Uvod: Ronjenje s komprimiranim plinovima je često praćeno stvaranjem plinskih mjehurića u krvi i hiperoksijom, što se smatra mogućim
uzrocima endotelne disfunkcije nakon ronjenja. Endotelna ...disfunkcija se povezuje s asimptomatskim kardiovaskularnim i neurološkim
promjenama nakon ronjenja s komprimiranim plinovima.
Cilj ove disertacije je istražiti fiziološke i biokemijske promjene vaskularne i endotelne funkcije, te ispitati ima li znakova narušenog
integriteta SŽS-a nakon ronjenja s komprimiranim plinskim smjesama. Pritom su testirane sljedeće hipoteze: asimptomatski zaroni s
komprimiranim zrakom će uzrokovati promjene biokemijskih parametara endotelne funkcije i oksidacijskog stresa; asimptomatski zaroni će
imati različiti utjecaj na vaskularnu funkciju s obzirom na vrstu korištene plinske smjese za disanje; asimptomatski zaroni će u prisutnosti
arterijalizacije plinskih mjehurića uzrokovati porast koncentracije humoralnih biljega oštećenja endotela i središnjeg živčanog sustava.
Metode i ispitanici: Istraživanje je obuhvatilo tri studije. U prvoj studiji na 15 ispitanika istražene su promjene sljedećih parametara nakon
ronjenja s komprimiranim zrakom: vWf-a, PAI-1-a, hsCRP-a kao pokazatelja endotelne funkcije, te adenozina, TBARS i FRAP kao
pokazatelja oksidacijskog statusa uz istovremeno praćenje stupnja stvaranja mjehurića. U drugoj studiji na 10 ispitanika uspoređivan je
utjecaj ronjenja sa zrakom i ronjenja s nitrox-om na vaskularnu funkciju ispitivanjem promjena pokazatelja arterijske elastičnosti: brzine
pulsnog vala i augmentacijskog indeksa; pokazatelja endotelne funkcije mjerenjem protokom posredovane dilatacije (FMD); te koncentracije
nitrita u plazmi. Usporedba je uključivala i stupanj stvaranja mjehurića nakon zarona. U trećoj studiji na 16 ispitanika istraženo je dovodi li
arterijalizacija mjehurića do promjene koncentracije endotelina-1, S100-B i NSE u krvi odnosno do pojave znakova narušenog integriteta
endotela i središnjeg živčanog sustava.
Rezultati: Ronjenje je uzrokovalo značajno stvaranje plinskih mjehurića u sve tri studije. Arterijalizacije su zabilježene u sve tri studije i bile
su praćene visokim stupnjem stvaranja mjehurića. Značajno veći stupanj stvaranja mjehurića i češće arterijalizacije uslijedili su nakon
ronjenja sa zrakom u odnosu na nitrox. Indikatori arterijske elastičnosti pokazali su oprečne promjene nakon ronjenja s obje plinske smjese.
Brzina pulsnog vala je porasla sugerirajući porast krutosti arterija, dok se augmentacijski indeks smanjio nakon ronjenja. Ronjenje s nitroxom
pokazuje veći učinak na protokom posredovanu dilataciju u odnosu na ronjenje sa zrakom upućujući na veći utjecaj hiperoksije na
endotelnu funkciju od plinskih mjehurića. Ronjenje sa zrakom u prvoj studiji nije polučilo promjenu oksidacijskog statusa izuzev blagog
porasta antioksidacijskog kapaciteta nakon ronjenja. Mjerenje endotelnih parametara nije otkrilo promjene izuzev PAI-1 u prvoj studiji koji
pokazuje pad aktivnosti nakon ronjenja. Promjenu humoralnih biljega oštećenja SŽS-a u vidu porasta nakon ronjenja otkriva samo S-100B,
ali bez dokaza o oštećenju integriteta SŽS-a budući se sličan porast očitovao i nakon kontrolnog zarona bez stvaranja plinskih mjehurića i
arterijalizacije.
Zaključak: Ovim istraživanjem je pokazano da asimptomatski zaroni sa zrakom ne uzrokuju promjene biokemijskih pokazatelja endotelnog
oštećenja unatoč prisutnosti plinskih mjehurića u krvi, te da je potaknuta antioksidacijska obrana mogući mehanizam zaštite od endotelne
disfunkcije. Značajniji negativni učinak nitrox-a na endotel u odnosu na zrak, unatoč manjem stvaranju plinskih mjehurića i njihovoj rjeđoj
arterijalizaciji, upućuje na endotelno oštećenje uzrokovano hiperoksijom i na potrebu za dodatnim istraživanjima mehanizama endotelnih
promjena.
Uvod: Ronjenje s komprimiranim plinovima je često praćeno stvaranjem plinskih mjehurića u krvi i hiperoksijom, što se smatra mogućim
uzrocima endotelne disfunkcije nakon ronjenja. Endotelna disfunkcija se povezuje s asimptomatskim kardiovaskularnim i neurološkim
promjenama nakon ronjenja s komprimiranim plinovima.
Cilj ove disertacije je istražiti fiziološke i biokemijske promjene vaskularne i endotelne funkcije, te ispitati ima li znakova narušenog
integriteta SŽS-a nakon ronjenja s komprimiranim plinskim smjesama. Pritom su testirane sljedeće hipoteze: asimptomatski zaroni s
komprimiranim zrakom će uzrokovati promjene biokemijskih parametara endotelne funkcije i oksidacijskog stresa; asimptomatski zaroni će
imati različiti utjecaj na vaskularnu funkciju s obzirom na vrstu korištene plinske smjese za disanje; asimptomatski zaroni će u prisutnosti
arterijalizacije plinskih mjehurića uzrokovati porast koncentracije humoralnih biljega oštećenja endotela i središnjeg živčanog sustava.
