Zašto izloženost bezazlenim i široko rasprostranjenim tvarima iz okoliša u nekih osoba izaziva neprimjereno jake reakcije imunološkog
sustava klinički prepoznatljive kao alergijske bolesti te na koji ...način ponavljana izloženost istim tim tvarima u obliku specifične imunoterapije preusmjerava imunološku reaktivnost, za sada su samo djelomično razjašnjena pitanja. Zbivanja tijekom alergijskog upalnog odgovora relativno su dobro poznata, kao i glavni mehanizmi kojima specifična imunoterapija, kao jedina uzročna metoda liječenja, mijenja taj odgovor. Ipak, svakodnevne nove spoznaje dopunjavaju slagalicu i naše razumijevanje ovih složenih zbivanja. Cilj je ovoga rada prikazati do danas upoznate mehanizme nastanka i liječenja alergijskih bolesti, uz poseban osvrt na ključnu ulogu T-limfocita u tim procesima.
Provider: - Institution: - Data provided by Europeana Collections- Cilj ovog doktorskog rada je procjeniti stanicama posredovanu imunost temeljem istraživanja prisutnosti, raspodjele i citotoksičnog ...potencijala posredovanog apoptotičkom molekulom granulizina izraženoj u limfocitima periferne krvi bolesnika s akutnim infarktom miokarda. Ispitanici, materijal i metode: U istraživanje su bili uključeni bolesnici s akutnim infarktom miokarda sa ST elevacijom (STEMI) kojima je u činjena primarna perkutana koronarna intervencija uz nastavak medikamentne terapije, bolesnici s akutnim infarktom miokarda bez ST elevacije(NSTEMI) koji su liječeni medikamentnom terapijom prema smjernicama Europskog kardiološkog društva. Obje skupine bolesnika bile su uključene u program rane stacionarne medicinske rehabilitacije. Zdravi ispitanici regrutirani tijekom rutinskih kardioloških pregleda predstavljali su kontrolnu skupinu. Prvog, 7-og, 14-og, 21-og i 28-og dana nakon akutnog koronarnog zbivanja svakom bolesniku uzimali smo 10-16 ml venske krvi za imunološka i biokemijska istraživanja. Mononuklearne stanice periferne krvi izdvojili smo centrifugiranjem na gradijentu gustoće, te neposrednim dvostrukim obilježavanjem površinskih biljega CD3 i CD56 i unutarstaničnog biljega granulizina metodom indirektne imunofluorescencije analizirali njihov fenotip protočnom citometrijom. Imunocitokemijom smo prikazali granulizin u limfocitima periferne krvi. Metodom imunohistologije smo obilježavali antigene CD3, CD56, granulizin, APAF-1(od engl. Apoptotic Protease Activating Factor 1) i MHC molekule razreda I u parafinskim rezovima miokarda bolesnika umrlih u prvom i petom tjednu nakon akutnog koronarnog zbivanja, te bolesnika umrlih od bolesti koje primarno nisu zahva ćale miokard. Citotoksičnost stanica NK (nekroza i apoptoza)prema K-562 ciljevima tijekom dugotrajnog (18-satnog) testa citotoksičnosti ispitivali smo protočnom citometrijom uz pomoć PKH26 testa i aneksina V u prisustvu samo medija, te neutralizirajućih protutijela usmjerenih prema perforinu i/ili granulizinu. Ispitanicima smo bilježili dob, spol, naviku pušenja cigareta, te ispitivali kompletnu krvnu sliku i laboratorijske biokemijske parametre. Rezultati upućuju na zaključak da granulizinom posredovana apoptoza bar djelomično sudjeluje u oštećenju miokarda tijekom i neposredno nakon akutnog koronarnog zbivanja, ali molekula granulizina može doprinositi rezoluciji leukocita i upalnog odgovora nastalog tijekom endogene ishemijske pro-upalne reakcije u miokardu zahvaćenom infarktom.- Objectives: The aim of this doctor thesis is to evaluate cell-mediated immunity of patients
with acute myocardial infarction based on investigation of the presence and distribution of
apoptotic molecule granulysin in peripheral blood lymphocytes, as well as estimation of their
cytotoxic potential.
Patients, material and methods: Patients with acute myocardial infarction with ST elevation
(STEMI), who underwent primary percutaneous coronary intervention with continued drug
treatment and patients with acute myocardial infarction without ST elevation (NSTEMI), who
were treated with drug therapy according to the guidelines of the European Society of
Cardiology were included in the investigation. Both groups of patients were involved in the
program of early stationary medical rehabilitation. Healthy subjects, which were recruited
during routine cardiac’s examination representd the control group. On days 1, 7, 14, 21 and on
day 28 after the acute coronary event 10-16 ml of venous blood were taken from the patients
for immunological and biochemical analyses. Mononuclear cells from peripheral blood were
separated by centrifugation on a density gradient. Their phenotype was analysed by flow
cytometry. Direct double-labeling of surface CD3 and CD56 markers was aplied in the
combination with intracellular granulysin labeling by indirect immunofluorescence.
