Association of serum levels of antibodies against ALDOA and FH4 with transient ischemic attack and cerebral infarction.

BACKGROUND: Ischemic stroke, including transient ischemic attack (TIA) and acute-phase cerebral infarction (aCI), is a serious health problem in the aging society. Thus, this study aimed to identify TIA and aCI biomarkers. METHODS: In 19 patients with TIA, candidate antigens recognized by serum IgG autoantibodies were screened using a human aortic endothelial cell cDNA library. Through amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA), serum antibody levels against the candidate antigens were examined in healthy donor (HD), TIA, and aCI cohorts (n = 285, 92, and 529). The plasma antibody levels in the Japan Public Health Center-based Prospective Cohort Study (1991-1993) were also examined. RESULTS: The candidate antigens were aldolase A (ALDOA) and fumarate hydratase (FH). In AlphaLISA, patients with TIA or aCI had higher anti-ALDOA antibody (ALDOA-Ab) and anti-FH antibody (FH-Ab) levels than the HDs (P < 0.05). In a multivariate logistic regression analysis, the ALDOA-Ab (odds ratio [OR]: 2.46, P = 0.0050) and FH-Ab (OR: 2.49, P = 0.0037) levels were independent predictors of TIA. According to the case-control study, the ALDOA-Ab (OR: 2.50, P < 0.01) and FH-Ab (OR: 2.60, P < 0.01) levels were associated with aCI risk. In a correlation analysis, both ALDOA-Abs and FH-Abs were well associated with hypertension, coronary heart disease, and habitual smoking. These antibody levels also correlated well with maximum intima-media thickness, which reflects atherosclerotic stenosis. CONCLUSIONS: ALDOA-Abs and FH-Abs can be novel potential biomarkers for predicting atherosclerotic TIA and aCI.

Serum anti-LRPAP1 is a common biomarker for digestive organ cancers and atherosclerotic diseases.

Some cancers are related to atherosclerotic diseases; therefore, these two types of disease may share some antibody biomarkers in common. To investigate this, a first screening of sera was performed from patients with esophageal squamous cell carcinoma (ESCC) or acute ischemic stroke (AIS) for serological identification of antigens using recombinant cDNA expression cloning (SEREX). The amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) method, which incorporates glutathione donor beads and anti-human IgG acceptor beads, was used to evaluate serum antibody levels. SEREX screening identified low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) as a target antigen of serum IgG antibodies in the sera of patients with ESCC or AIS. Antigens, including recombinant glutathione S-transferase-fused LRPAP1 protein, were prepared to examine serum antibody levels. AlphaLISA revealed significantly higher antibody levels against the LRPAP1 protein in patients with solid cancers such as ESCC and colorectal carcinoma and some atherosclerosis-related diseases such as AIS and diabetes mellitus compared with healthy donors. Correlation analysis revealed that the elevated serum antibody levels against LRPAP1 were associated with smoking, a well-known risk factor for both cancer and atherosclerosis. Serum LRPAP1 antibody is therefore a common marker for the early diagnosis of some cancers and atherosclerotic diseases and may reflect diseases caused by habitual smoking.

Investigation of novel biomarkers for predicting the clinical course in patients with ulcerative colitis.

BACKGROUND: The clinical course of ulcerative colitis (UC) is characterized by repeated episodes of relapse and remission. We hypothesized that biomarkers that help distinguish refractory UC patients who are in remission using strong anti-immunotherapy could contribute in preventing the overuse of corticosteroids for treatment. Here, we clarified novel autoantibodies for UC patients in remission as clinical indicators to distinguish between refractory and non-refractory UC. METHODS: Antigen proteins recognized by serum antibodies of patients with UC in remission were screened using the protein array method. To validate the results, AlphaLISA was used to analyze the serum antibody titers with candidate protein antigens. Serum samples from 101 healthy controls, 121 patients with UC, and 39 patients with Crohn's disease were analyzed. RESULTS: Of 66 candidate protein antigens screened by ProtoArray™, six were selected for this study. The serum titers of anti-poly ADP-ribose glycohydrolase (PARG), anti-transcription elongation factor A protein-like 1, and anti-proline-rich 13 (PRR13) antibodies were significantly higher in patients with UC than in healthy controls. Anti-PARG and anti-PRR13 antibody titers were significantly higher in patients with refractory UC than in patients with non-refractory UC. There were no significant differences in any antibody titer between the active and remission phases. CONCLUSIONS: The serum titers of anti-PARG, anti-transcription elongation factor A protein-like 1, and anti-PRR13 antibodies were elevated in patients with UC. Anti-PARG and anti-PRR13 antibody titers may be novel clinical indicators for detecting refractory UC in patients in remission.