Metode i ispitanici: Istraživanje je obuhvatilo tri studije. U prvoj studiji na 15 ispitanika istražene su promjene sljedećih parametara nakon
ronjenja s komprimiranim zrakom: vWf-a, PAI-1-a, hsCRP-a kao pokazatelja endotelne funkcije, te adenozina, TBARS i FRAP kao
pokazatelja oksidacijskog statusa uz istovremeno praćenje stupnja stvaranja mjehurića. U drugoj studiji na 10 ispitanika uspoređivan je
utjecaj ronjenja sa zrakom i ronjenja s nitrox-om na vaskularnu funkciju ispitivanjem promjena pokazatelja arterijske elastičnosti: brzine
pulsnog vala i augmentacijskog indeksa; pokazatelja endotelne funkcije mjerenjem protokom posredovane dilatacije (FMD); te koncentracije
nitrita u plazmi. Usporedba je uključivala i stupanj stvaranja mjehurića nakon zarona. U trećoj studiji na 16 ispitanika istraženo je dovodi li
arterijalizacija mjehurića do promjene koncentracije endotelina-1, S100-B i NSE u krvi odnosno do pojave znakova narušenog integriteta
endotela i središnjeg živčanog sustava.
Rezultati: Ronjenje je uzrokovalo značajno stvaranje plinskih mjehurića u sve tri studije. Arterijalizacije su zabilježene u sve tri studije i bile
su praćene visokim stupnjem stvaranja mjehurića. Značajno veći stupanj stvaranja mjehurića i češće arterijalizacije uslijedili su nakon
ronjenja sa zrakom u odnosu na nitrox. Indikatori arterijske elastičnosti pokazali su oprečne promjene nakon ronjenja s obje plinske smjese.
Brzina pulsnog vala je porasla sugerirajući porast krutosti arterija, dok se augmentacijski indeks smanjio nakon ronjenja. Ronjenje s nitroxom
pokazuje veći učinak na protokom posredovanu dilataciju u odnosu na ronjenje sa zrakom upućujući na veći utjecaj hiperoksije na
endotelnu funkciju od plinskih mjehurića. Ronjenje sa zrakom u prvoj studiji nije polučilo promjenu oksidacijskog statusa izuzev blagog
porasta antioksidacijskog kapaciteta nakon ronjenja. Mjerenje endotelnih parametara nije otkrilo promjene izuzev PAI-1 u prvoj studiji koji
pokazuje pad aktivnosti nakon ronjenja. Promjenu humoralnih biljega oštećenja SŽS-a u vidu porasta nakon ronjenja otkriva samo S-100B,
ali bez dokaza o oštećenju integriteta SŽS-a budući se sličan porast očitovao i nakon kontrolnog zarona bez stvaranja plinskih mjehurića i
arterijalizacije.
Zaključak: Ovim istraživanjem je pokazano da asimptomatski zaroni sa zrakom ne uzrokuju promjene biokemijskih pokazatelja endotelnog
oštećenja unatoč prisutnosti plinskih mjehurića u krvi, te da je potaknuta antioksidacijska obrana mogući mehanizam zaštite od endotelne
disfunkcije. Značajniji negativni učinak nitrox-a na endotel u odnosu na zrak, unatoč manjem stvaranju plinskih mjehurića i njihovoj rjeđoj
arterijalizaciji, upućuje na endotelno oštećenje uzrokovano hiperoksijom i na potrebu za dodatnim istraživanjima mehanizama endotelnih
promjena.
Background: Diving with compressed gases (SCUBA diving) is often associated with occurence of gas bubbles in the blood and hyperoxia,
which are possible causes of endothelial dysfunction after diving. Endothelial dysfunction is associated with asymptomatic cardiovascular
and neurological changes caused by SCUBA diving.
The aim of this study is to explore the physiological and biochemical changes of vascular and endothelial function, as well as to examine the
signs of impaired CNS integrity after diving with compressed gas mixtures. The following hypotheses were tested: asymptomatic dives with
compressed air will change the biochemical parameters of endothelial function and oxidative stress after dive; asymptomatic dives will have
different effects on vascular function considering different types of breathing gases; and, finally, asymptomatic dives will cause increased
levels of humoral markers of endothelial damage and CNS integrity due to arterializations of gas bubbles.
Methods: Three studies were performed. The first study including 15 participants examined changes of vWf, PAI-1 and hsCRP which
served as indicators of endothelial function, as well as adenosine, TBARS and FRAP as indicators of oxidative state while the bubble grades
were also monitored after dives with compressed air. In the second study that included 10 divers, the effects of diving with air and nitrox on
vascular function were compared by examining the indicators of arterial elasticity: pulse wave velocity and augmentation index; indicators
of endothelial function through assessment of flow-mediated dilation (FMD) and nitrite concentrations in plasma; and assessment of bubble
production. Third study which included 16 subjects examined whether bubble arterializations lead to increase in blood levels of endothelin-
1, S-100B and NSE, which would suggest an impaired endothelium and CNS integrity.
