- Email: [email protected]
INCREASED PLASMA LEVELS OF HEAT SHOCK PROTEIN 70 ASSOCIATED WITH SUBSEQUENT CLINICAL CONVERSION IN NORMAL ELDERLY
P672
Poster Presentations: P3
Colombia; 2Universidad Antonio Nari~no, Bogota, Colombia. Contact e-mail: [email protected] Background: Studies in humans have shown the potential role of alterations in DNA methylation in the pathogenesis of Late-Onset Alzheimer’s disease (LOAD). The epigenetic regulation of the genes involved in the Alzheimer’s pathogenesis may help to explain the complexity of the LOAD. Methods: DNA was extracted from peripheral blood mononuclear cells of 100 Colombian AD patients (mean age 77.56 5 years) and 100 healthy subjects (mean age 75.36 5,5years). DNA was bisulfite treated using EZ DNA Methylation Direct Kit (Zymo-research). Primers were manually designed for the 5’ region of the CpG Island in the APP promoter and APOE CpG Island on exon 4. The APP and APOE MS-HRMs were performed in a CFX 96 device (BioRad), using 14 ng of bisulfite treated DNA. The quantitative results were obtained by interpolation of the first derived RFU normalized data, generated by the linear regression of the standard curve. Results: The correlation coefficient of the linear regression of the standard curves was r2¼ 0.96 for APP gen and 0.898 for APOE gen The inter-assay and intra-assay reproducibilities were verified, having a coefficient of variance less than 0.5 between replicates. In a preliminary analysis we did not find differences in APP methylation levels between AD patients and control subjects (mean 2.14% +- 1.15 vs 2.05 +- 0.96). For APOE the difference was not significant in methylation levels (patients vs. Controls 73.01 vs 70.9) Conclusions: The present study tested changes in DNA methylation of in blood samples from Colombian LOAD patients, which showed no significant differences in comparison with control samples for APP and APOE genes. This is the first report of the analysis of APP and APOE DNA methylation levels in LOAD using the quantitative and cost-effective in Latin American AD patients. It would be important to analyze, in further studies, the methylation levels for additional candidate regions in several tissues from AD patients.
P3-120
STRUCTURAL ELUCIDATION OF THE GLYCOSYLATION PROFILES OF HUMAN PLASMA CLUSTERIN IN ALZHEIMER’S DISEASE PATIENTS
Hui-Chung Liang1, Claire Russell2, Raymond Chung3, Chantal Bazenet3, Abdul Hye4, Simon Lovestone4, Ian Pike5, Malcolm Andrew Ward1, 1 Proteome Sciences PLC, London, United Kingdom; 2Proteome Sciences PLC, London, United Kingdom; 3Institute of Psychiatry, King’s College London, London, United Kingdom; 4King’s College London, London, United Kingdom; 5Proteome Sciences PLC, Cobham, United Kingdom. Contact e-mail: [email protected] Background: Clusterin is a highly glycosylated secreted protein implicated in the pathogenesis of Alzheimer’s disease (AD). Expression of the clusterin gene is significantly elevated in AD brain (May et al., 1990) and levels of plasma clusterin correlate with AD progression (Thambisetty et al., 2010). Since glycosylation plays an important role in the physiological functions of clusterin (Stuart et al., 2007), we have developed an IP-LC/MS/MS workflow and performed detailed profiling of plasma clusterin glycosylation, including determination of glycosylation sites and structural characterisation of glycosylated peptides. In this study, glycosylation analysis of plasma clusterin is performed to investigate the correlation between its glycosylation pattern and AD progression. Methods: Plasma clusterin from 28 patients was enriched using our established immunoprecipitation workflow, and analysed via LC-MS/MS using nanoflow reverse-phase chromatography on both the Orbitrap Velos and Orbitrap Fusion (ThermoScientific, USA). Glycopeptides were manually identified by the presence of glycan-specific oxonium ion fragments, m/ z 204.08 for N-acetylhexosamine (HexNAc), m/z 366.14 for hexose-Nacetylhexosamine (Hex-HexNAc), and m/z 657.24 for N-acetylneuraminic acid-hexose-N-acetylhexosamine (NeuAc-Hex-HexNAc) in the MS/MS spectra. Results: Using our IP-LC/MS/MS workflow, all six known N-glycosylation sites were identified, three in the a subunit (a64N, a81N and a123N) and three in the b subunit (b64N, b127N, and b147N). In addition, we have determined the differences in the glycoforms associated at each of the different glycosylation sites. We are currently
