20.500.12556/RUL-136793 Merjenje aktivacije limfocitov B po spodbujanju s CpG pri diagnostiki specifičnih imunskih pomanjkljivosti Measurement of B cells activation after stimulation with CpG in the diagnosis of primary immunodeficiencies Več kot polovica primarnih imunskih pomanjkljivosti je povezana z motnjo tvorbe protiteles, pri čemer so mehanizmi za pojav bolezni še vedno zelo neraziskani. Prvi znaki se najpogosteje izrazijo že v otroštvu, zgodnja diagnoza pa je pomembna za začetek primerne terapije in s tem preprečitev pojava težjih okužb. Funkcijski testi aktivacije limfocitov B so pomembni za ocenjevanje stanja humoralnega imunskega sistema pri bolnikih s primarno imunsko pomanjkljivostjo. Na podlagi in vitro funkcijskega testa, s katerim določamo proliferacijski potencial, diferenciacijo v plazmablaste in izločanje imunoglobulinov po stimulaciji limfocitov B s CpG, lahko zanesljivo ocenimo sposobnost aktivacije limfocitov B. CpG aktivira limfocite B po od limfocitov T neodvisni poti. Proliferacijo določimo z merjenjem vgraditve fluorescenčno označenega EdU v DNA delečih se celic. Dokazali smo, da pri zdravih posameznikih limfociti B po stimulaciji proliferirajo, se diferencirajo v plazmablaste in izločajo imunoglobuline. Pri bolnikih z diagnozo primarne imunske pomanjkljivosti pa je proliferacija limfocitov B, diferenciacija v plazmablaste ali proizvodnja imunoglobulinov znižana ali popolnoma odsotna. Z namenom vpeljave in vitro funkcijskega testa v rutinsko izvajanje smo določili optimalne pogoje za stimulacijo limfocitov B s CpG. Določili smo normalne vrednosti proliferacije celokupnih, naivnih, spominskih limfocitov B in plazmablastov pri zdravih posameznikih za skupino odraslih in otrok. More than half of primary immune deficiencies are associated with impaired antibody production with mechanisms for disease being still largely unknown. First signs are most often expressed in childhood and early diagnosis is important for starting appropriate treatment and preventing severe infections. Functional activation tests of B cells are important to assess function of humoral immune system in patients with primary immune deficiency. Based on in vitro functional test for determination of proliferation potential, differentiation into plasmablast and immunoglobulin secretion after stimulation of B cells with CpG, we can reliably assess the ability of B cells to be activated. CpG activates B lymphocytes in a T cell-independent pathway. Proliferation is determined by measuring incorporation of fluorescently labelled EdU in DNA of dividing cells. We have shown that after stimulation B cells proliferate, differentiate into plasmablasts, and secrete immunoglobulins in healthy individuals. However, in patients with primary immune deficiency diagnosis, proliferation of B cells, plasmablast differentiation or immunoglobulin secretion is reduced or absent. To introduce in vitro function test in routine, we determined optimal conditions for stimulation of B cells with CpG. We also determined normal values of proliferation of B cells, naïve and memory B cells and plasmablasts in healthy individuals for adults and children. limfociti B proliferacija limfocitov B aktivacija limfocitov B CpG EdU pretočni citometer molekularna imunologija molekularna diagnostika lymphocyte B lymphocyte B proliferation lymphocyte B activation CpG EdU flow cytometer molecular immunology molecular diagnostic true false false [A. Kerin] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2022-05-21 07:15:12 2022-05-21 07:15:43 2022-09-15 04:14:20 0000-00-00 00:00:00 2022 0 Ljubljana 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577.27.083.3:616-097 203487 108815107 22939.pdf 22939.pdf 1 BEE644F8B9410633ED32D11519EC26C2 ab2c9f8a95f39110124f11685be6505f40665436394a63b3cb3bc9cdcc31216e f666d8e6-d8c4-11ec-8aca-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=156583 Biotehniška fakulteta 0 0 0