Functional Analysis of Primary Immunodeficiencies in vitro

S76 cells. In addition activated Cbl, an ubiquitin ligase that marks TCR for degradation, was observed early during synapse formation. Only a small fraction of Ob.1A12 and Ob.2F3 T-cells had visible pZAP-70 microclusters and recruitment of pCbl was not detected in any of these cells. These data suggest that the self-antigen specific T-cell clones have a defect in IS formation. doi:10.1016/j.clim.2010.03.227

F.6. Longitudinal Study of Childhood Celiac Disease Incidence over a 33-year Period in Estonia Krista Ress 1, Katrin Luts 2, Tiina Rägo 1, Oivi Uibo 1. 1 University of Tartu, Tartu, Estonia; 2Tallinn Children's Hospital, Tallinn, Estonia Introduction: Celiac disease (CD) is a wheat gluten and related prolamines-induced autoimmune disease with a frequency that is underestimated in many geographical regions and populations. The aim of the present study was to analyze trends in the incidence and clinical presentation of CD in Estonian children from 1976 to 2008. Methods: Study included all children up to 19 years of age diagnosed with small bowel biopsy-proven CD in the whole Estonia. Data were collected from both two referral CD hospitals. Results: CD has been diagnosed in 131 children (median age 1.8 years). During 1976-1980 the age-standardized incidence rate of CD was 0.10 per 100 000 person-years. After introduction of the gliadin and/or endomysium antibody screening and an active clinical search for CD the incidence increased from 0.48 in 1986-1990 to 1.55 per 100 000 personyears in 1991-1995 (patients were mainly b 5 year old with typical clinical picture). In 2000, hospital screening with anti-tissue transglutaminase antibodies was started. This and the screening for CD among all children with type 1 diabetes (started in 2005) increased the incidence from 1.59 in 20012005 to 2.79 in 2006-2008 (40% were asymptomatic, median age 6.7 years). Discussion: Until 1990 CD has been considered an extremely rare disease in Estonia. Our study shows almost 30-fold increase in incidence of CD Estonia during 1976-2008. Introduction of serological screening methods, active clinical search and rising the awareness among physicians have increased the detection rate of CD, although a true increase in incidence may have also occurred. doi:10.1016/j.clim.2010.03.228

F.7. Functional Analysis of Primary Immunodeficiencies in vitro Ester van Leeuwen, Paul Baars, Kirsty van Nierop, Ineke ten Berge, Taco Kuijpers, Rene van Lier. Academic Medical Center, Amsterdam, Netherlands In patients suspected to have a primary immunodeficiency, thorough analysis of the lymphocytes in vitro can give answers pointing towards the underlying defect. We use a combination

Abstracts of assays to characterize the phenotype and functionality of lymphocytes of these patients. Next to quantification of NK cells, T cells, and B cells, the latter two are further analyzed for the distribution of naïve/effector/memory (T cells) or naïve/non-switched memory/switched memory (B cells) subsets. This gives an indication of the immune history and differentiation capacities of these cells in vivo. To then further analyze the lymphocyte functionality, an in vitro culture is performed with CFSE-labeled cells allowing visualization of cell proliferation and differentiation. Using these assays we have detected a novel mutation in the cytokine receptor common gamma chain (cγ) in combined immunodeficiency (CID) patients. These patients had low-normal T cell numbers but both T and NK cells showed diminished in vitro proliferation upon stimulation with cγ cytokines like IL-2 and IL-15. In vitro analysis has also proven useful in studying patients with defects in immunoglobulin production due to perturbed B cell differentiation such as common variable immunodeficiency (CVID) patients. Analysis of B cell subsets and B cell responses to different T cell-(in)dependent stimuli in culture can provide information on the specific malfunctioning. Not only proliferation can be monitored but also differentiation of B cells into CD20lowCD38high plasmacells and IgG and IgM secretion in the supernatant. In conclusion, functional lymphocyte analysis is a powerful tool in the definition of cellular defects in immunodeficient patients. doi:10.1016/j.clim.2010.03.229

F.8. Evaluation of Lymphocyte Subsets in Peripheral Blood of Sarcoidosis Patients Reveals Frequent CD4, CD8 and CD19 Lymphopenia Rafah Salloum, Maria Badaracco, Marisa Alegre, Nadera Sweiss, Timothy Niewold. University of Chicago, Chicago, IL Objective: We evaluated lymphocyte subsets in peripheral blood in patients with sarcoidosis to determine the prevalence, severity, and clinical features associated with lymphopenia. Methods: We studied lymphocyte subsets in 28 patients with sarcoidosis. Results were compared between groups of patients defined by disease manifestations or medical treatment, and data were also compared to 100 healthy individuals who were tested by the clinical lab for assay standardization. Results: The majority of sarcoidosis patients had low lymphocyte subset counts (57% of patients had low CD4, 54% had low CD8, and 54% had low CD19 counts respectively, p b 4 × 10-10 for a difference compared to healthy controls for each subset). CD4, CD8, and CD19 lymphocyte subset counts were significantly correlated (p = 0.0017). Lymphocyte subset counts did not show any significant relationship to treatment with prednisone, traditional disease-modifying anti-rheumatic drugs, or tumor necrosis factor inhibitors (p N 0.5 for each comparison). Four patients not taking any medical therapy, had significantly low lymphocyte subset counts in all three subsets (p b 0.022 for each subset). Patients with severe organ system involvement including neurologic, cardiac, ocular, and advanced pulmonary involvement had lower lymphocyte subset counts than patients with milder disease manifestations (CD4 p = 0.0043,