Results: Diving resulted in significant production of bubbles in all three studies. Arterializations were recorded in all three studies and were
accompanied by high bubble grade. Diving with air in the first study did not change the oxidative status with the exception of a slight
increase in antioxidant capacity after diving. Assessment of endothelial parameters did not reveal any changes, except PAI-1, activity of
which declined after diving . Air dives resulted in sign
Stresni proteini (HSP) pripadaju skupini unutarstaničnih proteina koji su eksprimirani konstitutivno i
kao odgovor na fizikalni odnosno biološki stres. U staničnom odgovoru na stres HSP imaju ulogu ...stabiliziranja
proteina i peptida čime promoviraju preživljenje stanice. Hsp27 i Hsp70 inhibitori su različitih čimbenika
apoptoze, dok je glavna uloga Hsp90 osiguravanje aktivnost čimbenika uključenih u proliferaciju stanica.
Sposobnost stresnih proteina da zaustave proces programirane stanične smrti i potaknu proliferaciiju predstavlja
bitan aspekt njihove povezanosti s malignom proliferacijom.
Glavni cilj ovog istraživanja bio je procijeniti ulogu Hsp70 u zaštiti od programirane stanične smrti u
tumorskim stanicama. U tom smislu ispitana je uloga inducibilnog oblika Hsp70 u fiziološkim i stresnim
uvjetima. Hsp70, induciran stresom, pokazao je značajnu ulogu u zaštiti tumorskih stanica od stanične smrti
izazvane staurosporinom. U svrhu utišavanja ekspresije Hsp70 pri fiziološkim uvjetima korištena je tehnologija
koja počiva na RNA interferenciji, specifičnoj razgradnji ciljne mRNA pomoću kratke siRNA uklopljene u
nanočestice. Dizajnirane su kitozanske nanočestice za dostavu Hsp70 sljedno-specifične siRNA. U uvjetima in
vitro kitozanske nanočestice pokazale su nisku toksičnost, učinkovito uklapanje Hsp70 siRNA te učinkovito
utišavanje Hsp70. U staničnim linijama Jurkat i U251N utišavanje Hsp70 omogućeno pomoću Hsp70 siRNA
uklopljene u kitozanske nanočestice, prouzročilo je smanjenje vijabilnosti ovih tumorskih staničnih linija.
U radu je ispitana i antitumorska učinkovitost istovremene inhibicije Hsp90, pomoću celastrola, i
Hsp70 pomoću kitozanskih nanočestica s uklopljenom Hsp70 siRNA. Učinkovitost ovog pristup bila je značajna
u dvodimenzionalnim modelima tumorskih staničnih linija, dok su u trodimenzionalnim modelima bile potrebne
povećane koncentracije oba spoja/sustava kako bi se postiglo značajno smanjenje vijabilnosti tumorskih stanica.
Rad predstavlja temelje za daljnja istraživanja optimalnih nanosustava za dostavu sljedno-specifičnih
siRNA za HSP te moguća ispitivanja istovremene inhibicije Hsp70 i Hsp90 u tumorskim modelima in vivo.
Stress proteins (HSP) are intracellular proteins expressed constitutively but can also be induced by
various types of stress including environmental changes and non-stress conditions such as cell cycle, growth
factors, development and differentiation. Under physiological conditions these proteins function as molecular
chaperones that enable the function of different proteins. Hsp27 and Hsp70 appear to function at key regulatory
control points in apoptotic process, whereas the major role of Hsp90 is protection from degradation for the major
factors responsible for cell proliferation. The promotion of tumor cell survival is closely related to the ability of
Hsp to inhibit programmed cell death during malignant proliferation.
The purpose and the main goal of the proposed study was to explore Hsp70's role in protection against
programmed cell death in tumor cells. The role of stress induced Hsp70 and the role of Hsp70 within
physiological levels was explored. Stress induced Hsp70 was found to efficiently protect tumor cells against cell
death promoted with staurosporin. Hsp70 silencing was accomplished using the technology based on RNA
interference, specific degradation of mRNA after binding with siRNA delivered into the cells with nanoparticles.
Chitosan based nanoparticles for Hsp70 siRNA delivery were prepared. These showed low toxicity, efficient
siRNA entrapment and efficient Hsp70 silencing. In Jurkat and U251N cell lines Hsp70 silencing with Hsp70
siRNA delivered in chitosan nanoparticles significantly decreased cell viability.
The antitumor efficiency of simultaneous inhibition of Hsp90 with celastrol and Hsp70 with chitosan
nanoparticles entrapping Hsp70 siRNA was also explored. Significant reduction in cell viability was observed in
two-dimensional cell cultures, while for the induction of comparable effect in three-dimensional cell cultures,
U251N spheroids, higher concentration of both celastrol and Hsp70 siRNA were necessary.
The results of this work present important findings that could lead into the development of optimal
delivery systems for siRNA and possible in vivo research of Hsp70/Hsp90 inhibition approach.
