Cerebrovaskularni poremećaji (CVP) su značajan medicinski problem u djece i odraslih. Cilj istraživanja bio je ispitati razlike u učestalostima polimorfizama gena za HPA (-1, -2, -3, -5), PSEL ...(S290N, N562D, V599L, T715P), mutacija FV G1691A (FVL) i FII G20210A u bolesnika s CVP-om u odnosu na kontrolnu skupinu, te utvrditi njihovu povezanost s bolešću.
Ukupno je ispitano 300 bolesnika s CVP-om (ishemijski moždani udar-iCVI, TIA) (djece-100, odraslih-200) i 200 ispitanika kontrolne skupine (djece-100, odraslih-100), uz podatke za odrasle o standardnim čimbenicima rizika. Polimorfizmi HPA genotipizirani su PCR-SSP-om i PCR-om u stvarnom vremenu, PSEL-a PCR-SSP-om, a FVL i FII G20210A PCR-RFLP-om.
U Hrvata alelne učestalosti HPA odgovaraju podacima za Europljane. Multivarijantnom regresijskom analizom u odraslih potvrđena je povezanost anamneze, hipertenzije, šećerne bolesti i alkoholizma s CVP-om, ali ne s alelom HPA-2b. U djece značajno je učestaliji genotip HPA-5a/b i alel HPA-5b u oboljelih od TIA-e u odnosu na iCVI, a u djece s genotipom GA ili s alelom A FVL, 5 puta je veća vjerojatnost oboljenja od CVP-a. Značajno učestaliji genotip GA i alel A FVL dobiven je u djece u odnosu na odrasle s CVP-om, te iCVI-om. Pomoć u predviđanju CVP-a u odraslih imaju anamneza, hipertenzija, šećerna bolest i alkoholizam, a u djece FVL.
Cerebrovascular disorders (CVD) are a significant medical problem in children and adults. The aim of this research was to examine differences in the frequency of gene polymorphisms for HPA (-1, -2, -3, -5), PSEL (S290N, N562D, V599L, T715P), FV G1691A (FVL) and FII G20210A mutations in patients with CVD as compared to the control group, and to determine their correlation with the disease.
The research included 300 patients with CVD (ischemic stroke-iCVI, TIA) (children-100, adults-200), 200 control subjects (children-100, adult-100), as well as data for standard risk factors for adults. HPA polymorphisms were genotyped using PCR-SSP and real-time PCR, PSEL by PCR-SSP, and the FVL and FII G20210A by PCR-RFLP.
The allelic frequencies of HPA in Croats correspond to the data for Europeans. Multivariate regression analysis in adults confirmed the correlation between family history, hypertension, diabetes and alcoholism with CVD, but not with the HPA-2b allele. In children, HPA-5a/b genotype and HPA-5b allele is significantly more frequent in patients with TIA as compared to iCVI, and in children with GA genotype or with A allele FVL, there is a 5 times higher likelihood of CVD disease. Significantly more frequent GA genotype and A allele FVL was obtained in children than in adults with CVD and iCVI. Help in predicting CVD in adults is provided by family history, hypertension, diabetes and alcoholism, and in children by FVL.
Hemofilija A je rijetki nasljedni poremećaj zgrušavanja krvi koji nastaje zbog manjka FVIII u cirkulaciji i dijeli
se na teški, srednje teški i blagi oblik. Kod teškog oblika bolesti javljaju se ...spontana krvarenja, koja su kod
blažih oblika rijetka. Literaturni podaci ukazuju na heterogenost kliničkog izražaja hemofilije A. Cilj ovog
istraživanja bio je utvrditi mogući utjecaj drugih prokoagulacijskih i fibrinolitičkih čimbenika, osim FVIII, na
endogeni trombinski potencijal i na klinički izražaj bolesti. Ispitana je vrijednost analize reakcijske krivulje
APTV-a i analize stvaranja trombina u dijagnostici hemofilije A jer se ovim metodama mjeri ukupni hemostatski
potencijal plazme. Dobiveni rezultati potvrđuju povećanu aktivnost fibrinolitičkog sustava kod hemofilije A u
odnosu na zdravu populaciju, koja je izraženija kod teškog oblika bolesti. Pojačana fibrinoliza kod bolesnika s
težom kliničkom slikom nije nedvojbeno potvrđena. Potvrđena je vrijednost analize reakcijske krivulje APTV-a
u dijagnostici hemofilije A. Pokazana je dobra korelacija sa standardnim laboratorijskim metodama i kliničkim
pokazateljima te dobro razlikovanje stupnja bolesti. Jednostavnost i cijena metode, koja zahtijeva samo mjerenje
APTV-a, dodatne su prednosti. Ispitivanjem analize stvaranja trombina dobivena je slabija korelacija sa
standardnim laboratorijskim metodama i kliničkim pokazateljima, te slabije razlikovanje stupnja bolesti. U
postojećoj izvedbi metoda nije primjenjiva u rutinskoj laboratorijskoj dijagnostici hemofilije A.
