Protective immunity to leishmaniasis has been demonstrated in murine models to be mediated by T cells and the cytokines they produce. We have previously shown that resistance to experimental Leishmania infantum infection in the dog, a natural host and reservoir of the parasite, is associated with the proliferation of peripheral blood mononuclear cells (PBMC) to parasite antigen and to the production of interleukin-2 and tumour necrosis factor. In this study we show that PBMC from asymptomatic experimentally infected dogs produce interferon-gamma upon parasite antigen-specific stimulation, whereas lymphocytes from symptomatic dogs do not. In addition, we report for the first time the lysis of L. infantum-infected macrophages by PBMC from asymptomatic dogs and by parasite-specific T cell lines derived from these animals. These T cell lines were generated by restimulation in vitro with parasite soluble antigen and irradiated autologous PBMC as antigen-presenting cells. We show that lysis of infected macrophages by T cell lines is major histocompatibility complex restricted. Characterization of parasite-specific cytotoxic T cell lines revealed that the responding cells are CD8+. However, for some animals, CD4+ T cells that lyse infected macrophages were also found. In contrast to asymptomatic dogs, lymphocytes from symptomatic dogs failed to proliferate and produce interferon-gamma after Leishmania antigen stimulation in vitro and were not capable of lysing infected macrophages. These results suggest that both the production of interferon-gamma and the destruction of the parasitized host cells by Leishmania-specific T cells play an important role in resistance to visceral leishmaniasis.

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Background The determination of HLA-B27 antigen is a useful tool in the diagnosis of different inflammatory rheumatic diseases, being the association with ankylosing spondylitis (AS) the most frequent. The techniques used for this determination are microlymphocytotoxicity test, flow cytometry assay (FCM) and polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO), being the last one the most accurate determination. Objectives As FCM rends some indeterminate results, the objective of the present work is to identify by PCR-SSO, the alleles of HLA-B gene that give indeterminate results in FCM. Also, to study the most common diagnosisassociated with HLA-B27 in our cohort of patients. Methods In 1110 samples from patients studied in the Rheumatology Unit of Fundación Jiménez Díaz-Capio during a year, the determination of HLA-B27 was performed in whole peripheral blood by FCM in a FACS Canto II cytometer (BD Biosciences, USA), using two different monoclonal antibodies: HLA-B27 (FITC) / HLA-B7 (PE) from Beckman Coulter and HLA-B27 (FITC) from One Lambda. Grafics were analyzed with Infinicyt software (Cytognos, Spain) The HLA-B27 typing was performed by PCR-SSO (INNO-LIPA, HLA-B Innogenetics, Belgium). Results 13,4% (149) of patients were positive HLA-B27 by FCM. 1,9% (22) patients that were indeterminate by FCM were evaluated by PCR-SSO. Of these 22 patients, 12,8% (5) were HLA-B27 positive. The frequency of alleles found in HLA-B27 negative by PCR-SSO were: B44 (20,5%), B18 (10,3%), B35 (10,3%), B8 (7,7%), B51 (7,7%), B7 (5,1%), as well as B15, B37 and B49. Only 18% of HLA-B27 positive patients were diagnosed of ankylosing spondylitis (AS) followed by 14% with undifferentiated spondyloarthropathy, 12% with some type of pains, 9% with arthritis (inflammatory, microcrystalline or infectious), 8% of uveitis and the rest of patients with a miscellaneous. Conclusions The more frequents alleles of HLA-B that give indeterminate results in the determination of HLA-B27 by FCM, were B44 allele, B18 and B35. FCM is proposed as a useful assay in the screening of the HLA-B27 antigen in the daily practice due its high performance (98,01%). For borderline cases, PCR-SSO is the recommended tool. References A high-resolution polymerase chain reaction-sequence-specific primer HLA-B*27 typing set and its application in routine HLA-B27 testing. Downing J, Coates E, Street J, Hammond L, Rees TJ, Pepperall J, Darke C.Genet Test. 2006 Summer;10(2):98-103. Disclosure of Interest None Declared