Title SVOJSTVA CIRKULIRAJUĆIH POZITIVNIH STANICA VON WILLEBRANDOVA ČIMBENIKA U BOLESNIKA S AKUTNIM INFARKTOM MIOKARDA Title (english) SVOJSTVA CIRKULIRAJUĆIH POZITIVNIH STANICA VON WILLEBRANDOVA ČIMBENIKA U BOLESNIKA S AKUTNIM INFARKTOM MIOKARDA Author Marijana Rakić Mentor Gordana Laškarin (mentor)Mentor Viktor Peršić (komentor)Committee member Vlatka Sotošek Tokmadžić (predsjednik povjerenstva)Committee member Mirza Žižak (član povjerenstva)Committee member Alen Ružić (član povjerenstva)Committee member Gordana Laškarin (član povjerenstva)Committee member Viktor Peršić (član povjerenstva)Granter University of Rijeka Defense date and country 2020-12-17, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 616 - Pathology. Clinical medicine ThesaurusMESH - Medical Subject Headings )
Myocardial Infarction
von Willebrand Factor
Abstract Cilj istraživanja: utvrditi učestalost, fenotip te izražaj citokina i kemokina u pozitivnih stanica von Willebrandova čimbenika (vWF+) u perifernoj krvi bolesnika s akutnim infarktom miokarda (AIM) i rezultate usporediti s autolognim CD14+ stanicama i vWF+ stanicama zdravih ispitanika. Uspostaviti protokol za pročišćavanje vWF+ stanica u magnetskom polju i ispitati njihov utjecaj na održavanje perforina u autolognim limfocitima T te istražiti prisutnost perforin+CD3+ stanica u miokardu bolesnika umrlih od AIM.
Ispitanici i metode: Bolesnicima s AIM krv je uzorkovana 1. dana AIM (prije primarne PCI - od engl, percutaneous coronary intervention), potom 7. i 28. dana, a zdravim ispitanicima jednokratno. Uz kliničke i rutinske laboratorijske metode koristili smo imunološke metode
izdvajanje mononuklearnih stanica (MS) naslajanjem na gradijent gustoće, određivanje viabilnost vWF+ i CD14+ stanica tripanskim modrilom, obilježavanje biljega metodom indirektne i direktne imunofluorescencije (vWF, CD14, CD1a, HLA-DR, CD80, CD86, CCR7, perforina, CD3, CD56), obilježavanje unutarstaničnih citokina interleukina (IL)-15, interferona- gama (IFN-γ) i CC liganda (CCL) 17). Također smo pročistili stanične podvrste u magnetskom polju, za pokuse ko-kulture funkcijski netaknutih CD3+ limfocita T i vWF+, odnosno CD14+ stanica te dvostrukom imunofluorescencijom analizirali perforin+/CD3+ stanice u rezovima tkiva miokarda uklopljenih u parafin bolesnika umrlih od AIM.
Rezultati: U svih bolesnika s AIM udio vWF+ stanica nije se razlikovao s obzirom na površinsko ili unutarstanično obilježavanje i bio je viši prvog, sedmog i 28. dana u odnosu na zdrave ispitanike. U bolesnika s NSTEMI (od engl. non-ST-elevation myocardial infarction), koji su liječeni s PCI, dinamika vWF+ stanica je pratila dinamiku onih s STEMI (od engl. ST- elevation myocardial infarction) i primarnom PCI. Bolesnici s NSTEMI liječeni medikamentnom terapijom imali su najviši udio cirkulirajućih, viabilnih vWF+ stanica, koje pokazuju fenotip antigen predočnih stanica (APS). Prvog dana nakon AIM izražavali su HLA-DR i CD86. Nisu izražavali CCR7. vWF+ stanice su stvarale manje IL-15, IFN-γ i CCL17 u odnosu na autologne CD14+ stanice. Oplemenili smo vWF+ stanice i CD14+ stanice iz adherentne frakcije MS bolesnika s NSTEMI 1. dana po AIM do oko 80% (viabilnost >90%). U pokusu ko-kulture, vWF+ stanice nisu mogle statistički značajno povećati udio perforina u CD3+ limfocitima T, ali su povećale statistički značajno MFI (od engl. mean fluorescence intensity) u omjeru 1:2,5. CD14+ stanice učinkovito su održavale postotak i MFI za perforin u limfocitima T u omjeru 1:2,5. Na tkivnim rezovima miokarda bolesnika umrlog od AIM, broj stanica koje izražavaju perforin je bio oskudan, i sačinjavao je neznatan udio CD3+ limfocita.