Hemophilia A is a rare X-linked bleeding disorder characterized by decreased FVIII activity, and is classified as
severe, moderate or mild. The clinical phenotype of severe hemophilia A consists of spontaneous bleeding
episodes that are rare in non-severe types. Heterogeneity in bleeding pattern has been observed, but mechanisms
are poorly understood. The aim of this study was to identify whether activities of common prothrombotic and
fibrinolytic factors are associated with clinical phenotypes. New laboratory methods were evaluated that assess
overall clotting function, clot waveform analysis and thrombin generation test. Results showed enhanced
fibrinolytic activity in patients as compared to healthy subjects, as well as in severe hemophilia A patients
compared to those with mild disease. The enhanced fibrinolytic activity in patients with severe phenotype was
not confirmed with certainity. By using waveform analysis, good correlation was obtained to standard laboratory
methods and clinical parameters, as well as good discrimination of disease severity. Simplicity and cost benefit
of this method make this approach a promising tool for assessing coagulation in hemophilia patients. The
correlation to routine laboratory methods and clinical parameters in thrombin generation test was weaker and this
method did not fulfill the goals expected for a routine laboratory method.
Galektini-1 i -3 moduliraju mnoge stanične procese, a regulacijom migracije, fagocitoze i degranulacije
upalnih stanica, te sinteze citokina i medijatora upale, ovi su lektini uključeni u sve faze ...upalnih reakcija
uroĎenog i stečenog imunosnog odgovora. Djelovanje mu izrazito ovisi o lokalizaciji i tipu stanica, no galektin-1
općenito se smatra jakim protu-upalnim signalom, dok galektin-3 uglavnom potiče upalu. Ovim radom ispitana
je uloga galektina-3 u fiziologiji limfocita, te uloga i ekspresija galektina-1 i -3 u monocita, te klasično i
alternativno diferenciranih i aktiviranih makrofaga.
Humani limfociti i monociti izolirani su iz krvi zdravih dobrovoljnih davatelja. Uzgojem u hranidbenom
mediju uz dodatak granulocitno-makrofagnog, odnosno makrofagnog faktora stimulacije rasta kolonija, monociti
su diferencirani u makrofage tipa M1 i M2. Diferencirani makrofagi M1 aktivirani su klasično, dodatkom
lipopolisaharida i interferona-γ, ili alternativno u fenotip M2a/M2c, dodatkom interleukina (IL)-4/IL-10.
UtvrĎena je ekspresija galektina-1 i -3 na genskoj i proteinskoj razini, te njihova prisutnost na membrani stanica.
U hranidbenom mediju spomenutih stanica odreĎena je koncentracija galektina-3. UtvrĎen je učinak egzogeno
dodanog galektina-3 na fiziologiju limfocita, monocita i aktiviranih makrofaga.
Dobiveni rezultati upućuju na to da diferencijaciju i polarizaciju humanih monocita u klasično (M1) i
alternativno (M2a/M2c) aktivirane makrofage prate izrazite promjene razine ekspresije i proteolitičkog kidanja
galektina-3. Ekspresija i sekrecija galektina-3 usko su regulirane i znatno se razlikuju u klasično, u odnosu na
alternativno aktivirane makrofage, dok u galektina-1 razlike u razini ekspresije nisu toliko naglašene.
Zamijećene su izrazite razlike ekspresijskih profila i kidanja galektina-3 u istih podtipova makrofaga podrijetlom
od različitih donora krvi. U klasično aktiviranih makrofaga, temeljem intenziteta ekspresije membranskog
galektina-3, zamijećene su dvije odvojene populacije stanica. Humani monociti pokazuju izrazito velik kapacitet
vezanja egzogeno dodanog galektina-3, dok su u aktiviranih makrofaga receptori tog proteina u potpunosti
zasićeni. Egzogeno dodani galektin-3 ne utječe na izlučivanje citokina limfocita i aktiviranih makrofaga.
