T.7. Novel Dual-luminescence Biolayer Interferometry Assay to Measure the Damage Associated...
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T.6.5. Resetting Defective T Cell Homeostasis in Rheumatoid Arthritis (RA) by Targeting DNA Repair Mechanisms Lan Shao, Hiroshi Fujii, Ines Colmegna, Jorg Goronzy, Cornelia Weyand. Emory University, Atlanta, GA Background: Patients with RA have premature aging of their immune system, including naive T cells and hemato- poietic stem cells. With only some of the characteristics emblematic for physiologic aging, RA shares similarities with segmental aging syndromes. Genes implicated in genetic forms of segmental aging regulate DNA damage response pathways. Here, we have explored the intactness of DNA repair in RA T cells. Methods: CD4 T cells were isolated from untreated and treated RA patients (n = 85) and age/sex- matched controls (n = 76). Unstressed and stressed T cells (γ-irradiation) were tested for DNA repair by comet assay, and by quantifying, silencing and overexpressing the DNA repair enzyme ATM and the MRE11 complex (MRE11, NBS1, RAD50). T cell apoptosis was measured by flow cytometry. Results: In RA CD4 T cells, DNA damage was increased with 3-fold elevated tail moments (p = 0.0001). DNA repair after γ-radiation was blunted and delayed (p=0.01). ATM tran- scripts and protein were significantly lower than in controls. RA T cells produced less MRE11, NBS1 and RAD50, a defect mimicked by silencing ATM in healthy T cells. Forced ATM overexpression in RA T cells reconstituted expression of the MRE11 complex and restored DNA repair. Damaged DNA correlated with apoptosis susceptibility; reconstituting ATM rescued RA T cells from excessive death. ATM deficiency and DNA damage were unaffected by RA treatment. Con- clusion: RA T cells are deficient in the protein kinase ATM resulting in defective DNA repair, apoptosis and dispropor- tionate T cell loss. This defect imposes replicative stress upon the T cell pool straining homeostatic mechanisms, enforcing increased autoproliferation and biasing the T cell repertoire towards autoreactivity. Resetting T cell homeostasis by improving DNA repair emerges as a novel therapeutic target. doi:10.1016/j.clim.2009.03.134 T.7. Novel Dual-luminescence Biolayer Interferometry Assay to Measure the Damage Associated Molecular Pattern Molecule HMGB1 in Serum Jessica Turner, Louis Sparvero, Andrew Amoscato, Michael Lotze. University of Pittsburgh, Pittsburgh, PA HMGB1 is a multi-functional nuclear protein causing DNA binding and bending to allow access of various transcription factors to target DNA sequences. When actively secreted, it can act in conjunction with other factors to trigger recruitment and activation of cellular inflammatory media- tors. In the setting of damage, including cancer, transplan- tation, arthritis and sepsis, HMGB1 is released along with other factors from necrotic cells into the tissue micro- environment. In this setting, HMGB1 is recognized as a damage-associated molecular pattern (DAMP) molecule mediating inflammation and regulation of metabolism making it an important immunotherapeutic target. HMGB1 is detectable in the serum of patients with chronic in- flammation and malignant disease; increasing concentra- tions of this DAMP found in the serum directly correlates with disease progression and poor prognosis. The presence and amount of HMGB1 and other DAMPs found in patient serum may serve as valuable diagnostic markers to assess disease severity and/or treatment efficacy. In an effort to design a method to rapidly detect and measure HMGB1 in serum, we have assessed the utility of biolayer interferometry (BLI) technology to measure the binding kinetics of HMGB1- specific antibodies and HMGB1 and created an automated detection method based on dual-luminescence. Various con- centrations of HMGB1 in serum were detected in a sandwich ELISA-like fashion followed by dual-luminescence BLI. HMGB1 was detectable in 5- 50 % serum at concentrations of 3- 80 N ng/ml, final. This technique represents an auto- mated and sensitive system to detect DAMPs in patient bio- logical fluid samples. doi:10.1016/j.clim.2009.03.135 T.7.5. Development and Homeostasis of Langerhans Cells Laurent Chorro, Frédéric Geissmann. King's College London, London, UK Langerhans cells (LC) are located in multilayered epithe- lium, such as the epidermis. Their hematopoietic origin has been established in the 1970s. However, the mechanisms involved in their development and homeostasis are still not fully understood. It is currently believed that LC capture antigens in the skin and migrate to the lymph nodes to trigger or regulate T cell responses. In vitro, LC indeed develop into dendritic cells that stimulate T cells, but their function in vivo remains controversial. The purpose of our project is to define the mechanisms that control the establishment and homeostasis of the LC network through migration and proliferation of LC or their precursors in the mouse. Toward this aim, we investigate the role of candidate genes using reporter mice, mutant mice, flow cytometry, and intravital microscopy. These studies are performed in the perinatal period when the LC network develops, and in adults, both in homeostatic conditions and during the development of skin diseases such as atopic dermatitis, where the LC network undergoes dramatic changes. Our results should help to understand the molecular mechanisms that control the pro- liferation and migration of LC. doi:10.1016/j.clim.2009.03.136 T.8. Amelioration of Insulitis and Reversal of Diabetes in NOD Mice by Murine Anti-Thymocyte Globulin and Granulocyte-Colony Stimulating Factor Combination Therapy Matthew Parker, Song Xue, Clive Wasserfall, Martha Campbell-Thompson, Mark Atkinson. University of Florida, Gainesville, FL S48 Abstracts
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Transcript of T.7. Novel Dual-luminescence Biolayer Interferometry Assay to Measure the Damage Associated...
