S155Abstracts

NGF increased p-Akt expression in CD3+T lymphocytes. Herewe have observed two novel growth functions of NGF on Tcellbiology. NGF is mitogenic to T lymphocytes and promotessurvival of T lymphocytes by inhibiting TNF-α inducedapoptosis.

doi:10.1016/j.clim.2009.03.456

S.83. Th17, Th1 and Treg Subsets are Increased inSystemic Sclerosis (SSc) PatientsJanette Furuzawa-Carballeda, Luis Llorente, JavierCabiedes, Luis Fajardo-Hermosillo, María Inés Vargas-Rojas,Tatiana Rodriguez-Reyna. Instituto Nacional de CienciasMédicas y Nutrición, Mexico, Mexico

SSc is an autoimmune disease characterized by fibrosisand vasculopathy. A key feature of the inflammatory lesionsis the presence of T cells. High levels of IL-17 have beenreported. This study suggested that the T-cell activity in SSctakes place outside the conventional Th1/Th2 subsets. Aim.To quantify T cell subsets in peripheral blood from SScpatients. Methods. Blood samples from 153 consecutive SScpatients were obtained. Clinical evaluation and organinvolvement were determined using the Medsger severityscale. Controls were collected from age-and sex-matchedhealthy volunteers (n=12; mean age=30.3±8.5yrs); activeSLE patients(n=13; mean age=36.7±12.2yrs)and active RApatients(n=12; mean age=46.8±15.3yrs). Mononuclear cellswere analyzed by flow cytometry to determine the absolutenumber (cel/ml) of Th1 (CD4+/CD14-/IFN-γ+), Th2 (CD4+/CD14-/IL-4+), Th17 (CD4+/CD14-/CD4/IL-17+), Treg (CD4+/CD14-/FoxP3+) and T CD4+/IFN-γ+/IL17+subsets. Patientswere classified according to skin involvement in diffuse(n=63; age=44.4±13.2yrs; disease evolution=8.2±5.9yrs)and limited (n=90; age=46.8±13.5; disease evolution=11.1±10.1). Results. Th17, Th1 and Treg subsets were increased inSSc and active RA groups vs. healthy control and SLE groups(Th17: limited=135.4±20.0; diffuse=222.8±108.4; vs.healthy control=26.93±4.0; SLE=9.3±2.2; pb0.001. Th1:limited=97.5±22.0; diffuse=113.7±55.0; vs. healthy con-trol=7.9±3.9; SLE=3.3±1.6; pb0.006. Treg: limited=165.4±24.2; diffuse=157.6±33.3; vs. healthy control=81.0±9.3;SLE=45.0±9.6; pb0.04). There were no differences betweenactive RA and SSc patients. There were no statisticaldifferences in T CD4+/IFN-γ+/IL17+subset, by age, time ofdiseaseevolutionandtreatmentbetweenSScgroups.Conclusion.Th17, Th1 and Treg subsets are increased in patients with SSc.

doi:10.1016/j.clim.2009.03.457

S.84. Role of C-Chemokine Receptor 2 (Ccr2) inMurine Model of Kawasaki's DiseaseHernan Martinez1, Marlon Quinones1, Fabio Jimenez1,Carlos Estrada1, Kassandra Clark1, Kazuo Suzuki3, NorikoN. Miura2, Naohito Ohno2, Seema Ahuja1. 1UTHSCSA, SanAntonio, TX; 2Chiba University Graduate School of Medicine,Tokio, Japan; 3Tokyo University Pharmaceutical and LifeSciences, Tokyo, Japan

Mounting evidence suggests that immune responses trig-gered by unidentified infectious agent(s) may underlie thepathogenesis of Kawasaki Disease (KD); an illness character-ized by coronary vessel inflammation (CVI) mostly affectingyoung children and associated with high circulating levels ofthe CC chemokine ligand 2 (CCL2). We used a murine KDmodel based on exposure to Candida Albicans Water Solubleextract (CAWS) antigens as a trigger for development of CVI toelucidate the role of the CCL2′s receptor CCR2 in KD. Ccr2null mice (Ccr2-/-), Rag1-/-(T and B cell deficiency) andmatched controls (WT) received two 5-day cycles of dailyintraperitoneal CAWS injections and were euthanized after8 weeks. The incidence and severity of CVI after CAWSexposure was significantly attenuated in Ccr2-/-mice. Thisresistant phenotype was associated with reduced macro-phage infiltration and lower circulating levels of known KD-pathogenic factors such as anti-MPO antibodies and MMP9.Two lines of evidence suggested that resistance to CVI inCcr2-/-mice might be linked to effects on Tand B cells inter-actions. First, Rag1-/-mice became susceptible to CVI onlyafter being reconstituted with T+B cells from WT but notCcr2-/-mice. Second, genetic inactivation of CCR2 bluntedthe depletion of circulating regulatory T cells (CD4+, CD25+,and Foxp3+Tregs) observed in WT mice following exposure toCAWS. Collectively, our data suggest that CCR2 plays a criticalrole in the pathogenesis of CAWS induced CVI, possiblyorchestrating immune regulation through its effects on Tcellresponses.