Immunocytochemistry was used to visualize granulysin in peripheral blood lymphocytes.
Immunohistology was implemented for detection of CD3 and CD56 antigens, granulysin,
apoptotic protease activating factor 1 (APAF-1) and MHC class I molecules in paraffin
embedded myocardial tissue sections from patients who died in the first and the fifth week
after the acute coronary event, and from patients who died from non-cardiac causes. Flow
cytometry with PKH26 test and anexin V was applied to analyse long term (18 hours)
cytotoxicity (necrosis and apoptosis) of NK cells against K-562 targets in the presence of
medium only, neutralizing antibodies directed towards perforin and/or granulizin. Age,
gender, smoking habit, complete blood count and laboratory biochemical parameters (urea,
creatinine, Na+, K+, aspartate aminotransferase (AST), alanine transaminase (ALT), creatine
kinase (CK) and troponin I) were analysed in examinees. Ultrasound examination of the heart
was performed in examinees at the beginning and at the end of rehabilitation period.
Results: In patients with NSTEMI percentage of granulysin+ peripheral blood lymphocytes in
all subpopulations, including both subsets of NK cells (CD56+dim and CD56+bright) was
statistically significantly higher on days 1 and 7 after the acute coronary event when
compared with control group while their lowest frequency was on the 14th day after the acute
coronary event. In patients with STEMI percentage of granulysin+ lymphocytes including
both subsets of peripheral blood NK cells was almost negligible on days 7 and 14 after the
acute coronary event. Visualization of granulysin+ cells using immunocytochemistry in
peripheral blood lymphocytes of patients with NSTEMI and STEMI was followed by the
mean fluorescence intensities (MFIs), which were calculated with flow cytometry. In healthy
subjects, peripheral blood NK cells in the 18-hour cytotoxicity assay spontaneously kill and
induced apoptosis of target K562 cells, mainly using perforin mechanism. In patients with
NSTEMI on days 7 and 28 after the acute coronary event the apoptosis of K 562 targets was
statistically significantly decreased in the presence of anti-perforin antibody, anti-granulysin
antibody or their combination, but without additive effect. In the same patients, the 14th day
after acute coronary events the killing was significantly reduced and apoptosis becomes
negligible due to the low frequency of granulizin+ NK cells. Increased percentage of
granulysin+ NK cells on days 21 and 28 led to the higher apoptosis of K 562 cells in direct
contact. In patients with STEMI apoptosis of K 562 targets was negligible on the 7th day due
to low frequency of granulysin+ NK cells and MFI. Apoptosis, which was observed on 14th
and on 21th day after the acute coronary event was only 1-10% and it could be removed with
the addition of anti-perforin and anti-granulysin antibodies with additive effect, although each
antibody itself did not cause statistically significant reduction. Expression of MHC class I
molecules was significantly decreased in the center of the infarct necrosis, but MHC class I
molecules were present in the area surrounding the necrosis and in the healthy myocardium.
In the leukocyte infiltration, there were relatively rare CD3+ and CD56+ cells that expressed
granulysin in paraffin embeded myocardium tissue sections from patients who died in the 1st
week after the acute coronary event. CD3+ cells, CD56+ cells and granulysin+ cells were
found in the vicinity of APAF-1+ cardiomyocytes, especially in areas with myocardial
infarction and leukocyte infiltration, in contrast to patients who died in the fifth week after
acute coronary event, where only APAF-1+ cells were present. Ultrasound examination
showed mild recovery of ejection fraction at the end of rehabilitation period, although it was
not statistically significant.