comparing the clusterin glycoform distribution in plasma obtained from subjects of low atrophy (n¼14) and high atrophy (n¼14), and the results of this analysis will be presented at the meeting. Furthermore, we are developing a selected reaction monitoring (SRM) method with the aim of achieving a robust workflow suitable for rapid verification of specific clusterin glycoforms to aid AD biomarker discovery. Conclusions: A thorough characterisation of the clusterin glycopeptide profile using LC/MS/ MS and SRM will be a useful tool, allowing potential clusterin glycoform biomarkers of AD progression to be identified. P3-121
ASSOCIATION BETWEEN SERUM LIPIDS AND CEREBRAL AMYLOIDOSIS IN COGNITIVELY NORMAL ELDERLY
HyoJung Choi1, Min Soo Byun1, Young Min Choe1, Bo Kyung Sohn2, Dahyun Yi3, Ji Young Han3, Song E. Kim4, Hyewon Baek3, Eun Hyun Seo3, Jinsick Park5, Youngjin Lee6, Jong Inn Woo7, Dong Young Lee1, 1Seoul National University College of Medicine, Seoul, South Korea; 2Seoul Metropolitan Government-Seoul National University Boramae Medical Center, Seoul, South Korea; 3Seoul National University Hospital, Seoul, South Korea; 4Seoul National University Hospital, Seoul, South Korea; 5 Hanyang University, Seoul, South Korea; 6Department of Neurospychiatry, Seoul, South Korea; 7Seoul National University Medical Research Center, Seoul, South Korea. Contact e-mail: [email protected] Background: Cerebral amyloid-beta protein (Ab) deposition has been considered as a key initiating step in Alzheimer’s disease (AD) process. Although many preclinical studies have suggested the possible linkage between dyslipidemia and cerebral amyloidogenesis or amyloid deposition, the association between serum lipids and cerebral Ab deposition in human brain is still poorly known. We aimed to investigate such association in cognitively normal (CN) elderly individuals. Methods: Sixty two CN elderly individuals were included. All participants received comprehensive clinical and neuropsychological assessment based on the CERAD protocol, 11 C labeled Pittsburgh Compound B (PiB) positron emission tomography volumetric MRI, and quantification for serum lipid components including total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) with apolipoprotein A1 (APOA1) and B (APOB). Results: Global cerebral Ab deposition was defined as mean cortical PiB retention of the cortical regions including frontal, lateral temporal, lateral parietal and precuneus/posterior cingulate cortices. Univariate analyses showed significant positive association of mean cortical PiB retention with the levels of serum TG and APOB, and negative association with HDL-C and APOA1 levels. Multivariate statistical models that controlled age, education, gender, apolipoprotein E ε4 genotype revealed independent associations between the levels of TG and mean cortical 11 C-PiB retention. Higher serum TG level was associated with heavier cortical 11 C-PiB retention. No association was found between both total cholesterol and LDL-C, and cortical 11 C-PiB retention. Conclusions: Our findings strongly support the association between lipid profiles in serum, especially TG level, and the degree of cerebral amyloidosis in cognitively normal elderly people.The findings also suggest that the mechanisms through which serum lipid or lipoproteins affect cerebral Ab could provide novel targets of AD therapeutics. More specifically, various approaches known to reduce blood TG level, such as moderate exercise and omega-3 fatty acid intake, might be helpful for slowing Ab deposition and reducing AD occurrence. P3-122