Hrana je glavni izvor izloženosti ljudi esencijalnim i toksičnim metalima, uključujući kadmij (Cd). Cilj istraživanja je bio procijeniti učinke peroralne izloženosti Cd tijekom skotnosti štakorice na ...razdiobu kadmija i stanje mikronutrijenata u majčinom organizmu i fetusu te funkcije posteljice u prijenosu nutrijenata i sintezi steroidnih hormona. Hipoteze istraživanja su bile da se tijekom graviditeta povećava želučanocrijevna apsorpcija Cd koji se nakuplja u posteljici i može poremetiti prijenos nutrijenata do fetusa i sintezu posteljičnih hormona. Pokusne štakorice (Wistar) su nakon parenja izlagane dozi od 50 mg Cd/l (u obliku CdCl2xH2O) u vodi za piće od 1. do 19. ili 20. dana skotnosti. Neskotne štakorice su istodobno izlagane pod jednakim uvjetima izloženosti tijekom 20 dana. Posljednjeg dana pokusa je svim štakoricama u općoj anesteziji izvađena krv iz srca i uzorkovani unutrašnji organi, posteljice i fetusi, koji su pripremljeni za analize mikroelemenata (metodom AAS). Progesteron i testosteron su analizirani imunokemijski izravno u serumu (metodom IEMA) i u uzorcima pripremljenim iz posteljičnog tkiva (metodama IEMA i/ili ELISA). U svih izloženih štakorica su nađena povećanja Cd u svim izmjerenim uzorcima i cinka u jetri. Količine željeza u fetusu i cinka u fetusu i/ili posteljici su bile smanjene. U izloženih skotnih vs. izložene neskotne štakorice su bila izraženija povećanja koncentracije Cd u krvi i količina Cd u jetri i bubregu kao i smanjenja količina željeza u jetri i bubregu te cinka i bakra u bubregu. U svih skotnih vs. neskotne štakorice su bile veće koncentracije Cd u krvi i količine bakra u bubregu te manje količine željeza u jetri i bubregu i bakra u jetri. Nije bilo promjena u steroidnim hormonima ni u serumu, ni u posteljici, u kojoj su vrijednosti svakog hormona izmjerene dvjema imunokemijskim metodama značajno korelirale. U zaključku, ovim radom su dobiveni izvorni podaci o vrijednostima progesterona i testosterona (kao prekusora za sintezu estradiola u jajniku) u serumu i posteljici štakorice blizu roka okoćenja. Novi i izvorni znanstveni rezultati su da peroralna izloženost štakorica dozi od 6,5 mg Cd/kg tjelesne mase otopinom za napajanje tijekom skotnosti povećava razine Cd u krvi, jetri i bubregu s posljedičnim biokemijskim promjenama mikronutrijenata u većoj mjeri nego u neskotnih štakorica što istodobno s nakupljanjem Cd u posteljici remeti transplacentarni prijenos željeza i cinka te može predstavljati opasnost za rast i razvoj fetusa in utero.
Food is the main source of human exposure to essential and toxic metals, including cadmium (Cd). This investigation aimed to assess the effects of oral Cd exposure during rat pregnancy on Cd distribution and micronutrient status in a maternal organism and foetus and placental functions in nutrient transport and steroid hormone synthesis. Research hypotheses were that Cd gastrointestinal absorption increases during pregnancy and Cd accumulates in the placenta where it may interfere with nutrient transport to the foetus and placental hormone biosynthesis. Female rats (Wistar) were mated and exposed to 50 mg Cd/l (as CdCl2xH2O) in drinking water from gestation day 1 through 19 or 20. Non-pregnant rats were concurrently exposed during 20 days under the same exposure conditions. On the last experimental day, under general anaesthesia, blood was taken by cardiac puncture from all of the rats and internal organs, placentas and foetuses were dissected and prepared for element analysis (by AAS). Progesterone and testosterone were assayed by immunochemical methods directly in sera (by IEMA) and in placental tissue-derived samples (by IEMA and/or ELISA). All of the exposed rats exhibited increases in Cd in all of the analysed samples and zinc in the liver. Contents of iron in the foetus and zinc in the foetus and/or placenta were decreased. In exposed pregnant vs. exposed non-pregnant rats, more pronounced increases in Cd concentration in the blood and Cd contents in the liver and kidney, decreases in iron contents in the liver and kidney, and decreases in zinc and copper contents in the kidney were recorded. In all pregnant vs. non-pregnant rats, higher Cd concentrations in the blood and copper content in the kidney and lower iron contents in the liver and kidney and copper content in the liver were found. Steroid hormones did not change in either the serum or placenta; in the latter the values of either hormone measured by two immunochemical assays were correlated. In conclusion, this work provides original evidence on progesterone and testosterone (as the precursor for the ovarian oestradiol synthesis) in rat serum and placenta at term. New and original research results are that oral Cd exposure to 6.5 mg Cd/kg body mass in drink during rat pregnancy increases levels of Cd in the blood, liver and kidney with consequent biochemical changes of micronutrients more pronouncedly than in non-pregnant rats, which together with accumulation of Cd in the placenta disrupts the transplacental handover of iron and zinc and may put at risk foetal growth and development in utero.
Problemi u funkciji hoda u pacijenata nakon moždanog udara svode se na problem balansa, na smanjenje brzine hoda, smanjenje dužine i ciklusa koraka te oblik asimetričnog uzorka što smanjuje njihovu ...sposobnost u obavljanju svakodnevnih aktivnosti, a time i njihovo zadovoljstvo samom kvalitetom života.
Cilj istraživanja bio je utvrditi učinkovitost neurofacilitacijskog programa prema Bobath konceptu, kao i kombiniranog tretmana neurofacilitacijske terapije sa dodatnim tretmanom specifičnih mobilizacija na balans i funkciju hoda, te razliku u učinkovitosti provedenih programa.
Ispitanici su raspoređeni u dvije ispitivane skupine po 20 ispitanika. Prva skupina ispitanika bila je u programu neurofacilitacijske terapije kroz pet tjedana, dok je druga skupina ispitanika bila uključena u isti program s dodatnim tretmanom specifičnih mobilizacija mekih tkiva. Razlike kod ispitanika u pojedinim varijablama unutar grupa i između grupa ispitanika u inicijalnom i finalnom mjerenju obrađene su univarijantnom analizom varijance - ANOVA. Uspoređeni su rezultati učinaka dvaju programa neurofacilitacijskog tretmana sa i bez specifičnih mobilizacija mekih tkiva testovima „Berg balance scale“, „Timed up and go testa“ i aktivnog pokreta dorzalne fleksije stopala, fleksije i ekstenzije koljena.