Identification of serum anti-striatin 4 antibodies as a common marker for esophageal cancer and other solid cancers.

Solid cancers have a poor prognosis, and their morbidity and mortality after surgery is high. Even after radical surgery for esophageal cancer, there have been cases of early postoperative death. The present study therefore aimed to explore new tumor markers that can predict the early postoperative prognosis. To identify antibody markers, serological antigens were identified using recombinant cDNA expression cloning (SEREX). The results identified striatin 4 (STRN4) as the antigen recognized by serum IgG antibodies in patients with esophageal cancer. After performing an amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA), it was revealed that when compared with healthy donors, serum anti-STRN4 antibody (STRN4-Ab) levels were significantly higher not only in patients with esophageal cancer but also to lesser extent, in those with gastric cancer, colorectal cancer, lung cancer and breast cancer. Compared with STRN4-Ab-negative patients with esophageal cancer, STRN4-Ab-positive patients had a poorer postoperative prognosis at early stages, suggesting that STRN4-Abs may be useful for predicting poor early-stage prognoses of patients with esophageal cancer. The positive diagnosis rates of esophageal cancer using the STRN4-Ab marker and conventional markers, including squamous cell carcinoma antigen and p53 antibody alone, were 26.4, 35.2 and 19.1% respectively; a result that increased up to 59.1% by combining all three markers. Serum STRN4-Ab may serve as a novel marker of esophageal cancer.

The Neuropsychological Correlates of Brain Perfusion and Gray Matter Volume in Alzheimer's Disease.

BACKGROUND: Neuropsychological tests, structural neuroimaging, and functional neuroimaging are employed as diagnostic and monitoring biomarkers of patients with Alzheimer's disease (AD)Objective:We aimed to elucidate the similarities and differences in neuropsychological tests and neuroimaging with the use of the Mini-Mental State Examination (MMSE), Alzheimer's Disease Assessment Scale cognitive subscale (ADAS-cog), structural magnetic resonance image (MRI), and perfusion single photon emission computed tomography (SPECT), and parametric image analyses to understand its role in AD. METHODS: Clinically-diagnosed AD patients (n = 155) were scanned with three-dimensional T1-weighted MRI and N-isopropyl-p-[123I] iodoamphetamine SPECT. Statistical parametric mapping 12 was used for preprocessing images, statistical analyses, and voxel-based morphometry for gray matter volume analyses. Group comparison (AD versus healthy controls), multiple regression analyses with MMSE, ADAS-cog total score, and ADAS-cog subscores as variables, were performed. RESULTS: The AD group showed bilateral hippocampal volume reduction and hypoperfusion in the bilateral temporo-parietal lobe and posterior midline structures. Worse MMSE and ADAS-cog total score were associated with bilateral temporo-parietal volume loss and hypoperfusion. MMSE, but not ADAS-cog, was associated with the posterior midline structures. The ADAS-cog subscores were associated with the temporal volume, while perfusion analyses were linked to the left temporo-parietal region with the language function and right analogous region with the constructional praxis subscore. CONCLUSION: MMSE and ADAS-cog are associated with temporo-parietal regions, both in volume and perfusion. The MMSE score is associated with posterior midline structures and linked to an abnormal diagnostic AD pattern. Perfusion image analyses better represents the cognitive function in AD patients.

Association between serum anti­ASXL2 antibody levels and acute ischemic stroke, acute myocardial infarction, diabetes mellitus, chronic kidney disease and digestive organ cancer, and their possible association with atherosclerosis and hypertension.

The aim of the present study was to identify novel antibody markers for the early diagnosis of atherosclerosis in order to improve the prognosis of patients at risk for acute ischemic stroke (AIS) and acute myocardial infarction (AMI). A first screening involved the serological identification of antigens by recombinant cDNA expression cloning and identified additional sex combs­like 2 (ASXL2) as a target antigen recognized by serum IgG antibodies in the sera of patients with atherosclerosis. Antigens, including the recombinant glutathione S­transferase­fused ASXL2 protein and its synthetic peptide were then prepared to examine serum antibody levels. Amplified luminescence proximity homogeneous assay­linked immunosorbent assay, which incorporates glutathione­donor beads and anti­human­IgG­acceptor beads, revealed significantly higher serum antibody levels against the ASXL2 protein and its peptide in the patients with AIS, diabetes mellitus, AMI, chronic kidney disease, esophageal squamous cell carcinoma, or colorectal carcinoma compared with those in healthy donors. The ASXL2 antibody levels were well associated with hypertension complication, but not with sex, body mass index, habitual smoking, or alcohol intake. These results suggest that the serum ASXL2 antibody marker can discriminate between hypertension­induced atherosclerotic AIS and AMI, as well as a number of digestive organ cancers.

