Autoantibodies can be used in the early diagnosis and treatment of atherosclerosis-related diseases. Using ProtoArray® screening of samples from patients with atherosclerosis, the present study identified thiosulfate sulfurtransferase-like domain-containing 2 (TSTD2) as a novel atherosclerosis antigen. The serum TSTD2 antibody levels were then quantified using an amplified luminescent proximity homogeneous assay-linked immunosorbent assay. This demonstrated the levels of TSTD2 antibodies (TSTD2-Abs) to be significantly higher in patients with acute cerebral infarction or chronic kidney disease than in healthy donors. The TSTD2-Ab levels were also found to be higher in males, older adults, smokers, in those who consumed alcohol regularly, and in those with hypertension. Furthermore, Spearman's rank correlation analysis revealed TSTD2-Ab levels to be strongly associated with measures of atherosclerosis severity, including plaque scores, intima-media thickness of the carotid artery and the cardio-ankle vascular index. Thus, TSTD2-Abs may thus be a promising novel biomarker for atherosclerosis-related cerebral infarction and kidney disease.
Ischemic stroke is one of the most common vascular disorders worldwide and, despite notable advancements being made in treatments and diagnostic imaging techniques, it is still associated with high mortality and morbidity rates (
Serum samples were obtained from patients who suffered an ischemic stroke and from healthy donors (HDs). The present study analyzed 684 serum samples, including 275 from patients with acute ischemic stroke (AIS), 300 from patients with CKD and 109 from HDs. The AIS group consisted of 196 patients with acute cerebral infarction (aCI) and 79 patients with transient ischemic attack (TIA). The serum samples of the patients with aCI and TIA were collected at Chiba Prefectural Sawara Hospital, Chiba Rosai Hospital, and Chiba Aoba Municipal Hospital. The aCI samples were collected within 2 weeks of the diagnosis of atherothrombotic brain infarction. The median (range) of age in years of the HD, aCI and TIA subjects were 60 (45-90), 77 (58-85) and 73 (26-90), respectively. The subject information of the Sawara stroke cohort is summarized in
The CKD serum samples were obtained from the Kumamoto cohort (
The present study was conducted in accordance with the principles of the 1913 revision of the Declaration of Helsinki and with the approval of the Ethical Review Committee of Chiba University, Graduate School of Medicine and cooperative hospitals (approval no. 2018-320). The research on recombinant DNA was conducted with the permission of the Graduate School of Medicine, Chiba University, and following Japanese regulations. Each participant provided written informed consent to participation and publication.
The initial screening was performed using ProtoArray® Human Protein Microarrays v.4.0 (Thermo Fisher Scientific, Inc.), which are loaded with 9,480 proteins, as previously described (
The expression plasmid of the TSTD2 protein tagged with glutathione-S-transferase (GST) was constructed by recombining the cDNA sequence into a pGEX-4T-1 (Cytiva) plasmid vector. The competent
The GST and GST fusion proteins (0.3 µg) were separated by SDS-PAGE (11% polyacrylamide) and transferred to nitrocellulose membranes (cat. no. 1620112, Advantec Toyo Kaisha, Ltd.). The membranes were blocked with blocking solution [0.5% skim milk powder in a buffer consisting of 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween-20], and subjected to specific primary antibodies, i.e., 1:5,000-diluted antibodies against GST (cat. no. 600-101-200, Rockland Immunochemicals, Inc.) or 1:1,000-diluted sera from subjects. Following incubation with 1:30,000-diluted horseradish peroxidase (HRP)-conjugated secondary antibodies [donkey anti-goat (cat. no. sc-2056), or anti-human IgG (cat. no. sc-2453); both from Santa Cruz Biotechnology, Inc.], immunoreactivity was measured using Immobilon Western HRP Substrate (Merck KGaA) and LuminoGraph II (ATTO Co, Ltd.). The results were detected as previously described (
AlphaLISA was used for the quantitative measurement of serum antibodies to purified proteins. Subsequently, 2.5 µl serum diluted 1:100 in AlphaLISA buffer [25 mM hydroxyethyl piperazine ethane sulfonic acid (Thermo Fisher Scientific, Inc.), pH 7.4, 0.1% casein (Merck KGaA), 0.5% Triton X-100 (FUJIFILM Wako Pure Chemical Corporation), 1 mg/ml dextran-500 (Merck KGaA) and 0.05% Proclin-300 (Merck KGaA)] and 2.5 µl GST or GST-fused TSTD2 proteins (10 µg/ml) was placed in 384-well microtiter plates (white opaque OptiPlate, PerkinElmer, Inc.) and used for the experiments. The reaction mixture was incubated at room temperature for 6-8 h, after which anti-human IgG-conjugated acceptor beads (2.5 µl, 40 µg/ml; PerkinElmer, Inc.) and glutathione-conjugated donor beads (2.5 µl, 40 µg/ml; PerkinElmer, Inc.) were added. The mixture was incubated for 7-21 days at room temperature in the dark. Chemiluminescence was read on an EnSpire Alpha (PerkinElmer, Inc.) microplate reader (PerkinElmer, Inc.), as previously described (
The Dunn's multiple comparison test following a Kruskal-Wallis test was used to analyze continuous variables using JMP Pro 14.2.0 software (SAS Institute Inc.). Correlations between TSTD2 antibody levels and each clinical and demographic parameter were evaluated using Spearman's correlation analysis. The cutoff TSTD2 antibody level for predicting ischemic stroke was assessed to maximize the sum of the sensitivity and specificity rates using a receiver operating characteristic (ROC) curve analysis. Clinical and demographic factors, including sex, age, smoking and alcohol drinking habits, and the presence of medical conditions such as hypertension, diabetes, cardiovascular disease, hyperlipidemia and obesity [based on body mass index (BMI)] were examined in relation to the serum TSTD2 antibody levels using a Mann-Whitney U test. Spearman's correlation analyses, ROC analysis and analysis using the Mann-Whitney U test were performed using GraphPad Prism 5 software (GraphPad, Inc.). All the tests were two-tailed, and a P-value <0.05 was considered to indicate a statistically significant difference.
