- Email: [email protected]
P3-281
S458
Poster P3:: Tuesday Posters
erating, either AD or PD. Four different groups have been analyzed: AD and PD patients as well as healthy young and aged-matched non-demented individuals (n ⬎ 30 for all groups). Results: Our findings indicate that all elderly groups have a higher Treg (CD4⫹Foxp3⫹) frequency compared to the young group, without any difference between the elderly groups. In addition, we observed increasing Treg activity for all elderly groups compared to the young group. However, no difference between the aged healthy individuals and AD patients could be observed. In contrast, Treg activity in PD patients was significantly higher compared to the aged healthy donors. The increase of suppressive activity was thereby independent of the Foxp3 expression level. Conclusion: Our observations, of quantitative and qualitative differences already in the total pool of peripheral Treg between young and elderly groups, identify Treg as an immunecell type undergoing immunological senescence. Taking into account that changes in Treg function increase with a neurodegenerative phenotype present in PD patients, our data support the hypothesis of an existing beneficial autoimmune effector mechanism in these patients, which is suppressed by Treg. Furthermore, we cannot exclude that by analyzing a more specialized subset of Treg in AD patients a similar result would be obtained for this group. Consequently, it will be of special interest to analyze Treg specific for CNS antigens relevant to AD and PD, in particular amyloid-beta and alpha-synuclein. P3-281
ADVANCED GLYCATED END PRODUCTS PEPTIDES INDUCE A PATHOLOGIC RELEASE OF INFLAMMATORY CYTOKINES, EXPECIALLY DURING AMYLOID-BETA EXPOSURE, IN SUBJECTS WITH ALZHEIMER’S DISEASE
Sebastiano B. Solerte1, Annunziata Lapolla2, Eleonora Locatelli1, Valeria Mansi1, Roberto Bonacasa3, Nicola Schifino3, Concetta Paolucci1, Serena Sarra1, Ester Marazzi1, Marisa Fioravanti1, 1 University of Pavia, Pavia, Italy; 2Department of Medical Sciences, University of Padova, Padova, Italy; 3A.S.P.S. Margherita Geriatric Institute, Pavia, Italy. Contact e-mail: [email protected] Background: Advanced glycation end products/peptides (AGEs) can induce metabolic disorders, oxydative stress and inflammatory reaction in tissues and in particular into the brain. Therefore, AGEs could negatively be involed in Alzheimer’s disease physiopathology. Objectives: Within this pathogenetic context, we evaluate the proinflammatory pattern of circulating blood lymphomononuclear cells (BLMC) of AD subjects during a dose-dependent load of AGEs, together with plasma AGEs and the glyco-oxidative molecule Pentosidine. Methods: Twenty-seven mild to moderate sporadic AD subjects and 22 healthy old subjects were studied. Plasma AGEs and Pentosidine were determined by hs-mass spectrometry. BLMC, extracted by Ficoll/Hypaque and concentrated at 7,75 ⫻ 106 cells ⫻ mL, were incubated with glycated human serum albumin (GHSA: 5, 10, 20, 50 g/mL) and with GHSA-20 g/mL⫹A 1-42 fragment (0.5 g/mL) in order to evaluate the secretion of cytokines TNF-␣ and IFN-␥ (ELISA, R&D Systems) in the supernates of cultured BLMC. Results: Higher secretion of TNF-␣ and IFN-␥ by BLMC of AD subjects was found after incubation with GHSA than in healthy subjects; mean TNF-␣/IFN-␥: AD-group 230/300 pg/mL (baseline), 310/421 pg/mL (GHSA-5 g/mL), 416/502 pg/mL (GHSA-10 g/mL), 530/677 pg/mL (GHSA-20 g/mL), 757/936 pg/mL (GHSA-50 g/mL); Control-group 128/180 pg/ mL,p⬍0.001 (baseline), 148/219 pg/mL,p⬍001 (GHSA-5 g/mL), 186/ 282 pg/mL,p⬍0.001 (GHSA-10 g/mL), 237/341 pg/mL,p⬍0.001 (GHSA-20 g/mL), 297/416 pg/mL,p⬍0.001 (GHSA-50 g/mL). The co-incubation of GHSA with A further increase TNF-␣/IFN-␥ release in AD compared to healthy subjects 1180/1400 pg/mL vs 380/320 pg/mL, p⬍0.001. Finally, elevated plasma AGEs/Pentosidine levels were demonstrated in AD than in control subjects (mean: 14.5/196 g/mL and 8.1/110 pmol/mL respectively,p⬍0.001; while Pentosidine levels correlate with MMSE score (r⫽-0.79,p⬍0.001). Conclusions: Metabolic abnormalities involving AGEs and glyco-oxidative markers, i.e. Pentosidine, can trigger a proinflammatory reaction from BLMC of AD subjects This reaction is
