Oralni antikoagulans varfarin se već desetljećima koristi u terapiji i/ili prevenciji tromboembolijskih stanja različite
etiologije. Varfarin ima uzak terapijski raspon i pokazuje veliku ...interindividualnu varijabilnost u odgovoru na terapiju,
što može potencijalno rezultirati i po život opasnim komplikacijama uslijed prekomjernog antikoagulacijskog učinka i
krvarenja.
Cilj ovoga istraživanja bio je ispitati utjecaj polimorfizama VKORC1 -1639G>A, VKORC1 1173C>T, CYP2C9*2,
CYP2C9*3, CYP4F2*3 i GGCX 12970C>G te kliničkih i okolišnih čimbenika na stabilnu dozu održavanja i nuspojave
terapije varfarinom. U istraživanju su sudjelovala 204 bolesnika na terapiji varfarinom i 420 zdravih ispitanika. Rezultati
genotipizacije na zdravim ispitanicima pokazuju kako je učestalost polimorfizama VKORC1 1639G>A i VKORC1
1173>T u populaciji Hrvatske u skladu s učestalošću u europskim populacijama. Rezultati istraživanja na skupini
bolesnika na terapiji varfarinom ukazuju na statistički značajnu povezanost stabilne doze održavanja varfarina sa
sljedećim čimbenicima: genotipovima CYP2C9, VKORC1 i CYP4F2, zatim s fizičkom aktivnošću, tjelesnom
površinom, te uzimanjem lijekova koji utječu na metabolizam varfarina. Algoritam za predviđanje doze dobiven
višestrukom linearnom regresijom u stanju je objasniti 49,8 % varijabilnosti doze varfarina. Dokazana je statistički
značajna povezanost vremena potrebnog za postizanje stabilne doze varfarina s polimorfizmom CYP4F2*3 te
povezanost udjela vremena provedenog unutar terapijskog raspona i pojave nuspojava terapije varfarinom u obliku
prekomjerne antikoagulacije s polimorfizmima VKORC1 -1639G>A i/ili VKORC1 1173C>T. Isto tako, utvrđena je
statistički značajna povezanost VKORC1 -1639G>A i/ili VKORC1 1173C>T te CYP4F2*3 s nuspojavama terapije
varfarinom u obliku krvarenja.
Warfarin is the most commonly used oral anticoagulant worldwide. It is used for over a half of century for prevention
of thromboembolic disorders. Dosage of warfarin is difficult due to a very narrow therapeutic index (with lifethreatening
overdose complications) and wide interindividual variability.
Aim of this study was to investigate association of VKORC1 -1639G>A, VKORC1 1173C>T, CYP2C9*2, CYP2C9*3,
CYP4F2*3 and GGCX 12970C>G genetic polymorphisms, along with some clinical and environmental factors, on
warfarin maintenance dose and adverse effects of warfarin therapy. This study included 204 patients on warfarin therapy
and 420 healthy volunteers. Results of genotyping study on healthy population confirms that allele frequencies for
VKORC1 -1639G>A and VKORC1 1173C>T polymorphisms in Croatian population are in good agreement with other
populations of European ancestry. Results obtained on warfarin patient population indicate that warfarin maintenance
dose was significantly associated with following variables: CYP2C9, VKORC1 and CYP4F2 genotype, physical activity,
body surface area and concomitant usage of drugs that influence warfarin metabolism. Dosage prediction algorithm
obtained with multiple regression could explain 49,8 % of dose variability. Time to warfarin stable dose was significantly
associated with CYP4F2*3 genetic polymorphism. Time in therapeutic range was significantly associated with VKORC1
-1639G>A and VKORC1 1173C>T polymorphisms. VKORC1 -1639G>A and VKORC1 1173C>T polymorphisms were
also significantly associated with time to first overanticoagulation in warfarin therapy. VKORC1 -1639G>A, VKORC1
1173C>T and CYP4F2*3 polymorphisms were significantly associated with bleedings encountered during warfarin
therapy.
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.