INCREASED PLASMA LEVELS OF HEAT SHOCK PROTEIN 70 ASSOCIATED WITH SUBSEQUENT CLINICAL CONVERSION IN NORMAL ELDERLY
Hyunwoong Roh1, Sang Joon Son2, Chang Hyung Hong3, Kang Soo Lee4, Ki Jung Chang5, 1Ajou University Hospital, Suwon, South Korea; 2Myoungji Hospital, Goyang-si, South Korea; 3Ajou University School of Medicine, Suwon-si, South Korea; 4CHA Gangnam Medical Center, CHA University, Seoul, South Korea; 5CHA Gangnam Medical Center, CHA University, Suwon, South Korea. Contact e-mail: [email protected]
Poster Presentations: P3 Table 1 Demographic and clinical characteristics of subjectsy
Age baseline Gender female Education Follow up duration HTN DM BMI (kg/m2) Baseline Follow up KSGDS Baseline Follow up
NCI to NCI Non conversion (N¼36)
NCI to MCI conversion (N¼30)
70.0065.01 23 (63.89) 6.464.8 5.3660.68 21 (58.33) 2 (5.56)
69.9364.42 24 (80.00) 3.964.3 5.2360.56 17 (56.67) 5 (16.67)
0.06 2.07 2.18 0.82 0.02 2.13
0.955 0.180 0.033 0.417 0.545 0.231
25.6963.61 25.8763.81
25.3964.61 25.8364.61
0.26 0.04
0.794 0.972
5.1464.07 5.9765.12
6.3864.03 8.4366.74
-1.23 -1.69
0.224 0.097
t or
p
P673
Background: Heat shock proteins (Hsps) have been regarded as cytoprotectants that protect brain cells from damage encountered following cerebral ischemia or during the progression of neurodegenerative diseases. Methods: In this study, we assessed the plasma Hsp70 and Hsp27 levels in both not cognitively impaired (NCI) subjects with and without converting to mild cognitive impairment (MCI) groups (Non conversion group, N¼36; Conversion group, N¼30). Results: After three to four years follow-up period, comparison of the plasma Hsp70 and Hsp27 levels of the two groups revealed that only the plasma Hsp70 level was associated with an conversion to MCI after adjustment of age, gender, education year, follow-up duration, hypertension and diabetes (Repeated measure ANOVA: F¼9.33, p ¼0.003). Furthermore, increasing plasma Hsp70 level was associated with cognitive dysfunction in language and executive function (Generalized estimating equations: Korean Boston Naming Test, -4.23 (-7.80, -0.67), p ¼0.020; Stroop Test, -2.63 (-4.16, -0.91), p ¼0.002). Conclusions: These findings suggest that the plasma Hsp70 level may be related to cognitive decline in the elderly.
y
T-test or 2 test were tested. All values are mean6SD (continuous variables) or frequencies (N,%)(categorical variables). Abbreviation: NCI¼not cognitive impaired subject, HTN¼Hypertension, DM¼Diabetes Mellitus, BMI¼Body Mass Index, KSGDS¼ Short Form Geriatric Depression Scale Korean version. Table 2 Plasma heat shock protein 70 and 27 level at baseline and at the follow-upy
HSP70 (ng/ml) HSP27 (ng/ml)
Total Non conversion conversion Total Non conversion conversion
Baseline
Follow up
F
p
0.1560.03 0.1560.03 0.1660.02 0.3360.25 0.3160.24 0.3460.26
0.5160.46 0.3360.22 0.7160.57 0.6761.02 0.5560.87 0.8261.18
0.07
0.798
0.29
0.591
y Repeated measure ANOVA was tested after adjusting for age, gender, education year, follow-up duration. Abbreviation: HSP¼heat shock protein Table 3 Association between the change in heat shock protein level and cognitive function over the follow-up period y Dependent variables HSP 70 K-BNT SVLT-recall RCFT-copy COWAT Stroop test HSP 27 K-BNT SVLT-recall RCFT-copy COWAT Stroop test
B
Std. Err
95% C.I.
p
-4.23 -0.75 -1.46 -1.62 -2.53
1.82 0.39 2.71 0.79 0.83
[-7.80, -0.67] [-1.52, 0.02] [-6.76, 3.85] [-3.16, -0.08] [-4.16, -0.91]
0.020 0.056 0.591 0.039 0.002
-1.35 -0.10 0.25 -0.29 -0.13
0.73 0.26 0.90 0.30 0.49
[-2.78, 0.07] [-0.61, 0.41] [-1.52, 2.02] [-0.87, 0.29] [-1.09, 0.83]
0.063 0.692 0.783 0.328 0.791
y Generalized estimating equations was tested Adjusted by age, gender, education year, follow-up duration, hypertension and diabetes Abbreviation: K-BNT¼Korean Boston Naming Test, SVLT¼Seoul Verbal Learning Test, RCFT¼Rey-Complex Figure Test, COWAT¼Controlled Oral Word Association Test.
