Cell and molecular markers of systemic vasculitis and autoantibodies against serum amyloid A as physiological regulators of the inflammatory response

doctoral thesis

Tadeja Kuret (Avtor), Snežna Sodin-Šemrl (Mentor), Borut Božič (Član komisije za zagovor), Matjaž Jeras (Član komisije za zagovor), Peter Veranič (Član komisije za zagovor), Saša Čučnik (Komentor)

Povzetek

Giant cell arteritis and immunoglobulin A vasculitis represent a group of chronic rheumatic inflammatory diseases that are categorized as primary systemic vasculitides. They are associated with increased morbidity and earlier mortality as a result of severe cardiovascular complications and long-term use of immunosuppressive therapy. Setting the appropriate diagnosis of both giant cell arteritis and immunoglobulin A vasculitis can be challenging due to diverse and unspecific clinical signs and symptoms and, in some cases, atypical clinical presentation. The therapeutic options for patients with both types of vasculitides are scarce, with glucocorticoids being the first choice. However, glucocorticoids can be associated with severe adverse effects and patients may still experience a relapse when glucocorticoid tapering regimen is applied. Both types of vasculitides are characterized by an acute inflammatory response that can be extended for several years due to impaired or inadequate resolution leading to chronic systemic inflammation. Persistently high levels of acute phase proteins (such as serum amyloid A) during the inflammatory process can lead to accelerated development of atherosclerosis, which is the most common underlying pathological process of cardiovascular diseases. Consequently, patients with giant cell arteritis and immunoglobulin A vasculitis have an increased risk of developing cardiovascular disease compared to the general population. Understanding the mechanisms involved in the resolution of inflammation may thus support the development of alternative, inflammation-blocking therapeutic options for these patients, whereas understanding the role of inflammation and serum amyloid A might unveil new insights into the acceleration of the pathogenic mechanism of atherosclerosis. The work outlined in this thesis aimed to: i) improve the diagnostic, prognostic and therapeutic options of patients with giant cell arteritis and immunoglobulin A vasculitis, ii) explore the role of autoantibodies against serum amyloid A as physiological regulators of the inflammatory response and iii) advance our understanding of the interplay between chronic inflammation, serum amyloid A, glucocorticoid treatment and possible development of atherosclerosis. Within the doctoral thesis, we first focused on reviewing the literature to identify potential serological and tissue markers of giant cell arteritis. We examined the strengths and limitations of existing methodology and identified missing gaps in the current literature. In order to foster the up-to-date scientific knowledge on giant cell arteritis biomarkers, we consequently designed and performed three different research studies. In the first study, we discovered that tissue micro RNA-125b, expressed in the temporal artery biopsies, could be used as a biomarker to identify giant cell arteritis patients, in whom the typical histopathological changes in the temporal arteries are not observed and thus improve their diagnostic options. However, to measure micro RNA expression, performing a temporal artery biopsy is required, which is an invasive procedure. We therefore performed a second, cross-sectional study on giant cell arteritis patients where we focused on measuring serum analytes that could be used to better characterize treatment-naïve patients with giant cell arteritis and to predict disease-associated complications. Serum samples are easy to obtain with a minimally invasive procedure and can be a valuable source of