Istraživanje je pokazalo da je u prvoj skupini ispitanika koja je imala neurofacilitacijski tretman u 83,4% varijabli statistički značajnih rezultata u finalnom mjerenju na razini od p<0.05. U drugoj skupini ispitanika tretman neurofacilitacijske terapije kombiniran s tretmanom specifičnih mobilizacija mekih tkiva, pokazao se ključnim za poboljšanje aktivnosti u pokretima stopala i koljena što je značajno doprinijelo učinkovitosti onih varijabli koje u prvoj skupini nisu bile značajne, a to su hod, ustajanje, sjedanje, pa je tako učinkovitost bila značajna gotovo u svim varijablama statičkog i dinamičkog balansa.
Rezultati dobiveni ovim istraživanjem pokazali su značajan doprinos kombiniranog tretmana na sve varijable balansa, funkciju hoda kao i na povećanje fleksibilnosti mišića i tetiva koji su važni za funkciju koljena i skočnog zgloba, a time i na funkciju hoda.
Introduction: Problems in the function of the stroke patient gait are balance problems, slower walking speed, reducing the step length and the gait cycle, and the asymmetrical gait pattern. They all reduce the patient’s ability to perform everyday activities and result in the patient’s dissatisfaction with the quality of their life. Finding new programmes to improve the quality of the previously used rehabilitation procedures is imperative for members of the team included in movement and gait re-education of stroke patients.
Subjects and methods: The aim of this study is to determine the effectiveness of the neurofacilitaton programme according to the Bobath concept, and a combined treatment consisting of the neurofacilitation therapy and an additional treatment with specific mobilization to improve balance and gait, as well as determining the difference in the effectiveness of the implemented programmes.
40 subjects, successfully tested initially, at least 3 months after suffering a stroke, and after a one-year period, were diagnosed by magnetic resonance imaging. They were suffering from hemiparesis, classified as level 3 according to the Medical Research Council paresis classification. The subjects were randomly divided into two groups consisting of 20 patients. The first experimental group was in the programme of neurofacilitation therapy for 5 weeks, undergoing 45-minute sessions 5 times a week, while the second group was included in the same programme with an additional specific mobilization programme with 20-minute sessions 3 times a week, during 5 weeks. The STATISTICA for Windows ver. 10 StatSoft Inc. statistical software packet was used for data processing. Differences between the respondents in individual variables within groups and between groups in both initial and final measurements were processed by ANOVA, the univariate analysis of variance.
Results: Effects of the two neurofacilitation treatment programmes, with and without specific mobilization of soft tissues, were compared by the Berg Balance Scale, the Timed Up and Go Test and active dorsiflexion of the foot, flexion and extension of the knee. The study showed statistically significant results in the final measurement in 83.4% of variables at the level of p<0.05 in the tested group of patients who only had a neurofacilitation treatment. The variables which did not show significant results were complex activities, such as getting up, walking, rotation, and sitting down, which clearly required additional treatment. The neurofacilitation therapy combined with the specific mobilization of soft tissues, which the second tested group of patients underwent, greatly contributed to the improvement of foot and knee movements resulting in better walking, standing up and sitting down activities, as well as other variables of static and dynamic balance. Results also suggested that there are no statistically significant differences between the two tested groups in the final measurement of the given variables. However, the additional statistical analysis which took into account the period between the initial and final measurements demonstrated a statistical significance and a high tendency to significance in almost 61% of variables indicating the benefit of the combined therapy programme.
Conclusion: Results obtained in this study showed a significant contribution of the combined treatment to all the variables of the static and dynamic balance and gait function, as well as an increase in the muscle and tendon flexibility which is important for knee and ankle functions, thus influencing the gait function, too.
Analize često otkriju neujednačenost sastava tableta ecstasyja od čistoga 3,4-metilendioksimetamfetamina (MDMA) do mješavina njegovih derivata, amfetamina i drugih neutvrđenih tvari. Stoga je za ...kvalitetnu toksikološku analizu potreban uvid u sve korake sinteze MDMA, s obzirom na to da se ondje vjerojatno kriju izvori nečistoće (prekursori, katalizatori). Cilj ovog ispitivanja bio je sintetizirati derivate MDMA te napraviti njihovu kemijsko-fi zikalnu i biološku in vitro karakterizaciju. 3,4-metilendioksifenil-2-nitropropen (MDNP) dobiven je kondenzacijom piperonala u suvišku nitroetana uz
dodatak amonijeva acetata. Njegovom redukcijom s pomoću LiAlH3 dobiven je 3,4-metilendioksiamfetamin
(MDA). Svi spojevi iz pojedinih koraka sinteze karakterizirani su s pomoću tekućinske kromatografi je visoke djelotvornosti (HPLC) i spektroskopskih tehnika Ramanove spektroskopije, nuklearne magnetske rezonancije (NMR-a) te infracrvene spektroskopije (IR-a). Usto je ocijenjen i njihov biološki učinak in vitro mjerenjem (i) koefi cijenta raspodjele krvna stanica/puna krv, (ii) vezanja za bjelančevine u plazmi (Fbp) te (iii) adsorpcije na membranu. Kemijska je struktura utvrđena s pomoću fl uorescentnoga polarizacijskog imunokemijskog testa (FPIA). Analiza je u konačnim proizvodima utvrdila prisutnost krutih nečistoća, napose spojeva neurotoksičnog aluminija (Al3+). FPIA je prepoznao aminoetansku skupinu blizu supstituiranoga benzenskog prstena, ali ne i dva glavna prekursora za MDMA: MDNP i piperonal. Posebno je zanimljiva Ramanova spektroskopija budući da (i) pruža privlačnu alternativu za karakterizaciju sastava tableta ecstasyja te (ii) može otkriti stereoizomerne cis/trans-oblike spoja poput cis-MDNP-a odnosno
trans-MDNP-a, čiji se signal vidi na 1650 cm-1 odnosno 1300 cm-1.
