Title Stanična imunost i citotoksični potencijal u bolesnika s višestrukim ozljedama tijela Title (english) Cellular immunity and cytotoxic potential in multiple injured patients Author Nikola Gržalja Mentor Tedi Cicvarić (mentor)Mentor Vlatka Sotošek Tokmadžić (komentor)Committee member Gordana Laškarin (predsjednik povjerenstva)Committee member Drago Batinić (član povjerenstva)Committee member Alen Protić (član povjerenstva)Granter University of Rijeka Defense date and country 2018, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences ThesaurusMESH - Medical Subject Headings )
Immunity, Cellular
Multiple Trauma
Abstract Cilj istraživanja:
Višestruke ozljeda tijela povezane su s povećanim rizikom od nastanka komplikacija, što može biti povezano s intenzivnim proupalnim i protuupalnim odgovorom organizma na ozljedu. Iako su brojni radovi objasnili utjecaj višestrukih ozljeda na funkciju imunološkoga sustava, postoji vrlo malo podataka o promjenama u citolitičkom potencijalu imunokompetentnih stanica, uključujući i izražavanje citolitičkih posrednika kao što je perforin. Cilj ovog rada bio je istražiti i usporediti promjene u udjelu leukocitnih subpopulacija, kao i promjene u njihovom izražavanju perforina u perifernoj venskoj krvi bolesnika s izoliranom ozljedom donjih ekstremiteta, bolesnika s izoliranom ozljedom grudnog koša te bolesnika s ozljedom grudnog koša i pridruženom ozljedom donjih ekstremiteta.
Ispitanici, materijal i metode:
U istraživanje je bilo uključeno 90 bolesnika (30 bolesnika s izoliranom ozljedom donjih ekstremiteta, 30 bolesnika s izoliranom ozljedom grudnog koša te 30 bolesnika s ozljedom grudnog koša i pridruženom ozljedom donjih ekstremiteta), a kontrolnu skupinu činilo je 30 zdravih dobrovoljnih davaoca krvi koji su spolom i dobi odgovarali skupinama bolesnika. Uzorci periferne venske krvi bolesnika uključenih u ispitivanje uzimani su odmah po dolasku u bolnicu te su potom postupkom centrifugiranja na gradijentu gustoće izolirane mononuklearne stanice. Izolirane mononuklearne stanice su obilježene s monoklonskim protutijelima na površinske biljege kako bi se utvrdila njihova pripadnost pojedinom leukocitnim populacijama (limocitima T, stanicama NK, stanicama NKT) i njihovim subpopulacijama. Dodatno je izoliranim mononuklearnim stanicama permeabilizirana membrana te metodom izravne imunofluorescencije unutarstanično obilježena citotoksična molekula perforin i citokin interleukin (IL)-15. Uzorci koji su bili predviđeni za unutarstanično obilježavanje IL-15 prethodno su stimulirani s PMA (eng. phorbol myristate acetat), jonomicinom i monenzinom radi prikazivanja citokina u stanici. Stanice obilježene protutijelima su analizirane na protočnome citometru.
Koncentracije interleukina-4 (IL-4) i interferona-gama (IFNγ) u plazmi bolesnika određene su upotrebom ELISA (od eng. enzyme linked immunosobrent assay) komercijalnih kitova. Metodom pozitivne magnetske separacije iz mononuklearnih stanica periferne krvi izdvojene su stanice NK i određivana njihova citotoksična aktivnost prema K-562 NK-osjetljivoj staničnoj liniji korištenjem PKH-26 dvosatnog testa citotoksičnosti uz očitavanje protočnim citometrom.
Rezultati:
U bolesnika s ozljedom grudnih organa i pridruženom ozljedom donjih ekstremiteta postoji statistički značajan pad udjela limfocita T, stanica NK, stanica NKT i njihovih subpopulacija te značajan pad u izražaju perforina u perifernoj venskoj krvi u odnosu na kontrolnu skupinu i skupine bolesnika s izoliranom ozljedom donjih ekstremiteta i izoliranom ozljedom grudnog koša. U skupini bolesnika s ozljedom grudnog koša i pridruženom ozljedom donjih ekstremiteta utvrđene su povećane koncentracije IL-4 i IFNγ neposredno po ozljedi. Bolesnici s istovremenom ozljedom donjih ekstremiteta i grudnoga koša imaju statistički značajno manju citotoksičnost prema NK-osjetljivoj staničnoj liniji K-562 u odnosu na ostale skupine bolesnika. U skupini višestruko ozlijeđenih bolesnika s ozljedom grudnog koša i pridruženom ozljedom donjih ekstremiteta postoji značajna negativna korelacija između težine ozljede, određene ISS skorom (eng. Injury Severity Score) i postotka limfocita T, stanica NK i postotka stanica NKT. U skupini višestruko ozlijeđenih bolesnika s ozljedom grudnog koša i pridruženom ozljedom donjih ekstremiteta postoji značajna negativna korelacija između težine ozljede određene ISS-om te postotka perforin pozitivnih stanica NK i postotka perforin pozitivnih stanica NKT.
