S115Abstracts

METHODS:Peripheral blood mononuclear cells (PBMCs) wereisolated from a cohort of adult patients with 22qdel (ages 15-48) and controls (ages 20 to 78). PBMCs were incubated forfive hours at 37° C in RPMI after stimulation with either PMA5 ng/ml, ionomycin 500 ng/ml, and GolgiPlug 1 μl/ml (for IL-4 and IFN-γ staining); PMA 50 ng/ml, ionomycin 1 μg/ml andGolgiStop 1.5 μl/ml (for IL-17 staining); or GolgiPlug 1 μl/ml(control). PBMCs were stained with surface antibodiesagainst CD3 and CD4 and intracellular antibodies against IL-4, IFN-γ, and IL-17 and analyzed by flow cytometry.Statistical analysis was performed using the Mann-Whitneytest.RESULTS:There was a significantly elevated percentageof CD3+CD4+IL-4+ IFN-γ- lymphocytes in 22qdel patientscompared to controls (2.21% vs 1.09%, pb0.01). There was nosignificant difference in the percentage of CD3+CD4+IL-4-IFN-γ+ or CD3+CD4+IL-17+ lymphocytes in 22qdel patientscompared to controls (14.48% vs 11.68%, p=0.15 and 1.60% vs1.35%, p=0.44).CONCLUSIONS:22qdel patients had a higherpercentage of IL-4+CD4+ T-cells than controls consistent witha skewing towards a Th2 phenotype. This is consistent withthe clinical observation of increased atopic manifestations in22qdel patients and could be due to a skewing of T-cellsubsets during homeostatic proliferation.

doi:10.1016/j.clim.2009.03.337

F.80. The TransmembraneE3 Ligase, GRAILUbiquitinates and Degrades CD83 on CD4+ T cellsLeon Su, Hideyuki Iwai, Jack Lin, C. Garrison Fathman.Stanford University, Stanford, CA

Ubiquitination of eukaryotic proteins regulates a broadrange of cellular processes including T cell activation andtolerance. We have previously demonstrated that GRAIL, atransmembrane RING finger ubiquitin E3 ligase, is required forthe induction of T cell anergy. In this study, we show thatGRAIL can down modulate the expression of CD83, predomi-nantly described as a cell surface marker for mature dendriticcells, on CD4 T cells. GRAIL mediated down modulation ofCD83 is dependent on an intact GRAIL extracellular proteaseassociated (PA) domain and an enzymatically active cytosolicRING domain, and proceeds via an ubiquitin-dependent 26Sproteosome pathway. Furthermore, ubiquitin modification oflysine residues at position K168 and K183, but not K192 in thecytoplasmic tail of CD83 was shown to be necessary fordegradation of CD83. This study defines a unique mechanismof ubiquitination by a transmembrane RING E3 ligase, andidentifies CD83 as a new E3 substrate of GRAIL.

doi:10.1016/j.clim.2009.03.338

F.81. Regulatory T Cells in Pediatric RenalTransplant RecipientsJames Tong, Qizhi Tang, Paul Brakeman, Peter Stock.University of California at San Francisco, San Francisco, CA

Regulatory T cells (Tregs) are a subset of T cells that arehypoproliferative and can modulate the action of effector Tcells (Teff). In humans, this subset of Tcells represents 1-2% of

all Tcells and 5-10% of all CD4+ Tcells. Because of their abilityto suppress Teff, there has been increasingly more interest intheir role in autoimmunity and transplantation where thebalance of Teff overwhelmingly outweighs Tregs. NaturalTregs are defined as CD4+CD25+Foxp3+T cells. We used FACSanalysis of peripheral blood to profile T cells in a retro-spective cohort of pediatric renal transplant undergoingstandard immunosuppression of daclizumab, tacrolimus,mycophenolate mofetil, and steroids. We hypothesized thatat one year post transplantation, pediatric renal transplantrecipients without any episodes of acute cellular or humoralrejection would have higher percentages of natural Tregsamong CD4+ Tcells compared with those recipients who havehad previous episodes of rejection. At 12 months posttransplantation, rejection free patients had 6.8% (SD=1.8%,n = 10) Tregs in their peripheral blood compared to 3.17%(SD=0.4%, n=5) Tregs in thosewho had at least one episode ofrejection (p=0.0008). This result shows that pediatric kidneytransplant recipients who are rejection free have similarpercentages of Tregs compared to healthy controls and havesignificantly increased percentages of Tregs compared tothose with prior episodes of rejection. This data, ifconfirmed, could be a marker for those patients who maytolerate reduction in immunosuppression.