Novi dokazi ukazuju da je antinociceptivno djelovanje botulinum toksina tipa A (BT-A) središnjeg porijekla. U ovom doktorskom radu smo provjerili ovu pretpostavku u ranije nedovoljno istraženim ...oblicima boli, istraživali interakciju sa središnjim neurotransmitorima kao mogući mehanizam djelovanja te pokušali pobliže utvrditi mjesto djelovanja toksina u središnjem živčanom sustavu (SŽS).
Ispitivanja su izvedena na mužjacima Wistar štakora. U upalnoj, neuropatskoj i bilateralnoj mišićnoj boli ispitan je učinak selektivnih i neselektivnih antagonista opioidnih i GABAA receptora, primijenjenih sistemski, spinalno ili supraspinalno, na antinociceptivno djelovanje periferno (supkutano u šapu) primijenjenog BT-A. U tkivu kralješnične moždine ispitana je aktivacija neuronalnih i glija stanica, ekspresija mRNA proupalnih citokina i μ-opioidnih receptora te ekspresija Leu/Met-enkefalina metodama imunofluorescencije i lančane reakcije polimerazom s reverznom transkripcijom. Istraživano je antinociceptivno djelovanje BT-A nakon periferne, spinalne i supraspinalne primjene te je imunofluorescencijom ispitana enzimska aktivnost BT-A u tkivu SŽS-a.
Opioidni i GABAA antagonisti su ovisno o dozi, sistemski i intratekalno, ali ne i supraspinalno, poništili antinociceptivno djelovanje BT-A u svim ispitanim modelima. Učinak antagonista bio je kratkotrajan. BT-A je smanjio neuronalnu aktivaciju u dorzalnom rogu kralješnične moždine, što su antagonisti blokirali. BT-A je smanjio bol u dosad neistraženim modelima visceralne boli (peritonitis, kolitis). Bilateralno antinociceptivno djelovanje BT-A posljedica je prisutnosti toksina samo na ipsilateralnoj strani. BT-A je smanjio bol nakon periferne i intratekalne primjene, dok primijenjen supraspinalno (cisterna magna, moždane komore) nije djelovao, unatoč nalazu njegove enzimske aktivnosti u pojedinim regijama mozga uključenima u nocicepciju.
BT-A ima segmentalno antinociceptivno djelovanje spinalnoj razini, uz neizravnu aktivaciju endogenog opioidnog i GABA-ergičkog sustava. Ovi bi nalazi mogli biti važni za klinička ispitivanja potencijalno korisnih sinergističkih interakcija s konvencionalnim analgeticima i drugim lijekovima te usmjeriti klinička ispitivanja na nove indikacije i nove načine primjene, poput intratekalne.
Novel evidence suggests that the antinociceptive effect of botulinum toxin type A (BT-A) is of central origin. In this doctoral thesis, we verified this assumption in previously insufficiently investigated types of pain, investigated the interaction with central neurotransmitters as the possible mechanism of action and tried to determine the site of the toxin’s action within the central nervous system (CNS).
Male Wistar rats were used in experiments. We examined the effect of selective and nonselective opioid and GABAA antagonists, applied systemically, spinally and supraspinally, on antinociceptive effect of peripherally (subcutaneously into hind paw) applied BT-A in inflammatory, neuropathic, and bilateral pain. Neuronal and glial cells’ activation, proinflammatory cytokines’ and μ-opioid receptors mRNA expression, and Leu/Met-enkephalin protein expression were analyzed using immunofluorescence and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in lumbar spinal cord tissue. Further, we investigated the antinociceptive effect of BT-A following peripheral, spinal and supraspinal application. In parallel, enzymatic activity of BT-A in CNS tissue was examined using immunofluorescence.
Opioid and GABAA antagonists, applied systemically and intrathecally, dose-dependently abolished the antinociceptive effect of peripheral BT-A in all tested models, while no effect was observed following their supraspinal application. The effect of antagonists was short-lasting. BT-A reduced neuronal activation in dorsal horn, which was abolished by both, opioid and GABAA antagonist. BT-A diminished pain in, yet uninvestigated, models of visceral pain (peritonitis and colitis). BT-A’s bilateral antinociceptive action occurs after toxin’s presence on ipsilateral side only. BT-A reduced pain after peripheral and intrathecal application, but not after application in cisterna magna or cerebral ventricles, despite of its enzymatic activity in brain regions involved in nociception.
