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To elucidate the role of metallothioneins (MTs) in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MTs I+II and the tissue concentrations of Zn2+ and Cu2+ in the brain, spinal cord (SC) and in the liver during the periods of attacks and remissions in chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE). Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control rats were treated with CFA. The data, obtained by clinical assessment, immunohistochemistry and inductivity coupled plasma spectrometry, have shown that during the first attack (on the 12th day) MTs I+II were markedly upregulated in subarachnoid regions and perivascular space on astrocytes, microglia and on spinal neurons. Simultaneously, the concentrations of zinc in the SC and zinc and copper in the liver have found to be increased. During the second attack (on the 22nd day) a new overexpression of MTs was found in the cerebellum, in sulcus hippocampi, in spinal neurons and particularly in hepatocytes around the central vein. Concomitantly, in the brain and SC the concentration of copper increased. The data point to a neuroprotective role of MTs and to an important regulatory role of essential metals and hepatic MTs in the pathogenesis of CR-EAE

RATs provide targeted and site-specific pain relief, effectively managing acute pain in the surgical area. Those techniques can significantly reduce the need for systemic opioids, minimizing the risk of opioid-related side effects and complications. By controlling pain and minimizing opioid use, therefor minimizing the side effects like respiratory depression, sedation, nausea, and constipation, regional anesthesia contributes to enhanced postoperative recovery, shorter hospital stays, and faster return to normal activities. Effective pain management through RATs improves patient comfort, satisfaction, and overall experience during the perioperative period. Adequate pain control with regional anesthesia may decrease the risk of developing chronic pain conditions after surgery, improving long-term patient outcomes. Regional anesthesia allows for tailored pain management based on individual patient factors, such as surgical site, comorbidities, and patient preferences. The combination with other analgesic modalities, such as non-opioid medications, provides a comprehensive pain relief and optimizes patient outcomes. Effective pain control with regional anesthesia can lead to cost savings by reducing the length of hospital stays, postoperative complications, and the need for additional pain management interventions.

Dugotrajna izloženost niskim dozama mikotoksina predstavlja rizik za pojavu bubrežnih oboljenja. U javnozdravstvenom smislu najznačajniji su mikotoksini koje proizvode plijesni rodova Aspergillus, Penicillium i Fusarium, koji kontaminiraju namirnice za prehranu ljudi i životinja te uzrokuju pojavu mikotoksikoza. Ulazak mikotoksina u epitelne stanice proksimalnih kanalića (PK) bubrega posredovan je membranskim prijenosnicima organskih aniona (Oat), a nakupljanje mikotoksina u PK preduvjet je njihova toksičnog učinka. Kako su namirnice najčešće kontaminirane različitim mikotoksinima, cilj ovog rada je istražiti pojedinačne i združene učinke okratoksina A (OTA) i citrinina (CIT) nakon 21-dnevnog tretmana te mogući protektivni/reparatorni učinak resveratrola (RSV) na ekspresiju housekeeping (rNa/K-ATPaze i r-aktina) i Oat proteina (rOat1, rOat2, rOat3 i rOat5) u bubrezima štakora metodama western i imunocitokemijske analize. OTA (0,125 mg/kg tj.m. ili 0,250 mg/kg tj.m.) je u ovisnosti o dozi smanjio ekspresiju rOat1, ali ne rOat3 i rOat5 proteina, dok se ekspresija proteina rOat2 smanjila višom dozom OTA (0,250 mg/kg tj.m). Tretman CIT (20 mg/kg tj.m.) je smanjio je ekspresiju rOat2 i rOat5, ali ne rOat1 i rOat3 proteina. Smjesa mikotoksina (0,250 mg/kg tj.m. OTA+20 mg/kg tj.m. CIT) smanjila je ekspresiju rOat1, rOat2 i rOat5, a povećala ekspresiju rOat3 proteina. Tretman OTA ili CIT, kao i njihovom smjesom nisu mijenjali ekspresiju housekeeping proteina, izuzev smjese mikotoksina s višom dozom OTA koja je smanjila ekspresiju r-aktina. Tretman RSV (0,250 mg/kg tj.m. OTA+20 mg/kg CIT+20 mg/kg tj.m. RSV) nije umanjio nefrotoksične učinke mikotoksina. Dobiveni podatci ukazuju na važnost interakcija mikotoksina u regulaciji ekspresije proteina rOat u bubrezima štakora., Prolonged exposure to low doses of mycotoxins poses a risk for kidney disease. The most important mycotoxins which contaminate foodstuffs for human and animal consumption and cause mycotoxicosis are produced by molds of the genera Aspergillus, Penicillium and Fusarium. The entry of mycotoxins into the renal proximal tubules (PT) epithelial cells is mediated by organic anions transporters (Oat), and the accumulation of mycotoxins in the PT is a prerequisite for their toxic effect. The aim of this study was to investigate the individual and combined effects of ochratoxin A (OTA) and citrinin (CIT) after 21 day exposure and the possible protective/reparative effect of resveratrol (RSV) on expression pattern of housekeeping (rNa / K- ATPase and rβ-actin) and Oat protein (rOat1, rOat2, rOat3 and rOat5) in rat kidneys by western and immunocytochemical analysis. OTA (0.125 mg/kg bw or 0.250 mg/kg bw) reduced the expression of rOat1 but not rOat3 and rOat5 protein, depending on the dose, while the expression of rOat2 protein decreased with a higher dose of OTA (0.250 mg/kg bw). CIT treatment (2 mg/kg bw) reduced the expression of rOat2 and rOat5, but not rOat1 and rOat3 proteins. A mixture of mycotoxins (0.250 mg/kg bw OTA + 2 mg/kg bw CIT) decreased rOat1, rOat2 and rOat5 expression and increased rOat3 protein expression. Treatment with OTA or CIT, as well as their mixture, did not alter the expression of housekeeping protein, except for a mixture of mycotoxins with a higher dose of OTA that reduced rβ-actin expression. RSV treatment (0.250 mg/kg bw OTA + 2 mg/kg CIT + 20 mg/kg bw RSV) did not reduce the nephrotoxic effects of mycotoxins. The data obtained indicate the importance of mycotoxin interactions in the regulation of rOat protein expression in rat kidneys.

