Aktivnost serumske dipeptidil-peptidaze IV i ekspresija CD26 antigena na imunokompetentnim ćelijama periferne krvi kod bolesnica sa benignim i malignim promenama dojke

Abstract

Uvod: dipeptidil-peptidaza IV (DPPIV ili CD26) je serin-proteaza koja ima značajnu ulogu u brojnim biološkim procesima. Kalretikulin je protein koji reguliše brojne važne ćelijske funkcije. Cilj ovog rada je bio da se odredi serumska aktivnost DPPIV, procenat CD26+ belih krvnih ćelija i CD26+ limfocita, kao i srednja vrednost intenziteta fluorescencije ekspresije CD26 na limfocitima u bolesnica sa benignim i malignim lezijama dojke i u zdravih kontrola i da se utvrdi da li su intenzitet i lokalizacija povišene ekspresije kalretikulina u tumorskim ćelijama povezani sa povećanjem humoralnog imuniteta na kalretikulin u ispitivanim grupama. Takođe je analiziran procenat nekih limfocitnih subpopulacija, CD16+/56+ i CD16+/89+ kod bolesnica sa malignim i benignim promenama dojke, kao i kod zdravih kontrola. Metode: Uključeno je 37 bolesnica sa benignim i 68 sa malignim promenama dojke, pre hirurškog tretmana, kao i 37 zdravih kontrola. Serumska aktivnost DPPIV izmerena je kolorimetrijskim testom, dok je broj CD26+ćelija određena metodom protočne citomerije. Citoplazmatska i membranska ekspresija kalretikulina u malignim i benignim ćelijama parafinskih isečaka utvrđena je imunohistohemijski. Nivoi serumskih antikalretikulinskih antitela izmereni su ELISA metodom. Procenat ispitivanih limfocitni subpopulacija određen je metodom protočne citomerije. Rezultati: Značajan pad u procentu ukupnih CD26+ ćelija ustanovljen je u grupi bolesnica sa benignim i malignim promenama u poređenju sa zdravim kontrolama. Intenzitet fluorescencije CD26 ekspresije na limfocitima je značajno niži kod bolesnica sa malignim promenama u poređenju sa zdravim kontrolama. Statistički značajna razlika je utvrđena u serumskim nivoima antikalretikulinskih IgA antitela između bolesnica i zdravih kontrola. Relativna učestalost gCD16dim+/56bright+ subpopulacije imunokompetentnih ćelija je statistički značajno niža u bolesnica sa malignim, ali i benignim promenama dojke u odnosu na zdrave kontrole; statistički značajno niže vrednosti su utvrđene i u relativnoj učestalosti tCD16dim+/56bright+ ćelija u bolesnica sa malignim promenama dojke u odnosu na one u zdravih kontrola; niže vrednosti procenta gCD16bright+/56dim+imunskih ćelija su takodje karakteristika bolesnica sa malignim ili benignim promenama u odnosu na zdravie kontrole. Procenat CD89+ imunskih ćelija (uglavnom granulocita) značajno je povišen kako u bolesnica sa malignim promenama tako i u bolesnica sa benignim promenama u odnosu na zdrave kontrole...

Objective: Dipeptidyl peptidase IV (DPPIV or CD26) is a serine protease which has a significant role in a variety of biological processes. Calreticulin is a protein which regulates many important cellular responses. The aim of this work was to determine serum DPPIV activity as well as the percentage of CD26+ white blood cells and of CD26+ lymphocytes and the mean fluorescence intensity (MFI) of CD26 expression on lymphocytes in groups of patients with benign or malignant breast tumors and in healthy controls and to elucidate whether the intensity and location of calreticulin overexpression in tumor cells are related to the elevated humoral immunity to calreticulin in patients with benign or malignant breast disease. It was also analized the percentage of some subpopulations of lymphocytes, such as: CD16+/56+ i CD16+/89+ in patients with benign and malignant breast lesions and in healthy controls as well. Methods: This study involved 37 patients with benign and 68 patients with malignant breast tumors before surgical resection and 37 healthy volunteers. Serum DPPIV activity was determined by colorimetric test, while CD26+ cells were counted using flow cytometer. Cytoplasmatic or membranous calreticulin overexpression in malignant or benign cells in paraffinembedded tissues was determined using immunohistochemistry. Levels of the serum anti-calreticulin autoantibodies were detected by ELISA. Percentage of lymphocytes was determined using flow cytometry. Results: Significant decrease in the percentage of CD26+ total white blood cells was found in the group of breast cancer patients and in patients with benign breast tumors compared to healthy people. The MFI of CD26 expression on lymphocytes was significantly lower for cancer patients in comparison to healthy controls. Statistically significant differences between serum levels of IgA of anti-calreticulin antibodies in controls and patients with breast tumors, and between controls and patients with nonmalignant breast diseases were found. It was revealed significant decrease in the percentage of gCD16dim+/56bright+ cells in patients with benign and malignant breast lesions, as well as in the of percentage of tCD16dim+/56bright+ in malignant breast lesions compared with healthy controls. It was also found significant decrease in the of percentage of gCD16bright+/56dim+ cells in patients with benign and malignant breast lesions compared with healthy controls. Percentage of CD89+ cells was significant elevated in patients with benign and malignant breast lesions compared to healthy controls..