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Quantitative analysis of faecal calprotectin using a new chemiluminescent method for the Bio-Flash analyzer (Inova Diagnostics)
No full textFekalni kalprotektin prepoznat je kao specifičan i vrlo osjetljiv biomarker upale crijevne sluznice koji omogućuje razlikovanje upalnih bolesti crijeva od sindroma iritabilnog crijeva. Dijagnostički testovi pojedinih proizvođača temeljeni su na različitim imunokemijskim metodama te još uvijek ne postoji standardiziran postupak ekstrakcije uzorka, pohrane i analize što dovodi do velike varijacije u dobivenim rezultatima i otežava njihovu međusobnu usporedbu.
U ovome radu opisano je provođenje postupka verifikacije kvantitativne kemiluminiscentne metode QUANTA Flash® Calprotectin (INOVA Diagnostics, San Diego, CA, USA) za određivanje koncentracije fekalnog kalprotektina u uzorku stolice na analizatoru Bio-Flash (Biokit S.A.©, Španjolska) i usporedbe s postojećim dijagnostičkim testom u rutinskoj primjeni Bühlmann fCAL® turbo (Bühlmann, Schönenbuch, Switzerland) na analizatoru Abbott Architect c8000 (Abbott S.r.l., Roma, Italy) u KBC-u Sestre Milosrdnice. Mjerenja su vršena na 59 ostatnih uzoraka stolica pacijenata kod kojih je zatraženo određivanje fekalnog kalprotektina. Nepreciznost ispitivane metode zadovoljila je zadane kriterije što ju čini prihvatljivom za kliničku primjenu. Unatoč statistički značajnom odstupanju vrijednosti dobivenih ispitivanom i postojećom metodom, podudaranje u kategorizaciji rezultata u dvije interpretativne kategorije pokazalo se snažnim. Zaključeno je da se ispitivana metoda može koristiti u rutinskom radu, ali je preporučljivo za istog pacijenta koncentraciju fekalnog kalprotektina uvijek mjeriti istim testom kako bi rezultati bili međusobno usporedivi jer rezultati dobiveni različitim imunokemijskim metodama nisu međusobno usporedivi i zamjenjivi.Fecal calprotectin was recognized as a specific and sensitive biomarker of intestinal inflammation, making it easier to differentiate inflammatory bowel diseases from irritable bowel syndrome. Numerous existing assays based on different immunochemical methods, along with the lack of standardization of sample extraction, storage, and analysis, have led to significant variation in assays results and exacerbated assay comparability.
This thesis describes the verification process of quantitative chemiluminescent method QUANTA Flash® Calprotectin assay (INOVA Diagnostics, San Diego, CA, USA) on analyzer Bio-Flash (Biokit S.A.ã, Spain) and comparison to Bühlmann fCAL® turbo assay (Bühlmann, Schönenbuch, Switzerland) on Abbott Architect c8000 (Abbott S.r.l., Roma, Italy) analyzer which is routinely used within the Department of Clinical Chemistry, University Hospital Centre ''Sestre Milosrdnice'', Zagreb. Samples used for measurement were obtained from remainders of 59 stool samples of patients for whom the clinicians requested the analysis of fecal calprotectin. Assay imprecision met the set criteria, which makes it acceptable for clinical use. Despite statistically significant bias of obtained values, the classification of results of both methods into two categories (positive and negative) proved that the agreement between the two methods is reliable. In conclusion, QUANTA Flash® Calprotectin assay can be used routinely. For follow-up of the same patient, it is advisable always to use the same diagnostic test since the results obtained by different methods are not comparable or interchangeable
Analysis of serum calprotectin and CRP concentration data in patients with COVID-19
No full textCOVID-19 je bolest uzrokovana virusom SARS-CoV-2. U organizam čovjeka virus ulazi preko dišnog sustava. Pneumociti opterećeni virusima oslobađaju različite citokine i upalne biljege, a posljedica je ozljeda pluća te difuzno oštećenje alveola koje na kraju može kulminirati sindromom akutnog respiratornog distresa. Kalprotektin je protein kojeg otpuštaju stimulirani neutrofili i monociti. Prilikom virusne ili bakterijske infekcije dišnog sustava povećava se ekspresija kalprotektina. CRP je protein akutne faze upale. Stimulacijom proupalnih citokina raste koncentracija CRP-a u plazmi.
Cilj ovog rada je bio analizirati rezultate serumskog kalprotektina i CRP-a dobivene imunoturbidimetrijskim metodama kako bi se uočile razlike ovisno o težini kliničke slike kod COVID-19 bolesnika, odnosno odsustvu bolesti COVID-19.
Korišteni su ostatni uzorci 143 ispitanika. Ispitanici su podijeljeni u četiri skupine ovisno o težini kliničkog stanja, stanju nakon preboljenja ili kao skupina zdravih dobrovoljaca. Koncentracije serumskog kalprotektina i CRP-a su određivane imunoturbidimetrijskim metodama na analitičkom sustavu Beckman Coulter AU 5800 (Beckman Coulter, Brea, SAD).