Figure 1. Interactive effect between change in heat shock protein level and subject groupsy y Repeated measure ANOVA was tested Adjusted by age, gender, education year, follow-up duration, hypertension and diabetes (HSP70: F¼9.33 p¼0.003; HSP27:F¼0.60, p¼0.443) P3-123
HUMAN AUTOANTIBODIES TO TAU PROTEIN: HOW SPECIFIC ARE THEY?
Jan Ricny, Michala Kolarova, Ales Bartos, Daniela Ripova, Prague Psychiatric Center, Prague, Czech Republic. Contact e-mail: ricny@pcp. lf3.cuni.cz Background: Antibodies against tau protein are found in human sera and they might play a role in some pathological conditions (Alzheimer disease, tauopathies) and/or serve as disease-status indicators. ELISA technique is often used for the detection/quantification; however, concentration of these antibodies in sera represents just a tiny fraction of total immunoglobulins and therefore relatively low dilutions are used in such assays. Unspecific signal under such conditions can cause a significant distortion. Methods: Conventional ELISA procedure, wells coated with recombinant tau protein, negative controls obtained by several procedures (e.g. detergent inactivation, coating with calf serum, bovine serum albumin, gelatin, casein) was applied.We measured antibodies to tau in sera samples from patients with Alzheimer disease (fulfiling NIAAA criteria), multiple sclerosis and cognitively normal control subjects. Several procedures for negative controls were tried to distinguish specific and unspecific binding. Incubation under dissociating conditions, permitting the binding of just high-affinity antibodies were employed as well. Results: Our experiments showed quite high diversity in signals measured both in wells coated with tau proteins and in inactivated wells (e.g. blanks). The negative signals varied among sera and there was no correlation between unspecific signals and specific antibody titers. Signals obtained for different inactivation method varied as well, showing some "specificity" for a given inactivating protocol. Conclusions: Use of the negative control for each measured serum and dilution is a necessity for obtaining relevant information concerning specific antibody concentrations. The 13-26601S, P304/12/G069 and study was supported by grant GACR IGA NT 13183.
Poster Presentations: P3
Colombia; 2Universidad Antonio Nari~no, Bogota, Colombia. Contact e-mail: [email protected] Background: Studies in humans have shown the potential role of alterations in DNA methylation in the pathogenesis of Late-Onset Alzheimer’s disease (LOAD). The epigenetic regulation of the genes involved in the Alzheimer’s pathogenesis may help to explain the complexity of the LOAD. Methods: DNA was extracted from peripheral blood mononuclear cells of 100 Colombian AD patients (mean age 77.56 5 years) and 100 healthy subjects (mean age 75.36 5,5years). DNA was bisulfite treated using EZ DNA Methylation Direct Kit (Zymo-research). Primers were manually designed for the 5’ region of the CpG Island in the APP promoter and APOE CpG Island on exon 4. The APP and APOE MS-HRMs were performed in a CFX 96 device (BioRad), using 14 ng of bisulfite treated DNA. The quantitative results were obtained by interpolation of the first derived RFU normalized data, generated by the linear regression of the standard curve. Results: The correlation coefficient of the linear regression of the standard curves was r2¼ 0.96 for APP gen and 0.898 for APOE gen The inter-assay and intra-assay reproducibilities were verified, having a coefficient of variance less than 0.5 between replicates. In a preliminary analysis we did not find differences in APP methylation levels between AD patients and control subjects (mean 2.14% +- 1.15 vs 2.05 +- 0.96). For APOE the difference was not significant in methylation levels (patients vs. Controls 73.01 vs 70.9) Conclusions: The present study tested changes in DNA methylation of in blood samples from Colombian LOAD patients, which showed no significant differences in comparison with control samples for APP and APOE genes. This is the first report of the analysis of APP and APOE DNA methylation levels in LOAD using the quantitative and cost-effective in Latin American AD patients. It would be important to analyze, in further studies, the methylation levels for additional candidate regions in several tissues from AD patients.
