Stopa depresivnih poremećaja u pandemijskom je porastu. Većinu samoubojstava i pokušaja samoubojstva počine depresivne osobe. Zbog navedenog, neophodni su pouzdani biomarkeri individualizirane ...procjene rizika suicidalnog ponašanja. Mjerenje koncentracije elemenata u pojedinim biološkim uzorcima omogućuje uvid u promjenu stanja dinamike metabolizma normalnih i patoloških procesa. Metabolički status bioelemenata (BE) te razina hormona i aminokiselina koje sudjeluju u regulaciji metabolizma mogu biti potencijalni biomarkeri suicidalnog ponašanja. Glavni cilj istraživanja bio je ispitati povezanost i međuodnos elektrolita i elemenata u tragovima, aminokiselina i vazopresina u plazmi ispitanika oboljelih od depresije koji su pokušali samoubojstvo (n=38) u odnosu na kontrolnu skupinu (n=47). Istraživanjem nije potvrđena povezanost koncentracije natrija sa suicidalnošću, a rizik za suicid raste sa sniženom ili prosječnom koncentracijom joda (I), litija (Li), povišenom ili prosječnom koncentracijom barija (Ba) kao i s dobi iznad 43,5 godina. Utvrđena je statistički značajno niža koncentracija asparagina kod suicidalne skupine. Potvrđena je obrnuto proporcionalna povezanost vazopresina i suicidalnosti (p<0,001). Dobiveni rezultati mogli bi biti temelj za pronalazak neurospecifičnih biomarkera za depresiju u sklopu personalizirane medicine.
Most of the suicides and suicide attempts are result of depressive disorders. Due to a pandemic increase of such disorders, most suicides and suicide attempts are committed by depressive persons, reliable biomarkers of the suicide risk are needed. Measurement of elements concentration in specific biological samples gives us an insight into metabolic dynamic change in normal and pathological processes. Metabolic status of bioelements as well as hormones and amino acids levels could be the biomarkers of suicidal behavior.The aim of research was to analyze concentracions of electrolytes and trace elements, plasma amino acids and vasopressin in depressed hospitalized suicide attempters (n=38) and to correlate the results with those in control subjects (n=47). The main hypothesis of this research was that there is a significant difference in above mentioned parameters between suicide attempters with major depression disorder and healthy control subjects. Significantly higher concentration of sodium (Na) has not been observed in suicide attempter group. Suicide risk increases with decreased or average concentration of iodine (I) and lithium (Li), increased or average concentration of barium (Ba) as well as the age > 43.5 years. We determined significantly decreased concentration of asparagine in suicide attempters. Negative correlation between arginine vasopressin and suicidal behaviour was found (p<0,001). This study could be the basis for potential development of neuro-specific biomarkers in personalized medicine.
Alzheimerova bolest (engl. Alzheimer's disease, AD) je najčešća demencija karakterizirana stvaranjem plakova i
neurofibrilarnih spletova. Vaskularna demencija (VAD) je druga najučestalija vrsta ...demencije i nastaje uslijed
ishemijskih, hipoperfuzijskih ili hemoragičnih moždanih lezija. Ciljevi ovog rada su: ispitati diferencijalno
dijagnostičko značenje određivanja serumske koncentracije neurozina (humanog kalikreina 6, KLK6), klasterina
(CLU) i adiponektina (ADPN), te upalnog biljega interleukina-6 (IL-6) u razlikovanju AD i VAD; procijeniti
značenje ispitivanih biljega u razlikovanju kognitivno zdravih ispitanika iste dobi od oboljelih od demencije i
onih ispitanika iste dobi koji imaju blagi kognitivni poremećaj u odnosu na one s dijagnozom AD i VAD;
procijeniti korelaciju ispitivanih biljega sa standardnim pokazateljima kognitivnog deficita.
Ispitivanjem je obuhvaćeno 70 bolesnika s AD i 67 bolesnika s VAD koji su u neprekidnom slijedu pristizali na
redovnu neurološku obradu u Kliniku za neurologiju KBC-a Sestre milosrdnice u Zagrebu. Skupina kontrolnih
ispitanika iste dobi podijeljena je na kognitivno zdrave (N = 50) i one s blagim kognitivnim poremećajem (N =
48). U okviru rutinske neurološke obrade provedeni su neuropsihološki testovi Mini Mental State Examination
(MMSE) i Montreal Cognitive Assessment (MoCA). Korištene su slikovne tehnike: kompjuterizirana
tomografija mozga (MSCT), Colour Doppler Flow Imaging (CDFI) i transkranijska Doppler sonografija.
Rutinski biokemijski testovi izrađeni su na automatskom biokemijskom analizatoru. Koncentracije biljega KLK6
i CLU su određene ELISA metodom, a koncentracija ADPN je određena imunoturbidimetrijom na automatskom
biokemijskom analizatoru. Koncentracija IL-6 je određena na imunokemijskom analizatoru. Ovisno o vrsti
razdiobe dobivenih rezultata, za testiranje razlika korišteni su statistički testovi Kruskal Wallis i ANOVA. Za
analizu korelacije koristio se Spearmanov, odnosno Pearsonov test. Statistička analiza provedena je pomoću
programa MedCalc.
Kod ispitivanih skupina je nađena razlika za one testove koji se rutinski koriste u neurološkoj obradi dementnih
bolesnika. Koncentracije ispitivanih biljega KLK6, CLU i ADPN u serumu nisu se razlikovale između skupina
(P = 0,137, P = 0,178 i P = 0,268). Koncentracije upalnog biljega IL-6 značajno su se razlikovale između
ispitivanih skupina (P = 0,014), s najvećim medijanom koncentracije u skupini bolesnika s VAD (4,1 pg/mL) i
najmanjim medijanom koncentracije u skupini s MCI (2,3 pg/mL).
