OPTIMALIZÁCIA IN VITRO DIFERENCIÁCIE A AKTIVÁCIE MONOCYTOV PRED ANALÝZOU EXPRESIE GÉNU PLAUR

SLANINA, Peter, Julie ŠTÍCHOVÁ, Petra KULÍŠKOVÁ, Lucie BALLONOVÁ, Jan BAROŠ, Jiří LITZMAN, Marcela VLKOVÁ, Přemysl SOUČEK, Tomáš FREIBERGER and Roman HAKL. OPTIMALIZÁCIA IN VITRO DIFERENCIÁCIE A AKTIVÁCIE MONOCYTOV PRED ANALÝZOU EXPRESIE GÉNU PLAUR (OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION). In XXXIX sjezd českých a slovenských alergologů a klinických imunologů. 2022.

Basic information

• Original name: OPTIMALIZÁCIA IN VITRO DIFERENCIÁCIE A AKTIVÁCIE MONOCYTOV PRED ANALÝZOU EXPRESIE GÉNU PLAUR
• Name in Czech: OPTIMALIZÁCIA IN VITRO DIFERENCIÁCIE A AKTIVÁCIE MONOCYTOV PRED ANALÝZOU EXPRESIE GÉNU PLAUR
• Name (in English): OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION
• Authors: SLANINA, Peter (703 Slovakia, belonging to the institution), Julie ŠTÍCHOVÁ (203 Czech Republic, belonging to the institution), Petra KULÍŠKOVÁ (203 Czech Republic, belonging to the institution), Lucie BALLONOVÁ (203 Czech Republic, belonging to the institution), Jan BAROŠ (203 Czech Republic, belonging to the institution), Jiří LITZMAN (203 Czech Republic, belonging to the institution), Marcela VLKOVÁ (203 Czech Republic, belonging to the institution), Přemysl SOUČEK (203 Czech Republic, belonging to the institution), Tomáš FREIBERGER (203 Czech Republic, belonging to the institution) and Roman HAKL (203 Czech Republic, belonging to the institution).
• Edition: XXXIX sjezd českých a slovenských alergologů a klinických imunologů, 2022.

Other information

• Original language: Czech
• Type of outcome: Conference abstract
• Field of Study: 30102 Immunology
• Country of publisher: Czech Republic
• Confidentiality degree: is not subject to a state or trade secret
• RIV identification code: RIV/00216224:14110/22:00129722
• Organization unit: Faculty of Medicine
• Keywords (in Czech): Monocyty; PLAUR; izolace; diferenciace
• Keywords in English: Monocytes; PLAUR; isolation; differentiation
• Tags: rivok

Abstract

Cieľom práce bola optimalizácia procesu izolácie, diferenciácie a aktivácie monocytov. Snahou bolo dosiahnuť maximálne výťažky monocytov z odberu periférnej krvi, ich vysokú čistotu a najnižšiu možnú primárnu aktiváciu buniek. Výberom vhodnej koncentrácie cytokínov a inkubačnej doby sme sa snažili docieliť in-vitro diferenciáciu monocytov do makrofágov typu M1. V aktivovaných a plne diferencovaných monocytoch u pacientov s hereditárnym angioedémom, astmou a reumatoídnou artritídou bude následne sledovaná expresia génu PLAUR. Gén PLAUR kóduje proteín uPAR (CD87) ovplyvňujúci fibrinolýzu po jeho interakcii s urokinázovým aktivátorom plasminogénu (uPA), zrážanlivosť krvi a produkciu bradikinínu väzbou na FXII, ktorý sa aktivuje na povrchu buniek a moduluje bunečnú adhezivitu a migráciu cez interakcie s proteínmi extracelulárneho matrix. Z odberu periférnej krvi boli monocyty izolované metódou magnetickej separácie. Získané monocyty boli diferencované kombináciou cytokínov M-CSF a INFγ, ktoré podporili diferenciáciu do M1 makrofágov. Čistota izolovaných monocytov, ich diferenciácia a aktivácia bola stanovená mikroskopicky a pomocou prietokovej cytometrie. Vyššie výťažky monocytov boli dosiahnuté použitím dvojnásobne koncentrovaného roztoku PBS pričom sa priemerný výťažok monocytov pohyboval okolo 150 000 buniek z 1 ml periférnej krvi s čistotou nad 95 %. Nízka aktivácia monocytov bola docielená pri použití sklenených skúmaviek a udržaním nízkej teploty (< 8 ºC) bunečnej suspenzie počas celého procesu izolácie. Mikroskopickým pozorovaním bola stanovená najvhodnejšia dĺžka inkubácie monocytov a koncentrácie cytokínov MCSF a INFg, Diferenciácia a aktivácia monocytov bola stanovená meraniami na prietokovom cytometri na základe zvýšenej povrchovej expresie markerov CD86, CD38 a CCR7. Takto diferencované a aktivované monocyty sa ukázali byť vhodné pre ďalšie spracovanie. Výsledkom prevedených pokusov je optimalizovaný a verifikovaný postup izolácie a aktivácie monocytov potrebný na presnú analýzu expresie génu PLAUR.

Abstract (in English)

The aim of the work was to optimize the process of isolation, differentiation and activation of monocytes. The aim was to achieve maximum monocyte yields from peripheral blood collection, their high purity and the lowest possible primary cell activation. By selecting the appropriate cytokine concentration and incubation time, we aimed to achieve in-vitro differentiation of monocytes into M1-type macrophages. Subsequently, PLAUR gene expression will be monitored in activated and fully differentiated monocytes from patients with hereditary angioedema, asthma, and rheumatoid arthritis. The PLAUR gene encodes a uPAR protein (CD87) affecting fibrinolysis upon its interaction with urokinase plasminogen activator (uPA), blood coagulation and bradykinin production by binding to FXII, which is activated at the cell surface and modulates cell adhesion and migration through interactions with extracellular matrix proteins. Monocytes were isolated from peripheral blood samples by magnetic separation. The recovered monocytes were differentiated by a combination of the cytokines M-CSF and INFγ, which promoted differentiation into M1 macrophages. The purity of the isolated monocytes, their differentiation and activation were determined microscopically and by flow cytometry. Higher monocyte yields were obtained using a doubly concentrated PBS solution with an average monocyte yield of around 150,000 cells per 1 ml of peripheral blood with a purity of over 95%. Low monocyte activation was achieved by using glass tubes and by maintaining a low temperature (< 8 ºC) of the cell suspension throughout the isolation process. The most appropriate length of monocyte incubation and concentrations of the cytokines MCSF and INFg were determined by microscopic observation, Monocyte differentiation and activation were determined by flow cytometer measurements based on increased surface expression of the markers CD86, CD38 and CCR7. Thus differentiated and activated monocytes proved to be suitable for further processing. As a result of the performed experiments, an optimized and verified monocyte isolation and activation procedure necessary for accurate analysis of PLAUR gene expression has been established.

Links

• NU21-05-00438, research and development project: Name: Úloha alternativních forem uPAR v rozvoji imunopatologických reakcí

Investor: Ministry of Health of the CR, Subprogram 1 - standard