Elevated levels of autoantibodies against DNAJC2 in sera of patients with atherosclerotic diseases.

BACKGROUND: Serum antibody markers have been increasingly identified not only for cancer and autoimmune diseases but also for atherosclerosis-related diseases such as acute ischemic stroke (AIS), acute myocardial infarction (AMI), diabetes mellitus (DM), and chronic kidney disease (CKD). Biomarkers for transient ischemic attack (TIA) and non-ST segment elevation acute coronary syndrome (NSTEACS) are potentially useful for detection of early phase of atherosclerotic changes against AIS and AMI, respectively. METHODS: We utilized serological identification of antigens by recombinant cDNA expression cloning (SEREX) using a human aortic endothelial cell cDNA phage library and sera from patients with TIA or NSTEACS. Serum antibody levels were measured by amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using purified recombinant antigens. RESULTS: Screening of sera from patients with TIA identified DnaJ heat shock protein family (Hsp40) member C2 (DNAJC2) as a candidate antigen, which was also isolated by SEREX screening using sera of patients with NSTEACS. The validation cohort revealed significantly higher DNAJC2 antibody (DNAJC2-Ab) levels in the sera of patients with TIA or AIS than those in healthy donors (HDs). Multivariate logistic regression analysis indicated that the predictive odds ratios (OR) of DNAJC2-Ab levels for TIA and AIS were 2.54 (95% confidence interval [CI]: 1.36-4.74, p = 0.0034) and 2.14 (95% CI: 1.39-3.30, p = 0.0005), respectively. Serum DNAJC2-Ab levels were also higher in patients with AMI, DM, and CKD than those in HDs. CONCLUSION: Serum DNAJC2-Ab level may be useful for early detection of atherosclerotic lesions, which lead to AIS and AMI.

Elevated levels of autoantibodies against EXD2 and PHAX in the sera of patients with chronic thromboembolic pulmonary hypertension.

While circulating autoantibodies have been detected in patients with several cardiovascular diseases, such studies have not been performed for chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH). Here we investigated the production of certain auto-antibodies in CTEPH patients. Initial screening was performed in 5 CTEPH patients and 5 healthy donors (HDs) using a ProtoArray Human Protein Microarray v5.1 containing 9,375 human proteins, and we selected 34 antigens recognized by IgG antibodies more strongly in the sera of CTEPH patients than in the sera of HDs. In subsequent second/third analyses, we validated the auto-antibody level using amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in 96 CTEPH patients and 96 HDs as follows: At the second screening, we used 63 crude peptides derived from those selected 34 antigens and found that the serum levels of autoantibodies for 4 peptides seemed higher in CTEPH patients than in HDs. In third analysis, we used the purified peptides of those selected in second screening and found that serum antibodies against peptides derived from exonuclease 3'-5' domain-containing 2 (EXD2) and phosphorylated adaptor for RNA export (PHAX) were significantly higher in CTEPH patients than in HDs. The serum antibody levels to these antigens were also elevated in PAH patients. The titers against EXD2 peptide decreased after surgical treatment in CTEPH patients. These autoantibodies may be useful as biomarkers of CTEPH and PAH, and further investigations may provide novel insight into the etiology.

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.

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.

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.

Expression of nitric oxide synthases in the anterior horn cells of amyotrophic lateral sclerosis.

The authors investigated for a correlation between the expression of nitric oxide synthases (NOSs) with the severity of motor neuronal loss in the anterior horns of patients with amyotrophic lateral sclerosis (ALS). Spinal cords from six patients with ALS and from three normal controls were examined. The sections of cervical, lumbar, and sacral cord including Onuf's nucleus, which are seldom degenerated until the late stage, were stained with three antibodies against NOSs (anti-n-NOS, anti-e-NOS, and anti-i-NOS) using ABC methods. Perikarya of motor neurons in ALS, but not in controls, were immunoreactive against anti-n-NOS and e-NOS. Anti-i-NOS did not recognize the motor neurons of ALS or of controls. The immunoreactivity for n- and e-NOSs was approximately the same in the sections of cervical, lumbar, and sacral cord in ALS. No significant differences in immunoreactivity were observed among the patients with ALS. These results suggest that the expression of NOSs does not immediately affect neuronal loss in ALS.

