S102 Abstracts

F.83. Examination Profile of IL-6 and TNF-α inRF-positive Patients with Rheumatoid ArthritisDuring Infliximab TreatmentLudmila Siziakina, Tatiana Kalashnikova. Rostov StateMedical University, Rostov-on-Don, Russian Federation

Rheumatoid arthritis (RA) is a chronic autoimmune diseasewith destructive lesions of peripheral joints. It have beenshown that IL-1, IL-6 and TNF-α are the most importantinflammatory cytokines in rheumatoid synovium. The firstanti-TNF drug approved for clinical use was infliximab (Inx),unfortunately, 30% of the patients are primary non-respon-ders. The aim of our researchwas to examine the profile of IL-6 and TNF-α in RF-positive positive patients with rheumatoidarthritis during infliximab treatment. Materials andmethods.The study included 28 patients (22 females, 6 males) with RF-positive variant of RA, mean age of 42,6±2,8 years, durationof a disease is 7,2±1,4 years. All patients received combinedtherapy with methotrexate (MT-12,5-15 mg) and Inx whichwas injected at single dose of 3 mg/kg intravenously,followed by administration 2 and 6 weeks later, and repeatedevery 8 weeks (6 infusions). Efficiency of the therapy wasestimated according to DAS28 4V Index and improvementcriteria of EULAR. Regular examination of patients includeddynamics TNF-α and IL-6 of serum and supernatants whichwas determined in ELISA, the quantity of lymphocytesexpressing receptors to TNF-α (CD120a). Results. Two groupsof patients have been estimated depending on EULARimprovement criteria of therapy after 6th infusions: I groupwith a good response and II group with moderate response orno response. Conclusion. Non-responders have initial in-creased IL-6 and TNF-α in serum, it was initial increased PHA-stimulated production of TNF-α and IL-6 by PMBC andCD120a-lymphocytes not determined in a blood.

doi:10.1016/j.clim.2010.03.305

F.84. Impaired T Cell Receptor Activation inIRAK-4-Deficient PatientsDouglas McDonald1, Frederick Goldman3, Oscar Gomez2,Andrew Issekutz4, Dinakantha Kumararatne5, Raif Geha1.1Children's Hospital, Boston, Boston, MA; 2University ofIowa Children's Hospital, Iowa City, IA; 3Children's Hospitalof Alabama, Birmingham; 4Dalhousie University, Halifax,NS, Canada; 5Addenbrooke's Hospital, Cambridge, UnitedKingdom

IRAK4 is a protein kinase effector of the Toll-like receptor(TLR) and interleukin 1 receptor pathways which plays acritical role in innate immune responses. The role of IRAK-4 inadaptive immune functions in humans is incompletely under-stood. We evaluated T cell receptor (TCR) function in fourIRAK-4-deficient patients. We compared upregulation of CD25and CD69 on T cells and production of the cytokines IL-2, IL-6,and IFNγ in anti-CD3 plus anti-CD28 stimulated PBMCs fromfour IRAK-4-deficient patients and four normal controls.Upregulation of CD25 and CD69 on T cells was significantlyreduced in the IRAK-4-deficient patients compared to normalcontrols following cross-linking with anti-CD3 plus anti-CD28.

Production of IL-6 (patients=210 pg/ml vs. normals=7000 pg/ml, pb0.05) and IFNγ (patients=777 pg/ml vs. normals=72000 pg/ml, pb0.05), but not IL-2 (patients=5200 pg/ml vs.normals=7250 pg/ml, p=n.s.), by PBMCs was reduced in IRAK-4-deficient patients compared to normal controls followingcross-linking with anti-CD3 plus anti-CD28. IRAK-4-deficientpatients have defects in T cell activation and TCR-induced cy-tokine production. Defects in T cell activation may contributeto the infectious susceptibilities of IRAK-4-deficient patients.

doi:10.1016/j.clim.2010.03.306

F.85. Dexamethasone Pretreatment Followed byan Inoculation of Short-termLipopolysaccharide-stimulated Dendritic Cells Improvesthe Response of Murine Collagen-induced ArthritisJuan Aguillon, Maria Peña, Diego Catalan, Octavio Aravena,Nicole Rojas-Colonelli, David Garate. Universidad de Chile,Santiago, Chile