Osobe najveće životne dobi su bile u skupini bolesnika koji imaju najtežu kliničku sliku. Bolesnici s težim kliničkim slikama su imali visoke koncentracije serumskog kalprotektina i CRP-a, a ispitanici koji su preboljeli COVID-19 i ispitanici iz kontrolne skupine su imali niže koncentracije. Serumski kalprotektin je bio viši kod ispitanika koji su preboljeli COVID-19, nego u kontrolnoj skupini, iako ti ispitanici više nisu bili u akutnoj fazi upale. Koncentracije serumskog kalprotektina pokazale su značajnu korelaciju s CRP-om.
Veća distribucija težih oblika bolesti COVID-19 je kod osoba starije životne dobi. Koncentracije serumskog kalprotektina i CRP-a su visoke kod bolesnika s teškim kliničkim slikama. Serumski kalprotektin ostaje duže povišen kod ispitanika koji su preboljeli COVID-19, a više nisu u akutnoj fazi upale te time ne slijedi obrazac klasičnog biljega akutne faze kao što je CRP čija koncentracija nije povišena u stanjima bez akutne upale.COVID-19 is a disease caused by the SARS-CoV-2 virus. The virus enters the human body through the respiratory system. Virus-loaded pneumocytes release various cytokines and inflammatory markers, resulting in lung injury and diffuse alveolar damage that may eventually culminate in acute respiratory distress syndrome. Calprotectin is a protein released by stimulated neutrophils and monocytes. During viral or bacterial infection of the respiratory system, the expression of calprotectin increases. CRP is an acute phase reactant. Stimulation of proinflammatory cytokines increases the concentration of CRP in plasma.
The aim of this study was to analyze the results of serum calprotectin and CRP determined by immunoturbidimetric methods in order to detect differences depending on the severity of the clinical picture in COVID-19 patients or the absence of COVID-19 disease.
The remaining samples of 143 subjects were used. Subjects were divided into four groups depending on the severity of the clinical condition, the condition after recovery, or as a group of healthy volunteers. Serum calprotectin and CRP concentrations were determined by immunoturbidimetric methods on the Beckman Coulter AU 5800 analytical system (Beckman Coulter, Brea, USA).
The oldest persons were in the group of patients with the most severe clinical picture. Patients with more severe clinical pictures had high concentrations of serum calprotectin and CRP, while subjects who recovered from COVID-19 and subjects in the control group had lower concentrations. Serum calprotectin was higher in subjects who recovered from COVID-19 than in the control group, although these subjects were no longer in the acute phase of inflammation. Serum calprotectin concentrations showed a significant correlation with CRP.
There is a greater distribution of more severe forms of COVID-19 disease in the elderly. Serum calprotectin and CRP concentrations are high in patients with severe clinical pictures. Serum calprotectin remains elevated for a longer period in subjects who recovered from COVID-19 and are no longer in the acute phase of inflammation, thus not following the pattern of a classic acute phase marker such as CRP whose concentration is not elevated in conditions without acute inflammation
Medication errors in pediatric population reported to Agency for Medicinal Products and Medical Devices
No full textMedikacijske pogreške su nenamjerne greške u propisivanju, čuvanju, izdavanju, pripremi i primjeni
lijekova u kliničkoj praksi koje su uzrokovale ili imaju potencijal uzrokovati nuspojave. Pedijatrijska
populacija je posebno osjetljiva na takve greške s obzirom na nedostatak prikladnih farmaceutskih
formulacija za djecu, složenosti doziranja istih te s obzirom na različitu farmakokinetiku, farmakodinamiku,
djelotvornost i sigurnosni profil u odnosu na odrasle pacijenata. Cilj ovog rada bio je analizirati medikacijske
pogreške za sve mlađe od 18 godina prijavljene Agenciji za lijekove i medicinske proizvode do 31. prosinca
2021. godine te podignuti svijest o neispravnom korištenju lijekova. Zaprimljene prijave medikacijskih
pogrešaka analizirane su prema vrsti prijavitelja, dobi i spolu pacijenata, raspodjeli suspektnih lijekova prema
ATK klasifikaciji, raspodjeli nuspojava prema djelatnim tvarima, ozbiljnosti prijave i njihovom kriteriju
ozbiljnosti te prijavljenim medikacijskim pogreškama i nuspojavama. Dobiveni rezultati ovog diplomskog
rada nadopunit će postojeće informacije i dokaze o medikacijskim pogreškama u pedijatrijskoj populaciji te
doprinijeti smanjenju učestalosti istih.Medication error is unintended error in the prescribing, dispensing or administration of a medicinal
product in clinical practice that can cause or has potentional to cause harm. Pediatric population is
particularly vulnerable to such errors due to lack of suitable pharmaceutical formulations for children, the
complexity of dosing mediactions and different pharmacokinetics, pharmacodynamics, efficacy and safety
profile compared to adult patients. The aim of this study was to analyze medication errors for all under 18
years of age reported to the Agency for Medicinal Products and Medical Devices by 31 December 2021 and