BT-A has segmental antinociceptive effect at the spinal level, which involves an indirect activation of endogenous opioid and GABA-ergic systems. These findings might guide clinical investigations of potentially useful additive or synergistic effects with conventional analgesics and direct clinical trials for novel indications and the routes of application, like intrathecal.
U fiziološkom odgovoru na stres i kontroli stresnog odgovora uglavnom sudjeluju dvije komponente neuroendokrinog sustava, simpatičko-medularna (SAM) os, koja regulira prvi odgovor na stres i os ...hipotalamus-adenohipofiza-kora nadbubrežne žlijezde (HPAC), čija se aktivacija uočava 15 do 20 minuta nakon stresnog podražaja. Cilj ovog istraživanja bilo je ispitati bazalnu, fiziološku aktivnost SAM i HPAC osi te njihovu povezanost s nizom kovarijabli (spol, tip škole, turnus pohađanja nastave, ritam spavanja i budnosti te demografska, akademska i društvena obilježja) u zdravih učenika koji pohađaju završne razrede srednjih škola. Specifični ciljevi uključivali su ispitivanje koncentracije kortizola i aktivnosti alfa-amilaze kao salivarnih biljega odgovora na stres te istraživanje njihove povezanosti sa subjektivnim procjenama razine stresa i sa strategijama suočavanja sa stresom, kao i s polimorfizmima jednog nukleotida (SNP) gena za mineralokortikoidni i glukokortikoidni receptor (rs5522, rs6189, rs6190) te za vezni protein 5 za FK506 (rs1360780). U istraživanje su bila uključena 903 maturanta gimnazija i strukovnih škola iz četiri najveća grada u Hrvatskoj (Zagreb, Split, Rijeka i Osijek). Uzorci sline prikupljeni kod kuće, tijekom jednog radnog dana u tri vremenske točke (po buđenju, 30 do 45 minuta nakon buđenja i neposredno prije lijeganja), uzeti su za analizu koncentracija kortizola dok se aktivnost alfa-amilaze mjerila samo u jednom uzorku, neposredno po buđenju. Kako bi se ispitao cirkadijalni ritam lučenja kortizola analizirani su i indeksi lučenja kortizola (CAR, DCD, AUCG). Učenice su imale statistički značajno veće koncentracije kortizola u jutarnjim satima, dok se koncentracije kortizola neposredno prije lijeganja nisu razlikovale među spolovima. Učenice imaju različite obrasce priprema za izazove koji ih očekuju tijekom nadolazećeg dana u odnosu na učenike, imaju značajno viši CAR, izraženiji DCD, veći AUCG u odnosu na učenike koji su imali statistički značajno veću aktivnost alfa-amilaze. Turnus pohađanja nastave, koji svakako posljedično utječe na ritam spavanja i trajanje budnosti, pokazao se kao prediktor lučenja kortizola. Statistički značajne razlike većine varijabli kortizola potvrđene su između učenika koji pohađaju različite turnuse nastave (SCC30-45, CAR, DCD i AUCG). Učenici koji su se budili ranije i imali duže trajanje budnosti imali su veći CAR, niži DCD i veći AUCG. Nisu potvrđene statistički značajne razlike niti jedne ispitivane varijable u učenika koji pohađaju različite tipove škola (gimnazije vs. strukovne škole). Također, statistički značajne razlike između učenika i učenica bile su uočene i u subjektivnoj procjeni stresa, suočavanju te njihovoj povezanosti s koncentracijama i indeksima kortizola. Zabrinutost za budućnost su oba spola procijenila kao najstresniju domenu života, dok su učenice imale statistički značajno veću razinu ukupnog stresa u odnosu na učenike te su više koristile aktivno suočavanje sa stresom. U ovom su istraživanju pronađene statistički značajne razlike povezanosti alela G polimorfizma rs 5522 s višom koncentracijom kortizola po buđenju (P=0,036), nižim CAR-om (P=0,021), dok je povezanost s DCD-om bila granična (P=0,050). Može se pretpostaviti kako učenici s dokazanim alelom G imaju niži CAR te zbog toga pokazuju lošiju pripremljenost za stresne izazove u nadolazećem danu, unatoč nešto izraženijim vrijednostima DCD-a. Ovo istraživanje ukazuje na potrebu definiranja protokola prikupljanja uzoraka sline u nekliničkim uvjetima i metodoloških smjernica za populacijska istraživanja, ponajprije zbog postojanja niza kovarijabli koje utječu na mjerenje koncentracije kortizola i aktivnosti alfa-amilaze. Dobiveni rezultati doprinose saznanjima o spolnim razlikama u funkciji HPAC osi, koje bi se trebale primijeniti i tijekom provedbe preventivnih mjera u adolescenata te ranijeg otkrivanja skupina koje su posebno osjetljive na stresne poticaje.