Trenutno ne postoje farmakopejske ili standardne metode i aparature za ispitivanje oslobađanja djelatne tvari iz topikalnih mikročestica. Cilj ovog rada bio je razviti i validirati diskriminatornu in ...vitro metodu za ispitivanje oslobađanja djelatne tvari iz topikalnih mikročestica, uz korištenje imerzijske ćelije. Za potrebe rada pripremljene su i karakterizirane kitozanske i metakrilatne mikročestice s mupirocinom. Za izradu mikročestica korištena je tehnika sušenja raspršivanjem. Ispitana su sljedeća svojstva mikročestica: učinkovitost uklapanja lijeka, morfologija i veličina čestica, kristalno stanje, termička svojstva, spektralna svojstva, zeta potencijal, higroskopnost te sadržaj vode i ostatnih organskih otapala.
Kitozanske i metakrilatne mikročestice s mupirocinom korištene su kao modelni topikalni terapijski sustavi za razvoj in vitro metode za ispitivanje oslobađanja djelatne tvari. Imerzijska ćelija korištena je u kombinaciji s aparaturom s lopaticom. pH i temperatura medija (pH 5,5, 32°C) odabrani su u skladu s fiziološkim uvjetima na koži. Difuzija lijeka odvijala se preko membrane izrađene od smjese celuloznih estera, koja je pokazala nisku adsorpciju lijeka te nizak otpor difuziji lijeka. Nakon
početnog zastojnog vremena količina oslobođenog lijeka postala je proporcionalna korijenu iz vremena. Nagib u linearnom području krivulje oslobađanja lijeka korišten je kao mjera brzine oslobađanja. Varijacije u brzini okretanja lopatica (25 o/min, 50 o/min, 100 o/min), visini lopatica (1 cm, 2,5 cm) i volumenu medija za ispitivanje oslobađanja (100 ml, 200 ml) nisu značajno utjecale na brzinu oslobađanja. Analiza kitozanskih mikročestica izrađenih s kitozanima različitih molekulskih masa pokazala
je da se kod povećane molekulske mase kitozana smanjuje brzina oslobađanja. S druge strane, brzina oslobađanja bila je veća pri većoj koncentraciji lijeka unutar donorskog odjeljka. Na taj je način pokazana diskriminatornost metode prema razlikama u formulaciji, kao i prema razlikama u koncentraciji uzorka unutar donorskog odjeljka ćelije. Metodom je nadalje potvrđena sličnost serija istog sastava proizvedenih istim procesom. Metoda je validirana u skladu s ICH smjernicama. U sklopu validacije metode potvrđena je njena specifičnost, linearnost, točnost, preciznost i robusnost. Metoda je uspješno primijenjena kod kitozanskih i metakrilatnih čestica, čime je pokazan njen potencijal za karakterizaciju raznih tipova
topikalnih čestičnih terapijskih sustava. Razvijena metoda može biti koristan alat u razvoju formulacije kod takvih terapijskih sustava.
Currently there are no compendial or standard methods and apparatuses for in vitro release testing of topical microparticles. The aim of this study was to develop and validate a discriminative in vitro release method for topical microparticles using the immersion cell. For the purpose of this study chitosan-based and methacrylate-based microparticles with mupirocin were prepared by spray drying. The following characteristics of the microparticles were
examined: encapsulation efficiency, particle size and morphology, crystallinity, thermal properties, spectral properties, surface charge, hygroscopicity, residual organic solvents and water content. Chitosan-based and methacrylate-based microparticles with mupirocin were used as model topical delivery systems for in vitro release method development. The immersion cells were used in combination with paddle dissolution apparatus. The pH and temperature of the release medium (pH 5.5, 32°C) were selected to reflect the physiological skin conditions. Diffusion of the drug occured
across a mixed cellulose ester membrane, which demonstrated low drug adsorption and low diffusional resistance. After an initial lag phase the amount of drug released became proportional to the square root of time. The slope in the linear portion of the release curve was used as a measure of release rate. Variations in paddle rotation speed (25 rpm, 50 rpm, 100 rpm), paddle height (1 cm, 2.5 cm) and volume of release medium (100 ml, 200 ml) did not significantly alter the release rates. Appropriate discriminatory power of the method was confirmed as the method was able to detect differences in formulation, as well as differences in drug concentration inside the sample compartment. The analysis of chitosan-based microparticles prepared with chitosans of different molecular weights has shown that the release rate decreases with increasing molecular weight of chitosan. On the other hand, the release rate increased with increasing drug concentration inside the sample compartment. The method was further used to confirm sameness between batches of the same composition prepared by the same process. The method was validated for its specificity, linearity, accuracy, precision and robustness in line with International Conference on Harmonisation (ICH) guidelines. The method was successfully applied both for chitosan-based and methacrylate-based microparticles, which demonstrates its potential application for various types of topical particulate delivery system.