biomarkers. We found that therapy-naïve giant cell arteritis patients had significantly higher serum levels of pro-inflammatory cytokines (interleukins-1β, -6, -18, -23, -27 and -31), chemokines (e.g. interleukin-8), acute phase proteins (serum amyloid A, α1-acid glycoprotein), vascular cell adhesion molecule-1 and matrix metalloproteinases-1 and -9, while levels of interleukin-13, matrix metalloproteinase-2 and α-fetoprotein were significantly lower compared to healthy blood donors. The highest fold change elevations between patients with giant cell arteritis and healthy blood donors were observed for concentrations of serum amyloid A (83-fold change), interleukin-23 (58-fold), and interleukin-6 (11-fold). Giant cell arteritis patients with visual disturbances had lower levels of acute phase parameters serum amyloid A, C-reactive protein, haptoglobin and erythrocyte sedimentation rate, compared to patients without visual disturbances. In contrast, the levels of SAA, C-reactive protein, and erythrocyte sedimentation rate were increased in therapy-naïve giant cell arteritis patients with future relapse, compared to the patients without future relapse. Our third, longitudinal giant cell arteritis study, reported on an increased neutrophil surface expression of adhesion molecule L-selectin in peripheral blood of treatment-naïve patients with active disease. The expression of L-selectin rapidly decreased after initiation of glucocorticoid treatment, followed by a progressive increase to week 48, when patients received lower doses of glucocorticoid monotherapy. This progressive increase of L-selectin was not observed in patients under combinatory therapy with leflunomide, which indicates that the addition of leflunomide might have a beneficial long-term effect on the control of neutrophil adhesion. Since erythrocyte sedimentation rate and levels of C-reactive protein are usually used to monitor giant cell arteritis activity but are not always reliable markers, our study also focused on alternative serum biomarkers that might be useful in this regard. Prior to treatment, the relapsing patients had significantly higher median levels of interleukin-23, compared to patients in the responder group. Serum levels of interleukin-23 were also higher in all three relapsing patients, at the time closest to relapse, compared to the last time point before relapse. Levels of interleukin-23 decreased again after relapse and concurrent administration of leflunomide. Interleukin-23 could therefore represent a viable biomarker to monitor giant cell arteritis activity. Similar to patients with giant cell arteritis, therapy-naïve adult patients with immunoglobulin A vasculitis also exhibited higher serum levels of serum amyloid A, interleukin-6 and interleukin-8 compared to healthy individuals, however these increases were not as high as those observed in giant cell arteritis. The inflammatory response, together with increased serum concentrations of immunoglobulin A and higher percentages of neutrophils determined in peripheral blood of immunoglobulin A vasculitis patients in our study, go in line with the theory of existing cross-talk between immunoglobulin A, endothelial cells and neutrophils in the pathogenesis of immunoglobulin A vasculitis that ultimately leads to substantial vascular damage. Our study confirmed the association of neutrophil to lymphocyte ratio with clinical manifestations involving gastrointestinal tract in patients with immunoglobulin A vasculitis. We also found that patients with gastrointestinal tract involvement had a lower expression of neutrophil adhesion molecule integrin αM, compared to patients with skin-limited presentation that would need further validation to be used as a marker of systemic involvement. Our results show that high- or medium-grade inflammatory response is present in therapy-naïve patients with giant cell arteritis and