Koncentracije ispitivanih biljega KLK6, CLU i ADPN nisu se značajno razlikovale između oboljelih od AD i
VAD. Također, ispitivani biljezi ne pokazuju zadovoljavajuću mogućnost razlikovanja kognitivno zdravih
ispitanika iste dobi od bolesnika s demencijom, kao što ne pokazuju diskriminacijski potencijal kada je u pitanju
razlikovanje skupine iste dobi s dijagnozom blagog kognitivnog poremećaja od skupine s dijagnozom AD,
odnosno VAD. Određivanje koncentracije upalnog biljega IL-6 se pokazalo korisnim u razlikovanju ispitivanih
skupina. Koncentracije ispitivanih biljega nisu korelirale sa rezultatima standardnih pokazatelja kognitivnog
oštećenja.
Alzheimer's disease (AD) is the most frequent dementia characterized by formation of plaques and neurofibrilary
tangles. Vascular dementia (VAD) is the second most frequent type of dementia, which is caused by ischemic,
hypoperfusive or hemorrhagic brain lesions. The aims of this study are: assessment of potential serum
biomarkers neurosin (human kallikrein 6, KLK6), clusterin (CLU), adiponectin (ADPN) and inflammatory
marker interleukin – 6 (IL-6) in differential diagnostics of AD and VAD; assessment of potential of KLK6,
CLU, ADPN and IL-6 to separate age-matched cognitively healthy individuals from those who are demented and
to separate those individuals with symptoms of mild cognitive impairment (MCI) from those with overt AD or
VAD; assessment of correlation of KLK6, CLU, ADPN and IL-6 with results of parameters regularly used for
determination of cognitive deficit.
70 patients with diagnosis of AD and 67 patients with VAD were included in study in consecutive order during
their routine neurological follow-up in University Department of Neurology in Medical School University
Hospital Sestre milosrdnice, Zagreb. Control group of age-matched individuals consisted of cognitively healthy
individuals (N = 50) and those with mild cognitive impairment (MCI) (N = 48). Neuropsychological tests Mini
Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) were done as part of routine
neurological examination. Neuroimaging techniques multi slice computed tomography (MSCT), Colour Doppler
Flow Imaging (CDFI) and transcranial Doppler sonography were used. Routine biochemistry tests were
determined on automated biochemistry analyzer. Concentrations of potential biomarkers were measured using
ELISA method for KLK6 and CLU, and concentration of ADPN was measured using immunoturbidimetry on
automated biochemistry analyzer. Concentration of IL-6 was determined using immunochemistry analyzer.
Depending on data distribution, statistic tests Kruskal Wallis and ANOVA were used for difference testing. For
correlation analysis Spearman and Pearson tests were performed. Statistical analysis was done with MedCalc
program.
Difference between tested groups was found for those tests which are routinely used for neurological follow-up
of demented patients. Concentrations of potential biomarkers KLK6, CLU and ADPN did not differ between
tested participant groups (P = 0,137, P = 0,178 and P = 0,268). Concentrations of IL-6 were significantly
different for tested groups (P = 0,014), with highest median of concentration in VAD group (4,1 pg/mL) and
lowest median of concentration in MCI group (2,3 pg/mL).
Concentrations of investigated biomarkers KLK6, CLU and ADPN did not differ significantly between AD and
VAD patient group. Also, capability of tested biomarkers to differentiate age-matched cognitively healthy
participants from patients with diagnosis of dementia was not sufficient, as well as their discriminating potential
for age-matched participants with MCI compared to AD and VAD patient groups. Measurement of concentration
of inflammatory marker IL-6 proved to be useful in discriminating between tested groups. Concentrations of
potential biomarkers did not correlate with results of standard indicators of cognitive deficiency.
Antitimocitni globulini su poliklona protutijela koja dovode do smanjenja broja T stanica. Često se koriste za imunosupresiju primatelja tijekom transplantacije bubrega. Timoglobulin su IgG ...protutijela dobivena imunizacijom zečeva timocitima ljudskog fetusa. Timoglobulin dovodi do smanjenja broja T-limfocita putem razgradnje stanica mehanizmom ovisnim o komplementu te apoptozom. Da bismo dokazali direktan učinak timoglobulina na stanice, koristili smo ljudske embrionalne stanice porijeklom iz bubrega (HEK-293) u staničnoj kulturi. Mjerili smo membranski potencijal stanice metodom prikovanih potencijala. Depolarizacijski učinak timoglobulina na membranski potencijal HEK-293 stanica je ovisan o koncentraciji timoglobulina te o početnom membranskom potencijalu stanica. Učinak timoglobulina u hipoksično-reoksigenacijskih uvjetima utvrđen je određivanjem stanične smrtnosti te stanične migracije testom grebanja. Timoglobulin je smanjio staničnu smrtnost u hipoksijskim uvjetima nakon kojih je slijedila reoksigenacija. Ovo je prvo istraživanje koje pokazuje direktan učinak timoglobulina na HEK-293 stanice. Rezultati ovog istraživanja potiču nova istraživanja o djelovanju timoglobulina na stanice transplantiranih bubrega.
Antithymocyte globulines are polyclonal T cell-depleting immunoglobulins used in induction of immunosuppression in kidney transplant recipients. Thymoglobuline, is purified rabbit IgG, obtained by immunization of rabbits with fetal human thymi, which depletes T lymphocytes by complement-dependent lysis and apoptosis. To determine possible direct effects of Thymoglobuline on the cells, we used Human Embryonic Kidney cell line (HEK-293) in culture. We measured membrane potential of the cells using slow whole patch clamp technique. Depolarizations of HEK-293 cells caused by Thymoglobuline were concentration- and membrane potential- dependent, showing direct effect of Thymoglobuline on the HEK-293 cells. The effects of Thymoglobuline in hypoxia/reoxigenation were detected by calculating cell death and determining the cell migration using scratch test. Thymoglobuline prevented the cell death induced by hypoxia and reoxigenation conditions. This is first study showing direct effect of thymoglobulin on HEK-293 cells. The results of this study encourage the research on epithelial cells in transplanted kidneys.