Zaključak:
Iz dobivenih rezultata može se zaključiti da je pad u udjelu limfocita periferne venske krvi, kao i smanjenje njihovog citotoksičnog potencijala povezan s težinom ozljede, što može pridonijeti povećanoj pojavi komplikacija koje se javljaju u skupini višestruko ozlijeđenih bolesnika.
Abstract (english) Objectives:
Patients with multiple body injuries are associated with increased risk of complications and this may be related to the intense pro- and anti-inflammatory patient response to the injury.
Although the impact of multiple injuries to the immune system at the cellular and molecular level is explained by numerous papers, there is little information about the changes in the cytolytic potential of immunocompetent cells, including expression of cytolytic mediators such as perforin. The aim of this study was to analyse and compare changes in the frequency of leukocyte subpopulations as well as changes in their expression of perforin in peripheral venous blood of patients with isolated lower limb fractures, patients with isolated chest injuries and patients with chest injuries and associated lower limb fractures.
Patients and Methods:
Ninety patients (30 patients with isolated lower limb fractures, 30 patients with isolated chest injuries and 30 patients with chest injuries and associated lower limb fractures) were included in this study. Peripheral venous blood of 30 sex- and age-matched healthy blood donors served as the control group. Peripheral venous blood samples were taken from all patients included in the study immediately after arrival in the hospital and mononuclear cells were isolated by gradient centrifugation. Isolated mononuclear cells were labeled with antibodies against specific surface antigens to determine different leukocytes population (T lymphocytes, NK cells, NKT cells) and their subpupulations. Isolated mononuclear cells were perameabilized for intracellular staining of perforin and interleukin (IL)-15. Cell samples that were scheduled for intracellular cytokine staining, were previously stimulated with PMA, ionomycine and monensim which are necessary for cytokine visualisation. Labelled cells were anlysed by flow cytometry.
Plsma concentrations of IL-4 and interferon gama (IFNγ) were detected by ELISA (enzyme linked immunosobrent assay) according to the manufacturer instructions. NK cells were isolated from peripheral blood mononuclear cells by positive magnetic cell separation method. The cytotoxic activity of separated CD56+ NK cells against NK sensitive cell K-562 cell line was analysed by PKH-26 (red) cell linker kit and flow cytometry.
Results:
In patients with chest pain and associated lower limb injury there was a statistically significant decrease in frequency of lymphocyte T, NK cells, NKT cells and their subpopulations, as well as a significant decrease in perforin expression in peripheral venous blood when compared to the control group and the group of patients with isolated lower limb and thoracic injury. In the group of patients with chest pain and associated lower limb injury, increased levels of IL-4 and IFNγ were determined directly after injury. Patients with concomitant lower extremity and chest pain had statistically significantly lower cytotoxicity to the NK-sensitive K-562 cell line compared to other patients. A significant negative correlation were found between ISS (Injury Severity Score) and the percentage of T lymphocytes, NK cells and NKT cells in the patients with chest trauma and injury associated lower limb injury. A significant negative correlation between ISS and percentage of positive cells in n the patients with chest trauma and injury associated lower limb injury.
Conclusion:
From the obtained results it can be concluded that the decrease in the frequency and cytotoxic potential of peripheral blood lymphocytes is related to the severity of trauma injury, which can explain the underlying mechanism contributing to the increased occurrence of complications in multiple injured patients.
Keywords
Bolesnik s višestrukim ozljedama tijela
perforin
stanice NK
stanice NKT
stanična imunost
Keywords (english)
Cellular Immunity
Multiple Injured Patient
NK Cells
NKT Cells
Perforin
Language croatian URN:NBN urn:nbn:hr:184:738252 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 Created on 2019-04-23 13:05:51
