI: 0.940-1.00), respectively. If the cut-off value of the bRPA2-132-Ab level was determined to be 0.334, the sensitivity and specificity of the antibody level as the diagnostic marker for stroke were 0.323 (95% CI: 0.209-0.453) and 1.00 (95% CI: 0.713-1.00), respectively.ConclusionsSEREX identified RPA2 as the antigen associated with ischemic stroke and serum auto-antibodies against RPA2 elevates in stroke patients. RPA2-Abs could become a biomarker for the evaluation of ischemic stroke at risk.

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.

Cathepsin D is an aspartyl protease involved in protein catabolism and tissue remodeling which can be secreted from cancer cells. To identify a potential serum marker for gliomas, we investigated the gene expression levels of cathepsin D in 87 tissue samples and measured the protein concentrations in sera of glioma patients. The tissue samples consisted of 43 glioblastomas, 13 anaplastic astrocytomas, 22 astrocytomas, and 9 normal brain tissues. The results of real-time quantitative reverse transcription-PCR analysis showed that cathepsin D transcript levels became significantly higher as the glioma grade advanced (P = 0.0466, glioblastoma and anaplastic astrocytoma; P = 0.0008, glioblastoma and astrocytoma; P = 0.0271, glioblastoma and normal brain tissue; unpaired t test). Immunohistochemical analysis with anticathepsin D antibody revealed dense and spotty staining in the tumor cells with high transcript levels. The low expression of cathepsin D significantly correlated with long survival of the glioma patients. Furthermore, the glioblastoma patients with high gene expression of cathepsin D lived significantly shorter than those with low expression (P = 0.0104, Cox-Mantel logrank test) and frequently had leptomeningeal dissemination (P = 0.0016, C 2 test). The multivariate analysis confirmed that the cathepsin D expression level was an independent predictor for short survival (P = 0.0102, Cox proportional hazard regression model). Measurement of the serum cathepsin D concentrations by ELISA showed a significant increase in the patients with high-grade gliomas as compared with the lowgrade tumors (P = 0.0081, C 2 test). These results collectively suggest that cathepsin D could be a potential serum marker for the prediction of aggressive nature of human gliomas. (Cancer Res 2005; 65(12): 5190-4)

Cerebral infarction (CI), cardiovascular disease (CVD), diabetes mellitus (DM) and chronic kidney disease (CKD) are atherosclerosis-related diseases, which are major causes of health damage. For early and sensitive diagnosis, development of novel biomarkers is expected and of significant practical importance. First screening was carried out by phage expression cloning to identify antigen proteins recognized by serum IgG antibodies in patients with atherosclerosis. RPA2, LRPAP1, EEF1A1, SPOCK1, LOC729260, tubulin beta 2C (TUBB2C) and KIAA0020 markers were identified. We then compared the serum antibody levels against the candidate proteins between healthy donors (HD) and patients with CI, CVD, DM, or CKD by Alpha (amplified luminescent proximity homogeneous assay)-LISA method. The results showed that the serum TUBB2C antibody levels were significantly higher in patients with CI, DM, or CKD than those in HD. Using the average + 2SD of HD as the cut-off value, the positive thresholds of TUBB2C antibody markers were 14.8% in CI, 25.8% in DM, and 18.3% in CKD. TUBB2C antibody levels were well correlated with artery stenosis degrees such as plaque score, maximum intima-media thickness and cardio ankle vascular index. Consequently, TUBB2C antibody markers are useful to diagnose atherosclerosis, DM, and CKD, and can be applied to the prediction of the onset of CI. The serum anti-TUBB2C antibody markers are useful for the diagnosis of DM and CKD.

The rupture of an intracranial aneurysm (IA) results in subarachnoid hemorrhage, a catastrophic neurological condition with high morbidity and mortality. Following-up on our previous genome-wide linkage study in Japanese population, we extensively analyzed a 4.6 Mb linkage region around D7S2472 on 7q11 by genotyping 168 single nucleotide polymorphisms (SNPs). SNP association and window scan haplotype-based association studies revealed a susceptibility locus for IA on a single LD block covering the 3'-untranslated region (3'-UTR) of ELN and the entire region of LIMK1. An association study with 404 IA patients and 458 non-IA controls revealed that the ELN 3'-UTR G(+659)C SNP has the strongest association to IA (P=0.000002) and constitutes a tag-SNP for an at-risk haplotype, which contains two functional SNPs, the ELN 3'-UTR (+502) A insertion and the LIMK1 promoter C(-187)T SNP. These allelic and haplotype-based associations were confirmed in a Korean population. Ex vivo and in vitro analyses demonstrate that the functional impact of both SNPs is the decrease of transcript levels, either through accelerated ELN mRNA degradation or through decreased LIMK1 promoter activity. Elastin and LIMK1 protein are involved in the same actin depolymerization signaling pathway; therefore, these lines of evidence suggest a combined effect of the SNPs in the at-risk haplotype possibly by weakening the vascular wall and promoting the development of IA.

BackgroundDisease 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.MethodsTo 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).ResultsFirst 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).ConclusionSerum PDCD11-Ab level may serve as a potential biomarker for TIA.