to raise awareness about the misuse of medicines. The received reports of medication errors were analyzed
according to the type of applicant, age and sex of patients, distribution of suspected drugs according to ATK
classification, distribution of side effects according to suspected ingredient, seriousness of reports and their
seriousness criteria, and reported medication errors and side effects. The obtained results of this thesis
supplement the existing information and evidence of medication errors in the pediatric population and
contribute to reducing their frequency
Influence of glycosylation, synergistic ion and pH on iron binding to human serum transferrin
No full textU sklopu ovog diplomskog rada proučavan je utjecaj promjene glikozilacije transferina, sinergističkog aniona te pH pufera na konstantu vezanja željeza. Ispitivanja su se provodila u MES puferu pri pH 5,6 i temperaturi 25°C uz dodatak karbonata ili oksalata kao sinergijskih aniona. Korištene su tehnike UV-Vis spektrofotometrije i fluorescencije. Za izračun konstanti K1 i K2, koje opisuju nastanak monoferičnog i diferičnog transferina, korišten je program ReactLab™ EQUILIBRIA. Dobivene konstante i titracijske krivulje ukazuju na to da se prvo željezo veže s najvećim afinitetom na sijalizirani transferin u prisutnosti karbonata. Također, željezo se veže većim afinitetom na sijalizirani transferin s karbonatom kao sinergističkim anionom u odnosu na sijalizirani transferin s oksalatom. U prisutnosti oksalata kao sinergističkog aniona željezo se jače veže na desijalizirani transferin. Usporedbom konstanti ravnoteže pri pH 5,6 i pH 7,4 zaključujemo da se željezo jače veže na transferin pri fiziološkom pH. Promjenom glikozilacije, sinergističkog iona te pH, mijena se afinitet vezanja željeza na transferin, a time i koncentracija slobodnog i potencijalno toksičnog željeza u krvi.As part of this thesis, it was investigated how does the change in glycosylation pattern, synergistic anion and pH of a buffer effect iron binding constant. All experiments were carried out by UV-Vis and fluorescence spectroscopy in MES buffer, with carbonate or oxalate, at pH 7.4 and 25° C. The ReactLab ™ EQUILIBRIA program was used to calculate the constants K1 and K2, which describe the formation of monoferric and diferric transferrin. The obtained constants and titration curves indicate that the first iron binds with the highest affinity to sialylated transferrin with carbonate. Also, iron binds with higher affinity to sialylated transferrin with carbonate as a synergistic anion compared to sialylated transferrin with oxalate, and in the presence of oxalate as a synergistic anion, iron binds more strongly to desialized transferrin. By comparing the equilibrium constants at pH 5.6 and pH 7.4, we conclude that iron is more strongly bound to transferrin at physiological pH. By changing glycosylation, synergistic ion and pH, the binding affinity of iron to transferrin changes and thus the concentration of free, potentially toxic, iron in the blood
Kemijski sastav, biološka aktivnost i primjena smilja u medicini i farmaciji
No full textSmilje (Helichrysum italicum (Roth) G. Don) je mediteranska
biljka iz porodice Asteraceae koja se od davnina primjenjuje u tradicionalnoj
medicini zbog svojih ljekovitih i aromatičnih svojstava. Smilje sadrži širok spektar
različitih kemijskih spojeva, a njihova prisutnost i sadržaj u biljci ovise o
raznim okolišnim čimbenicima. U radu je dan pregled nekoliko studija koje su
ispitale sastav i količinu eteričnog ulja smilja i njegovu biološku aktivnost, kao i
biološku aktivnost različitih ekstrakata. Rezultati navedenih istraživanja potvrdili
su brojna pozitivna farmakološka djelovanja eteričnog ulja i organskih
ekstrakata smilja u modelima in vitro. Potvrda tih rezultata in vivo otvara mogućnost
uspješne i sigurne primjene preparata smilja u farmaceutskoj i kozmetičkoj
industriji
Phytochemical composition and antioxidant activity of leaf and fruit of Arbutus unedo L. (Ericaceae)
No full textOvim diplomskim radom analiziran je sastav te je ispitano antioksidacijsko djelovanje listova i
plodova biljke Arbutus unedo L., ubranih 2020.-te godine na području Istre. Identifikacija
fenolnih kiselina (ružmarinske i klorogenske), flavonoida (kvercitrina, izokvercitrina i rutina),
trjeslovina (galne kiseline), saponina te triterpenskih kiselina i fitosterola (stigmasterola i betasitosterola)
provedena je primjenom prikladne metode tankoslojne kromatografije (TLC). U
listu je utvrđena prisutnost ružmarinske kiseline, klorogenske kiseline, kvercitrina i galne
kiseline. U plodu je nađena ružmarinska kiselina i stigmasterol. Kvantifikacija fenolnih kiselina
(ružmarinske i klorogenske), flavonoida (izokvercitrozida), trjeslovina (pirogalola) i antocijana
(cijanidin-3-glukozid klorida) provedena je spektrofotometrijskim metodama. U listu je udio
fenolnih kiselina 1,99% (ružmarinska kiselina) i 3,85% (klorogenska kiselina),a u plodu 0,59%
(ružmarinska kiselina) i 1,20% (klorogenska kiselina). Sadržaj flavonoida izražen kao
izokvercitrozid u listu iznosi 0,79%, a u plodu 0,025%. Udio trjeslovina u listu iznosi 0,0036%,
a u plodu 0,0011%. U plodu je udio antocijana 0,1748%, dok u listu oni nisu utvrđeni.