P3-120
STRUCTURAL ELUCIDATION OF THE GLYCOSYLATION PROFILES OF HUMAN PLASMA CLUSTERIN IN ALZHEIMER’S DISEASE PATIENTS
Hui-Chung Liang1, Claire Russell2, Raymond Chung3, Chantal Bazenet3, Abdul Hye4, Simon Lovestone4, Ian Pike5, Malcolm Andrew Ward1, 1 Proteome Sciences PLC, London, United Kingdom; 2Proteome Sciences PLC, London, United Kingdom; 3Institute of Psychiatry, King’s College London, London, United Kingdom; 4King’s College London, London, United Kingdom; 5Proteome Sciences PLC, Cobham, United Kingdom. Contact e-mail: [email protected] Background: Clusterin is a highly glycosylated secreted protein implicated in the pathogenesis of Alzheimer’s disease (AD). Expression of the clusterin gene is significantly elevated in AD brain (May et al., 1990) and levels of plasma clusterin correlate with AD progression (Thambisetty et al., 2010). Since glycosylation plays an important role in the physiological functions of clusterin (Stuart et al., 2007), we have developed an IP-LC/MS/MS workflow and performed detailed profiling of plasma clusterin glycosylation, including determination of glycosylation sites and structural characterisation of glycosylated peptides. In this study, glycosylation analysis of plasma clusterin is performed to investigate the correlation between its glycosylation pattern and AD progression. Methods: Plasma clusterin from 28 patients was enriched using our established immunoprecipitation workflow, and analysed via LC-MS/MS using nanoflow reverse-phase chromatography on both the Orbitrap Velos and Orbitrap Fusion (ThermoScientific, USA). Glycopeptides were manually identified by the presence of glycan-specific oxonium ion fragments, m/ z 204.08 for N-acetylhexosamine (HexNAc), m/z 366.14 for hexose-Nacetylhexosamine (Hex-HexNAc), and m/z 657.24 for N-acetylneuraminic acid-hexose-N-acetylhexosamine (NeuAc-Hex-HexNAc) in the MS/MS spectra. Results: Using our IP-LC/MS/MS workflow, all six known N-glycosylation sites were identified, three in the a subunit (a64N, a81N and a123N) and three in the b subunit (b64N, b127N, and b147N). In addition, we have determined the differences in the glycoforms associated at each of the different glycosylation sites. We are currently
comparing the clusterin glycoform distribution in plasma obtained from subjects of low atrophy (n¼14) and high atrophy (n¼14), and the results of this analysis will be presented at the meeting. Furthermore, we are developing a selected reaction monitoring (SRM) method with the aim of achieving a robust workflow suitable for rapid verification of specific clusterin glycoforms to aid AD biomarker discovery. Conclusions: A thorough characterisation of the clusterin glycopeptide profile using LC/MS/ MS and SRM will be a useful tool, allowing potential clusterin glycoform biomarkers of AD progression to be identified. P3-121
ASSOCIATION BETWEEN SERUM LIPIDS AND CEREBRAL AMYLOIDOSIS IN COGNITIVELY NORMAL ELDERLY
HyoJung Choi1, Min Soo Byun1, Young Min Choe1, Bo Kyung Sohn2, Dahyun Yi3, Ji Young Han3, Song E. Kim4, Hyewon Baek3, Eun Hyun Seo3, Jinsick Park5, Youngjin Lee6, Jong Inn Woo7, Dong Young Lee1, 1Seoul National University College of Medicine, Seoul, South Korea; 2Seoul Metropolitan Government-Seoul National University Boramae Medical Center, Seoul, South Korea; 3Seoul National University Hospital, Seoul, South Korea; 4Seoul National University Hospital, Seoul, South Korea; 5 Hanyang University, Seoul, South Korea; 6Department of Neurospychiatry, Seoul, South Korea; 7Seoul National University Medical Research Center, Seoul, South Korea. Contact e-mail: [email protected] Background: Cerebral amyloid-beta protein (Ab) deposition has been considered as a key initiating step in Alzheimer’s disease (AD) process. Although many preclinical studies have suggested the possible linkage between dyslipidemia and cerebral amyloidogenesis or amyloid deposition, the association between serum lipids and cerebral Ab deposition in human brain is still poorly known. We aimed to investigate such association in