Conclusion: Granulysin mediated apoptosis is, at least partially, involved in myocardial
injury during and immediately after the acute coronary event, but granulysin could contribute
to the resolution of the leukocytes and inflammatory response generated during ischemic
endogenous pro-inflammatory response in the field of myocardial infarction- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
Provider: - Institution: - Data provided by Europeana Collections- Cilj istraživanja bio je utvrditi povezanost između ekspresije čimbenika stanične smrti – Fas, FasL, perforina i HSP70 i bioloških ...karakteristika neuroblastoma. Uključena su sva djeca s dijagnozom neuroblastoma operirana na KBC Zagreb u razdoblju od 1994. do 2013. godine, a kriterije je zadovoljilo 42 ispitanika. Na parafinskim blokovima izuzetim prije početka kemoterapije imunohistokemijskim bojanjem određen je izražaj Fas i FasL molekule na stanicama neuroblastoma, broj citotoksičnih T limfocita u okolini stanica neuroblastoma, izražaj perforina na citotoksičnim T limfocitima te izražaj HSP70 na stanicama neuroblastoma. Utvrđeno je da je Fas smanjeno izražen na metastatskim stadijima tumora pa se može pretpostaviti da tumor smanjenjem ekspresije Fas-a na svojoj površini može izbjeći lokalni odgovor imunološkog sustava i tako metastazirati. Također je utvrđena povezanost ekspresije HSP70 i stadija neuroblastoma. Viši stadiji imaju smanjenu ekspresiju što je u suprotnosti s većinom tumora koji su do sada istraživani, no s druge strane se slaže s istraživanjima na nefroblastomu, drugom najčešćem solidnom tumoru dječje dobi. Najvjerojatnije objašnjenje, prema dosadašnjim istraživanjima, je da kod tumora dječje dobi sudjelovanje HSP70 u inhibiciji apoptoze nije toliko značajno kao sudjelovanje u diferencijaciji tumora te da njegova pojačana ekspresija u neuroblastoma dovodi do sazrijevanja tumora.- The aim of this study was to investigate the correlation between the expression of cell death factors - Fas, FasL, perforin and HSP70 and biological characteristics of neuroblastoma. All children diagnosed with neuroblastoma operated at KBC Zagreb in the period since 1994. to 2013. were included and 42 of them met our criteria. On paraffin blocks excluded before the start of chemotherapy through immunohistochemical staining the expression of Fas and FasL molecules on neuroblastoma cells, the number and expression of perforin on cytotoxic T lymphocytes in the vicinity of neuroblastoma cells and expression of HSP70 in neuroblastoma cells was analyzed. Reduced Fas expression was found in metastatic tumor stages so it can be assumed that by lowering expression of Fas on its surface, tumor can avoid the local immune response and thus metastasize. The connection of HSP70 expression and stage neuroblastoma is also found. Higher stages have decreased expression of which is in contrast with the majority of tumors that have been studied, but on the other hand agrees with research on nephroblastoma, the second most common solid tumor in children. The most likely explanation for that, according to available data and considering pediatric tumors, is that the role of HSP70 in inhibition of apoptosis is not as important as participation in the differentiation of the tumor and its overexpression in neuroblastoma leads to the maturation of the tumor.- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
Provider: - Institution: - Data provided by Europeana Collections- Uvod
Psorijaza je upalna sustavna Th1 citokinska kožna bolest. U psorijatičnom
epidermisu je vodeća promjena keratinocitna ...proliferacija povezana s upalnim
promjenama, a očita je i uloga imunološkog sustava (prisutnost velikog broja
aktiviranih limfocita T u epidermisu i dermisu, kao i povezanost psorijaze s razredom
I HLA sustava). Drži se da je antigena stimulacija limfocita T u epidermisu preko
razreda I HLA unutar kojeg se određuje vezivanje peptida i predodžba T stanicama
ključna u imunosnom odgovoru (CD4+ i CD8+). Aktivirane T-stanice izlučuju brojne
Th1 citokine (interleukin 2-IL-2, γ interferon –INF-γ, tumor necrosis factor α – TNF-α,
interleukin 6 – IL-6) koji potiču keratinocitnu proliferaciju. Nova istraživanja govore o
važnosti adhezijskih molekula ICAM-1 i VCAM-1 u nastajanju psorijaze. U ovom radu
dokazana je njihova pojavnost u psorijatičnoj promijenjenoj i nepromijenjenoj koži.
Unatrag deset godina primjenjuje se i imunoterapija protutijelima protiv odgovarajućih
molekula koje sudjeluju u lancu imunoloških zbivanja u nastanku psorijaze.
Cilj istraživanja
Usporediti pojavnost aktiviranih limfocita T te makrofaga 68 i adhezijskih molekula:
intercelularne adhezijske molekule-1 (ICAM-1), vaskularne adhezijske molekule-1
(VCAM-1) te interleukin-2 receptora (IL-2r), u promijenjenoj i nepromijenjenoj koži
ispitanika sa psorijazom s nalazom u koži zdravih ispitanika, kao kontrolne skupine.
Na temelju tih nalaza, ukoliko ima razlike u pojavnosti u odnosu na zdravu kožu,
dokazati njihovu ulogu u etiopatogenezi psorijaze.
Materijal i metode
U ovome radu učinjena je probatorna biopsija kože 50 ispitanika sa psorijazom, kod
kojih je uzet uzorak promijenjene kože i nepromijenjene kože najmanje 5 cm
udaljene od ruba promijenjene kože, bolesnika koji četrnaest dana nisu koristili
sustavnu niti lokalnu terapiju protiv psorijaze. Uzet je uzorak kože kontrole skupine
50 ispitanika sa zdravom kožom. Potom su svježe smrznuti rezovi u fiziološkoj
otopini obrađeni u Zavodu za patologiju Medicinskog fakulteta Sveučilišta u Rijeci.