Physiological response to stress and the control of the stress response is mediated by two major components of the neuroendocrinological systems, sympathetic nervous (SAM) system and hypothalamic-pituitary-adrenal (HPA) axis. In the stress response, the SAM axis is first activated. About fifteen to twenty minutes following the SAM activation, the HPA axis is activated. The aim of the study was to examine basal, physiologically activity of the SAM and HPAC axis activity and to determine associations of various covariates (gender, school type, school shift, sleep-wake rhythm, demographic, academic and life style characteristics) with altered daily salivary cortisol profiles in healthy students attending finishing grades of secondary schools. Specific aims of the study were to analyse concentration of cortisol and activity of alpha- amylase, as salivary markers of stress reaction, to determine association of salivary markers of stress with stress perception and coping, and to determine associations of salivary markers of stress with single nucleotide polymorphisms (SNP) in the genes for the mineralocorticoid and glucocorticoid receptors (rs5522, rs6189, rs6190) and FK506 binding protein 5 (rs1360780). The study included 903 secondary school students enrolled in the finishing classes of gymnasiums and vocational schools from the four largest cities in Croatia (Zagreb, Split, Rijeka and Osijek). The saliva samples were collected at students’ homes, over the course of one weekday. Salivary cortisol was sampled at three time points: at awakening, 30 to 45 after wakening and at bedtime. Salivary alpha-amylase was sampled at awakening. In order to analyse the circadian rhythm of salivary cortisol secretion, three indexes were analysed (CAR, DCD and AUCG).Females had higher morning concentrations of salivary cortisol than males, whereas bedtime cortisol concentrations were not different in females and males. Females had different salivary cortisol profile and different mechanisms for preparing for the anticipated stress of the upcoming day, higher CAR, steeper DCD and larger AUCG than males. On the other side, males had higher activity of salivary alpha-amylase. School shift, and therefore the sleep-wake up rhythm, was an important predictor of the secretion of salivary cortisol. There were statistically significant differences in two school shifts with respect to the majority of salivary cortisol measures (SCC30-45, CAR, DCD and AUCG). Students who woke-up earlier and were longer awake had larger CAR, flatter DCD and larger AUCG. Students from two types of schools (gymnasiums vs. vocational schools) did not differ in any of the measured salivary variables. Furthermore, female and male students differed significantly in stress perception, coping and associations between stress perception, coping and salivary cortisol concentrations and indexes. Future was rated as the most stressful problem domain in both sexes. The perception of total stress was higher in females than males, and females used more active coping than males. Finally, we found statistically significant differences in associations between G allele of rs 5522 polymorphism and higher cortisol concentration at awakening (P=0.036), lower CAR (P=0.021) and higher DCD (P=0.050), but at borderline. It can be hypothesized that the G allele is associated with the lower CAR and therefore, decreased ability of preparing for the anticipated stress (despite borderline association with the steeper DCD). This study points to the need for defining the protocol for salivary sampling in non-clinical conditions and developing methodological guidelines for population-based research, primarily due to the number of co-variables that affect the concentration of salivary cortisol and the activity of salivary alpha-amylase. Obtained results contribute to better understanding of sex differences in the functioning of the HPA axis, which should be considered in the prevention of various psychopathological conditions in adolescence and used for earlier identification of vulnerable groups at high risk of stress-related health problems.
PDF