Serotoninski prijenosnik (5HT prijenosnik, 5HTT) predstavlja ciljnu molekulu djelovanja antidepresivnih lijekova iz skupine selektivnih inhibitora povratnog unosa serotonina (SSRI). Iako često ...korišteni, ovi lijekovi ostvaruju terapijski učinak samo kod manjeg broja (do 50 %) bolesnika, a razlozi za to nisu poznati. Nalaz povezanosti terapijskog odgovora s polimorfizmom gena za 5HTT uputio je na moguću ulogu konstitucijske serotoninske homeostaze u kliničkom odgovoru na SSRI. Primjenom originalnog životinjskog modela, Wistar-Zagreb 5HT štakor, čije sublinije imaju konstitucijski pojačanu (5HT-visoki) ili smanjenu (5HT-niski) aktivnost trombocitnog 5HTT, u ovom radu je istražen odnos između konstitucijske 5HT homeostaze i odgovora na tretman SSRI-om. Farmakološka aktivacija 5HT sustava izazvana je jednokratnom ili kroničnom primjenom SSRI-a fluoksetina, a cilj je bio usporediti sublinije s obzirom na učinke fluoksetina na (i) aktivnost 5HTT i razinu 5HT u mozgu i na periferiji, (ii) razinu mRNA za 5HTT i receptor 5HT1A u mozgu te (iii) odabrana ponašanja modulirana serotoninom. Jednokratni tretman fluoksetinom doveo je do inhibicije aktivnosti trombocitnog 5HTT, koja je bila jače izražena kod životinja iz 5HT-visoke sublinije, te do porasta razine plazmatskog 5HT, koji je bio veći kod životinja iz 5HT-niske sublinije. Kronična primjena lijeka dovela je do značajnog pada razine trombocitnog serotonina, koji je bio veći kod životinja iz 5HT-niske sublinije, te do pada plazmatskog serotonina, koji je bio veći kod životinja iz 5HT-visoke sublinije. Smanjenje ponašanja nalik na anksioznost, kao i porast istraživačke i lokomotorne aktivnosti nakon kroničnog tretmana fluoksetinom bilo je prisutno samo kod životinja iz 5HT-visoke sublinije, a praćeno je promjenama u mozgu koje su bile vidljive kao tendencija povećanja ekspresije gena za 5HT1A receptor u hipokampusu te ekspresije gena za 5HTT u regiji jezgara rafe. S druge strane, kod životinja iz 5HT-niske sublinije, kronična primjena fluoksetina dovela je do pada kortikalne ekspresije mRNA za 5HTT i receptor 5HT1A, te tendencije pada razine 5HT u moždanoj kori, dok učinci na ponašanje nisu bili značajni. Rezultati istraživanja pokazuju da je konstitucijska serotoninska homeostaza važan čimbenik odgovora na tretman fluoksetinom. Sublinije Wistar-Zagreb 5HT štakora mogu biti koristan model u istraživanju neurobiološke osnove različitog odgovora pojedinaca na terapiju antidepresivima, kao i za vrednovanje trombocita kao perifernog modela za predviđanje terapijskog odgovora.
Serotonin transporter (5HT transporter, 5HTT) represents the target molecule of selective serotonin reuptake inhibitors (SSRIs) acting as antidepressant drugs. Although commonly used, these drugs produce a therapeutic effect only in a small number of patients (up to 50 %), and the reasons for that are not known. The association between therapeutic response and 5HTT gene polymorphism has pointed out to possible role of the constitutional serotonin homeostasis in clinical response to SSRI. Using the original animal model, Wistar-Zagreb 5HT rats, whose sublines have constitutional high (high-5HT) or low (low-5HT) of platelet 5HTT activity, we have investigated the relationship between constitutional 5HT homeostasis and responses to SSRI treatment. Following pharmacological activation of 5HT system, induced by single or repeated doses of SSRI fluoxetine, we have compared these sublines with respect to (i) the 5HTT activity and the 5HT levels in the brain and blood, (ii) the expression of 5HTT and 5HT1A receptor genes in the brain, and (iii) the selected behaviors modulated by serotonin. Acute treatment with fluoxetine led to inhibition of platelet 5HTT activity, which was more pronounced in animals from the high-5HT subline, and to increase of plasma 5HT level, which was higher in animals from the low-5HT subline. Chronic administration of the drug caused a significant decrease of platelet serotonin levels, which was higher in animals from the low-5HT subline, and a decrease of plasma 5HT levels, which was higher in animals from the high-5HT subline. The reduction of anxiety-like behavior, along with the increase of exploratory and locomotor activity after chronic treatment with fluoxetine was observed only in animals from the high-5HT subline, and was accompanied by the tendency towards increase of 5HT1A receptor gene expression in hippocampus and the tendency towards increased expression of 5HTT gene in raphe nuclei. On the other hand, in animals from the low-5HT subline, chronic application of fluoxetine led to decrease in 5HTT and 5HT1A receptor mRNA levels and tendency towards decrease in 5HT in the cerebral cortex. The obtained results show that constitutional serotonin homeostasis is an important factor in response to fluoxetine treatment. Furthermore, Wistar-Zagreb 5HT sublines may represent a useful model in studding the neurobiological basis of a different individual response to antidepressants, as well as in evaluating platelets as a peripheral model for predicting the therapeutic response.