Pharmacologically modulated dendritic cells (DCs) havebeen shown to restore tolerance in collagen (CII)-inducedarthritis (CIA). We examined the effect of dexamethasone(DXM) administration as a conditioning agent, followed by aninjection of lipopolysaccharide (LPS)-stimulated and CII-loadedDCs on the CIA course. After CIA induction, mice pre-treatedwith DXM were injected with 4-hour LPS-stimulated bone mar-row derived-DCs loaded with CII (DXM/4hLPS/CII/DCs) or leftunloaded (DXM/4hLPS/DCs). Mice inoculated with DXM/4hLPS/CII/DCs displayed significantly less severe clinical diseasecompared to animals receiving 4hLPS/CII/DCs alone or thosein which only DXM was given. Cytokine profile evaluation dem-onstrated that CD4+ T lymphocytes from DXM/4hLPS/CII/DCsand 4hLPS/CII/DCs groups released higher IL-10 levels thanthose of the DXM group or those of the CIA group. CD4+ T cellsfrom all DC-treated groups showed less IL-17 release whencompared to the CIA group. On the contrary, CD4+ T lym-phocytes from 4hLPS/CII/DCs and DXM/4hLPS/CIIC/DCs groupsreleased higher IFN-γ levels than those from DXM (Pb0.01) orCIA groups. Finally, we conclude that a combined treatment, in-cludingDXMpreconditioning followedbyan inoculation of short-term LPS-stimulated DCs, provides an improved strategy formodulating CIA. The benefit is driven by IL-10 producing CD4+ Tlymphocytes, which interfere with IL-17 production.

Supported by Fondecyt-Chile 107-0553 and MillenniumNucleus on Immunology and Immunotherapy-Chile P07-088-F.

doi:10.1016/j.clim.2010.03.307

F.86. Nascent Multiple Sclerosis Lesions areAssociated with Early Disturbances in theBlood–brain BarrierJorge Alvarez, Alisha Godschalk, Simone Terouz, AlexandrePrat. University of Montreal, Montreal, QC, Canada

Early changes in the normal appearing white matter of MSpatients precede the appearance of gadolinium-enhancing

S103Abstracts

lesions on MRI scans. Although these MRI studies suggest thebreakdown of the blood-brain barrier (BBB) as an importantfeature in MS pathogenesis, limited information is availableon the pathological changes occurring at the BBB duringlesion genesis. Thus, this study was focused to characterizethe differential changes of BBB components, particularly atthe early stages of lesion formation. MS lesions werecategorized as nascent, active, chronic active and chronicinactive. Sections were immunostained for distinct leuko-cyte markers and BBB disruption was determined byevaluating the expression pattern of fibrinogen, laminin,GFAP and the junctional proteins occludin, claudin-5, ZO1,VE-cadherin, α-catenin and p120-catenin. In addition, tocorrelate BBB disruption and endothelial activation, theexpression of ICAM-1, VCAM-1 and ALCAM was studied.Nascent lesions displayed moderate leukocyte infiltration,very limited demyelination, moderate gliosis, discretebasement membrane abnormalities, considerable changesin the expression of junctional proteins and increasedexpression of adhesion molecules. BBB junctional proteinsand basement membrane components were more severelydisrupted in active lesions and the expression pattern of mostjunctional components was partially reestablished in chronicactive and inactive lesions. Our pathological findingsindicate the presence of early disturbances in junctional,cell adhesion and basement membrane proteins at the BBBlevel in non-demyelinating MS specimens. These earlyvascular changes coincide with perivascular leukocyteinfiltration and bring pathological support for importantBBB disruption in the initial stages of MS lesion formation.

doi:10.1016/j.clim.2010.03.308

F.87. Sublingual Desensitization or ImmuneModulation with Dialyzable Leucoyte ExtractsImproves theClinicalOutcome inAllergic ConjunctivitisMaria Carmen Jimenez-Martinez2, Gustavo Aguilar1,Concepcion Santacruz1, Atzin Robles-Contreras1, Julio Ayala1,Pedro Hernandez4, Raul Chavez2, Sergio Estrada–Parra3, MayraPerez–Tapia3. 1Institute of Ophthalmology, Mexico City,Mexico; 2UNAM, Mexico City, Mexico; 3National School ofBiological Sciences, Laboratory of Molecular Immunology-II,Mexico City, Mexico; 4National Autonomous University ofMexico, Mexico City, Mexico