Razlike u razini i obrascu ekspresije galektina-3 u različito diferenciranih/aktiviranih makrofaga u odnosu na
galektin-1 su značajne. Stoga, specifičan uzorak ekspresije i sekrecije ovog proteina u diferenciranih i aktiviranih
makrofaga pridonosi boljem razumijevanju uloge i regulacije galektina-3 u tim stanicama. Novi uvid u biološke
karakteristike različito diferenciranih i aktiviranih makrofaga, te limfocita baca svjetlo na galektin-3 kao
modulator ovih stanica.
Galectins-1 and -3 modulate many cellular processes, and through regulation of cell migration, phagocytosis,
immune cell degranulation and modulation of cytokine and inflammatory mediator production, these lectins are
intimately involved in all phases of inflammatory reactions of innate and acquired immunity. Galectin-1 is
generally considered a strong anti-inflammatory and galectin-3 a pro-inflammatory signal, but their effects
tremendously vary with respect to their localization and the cell type. In this work we address the role of
galectin-3 in the physiology of lymphocytes and the role and expression of galectins-1 and -3 in human
monocytes and classically or alternatively differentiated and activated macrophages.
Human lymphocytes and monocytes were isolated from the blood of healthy donors and differentiated into
M1 and M2 subtype of macrophages by cultivation in culture media supplemented with granulocyte-macrophage
or macrophage colony-stimulating factor, respectively. M1 macrophages were activated classically by
lipopolysaccharide and interpheron-γ or alternatively into M2a/M2c phenotypes by interleukin (IL)-4/IL-10,
respectively. The expression of galectins-1 and -3 was determined on gene and protein levels, and the amount of
galectins bound to the cell membranes was estimated. Culture media were probed for secreted galectin-3. The
effect of exogenously added galectin-3 on the physiology of lymphocytes, monocytes and activated macrophages
was investigated.
Obtained results imply that differentiation and activation of monocytes into classically (M1) and alternatively
(M2a/M2c) activated macrophages is followed by marked changes of expression and proteolitic cleavage of
galectin-3. Expression and secretion of galectin-3 was tightly regulated and significantly differed among
classically and alternatively activated macrophages, while the differences of galectin-1 expression profiles were
not pronounced. Significant differences in galectin-3 expression profiles were observed between the same
macrophage subtypes obtained from different blood donors. Moreover, classically activated macrophages
polarize into two distinct populations with respect to the expression of membrane galectin-3. Human monocytes
exhibited high amount of free galectin-3 receptors, while on both types of activated macrophages the receptors
were fully saturated. Exogenously added galectin-3 does not affect cytokine secretion of lymphocytes or
activated macrophages.
Galectin-3 is more distinctive descriptor of macrophage differentiation/activation than galectin-1. Its specific
expression and secretion pattern in M1 vs. M2a/M2c macrophages contributes to better understanding of its role
and regulation in these cells and provides a new insight on biological characteristics of these cells.
Periferna arterijska bolest klinička je manifestacija ateroskleroze sa značajnim morbiditetom i mortalitetom.
Upala i angiogeneza imaju značajnu ulogu u njenom razvoju, progresiji i komplikacijama. ...Cilj rada bio je ispitati
postoji li povezanost izmeĎu katalitičkih koncentracija i genetskog polimorfizma paraoksonaze (PON1),
katalitičkih koncentracija acetil hidrolaze trombocitnog čimbenika aktivacije (PAF-AH), te koncentracija
vaskularnog endotelnog čimbenika rasta (VEGF), angiopoietina 2 (Ang-2) i njegovog Tie-2 receptora, kao novih
biomarkera u procesu upale i angiogeneze, sa pojavnošću i stupnjem stenotično/okluzivnih promjena perifernih
arterija. Snižene katalitičke koncentracije PON1 i polimorfizmi Q192R i -108C>T pon1 gena mogli bi imati
ulogu u razvoju periferne arterijske bolesti. Razlike u frekvenciji Q i R alela izmeĎu ispitanika i kontrolne
skupine mogu biti jedan od uzroka sniženih katalitičkih koncentracija PON1, što može doprinosti smanjenoj
zaštitnoj ulozi HDL-kolesterola i povećanom riziku od prijevremene ateroskleroze kod rizičnih skupina
bolesnika. Povećane koncentracije Ang-2 i Tie-2 receptora mogle bi ukazivati na povećano vaskularno
remodeliranje kao odgovor na prisutne čimbenike rizika, te bi se mogli smatrati novim biomarkerima
angiogeneze koji ukazuju na prisustvo periferne arterijske bolesti. Nepostojanje povezanosti izmeĎu
koncentracija istraživanih biokemijskih parametara i angiografskog sustava bodovanja, kao mjere anatomske
proširenosti aterosklerotskih promjena perifernih arterija ukazuje na važnost drugih čimbenika u progresiji
bolesti.