Cilj ovog istraživanja je prikazati učinke i razlike u toksičnosti te prooksidativne i antihemostatske mehanizme triju biljnih formulacija namijenjenih ljudskoj uporabi kao dodaci prehrani, a koja ...sadrže pripravak biljke Ginkgo biloba, dostupne na području Republike Hrvatske. Istražen je i njihov učinak u kombinaciji s antihemostatskim lijekovima, acetilsalicilnom kiselinom i varfarinom. Istraživanje je provedeno na životinjskom modelu štakora. Testovima agregacije i koagulacije dokazano je da svi korišteni biljni pripravci produžuju vrijeme agregacije trombocita dok na koagulaciju nemaju statistički značajan učinak. Dokazano je da korištenje biljnih pripravaka u kombinaciji s lijekovima mijenja vrijednosti mjerenih biljega oksidativnog stresa, aktivnost enzima superoksid dismutaze (SOD) i katalaze te koncentraciju reduciranog glutationa (GSH) i malondialdehida (MDA) u tkivu jetre, bubrega, slezene i mozga, te u krvi. Dobiveni rezultati ukazuju na potrebu veće pozornosti prilikom upotrebe pripravka ginka i različitih antihemostatskih lijekova u svakodnevnoj kliničkoj praksi.
The aim of this study was to demonstrate the effects and the differences in toxicity and prooxidant and antihemostatic mechanisms of three herbal dietary supplements, containing extract of Ginkgo biloba, that are available in Croatia. The goal was also to investigate their efect in combination with antihemostatic drugs, acetylsalicylic acid and warfarin. The study was conducted on animal model. Platelet aggregation and coagulation tests proved that all used herbal products prolong platelet aggregation, while on coagulation they had no statistically significant effect. It has been shown that the use of herbal dietary supplements in combination with medicaments changes the value of the measured biomarkers of oxidative stress, the activity of superoxide dismutase (SOD) and catalase and concentration of glutathione (GSH) and malondialdehyde (MDA) in tissues of liver, kidney, spleen and brain, and also in blood. The results suggested that the greater attention should be paid to the usage of these products in clinical practice.
Prekomjerno stvaranje reaktivnih kisikovih spojeva (ROS) u stanicama uzrokuje ireverzibilne modifikacije proteina, kao što je na primjer karbonilacija, koje utječu na njihovu strukturu i funkciju te ...na stabilnost proteoma. Istraživanja provedena na bakterijama i beskralješnjacima su pokazala postojanje korelacije između oštećenja proteoma i stanične smrti. Cilj ovog rada bio je utvrditi postoji li takva poveznica između stanične smrti i oštećenja proteina imortalnih staničnih linija dobivenih iz primarnog tumora (SW480) i metastaze (SW620) adenokarcinoma debelog crijeva. Izlaganjem staničnih linija oksidativnom stresu utvrdili smo da stanična linija SW620 ima nižu razinu oštećenih proteina od stanica SW480 te je otpornija na oštećenja proteoma izazvanih UVC zračenjem. Razlog povećane otpornosti proteoma stanica SW620 mogla bi biti niska razina ROS-a u odnosu na stanice SW480 i veća stabilnost proteoma. Usporednom analizom proteoma staničnih linija SW480 i SW620 u trenutku kada je smrtnost u populaciji iznosila 90% utvrdili smo da su mogući razlozi povećane osjetljivosti stanica SW480 narušene funkcije sustava za održavanje proteostaze i odstranjivanja oštećenih proteina te smo identificirali proteine koji bi mogli biti potencijalne mete u razvoju protutumorskih terapija.
The excessive production of reactive oxygen species (ROS) in cells induces irreversible protein modifications, carbonylation, that impairs protein structure and function, as well as proteome stability. The correlation between proteome damage and cell death is well established in bacteria and invertebrates and the aim of this research was to investigate the relationship between mortality and protein damage of immortal cell lines derived from primary tumor adenocarcinoma SW480 and its metastasis SW620. We found that SW620 cell line is more resistant to UVC-induced oxidative damage relative to SW480 cells. The reason for greater resistance of SW620 cells could be lower ROS level compared to SW480 and greater proteome stability. The differential analysis of proteomes of two cell lines after 90% UVC-induced mortality shows that SW480 displays greater sensitivity due to the damage of proteome quality system and the ability to remove oxidative damaged proteins. Also, we identified proteins that could be possible targets in anti-cancer therapy.
RSV (engl. respiratory syncytial virus) je najčešći uzročnik respiratornih infekcija djece do druge godine. Razvoj težih oblika bolesti i česte reinfekcije mogu se povezati s istovremenim razvojem ...antivirusne Th1 i nepoželjne Th2 imunoreakcije. Th1 imunoreakcija je povezana s povišenom proizvodnjom IFN-γ te kemokina CXCL10 i CXCL9, dok je Th2 povezana s proizvodnjom IL-4 odnosno CCL17 i CCL22. U djece s akutnom RSV-infekcijom smo lokalno i sistemski odredili kemokinski profil obzirom na tip-1 i tip-2 imunoreakcije te ekspresiju specifičnih kemokinskih receptora (CXCR3 i CCR4) na T-limfocitima. Radi boljeg uvida u usmjeravanje diferencijacije T-limfocita u tip-2, određena je ekspresija CRTh2-receptora. Rezultati su pokazali da RSV specifično podiže razinu CXCL10 i CCL17 tijekom akutne infekcije. Neodgovarajući razvoj memorijske imunosti popraćen je povećanim postotkom TEM-limfocita tipa-1 i tipa-2, te izostankom razvoja dugoživućih memorijskih T-limfocita tipa-1. Nemogućnost razvoja odgovarajuće memorijske imunosti u kombinaciji s istovremenim razvojem oba tipa imunosnog odgovora, mogao bi igrati veliku ulogu u patogenezi RSV-a.