immunoglobulin A vasculitis, respectively, with upregulated levels of acute phase proteins, especially serum amyloid A. If the inflammatory response does not resolve, it can lead to chronic inflammation with significant organ damage and accelerated development of atherosclerosis. One of the potential mechanisms involved in the resolution of inflammation may include autoantibodies against acute phase proteins that could neutralize the pro-inflammatory activities of their respective antigens or promote their clearance. To check this hypothesis, we optimized an in house enzyme-linked immunosorbent assay for the detection of immunoglobulin G autoantibodies against human recombinant serum amyloid A1α (endogenous human protein) and human recombinant serum amyloid A (a hybrid between forms A1α and A2β). The presence of both autoantibodies was detected in serum samples of 300 healthy blood donors. The peak levels of autoantibodies against serum amyloid A and serum amyloid A1α were determined in individuals between 41 and 50 years. We also found that autoantibodies against both proteins are present in the therapeutic preparation of intravenous IgG and could be isolated using affinity chromatography. The isolated immunoglobulin G fractions of anti-serum amyloid A and anti-serum amyloid A1α autoantibodies decreased interleukin-6 release from serum amyloid A- or serum amyloid A1α-stimulated peripheral blood mononuclear cells in a dose-dependent manner. This could represent a novel endogenous mechanism that could regulate excessive pro-inflammatory functions of serum amyloid A. To confirm the presence of anti-serum amyloid A1α autoantibodies with another method, we developed a novel bead-based duplex immunoassay for simultaneous detection of anti-serum amyloid A1α and anti-α1-acid glycoprotein autoantibodies in human sera. We showed that autoantibodies against both serum amyoid A1α and α1-acid glycoprotein can be detected in serum samples of healthy blood donors and patients with systemic autoimmune diseases (giant cell arteritis, immunoglobulin A vasculitis, systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis and antiphospholipid syndrome) as well as intravenous immunoglobulin G. When tested for detection in human subjects the highest levels of both autoantibodies were determined in patients with giant cell arteritis. No significant differences were observed between healthy blood donors and any group of patients with systemic autoimmune diseases. Our method could be further optimized and developed to increase the number of autoantibodies against different acute phase proteins that can be simultaneously detected. Chronic inflammation is closely linked to the onset and development of atherosclerosis and cardiovascular disease with serum amyloid A playing a contributory role. Since atherosclerotic lesions typically develop in coronary arteries and the activation of endothelium represents one of the crucial early steps, we used an in vitro culture of human coronary endothelial cells to study endothelial activation and intracellular synthesis of SAA. We found upregulated messenger RNA expression of both serum amyloid A1 and serum amyloid A2 in interleukin-1β-stimulated coronary endothelial cells, while confirming the constitutive expression of serum amyloid A4, unchanged upon stimulation. No increase in serum amyloid A1 or serum amyloid A2 messenger RNA synthesis was observed when coronary endothelial cells were stimulated with interleukin-6 or glucocorticoids. We detected all three serum amyloid A proteins localized around the nuclei of interleukin-1β-stimulated, as well as unstimulated coronary endothelial cells, and in tunneling membrane nanotubes that connect the cells. The detection of serum amyloid A1 in tunneling membrane nanotubes led us to analyze potential serum amyloid A1 transport between cells. The intercellular exchange of serum amyloid