Purpose: To evaluate the clinical outcome(CO), and tear-cytokines(TC), before and after 6 m of Sublingual immu-notherapy(SLIT) or Dialyzable Leukocyte Extracts(DLE) inOcular Allergy(OA). Methods: Patients were divided into 3groups according to allergen skin reactivity (ASR). Group A(GA) were ASR+ patients treated with SLIT; Group B(GB) wereASR+ patients treated with DLE; Group C(GC) were ASR-patients treated with DLE. Ophthalmologic data wereassessed by developing an analogue-visual table. Tearssamples were collected to determine Th1/Th2 cytokines bycytometric arrays. CO and TC were evaluated beforetreatment (T0), and after 3 m and 6 m. Statistical analysiswas performed with ANOVA test. Reuslts: TC results: IL-2GAT0 6.2 pg/mL, 6 m 3.8 pg/mL; IL-2 GBT0 4.1 pg/mL, 6 m

3.4 pg/mL; IL-2 GCT0 4.6 pg/mL, 6 m 1.8 pg/mL; IL-4 GAT07.8 pg/ml, 6 m 5.1 pg/mL; IL-4 GBT0 3.5 pg/mL, 6 m 4.7 pg/mL; IL-4 GCT0 9.4 pg/ml, 6 m 2.6 pg/mL; IL-5 GAT0 3.9 pg/mL, 6 m 2.8 pg/mL, GBT0 4.3 pg/mL, 6 m 3.3 pg/mL; GCT07.6 pg/mL, 6 m 2.9 pg/mL; IL-10 GAT0 6.9 pg/mL, 6 m5.7 pg/mL, GBT0 5 pg/mL, 6 m 10.7 pg/mL; GCT0 9.5 pg/mL, 6 m 3.7 pg/mL; IFN-g GAT0 10.7 pg/mL, 6 m 5.5 pg/mL,GBT0 8.5 pg/mL, 6 m 6.7 pg/mL, GCT0 15.1 pg/mL, 6 m notdetected; TNFGAT0 3.8 pg/mL, 6 m 4.9 pg/mL, GBT0 3.7 pg/mL, 6 m 3.6 pg/mL, GCT0 6.2 pg/mL, 6 m 2.7 pg/mL. COResults: Clinical improve was observed in 60% of GApatients,33% of GCpatients and 14% of GBpatients. Conclusions: SLITwith the triggering Ag was the best treatment to OApatientsand ASR; DLE was the best option to OApatients without ASR.We did not find statistical correlations between the CO andTC. It is possible the involvement of other regulatorycytokines in the clinical improvement of patients. Acknowl-edgements: CONACyT 71291.

doi:10.1016/j.clim.2010.03.309

F.88. Predicting Autoimmunity FollowingTreatment of Multiple Sclerosis with AlemtuzumabJoanne Jones, Alastair Compston, Alasdair Coles. Universityof Cambridge, Cambridge, United Kingdom

The lymphocyte-depleting humanisedmonoclonal antibodyalemtuzumab is highly effective in the treatment of earlyrelapsing-remitting multiple sclerosis. However 30% ofpatients develop autoimmunity, months to years aftertreatment, usually targeting the thyroid gland and rarelyblood components. Autoimmunity arising in the context oflymphopenia is well recognised but not fully understood. Wehave hypothesised that autoimmunity occurs, not as a result oflymphopenia itself, but due to the individual's homeostaticresponse to it. In support of this we have shown thatautoimmunity arises in those patients with higher rates of Tcell cycling driven by genetically determined higher levels ofserum IL-21, detectable even prior to alemtuzumab treatment(JCI 2009). We now show that pre-treatment serum levels oftwo further homeostatic cytokines, IL-7 and CCL21, alsopredict autoimmunity following alemtuzumab; with higherserum IL-7 protecting against autoimmunity (7.5 pg/mL vs.3.4 pg/mL; pb0.001) and higher serum CCL21 associating withit (492 pg/mL vs. 432 pg/mL; pb0.001).We demonstrate how,in combination, pre-treatment serum levels of IL-21, IL-7, andCCL21, can be used to accurately model the risk of developingautoimmunitymonths to years after lymphodepletion. This hassignificant implications for counselling patients with multiplesclerosis considering treatment with alemtuzumab. Theconcept that one can predict the long-term consequences ofan immunosuppressive agent and tailor treatment accordinglyis entirely novel. This approach may offer a new way ofthinking about, and dealing with, the increasingly complexrisk-benefit analysis required in the use of biologic agents inthe treatment of multiple sclerosis.

doi:10.1016/j.clim.2010.03.310