amplified by A 1-42 fragment, so suggesting a role for glyco-oxidative mechanism in AD immunopathogenesis. (Supported by a grant of the University of Pavia, Italy; FAR 2006 S.B. Solerte). P3-282
EXPRESSION OF COMPLEMENT SYSTEM COMPONENTS DURING AMYLOID DEPOSITION IN APP TRANSGENIC MICE
Julia Reichwald, Simone Danner, Karl-Heinz Wiederhold, Matthias Staufenbiel, Novartis Institutes for BioMedical Research, Basel, Switzerland. Contact e-mail: [email protected] Background: A role of the complement system in Alzheimer’s disease pathogenesis has been postulated. Using histology, complement components have been detected in association with amyloid deposits in AD brain which included markers for activation up to the membrane attack complex. In vitro, fibrillar A was demonstrated to activate complement suggesting an induction of the system by amyloid deposits in vivo. However, histological studies of amyloid containing APP transgenic mice have not replicated the findings in human brain, which may be due to a lack of expression, activation or antibody detection of mouse complement. On the other hand, genetic modification of the complement system in mice led to complex results regarding its role in the disease process. Objective: The expression of complement components and their induction by amyloid deposition was analyzed in mouse brain to better understand the observed differences to human AD brain. We also analyzed for complement activation. Methods: APP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 and BUB/BnJ mice were used at different ages. mRNA was quantified by Realtime PCR. Western blotting and histology was according to standard protocols. Results and Conclusions: While high mRNA levels were detected for early (in particular C1q) and several inhibitory complement components, expression of downstream components (in particular C9) was low. No major difference was found between the mouse strains and similar expression levels were observed with human brain material. Expression of most complement components increased with age in control mice. Several of these, most notably C1q and C3, showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was also detected on the protein level using Western blotting. In line with these results histology demonstrated amyloid plaque-associated C1q and C3 as well as C3d indicating activation of C3 convertase. These data show that the mouse complement system responds to amyloid deposition with increased expression and activation. The failure to detect activation of downstream components may be due to the lack of suited antibodies or a block of complete activation e.g. by the abundant inhibitory components. P3-283
DIFFERENTIAL REGULATION OF MICROGLIAL CYTOKINE PRODUCTION, PHAGOCYTOSIS AND MIGRATION BY NOREPINEPHRINE: HOW LOCUS CERULEUS DEGENERATION AFFECTS ALZHEIMER DISEASE
Michael T. Heneka1, Uwe Leimer1, Fred Van Leuven2, Magdalena Sastre1, Lucia Dumitrescu-Ozimek1, Douglas L. Feinstein3, 1 University of Mu¨nster, Mu¨nster, Germany; 2University of Leuven, Leuven, Belgium; 3University of Illinois at Chicago, Chicago, IL, USA. Contact e-mail: [email protected] Background: The locus ceruleus (LC) is the main site of cerebral norepinephrine (NE) production. Profound degeneration of LC subregions which predominantly project to the neocortex and hippocampus occurr early in Alzheimer’s disease (AD). Objective(s): Subsequently, cortical and hippocampal NE levels are decreasing. Since NE not only acts as a classical neurotransmitter but also modulates neuroinflammation, we evaluated NE effects on microglial functions including cytokine generation, migration and phagocytosis in vitro and in vivo. Meth-
