signaling may regulate the pathogenesis of adoptive trans-ferred myocarditis via a CCR3-dependent pathway. In viralmyocarditis, CD8+ T-cells have been speculated to be one ofthe initiators of irreversible myocardiocyte injury.

doi:10.1016/j.clim.2006.04.294

Sa.63. Successful Identification of Necrotic CellDeath at the Single Cell Level: Flow CytometricAnalysis of High Mobility Group B1 and Histone H1.Norimasa Ito, Richard DeMarco, Michael Lotze. Surgery andMolecular Medicine, University of Pittsburgh School ofMedicine, Pittsburgh, PA.

Necrosis, or Type III death, is distinguished on amorphologic basis from apoptotic [Type I] and autophagic[Type II] death. High-mobility group B 1 protein (HMGB1) isa chromatin-binding protein released when cells diefollowing necrotic cell death and also, but sequestered incells during apoptotic, autophagic, or platinum-induceddeath. We developed a highly sensitive method to analyzenecrotic cell death by detecting both intracellular HMGB1and Histone H1 using flow cytometry. Methods: We induced1) Apoptosis by UV irradiation, 2) Necrosis by detergent inhuman melanoma cell lines (451Lu, WM3248 or WM9).Annexin-V and Sytox Orange is used for detection ofapoptosis and necrosis. HMGB1, LDH, ATP, Uric Acid in thesupernatant were also measured. We also induced tumorcytolysis by PBMC, CD4 cells, CD8 cells, or NK cells with orwithout IL-2 stimulation. Results: 4-16 ng/ml of HMGB1were detected with detergent lysis at 1 � 106 cells/ml, butbelow the lower detection limit in an ELISA [b2—5 ng/ml] inthe supernatant of untreated cells. Conversely, by flowcytometric analysis, HMGB1 release could clearly beidentified with detergent lysis, but not by UV irradiation.Following detergent lysis, LDH, ATP, Uric Acid was alsoreleased but HMGB1 below the limits of detection in thesupernatant, Sytox Orange staining was positive, andAnnexin V negative. Thus, flow cytometry was very usefulin detection of HMGB1 release from tumor induced by IL-2activated PBMC. LDH, ATP, was released into the superna-tant, but Uric Acid was not released. CD4+ T-cells were theprimary cells inducing HMGB1 release. Conclusions: Dualstaining with HMGB1 and Histone H1 can be applied todetect necrotic death at a single cell level. We coulddetect induction of necrotic cell death of tumor cells by IL-2 activated lymphocytes.

doi:10.1016/j.clim.2006.04.295

Sa.64. Chemokines Regulate Innate Immunity andChemotaxis Through Suppressor of CytokineSignalling (SOCS) Proteins.Nigel Stevenson,1 Mark Addely,2 David Greaves,2

James Johnston.1 1Immunology, Queen’s University ofBelfast, Belfast, United Kingdom; 2Sir William Dunne Schoolof Pathology, University of Oxford, Oxford, UnitedKingdom.

Suppressors Of Cytokine Signalling (SOCS) are encoded byimmediate early genes that are known to inhibit cytokineresponses. These proteins are induced by a large number ofcytokines and subsequently inhibit signalling, as part of afeedback loop. Although SOCS gene expression has beenshown to be induced by a number of cytokines, growthfactors and innate immune stimuli such as lipopolysaccha-ride (LPS), whether their expression is induced by chemo-tactic stimuli has not been widely investigated. In this studywe report that the chemoattractants eotaxin, interleukin-8 (IL-8) and N-formyl-Methionyl-Leucyl-Phenylalanine(FMLP) regulate SOCS expression in primary human cells.We show that chemoattractant induced SOCS-1 expressionsuppressed both the LPS and Granulocyte-Colony Stimulat-ing Factor (G-CSF) signal transduction pathways. Further-more, we demonstrate that both SOCS-1 and SOCS-3 areupregulated in response to eotaxin and that these regulateeotaxin induced migration. These results not only implicateSOCS proteins in cross-talk between the chemoattractantsignal transduction pathway and both bacterial product andcytokine pathways, but also show their importance incontrolling migration of cells along a chemotactic gradient.

doi:10.1016/j.clim.2006.04.296

Sa.65. Influence of Eotaxin 67gNa Polymorphism onPlasma Eotaxin Concentrations in MyocardialInfarction Survivors and Healthy Controls.Yuri Sheikine,1 Birgitta Olsen,2 Baback Gharizadeh,3

Ken Jatta,2 Per Tornvall,4 Mehran Ghaderi.5 1Center forMolecular Medicine, Karolinska University Hospital,Stockholm, Sweden; 2Department of Clinical Medicine,University of Orebro, Orebro, Sweden; 3Stanford GenomeTechnology Center, Stanford University, Palo Alto, CA;4Department of Cardiology, Karolinska University Hospital,Stockholm, Sweden; 5Department of Clinical Pathology andCytology, Karolinska University Hospital, Stockholm,Sweden.

Objectives: Eotaxin (CCL11) is a CC chemokine, whosesystemic levels might be associated with coronary arterydisease (CAD) and genetic variants predispose to myocardialinfarction (MI). This study aimed to investigate thepotential relationship between genetic variants of eotaxinand its plasma concentrations in CAD patients. Methods:Using Pyrosequencing technology we genotyped 311patients, who survived a first MI and 338 healthy controlsfor a 67GNA single nucleotide polymorphism (SNP) in theeotaxin gene. By measuring plasma eotaxin concentrationsin those subjects we related the former to the presence of67GNA SNP. Results: There were no differences in eotaxingenotype frequencies between patients and controls.Patient G/G carriers had higher circulating eotaxin levelscompared both to G/A and A/A patients (P = 0.046) and G/G controls (P = 0.028), which might indicate the influenceof additional factors (e.g. inflammatory mediators) oneotaxin secretion in those patients. At the same time,eotaxin levels did not differ between patients and controlsirrespective of the 67GNA SNP variants they carried. Therewere no associations between plasma eotaxin levels,

Abstracts S127