A1 was more pronounced when coronary endothelial cells were stimulated with IL-1β. An inflammatory stress signal (e.g. interleukin-1β) can therefore increase intracellular serum amyloid A1 and serum amyloid A2 synthesis and promote serum amyloid A1 transport between cells. Taken together, we determined cell and molecular markers of primary systemic vasculitis, specifically giant cell arteritis and immunoglobulin A vasculitis, that could complement current diagnostics and might be used to monitor giant cell arteritis activity and response to treatment. We discovered that immunoglobulin G autoantibodies against serum amyloid A could function as physiological regulators of the inflammatory response by decreasing interleukin-6 release from serum amyloid A-stimulated peripheral blood mononuclear cells. Lastly, we showed that pro-inflammatory mediator interleukin-1β increased intracellular messenger RNA synthesis of serum amyloid A1 and serum amyloid A2 in human coronary artery endothelial cells, as well as promoted intercellular serum amyloid A1 transport.

Ključne besede

systemic vasculitis;autoantibodies;cell and molecular markers;

Podatki

Jezik:	Angleški jezik
Leto izida:	2020
Tipologija:	2.08 - Doktorska disertacija
Organizacija:	UL FFA - Fakulteta za farmacijo
Založnik:	[T. Kuret]
UDK:	616-002
COBISS:	46686979
Št. ogledov:	166
Št. prenosov:	0
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Slovenski jezik
Sekundarni naslov:	Celični in molekulski označevalci sistemskih vaskulitisov ter avtoprotitelesa proti serumskemu amiloidu A kot fiziološki regulatorji vnetnega odziva
Sekundarni povzetek:	Gigantocelični arteritis in vaskulitis imunoglobulina A sta primarna sistemska vaskulitisa, ki sodita v skupino kroničnih vnetnih revmatičnih bolezni. Povezana sta z večjo obolevnostjo in zgodnejšo umrljivostjo zaradi srčno-žilnih zapletov in dolgotrajnega zdravljenja z imunosupresivno terapijo. Postavitev diagnoze tako giagantoceličnega arteritisa kot tudi vaskulitisa imunoglobulina A je lahko zahtevna predvsem zaradi nespecifičnih in raznolikih znakov in simptomov bolezni ter v nekaterih primerih atipične klinične slike. Za farmakoterapijo obeh vaskulitisov se priporoča uporaba glukokortikosteroidov, ki povzročajo številne hude neželene učinke. Kljub zdravljenju se pri bolnikih z gigantoceličnim arteritisom še vedno lahko pojavi kasnejše poslabšanje oz. ponoven zagon bolezni ob zniževanju odmerka glukokortikosteroidov. Za bolnike z gigantoceličnim arteritisom in vaskulitisom imunoglobulina A je značilen sistemski vnetni odziv, ki dolgoročno ne izzveni in tako vodi v razvoj kroničnega vnetja. Kronično vnetje spremljajo trajno povišani serumski nivoji proteinov akutne faze, zlasti serumskega amiloida A. Pri tem sta pomembni dve obliki serumskega amiloida A, in sicer serumski amiloid A1 (podtipi α, β in γ) ter serumski amiloid A2 (podtipa α in β), medtem ko se koncentracija serumskega amiloida A4 med vnetjem ne spremeni. Serumski amiloid A lahko s svojim vnetnim delovanjem pospeši nastanek in razvoj ateroskleroze, enega izmed najpogostejših patoloških procesov, ki vodijo do nastanka bolezni srca in ožilja. Posledično imajo bolniki z gigantoceličnim arteritisom in vaskulitisom imunoglobulina A povečano tveganje za razvoj srčno-žilnih zapletov v primerjavi s splošno populacijo. Razumevanje mehanizmov, ki so vpleteni v umiritev vnetnega odziva, lahko tako vodi do razvoja alternativnih možnosti terapije za bolnike s kroničnimi vnetnimi boleznimi, medtem ko razumevanje vloge vnetja in serumskega amiloida A lahko razkrije nove možne vpoglede v mehanizem razvoja ateroskleroze. Raziskovalno delo v okviru doktorske disertacije je bilo tako namenjeno: i) iskanju bioloških označevalcev, ki bi lahko izboljšali diagnostične, prognostične in terapevtske možnosti bolnikov z gigantoceličnim arteritisom in bolnikov z vaskulitisom imunoglobulina A, ii) določitvi