Antioksidacijsko djelovanje definirano je spektrofotometrijskim metodama : određivanjem
sposobnosti hvatanja DPPH slobodnog radikala (list : IC50 = 3,38±0,17μg/mL, plod : IC50 =
25,91±2,42μg/mL), određivanjem sposobnosti hvatanja NO radikala (list : IC50 = 301,95±4,83
μg/mL) i određivanjem sposobnosti redukcije željeza 3+ u željezo 2+ (list : IC50 =
6,53±0,33μg/mL , plod : IC50 = 93,03±9,29μg/mL). Ovaj rad doprinosi utvrđivanju fitoterapijske
vrijednosti vrste Arbutus unedo L.In this graduation thesis were examined the composition and antioxidant activity of leaves and fruits
of the plant Arbutus unedo L., harvested in 2020 in the area of Istria. The identification of phenolic
acids (rosmarinic and chlorogenic), flavonoids (quercitrin, isoquercitrin and rutin), tannins (gallic
acid), saponins and triterpenic acids and phytosterols (stigmasterol and beta-sitosterol) was carried
out using a suitable thin-layer chromatography (TLC) method. Leaves show the presence of
rosmarinic acid, chlorogenic acid, quercitrin and gallic acid. In fruit were found rosmarinic acid and
stigmasterol. Quantification of phenolic acids (rosmarinic and chlorogenic), flavonoids
(isoquercitroside), tannins (pyrogallol) and anthocyanins (cyanidin-3-glucoside chloride) was
carried out by spectrophotometric methods. The amount of phenolic acids in leaf is 1.99%
(rosmarinic acid) and 3.85% (chlorogenic acid), and in fruit 0.59% (rosmarinic acid) and 1.20%
(chlorogenic acid). The content of flavonoids expressed as isoquercitroside in leaf is 0.79%, and in
fruit 0.025%. The portion of tannins in leaf is 0.0036%, and in fruit 0.0011%. The amount of
anthocyanins in fruit is 0.1748%, while in leaf they are not determined. The antioxidant activity was
defined by spectrophotometric methods: by determining the ability to capture DPPH free radical
(leaf : IC50 = 3.38±0.17μg/mL, fruit: IC50 = 25.91±2.42μg/mL), by determining the ability to
capture NO radical (leaf : IC50 = 301.95±4.83 μg/mL) and by determining the ability to reduce iron
3+ into iron 2+ (leaf : IC50 = 6.53±0.33μg/mL , fruit : IC50 = 93.03±9, 29 μg/mL). This work
contributes to the determination of phytotherapeutic values of Arbutus unedo L
Suitability assessment of selected cyclodextrins as solubilizers in fluticasone propionate in vitro permeation studies
No full textLoša topljivost flutikazonpropionata u vodi predstavlja veliki izazov u razvoju i karakterizaciji novih formulacija
lijeka. U in vitro ispitivanjima oslobađanja i permeabilnosti nužno je uspostaviti uvjete osigurane topljivosti. U odnosu
na organska suotapala i površinski aktivne tvari, ciklodekstrini pružaju prednost izrazite biokompatibilnosti sa staničnim
modelima, što ih čini prikladnim solubilizatorima za tu namjenu.
Cilj ovog rada bio je razviti metodu tekućinske kromatografije visoke djelotvornosti za pouzdano određivanje
sadržaja flutikazonpropionata u vodenim otopinama α- i hidroksipropil-α-ciklodekstrina, te potom ispitati učinak
ciklodekstrina na povećanje topljivosti lijeka pri uvjetima definiranim potrebama provođenja ispitivanja permeabilnosti
na Calu-3 stanicama.
U ovom diplomskom radu uspješno je razvijena metoda obrnutofazne razdjelne kromatografije za kvantitativnu
analizu flutikazonpropionata. Analiza je provedena uz izokratnu eluaciju pri stalnom omjeru acetonitrila, metanola i
fosfatnog pufera pH 3,5 u mobilnoj fazi (50:15:35, V/V/V). Volumen injektiranja bio je 50 μl, temperatura kolone 40⁰C,
a brzina protoka mobilne faze iznosila je 1 ml/min. Pri navedenim uvjetima, područje linearnosti metode potvrđeno je
za koncentracijski raspon lijeka od 1 μg/ml do 30 μg/ml (koeficijent korelacije 0,9998). Granice dokazivanja i
određivanja metode iznosile su 0,24 μg/ml te 0,74 μg/ml. Uz ovu metodu uspješno su analizirani uzorci ispitivanja fazne
topljivosti kompleksa flutikazonpropionata s α- te hidroksipropil-α-ciklodekstrinom provedenih pri 37⁰C u Hankovoj
otopini soli koja sadrži ione kalcija (pH 7,4). Primjenom α- te hidroksipropil-α-ciklodekstrina pri koncentracijama od
50 mM, povećana je topljivost flutikazonpropionata za 19,5, odnosno 27,3 puta u odnosu na intrinzičnu topljivost lijeka.