doi:10.1016/j.clim.2009.03.458

S.85. Chronic GVHD Appears to Cause GenomicInstability in Oral Mucosal Cells After AllogeneicHematopoeitic Cell Transplantation (HCT)Faisal Khan, Sarah Sy, Polly Louie, Douglas Stewart, JamesRussell, Jan Storek. University of Calgary, Calgary, AB,Canada

Genomic Instability (GI) is typically a precancerous/can-cerous state but is also reported in non-neoplastic chroni-cally inflamed tissues. Chronic Graft-versus-host-disease(GvHD) after allogeneic HCT may produce chronicallyinflamed tissues. Since chronic GvHD and second malignancyare frequent in oral and rare in nasal mucosa, we determinedwhether GI occurs in oral and nasal mucosal cells and, if yes,whether it is related to chronic GVHD. We examinedepithelial cells from oral and nasal mucosa of 85 subjectsthat include long-term (4-22 yrs, n=25) and short-term (1-3 months, n=27) survivors of allo-HCT. Controls includedsurvivors of auto-HCT (n=18), patients treated with inten-sive chemotherapy without HCT (n=5) and healthy volun-teers (n=10). DNA extracted from blood leukocytes and cellsof nasal and oral mucosa was PCR amplified for 15microsatellite markers. Fragment size analysis was done toidentify novel allele peaks (GI). GI was detected in 68% oforal specimens and 4% of nasal specimens in long-term allo-HCT survivors (pb0.001). Number of microsatellites showingGI in oral mucosa was higher in patients with than withouthistory of clinical oral GVHD (p=0.02). GI was found in oralmucosa of only 3.7% short-term allo-HCT survivors and was

S156 Abstracts

not found in their nasal mucosa. None of the controls showedGI. No GI was observed in blood leukocytes of any subject.We conclude that GI in long-term allo-HCT survivors occursfrequently in oral but rarely in nasal mucosal cells. The causeof GI is not cytotoxic therapy but likely it is due to chronicgraft-vs-host-oral-mucosal reaction.

doi:10.1016/j.clim.2009.03.459

S.86. Allergen-specific B Cell Quantity is Similar butAllergen-specific T Cell Quantity is Higher in AllergicCompared to Nonallergic IndividualsFaisal Khan1, Aito Ueno-Yamanouchi1, Bazir Serushago1,Tom Bowen1, Jan Storek1, Joanne Luider2. 1University ofCalgary, Calgary, AB, Canada; 2Calgary Laboratory Services,Calgary, AB, Canada

Allergen-specific IgE production is a hallmark of allergicdiseases like asthma, rhinitis or eczema. This could be due to ahigh number of allergen-specific B cells, Tcells (helping B cellsdifferentiate into allergen-specific IgE-producing plasma cells)or other mechanisms. Here we compared the number ofallergen-specific B cells and CD4 T (Th) cells between 48patients with allergic asthma/rhinitis/eczema (allergic indi-viduals) and 34 nonallergic individuals (asymptomatic andwithnegative skin prick test (SPT)). Allergen-specific B and Th cellswere enumerated by culturing CFSE-loaded PBMNCs for 7 dayswith allergen (cat, dog, D.pteronyssimus, timothy or birch),and determining by flow cytometry the number of B or Th cellsthat had proliferated (diluted CFSE). The quantities of B cellsspecific for each of the 5 allergens were similar in individualsallergic to the allergen (per SPT) compared to nonallergicindividuals. The quantity of allergen-specific Th cells fortimothy (p=0.003), cat (p=0.05) and birch (p=0.04) wassignificantly higher in timothy-, cat-and birch-allergic indivi-duals compared to nonallergic individuals. In contrast, thequantity of allergen-specific Th cells for dog andDPwas similarin allergic and nonallergic individuals. We conclude that forsome allergens (eg, timothy, cat and birch), a high number ofallergen-specific Th cells, but not B cells,may play a role in thepathogenesis of allergic asthma/rhinitis/eczema. For otherallergens (eg, dog and DP), the pathogenesis is likely different.