vloge avtoprotiteles, usmerjenih proti serumskemu amiloidu A, pri regulaciji vnetnega odziva in iii) doprinosu k boljšemu razumevanju povezave med kroničnim vnetjem, serumskim amiloidom A, imunosupresivno terapijo z glukokortikosteroidi in možnim razvojem ateroskleroze. V okviru doktorske naloge smo se najprej usmerili v pregled obstoječe literature o serumskih in tkivnih označevalcih pri bolnikih z gigantoceličnim arteritisom. Preučili smo prednosti in pomanjkljivosti uporabljenih metod in prepoznali manjkajoče vrzeli v obstoječih študijah. Na podlagi spoznanj smo zasnovali in izvedli tri različne raziskovalne študije. V prvi raziskavi smo odkrili, da bi lahko z merjenjem izražanja molekule mikro RNA-125b v tkivu temporalnih arterij prepoznali tiste bolnike z gigantoceličnim arteritisom, pri katerih ne opazimo značilnih histopatoloških sprememb v temporalnih arterijah. Na ta način bi izboljšali diagnostične možnosti bolnikov z gigantoceličnim arteritisom z neznačilno histopatološko sliko. Odvzem tkiva temporalnih arterij za histološko preiskavo ali merjenje izražanja molekul mikro RNA predstavlja za bolnike invaziven postopek. V naši drugi, presečni študiji smo se zato osredotočili na iskanje serumskih analitov, ki bi bili uporabni za pomoč pri postavitvi diagnoze gigantoceličnega arteritisa in napovedovanje zapletov, povezanih s samo boleznijo. Serumski vzorci se namreč pridobijo na enostaven način, z minimalno invazivnim postopkom in lahko predstavljajo dragocen vir bioloških označevalcev. Bolnikom z gigantoceličnim arteritisom smo serumske vzorce odvzeli, še preden so prejeli odmerek glukokortikosteroidov, koncentracije serumskih analitov pa primerjali s koncentracijami pri zdravih krvodajalcih. Ugotovili smo, da imajo bolniki z gigantoceličnim arteritisom značilno povišane serumske nivoje vnetnih citokinov (interlevkin-1β, -6, -18, -23, -27 in -31), kemokinov (npr. interlevkin-8), proteinov akutne faze (npr. serumski amiloid A, α1-kisli glikoprotein), vaskularne celične adhezijske molekule-1 in metaloproteinaz matriksa-1 ter -9, medtem ko so bile koncentracije interlevkina-13, metaloproteinaze matriksa-2 in α-fetoproteina značilno znižane v primerjavi z zdravimi posamezniki. Največje razlike med bolniki z gigantoceličnim arteritisom in zdravimi krvodajalci smo opazili pri koncentracijah serumskega amiloida A (83-krat), interlevkina-23 (58-krat) in interlevkina-6 (11-krat), kar jih uvršča med možne terapevtske tarče za zdravljenje gigantoceličnega arteritisa. Bolniki z gigantoceličnim arteritisom in motnjami vida so imeli nižje koncentracije serumskega amiloida A, C-reaktivnega proteina in zmanjšano hitrost sedimentacije eritrocitov v primerjavi z bolniki brez motenj vida. Bolniki s kasnejšim poslabšanjem bolezni so imeli v času pred uvedbo terapije povišane serumske nivoje serumskega amiloida A in C-reaktivnega proteina ter zvečano hitrost sedimentacije eritrocitov v primerjavi z bolniki brez kasnejšega poslabšanja bolezni. V tretji, longitudinalni študiji pri bolnikih z gigantoceličnim arteritisom smo poročali o povečanem izražanju adhezijske molekule L-selektina na nevtrofilnih granulocitih v periferni krvi bolnikov z aktivno, nezdravljeno boleznijo. Izražanje molekule L-selektina se je po začetku zdravljenja z glukokortikosteroidi zmanjšalo in se postopno spet povečalo v 48. tednu po začetku zdravljenja, ko so bolniki že prejemali nižje odmerke glukokortikosteroidov. Pri bolnikih z gigantoceličnim arteritisom, ki so prejemali kombinirano terapijo glukokortikosteroidov in leflunomida, ni bilo opaziti progresivnega zvečanja izražanja L-selektina, kar kaže, da bi lahko dodatek leflunomida dolgoročno vplival na uravnavanje adhezije nevtrofilnih granulocitov. Običajno se za spremljanje aktivnosti gigantoceličnega arteritisa določata hitrost sedimentacije eritrocitov in koncentracija C-reaktivnega proteina, ki pa nista vedno najzanesljivejša