Standardnim imunohistokemijskim postupkom izvršeno je bojenje tkiva kitovima
tvrtke DAKO, za adhezijske molekule ICAM-1 i VCAM-1, a zatim očitavanje i limfocita
CD4 i CD8, te CD68+ makrofaga , te IL-2r kitovima s Medicinskog fakulteta Sveučilišta u Rijeci. Statističku obradu podataka izvršili su statističari Medicinskog
fakulteta Sveučilišta u Rijeci.
Rezultati
Prema rezultatima ovoga rada utvrđena je statistički značajna razlika u pojavnosti
ICAM-1 molekule iz promijenjene i nepromijenjene kože ispitanika sa psorijazom
(p<0,001) u odnosu na (zdravu) kožu kontrolne skupine. Također je statistički
značajna razlika u pojavnosti molekule VCAM-1 u odnosu na kožu kontrole (zdravih
ispitanika) (p<0,0001). Isto tako, uočena je značajna razlika u usporedbi IL-2r u
promijenjenoj koži ispitanika sa psorijazom u odnosu na kontrolnu skupinu (zdravih)
ispitanika (p<0,001).
Limfociti CD4 i CD8 su nađeni u većem broju ukupno u epidermisu i dermisu u
promijenjenoj koži ispitanika sa psorijazom u usporedbi s kontrolom (p<0,001). Isto
tako uočena je povećana izražajnost CD68+ makrofaga u promijenjenoj koži
ispitanika sa psorijazom u usporedbi s kontrolom (p<0,001).
Zaključak
Prema rezultatima rada kao i prema nekim drugima autorima, utvrđena je statistički
značajna razlika u pojavnosti adhezijskih molekula u koži ispitanika sa psorijazom i to
u promijenjenoj i nepromijenjenoj koži u usporedbi sa kontrolom, čime je dokazana
njihova važnost u etiopatogenezi psorijaze i u slijedu imunoloških zbivanja u koži
ispitanika sa psorijazom. Važno je istaknuti da se nepromijenjena koža ispitanika sa
psorijazom nalazi u «stanju pripravnosti» i u svakom trenutku može nastati promjena
na koži zbog visokih nalaza adhezijskih molekula u nepromijenjenoj koži ispitanika sa
psorijazom. Smatramo da bi odgovarajuća imunološka terapija protutijelima mogla
biti učinkovita u liječenju psorijaze.- Introduction
Psoriasis vulgaris is a systemic Th1 cytokine-mediated skin disease. In psoriatic
epidermis, the leading change is proliferation of keratinocytes associated with
inflammation. The role of immunologic reactions is obvious through the presence of
many activated T lymphocytes in epidermis and dermis and also association with
HLA class I antigen. Stimulation of T lymphocytes in epidermis by peptide antigens
presented in connection with HLA class I molecules is assumed to be the key event
in immune response (CD4 and CD8) activation. Activated T lymphocytes secret
many cytokines that stimulate keratinocyte proliferation such as interleukin 2 -IL-2, γ
interferon -IFN-γ, tumor necrosis factor α -TNF-α, interleukin 6 -IL-6. Recent research
data suggest the significance of adhesion molecules ICAM-1 and VCAM-1 in the
development of psoriasis. In this study we have shown their expression in psoriatic
lesional and non-lesional skin. During the last ten years immunotherapy with
monoclonal antibodies directed against molecules involved in immunologic process
has been used successfully in these patients.
Aim
The aim of this study was to determine the presence of activated T lymphocytes,
macrophage 68 and adhesion molecules: intercellular adhesion molecule-1 (ICAM-
1), vascular adhesion molecule-1 (VCAM-1) and interleukin-2 receptor (IL-2R) in
lesional and non-lesional psoriatic skin as compared with healthy skin in control
group in order to evaluate the possible differences as compared with healthy skin
and determine the role of adhesion molecules in etiopathogenesis of psoriasis.
Materials and methods
Skin biopsies of lesional and non-lesional skin at least 5 cm from lesion were
performed in 50 patients with psoriasis that have used no topical or systemic therapy
during at least two weeks prior biopsy. Skin biopsies of healthy skin were performed
in 50 healthy volunteers. All fresh frozen sections were examined histologically and
immunohistochemically at the Department of Pathology Medical Faculty University of
Rijeka. Standard immunohistochemical procedure using DAKO kits and IL-2R kit of
Medical Faculty University of Rijeka followed by the molecule expression reading in
order to evaluate the expression of lymphocyte markers, IL-2R and adhesion molecules (Olympus BX 51). Statistical analyses were performed at Medical Faculty
University of Rijeka.
Results
We have detected statistically significant difference of ICAM-1 expression in lesional
and non-lesional skin in psoriatic patients (p<0,001) in comparison with healthy skin
of our volunteers. Also, we have found statistically significant difference in adhesion
molecule VCAM-1 expression in comparison with healthy volunteers (p<0,0001).