Uzevši u obzir strukturne značajke ciklodekstrina i lijeka te profil izotermi topljivosti, zaključeno je da je povećanje
topljivosti flutikazonpropionata posljedica uklapanja lijeka u agregate kompleksa lijek/ciklodekstrin i/ili samih
ciklodekstrina neinkluzijskim mehanizmima.
Temeljem dobivenih rezultata, procijenjeno je kako solubilizacijski učinak α- te hidroksipropil-α-ciklodekstrina nije
dostatan za primjenu u in vitro ispitivanju permeabilnosti flutikazonpropionata na staničnim modelima. Rezultati ovog
rada mogu poslužiti kao temelj za daljnja istraživanja s drugim vrstama ciklodekstrina koji bi mogli imati bolji
solubilizacijski učinak na flutikazonpropionat.Low aqueous solubility of fluticasone propionate poses a great challenge in new formulation development and
characterization. When conducting in vitro dissolution and permeation studies it is important to ensure that sink
conditions are met. Compared to organic cosolvents and surfactants, cyclodextrins offer the advantage of distinct
biocompatibility with cell lines, what makes them a suitable choice for solubilization purposes.
The aim of this study was to develop a reliable high performance liquid chromatography method for quantification
of fluticasone propionate in aqueous solutions containing α- and hydroxypropyl-α-cyclodextrin, as well as to investigate
the solubilization potential of these cyclodextrins at conditions defined by requirements of permeability tests on Calu-3
cell lines.
In this diploma thesis, a reverse-phase chromatographic method for quantitative analysis of fluticasone propionate
was successfully developed. The analysis was carried out with isocratic flow, at constant acetonitrile, methanol and
phosphate buffer pH 3.5 ratio within the mobile phase (50:15:35, V/V/V). The injection volume was 50 μl, column
temperature 40⁰C and flow rate 1 ml/min. At these conditions, the linearity range was confirmed within drug
concentration range of 1 μg/ml to 30 μg/ml (correlation coefficient 0.9998). The limit of detection and quantification
were found to be 0.24 μg/ml and 0.74 μg/ml, respectively. Using this method, samples obtained in phase solubility
studies of fluticasone propionate with α- and hydroxypropyl-α-cyclodextrin carried out at 37⁰C in Hank's balanced salt
solution containing calcium ions (pH 7.4), were successfully analysed. Using α- and hydroxypropyl-α-cyclodextrin at
50 mM concentration, the intrinsic solubility of fluticasone propionate was increased by 19.5 and 27.3 times,
respectively. Considering the structural characteristics of cyclodextrins and the drug, as well as the obtained phase
solubility diagram profiles, it was concluded that the enhancement in fluticasone propionate's solubility was the product
of its entrapment inside cyclodextrin and/or drug/cyclodextrin complex aggregates, through non-inclusion mechanisms.
The results obtained showed that the solubilization effect of α- and hydroxypropyl-α-cyclodextrin was not sufficient
enough to be applied for in vitro permeation studies of fluticasone propionate using cell models. The results of this study
could serve as a basis for further research of other cyclodextrins with better solubilization effect on fluticasone
propionate
Biomarkers of multiple sclerosis
No full textMultipla skleroza (engl. multiple sclerosis, MS) je kronična upalna demijelinizacijska bolest
središnjeg živčanog sustava (SŽS). Uzrok MS je nepoznat, no smatra se da je MS multikomponentna
bolest do koje dolazi prisutnošću genske predispozicije i autoimunosnim odgovorom koji je potaknut
infekcijom. Razvoj novih metoda i biljega omogućuje uvid u dosad nepoznatu patogenezu bolesti.
Standardi za dijagnostiku MS su oligoklonalne vrpce, IgG indeks i MRZ reakcija u kliničkoj praksi.