označevalca. V naši raziskavi smo se zato osredotočili na iskanje alternativnih serumskih označevalcev. Nezdravljeni bolniki z gigantoceličnim arteritisom, ki so kasneje doživeli poslabšanje bolezni, so imeli povišane srednje vrednosti serumskih nivojev interlevkina-23 v primerjavi z bolniki brez kasnejšega poslabšanja bolezni. Koncentracije interlevkina-23 so bile višje tudi pri vseh treh bolnikih s poslabšanjem bolezni v serumskih vzorcih, odvzetih ob času poslabšanja, v primerjavi z zadnjim odvzemom pred poslabšanjem. V vzorcih, odvzetih 12 tednov po poslabšanju bolezni in sočasni terapiji z leflunomidom, so se serumske vrednosti interlevkina-23 ponovno znižale. Merjenje serumskega interlevkina-23 bi nam tako lahko služilo za spremljanje aktivnosti gigantoceličnega arteritisa in morda tudi za napoved odziva na zdravljenje. Podobno kot pri bolnikih z gigantoceličnim arteritisom, smo tudi pri odraslih bolnikih z vaskulitisom imunoglobulina A še pred uvedbo terapije določili višje nivoje serumskega amiloida A, interlevkina-6 in interlevkina-8 v primerjavi z zdravimi krvodajalci, vendar ta razlika ni bila tako izrazita kot pri bolnikih z gigantoceličnim arteritisom. Bolniki z vaskulitisom imunoglobulina A, ki so bili vključeni v našo študijo, so imeli tudi višje izmerjene serumske koncentracije imunoglobulina A in višji delež nevtrofilnih granulocitov v krvi v primerjavi z zdravimi krvodajalci. S tem smo potrdili teorijo nastanka bolezni, v kateri sodelujejo imunoglobulini A, endotelijske celice v stenah žil in nevtrofilni granulociti, kar privede do poškodbe žilne stene. Naša raziskava je potrdila tudi povezavo razmerja med nevtrofilnimi granulociti in limfociti v krvi bolnikov z razvojem kliničnih znakov na prebavnem traktu. Ugotovili smo, da so imeli bolniki z vaskulitisom imunoglobulina A in prizadetostjo prebavnega trakta zmanjšano izražanje adhezijske molekule integrin αM na nevtrofilnih granulocitih v periferni krvi v primerjavi z bolniki z omejeno kožno prizadetostjo. Molekula integrin αM bi lahko služila kot označevalec razvoja prizadetosti prebavnega trakta, vendar bi za to potrebovali še potrditveno študijo na večji skupini bolnikov z vaskulitisom imunoglobulina A. Naši rezultati kažejo, da je vnetna komponenta lahko bolj ali manj izražena pri nezdravljenih bolnikih z gigantoceličnim arteritisom ali vaskulitisom imunoglobulina A, pri katerih so serumski nivoji proteinov akutne faze, še zlasti serumskega amiloida A, povišani. Če pri teh bolnikih ne pride do umiritve vnetnega odziva, se lahko pojavijo poškodbe tkiv in organov, razvoj ateroskleroze pa je pospešen. Eden od možnih mehanizmov, ki bi lahko sodeloval pri umiritvi vnetnega odziva, vključuje avtoprotitelesa proti proteinom akutne faze, ki lahko bodisi nevtralizirajo vnetno delovanje samih proteinov bodisi spodbujajo njihovo odstranitev iz telesa. Da bi preverili to hipotezo, smo optimizirali hišno encimsko-imunsko metodo na trdnem nosilcu za določanje prisotnosti in nivojev avtoprotiteles imunoglobulina G proti dvema rekombinantno pridobljenima oblikama serumskega amiloida A: serumskemu amiloidu A1α (aminokislinsko zaporedje je enako kot pri endogeni človeški obliki proteina) in serumskemu amiloidu A (aminokislinsko zaporedje vsebuje dele aminokislinskega zaporedja dveh endogenih človeških oblik, in sicer 1α in 2β). V serumskih vzorcih 300 zdravih krvodajalcev smo z omenjenima metodama odkrili prisotnost obeh avtoprotiteles. Najvišje nivoje avtoprotiteles proti serumskemu amiloidu A in proti obliki A1α smo določili pri posameznikih, starih med 41 in 50 let. Ugotovili smo tudi, da so avtoprotitelesa razreda G proti obema proteinoma prisotna v terapevtskih pripravkih intravenskih imunoglobulinov G in smo jih lahko izolirali z afinitetno kromatografijo. Izolirane frakcije imunoglobulinov G so zmanjšale sproščanje interlevkina-6 iz perifernih mononuklearnih celic, izoliranih