Significant difference was detected in IL-2R expression between lesional psoriatic
skin and healthy skin of examined volunteers (p<0,001).
CD4 and CD8 lymphocytes were found in epidermis and dermis in augmented
number in lesional skin of psoriatic patients as compared with healthy volunteers
(p<0,001). Also, increased expression of CD68+ molecule, a macrophages marker,
was found in lesional skin of psoriatic patients in comparison with skin of healthy
volunteers (p<0,001).
Conclusion
According to obtained results and previously published studies, we have established
a significant difference in expression of adhesion molecules between lesional skin
and in non-lesional skin of psoriatic patients in comparison with healthy skin. These
results prove their importance in etiopathogenesis and immunologic events involved
in development of psoriatic lesions. It is important to emphasize that non-lesional skin
in psoriatic patients is in «stand by» position and in each moment could become
lesional, due to high levels of adhesion molecules expressed in non-lesional skin of
these patients. We can assume that targeted biologic therapy with specific
monoclonal antibodies could improve the treatment of psoriasis vulgaris.- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
Provider: - Institution: - Data provided by Europeana Collections- Cilj istraživanja:
NK-stanice imaju ključnu ulogu u ranom nadzoru virusnih infekcija i tumora. Kod mišjeg soja C57BL/6 NK-stanice ...izražavaju aktivacijski receptor Ly49H koji prepoznaje virusni protein m157 izražen na površini stanica inficiranih mišjim citomegalovirusom (MCMV). Aktivacija NK-stanica putem Ly49H-m157 međudjelovanja dovodi do ranog zaustavljanja virusne replikacije. Preliminarni rezultati ovog istraživanja pokazali su da u slučaju izostanka rane aktivacije NK-stanica putem Ly49H-m157 međudjelovanja dolazi do pojačane aktivacije CD8+ limfocita T u akutnoj fazi MCMV-infekcije. Vođeni ovim nalazom cilj nam je bio utvrditi mehanizam regulacije nastajanja i intenziteta specifičnog CD8+ T-limfocitnog odgovora u uvjetima izostanka rane aktivacije NK-stanica putem receptora Ly49H.
Materijali i metode:
Ly49H+ i Ly49H– mišje sojeve inficirali smo divljim tipom virusa (WT MCMV) ili virusnom mutantom kojoj je uklonjen gen za m157 protein (m157 MCMV). Važnost pojedinih limfocitnih subpopulacija u kontroli MCMV-infekcije pratili smo nakon uklanjanja ovih subpopulacija monoklonskim protutijelima te usporedbom rasta virusa u tkivima testom virusnih čistina. Fenotipski i funkcionalni status imunih stanica tijekom MCMV-infekcije testirali smo protočnom citometrijom te određivanjem razine citokina u serumu.
Rezultati:
Istraživanjem je pokazano da se intenzitet CD8+ T-limfocitnog odgovora na MCMV razlikuje kod Ly49H+ i Ly49H– mišjih sojeva. Rezultati na mišjem soju C57BL/6 pokazuju kako je CD8+ T-limfocitni odgovor u prvom tjednu MCMV-infekcije određen angažmanom receptora Ly49H i kapacitetom NK-stanica da ograniče virusnu replikaciju tijekom prva dva dana nakon infekcije.
WT MCMV-infekcija rezultirala je snažnom aktivacijom NK-stanica, te posljedično slabijim specifičnim CD8+ T-limfocitnim odgovorom. Važnost aktivacije NK-stanica putem receptora Ly49H u modulaciji CD8+ T-limfocitnog odgovora na MCMV potvrđena je korištenjem virusne mutante m157 MCMV. Nemogućnost NK-stanica da ograniče replikaciju m157 MCMV-a rezultirala je visokim titrom virusa u organima, povećanom infekcijom konvencionalnih dendritičkih stanica i izlučivanjem velikih količina proupalnih citokina, a svi ovi čimbenici pridonijeli su razvoju snažnog CD8+ T-limfocitnog odgovora. Na modelu imunodeficijentnih miševa pokazano je da osim specifične aktivacije putem Ly49H-m157 međudjelovanja, imunoregulacijska uloga NK-stanica ovisi i o perforinskom citolitičkom mehanizmu.