RSV (respiratory syncytial virus) is the most common cause of respiratory infections in infants. Development of severe lower respiratory tract infections and frequent reinfections could relate with the simultaneous development of protective Th1 and pathogenic Th2 immune responses. Th1 is represented by predominant IFN-γ and associated with increased CXCL10 and CXCL9 production. Contrary, Th2 is represented by increased IL-4 and CCL17/CCL22 production. We have determined the chemokine profile of children with severe RSV infection, in addition to specific chemokine receptors CXCR3 and CCR4 expression on memory T cells. To confirm type-2 T-cell polarization, we also analyzed CRTh2 receptor expression. Results showed RSV specifically increases CXCL10 and CCL17 levels in acute infection, followed by inadequate immune memory development in terms of higher percentages of type-1 and type-2 TEM cells, and lower percentages of long-living type-1 memory T cells. Failure in the development of appropriate immune memory, together with the presence of both type-1 and type-2 immune response, could play an important role in RSV pathogenesis and reinfections.
Kosti su stalno u procesu trošenja i obnavljanja, a ravnoteža razgradnje i izgradnje
preduvjet je za zdravo koštano tkivo. Slabljenje kvalitete kosti posljedica je poremećaja te
ravnoteže. Za ...razgradnju su odgovori osteoklasti dok su za izgradnju odgovorni osteoblasti.
Osteopenija je stanje smanjene gustoće kosti, a do nje može doći i zbog dugotrajne primjene
glukokortikosteroida.
Glukokortikosteroidi su protuupalni lijekovi koji se, između ostalog, koriste za
sprečavanje upale dišnih puteva kod astme. Budući je astma česta kronična bolest u djece
potrebno je pratiti metaboličke i koštane učinke glukokortikosteroida tijekom terapije. Cilj
ovog rada je uspostava modela hipoplazije fize izazvane glukokortikosteroidima i odabir
optimalnih biljega njihovih metaboličkih i koštanih učinaka.
Istražen je učinak 3 glukokortikosteroida na rast i promjenu metabolizma kosti te na
ostale sustavne učinke u štakora. Beklometazon dipropionat, prednizolon i ciklezonid, davani
mladim muškim Sprague-Dawley štakorima 7 dana u dozama od 0,3-10 mg/kg dnevno, s.c.,
su ovisno o dozi inhibirali indeks tjelesne mase timusa (za 57%, 44% i 76% s 3 mg/kg).
Ciklezonid i manje učinkovit prednizolon su utjecali na ploču rasta glave femura inhibirajući
rast femura (za 41% i 18% s 10 mg/kg), značajno smanjujući povećanje tjelesne mase (oboje
za 100% s 10 mg/kg), te serumske koncentracije kisele fosfataze i tartarat rezistentne kisele
fosfataze (za >30% s 10 mg/kg); oba su povećala serumske razine glukoze i triglicerida.
Beklometazon dipropionat je imao slab učinak na ove dodatne varijable. Ciklezonid pokazuje
izraženo inhibirajuće djelovanje na rast kosti u štakora. Možemo zaključiti da je ovo dobar
model za ispitivanje utjecaja glukokortikosteroida na metabolizam kosti.
Bone in children is structurally different from adult bone. It is weaker but less brittle.
Bone growth starts with cartilage formation. Then vessels invade the cartilage, delivering
pluripotent stem cells, which initiate the formation of a primary center of ossification.
Secondary ossification centers are formed at each end of long bone, and between the primary
and secondary ossification centers the growth plate, or physis, develops. Bone grows as
secondary and primary ossification centers unite.
Bone tissue is in dinamic process of constant deteriorating and regeneration.
Weakening of bone quality is a result of imbalance in a process of bone remodelling. Bone
remodelling has two stages: bone resorption with the osteoclasts, bone cells which resorb the
bone, and bone formation with osteoblasts, bone cells which form the bone. The bone
remodelling cycle ends with bone mineralisation. The content of mineral in bones is defined
as bone density. Osteopenia is a condition with decreased bone density. Apart being a sign of
normal aging, osteopenia can be induced with prolonged use of glucocorticosteroids.
Glucocorticosteroids are antiinflammatory medications prescribed, among others, for
reducing and prevention inflammation of respiratory pathways in asthma. Since asthma is the
most common chronic disease in children, need for monitoring metabolic and bone effects of
glucocorticosteroids during therapy is appearing. Although inhaled glucocorticoids are known
to have systemic effects on bone metabolism, there is little comparative information on their
relative potencies.
The goal of this work is establishment of glucocorticosteroid induced hypoplasia of
the physis and finding the optimal markers of their metabolic and bone effects.
The effects of three standard glucocorticoids, beclomethasone dipropionate,
prednisolone and ciclesonide, in causing changes in bone metabolism and growth were
investigated in relation to other systemic effects in the rat.
Male, specific pathogen-free, Sprague-Dawley rats, 4,5–5,5 weeks old (at the
beginning of the experiments), were used in the study.
The rat femur model of glucocorticosteroid-induced hypoplasia of the physis was
established according to Belvisi et al., using subcutaneous (s.c.) drug administration to allow
for future parenteral comparison with novel compounds. Briefly, rats were randomly assigned
to experimental groups of 8 animals each. In total, three experiments were performed for each
glucocorticoid; beclomethasone dipropionate, prednisolone and ciclesonide, at doses of 0,3–
10 mg/kg daily for 7 days. Animals in the control groups received s.c. the volume of 10 ml/kg
of vehicle (4% DMSO in 0,125% CMC) daily, for 7 days. Twenty-four hours after the last
treatment, animals were anaesthetized with sodium thiopental and the blood was collected at
exsanguination in order to obtain serum. Also, thymus weights were recorded and the femoral
bones removed (for measurement of the thickness of the proliferating zone). Animal body
weights were correspondingly documented at the beginning and at the end of each
experiment. Body weight gain was calculated as the change in body weight from day 1 until
24 h after treatment on day 7.