The screening of ProtoArray® loaded with 9,480 proteins identified TSTD2 antibodies (accession no. NM_139246.3) in eight of the 10 serum samples from patients with atherosclerosis and in two of the 10 serum samples from the HDs. The expression of GST-fused TSTD2 protein was successively induced in
To investigate the association between TSTD2 autoantibodies and AIS, serum TSTD2 antibody levels were examined in the HD, aCI and TIA groups using AlphaLISA. Compared with the HD group, the aCI group exhibited significantly higher TSTD2 antibody levels (P=0.0191); however, no significant difference was observed between the TIA and HD groups (P=0.2030), or between the TIA and aCI groups (P=0.2589) (
The present study then examined antibody levels in the sera of patients with CKD. This group was divided into three subgroups according to CKD type as follows: Type-1 (diabetic kidney disease), type-2 (nephrosclerosis) and type-3 (glomerulonephritis). The type-1 and type-2 CKD subgroups had significantly higher serum TSTD2-Ab levels than the HDs. The type-3 CKD subgroup exhibited higher levels than the HD group, although the difference was not significant (P=0.0826) (
A ROC analysis of the TSTD2 antibody levels in aCI and type-1 and type-2 CKDs was also performed. The cutoff values for these levels in aCI and type-1 and type-2 CKDs were 28,954 (sensitivity, 37.4%; specificity, 79.8%), 11,5748 (sensitivity, 31.03%; specificity, 91.67%) and 12,1078 (sensitivity, 46.88%; specificity, 94.05%), respectively (
Spearman's correlation analyses to identify the correlations between the TSTD2 antibody levels, and the clinical and demographic variables in the Sawara stroke cohort. The patient information is presented in
Antibody levels were compared between two groups using the Mann-Whitney U test using the cutoff values determined by ROC analysis, and were found to be significantly higher in males than in females (P=0.036), those who were ≥65 years of age compared with those <65 of age (P=0.047), in those with hypertension compared with those without this condition (P=0.036), in smokers compared with non-smokers (P=0.003), and in those who consumed alcohol compared with those who did not (P=0.042) (
Subsequently, Spearman's correlation analysis was performed to identify the correlations between the serum TSTD2 antibody levels and the clinical features of patients in the Kumamoto CKD cohort. The patient information is presented in
Western blot analysis was performed to determine the presence of anti-TSTD2 antibodies in the serum samples. Both GST and GST-TSTD2 protein reacted with commercial anti-GST antibodies whereas GST-TSTD2, but not GST was recognized by antibodies in the sera of patients with aCI (
Atherosclerosis is one of the major causes of ischemic stroke (
Subsequently, the correlation between clinical factors and anti-TSTD2 antibody levels was examined in the aCI cohort. A significant elevation in antibody levels was observed in males, and in those with hypertension, who were older, and with a smoking history and a history of alcohol consumption (
Spearman's correlation analysis revealed that blood sugar, a typical diabetes mellitus (DM) marker, was related to the serum TSTD2 antibody levels (
TSTD2 is a thiosulfate sulfurtransferase (
Reactive oxygen species (ROS) are considered to contribute to vascular inflammation in atherosclerosis as a result of mediating various signaling pathways (
The early stages of atherosclerosis are usually accompanied by elevated autoantibody levels (
Not applicable.