S48 Abstracts
T.6.5. Resetting Defective T Cell Homeostasis inRheumatoid Arthritis (RA) by Targeting DNARepair MechanismsLan Shao, Hiroshi Fujii, Ines Colmegna, Jorg Goronzy,Cornelia Weyand. Emory University, Atlanta, GA
Background: Patients with RA have premature aging oftheir immune system, including naive T cells and hemato-poietic stem cells. With only some of the characteristicsemblematic for physiologic aging, RA shares similarities withsegmental aging syndromes. Genes implicated in geneticforms of segmental aging regulate DNA damage responsepathways. Here, we have explored the intactness of DNArepair in RA T cells. Methods: CD4 T cells were isolated fromuntreated and treated RA patients (n=85) and age/sex-matched controls (n=76). Unstressed and stressed T cells(γ-irradiation) were tested for DNA repair by comet assay,and by quantifying, silencing and overexpressing the DNArepair enzyme ATM and the MRE11 complex (MRE11, NBS1,RAD50). T cell apoptosis was measured by flow cytometry.Results: In RA CD4 T cells, DNA damage was increased with3-fold elevated tail moments (p=0.0001). DNA repair afterγ-radiation was blunted and delayed (p=0.01). ATM tran-scripts and protein were significantly lower than in controls.RA T cells produced less MRE11, NBS1 and RAD50, a defectmimicked by silencing ATM in healthy T cells. Forced ATMoverexpression in RA T cells reconstituted expression of theMRE11 complex and restored DNA repair. Damaged DNAcorrelated with apoptosis susceptibility; reconstituting ATMrescued RA T cells from excessive death. ATM deficiencyand DNA damage were unaffected by RA treatment. Con-clusion: RA T cells are deficient in the protein kinase ATMresulting in defective DNA repair, apoptosis and dispropor-tionate Tcell loss. This defect imposes replicative stress uponthe T cell pool straining homeostatic mechanisms, enforcingincreased autoproliferation and biasing the T cell repertoiretowards autoreactivity. Resetting T cell homeostasis byimproving DNA repair emerges as a novel therapeutic target.
doi:10.1016/j.clim.2009.03.134
T.7. Novel Dual-luminescence BiolayerInterferometry Assay to Measure the DamageAssociated Molecular Pattern Molecule HMGB1in SerumJessica Turner, Louis Sparvero, Andrew Amoscato, MichaelLotze. University of Pittsburgh, Pittsburgh, PA
HMGB1 is a multi-functional nuclear protein causing DNAbinding and bending to allow access of various transcriptionfactors to target DNA sequences. When actively secreted,it can act in conjunction with other factors to triggerrecruitment and activation of cellular inflammatory media-tors. In the setting of damage, including cancer, transplan-tation, arthritis and sepsis, HMGB1 is released along withother factors from necrotic cells into the tissue micro-environment. In this setting, HMGB1 is recognized as adamage-associated molecular pattern (DAMP) moleculemediating inflammation and regulation of metabolism
making it an important immunotherapeutic target. HMGB1is detectable in the serum of patients with chronic in-flammation and malignant disease; increasing concentra-tions of this DAMP found in the serum directly correlates withdisease progression and poor prognosis. The presence andamount of HMGB1 and other DAMPs found in patient serummay serve as valuable diagnostic markers to assess diseaseseverity and/or treatment efficacy. In an effort to design amethod to rapidly detect and measure HMGB1 in serum, wehave assessed the utility of biolayer interferometry (BLI)technology to measure the binding kinetics of HMGB1-specific antibodies and HMGB1 and created an automateddetection method based on dual-luminescence. Various con-centrations of HMGB1 in serum were detected in a sandwichELISA-like fashion followed by dual-luminescence BLI.HMGB1 was detectable in 5- 50 % serum at concentrationsof 3- 80Nng/ml, final. This technique represents an auto-mated and sensitive system to detect DAMPs in patient bio-logical fluid samples.
doi:10.1016/j.clim.2009.03.135
T.7.5. Development and Homeostasis ofLangerhans CellsLaurent Chorro, Frédéric Geissmann. King's CollegeLondon, London, UK
Langerhans cells (LC) are located in multilayered epithe-lium, such as the epidermis. Their hematopoietic origin hasbeen established in the 1970s. However, the mechanismsinvolved in their development and homeostasis are still notfully understood. It is currently believed that LC captureantigens in the skin and migrate to the lymph nodes to triggeror regulate T cell responses. In vitro, LC indeed develop intodendritic cells that stimulate T cells, but their function invivo remains controversial. The purpose of our project is todefine the mechanisms that control the establishment andhomeostasis of the LC network through migration andproliferation of LC or their precursors in the mouse. Towardthis aim, we investigate the role of candidate genes usingreporter mice, mutant mice, flow cytometry, and intravitalmicroscopy. These studies are performed in the perinatalperiod when the LC network develops, and in adults, both inhomeostatic conditions and during the development of skindiseases such as atopic dermatitis, where the LC networkundergoes dramatic changes. Our results should help tounderstand the molecular mechanisms that control the pro-liferation and migration of LC.
doi:10.1016/j.clim.2009.03.136
T.8. Amelioration of Insulitis and Reversal ofDiabetes in NOD Mice by Murine Anti-ThymocyteGlobulin and Granulocyte-Colony Stimulating FactorCombination TherapyMatthew Parker, Song Xue, Clive Wasserfall, MarthaCampbell-Thompson, Mark Atkinson. University of Florida,Gainesville, FL