Biochemical analyses were performed on rat sera. Serum concentrations of glucose
and triglycerides, alkaline and acid phosphatases, and tartrate-resistant acid phosphatase were
determined on the biochemical analyzer. Concentration of osteocalcin, as a biochemical
marker for bone formation, and TRACP 5b, as a biochemical marker for bone resorption,
were also determined using ELISA.
The thymus was dissected free of connective tissue and immediately weighed.
Thymus body mass index (BMI) was calculated according to the following formula: BMI
(thymus)=thymus weight (mg)/body mass (mg). The left femur was exposed and removed
with the head intact in the acetabulum by cutting through the pelvic girdle and through the
femur shaft above the knee joint. The tissue was then fixed in 10% neutral buffered formalin
for histological assessment.
For the purpose of quantitative histology of the femoral head proliferating zone femurs
were fixed, decalcified and processed to paraffin using the unit for tissue processing. Threemicrometer-
thick sections were cut in a way to include femoral head and stained. The femoral
head growth plate was examined under a light microscope. Images of the growth plate were
captured onto a computer. One image was captured from each tissue section, five
measurements of the growth plate width being obtained from each calibrated image.
Measurements involved drawing a line perpendicular to the growth plate between the edge of
the hypertrophic zone, distal to the articular cartilage and the end of the proliferating zone.
Daily treatment for 7 days with standard glucocorticoids resulted in significant
increases in serum glucose and triglycerides concentrations at the highest doses (10 mg/kg) of
prednisolone and ciclesonide. The most pronounced changes were observed with ciclesonide,
which also significantly increased serum triglycerides at a daily dose of 3 mg/kg.
None of the standard glucocorticoids had any significant effect on serum ALP, over
the tested dose range, given daily for 7 days. However, at the highest dose (10 mg/kg),
prednisolone and ciclesonide significantly inhibited both serum ACP and TRACP. The most
pronounced changes were observed with ciclesonide, which also significantly decreased
serum ACP and TRACP at a daily dose of 3 mg/kg. Beclomethasone dipropionate was less
effective, causing a slight but significant decrease in serum ACP (but not TRACP) at doses of
0,3 and 1 mg/kg.
The comparative potency of the three glucocorticoids in influencing non-specific
serum parameters of bone metabolism was also reflected in their effects on the proliferating
zone thickness of the femoral bone head. While beclomethasone dipropionate had no
significant effect, ciclesonide and prednisolong decreased physeal growth plate width.
Prednisolone reduced median bone growth by 18% at the highest dose (10 mg/kg) and
ciclesonide caused a dosedependent reduction in median bone growth, with significant
inhibition of up to 41% over the whole tested dose range (0,3–10 mg/kg).
All three standard glucocorticoids exerted significant, dose related inhibitory effects
on median body weight gain and thymus body mass indices after daily treatment for 7 days.
Beclomethasone dipropionate was the least growth inhibitory; although it caused statistically
significant inhibition of thymus BMIs by 50% at dose of 1 mg/kg per day, it caused
statistically significant 23% inhibition of median body weight gain only at 10 mg/kg per day.
Prednisolone affected thymus BMIs causing statistically significant inhibition by 44% at dose
of 3 mg/kg per day and body weight gain causing statistically significant inhibition by 33% at
dose of 1 mg/kg per day. Ciclesonide exerted the most pronounced inhibition of body weight
gain and thymus BMIs, causing statistically significant inhibition by 34% and 55%,
respectively, already at the lowest dose of 0,3 mg/kg per day.
In order to see how examined glucocorticoids effected bone markers results showed
that prednisolone had no statistical effect on bone formation while ciclesonide significantly
reduced osteocalcin concentration in doses of 3 and 10 mg/kg per day (157 ng/mL and 88
ng/mL, respectively) vs. control (453 ng/mL).
On the other hand, ciclesonid showed no significant effect on bone resorption while
prednisolone significantly reduced TRACP 5b concentration at the highest dose of 10 mg/kg
per day compared to negative control group (10,6±0,9 U/L vs. 19,7±3,1 U/L).
Ciclesonide, although a pro-drug, still has potent systemic activity in the rat, causing
typical glucocorticoid effects, including inhibition of bone growth. Prednisolone exhibits a
similar, though less potent, spectrum of systemic activity, while beclomethasone dipropionate
has weak activity in causing systemic metabolic effects, but retains thymus inhibiting
potency. However, although the distinction between the effect of glucocorticoids on bone
growth was observed in this study, the model can provide the toxic effect dose titration as
well as
Uvod i cilj
Nedostatak S-adenozilhomocistein hidrolaze (AHCY) je poremećaj uzrokovan mutacijama u genu ahcy čime je smanjena aktivnost proteina AHCY. AHCY ima ključnu ulogu u pravilnom odvijanju ...ciklusa aminokiseline metionina u stanici, stoga nedostatak njegove funkcije uzrokuje težak metabolički poremećaj. Kao jedini enzim koji hidrolizira SAH, snažni inhibitor staničnih metiltrasferaza, AHCY ima i indirektnu ulogu u održavanju metilacijskog statusa stanice. Klinička slika ovog potencijalno letalnog oboljenja je karakterizirana kombinacijom mišićnih, neuroloških i jetrenih poremećaja. Unatoč dokazanoj esencijalnosti AHCY, promjene u molekularnim mehanizmima stanice u stanju nedostatka AHCY su dosada nedovoljno istražene. Cilj ove doktorske disertacije je analiza i razumijevanje molekularnih i staničnih uloga AHCY s krajnjom svrhom probira potecijalnih biomarkera oboljenja.