Uvod: Glavni regulator sistemske homeostaze željeza je hepcidin, čiju sintezu reguliraju status željeza, upalni citokini, eritropoetska aktivnost, hipoksija i anemija. Cilj ovoga rada bio je ispitati ...povezanost između serumskog hepcidina i čimbenika koji ga reguliraju u skupini bolesnika s KOPB-om, da bi se ispitalo kako ovi čimbenici utječu na koncentraciju hepcidina i osiguravanje opskrbe željezom neophodnim za eritropoezu. Hipoteze istraživanja: koncentracija hepcidina u serumu bolesnika s KOPB-om promijenjena je u odnosu na kontrolnu skupinu i mijenja se tijekom egzacerbacije u odnosu na stabilnu fazu bolesti; na koncentraciju hepcidina u KOPB-u utječu upala i/ili hipoksija.
Metode i ispitanici: U istraživanje je bilo uključeno 40 bolesnika s KOPB-om i 30 zdravih ispitanika. U KOPB-u skupini parametri su longitudinalno praćeni u tri vremenske točke: u egzacerbaciji, fazi rezolucije i stabilnoj fazi bolesti. Svim ispitanicima određena je koncentracija hepcidina, pokazatelja statusa željeza (serumsko željezo, TIBC, saturacija transferina, feritin); pokazatelja upale (broj leukocita i neutrofilnih granulocita, IL-6 i CRP-a), eritropoetske aktivnosti (broj retikulocita, topivi transferinski receptor i eritropoetin) i koncentracija hemoglobina. Svim bolesnicima s KOPB-om određeni su parcijalni tlak kisika i saturacija hemoglobina kisikom kao pokazatelji hipoksije.
Rezultati: Serumska koncentracija hepcidina bila je povišena u egzacerbaciji i stabilnoj fazi KOPB-a u odnosu na kontrolnu skupinu, te je pozitivno korelirala s IL-6 i CRP-om. Koncentracija hepcidina je bila pozitivno povezana s feritinom i negativno s TIBC-om. Eritropoetska aktivnost, mjerena apsolutnim brojem retikulocita bila je u svim fazama istraživanja niža od kontrolne skupine, a negativna povezanost je pokazana u egzacerbaciji. Koncentracija hepcidina nije bila povezana s parametrima hipoksije. U kontrolnoj skupini hepcidin je korelirao samo s pokazateljima statusa željeza, negativno s TIBC-om i pozitivno s feritinom.
Zaključak: Istraživanje je pokazalo da prisutna sistemska upala i povišena razina IL-6 u egzacerbaciji i stabilnoj fazi KOPB-a mogu biti odgovorne za opaženi porast koncentracije hepcidina. Porast koncentracije hepcidina mogao bi biti povezan s restriktivnom eritropoezom koja se očituje sniženim brojem retikulocita u svim fazama istraživanja, kao i snižavanju koncentracije hemoglobina u stabilnoj fazi bolesti. Dobiveni rezultati pružaju uvid u dinamičke promjene metabolizma željeza i povezanosti s razinom hepcidina. Sistemska upala prisutna kod velikog broja bolesnika s KOPB-om jača kako bolest napreduje, te bi porast razine IL-6 mogao voditi do daljnjeg porasta koncentracije hepcidina.
Background: Hepcidin is the main regulator of systemic iron homeostasis, and its expression is modulated by iron status, hypoxia, erythroid factors and inflammation. The aim of this study was to examine a relationship between level of hepcidin and iron status, erythropoietic activity, hypoxia and inflammation in exacerbations and stable COPD. We hypothesized that hepcidin concentration is changed compared to control group and is changing in acute exacerbation compared to stable COPD; hepcidin concentration is substantially influenced by inflammation and/or hypoxia.
Methods: The study included 40 COPD patients and 30 healthy subjects. In COPD group parameters were longitudinally monitored at three time points: at exacerbation, on resolution and in stable disease. We determined concentration of hepcidin and hemoglobin; parameters of iron status: serum iron, total iron binding capacity (TIBC), ferritin and calculated transferrin saturation. Soluble transferrin receptors, reticulocyte number (Rtc), and regulatory hormone erythropoietin were measured as indicators of erythropoietic activity. Systemic inflammation was assessed by determination of CRP, IL-6, and number of white blood cells and neutrophils. In COPD group partial oxygen pressure and haemoglobin oxygen saturation were determined.
Results: Hepcidin was elevated in exacerbations and in a stable phase compared to the control group and we found positive correlations of hepcidin with inflammatory markers IL-6 and CRP. Hepcidin also correlated positively with ferritin and inversely with TIBC. Erythropoietic activity, measured by absolute Rtc number, was significantly reduced in COPD compared to the control group in all study phases, and negative correlation with hepcidin was established in exacerbation. In exacerbation and stable disease hepcidin correlated with ferritin and TIBC. No correlations were observed with indices of hypoxia. In the control group, positive associations were observed only with indices of iron status, positive with ferritin and negative one with TIBC.
Conclusion: This study shows that elevated values of IL-6 present in exacerbations and stabile COPD might be responsible for the observed increased hepcidin level. These might be associated with restricted erythropoiesis as shown by lower number of reticulocytes and decreased level of haemoglobin at the stable phase. The results obtained might provide new insights into dynamic changes of iron metabolism and its relation to hepcidin level in COPD. Systemic inflammation present in majority of COPD patients increases over time as disease progresses so raising of IL-6 level could lead to further up-regulation of hepcidin.
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.
