report highlighted by a “traffic light system” and pictorialpatient reports in MS-WORD, after analysis by especiallydesigned software incorporating MS-EXCEL macros enhancedby Visual Basic programming. B cell phenotyping correlatedwith pre versus post-immunisation serum pneumococcal anti-body levels in cases investigated for primary and secondaryimmunodeficiency. Discussion: MPA of cells and serum on asingle standard flow cytometer with powerful software appliesexisting expertise and resources in a flexible cost-effectiveapproach suitable for high-throughput testing and clinicallaboratories.

doi:10.1016/j.clim.2007.03.338

F.127 Catalytic Anti-dsDNA Activity from SLE Sera isAssociated with Remission Phases of the DiseaseGenevieve Servais, Responsible Autoimmunity LAB, Canada,Canada, CHU Brugmann, Immunology, Bruxelles, Belgium,Mimouna El Baz, Pharmacist, CHU Brugmann Immunology,Bruxelles, Belgium, Rafik Karmali, MD, CHU BrugmannInternal Medicine, Bruxelles, Belgium, Marie PauleGuillaume, MD, CHU Brugmann Internal Medicine,Bruxelles, Belgium, Jean Duchateau, MD PHD, CHUBrugmann Immunology, Bruxelles, Belgium

SLE is characterized by the presence of circulating anti-DNA antibodies, one of the eleven ACR criteria for theclassification of SLE, and they play an important role in thepathogenesis of SLE. Anti-DNA antibodies display differentspecificities, some being restricted to nuclear doublestranded DNA (dsDNA), or single stranded DNA (ssDNA), ormembrane associated DNA (mDNA) or nucleosomes. Someare sharing dual specificities. Autoantibodies displaying acatalytic activity, abzymes, were reported in patient’s sera,including catalytic anti-DNA antibodies. They have not beenreported in healthy control subjects. Catalytic activity variesin different pathologies and changes during the differentphases of the disease. The biological role of the catalyticanti-DNA present in serum of SLE is complex, as it seems thatsome may play positive and others negative roles, from aclearance role to a lowering of DNAse activity in SLEserum. We show here that circulating catalytic anti-DNA isfound in SLE and RA patients but also in healthy controlsthough with a higher percentage in SLE than in controls,where they can be unraveled when the sera are dilutedat 1/10. Their presence is inversely correlated with theclinical activity of the disease and with the titer ofcirculating autoantibodies to dsDNA, mDNA but not anti-nucleosomes antibodies.

doi:10.1016/j.clim.2007.03.339

F.128 Fungal Hypersensitivity is Not Type I (IgE)AllergyVincent A. Marinkovich, MD, Inc., Redwood City, CA

One hundred adult patients undergoing clinical evalua-tions for symptoms developing during chronic exposure to

high ambient fungal antigen levels in water damagedhomes were tested for specific IgE (RAST) and specific IgG(ELISA) levels to a number of fungi commonly found inmold-amplified environments. The tests were standardruns in a State licensed Physician’s Office Laboratoryusing test materials provided by Hitachi Chemical Diag-nostics for specific IgE and specific IgG in a multi antigenpanel format. The panels contain aspergillus, penicillium,alternaria, cladosporium and mucor. Results read aspositive when greater than 2 standard deviations greaterthan the mean using reference ranges previously deter-mined by Hitachi. Seven patients had elevated specificIgE levels to at least one of the fungi tested (7%). Eightynine patients showed elevated IgG titers to at least oneof the molds. The sera were then exposed to anadditional eight common molds, botrytis, candida, epi-coccum, fusarium, helminthosporium, pullularia, rhizopusand stemphylium, on an IgG panel and all patients (100%)showed elevated titers to at least one of the molds. Thecommon symptoms reported were remarkably consistentamong patients: fatigue, nasal congestion, headaches,cough, memory loss and chronic flu-like symptoms. All thepatients showed signs of chronic rhinosinusitis suggestingfungal colonization and eosinophilic degranulation. Sys-temic symptoms are likely the result of circulatingimmune complexes under conditions of functional asplenia(i.e., IC overload). The neurological symptoms may be theresult of local uptake of eosinophilic neurotoxin via thecribriform plate.

doi:10.1016/j.clim.2007.03.340

F.129 Automation for Isolation of Peripheral BloodMononuclear Cells (PBMC)Carlos Aparicio, Scientist, Beckman Coulter, Inc., Miami, FL,Enrique Rabelli, Director, Beckman Coulter, Inc., Miami, FL,Edward Jachimowicz, Scientist, Beckman Coulter, Inc.,Miami, FL, Sybil D’Costa, Scientist, Beckman Coulter, Inc.,Miami, FL, Mahsa Aspsater, Scientist, Beckman Coulter,Inc., Miami, FL, Julie Wilkinson, Scientist, BeckmanCoulter, Inc., Miami, FL, Wade Bolton, VP, BeckmanCoulter, Inc., Miami, FL, Enrique Rabelli, Director,Beckman Coulter, Inc., Miami, FL

Peripheral blood mononuclear cells (PBMC) are con-sidered appropriate target populations to immunologicallyassess the various subsets of blood lymphocytes. Purifica-tion of PBMC by density gradient centrifugation is alaborious and time consuming manual process and, thus,limiting factor for processing large number specimens.Generally, the media–gradient interface is generated bycarefully pipetting the blood cell suspension onto adensity gradient solution without disturbing the interface.Disruption of the interface can jeopardize the overallyield, purity and viability of the mononuclear cellpreparation. The study described herein focuses on analternative semi-automated strategy to harvest PBMCusing the Ficoll-Paque™ density gradient. This processintegrates the use of an automated liquid handler and acentrifuge to minimize the number of steps and time