doi:10.1016/j.clim.2009.03.339

F.82. Modulation of T Cell Unit in PersistentRecurrent Herpes Virus InfectionsMichael Rudenko1, Andrey Simbirtsev2, Irene Rudenko1,Andrey Zabelev2, Andrey Shemshura3. 1Clinical CenterEuroDon, Rostov-on-Don, Russia; 2State Research Instituteof Highly Pure Biopreparations, Saint Petersburg, Russia;3Parasitology, Rostov-on-Don, Russia

With the aim to prove the efficacy of the new patentedscheme of treatment in acute phase of persistent recurrentherpes virus infections a prospective blinded placebocontrolled cohort study was designed. 800 patients in acutephase of herpes virus infection were randomly recruited intotwo arms. The age distribution was 24-32 y.o. -43%, 33-42 y.o.-32%, 42-49 y.o. -25%, 43% of themweremen and 57 - women.No significant bias was found between arms. Patients in thefirst arm (n=400) received immune adjuvant D-glutaminil-L-tryptophan according to patented scheme: intranasally100 μg daily, valacyclovir 500 mg per os and penciclovirlocally during 10 days (priority patent certificate for themethod of treatment #2008113617/14(014798) from11.04.2008). Patients in the second arm received placeboand the same antiviral treatment as in arm one. Eachpatient underwent careful immune examination: {(PCR(blood, saliva, vesicles), ELISA (IgG, IgM, cytokines), flowcytometry (CD3+; CD4+; CD8+; CD16+; CD3+,CD25+; CD3+,CD95+; CD3+,HLADR+; CD20+)), assessment of phagocytosis,immunoglobulins A, M, G, circulating immune complexes}.After the treatment in the first arm immune deviations ofT-cell unit were optimized: T-cells were increased (64.08±5.88 vs. 77.7±8.58: before vs. after treatment) NK cells(10.38±4.68 vs 13.74±4.72), markers of late negativeactivation of T cells decreased (8.22±4.27vs. 5.21±4.18),

S116 Abstracts

and early activation increased (3.44±2.03 vs. 6.51±2.24)pb0.05. No statistically significant positive changes inimmune system were observed in the second arm. Accordingto the statistic analyzes of data obtained 2 weeks after theend of the treatment, D-glutaminil-L-tryptophan improvesimmune parameters of T-cell unit and can be used in complextreatment of patients with acute herpes virus infections.

doi:10.1016/j.clim.2009.03.340

F.83. in utero Exposure to the EnvironmentalEstrogen, Bisphenol A (BPA), Promotes AllergicSensitization and Airway Hyperresponsiveness(AHR) in an Animal Model of AsthmaTerumi Midoro-Horiuti, Youmin Lin, Randall Goldblum.University of Texas Medical Branch, Galveston, TX

We have reported that several environmental estrogensinduce direct mast cell degranulation and enhance IgE-mediated degranulation in vitro and that perinatal exposureto BPA promotes the development of asthma in a mousemodel.The goal of the current studywas to define the critical period(s)of BPA of exposure. BALB/c dams received 10 μg/ml BPA in theirdrinking water. Some of the pups were transferred from BPA-loaded mother to unexposed mother, or vise versa. The pupswere sensitized by intraperitoneal injection of 5 μg ofovalbumin (OVA) on day 4 and inhalation of 3% OVA on days 18to 20.Onday 22, serum IgEanti-OVAwerequantifiedandairwayinflammation and AHR assessed by enumerating eosinophils inbronchoalveolar lavage fluid and flexiVent analysis. BPA burdenin the pups were quantified by GC/MS. Pups exposed to BPAboth in utero and through breast milk, and in utero onlyresponded to this “suboptimal” sensitization, whereas unex-posed pups or pups exposed to BPA only from breast milk didnot. Therewas no difference in the responsiveness between thepups exposed in utero only and those exposed in utero and viabreastmilk. BPA content in the exposed pupswas similar to thatreported for human samples. In utero exposure may be themost critical period for BPA enhancement of allergic sensitiza-tion, bronchial inflammation and AHR in genetically susceptiblemice. The extensive exposure of women to low doses ofenvironmental estrogens might contribute to the increasingprevalence of childhood asthma.