iz polne krvi zdravih krvodajalcev in stimuliranih s serumskim amiloidom A ali A1α. Avtoprotitelesa proti serumskemu amiloidu A bi lahko predstavljala fiziološki mehanizem uravnave pretirane in škodljive vnetne funkcije samega proteina oz. prekomernega vnetnega odziva in možnost alternativne terapije za bolnike s kroničnimi vnetnimi boleznimi. Da bi potrdili prisotnost avtoprotiteles proti serumskemu amiloidu A1α še z drugo metodo, smo razvili nov imunološki test za hkratno določanje prisotnosti in nivojev avtoprotiteles proti serumskemu amiloidu A1α in α1-kislemu glikoproteinu v serumskih vzorcih. Pri testu gre za vezavo primarnih aminov antigena na karboksilne skupine, izpostavljene na površini magnetnega delca, in specifično vezavo med antigenom na magnetnemu delcu ter protitelesom v vzorcu. Detekcijski sistem pa meri jakost fluorescence sekundarnega, s fluoroforom označenega protitelesa. Pokazali smo, da lahko avtoprotitelesa, usmerjena proti serumskemu amiloidu A1α in α1-kislemu glikoproteinu, s to metodo določimo v serumskih vzorcih zdravih posameznikov, bolnikov s sistemskimi avtoimunskimi boleznimi (gigantocelični arteritis, vaskulitis imunoglobulina A, sistemski lupus eritematozus, sistemska skleroza, revmatoidni artritis in antifosfolipidni sindrom) in v intravenskih pripravkih imunoglobulinov G. Kronično vnetje je tesno povezano z nastankom in razvojem ateroskleroze ter bolezni srca in ožilja, pri čemer ima vlogo tudi serumski amiloid A. Aterosklerotične lehe se običajno razvijejo v koronarnih arterijah, kjer aktivacija endotelija predstavlja enega ključnih in zgodnjih korakov. Za proučevanje mehanizmov endotelne aktivacije in znotrajcelične sinteze serumskega amiloida A smo zato uporabili in vitro kulturo humanih koronarnih endotelijskih celic. Ugotovili smo, da se izražanje podtipov serumskega amiloida A1 in A2 na nivoju informacijske RNA poveča v koronarnih endotelijskih celicah po stimulaciji z vnetnim citokinom interlevkinom-1β, medtem ko se izražanje podtipa A4 ne spremeni. Obenem pa stimulacija koronarnih endotelijskih celic z interlevkinom-6 ali glukokortikosteroidi ni vplivala na spremenjeno sintezo serumskih amiloidov A1, A2 in A4. Vse tri oblike proteina so bile lokalizirane okoli jeder tako v nestimuliranih kot tudi v koronarnih endotelijskih celicah, predhodno stimuliranih z interlevkinom-1β. Opazili smo tudi, da se vse tri oblike nahajajo v tunelskih membranskih nanocevkah, ki povezujejo celice, kar nas je vodilo v nadaljnje raziskovanje morebitnega medceličnega prenosa serumskega amiloida A1. Tako smo dokazali, da se lahko serumski amiloid A1 med celicami prenaša, kar je bilo izrazitejše po stimulaciji z interlevkinom-1β. Predvidevamo, da lahko prisotnost stresnega signala (npr. prisotnost vnetja in zvišan nivo interlevkina-1β) poveča sintezo znotrajceličnih serumskih amiloidov A1 in A2 ter pospeši medcelični prenos serumskega amiloida A1. V doktorski nalogi smo določili celične in molekulske označevalce gigantoceličnega arteritisa in vaskulitisa imunoglobulina A, ki bi nam lahko služili za pomoč pri postavitvi diagnoze in bi jih lahko določali pri spremljanju aktivnosti gigantoceličnega arteritisa ter odzivu na zdravljenje. Odkrili smo, da lahko avtoprotitelesa razreda G proti serumskemu amiloidu A delujejo kot fiziološki regulatorji vnetnega odziva, tako da zmanjšajo sproščanje interlevkina-6 iz perifernih mononuklearnih celic, stimuliranih s serumskim amiloidom A. Nazadnje smo pokazali, da vnetni citokin interlevkin-1β v koronarnih endotelijskih celicah poveča znotrajcelično sintezo serumskih amiloidov A1 in A2, obenem pa tudi spodbuja medcelični prenos serumskega amiloida A1.
Sekundarne ključne besede:	sistemski vaskulitisi;avtoprotitelesa;celični in molekulski označevalci;
Vrsta dela (COBISS):	Doktorska disertacija
Komentar na gradivo:	Univ. v Ljubljani, Fak. za farmacijo
Strani:	XII, 203 str.
ID:	15504127