Materijali i metode
U sklopu ove disertacije su rađene studije novootkrivene Y328D mutante potvrđene u pacijentici oboljeloj od nedostaka funkcije AHCY, a za dodatna istraživanja na raspolaganju su bili i primarni fibroblasti pacijentice. Zbog ograničenja u radu s primarnim fibroblastima, tijekom izrade doktorske disertacije su se pripremili, validirali i istraživali modelni stanični sustavi za što se koristila kombinacija metoda klasične biologije i različitih omiks i visokoprotočnih tehnika.
Rezultati
Po prvi puta je tijekom istraživanja AHCY korištena metoda kojom se na temelju fluorescencije dviju komplementiranih podjedinica proteina Venus s kojima su označeni proteini od interesa može pratiti njihova interakcija u ljudskim stanicama. Dokazano je da Y328D mutanta gubi sposobnost homodimerizacije, a metoda je također uspješno prilagođena na visokoprotočno skeniranje interaktora proteina AHCY. Nadalje, uočena je smanjena razina mutiranih AHCY mRNA i proteina u fibroblastima pacijenta i u stanicama sa stabilno eksprimiranim mutiranim AHCY. U istim stanicama je također mikroskopijom pokazano da mutacije uzrokuju povećanu ili smanjenu količinu proteina u jezgri stanica u odnosu na divlji tip. U stanicama hepatocelularnog karcinoma s utišanim AHCY smanjena je proliferacija i migracija te su aktivirani stanični putevi koji signaliziraju oštećenje DNA. Tijekom analize transkriptoma i proteoma fibroblasta pacijenta i stanica hepatocelularnog karcinoma s utišanim AHCY, uočena je moguća veza proteina AHCY s patologijom različitih neuroloških, jetrenih i mišićnih oboljenja, a predložena su i dva potencijalna biomarkera oboljenja.
Zaključci
Promjene razine AHCY mRNA i proteina, kao i lokalizacije AHCY u stanici koje su uočene u svih istraživanih mutanti proteina AHCY mogu biti odgovorne ili pridonositi patologiji kod oboljenja nedostatka AHCY. Prema predloženom mehanizmu AHCY ima utjecaj na oštećenje DNA i stanični ciklus. Dva potencijalna biomarkera su predložena s ciljem nastavka njihovog budućeg istraživanja u svrhu prognostike i dijagnostike oboljenja nedostatka AHCY.
Introduction and aims
S-adenosylhomocysteine hydrolase (AHCY) deficiency is a disorder caused by lowered enzymatic activity of AHCY protein due to the mutations in ahcy gene. AHCY has a key role in proper functioning of the methionine cycle in cell, therefore the lack of AHCY function causes severe metabolic disorder. Since AHCY is a single enzyme that hydrolyses SAH, a strong inhibitor of cellular metiltransferazes, it has a central role in maintaining the methylation status of the cell. Clinical presentation of this potentially lethal disorder includes a combination of muscular, neurological and hepatic disorders. Despite the essential activity of AHCY, changes in molecular and cellular mechanisms in the state of AHCY deficiency have so far been poorly investigated. The aim of this doctoral thesis is the analysis and understanding of molecular and cellular roles of AHCY and prediction of potential disease biomarkers.
Materials and methods/methodological approach
In this thesis, we performed studies of newly discovered Y328D mutant confirmed in the recent case of AHCY deficiency. Patient fibroblasts were obtained from the mentioned case, however, due to the limitations in experimental work with primary fibroblasts, we prepared, validated and investigated model cellular systems by combining methods ranging from classical biology approach to different omics and high throughput techniques.
Results
A method that enables visualization of protein interactions in human cells based on fluorescence of two complemented parts of Venus protein fused to proteins of interest was used for the first time to research AHCY protein. It is proven that Y328D mutant lacks the ability to form homodimes in human cells, and the method has been also successfully adapted to high throughput screening of AHCY protein interactors. Further, we report lower levels of mutant AHCY mRNA and protein in patient fibroblasts as well as cells that stably express AHCY mutant proteins. Microscopy revealed higher or lower amounts of mutant AHCY protein in cell nuclei in mentioned cells when compared to wild type. In hepatocellular carcinoma cells with a silenced AHCY expression we report lowered proliferation and migration as well as activation of DNA damage induced cellular pathways. While analyzing the transcriptome and proteome of patient fibroblasts and hepatocellular carcinoma cells with silenced AHCY expression, potential implications of AHCY protein in the pathology of various neurological, hepatic and muscular diseases are recognized, and two potential disease biomarkers are suggested.
Conclusions
Changes in levels of mutant AHCY mRNA and protein, as well as changed cellular localization of mutant AHCY could be responsible for or contribute to the pathology of the AHCY deficiency. As proposed by our mechanism, AHCY can imapact cellular DNA damage and cell cycle. Two biomarkers are suggested for further research and usage in prognostics and diagnostics of AHCY deficiency.
Kronična opstrukcijska plućna bolest (KOPB) je složeni poremećaj koji zahvaća pluća, ali
i sistemski odjeljak. Naše se istraživanje temeljilo na hipotezi da je u bolesnika s KOPB-om
prisutna ...sistemska upala i sistemski oksidacijski stres. Sukladno tome, cilj ovog istraživanja
bio je istražiti sistemske pokazatelje oksidacijskih i upalnih promjena u bolesnika sa stabilnim
KOPB-om te ispitati povezanost sistemskih antioksidansa s poremećajem funkcije pluća i
biljezima sistemske upale. Ispitali smo i dijagnostičku učinkovitost biljega sistemskog
oksidacijskog stresa u razlikovanju zdravih i oboljelih od KOPB-a.
