Prethodna istraživanja pokazala su da BPC 157 sprječava nastanak okluzivnog tromba i ubrzava razgradnju već stvorenog ugruška nakon formiranja anastomoze aorte, a da, s druge strane, smanjuje vrijeme ...krvarenja kod štakora tretiranih varfarinom, heparinom i aspirinom. Temeljem toga, nametnula se potreba određivanja njegova utjecaja na agregaciju trombocita i viskoelastična svojstva krvnog ugruška. Za ispitivanje eventualne uključenosti NO sustava u djelovanje BPC 157 korišten je selektivni inhibitor topljive gvanilil ciklaze, ODQ. Životinje (n=60) su najprije podijeljene u skupine ovisno o tome koji antiagregacijski lijek primaju (1.aspirin, 2.klopidogrel, 3.cilostazol), a te skupine su tada nasumično podijeljene u četiri podskupine koje su primale a) fiziološku otopinu, b) BPC 157, c) ODQ, d) BPC 157 + ODQ. Mjerenja su izvršena impendancijskom agregometrijom s 4 različita agonista (ADP, AA, AA/PGE1, kolagen), te rotacijskom tromboelastometrijom s 3 različita agonista (preko vanjskog i unutarnjeg puta zgrušavanja, te bez doprinosa trombocita). Iz rezultata dobivenih usporedbom podskupina a i b, te b i d unutar svake skupine možemo zaključiti da BPC 157 oporavlja inhibiranu agregaciju trombocita, ali da nema utjecaja na viskoelastična svojstva krvnog ugruška u ispitivanih štakora.
As a natural extension of previous research that confirmed the role of BPC 157 in the prevention of obstructive thrombus formation and rapid destruction of an already formed one after aortic anastomosis, but also shortening of the bleeding time in rats treated with anticoagulants and aspirin, there was a necessity to determine how BPC 157 influences platelet aggregation and viscoelastic properties of the blood clot. To assess its relation to NO system, sCG selective inhibitor (ODQ) was used. Rats (n=60) were divided into groups depending on the antiaggregatory drug they were treated with (1.aspirin, 2.clopidogrel, 3.cilostazol). Groups were further divided into four subgroups treated with a) normal saline, b) BPC 157, c) ODQ, d) BPC 157 + ODQ. Impendance aggregomery measurements with four agonists (ADP, AA, AA/PGE1 and collagen), and also rotational thromboelastometric measurements with 3 agonists (for initiating external and internal coagulation pathway and without platelet contribution) were performed. Based on the results obtained by comparing subgroups a versus b, and b versus d in each group, we can conclude that BPC 157 rescues inhibited platelet aggregation, but it has no effect on viscoelastic properties of the blood clot.
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.
Biološka aktivnost roda Centaurea pripisuje se spojevima iz skupine terpena i flavonoida čija su antioksidacijska, antibakterijska, citotoksična i slična ljekovita svojstva dokazana. Cilj rada je ...odrediti sadržaj i sastav fitokemikalija ekstrakata hrvatske endemične biljne vrste Centaurea ragusina L. sakupljene na prirodnom lokalitetu, uzgojene u uvjetima in vitro i aklimatizirane na vanjske uvjete kao i njihovu antioksidacijsku, antibakterijsku i citotoksičnu aktivnost. U svrhu razjašnjenja biološke aktivnosti proučavane su interakcije ekstrakata, frakcija i spojeva s polinukleotidima. U etanolno vodenim ekstraktma izmjeren je najviši sadržaj fenola, flavonoida i hidroksicimetnih kiselina. Etanolno vodeni i etanolno etanolni ekstrakti pokazuju značajnu antioksidacijsku aktivnost neovisno o podrijetlu biljaka. Ekstrakti listova priređeni u etanolu, većina ispitivanih frakcija kao i dva izolirana seskviterpenska laktona pokazali su značajnu antibakterijsku aktivnost na Staphylococcus aureus ATCC 25923. Svi vodeni vodeni ekstrakti pokazuju značajni stabilizacijski učinak na DNA i RNA. Gotovo svi testirani ekstrakti te frakcije CRE01_06-10 pokazuju značajnu citotoksičnu aktivnost na testiranim tumorskim staničnim linijama.
Biological activity of Centaurea genus is mainly attributed to compounds of the terpenoid and flavonoid type which showed antioxidant, antimicrobial, cytotoxic and other therapeutic properties. The goal of this work was to determine polyphenol content and composition of phytochemicals in Croatian endemic plant species Centaurea ragusina L. collected at natural habitats, cultivated in vitro and acclimated plants as well as to estimate their antioxidant, antibacterial and cytotoxic activity. Ethanolic/aqueous extracts showed the highest level of total phenols, flavonoids and hydroxycinnamic acid. Ethanolic/aqueous and ethanolic/ethanolic extracts showed significant antioxidant activity regardless of the plant origin. Crude ethanolic/ethanolic extract of leaves, almost all tested fractions and two isolated sesquiterpene lactones showed significant antibacterial activity against Staphylococcus aureus ATCC 25923. All aqueous/aqueous extracts showed significant stabilisation effects of DNA and RNA. Almost all tested extracts and fractions CRE01_06-10 exhibited significant inhibitory activities against all tested cancer cell lines.
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.
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.
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.
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.