Poster P3:: Tuesday Posters
erating, either AD or PD. Four different groups have been analyzed: AD and PD patients as well as healthy young and aged-matched non-demented individuals (n ⬎ 30 for all groups). Results: Our findings indicate that all elderly groups have a higher Treg (CD4⫹Foxp3⫹) frequency compared to the young group, without any difference between the elderly groups. In addition, we observed increasing Treg activity for all elderly groups compared to the young group. However, no difference between the aged healthy individuals and AD patients could be observed. In contrast, Treg activity in PD patients was significantly higher compared to the aged healthy donors. The increase of suppressive activity was thereby independent of the Foxp3 expression level. Conclusion: Our observations, of quantitative and qualitative differences already in the total pool of peripheral Treg between young and elderly groups, identify Treg as an immunecell type undergoing immunological senescence. Taking into account that changes in Treg function increase with a neurodegenerative phenotype present in PD patients, our data support the hypothesis of an existing beneficial autoimmune effector mechanism in these patients, which is suppressed by Treg. Furthermore, we cannot exclude that by analyzing a more specialized subset of Treg in AD patients a similar result would be obtained for this group. Consequently, it will be of special interest to analyze Treg specific for CNS antigens relevant to AD and PD, in particular amyloid-beta and alpha-synuclein. P3-281
ADVANCED GLYCATED END PRODUCTS PEPTIDES INDUCE A PATHOLOGIC RELEASE OF INFLAMMATORY CYTOKINES, EXPECIALLY DURING AMYLOID-BETA EXPOSURE, IN SUBJECTS WITH ALZHEIMER’S DISEASE
Sebastiano B. Solerte1, Annunziata Lapolla2, Eleonora Locatelli1, Valeria Mansi1, Roberto Bonacasa3, Nicola Schifino3, Concetta Paolucci1, Serena Sarra1, Ester Marazzi1, Marisa Fioravanti1, 1 University of Pavia, Pavia, Italy; 2Department of Medical Sciences, University of Padova, Padova, Italy; 3A.S.P.S. Margherita Geriatric Institute, Pavia, Italy. Contact e-mail: [email protected] Background: Advanced glycation end products/peptides (AGEs) can induce metabolic disorders, oxydative stress and inflammatory reaction in tissues and in particular into the brain. Therefore, AGEs could negatively be involed in Alzheimer’s disease physiopathology. Objectives: Within this pathogenetic context, we evaluate the proinflammatory pattern of circulating blood lymphomononuclear cells (BLMC) of AD subjects during a dose-dependent load of AGEs, together with plasma AGEs and the glyco-oxidative molecule Pentosidine. Methods: Twenty-seven mild to moderate sporadic AD subjects and 22 healthy old subjects were studied. Plasma AGEs and Pentosidine were determined by hs-mass spectrometry. BLMC, extracted by Ficoll/Hypaque and concentrated at 7,75 ⫻ 106 cells ⫻ mL, were incubated with glycated human serum albumin (GHSA: 5, 10, 20, 50 g/mL) and with GHSA-20 g/mL⫹A 1-42 fragment (0.5 g/mL) in order to evaluate the secretion of cytokines TNF-␣ and IFN-␥ (ELISA, R&D Systems) in the supernates of cultured BLMC. Results: Higher secretion of TNF-␣ and IFN-␥ by BLMC of AD subjects was found after incubation with GHSA than in healthy subjects; mean TNF-␣/IFN-␥: AD-group 230/300 pg/mL (baseline), 310/421 pg/mL (GHSA-5 g/mL), 416/502 pg/mL (GHSA-10 g/mL), 530/677 pg/mL (GHSA-20 g/mL), 757/936 pg/mL (GHSA-50 g/mL); Control-group 128/180 pg/ mL,p⬍0.001 (baseline), 148/219 pg/mL,p⬍001 (GHSA-5 g/mL), 186/ 282 pg/mL,p⬍0.001 (GHSA-10 g/mL), 237/341 pg/mL,p⬍0.001 (GHSA-20 g/mL), 297/416 pg/mL,p⬍0.001 (GHSA-50 g/mL). The co-incubation of GHSA with A further increase TNF-␣/IFN-␥ release in AD compared to healthy subjects 1180/1400 pg/mL vs 380/320 pg/mL, p⬍0.001. Finally, elevated plasma AGEs/Pentosidine levels were demonstrated in AD than in control subjects (mean: 14.5/196 g/mL and 8.1/110 pmol/mL respectively,p⬍0.001; while Pentosidine levels correlate with MMSE score (r⫽-0.79,p⬍0.001). Conclusions: Metabolic abnormalities involving AGEs and glyco-oxidative markers, i.e. Pentosidine, can trigger a proinflammatory reaction from BLMC of AD subjects This reaction is