Zaključak:
Dobiveni rezultati proširili su spoznaje o imunoregulacijskoj ulozi NK-stanica u modulaciji CD8+ T-limfocitnog odgovora na MCMV. Specifična aktivacija NK-stanica putem Ly49H-m157 međudjelovanja i perforinskog citolitičkog mehanizma ključni su faktori u određivanju kinetike i intenziteta MCMV-specifičnog CD8+ T-limfocitnog odgovora tijekom akutne MCMV-infekcije.- Objectives:
NK cells play a key role in the early defense against viral infections and tumors. In C57BL/6 mice NK cells express Ly49H activating receptor which specifically recognizes virally encoded protein m157 on the surface of cells infected with mouse cytomegalovirus (MCMV). The activation of NK cells through Ly49H-m157 interaction results in the early restriction of virus replication. The preliminary data from our study showed that the absence of an early NK cell activation through Ly49H-m157 interaction results in higher activation of CD8+ T cells in acute phase of the MCMV infection. Guided by these findings, our goal was to determine the mechanism that regulates the development and intensity of the specific CD8+ T-cell response concerning the lack of early activation of NK cells via Ly49H receptor.
Materials and methods:
Ly49H+ and Ly49H– mice were infected with wild-type virus (WT MCMV) or mutant virus lacking m157 gene (m157 MCMV). The contribution of individual lymphocyte subsets in the control of MCMV infection was determined after depletion of these subsets with monoclonal antibodies, and by comparing the growth of the virus in different tissues of MCMV infected mice by standard plaque assay. Phenotypic and functional status of immune cells during MCMV infection was tested by flow cytometry and by determination of cytokine levels in the sera of infected mice.
Results:
This study showed that the strength of CD8+ T-cell response to MCMV differs in Ly49H+ and Ly49H– mice. Our results in C57BL/6 mice demonstrated that CD8+ T-cell response in the first week of MCMV infection is determined by the engagement of Ly49H receptor and the capacity of NK cells to control virus replication during the first two days. Infection with WT MCMV resulted in strong NK cell activation that restricted virus replication, and consequently impaired MCMV-specific CD8+ T-cell response. The importance of specific activation of NK cells through Ly49H receptor in the regulation of intensity of CD8+ T-cell response was confirmed by using mutant virus m157 MCMV. The inability of NK cells to restrict the replication of m157 MCMV resulted in high viral titers in organs, increased frequency of infected conventional dendritic cells and high levels of proinflammatory cytokines, and all these factors contributed to the development of strong CD8+ T-cell response. By using model of immunodeficient mice, we have shown that, besides specific activation through Ly49H-m157 interaction, the immunoregulatory role of NK cells also depends on perforin mediated cytolytic mechanism.
Conclusion:
The results of this study further expanded our knowledge about immunoregulatory role of NK cells in the modulation of CD8+ T-cell response to MCMV. The specific activation of NK cells through Ly49H-m157 interaction and perforin mediated cytolytic mechanism were shown to be the key factors in determining the kinetics and intensity of MCMV-specific CD8+ T-cell response during acute infection.- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
Babezioza pasa prouzročena parazitom B. canis čest je uzrok uginuća pasa u Hrvatskoj. Leukociti imaju ključnu ulogu u nastanku sustavnoga upalnoga odgovora koji je temeljni patofiziološki mehanizam u ...babeziozi. Cilj ovoga istraživanja bio je odrediti promjene u ukupnom broju leukocita te odnos između neutrofila i limfocita u jednostavnoj i kompliciranoj babeziozi pasa, prouzročenoj vrstom B. canis canis. Istraživanje je provedeno
na dvije skupine prirodno invadiranih pasa. Unutar prve skupine bilo je 30 pasa oboljelih od nekomplicirane, a unutar druge 30 pasa oboljelih od različitih oblika komplicirane babezioze. Uzorci krvi prikupljani su trokratno; na dan pregleda te drugi i sedmi dan nakon primjene antibabezijskoga sredstva (imidokarb dipropionat). Ukupan broj leukocita određen je hematološkim brojačem (System 9120; Serono Baker Diagnostic). Diferencijalna
krvna slika dobivena je brojenjem udjela segmentiranih i nesegmentiranih neutrofilnih granulocita, eozinofilnih i bazofilnih granulocita, monocita i limfocita na 100 leukocita u krvnim razmascima. Omjer neutrofila i limfocita izračunat je iz postotka neutrofila i limfocita izbrojenih u obojenom krvnom razmasku. Postotak parazitemije određen je brojenjem parazitiranih eritrocita na tisuću eritrocita. Ukupni broj leukocita, postotak neutrofila i
limfocita, omjer neutrofila i limfocita i postupak parazitemije između istraživanih skupina uspoređen je Mann-Whitney-evim U testom. Ukupni broj leukocita bio je značajno viši sedmi dan nakon antibabezijske terapije u skupini pasa oboljelih od kompliciranoga oblika bolesti. Udio neutrofila bio je značajno viši drugi i sedmi dan nakon terapije, dok je udio limfocita bio značajno niži drugi dan nakon terapije, isto u skupini pasa oboljelih od kompliciranoga oblika bolesti. Međusobni omjer neutrofila i limfocita bio je značajno viši drugi dan nakon terapije u pacijenata u kojih se razvio komplicirani oblik babezioze. Isto tako je i postotak parazitemije bio značajno viši u pacijenata s komplikacijama. Zaključno možemo ustvrditi da u pasa oboljelih od babezioze postoji signifikantna koleracija između težine kliničkih znakova te neutrofilije i limfopenije. Međusobni odnos neutrofila i limfocita je dostupan i lako odrediv pokazatelj koji odražava jačinu oštećenja i može poslužiti kao dobar prognostički pokazatelj.