Prognostički biomarkeri za MS mogu pružiti informacije o tijeku aktivnosti bolesti i ukazati na
konverziju u drugi oblik MS (detekcija oligoklonalnih vrpca IgG u likvoru, detekcija oligoklonalnih
vrpca IgM u likvoru, CHI3L1, NF, N-CAM, MMP). Biomarkeri terapije (CXCL13, NFL, vitamin
D, BDNF) u postupku su istraživanja i implementacije. U istraživanjima je pokazana mogućnost
primjene novih biomarkera, cfDNA i miRNA, prilikom dijagnoze, praćenja tijeka bolesti i terapije
MS koji bi kao takvi bili neinvazivni biomarkeri. Razvoj metode sekvenciranja nove generacije,
omogućio je otkrivanje novih čimbenika uključenih u patogenezi MS, potencijalnih biomarkera gena
i novih meta za razvoj lijekova.Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous
system (CNS). The cause of MS is unknown, but MS is thought to be a multicomponent disease
caused by the presence of a genetic predisposition and an autoimmune response triggered by
infection. The development of new methods and markers provides insight into the unknown
pathogenesis of the disease. Standards for the diagnosis of MS are oligoclonal bands, IgG index
and MRZ reactions in clinical practice. Prognostic biomarkers for MS can provide information
on the course of disease activity and indicate conversion to another form of MS (detection of
oligoclonal IgG bands in cerebrospinal fluid, detection of oligoclonal bands IgM in cerebrospinal
fluid, CHI3L1, NF, N-CAM, MMP). Biomarkers of therapy (CXCL13, NFL, vitamin D, BDNF)
are in the process of research and implementation. Research has shown the possibility of using
new biomarkers, cfDNA and miRNA, in the diagnosis, monitoring of the course of the disease
and therapy of MS, which as such would be non-invasive biomarkers. The development of a new
generation sequencing method has made it possible to discover new factors involved in the
pathogenesis of MS, potential gene biomarkers and new targets for drug development
Farmakogenomička testiranja kandidata za transplantaciju bubrega i terapijski ishodi
No full textCilj istraživanja: Cilj ovoga rada jest procijeniti postoji li i koliki je potencijal farmakogenetike u području
optimizacije imunosupresivne terapije u transplantaciji bubrega. Nadalje, cilj je i procijeniti ulogu
farmakogenetike u ovom kliničkom području. Hipoteza je da genetičko nasljeđe može značajno izmijeniti
međusobne interakcije imunosupresivnih lijekova i organizma.
Materijali i metode: Učinjen je pregled dostupne relevantne literature, a posebna je pozornost pri tome
bila posvećena istraživanju udžbeničke literature i stručnih smjernica. Potom je slijedilo pretraživanje
bibliografskih baza podataka (PubMed) i baza podataka sintetiziranog znanja (PharmGKB), koje je
uključivalo sljedeće ključne pojmove u različitim kombinacijama: farmakogenomika, imunosupresivni
lijekovi, transplantacija bubrega, polimorfizam, mikofenolat, CNI, ciklosporin, takrolimus, mTOR
inhibitor, sirolimus, everolimus, CYP3A4, CYP3A5, farmakodinamika, prijenosnici. U obzir je uzimana
literatura objavljena u razdoblju od 2010. do 2020., uz manje iznimke vezane za udžbenike i pojedina
pionirska istraživanja.
Rezultati: Pregledana su značajnija istraživanja u kojima je metodologija temeljena na kombiniranju
informacije o dozi lijeka s podacima dobivenima genotipizacijom primatelja bubrežnog presatka za
polimorfizme pojedinačnih nukleotida (engl. Single-Nucleotide Polymorphism, SNP) gena koji kodiraju
metaboličke enzime i prijenosnike imunosupresivnih lijekova, kao i pregledni radovi koji se na takva i
slična istraživanja referiraju. Pronađeno je kako smjernice konzorcija za implementaciju kliničke
farmakogenetike (engl. Clinical Pharmacogenetics Implementation Consortium, CPIC) otvaraju
mogućnost da genotipom gena natporodice citokroma P450, porodica 3, potporodica A, člana 5 (engl.
Cytochrome P450 Family 3 Subfamily A Member 5, CYP3A5) usmjeravano doziranje takrolimusa poboljša
kliničke ishode jer se njime može brže nakon transplantacije dostići ciljne koncentracije takrolimusa u
krvi, čak i kada se primjenjuje titracija terapijskim praćenjem lijeka (engl. Therapeutic Drug Monitoring,
TDM). Brže postizanje ciljnih koncentracija može potencijalno umanjiti rizik odbacivanja presatka uslijed
premale ili toksičnost uslijed prevelike izloženosti lijeku. U smjernicama se oprezno pristupa ideji
zamjenjivanja TDM-a genotipizacijom CYP3A5, zbog toga što drugi faktori (poput demografskih,
interakcija lijek-lijek, genetičkih varijacija koje bi utjecale na farmakodinamiku) također mogu utjecati na
doziranje takrolimusa. Takrolimus je jedini takav imunosupresiv, ukoliko zanemarimo smjernice za
doziranje azatioprina (prema genotipu tiopurin metiltransferaze (engl. thiopurine S-methyltransferase,
TPMT)) čija je primjena u transplantaciji već dugi niz godina zanemariva. Daleko je manje
farmakogenetički istraživana farmakodinamika imunosupresiva primjenjivanih u transplantaciji bubrega,
a po pitanju jedinog farmakodinamičkog polimorfizma koji je značajnije istraživan, onoga gena koji
kodira enzim inozin-5'-monofosfat-dehidrogenazu (engl. inosine-5'-monophosphate dehydrogenase,
IMPDH), nije postignut širi konsenzus po pitanju odabira farmakogenetičkog biljega i njegove kliničke
primjene.
