Apoptosis in Autoimmune Lymphoproliferative Syndrome Suspected Patients with Clinical and Laboratory Findings

Objectives: This study aims to investigate whether there is an impairment of Fas-mediated lymphocyte apopotosis mechanism in patients with autoimmune lymphoproliferative syndrome (ALPS) patients. Patients and methods: Twenty seven patients (15 males, 12 females; mean age 12.9±2.6 years; range 4 to 45 years) who were monitored at our Immunology Unit of Hacettepe University Ýhsan Doðramacý Children's Hospital with suspected diagnosis of ALPS based on clinical and laboratory signs and four patients (mean age 12±11.2 years; range 3 to 27 years) with confirmed heterozygous Fas mutation who were diagnosed with ALPS-Fas, and 30 healthy individuals were included. Peripheral mononuclear cells were isolated from freshly drawn heparinized blood using ficoll-histopaque density gradient centrifugation method. These cells were activated for 12 days with phytohemagglutinin (PHA) and interleukin-2. Subsequently, they were cultured with anti-Fas monoclonal antibody for additional 48 hours. The cells were stained with trypan blue dye and apoptotic cells were counted under light microscope. Results: It was found that Fas-induced lymphocyte apoptosis was defective in four ALPS-Fas patients. The patients with suspected ALPS and controls had normal Fas-induced apoptosis. Conclusion: Our results indicate that this functional apoptosis test is useful for identification of the patients with Fas mutations. It can also be used for selecting patients for further analyses.

Klinik ve Laboratuvar Özellikleri ile Otoimmün Lenfoproliferatif Sendrom Düþünülen Hastalarda Apoptoz

Amaç: Bu çalýþmada, otoimmün lenfoproliferatif sendrom (ALPS) düþünülen hastalarda Fas aracýlý lenfosit apoptoz mekanizmasýnda bozukluk olup olmadýðý araþtýrýldý. Hastalar ve yöntemler: Hacettepe Üniversitesi Ýhsan Doðramacý Çocuk Hastanesi’nin Ýmmünoloji Ünitesi tarafýndan takip edilen, klinik ve laboratuvar bulgularý ile olasý ALPS tanýsý ile izlenen 27 hasta (15 erkek, 12 kýz; ort. yaþ 12.9±2.6 yýl; daðýlým 4-45 yýl) ile heterozigot Fas mutasyonu kanýtlanmýþ ve ALPS-Fas tanýsý konulmuþ dört kýz hasta (ort. yaþ 12±11.2 yýl; daðýlým 3-27 yýl) ve 30 saðlýklý birey çalýþmaya alýndý. Periferik mononükleer hücreler yeni alýnmýþ kandan heparinli ficoll-histopaque yoðunluk gradyaný santrifüj yöntemi kullanýlarak izole edildi. Bu hücreler 12 gün boyunca phytohemaglutinin (PHA) ve interlökin-2 ile aktive edildi. Takiben ek 48 saat için anti-Fas monoklonal antikoru kullanýlarak hücre kültürü yapýldý. Kýrk sekiz saat sonunda hücreler tripan mavisi ile boyanarak ýþýk mikroskobunda apoptotik hücre sayýmý yapýldý. Bulgular: Otoimmün lenfoproliferatif sendrom-Fas tanýlý dört hastada Fas aracýlý lenfosit apoptoz mekanizmasýnýn defektif olduðu saptandý. Olasý ALPS þüphesi ile izlenen hastalar ve kontrollerde ise Fas aracýlý apoptoz normal idi. Sonuç: Elde ettiðimiz sonuçlara göre, fonksiyonel apoptoz testi, Fas mutasyonu olan hastalarýn belirlenmesinde ve bir ileri basamakta moleküler testlerin uygulanacaðý hastalarýn seçiminde kullanýlabilir.