Zaključak: Udio cirkulirajućih vWF+ stanica značajno je viši u svih bolesnika nakon AIM nego u zdravih ispitanika. Viabilne vWF+ stanice u bolesnika s NSTEMI prvi dan nakon AIM pokazuju pokazuju fenotip stanica koje predočavaju antigen sa smanjenom mogućnošću kostimulacije limfocita T iz periferne krvi u pokusima održavanja perforina, što ukazuje na njihovu ulogu u nishodnom podešavanju imunološkog odgovora tijekom akutnog AIM.
Abstract (english) Aim of the study: to determine frequency, phenotype and expression of cytokines and chemokines in circulating vWF+ cells in patients suffering from acute myocardial infarction (AMI) and compare the results with autologous CD14+ cells and vWF+ cells of the healthy control group; to establish a purification protocol of vWF+ cells and investigate their influence on perforin maintenance in co-culture with autologous T cells and analyze frequency of perforin+CD3+ cells in the myocardium of patients who died from AMI.
Material and Methods: peripheral blood was sampled from patients with AIM on the first day after AIM (before primary PCI-percutaneous coronary intervention) and then again on 7th and 28th day, while only once in healthy subjects. Besides clinical (patient examination, ECG) and laboratory methods (detection of inflammatory parameters, blood cell counts, liver and kidney parameters) we used immunological methods as follows: gradient density centrifugation for isolation of mononuclear cells (MC), determination of cell-viability by trypan blue, phenotype analysis by labeling surface or intracellular antigens (vWF, CD14, CD1a, HLA-DR, CD80, CD86, CCR7, perforin, CD3, CD56, interleukin (IL)-15, interferon-gamma (IFN-γ) and CC ligand (CCL)-17) by flow cytometry. We used magnetic cell separation method to enrich vWF+ cells and CD14+cells, and co-cultured them with enriched and functionaly untouched cognate CD3+ cells. We analyzed presence of perforin+/CD3+ cells by immnofluorescence in parafin embedded myocardium from patients who died from AMI.
Results: In all patients who suffered AIM, the proportion of vWF+ cells was higher then in healthy control, irrespectively of intracellular or surface vWF labeling. Dynamics of circulating vWF+ cells in patients with NSTEMI (non-ST elevation myocardial infarction) treated with PCI was similar to the dynamics in patients with STEMI (ST elevation myocardial infarction) and primary PCI. The highest proportion of circulating, viable vWF+ cells was found in patients with NSTEMI treated with anti-ishaemic drugs on the first day after AMI. They mostly expressed HLA-DR, while CD86 and CD80 are expressed less. CCR7 is absent. They produced less IL- 15, IFN-γ and CCL17 when compared with autologous CD14+ cells. Enriched vWF+ cells (80%) of viability 90% significantly increased MFI (mean fluorescence intensity) for perforin in CD3+ cells in a cell ratio of 1:2,5, while equaly enriched CD14+ cells increased MFI for perforin in T cells at cell ratios 1:2,5 and 1:5, and the proportion at 1:2,5 ratio. The number of perforin+ cells was scarce in the myocardium of patients deceased after AMI and represented negligible proportion od CD3+ cells infiltrating myocardium.
Conclusion: the proportion of vWF+ cells was significantly higher in all patients with AIM when compared to healthy control group. Viable vWF+ cells in patients with NSTEMI on the first day after AIM demonstrated the phenotype of APC with decreased possibility for peripheral blood T cells costimulation in perforin maintenance experiment which shows their role in downregulating of immune response during AIM.
Keywords
infarkt miokarda
akutni
limfocit T
perforin
stanice
antigen predočne
stanice
endotelne
von Willebrand faktor
Keywords (english)
antigen presenting
cells
endothelial
cells
myocardial infarction
acute
T cells
monocytes
von Willebrand factor
Language croatian URN:NBN urn:nbn:hr:184:997793 Promotion 2021 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Repository Repository of the University of Rijeka, Faculty of Medicine Created on 2021-03-02 23:26:06