All results of the ProtoArray® Human Protein Microarrays are available in the Figshre database (
MK, TMac, HK, YI and TH conceived and designed the study. MK, BSZ, SYL, HW and AAd performed the experiments and acquired the data. YY, TMac, SM, IK, TW, AAo, KK and HT contributed the reagents, materials, analysis tools or patient data. YY, AAd and TMat analyzed and interpreted the data. BSZ, TMat and HK performed the statistical analyses. MK, BSZ, SYL, TMac, YI and TH drafted the manuscript. SM, TW and HT confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The present study was conducted in accordance with the principles of the 1913 revision of the Declaration of Helsinki and with the approval of the Ethical Review Committee of Chiba University, Graduate School of Medicine and cooperative hospitals (approval no. 2018-320). The research on recombinant DNA was conducted with the permission of the Graduate School of Medicine, Chiba University, and following Japanese regulations. Each participant provided written informed consent to participation and publication.
Not applicable.
The present study was performed in collaboration with Fujikura Kasei Co., Ltd., and HK is an employee of Fujikura Kasei Co., Ltd.
Western blot analysis.
Comparison of the serum levels of TSTD2 antibodies in HDs, and in patients with aCI and TIA. (A) The figure illustrates the levels of serum TSTD2-Abs examined using amplified luminescence proximity homogeneous assay (Alpha)-linked immunosorbent assay. Antibody levels are represented by Alpha photon counts and shown in a box-whisker plot. The horizontal lines represent medians, and the boxes represent the 25 and 75th percentiles. The whiskers represent the 10 and 90th percentiles, and the dots represent outliers. P-values were calculated using the Kruskal-Wallis test. A ROC curve analysis was performed to assess the ability of serum TSTD2-Abs to detect aCI. (B) The numbers in the graph are the AUC, specificity, sensitivity and cutoff values for the marker levels. TSTD2, thiosulfate sulfurtransferase-like domain-containing 2; TSTD2-Ab, TSTD2 antibody; HD, healthy donor; aCI, acute cerebral infarction; TIA, transient ischemic attack; ROC, receiver operating characteristic; AUC, area under the curve.
Comparison of serum TSTD2 antibody levels in HDs, and patients with type-1, -2 and -3 CKD. (A) Serum TSTD2 antibody levels were quantified using amplified luminescence proximity homogeneous assay-linked immunosorbent assay and compared between the HD group and the type-1 (diabetic kidney disease), type-2 (nephrosclerosis) and type-3 (glomerulonephritis) CKD subgroups. TSTD2 antibody levels are depicted in a box-whisker plot. (B and C) The TSTD2 antibody levels were analyzed using a ROC curve to compare type-1 and type-2 CKDs. The numbers in the graphs indicate the AUC, specificity, sensitivity and cutoff values. TSTD2, thiosulfate sulfurtransferase-like domain-containing 2; CKD, chronic kidney disease; ROC, receiver operating characteristic; AUC, area under the curve.
Associations between TSTD2 antibody levels and clinical and demographic variables in the serum of acute cerebral infarction patients. The associations between TSTD2 antibody levels and (A) sex, (B) age, (C) hypertension, (D) smoking status, (E) alcohol consumption, (F) diabetes, (G) cardiovascular disease, (H) hyperlipidemia, and (I) BMI were examined in the acute cerebral infarction cohort. The TSTD2 antibody levels obtained using amplified luminescence proximity homogeneous assay-linked immunosorbent assay are shown in box-whisker plots. The P-values were calculated using Mann-Whitney U tests. TSTD2, thiosulfate sulfurtransferase-like domain-containing 2; BMI, body mass index.
Spearman's correlation analysis for the correlation between the serum TSTD2 antibody levels and clinical features of patients with aCI.