cognitively normal (CN) elderly individuals. Methods: Sixty two CN elderly individuals were included. All participants received comprehensive clinical and neuropsychological assessment based on the CERAD protocol, 11 C labeled Pittsburgh Compound B (PiB) positron emission tomography volumetric MRI, and quantification for serum lipid components including total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) with apolipoprotein A1 (APOA1) and B (APOB). Results: Global cerebral Ab deposition was defined as mean cortical PiB retention of the cortical regions including frontal, lateral temporal, lateral parietal and precuneus/posterior cingulate cortices. Univariate analyses showed significant positive association of mean cortical PiB retention with the levels of serum TG and APOB, and negative association with HDL-C and APOA1 levels. Multivariate statistical models that controlled age, education, gender, apolipoprotein E ε4 genotype revealed independent associations between the levels of TG and mean cortical 11 C-PiB retention. Higher serum TG level was associated with heavier cortical 11 C-PiB retention. No association was found between both total cholesterol and LDL-C, and cortical 11 C-PiB retention. Conclusions: Our findings strongly support the association between lipid profiles in serum, especially TG level, and the degree of cerebral amyloidosis in cognitively normal elderly people.The findings also suggest that the mechanisms through which serum lipid or lipoproteins affect cerebral Ab could provide novel targets of AD therapeutics. More specifically, various approaches known to reduce blood TG level, such as moderate exercise and omega-3 fatty acid intake, might be helpful for slowing Ab deposition and reducing AD occurrence. P3-122
INCREASED PLASMA LEVELS OF HEAT SHOCK PROTEIN 70 ASSOCIATED WITH SUBSEQUENT CLINICAL CONVERSION IN NORMAL ELDERLY
Hyunwoong Roh1, Sang Joon Son2, Chang Hyung Hong3, Kang Soo Lee4, Ki Jung Chang5, 1Ajou University Hospital, Suwon, South Korea; 2Myoungji Hospital, Goyang-si, South Korea; 3Ajou University School of Medicine, Suwon-si, South Korea; 4CHA Gangnam Medical Center, CHA University, Seoul, South Korea; 5CHA Gangnam Medical Center, CHA University, Suwon, South Korea. Contact e-mail: [email protected]
Poster Presentations: P3 Table 1 Demographic and clinical characteristics of subjectsy
Age baseline Gender female Education Follow up duration HTN DM BMI (kg/m2) Baseline Follow up KSGDS Baseline Follow up
NCI to NCI Non conversion (N¼36)
NCI to MCI conversion (N¼30)
70.0065.01 23 (63.89) 6.464.8 5.3660.68 21 (58.33) 2 (5.56)
69.9364.42 24 (80.00) 3.964.3 5.2360.56 17 (56.67) 5 (16.67)
0.06 2.07 2.18 0.82 0.02 2.13
0.955 0.180 0.033 0.417 0.545 0.231
25.6963.61 25.8763.81
25.3964.61 25.8364.61
0.26 0.04
0.794 0.972
5.1464.07 5.9765.12
6.3864.03 8.4366.74
-1.23 -1.69
0.224 0.097
t or
p
P673
Background: Heat shock proteins (Hsps) have been regarded as cytoprotectants that protect brain cells from damage encountered following cerebral ischemia or during the progression of neurodegenerative diseases. Methods: In this study, we assessed the plasma Hsp70 and Hsp27 levels in both not cognitively impaired (NCI) subjects with and without converting to mild cognitive impairment (MCI) groups (Non conversion group, N¼36; Conversion group, N¼30). Results: After three to four years follow-up period, comparison of the plasma Hsp70 and Hsp27 levels of the two groups revealed that only the plasma Hsp70 level was associated with an conversion to MCI after adjustment of age, gender, education year, follow-up duration, hypertension and diabetes (Repeated measure ANOVA: F¼9.33, p ¼0.003). Furthermore, increasing plasma Hsp70 level was associated with cognitive dysfunction in language and executive function (Generalized estimating equations: Korean Boston Naming Test, -4.23 (-7.80, -0.67), p ¼0.020; Stroop Test, -2.63 (-4.16, -0.91), p ¼0.002). Conclusions: These findings suggest that the plasma Hsp70 level may be related to cognitive decline in the elderly.