Zaključak: Trenutno postoje ograničenja za rutinsku kliničku primjenu farmakogenomike u odabiru
imunosupresivnog liječenja za bolesnike kandidate za transplantaciju bubrega. Izazov u
farmakogenomici imunosupresivnih lijekova nepostojanje je dostatnih kliničkih dokaza o prednosti
provedbe doziranja na temelju testova genotipizacije. Da bi se znanstvene spoznaje o važnosti
polimorfizama primijenile u individualizaciji terapije nužno je provesti dodatne kliničke studije o utjecaju
farmakogenetike na preživljenje presatka ili na opće stanje bolesnika. Objašnjenje za poziciju koju
farmakogenomika trenutno zauzima u području imunosupresivne terapije u transplantaciji bubrega,
djelomično može biti u tomu da se većina najčešće upotrebljavanih imunosupresiva metabolizira putem
enzima natporodice citokroma P450, porodica 3, potporodica A, član 4 (engl. Cytochrome P450 Family 3
Subfamily A Member 4, CYP3A4) čija je ekspresija neovisno o genotipu vrlo varijabilna inter- i
intraindividualno te je složenost predviđanja farmakokinetike neminovna. Na to, između ostalog, utječu
lijek-lijek interakcije, varijacije u genima koji utječu na aktivnost CYP3A te epigenski faktori.Objectives: The aim of this thesis is to assess whether there is, and to what extent, a potential for
pharmacogenetics in the field of immunosuppressive therapy optimization in kidney transplant
recipients. The aim is also to determine what role pharmacogenetics currently plays in this area. The
hypothesis is that genetic inheritance can significantly alter the interactions between
immunosuppressive drugs and the organism.
Materials and methods: A review of the available relevant literature was made, and special attention
was paid to the research of textbook literature and professional guidelines. This was followed by a
search of bibliographic databases (PubMed) and synthesized knowledge databases (PharmGKB), which
included the following key terms in different combinations: pharmacogenomics, immunosuppressive
drugs, kidney transplantation, polymorphism, mycophenolate, CNI, cyclosporine, tacrolimus, mTOR
inhibitor, sirolimus, everolimus, CYP3A4, CYP3A5, pharmacodynamics, transporters. The literature
published in the period from 2010 to 2020 was taken into account, with minor exceptions related to
textbooks and individual pioneering research.
Results: Significant studies in which the methodology is based on combining drug dose information with
data obtained by renal graft recipients genotyping for Single-Nucleotide Polymorphisms (SNPs) in genes
encoding metabolic enzymes and transporters of immunosuppressive drugs have been reviewed, as well
as review papers referring to such and similar studies. Clinical Pharmacogenetics Implementation
Consortium (CPIC) guidelines have been found to open the possibility that Cytochrome P450 Family 3
Subfamily A Member 5, CYP3A5 genotype-directed dosing of tacrolimus improves clinical outcomes
because it could help reach the target tacrolimus blood concentrations faster after transplantation, even
when therapeutic drug monitoring (TDM) titration is applied. Achieving target concentrations faster can
potentially reduce the risk of graft rejection due to too low or toxicity due to too high drug exposure.
The guidelines cautiously approach the idea of replacing TDM with CYP3A5 genotyping, because other
factors (such as demographics, drug-drug interactions, genetic variations that would affect the
pharmacodynamics) may also affect tacrolimus dosing. Tacrolimus is the only immunosuppressive drug
for which guidelines on dosing according to genotype exist, disregarding the guidelines for dosing of
azathioprine (according to thiopurine S-methyltransferase (TPMT) genotype), use of which in
transplantation has been negligible for many years. The pharmacodynamics of immunosuppressants
used in kidney transplantation is far less researched, and for the only pharmacodynamic polymorphism
that has been significantly researched, the inosine-5'-monophosphate dehydrogenase gene (IMPDH), a
consensus on the choice of pharmacogenetic marker and its clinical application has yet to be reached.
Conclusion: Currently, significant limitations exist for the routine application of pharmacogenomics in
the selection of immunosuppressive treatment for kidney transplant recipients. A challenge in the
pharmacogenomics of immunosuppressive drugs is the lack of sufficient clinical evidence on the benefit
of dosing based on genotyping tests. In order to apply scientific knowledge about the importance of
polymorphisms in therapy individualization of kidney transplant candidates, it is necessary to conduct
additional clinical studies on the impact of pharmacogenetics on graft survival or general patient
condition. The explanation for the current position of pharmacogenomics in the field of
immunosuppressive therapy in renal transplantation may be partly consequence of the fact that most
commonly used immunosuppressants are metabolized by the enzyme Cytochrome P450 Family 3
Subfamily A Member 4, CYP3A4, since expression of this enzyme is, regardless of genotype, highly
variable inter- and intraindividually. This is influenced, among other things, by drug-drug interactions,
variations in genes that affect CYP3A activity, and epigenetic factor
Macroevolutionary history of glycosylation and biostatistical analysis of environmental effects on N-glycosylation of immunoglobulin G
No full textGlikozilacija je prisutna u svim živućim organizmima – od bakterija i arheja pa sve do čovjeka. Struktura
glikana nije izravno zapisana u genomu nego je rezultat precizno kontrolirane aktivnosti proteina zaduženih
za biosintezu glikana. Strukturne razlike glikana između vrsta su odgovor na selektivni pritisak, a svaka
promjena koja je postala konzervirana u evoluciji nosi važnu ulogu. S druge strane, uočena je varijabilnost
glikana unutar organizama jedne vrste što je dijelom rezultat okolišnih čimbenika i stanja pojedine jedinke.
