IL-2, IL-5, TNF-α and IFN-γ mRNA expression in epidermal keratinocytes of systemic lupus erythematosus skin lesions

OBJECTIVE: To analyze cytokine gene expression in keratinocytes from patients with systemic lupus erythematosus (SLE). INTRODUCTION: Keratinocytes represent 95 percent of epidermal cells and can secrete several cytokines. METHODS: Keratinocytes were obtained by laser microdissection from 21 patients with SLE (10 discoid and 11 acute lesions) at involved and uninvolved sites. All patients were receiving a low/moderate prednisone dose and 18 were receiving chloroquine diphosphate. IL-2, IL-5, TNF-α and IFN-γ gene expression was evaluated by real-time PCR and expressed as the ratio (R) to a pool of skin samples from 12 healthy volunteers. RESULTS: Heterogeneity in cytokine gene expression was found among patients with SLE. Eighteen of 38 valid SLE samples (47 percent) presented overexpression (R>1) of at least one cytokine. Lesional skin samples tended to show higher cytokine expression than samples from uninvolved skin (p = 0.06). IL-5 and IFN-γ were the most commonly overexpressed cytokines. Samples with cytokine overexpression corresponded to more extensive and severe lesions. Prednisone dose did not differ between samples without cytokine overexpression (15.71±3.45 mg/day) and those with overexpressed cytokines (12.68±5.41 mg/day) (p = 0.216). Samples from all patients not receiving diphosphate chloroquine had at least one overexpressed cytokine. CONCLUSIONS: The heterogeneous keratinocyte cytokine gene expression reflects the complex immunological and inflammatory background in SLE. Patients with severe/extensive skin lesions showed a higher frequency of cytokine gene overexpression. Increased IFN-γ and IL-5 expression suggests that Th1 and Th2 cells are involved in SLE skin inflammation. The possibility that prednisone and antimalarial drugs may have contributed to low cytokine gene expression in some samples cannot be ruled out.

Genotyping of Kell, Duffy, kidd and RHD in patients with B Thalassemia

A determinaçäo dos fenótipos Rh, Kell, Duffy e Kidd, associada ao ABO é utilizada para prevenir a aloimunizaçäo a antígenos eritrocitários e participam também no processo de identificaçäo de anticorpos nos pacientes com B talassemia. Todavia, a fenotipagem desses pacientes é trabalhosa e de difícil interpretaçäo. Nesta situaçäo, deve ser avaliada uma alternativa ao teste de hemaglutinaçäo para determinar o padräo antigênico dos pacientes. Utilizamos para tal fim o método PCR-RFLP. Foram preparados DNAs de 50 pacientes com Btalassemia que haviam sido anteriormente fenotipados pela hamglutinaçäo e testados para Kell, Kidd, Duffy/GATA mutaçäo por PCR-RFLP. RHD/näo-D foi analisado pelo tamanho do produto, do PCR associado à seqüência do gene RHD no intron 4 e exon 10/3' UTR. Os testes de genotipagem foram realizados sem o conhecimento dos resultados dos fenótipos. Para os RHD/näo-D, 47 foram RHD+ e RHD+/RHCE+, e 3 foram RHD- e RHD-/RHCE+. Para o Kell, 48 kk foram K2K2 e 2 Kk foram K1K2. Para o Duffy, das 44 amostras que haviam sido normais, GATA box, 8 Fy(a+b-)foram FYA/FYA, 15Fy(a+b-) foram FYB/FYB; e 19 Fy(a+b+) foram FYA/FYB; das outras 4 amostras, 3 foram FYA/FYB; homozigoto GATA mutaçäo. Duas amostras fenotipadas como Fy(a+b-), que eram normais GATA, apresentavam as mutaçöes 265T/298a e 2 amostras fenotipadas como Fy(a+b+) haviam sido genotipadas como FYA/FYB. Para o Kidd, 15Jk(a+b+) foram JKA/JKA, 12jk(aa-b+) foram JKB/JKB, e 20 jk(a+b+) haviam sido genotipadas como JKB/JKB. A genotipagem é mais acurada que a fenotipagem para determinaçäo de grupos sangüíneos em pacientes portadores de B talassemia poli transfundidos. A genotipagem nesses pacientes pode ser importante para selecionar hemácias antigenicamente negativas para transfusäo de glóbulos vermelhos.