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requiring operator intervention in current laboratoryprocedures. PBMC harvested by this automated processhad a viability over 95%, and when compared to themanual procedure showed: (1) cell recovery (N95%) withno selective loss as judged by phenotyping and functionalresponse by the various lymphocytes. The goal of thiswork is to provide a fully automated and standardizedmethod for preparing PBMC that can facilitate supportcell based-assay testing strategies in clinical trials byimproving overall quality and efficiency of the process aswell as reducing labor and cost.

doi:10.1016/j.clim.2007.03.341

F.130 Interleukin 32 Expression is Associated with aUnique Subset of Human Dendritic Cells Receptiveto the Venezuelan Equine EncephalitisVirus-derived Replicon VectorKevin Nishimoto, Graduate student, University of CaliforniaIrvine, Department of Microbiology and Biochemistry,Irvine, CA, Charles Dinarello, Professor of Medicine,University of Colorado Health Sciences Center, Departmentof Medicine, Denver, CO, Stephen Hou, PhD, University ofCalifornia Irvine, Department of Medicine, Division ofHematology/Oncology, School of, Irvine, CA, EdwardNelson, Assistant Professor of Medicine, University ofCalifornia Irvine Department of Medicine, Division ofHematology/Oncology, School of Medicine, Irvine, CA

Establishing successful immunotherapeutic strategiesutilizing dendritic cells (DC), the most potent antigenpresenting cell, largely depends on understanding therequirements necessary for optimal DC function. We andothers have reported that the Venezuelan Equine Ence-phalitis (VEE) replicon particle (VRP) vector system hasconserved tropism for murine, rat, and human DCs.However the DC subset(s) and functional capacities arenot fully understood. The VRP vector system has in vitrotropism for a subset of human immature monocyte-derived DCs (M-DCs); therefore, we developed a noveltechnique to isolate this VRP receptive DC subset forfurther characterization. An evaluation between VRPreceptive and non-receptive M-DC subsets by microarrayrevealed distinct differences in gene expression profileswith N700 significantly expressed genes (0.001 e P value,0.98 d PPDE) between cellular subsets. Notably in thereceptive DC population, the NK4 transcript was signifi-cantly increased and accompanied by protein expression.The NK4 gene has recently been identified as encoding arecently described cytokine, interleukin 32. These dataprovide the initial characterization of a new subset ofmyeloid DCs targeted by this vector, and provide insightsinto human DC biology allowing explicit study of themechanisms employed by this exceptionally potent immu-notherapeutic vector system.

doi:10.1016/j.clim.2007.03.342

F.131 Retinoic Acid Promotes Development ofHuman Macrophages Over Dendritic CellDifferentiationJuliana Sousa-Canavez, Researcher, Genoa Biotech, SãoPaulo, Brazil, Cristina Massoco, Researcher, GenoaBiotech, São Paulo, Brazil, Elaine Corneta, Student,Genoa Biotech, São Paulo, Brazil, Katia Leite, Pathologist,Genoa Biotech, São Paulo, Brazil, Tatiane Marisis, Student,Genoa Biotech, São Paulo, Brazil, Luiz Camara-Lopes,Pathologist, Genoa Biotech, São Paulo, Brazil, AntonioMisiara, MD, Genoa Biotech, São Paulo, Brazil, DewtonMoraes-Vasconcelos, Professor, Genoa Biotech, São Paulo,Brazil

Previous studies showed that all vitamins includingvitamin A are fundamental for immune system properlyfunctioning. All trans retinoic acid (atRA) at physiologicalor near-physiological concentrations appears to affectTh1–Th2 differentiation and it is believed that effectsof retinoids on immune responses might also be mediatedby dendritic cells (DC). Nonetheless, effects of atRA in DCdifferentiation have been showing contradictory resultssince it was observed either induction or inhibition ofdifferentiation. Our work shows effects of atRA on humanmonocyte derived DC by phenotype and phagocytosisstudies. DC (positive control group) was differentiatedwith cytokines (GM-CSF and IL-4) and maturation inducedby TNF-α. We have tested pharmacological (10 1/4M and1 1/4M) and physiological (10 nM) doses of atRA with orwithout cytokines. Cell phenotype was analyzed by flowcytometry using CD80, CD86 and CD14 antibodies. We alsoinvestigated functional performance analyzing phagocyto-sis and respiratory burst (ROS) in all culture conditionsmentioned above. Our data demonstrate that effects ofatRA depend on the dose used, as pharmacological dosesinhibited expression of all phenotypic markers tested,while a physiological one caused cell differentiation.Physiologic concentration of atRA especially in combina-tion with GM-CSF blocked differentiation of DC populationcorroborating a macrophage preferable transformation.Moreover phagocytosis and ROS were approximately threetimes greater in cells cultivated with atRA compared topositive control group. On the other hand, IL-4 and atRAapparently induced differentiation to an immature DCphenotype when TNF-α stimulus was added.

doi:10.1016/j.clim.2007.03.343

F.132 OTC Allergy TestingChris Brown, ImmuneTech, Inc., Menlo Park, CA, Vincent A.Marinkovich, MD, Inc. Redwood City, CA

Quantitative, comprehensive in vitro, specific IgE testsfor allergy have not hitherto been introduced into theover the counter (OTC) market because of their high costsand the need for a large sample of blood requiring avenapuncture. Recently the U.S. Food and Drug Adminis-tration cleared for OTC distribution a new allergen-specific IgE test which has two major advantages over

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