Cilj ovog rada je odrediti makroevolucijske trendove glikozilacije i analizirati utjecaj okolišnih čimbenika
na N-glikom imunoglobulina G (IgG). Genomskom je filostratigrafijom određena starost gena koji kodiraju
za glikoproteine i gena koji kodiraju za proteine koji sudjeluju u glikozilaciji. Dobiveni rezultati su pokazali
da proteini koji sudjeluju u glikozilaciji vuku korijene još iz zajedničkog pretka svih staničnih organizama,
uz pojedine inovacije u zajedničkom pretku svih eukariota, te manje u zajedničkom pretku pravih životinja.
Unatoč starosti, glikozilacija u svoje zlatno doba ulazi razvojem višestaničnih životinja s ciljem razvoja
međustanične komunikacije i interakcije stanica s okruženjem kada u većoj mjeri nastaju novi geni koji
kodiraju za polipeptidne lance glikoproteina. S ciljem procjene utjecaja raznih čimbenika i stanja analizirana
je povezanost podataka dostupnih u biobazi „10001 Dalmatinac“ s različitim deriviranim svojstvima Nglikoma
IgG-a kao najproučavanijeg glikoproteina. Rezultati dobiveni analizom preko 3500 ljudi su pokazali
da postoji utjecaj prehrane (učestalost konzumacije voćnih derivata i slatke hrane), hipertenzije, artritisa,
regularnosti menstrualnog ciklusa, debljine, krvnog tlaka, koncentracije gliciranog hemoglobina, fibrinogena
i inzulina na sintezu agalaktoziliranih glikana IgG-a. Zatim, digalaktozilacija IgG-a je povezana s
hipertenzijom, artritisom, redovitošću menstrualnog ciklusa, debljinom, krvnim tlakom, mineralnom
gustoćom kostiju, prehranom (voćni derivati, slatka hrana), koncentracijama urata, triglicerida, fibrinogena,
inzulina, kolesterola i HDL kolesterola. Sijalinizacija IgG-a je povezana s regularnošću menstrualnog
ciklusa, debljinom, mineralnom gustoćom kostiju, krvnim tlakom, koncentracijama triglicerida i inzulina.
Udio struktura s račvajućim N-acetilglukozamin u N-glikomu IgG-a ovisi o mineralnoj gustoći kostiju,
redovitošću menstrualnog ciklusa, pušenju, te koncentracijama triglicerida i kolesterola.Glycosylation is present in all living organisms - from bacteria and archaea to humans. The structure of
glycans is not directly written in the genome but is the result of the precisely controlled activity of proteins
responsible for glycan biosynthesis. The structural differences of glycans between species are a response to
selective pressure, and any change that has become conserved in evolution plays an important role. On the
other hand, variability of glycans within the organism of one species was observed, which is partly the result
of environmental factors and the state of individual individuals.
This thesis aims to determine the macroevolutionary trends of glycosylation and to analyse the influence of
environmental factors on the N-glycome of immunoglobulin G.
Genomic phylostratigraphy determined the evolutionary age of genes encoding glycoproteins and genes
encoding proteins involved in glycosylation eukaryotes. Obtained results pointed out that the genes involved
in glycosylation which we find today in humans, were present in all cellular organisms, with some
innovations in the last common ancestor of all eukaryotes and less in the common ancestor of eumetazoans.
Despite its ancient evolutionary age, glycosylation in its golden era enters with the development of
multicellular animals when new genes encode polypeptide glycoprotein chains are increasingly emerging,
probably intending to develop tools for cell-cell and cell-environment interaction.
To assess the influence of various factors and conditions, the correlation of data available in the 10001
Dalmatian database with different derived properties of N-glycome immunoglobulin G (IgG), as the most
studied glycoprotein, was analysed. The results obtained from the analysis of over 3500 people showed that
there is an impact of diet (frequency of consumption of fruit derivatives and sugary foods), hypertension,
arthritis, menstrual regularity, obesity, blood pressure and glycated haemoglobin, fibrinogen, and insulin
concentration on IgG agalactosylation. Then, IgG digalactosylation is associated with hypertension, arthritis,
menstrual regularity, obesity, blood pressure, bone mineral density, diet (fruit derivatives, sugary foods),
urate, triglyceride, fibrinogen, insulin, cholesterol, and HDL cholesterol levels. Sialylation of IgG is
associated with menstrual cycle regularity, obesity, bone mineral density, blood pressure, triglyceride, and
insulin concentrations. The abundance of structures with branching N-acetylglucosamine in N-glycome IgG
depends on bone mineral density, regularity of the menstrual cycle, smoking, and the concentration of
triglycerides and cholesterol