Provider: - Institution: - Data provided by Europeana Collections- Cilj istraživanja: Granulizin je citolitičko/apoptotička molekula sposobna ubiti različite patogene i tumorske stanice. Malo je ...poznato o ulozi granulizina na majčino-fetalnom spoju. Cilj našeg istraživanja bio je ispitati: (1) izraženost i unutarstanični smještaj granulizina u decidui i perifernoj krvi rane trudnoće, (2) utjecaj molekula HLA-G i HLA-C na izraženost, lučenje i unutarstanični smještaj granulizina, (3) sposobnost stanica NK da izlučuju granulizin, (4) mehanizme regulacije izraženosti granulizina Th1 citokinima, antigen predočnim stanicama (APS) i progesteronom.
Materijal i metode: Uzorke decidualnog tkiva i periferne krvi dobivali smo sa Klinike za ginekologiju i porodništvo KBC-a Rijeka nakon namjernog prekida normalne trudnoće (6.-10. tjedan). Različite stanične subpopulacije pročistili smo metodom pozitivne magnetske separacije. Izraženost granulizina u decidui i perifernoj krvi analizirali smo metodom imunohistokemije i imunofluorescencije uz očitavanje protočnim citometrom ili konfokalnim mikroskopom. Izlučivanje granulizina iz pročišćenih stanica NK ispitali smo imunoenzimskim testom ELISA. Gensku izraženost granulizina i ostalih citolitičkih medijatora analizirali smo metodom lančane reakcije polimeraze u stvarnom vremenu.
Rezultati: Granulizin je obilno izražen u decidui. Više od 85% decidualnih stanica NK izražava granulizin. Decidualni limfociti T, u odnosu na limfocite T iz periferne krvi, u značajno većem postotku izražavaju granulizin. Decidualne APS također izražavaju granulizin. Izraženost granulizina na genskoj razini u decidualnim staničnim subpopulacijama (stanice NK, NKT, limfociti T, nezrele dendritične stanice, makrofagi) je značajno viša u odnosu na ostale citolitičke medijatore (perforin, FasL, TRAIL). U usporedbi s perforinom, granulizin se u stanicama nalazi pretežno u granulama koje su smještene uz staničnu membranu dok se perforin nalazi u centralnije smještenim granulama. Kultivirane u mediju, decidualne stanice NK spontano luče velike količine granulizina. Lučenje se može potaknuti HLA-C molekulama, a potisnuti HLA-G molekulama koje potiskuju i proteinsku izraženost granulizina u stanicama NK. Decidualne APS i IL-15 također sudjeluju u kontroli izraženosti granulizina u stanicama NK.
Zaključak: Obilna izraženost granulizina u imunokompetentnim stanicama, kao i sposobnost stanica NK da spontano izlučuju velike količine granulizina upućuje na ulogu granulizina kao zaštitne i imunomodulacijske molekule na majčino-fetalnom spoju. Klasične i neklasične HLA molekule I. razreda izražene na stanicama ekstraviloznog trofoblasta sudjeluju u regulaciji lučenja i izraženosti granulizina u stanicama NK te time vjerojatno doprinose očuvanju trudnoće.- Objectives: Granulysin is a novel cytolytic protein lytic against a variety of tumour cells and microbes. The role(s) of granulysin during pregnancy have not been extensively explored. The aim of our study was to examine: (1) granulysin expression and localization in the first trimester pregnancy peripheral blood (PB) and decidua, (2) the influence of HLA-G and HLA-C molecules on granulysin expression, secretion and intracellular localization, (3) the ability of NK cells to secrete granulysin, and (4) the role of antigen presenting cells (APC), Th1 cytokines and progesteron in the regulation of granulysin expression.
Material and methods: Decidual tissue samples and peripheral blood were obtained from Department of Gynecology and Obstetrics, Clinical Hospital Center, Rijeka after an elective pregnancy termination of normal 6.-10. week old pregnancies. Different cell subpopulations were purified by positive magnetic separation. Granulysin expression was analyzed using cell permeabilization method, flow cytometry, confocal microscopy and immunohistochemistry. Granulysin secretion by purified NK cells was detected by ELISA method. Granulysin, perforin, FasL and TRAIL gene expression was analyzed by quantitative real-time PCR.