Relationship between metabolic disorders and relative risk values of brain infarction estimated by protein-conjugated acrolein, IL-6 and CRP together with age.

BACKGROUND: We have recently found that the median relative risk value (RRV) (0-1) of brain infarction estimated by protein-conjugated acrolein (PC-Acro), IL-6 and CRP together with age was in the order silent brain infarction (SBI) (0.80)>carotid atherosclerosis (CA) (0.76)>white matter hyperintensity (WMH) (0.46)>control (0.14). We clarified how metabolic disorders [hypertension (HT), hyperlipidemia (HL) and hyperglycemia (HG)] are correlated with RRV. METHODS: The levels of PC-Acro, IL-6 and CRP in plasma were measured by ELISA. SBI and WMH were evaluated by MRI, and CA was evaluated by duplex carotid ultrasonography. RESULTS: The median RRV of metabolic disorders was in the order HT+HG (0.84)>HT+HL (0.73)>HT (0.65)≈HG (0.65)>HL (0.61)>HL+HG (0.48)>no metabolic disorder (0.24)>normal (0.11). Correlation with SBI was in the order HT+HG (52%)>HT+HL (42%)>HT (40%)>HG (34%)≈HL(33%)>HL+HG (14%)≈no metabolic disorder (14%). CONCLUSION: The results indicate that HT is the most strongly associated factor with SBI among metabolic disorders and that the seriousness of metabolic disorder estimated by RRV was well correlated with SBI.

Correlation between images of silent brain infarction, carotid atherosclerosis and white matter hyperintensity, and plasma levels of acrolein, IL-6 and CRP.

OBJECTIVE: We found previously that the measurement of plasma levels of protein-conjugated acrolein (PC-Acro) together with IL-6 and CRP can be used to identify silent brain infarction (SBI) with high sensitivity and specificity. The aim of this study was to clarify how three biochemical markers are correlated to SBI, carotid atherosclerosis (CA) and white matter hyperintensity (WMH). METHODS: The levels of PC-Acro, IL-6 and CRP in plasma were measured by ELISA. SBI and WMH were evaluated by MRI, and CA was evaluated by duplex carotid ultrasonography. RESULTS: A total of 790 apparently healthy volunteers were classified into 260 control, 214 SBI, 263 CA and 245 WMH subjects, which included 187 subjects with two or three pathologies. When the combined measurements of PC-Acro, IL-6 and CRP were evaluated together with age, using a receiver operating characteristic curve and artificial neural networks, the relative risk value (RRV), an indicator of tissue damage, was in the order SBI with CA (0.90)>SBI (0.80)>CA (0.76)>WMH with CA (0.65)>WMH (0.46)>control (0.14). RRV was also correlated with severity in each group of SBI, CA and WMH. CONCLUSION: The RRV supports the idea that the degree of risk to develop a stroke is in the order SBI>CA>WMH.

Astrocytic tau pathology positively correlates with neurofibrillary tangle density in progressive supranuclear palsy.

Tufted astrocytes (TAs) are considered reliable, specific markers for the neuropathologic diagnosis of progressive supranuclear palsy (PSP). It is known that neurofibrillary tangles (NFTs) may relate directly to neurodegeneration, but the role of glial tau pathology is not well determined. To examine the hypothesis that TAs are as pathogenic as NFTs and that both might have a common accumulation, we evaluated the topographic relationship between TAs and NFTs in 12 cases of PSP. The sections of 13 different parts of the brain were stained using the Gallyas-Braak method, and TAs and NFTs were counted and compared statistically. The number of TAs significantly correlated with that of NFTs in the central gray matter, pontine nuclei, and tegmentum, which are responsible for the main symptoms in PSP. In the examined allocortex, however, NFTs were abundant without accompanying TAs. Staining with the specific antibody for 4-repeat tau (RD4) and 3-repeat tau (RD3) was performed to clarify this discrepancy from the standpoint of tau isoforms. NFTs in the entorhinal cortex were stained with both RD3 and RD4, but NFTs in the premotor cortex were stained with only RD4. The nature of NFTs in the allocortical area was different from that of the isocortex in PSP. TAs in the isocortex may share the same pathologic cascade with NFTs stained only by RD4. These results suggest that TAs are part of the same pathologic process as NFTs in PSP.