S93Abstracts

caspase axes (3 and 9) in tolerized Ti. In addition, the an-tiapoptotic genes survivin (2.24-fold, p=0.0285), COX-2(7.20-fold, p=0.0056), and the p21-activated kinase Pak7(3.59-fold, p=0.0398) were upregulated. Interestingly, thesurvivin and COX-2 genes are targets of the antiapoptotictranscription factor NF-κB, and caspase-3 and -9 signalingpathways are intimately involved in NF-κB regulation. Weare currently delineating the roles Apaf1, COX-2, andselectin play in NF-κB-driven Ti apoptosis pathways withRNA interference, pharmacological inhibition, and proteinstability assays.

doi:10.1016/j.clim.2008.03.259

Sa.39. Reduced Levels of FasL and ApoptosisResistance in SJIA MonocytesShivani Srivastava, Claudia Macaubas, Tzielan Lee,Christy Sandborg, Elizabeth Mellins. Stanford University,Stanford, CA

Systemic juvenile idiopathic arthritis (SJIA) is a chronicinflammatory childhood disease characterized by a combi-nation of systemic features and arthritis. Microarrayanalysis of PBMC in paired samples from patients (n=16) atflare and quiescence showed both anti- and pro-apoptosisgenes were overexpressed in SJIA flare. We therefore ana-lyzed apoptosis in separated monocytes and lymphocytes.At flare, anti-apoptotic genes were overexpressed in mo-nocytes relative to lymphocytes, while pro-apoptotic geneswere overexpressed in lymphocytes relative to monocytes.Incubation in low-serum media showed that SJIA mono-cytes were more resistant to apoptosis induction than con-trol monocytes (p=0.02), even in cells obtained at diseasequiescence. To determine the contribution of Fas/FasLpathways, we measured surface Fas and FasL levels in 13SJIA and 9 control monocyte samples immediately ex vivoand 9 SJIA and 6 control monocytes samples after 24-hourincubation in low-serum media. FasL levels were compar-able in SJIA and control monocytes ex vivo but were sig-nificantly lower in SJIA monocytes after apoptosis induction(p=0.0076). To determine whether the downstream Fas/FasL pathway was defective, we incubated 9 SJIA and 9control monocytes samples with anti-Fas antibody and mea-sured apoptosis via 7-AAD staining. Apoptosis levels werenot significantly different between SJIA and control mono-cytes, arguing that the intracellular Fas/FasL pathway isintact in SJIA monocytes. These data indicate that failure toupregulate FasL is the primary defect in the Fas/FasL path-way contributing to apoptosis resistance in SJIA monocytes.Notably, initial studies suggest that alterations in the mi-tochondrial pathway contribute to apoptosis resistance inSJIA monocytes.

doi:10.1016/j.clim.2008.03.260

Sa.40. The Role of Resting Cells on Anergy Inductionin Peripheral Blood Mononuclear Cells fromSystemic Lupus Erythematosus Patients

Diana Gómez-Martín, Mariana Díaz-Zamudio, María InésVargas, Jorge Alcocer-Varela. Instituto Nacional de CienciasMedicas y Nutricion, Salvador Zubiran, Mexico City, Mexico

Discrepancy exists among the defects shown by T cellsfrom Systemic Lupus Erythematosus (SLE) patients, whichdisplay features of anergic cells, although a resistant toanergy phenotype has been evidenced. This may be relatedto intrinsic defects or microenvironmental changes, whichconvey a high threshold for in vitro activation and anergyinduction. The aim of this study was to evaluate the impactof rest on the proliferative and tolerizing response from SLEpatients mononuclear cells. Five patients with inactive SLEand five controls were included. Peripheral blood mono-nuclear cells (PBMC) were isolated by Ficoll Hypaquemethod. Six experimental conditions were defined asfollowed: activation (plate bound anti-CD3 + soluble anti-CD28), anergy (ionomycin), without stimuli (RPMI media);each one, with or without previous rest (72 hs.). Cellularproliferation was addressed by Carboxy-fluorescein diace-tate, succinimidyl ester and flow cytometry. PBMC from SLEpatients shown a lower proliferation index in response toactivation protocol when compared to controls (11.54±340.96 vs 111.7±516.1, p=0.047; 83.34±116.1 vs 276.72±242.18, p=0.028).This difference was higher for the PBMCwith previous rest. PBMC from SLE patients displayed aresistant to anergy phenotype, even in the presence of pre-vious rest (1.3±5.54 vs 0.62±13.04, pN0.05). These findingssuggest that PBMC from SLE patients show an intrinsicdefect related to the modulation of the T cell receptorthreshold to proliferative and tolerizing conditions. Thus,microenvironmental changes might not be able to reversethe intrinsic defects shown by PBMC from SLE patients,denoting the possible role of alterations in the anergyinduced genetic program.

doi:10.1016/j.clim.2008.03.261

Sa.41. The Effect of Chloroquine Add-on Therapy onTLR9 Expression in Systemic Lupus ErythematosusAlma-Martina Cepika,1 Dragica Soldo-Juresa,2 JadrankaMorovic-Vergles,2 Branko Malenica,3 Alenka Gagro.11Institute of Immunology, Zagreb, Croatia; 2Dubrava ClinicalHospital, Zagreb, Croatia; 3Zagreb University HospitalCenter, Zagreb, Croatia

Antimalarial agents (chloroquine and others) are exten-sively used in treatment of systemic lupus erythematosus(SLE), although their precise mode of action is unclear. Re-cently, studies on murine SLE models demonstrated thatendogenous DNA-containing immune complexes can induceautoantibody production via TLR9. This provided a possibleexplanation for chloroquine's efficacy in SLE treatment, aschloroquine blocks endosomal acidification and preventsTLR9 signaling. Therefore, we investigated the possible in-fluence of chloroquine administration on TLR9 expression in11 newly-discovered patients with SLE using flow cytometry.Patients were analyzed before therapy, after initial gluco-corticoid treatment, and again after 3 months, when chlo-roquine was added and glucocorticoid dose reduced. The