doi:10.1016/j.clim.2009.03.341

F.84. Influence of Vitamin D Deficiency on HumoralAutoimmunity and Antigen Specific Immunity inSystemic Lupus ErythematosusLauren Cole1, Virginia Roberts1, Amy Dedeke2, WendyKlein1, Sherry Crowe1, Judith James2. 1Oklahoma MedicalResearch Foundation, Oklahoma City, OK; 2University ofOklahoma Health Sciences Center, Oklahoma City, OK

Autoimmune diseases have complex etiologies, likelyincluding variable interactions between genetic predisposi-tions and environmental risks. Vitamin D deficiency hasrecently been linked with many autoimmune disorders,

including systemic lupus erythematosus (SLE). Vitamin Dinhibits antibody secretion and autoantibody production in Bcells of animal models; however, the influence of vitamin Ddeficiency on autoantibody production and antigen specificimmune responses in SLE patients is unknown. Our study testswhether vitamin D deficiency is associated with SLE B cellhyperreactivity or improved antigen-specific antibodyresponses. 25-hydroxyvitamin D (25OHD) levels, lupus-asso-ciated autoantibodies (Ro, La, Sm, nRNP, ribosomal P, dsDNA,ANAs and phospholipid antibodies) and influenza humoralimmune responses (native/denatured ELISA responses, rela-tive affinities and hemaglutinnation inhibition) were mea-sured in plasma from European-American female SLE patientsand matched controls. SLE patients and autoantibody-positive controls were more likely to have deficient 25(OH)D levels compared to autoantibody-negative healthy controls(SLE [n=32] 16.43 ng/ml (deficiency≤20 ng/ml); autoanti-body-positive controls [n=15] 22.91 ng/ml; autoantibody-negative controls [n=16] 27.57 ng/ml (p=0.0172, one-wayANOVA). Surprisingly, no association was seen betweenvitamin D status and the ability to mount a strong influenzavaccination antibody response (high responders were 50% 25(OH)D sufficient vs 37% insufficient (p=0.49, Fisher's test).These data support a role for vitamin D deficiency in human Bcell hyperreactivity and autoantibody production; however,this defect does not appear to influence antigen-specificantibody production.

doi:10.1016/j.clim.2009.03.342

F.85. Foxp3 Expression In Human T Cells does notRequire Thymic FunctionIvan Chinn, Mary Marker. Duke University Medical Center,Durham, NC

Rationale: Studies of Foxp3 expression in human T cellshave focused upon thymically-derived cells, especially CD4+

regulatory Tcells. Children with atypical complete DiGeorgeanomaly (cDGA) have circulating oligoclonal T cells but nothymic function before allogeneic thymus transplantation.We hypothesized that Foxp3 expression would be absent inthese T cells. Methods: Seven infants with atypical cDGAwere studied before thymus transplantation. The subjectshad oligoclonal expansions of T cells and no thymic function(defined as circulating CD45RA+CD62L+ Tcells b5% of total Tcells or b50 cells/mm3). Foxp3 expression in CD4+ Tcells wasassessed with quantitative real-time PCR and flow cytome-try; TCRVβ expression was assessed by flow cytometry.Results: Four of the 7 subjects demonstrated clear evidenceof Foxp3 expression in CD4+ T cells. Foxp3 expression wasobserved in 2 of 4 subjects by flow cytometry. Two othersubjects had low initial Foxp3 mRNA expression by quanti-tative real-time RT-PCR, but flow cytometry over 3 monthslater showed Foxp3 expression in greater than 10% of CD4+ Tcells. Subsequent simultaneous quantitative real-time RT-PCR and flow cytometry testing confirmed Foxp3 expression.TCRVβ analyses in these 2 subjects demonstrated significantoligoclonality of the CD4+Foxp3+ T cells that differed fromthe overall oligoclonality of the CD4+ T cells. Foxp3+ cellsappeared to have expanded within specific TCRVβ families.