www.medenosrce.net/arhimed   arhimed(a-t>medenosrce.net   [23/04/2026 12:35:10] http://www.medenosrce.net/arhimed/poglej.asp?id=121 Aktivacija molekul CD69 in CD25 na za antigene bakterije Borrelia afzelli specificnih CD4+, CD8+ in CD19+ limfocitnih bolnikov z erythema migrans Avtor: Matevž Jan, Sergeja Gregorcic Mentor: prof. dr. Vladimir Kotnik IZHODIŠCE: Lymska borelioza je v Sloveniji endemicna bolezen. Povzroca jo Borrelia burgdorferi sensu lato, prenašajo pa jo klopi. Iz kožnih sprememb v Sloveniji najpogosteje osamimo B. afzelii. Laboratorijsko skušamo potrditi bolezen z dokazom navzocnosti protiteles, specificnih za borelijske antigene, in/ali izolacijo povzrocitelja. V primerih zgodnje lokalizirane borelioze je protitelesni imunski odziv navadno skromen ali pa ga ni. Predvidevamo, da bi lahko takrat uspešneje sklepali na prisotnost in aktivnost bolezni z merjenjem celicnega imunskega odziva. NAMEN: V raziskavi smo merili izražanje molekul CD25 in CD69, ki se pojavijo na površni razlicnih vrst in podvrst aktiviranih limfocitov. Želeli smo ugotoviti, ali bi lahko metodo iz raziskave prenesli v laboratorijsko diagnostiko zgodnje borelijske okužbe. HIPOTEZA: Pricakovali smo, da bomo pri bolnikih z erythema migrans glede na zdrave preiskovance, ki niso bili v stiku z borelijami, dokazali vecji delež limfocitov, ki po in vitro spodbudi z borelijskimi antigeni izražajo aktivacijske oznacevalce CD25 in CD69. METODE: Spremembe smo merili v krvi devetih bolnikov z erythema migrans in sedmih zdravih preiskovancev. Kri smo po odvzemu najprej 24 ur inkubirali z antigeni B. afzelii ter nato s pretocno citometrijo ugotavljali izražanje aktivacijskih oznacevalcev CD25 in CD69 na vrstah in podvrstah limfocitov. Obenem smo izmerili koncentracijo INF-a v plazemskem preostanku po in vitro spodbudi limfocitov z borelijskimi antigeni. Bolnikom smo izmerili tudi titer specificnih protiteles proti B. burgdorferi sensu lato. Rezultate smo statisticno vrednotili z Studentovim dvorepim, dvovzorcnim testom t. Korelacijo smo izrazili s koeficientom korelacije po Pearsonu (r). REZULTATI: Ugotovili smo, da je razlika izražanja molekul CD69 med zdravimi preiskovanci in bolniki statisticno znacilna na limfocitih CD4+ (p=0,046) in CD8+ (p=0,004) po spodbudi s 5,0 µg/mL antigena B. afzelii. Na spodbudo s 2,5 µg/mL antigenov B. afzelii so se odzvali samo limfociti CD8+ (p=0,012). Ravno tako se izražanje molekul CD25 statisticno znacilno razlikuje med zdravimi preiskovanci in bolniki na limfocitih CD3+ (p=0,036) po spodbudi s 5,0 µg/mL antigenov. Pri zdravih preiskovancih v primerjavi z bolniki smo ugotovili šestkrat vecji porast koncentracije IFN-a v plazemskem preostanku po spodbudi s 5,0 µg/mL antigenov B. afzelii. Ugotovitev je pomembna, kljub temu da razlika ni statisticno znacilna (p=0,098), saj nakazuje verjetnost, da vsaj pri nekaterih preiskovancih sprožijo antigeni B. afzelii imunski odziv po tipu Th 2. ZAKLJUCKI: Raziskavo zakljucujemo s sklepom, da smo odkrili z limfociti CD4+ in CD8+ posredovan zgodnji celicni imunski odziv na okužbo z B. afzelii. Ugotavljamo, da lahko merjenje izražanja aktivacijskega oznacevalca CD69 na limfocitih CD4+ in CD8+ olajša diagnosticne dileme pri bolnikih z nejasno klinicno sliko zgodnje lokalizirane borelioze. «» [Abstract / English version] Aktivacija molekul CD69 in CD25 na za antigene bakterije Borrelia afzelli specificnih CD4+, CD8+ in CD19+ limfocitnih bolnikov z erythema migrans Author: Matevž Jan, Sergeja Gregorcic Mentor: prof. dr. Vladimir Kotnik BACKGROUND: Lyme borreliosis is endemic in Slovenia. It is a disease caused by Borrelia burgdorferi sensu lato and is transmitted by ticks. The most frequently isolated borrelia from skin manifestations of patients with borreliosis in Slovenia is B. afzelii. Diagnosis of borreliosis is based on clinical and laboratory findings. Laboratory diagnosis is aimed toward detecting the presence of antibodies specific to borrelial antigens. Sometimes isolation of borrelia is used. Humoral immune response is usually poor or absent in cases of early localized borreliosis. We assume that in the early phase of borreliosis the presence and the activity of the disease could be detected by measuring cellular immune response. AIM: We measured the presence of CD25 and CD69 molecules that express on surfaces of activated lymphocytes. We also reflected the possibility of transfering our research method to laboratory diagnostics of early localized borreliosis. HYPOTHESIS: We expected that after in vitro stimulation with borrelial antigens patients with erythema migrans will have higher proportion of lymphocytes expressing activation markers CD25 and CD69 than healthy individuals, which were not infected with borrelia. METHODS: Whole blood of nine patients with erythema migrans and seven healthy induviduals was used in the research. Whole blood was incubated with antigens of B. afzelii for 24 hours and then we measured the expression of activation markers CD25 and CD69 on surfaces of different populations and subpopulations of lymphocytes using flow cytometry. We also measured IFN-a concentration in the plasma remnant after in vitro stimulation of lymphocytes with borrelial antigens. Patients had their titre of B. burgdorferi sensu lato specific antibodies measured. All results were statistically evaluated using Student's t-test. Correlation was expressed as Pearson's correlation coefficient (r). RESULTS: We discovered that the difference in CD69 expression between healthy individuals and patients was statistically significant, when measured on CD4+ (p=0,046) and CD8+ (p=0,004) lymphocytes after stimulation with concentration of 5,0 µg/mL of B. afzelii antigens, and also when measured on CD8+ (p=0,012) lymphocytes after stimulation with concentration of 2,5 µg/mL of B. afzelii antigens. We also discovered that the difference in CD25 expression between healthy individuals and patients was statistically significant, when measured on CD3+ (p=0,036) lymphocytes after stimulation with concentration of 5,0 µg/mL of B. afzelii antigens. We discovered a sixfold increase of IFN-a concentration in plasma after stimulation with concentration of 5,0 µg/mL of B. afzelii antigens in healthy individuals compared to patients. This finding is important despite the fact that the difference was not statisticaly significant (p=0,098), because it shows the possibility that Th 2 type immune response is triggered by B. afzelii antigens. CONCLUSIONS: We discovered CD4+ and CD8+ mediated early immune response to infection with B. afzelii. We established that measuring activation marker CD69 expression on CD4+ and CD8+ lymphocytes could be used for diagnostic purposes in cases of clinically uncharacteristic early localized borreliosis.