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
Učinak sekundarnog metabolita masline (Olea europaea L.) oleuropeina, njegovog razgradnog produkta hidroksitirosola i vodenog ekstrakta maslinova lista na staničnu vijabilnost medicinski značajne ...gljivične vrste Candida albicans ispitan je in vitro testovima. Metodom mikrodilucije određene su minimalne inhibitorne koncentracije (MIK) koje uzrokuju 80 % smanjenja stanične vijabilnosti i koje su iznosile 12,5 mg/ml za oleuropein, 6,25 mg/ml za hidroksitirosol i 25 mg/ml za vodeni ekstrakt maslinova lista. Kvantitativna analiza s fluorescentnim bojanjem upućuje na zaključak da je apoptoza primarni način stanične smrti u uzorcima tretiranim sa sub-MIK koncentracijama ovih tvari. Budući da je patogenost vrste određena virulentnim čimbenicima, ispitani su učinci oleuropeina i hidroksitirosola na najznačajnije virulentne čimbenike vrste C. albicans. Utvrđen je inhibitorni učinak ovih fenolnih tvari na promjenu u hifalni oblik rasta kod vrste C. albicans induciranu u uvjetima staničnog gladovanja. Pri sub-MIK koncentracijama, oleuropein i hidroksitirosol nisu pokazali inhibitorno djelovanje na stvaranje biofilma ove vrste. Pri subinhibitornim koncentracijama nakon 24 sata inkubacije, oleuropein i hidroksitirosol su uzrokovali modulaciju stanične površinske hidrofobnosti koja se dovodi u svezu s adherencijom vrste C. albicans na biomaterijale. Uzgoj vrste C. albicans u prisutnosti oleuropeina i hidroksitirosola je uzrokovao inhibiciju aktivnosti kandidinih hidrolitičkih enzima, aspartil-proteaza (Sap) i α-glukozidaza. Tretiranjem vrste C. albicans s oleuropeinom i hidroksitirosolom utvrđeno je višestruko djelovanje ovih tvari uključujući oštećenje stanične stijenke, stanične membrane i otpuštanje citoplazmatskog sadržaja (DNA i proteina). S obzirom da neki antimikotici djeluju kao inhibitori biosinteze ergosterola, provedeno je ispitivanje učinka oleuropeina i hidroksitirosola na modulaciju ergosterola kojima je utvrđeno smanjenje ovog sterola u ovisnosti o primijenjenoj koncentraciji. Uz testove određivanja djelovanja na gljivičnu vrstu, ispitani su mogući citotoksični i genotoksični učinci na stanice nositelja. Navedene tvari nisu pokazale hemolitičku aktivnost na humanim eritrocitima pri testiranim koncentracijama u rasponu 3-60 μM. Ispitivanje učinka na humanim limfocitima periferne krvi provedeno kometnim testom pokazalo je protektivno djelovanje ovih tvari na oštećenja DNA inducirana s mutagenom H2O2, pri čemu je hidroksitirosol imao bolji protektivni učinak u usporedbi s oleuropeinom nakon 120 min predtretmana s ispitivanim tvarima u koncentracijama 1, 5 i 10 μM. Navedeni genoprotektivni učinci su posljedica snažnog antioksidativnog djelovanja ovih fenolnih spojeva utvrđenih ABTS, FRAP i CUPRAC metodom.
Activity of oleuropein, a phenol secoiridoid present in olive tree products, its derivative hydroxytyrosol and water extract of olive leaf were investigated using in vitro tests against opportunistic fungal pathogen Candida albicans. Antifungal susceptibility testing was used to estimate the drop of viability up to 80 % in comparison to the control (untreated cells). Minimal inhibitory concentration (MIC) against C. albicans for oleuropein was 12,5 mg/ml, for hydroxytyrosol was 6,25 mg/ml and for water extract of olive leaf was 25 mg/ml. Morphological changes in the nuclei after staining with fluorescent DNA-binding dyes revealed that apoptosis was a primary mode of cell death in analyzed samples treated with sub-MIC concentrations of tested compounds. In order to understand mode of action of oleuropein and hydroxytyrosol, their effect on C. albicans virulence factors essential for development of infection in the host have been investigated. Both oleuropein and hydroxytyrosol modulate morphogenetic coversion and inhibit filamentation of C. albicans induced in the conditions of reduced nutrient availability. On the other hand, treatment with sub-MIC concentractions of these phenolic compounds did not affect C. albicans biofilm formation. The hydrophobicity assay showed that both compounds, in sub-MIC values, have decreased the cellular surface hydrophobicity, a factor associated with adhesion to epithelial cells. Cultivation of C. albicans in the presence of oleuropein and hydroxytyrosol also inhibits, in the dose-dependent manner, acitivity of aspartyl-proteases (Sap), hydrolytic enzymes important in adherence, tissue penetration, invasion and destruction of host tissue. Most therapies for fungal infections target the ergosterol biosynthesis pathway or its end product ergosterol which is necessary for growth and normal function of fungal cells. At subinhibitory concentrations tested compounds have altered sterol content and subsequently affected the cell membrane of C. albicans. Both compounds induced dose-dependent loss of intracellular material to outer space (DNA, protein leakage) and caused membrane depolarisation indicating their direct effect on the membrane. In addition, cytotoxicity and genotoxicity assays were performed to elucidate the effect of oleuropein and hydroxytyrosol on the host cells. Oleuropein and hydroxytyrosol exhibited no hemolytic activity on human erythrocytes at tested concentrations (3-60 μM). Antigenotoxic effects of oleuropein and hydroxytyrosol against H2O2-induced damage in human lymphocytes were evaluated in vitro by alkaline comet assay. Pretreatment of human lymphocytes with each of the substance for 120 min produced dose-dependent reduction of primary DNA damage in the tested cell type. At tested concentrations (1, 5, 10 μM), hydroxytyrosol showed better protective effect than oleuropein against H2O2-induced DNA breaks. Our results suggest that genoprotective effects of these phenolic compounds could be attributed to their radical scavenging and metal ions chelating activity which were determined using ABTS, FRAP and CUPRAC assays.
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