Peripheral artery disease is a clinical manifestation of atherosclerosis with significant morbidity and mortality.
Inflammation and angiogenesis play an important role in its development, progression and complications. The
aim of this thesis was to investigate the association of the catalytic concentrations and genetic polymorphism of
paraoxonase (PON1), the catalytic concentrations of platelet activating factor acetylhydrolase (PAF-AH), the
concentrations of vascular endothelial growth factor (VEGF), angiopoietin 2 (Ang-2) and its receptor Tie-2, as
novel biomarkers of inflammation and angiogenesis with the appearance and degree of stenotic-occlusive
changes in the periphearal arteries. Lower catalytic concentrations of PON1 and polymorphisms Q192R and -
108C>T pon1 gene could play a role in the development of peripheral arterial disease. Differences in the
frequency of Q and R alleles between patient and control groups may be one of the causes of the reduced
catalytic concentrations of PON1, which may contribute to a reduced protective role of HDL-cholesterol and
increased risk of early atherosclerosis in the risk group of patients. An increased concentration of Ang-2 and
Tie-2 receptor could indicate increased vascular remodeling in response to the presence of risk factors and could
be considered new biomarkers of angiogenesis which indicate the presence of peripheral arterial disease. The
absence of significant correlation between the concentrations of the biochemical parameters investigated and the
angiographic score as a measure of the anatomic extent of atherosclerotic lesions to the peripheral arteries,
suggests that other factors are more important in the progression of the disease.
Bolesnici zaraženi HIV-om skloni su ranoj pojavi ateroskleroze zbog upale uzrokovane samim virusom, kao i
zbog nuspojava antiretrovirusnog liječenja. Antiaterogeno djelovanje ekstra djevičanskog ...maslinovog ulja
(EVOO) moglo bi umanjiti rizik od kardiovaskularnih bolesti u osoba zaraženih HIV-om. 39 HIV-om zaraženih
muških ispitanika uključeno je u križni klinički pokus. Ispitanici su konzumirali EVOO i rafinirano maslinovo
ulje (ROO) - placebo. Više od 50% ispitanika imalo je izražene čimbenike za aterosklerozu, a više od 90%
ispitanika imalo je povećanu koncentraciju malondialdehida i povećanu katalitičku koncentraciju superoksid
dismutaze. Udio rizičnih čimbenika znatno je veći u ispitanika koji se liječe inhibitorima proteaze ili
abakavirom. Konzumacija ROO smanjila je koncentraciju malondialdehida za 18% u svih ispitanika, dok je
konzumacija EVOO smanjila koncentracije hsCRP za 151% i brzinu sedimentacije eritrocita za 62% u ispitanika
liječenih inhibitorima proteaze. Koncentracije hsCRP-a značajno su smanjene nakon konzumacije EVOO samo u
ispitanika čija je suradljivost bila veća od 90%. Konzumacija maslinovih ulja nije smanjila rizik za koronarnu
bolest srca i metabolički sindrom. Rizične čimbenike za kardiovaskularne bolesti potrebno je definirati i pratiti
tijekom antiretrovirusnog liječenja. Učinkovitost EVOO trebalo bi potvrditi na većem broju ispitanika.
Premature atherosclerosis in HIV infected patients is associated with chronic infection by itself and side effects
of antiretroviral therapy. Beneficial effect of extra virgin olive oil (EVOO) could decrease the risk for
cardiovascular disease in HIV-infected patients. 39 HIV positive male participants were included in randomized
crossover controlled trial. They consumed EVOO and refined olive oil (ROO) - placebo. The atherosclerosis risk
factors were affected in more than 50% of participants receiving antiretroviral therapy. In more than 90% of
participants malondialdehyde concentrations and superoxide dismutase cathalitic concentrations were increased.
Risk factors were more affected in participants using protease inhibitors and abacavir containing antiretroviral
therapy. After ROO administration, MDA concentration was 18% higher when compared to basic values. After
EVOO administration, in participants using protease inhibitors containing antiretroviral therapy, erythrocyte
sedimentation rate and hsCRP concentrations were 62% and 151% lower when compared to basic values,
respectively. hsCRP concentrations showed a significant decrease after EVOO administration only in
participants with >90% compliance. Olive oil consumption did not affect the risk for coronary heart disease and
the metabolic syndrome diagnosis. Cardiovascular disease risk factors should be recognized and followed in all
HIV patients receiving antiretroviral therapy. EVOO effects should be confirmed on a larger sample size.