y
T-test or 2 test were tested. All values are mean6SD (continuous variables) or frequencies (N,%)(categorical variables). Abbreviation: NCI¼not cognitive impaired subject, HTN¼Hypertension, DM¼Diabetes Mellitus, BMI¼Body Mass Index, KSGDS¼ Short Form Geriatric Depression Scale Korean version. Table 2 Plasma heat shock protein 70 and 27 level at baseline and at the follow-upy
HSP70 (ng/ml) HSP27 (ng/ml)
Total Non conversion conversion Total Non conversion conversion
Baseline
Follow up
F
p
0.1560.03 0.1560.03 0.1660.02 0.3360.25 0.3160.24 0.3460.26
0.5160.46 0.3360.22 0.7160.57 0.6761.02 0.5560.87 0.8261.18
0.07
0.798
0.29
0.591
y Repeated measure ANOVA was tested after adjusting for age, gender, education year, follow-up duration. Abbreviation: HSP¼heat shock protein Table 3 Association between the change in heat shock protein level and cognitive function over the follow-up period y Dependent variables HSP 70 K-BNT SVLT-recall RCFT-copy COWAT Stroop test HSP 27 K-BNT SVLT-recall RCFT-copy COWAT Stroop test
B
Std. Err
95% C.I.
p
-4.23 -0.75 -1.46 -1.62 -2.53
1.82 0.39 2.71 0.79 0.83
[-7.80, -0.67] [-1.52, 0.02] [-6.76, 3.85] [-3.16, -0.08] [-4.16, -0.91]
0.020 0.056 0.591 0.039 0.002
-1.35 -0.10 0.25 -0.29 -0.13
0.73 0.26 0.90 0.30 0.49
[-2.78, 0.07] [-0.61, 0.41] [-1.52, 2.02] [-0.87, 0.29] [-1.09, 0.83]
0.063 0.692 0.783 0.328 0.791
y Generalized estimating equations was tested Adjusted by age, gender, education year, follow-up duration, hypertension and diabetes Abbreviation: K-BNT¼Korean Boston Naming Test, SVLT¼Seoul Verbal Learning Test, RCFT¼Rey-Complex Figure Test, COWAT¼Controlled Oral Word Association Test.
Figure 1. Interactive effect between change in heat shock protein level and subject groupsy y Repeated measure ANOVA was tested Adjusted by age, gender, education year, follow-up duration, hypertension and diabetes (HSP70: F¼9.33 p¼0.003; HSP27:F¼0.60, p¼0.443) P3-123
HUMAN AUTOANTIBODIES TO TAU PROTEIN: HOW SPECIFIC ARE THEY?
Jan Ricny, Michala Kolarova, Ales Bartos, Daniela Ripova, Prague Psychiatric Center, Prague, Czech Republic. Contact e-mail: ricny@pcp. lf3.cuni.cz Background: Antibodies against tau protein are found in human sera and they might play a role in some pathological conditions (Alzheimer disease, tauopathies) and/or serve as disease-status indicators. ELISA technique is often used for the detection/quantification; however, concentration of these antibodies in sera represents just a tiny fraction of total immunoglobulins and therefore relatively low dilutions are used in such assays. Unspecific signal under such conditions can cause a significant distortion. Methods: Conventional ELISA procedure, wells coated with recombinant tau protein, negative controls obtained by several procedures (e.g. detergent inactivation, coating with calf serum, bovine serum albumin, gelatin, casein) was applied.We measured antibodies to tau in sera samples from patients with Alzheimer disease (fulfiling NIAAA criteria), multiple sclerosis and cognitively normal control subjects. Several procedures for negative controls were tried to distinguish specific and unspecific binding. Incubation under dissociating conditions, permitting the binding of just high-affinity antibodies were employed as well. Results: Our experiments showed quite high diversity in signals measured both in wells coated with tau proteins and in inactivated wells (e.g. blanks). The negative signals varied among sera and there was no correlation between unspecific signals and specific antibody titers. Signals obtained for different inactivation method varied as well, showing some "specificity" for a given inactivating protocol. Conclusions: Use of the negative control for each measured serum and dilution is a necessity for obtaining relevant information concerning specific antibody concentrations. The 13-26601S, P304/12/G069 and study was supported by grant GACR IGA NT 13183.