Sistemski biljezi oksidacijskog stresa (albumin, transferin, ceruloplazmin, ukupni bilirubin,
slobodne tiolne skupine, malondialdehid (MDA), paraoksonazna i arilesterazna aktivnost
paraoksonaze 1 (PON1), te ekspresija i aktivacija unutarstaničnih signalnih molekula Hsp27,
Hsp70, ERK, JNK i p38) određeni su u 106 bolesnika sa stabilnim KOPB-om i 45 zdravih
ispitanika. Ispitane su i njihove povezanosti s biljezima sistemske upale (CRP, fibrinogen,
ukupni leukociti), pušenjem i pokazateljima funkcije pluća (FEV1 i FEV1/FVC).
Bolesnici s KOPB-om su imali povišene koncentracije ceruloplazmina i MDA, snižene
koncentracije albumina, transferina i tiola, te snižene obje ispitane aktivnosti PON1.
Koncentracije ukupnog bilirubina nisu se razlikovale usporedbom ispitivanih skupina.
Ceruloplazmin je pokazao pozitivnu korelaciju s CRP-om i fibrinogenom. Albumin i
transferin su pokazali negativnu korelaciju s CRP-om, te pozitivnu korelaciju sa slobodnim
tiolima. Transferin je negativno korelirao s fibrinogenom. Jedini pokazatelj povezan s
funkcijom pluća bio je MDA. Usporedbom pušača, bivših pušača i nepušača iz skupine
bolesnika s KOPB-om nisu nađene razlike u koncentracijama promatranih biljega
oksidacijskog stresa i upale. Povišene koncentracije ceruloplazmina su najsnažniji prediktor
prisutnosti KOPB-a. Model koji uključuje ceruloplazmin, albumin, MDA i arilesteraznu
aktivnost PON1 te biljege sistemske upale pokazao je najbolju dijagnostičku učinkovitost u
predviđanju KOPB-a (AUC (95%CI) = 0,96 (0,92 – 0,99)). Predloženi bi model mogao
ispravno predvidjeti prisutnost KOPB-a u 89% bolesnika. Također, naši su rezultati ukazali da
je razina ekspresije Hsp27 i Hsp70 u leukocitima periferne bila je najniža u pušača s KOPBom.
Ekspresija svih ispitivanih MAPK (ERK, JNK i p38) u perifernim leukocitima bolesnika
s KOPB-om nije se razlikovala u usporedbi sa zdravim ispitanicima. Aktivacija ERK bila je
značajnija kod zdravih i bolesnih nepušača, dok je aktivacija JNK i p38 bila najizraženija u
pušača s KOPB-om.
Rezultati su ukazali da prisutnost bolesti i pušenje utječu na ispitane unutarstanične
signalne molekule. Bolje razumijevanje ovih molekularnih mehanizama moglo bi pomoći u
dijagnozi i pronalaženju novih terapijskih meta za KOPB. Dijagnostičke karakteristike
predloženog modela koji kombinira koncentracije biljega sistemske upale i sistemskog
oksidacijskog stresa ukazuju da bi se on mogao koristiti kao vrijedan alat u razlikovanju
zdravih ispitanika i bolesnika s KOPB-om.
Background: Chronic obstructive pulmonary disease (COPD) is a complex disorder
affecting the lungs and the systemic compartment. We hypothesized that systemic
inflammation and systemic oxidative stress are present in patients with COPD. Therefore, we
aimed to investigate markers of systemic oxidative and inflammatory alterations in patients
with stable COPD, and to test the association of systemic antioxidants with indicators of lung
function and systemic inflammation. The diagnostic accuracy of systemic oxidative stress
parameters in distinguishing between healthy subjects and patients with COPD was also
evaluated.
Materials and methods: Systemic oxidative stress markers (albumin, transferrin,
ceruloplasmin, total bilirubin, thiols, malondialdehyde (MDA), paraoxonase and arylesterase
activity of paraoxonase 1 (PON1), and the expression and activation of intracellular signalling
molecules Hsp27, Hsp70, ERK, JNK i p38) were assessed in 106 stable COPD patients and
45 healthy subjects. Their association with systemic inflammatory markers (CRP, fibrinogen,
total leukocytes), smoking status and lung function parameters (FEV1 i FEV1/FVC) was
investigated.
Results: Higher ceruloplasmin and MDA concentrations, and lower albumin, transferrin,
thiols and PON1 activities (paraoxonase and arylesterase) were found in patients with COPD.
Total bilirubin concentrations were similar in the studied groups. Ceruloplasmin showed a
positive correlation with CRP and fibrinogen. Albumin and transferrin showed a negative
correlation with CRP, and a positive corelation with thiols. Transferrin negatively correlated
with fibrinogen. Only MDA showed an association with pulmonary function. No differences
were found comparing concentrations of oxidative stress and inflammatory markers between
COPD patients subdivided according to their smoking status. Ceruloplasmin was the strongest
single predictor of COPD. The model combining ceruloplasmin, albumin, MDA, arylesterase
PON1 activity, and markers of systemic inflammation demonstrated very good diagnostic
performances (AUC (95%CI) = 0,96 (0,92 – 0,99)). The proposed model correctly identifies
89% of patients with COPD. In addition, our results showed that the decrease in expression of
peripheral blood leukocytes' Hsp27 and Hsp70 was the most prominent in COPD smokers.
Expression levels of all three MAPKs investigated was not altered in leukocytes of COPD
patients compared to healthy subjects. However, ERK activation was stimulated in healthy and COPD non-smokers, while JNK and p38 activation was the most pronounced in COPD
smokers.
Conclusions: Our results showed that COPD and smoking affect the intracellular
signalling pathways investigated. Improved understanding of these molecular mechanisms
could help identify novel targets for diagnosis and therapeutic interventions in COPD.
Diagnostic characteristics of the proposed model, obtained by combining markers of systemic
inflammation and systemic oxidative stress, suggest its potential value as an additional tool in
COPD diagnosis.