Results: Granulysin is abundantly expressed at the maternal-fetal interface in early pregnancy. Over 85% of decidual NK cells express granulysin. Decidual T lymphocytes, in comparison to PB T lymphocytes, express significantly higher levels of granulysin. Decidual APC also express granulysin. In comparison to perforin and other cytolytic mediators, granulysin mRNA is present at significantly higher levels in NK cells, NKT cells, macrophages and immature dendritic cells, but not in T lymphocytes. Intracellularly, granulysin is mostly stored in the granules near the cell membrane and rarely colocalizes with perforin which is stored in more centrally positioned granules. When cultured, decidual NK cells spontaneously release high quantities of granulysin. HLA-C molecules enhance granulysin secretion, whereas HLA-G molecules suppress granulysin secretion and protein expression in NK cells. Decidual APC participate in the control of granulysin expression in NK cells as well.
Conclusion: Abundant expression of granulysin by decidual immunocompetent cells and the capacity of decidual NK cells to spontaneously secrete high quantities of granulysin points to important protective and immunomodulatory role(s) that this molecule could play at the maternal-fetal interface. Classical and non-classical HLA class I molecules expressed by extravillous trophoblast cells contribute to the maintenance of pregnancy by regulation of granulysin expression and secretion in NK cells.- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
Uvod: Patogeneza Dupuytrenove bolesti je nejasna, no mogu se pretpostaviti upalni mehanizmi u njenoj pozadini.
Materijali i metode: Mjerili smo udjele različitih podvrsta monocita i lim-focita u ...perifernoj krvi prema stadiju bolesti kod 39 bolesnika oboljelih od Dupuytrenove bolesti. Rezultate smo usporedili s rezultatima 29 zdravih kontrolnih ispitanika iz iste dobne skupine. Mjerenja su napravljena pomoću protočne citometrije.
Rezultati: U aktivnom su stadiju bolesnici imali bitno povišen udio monocita, NK-stanica sličnih T-limfocitima, dok je udio B-limfocita bio značajno snižen, uključujući CD5+ B-limfocite. Udjeli CD4+, CD8+ T-limfocita i B-limfocita nisu se znatno promijenili. U naprednom su stadiju bolesti udjeli monocita i B-limfocita ostali konstantnima, no značajno su se snizili udjeli T-limfocita. U četiri slučaja među bolesnicima koji boluju od Dupuytrenove bolesti pojavile su se limfoidne neoplazme zrelih B- ili T-limfocita.
Zaključci: Naši rezultati podupiru prijašnja mišljenja da su limfociti povezani s patogenezom Dupuytrenove bolesti te pojačavaju ulogu monocita, NK-stanica sličnih T-limfocitima i promjenu imunosnog odgovora između stadija bolesti.
Sve veća uporaba i stvaranje procjednih voda iz nekontroliranih odlagališta krutoga otpada postali su ozbiljna prijetnja vodenom okolišu. Cilj je ovog istraživanja bio procijeniti genotoksičnost ...takvih procjednih voda te podzemnih voda uzorkovanih iz bunara u blizini odlagališta otpada grada Settata u Maroku. U tu svrhu rabili smo mikronukleusni test in vitro i usporedno istražili kinetiku proliferacije limfocita periferne krvi zdrave dobrovoljne ispitanice. Osim toga, analizirano je više fizikalno-kemijskih parametara (nitrati, ortofosfati, nitriti, pH, otopljeni kisik, kemijska potrošnja kisika, temperatura, zamućenost vode). Te su analize procjednih voda i vode iz bunara pokazale brojna odstupanja od propisanih marokanskih standarda. Usto je u limfocitima izlaganim ovim vodama utvrđena i povišena učestalost mikronukleusa. Preliminarni nalazi pokazuju da su obje vrste voda genotoksične i da su mogući izvor rizika za okoliš i ljudsko zdravlje.
Perforin je protein smješten u sekretornim granulama citotoksičkih T-limfocita. Zajedno s ostalim komponentama granula nužan je za lizu stanica organizma inficiranih unutarstaničnim patogenima. ...Ispitali smo izražaj perforina u citotoksičkim T-limfocitima dojenčadi s bronhiolitisom uzrokovanim respiracijskim sincicijskim virusom (RSV). Uzorci periferne krvi prikupljeni su za vrijeme primarne infekcije i rekonvalescencije u inficirane dojenčadi (n=12) i od kontrolne skupine zdrave djece koja su odgovarala po dobi i spolu. Analiza prikupljenih podataka pokazala je približno jednak postotak citotoksičkih T-limfocita koji izražavaju perforin u sve tri skupine ispitanika. Međutim, pronađene su razlike u razini izražaja perforina po pojedinom limfocitu između rekonvalescentne djece i zdravih kontrola, što upućuje na trajniji poremećaj stanične imunosti nakon prestanka infekcije RSV-om.
PDF