Variables | Spearman's rank correlation coefficient (Rho) | P-value |
---|---|---|
Age, years | 0.0954 | 0.0624 |
BMI | 0.0193 | 0.7075 |
Maximum IMT | 0.1703 | |
AST | 0.0054 | 0.9161 |
ALT | -0.0428 | 0.4051 |
ALP | 0.0647 | 0.228 |
LDH | 0.086 | 0.0986 |
tBil | -0.0226 | 0.6642 |
CHE | -0.1960 | |
γ-GTP | 0.0586 | 0.2677 |
TP | -0.0910 | 0.0797 |
ALB | -0.1727 | |
BUN | 0.034 | 0.5082 |
Creatinine | -0.0079 | 0.8783 |
eGFR | -0.0124 | 0.8154 |
UA | 0.069 | 0.2558 |
AMY | -0.0764 | 0.2537 |
T-CHO | -0.1567 | 0.0046 |
HDL-C | -0.0673 | 0.3106 |
TG | -0.1109 | 0.0814 |
Na | -0.0139 | 0.7877 |
K | -0.0994 | 0.054 |
Cl | -0.0989 | 0.055 |
CRP | 0.1395 | |
WBC | 0.0905 | 0.0783 |
RBC | -0.0673 | 0.1912 |
HGB | -0.0564 | 0.2737 |
HCT | -0.0626 | 0.2243 |
PLT | -0.0481 | 0.3503 |
BS | 0.1542 | |
HbA1c | -0.0250 | 0.6695 |
Smoking duration (years) | 0.1938 | |
Frequency of alcohol consumption (times/week) | 0.1174 |
A correlation analysis was performed to identify the correlation between TSTD2 antibody levels and the clinical features of patients with ischemic stroke. Correlation coefficients (Rho) and P-values were calculated using Spearman's correlation analysis. Significant correlations (P<0.05) are marked in bold font. aCI, acute cerebral infarction; TSTD2, thiosulfate sulfurtransferase-like domain-containing 2; BMI, body mass index; IMT, intima-media thickness; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; tBil, total bilirubin; CHE, choline esterase; γ-GTP, γ-glutamyl transpeptidase; TP, total protein; ALB, albumin; BUN, blood urea nitrogen; eGFR, estimated glomerular filtration rate; UA, uric acid; AMY, amylase; T-CHO, total cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride; CRP, C-reactive protein; WBC, white blood cell count; RBC, red blood cell count; HCT, hematocrit; PLT, platelet count; BS, blood sugar; HbA1c, hemoglobin A1c.
Spearman's correlation analysis of the correlation between the serum TSTD2 antibody levels and clinical features of patients with CKD.
Variables | Spearman's rank correlation coefficient (Rho) | P-value |
---|---|---|
Age, years | 0.0545 | 0.3468 |
Height | -0.0044 | 0.9401 |
Weight | -0.1152 | 0.0461 |
BMI | -0.1509 | |
Plaque score | 0.1608 | |
Maximum IMT | 0.1266 | |
ABI (right) | -0.0122 | 0.8353 |
ABI (left) | -0.0124 | 0.8319 |
CAVI (right) | 0.1430 | |
CAVI (left) | 0.1472 | |
HbA1c | -0.0077 | 0.9260 |
PTH | 0.1539 | |
Fe | -0.0984 | 0.0889 |
Ferritin | 0.1110 | 0.0547 |
TSAT ratio | -0.0615 | 0.2882 |
Kt/V | -0.0874 | 0.1308 |
RBC | -0.0680 | 0.2406 |
PLT | -0.0743 | 0.1994 |
TP | -0.0568 | 0.3272 |
ALB | -0.0758 | 0.1907 |
UA | -0.0153 | 0.7912 |
Na | 0.1010 | 0.0807 |
K | -0.0112 | 0.8462 |
Cl | 0.0522 | 0.3676 |
Ca | 0.0130 | 0.8223 |
IP | -0.0017 | 0.9770 |
Mg | 0.0544 | 0.3476 |
AST | 0.1654 | |
ALT | 0.0975 | 0.0920 |
LDH | 0.1749 | |
γ-GTP | 0.0895 | 0.1219 |
AP | 0.0557 | 0.3366 |
tBil | -0.0105 | 0.8559 |
AMY | -0.0448 | 0.4394 |
Creatinin | -0.0354 | 0.5415 |
T-CHO | -0.0300 | 0.6047 |
HDL-C | -0.0741 | 0.2004 |
LDL-C | -0.0058 | 0.9197 |
TG | 0.0383 | 0.5086 |
CRP | 0.1638 |
A correlation analysis was performed to identify the correlation between TSTD2 antibody levels and the clinical features in patients with CKD. Correlation coefficient (Rho) and P-values were calculated using Spearman's correlation analysis. Significant correlations (P<0.05) are marked in bold font. TSTD2, thiosulfate sulfurtransferase-like domain-containing 2; CKD, chronic kidney disease; BMI, body mass index; maximum IMT, maximum intima-media thickness; ABI, ankle brachial pressure index; CAVI, cardio-ankle vascular index; HbA1c, glycated hemoglobin; W-PTH, whole parathyroid hormone; ARB, angiotensin II receptor blocker; ACE, angiotensin converting enzyme; PTA, prothrombin; TSAT ratio, transferrin saturation ratio; Kt/V, standardized urea clearance; RBC, red blood cell number; PLT, platelet number; TP, total protein; ALB, albumin; UA, uric acid; HGB, hemoglobin; HCT, hematocrit; UN, urea nitrogen; CRE, creatinine; IP, inorganic phosphate; AST, aspartate aminotransferase; ALT, alanine amino transferase; LDH, lactate dehydrogenase; γ-GTP, γ-glutamyl transpeptidase; AP, alkaline phosphatase; tBil, total bilirubin; AMY, amylase; T-CHO, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglyceride; and CRP, C-reactive protein.