doi:10.1016/j.clim.2009.03.460

S.87. A Phase-II Clinical Trial of Rituximab Therapyafter Allogeneic HCT: Decreased Alloreactive B cellsResponses and Chronic GVHD IncidenceBita Sahaf1, Sally Arai2, George Chen1, Kartoosh Heydari3,Julie Boiko1, David Miklos1. 1Bone Marrow Transplantation,Stanford, CA; 2Medical Center, Stanford, CA; 3StanfordUniversity School of Medicine, Palo Alto, CA

Our previous work shows allo-antibody responses tominor-histocompability antigens (mHA) develop in association withcGVHD suggesting allogeneic B-cell role in cGVHD pathogen-esis. Anti B-cell therapy (rituximab) is effective in steroid

refractory cGVHD. We hypothesized that rituximab treat-ment two months post-HCT would deplete donor derivedalloreactive B-cells preventing cGVHD. Thirty-six CLL andMCL patients were conditioned with TLI (80 cGy)/ATG(1.5 mg/kg/day). PBPC were infused on day 0. Rituximabinfusion was (375 mg/m2/week x4) on days 56, 63, 70 and 77.B-cell reconstitution was studied by polychromic FACS inbone-marrow aspirates from 36 patients (rituximab/RIC) and16 (RIC only) on days 56, 90, 270 and 365 post-HCT. By day 56we detected B-cells in the human bone-marrow. Phenotypeswere as follows: common lymphoid-progenitor (CD34+CD117+CD7+), Pro-B-cells (CD34+CD19-CD10+), pre-B-cells (CD34-CD19-CD10+), immature-B-cell (CD19-CD38-IgM+IgDlow/neg),mature (CD19+CD38+IgD+IgM+), and CD38+CD138+plasma-cells. Rituximab purged the bone-marrow B-cells expressingCD20. Early progenitors, pro B-cells and plasma-cellsremained unchanged (n=28). B-cells in the bone-marrowemerged again 270 days post-HCT. In line with these results,the allo-responses to mHA were decreased in rituximab/RICcompared to RIC only patients (p=0.028). The incidence ofcGVHD was 18% compared to 50%. Peripheral B-cells weredetected in 17 patients prior to rituximab. Followingrituximab, B-cells were detected 1-1.5 years post-HCT.Naïve B-cells are the first subset to circulate. At 1.5 years,dominating subset circulating is the transitional B-cells (CD19+IgD+CD38+). In sum, rituximab/RIC combination therapyresulted in low cGVHD incidence with decreased allo-anti-body responses that persists following delayed B-cellreconstitution.

doi:10.1016/j.clim.2009.03.461

S.88. Prolactin Deregulates the Function of T CD4+CD25+LymphocytesVictoria Legorreta, Karina Chávez, Cervera Hernando,Luis Chavez-Sanchez, Eduardo Montoya, FranciscoBlanco-Favela. IMSS, Mexico City, Mexico

Introduction: The T regulatory lymphocytes (Treg) CD4+CD25+have an inhibitor effect on T CD4+CD25-. Someevidences show that prolactin has a role in immune responseprobable like a cytokine. However, the way throughout whichtakes part in the regulation of T cells still is unknown. Tregconstitute an important mechanism of peripheral tolerance,because their elimination drives to autoimmune diseases. Weaddress our research in order to know the prolactin effect onthe Treg function. Aim: To demonstrate if the prolactindecreases the function of the human Treg lymphocytes.Material and Methods: CD4+CD25+ and CD4+CD25-werepurified by sort and the relative expression of PRL's receptorRNAm was measured by real time RT-PCR. The functionalactivity was evaluated by means of proliferation assays andcytokine secretion in cells activated with CD3/CD28, in PRL'spresence or absence. Results: We found that human Treginduce 35.8% (p=0.04) of inhibition in the proliferation fromCD4+CD25-cells. This effect was reverted when we add PRL,founding that CD4+CD25-cells recover her proliferationcapacity (p=0.03). In other results, we quantified theexpression of PRL-R-mRNA in different sub populations; wefound that the R-PRL messenger is constitutive in Treg.