amplified by A 1-42 fragment, so suggesting a role for glyco-oxidative mechanism in AD immunopathogenesis. (Supported by a grant of the University of Pavia, Italy; FAR 2006 S.B. Solerte). P3-282
EXPRESSION OF COMPLEMENT SYSTEM COMPONENTS DURING AMYLOID DEPOSITION IN APP TRANSGENIC MICE
Julia Reichwald, Simone Danner, Karl-Heinz Wiederhold, Matthias Staufenbiel, Novartis Institutes for BioMedical Research, Basel, Switzerland. Contact e-mail: [email protected] Background: A role of the complement system in Alzheimer’s disease pathogenesis has been postulated. Using histology, complement components have been detected in association with amyloid deposits in AD brain which included markers for activation up to the membrane attack complex. In vitro, fibrillar A was demonstrated to activate complement suggesting an induction of the system by amyloid deposits in vivo. However, histological studies of amyloid containing APP transgenic mice have not replicated the findings in human brain, which may be due to a lack of expression, activation or antibody detection of mouse complement. On the other hand, genetic modification of the complement system in mice led to complex results regarding its role in the disease process. Objective: The expression of complement components and their induction by amyloid deposition was analyzed in mouse brain to better understand the observed differences to human AD brain. We also analyzed for complement activation. Methods: APP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 and BUB/BnJ mice were used at different ages. mRNA was quantified by Realtime PCR. Western blotting and histology was according to standard protocols. Results and Conclusions: While high mRNA levels were detected for early (in particular C1q) and several inhibitory complement components, expression of downstream components (in particular C9) was low. No major difference was found between the mouse strains and similar expression levels were observed with human brain material. Expression of most complement components increased with age in control mice. Several of these, most notably C1q and C3, showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was also detected on the protein level using Western blotting. In line with these results histology demonstrated amyloid plaque-associated C1q and C3 as well as C3d indicating activation of C3 convertase. These data show that the mouse complement system responds to amyloid deposition with increased expression and activation. The failure to detect activation of downstream components may be due to the lack of suited antibodies or a block of complete activation e.g. by the abundant inhibitory components. P3-283
DIFFERENTIAL REGULATION OF MICROGLIAL CYTOKINE PRODUCTION, PHAGOCYTOSIS AND MIGRATION BY NOREPINEPHRINE: HOW LOCUS CERULEUS DEGENERATION AFFECTS ALZHEIMER DISEASE
Michael T. Heneka1, Uwe Leimer1, Fred Van Leuven2, Magdalena Sastre1, Lucia Dumitrescu-Ozimek1, Douglas L. Feinstein3, 1 University of Mu¨nster, Mu¨nster, Germany; 2University of Leuven, Leuven, Belgium; 3University of Illinois at Chicago, Chicago, IL, USA. Contact e-mail: [email protected] Background: The locus ceruleus (LC) is the main site of cerebral norepinephrine (NE) production. Profound degeneration of LC subregions which predominantly project to the neocortex and hippocampus occurr early in Alzheimer’s disease (AD). Objective(s): Subsequently, cortical and hippocampal NE levels are decreasing. Since NE not only acts as a classical neurotransmitter but also modulates neuroinflammation, we evaluated NE effects on microglial functions including cytokine generation, migration and phagocytosis in vitro and in vivo. Meth-