Endemska nefropatija kronična je tubulointersticijska bolest s latentnim početkom i sporim napredovanjem do završnog stadija bubrežne bolesti. Pogađa podjednako muškarce i žene koji žive u određenim endemskim ruralnim mjestima smještenih oko pritoka Dunava u Rumunjskoj, Bugarskoj, Hrvatskoj, Bosni i Hercegovini, Kosovu i Srbiji. Procjenjuje se da je gotovo sto tisuća ljudi u opasnosti za razvoj bolesti dok trenutno ima više od dvadeset pet tisuća oboljelih, a prevalencija iznosi oko 2 – 5 %. Prirodni tijek bolesti karakteriziraju dugo razdoblje inkubacije, nespecifični i teško prepoznatljivi klinički znakovi i simptomi te visoka incidencija za razvoj zloćudnog tumora prijelaznog epitela gornjeg dijela mokraćnog sustava s učestalosti čak do 50 %. Naglašeno obilježje obuhvaća obiteljski obrazac u združenim kućanstvima u kojim nekoliko članova istog domaćinstva, koji nisu nužno u krvnom srodstvu, mogu razviti bolest. Tijekom godina predložen je veliki broj potencijalnih etioloških čimbenika od kojih je većina bila opovrgnuta. Glavni okolišni uzročnik nefropatije i urotelijalnog karcinoma je kronično trovanje aristolohičnom kiselinom koja se nalazi u sjemenkama biljke vučje stope (Aristolochia clematitis L.). Specifični biomarkeri koji služe za procjenu izloženosti kancerogenoj i nefrotoksičnoj aristoholičnoj kiselini su aristolaktamski DNA adukti detektirani u bubrežnom tkivu i karcinomima urinarnog trakta. Također induciraju specifične mutacije u tumor supresorkom genu p53 koji će dalje utjecati na faze kancerogeneze. Genetska predispozicija ugroženih pojedinaca ima sekundarnu ulogu kao endogeni etiološki čimbenik. Danas postoje jednostavne mjere za kontroliranje i smanjenje rasta vučje stope s ciljem sprječavanja ili eliminiranja kontaminacije aristolohične kiseline., Endemic nephropathy is a chronic tubulointerstitial disease with a latent onset and slow progression to end-stage renal disease. It affects both men and women living in certain endemic rural areas located around the tributaries of the Danube in Romania, Bulgaria, Croatia, Bosnia and Herzegovina, Kosovo and Serbia. It is estimated that almost one hundred thousand people are at risk of developing the disease, while there are currently more than twenty-five thousand patients, and the prevalence is about 2-5 %. The natural course of the disease is characterized by a long incubation period, nonspecific and difficult to recognize clinical signs and symptoms, and a high incidence of the development of a malignant tumor of the transitional epithelium of the upper part of the urinary tract with a frequency of up to 50 %. The highlighted feature includes a family pattern in conjointed households in which several members of the same household who are not necessarily related by blood can develop the disease. Over the years, a large number of potential etiological factors have been proposed, most of which have been disproved. The main environmental cause of nephropathy and urothelial carcinoma is chronic poisoning with aristolochic acid, which is found in the seeds of the birthwort plant (Aristolochia clematitis L.). Specific biomarkers used to assess exposure to carcinogenic and nephrotoxic aristocholic acid are aristolactam DNA adducts detected in kidney tissue and urinary tract cancers. They also induce specific mutations in the p53 tumor suppressor gene that will further affect the stages of carcinogenesis. The genetic predisposition of vulnerable individuals has a secondary role as an endogenous etiological factor. Today, there are simple measures to control and reduce the growth of birthwort with the aim of preventing or eliminating aristolochic acid contamination.

Multipla skleroza (MS) je autoimuna bolest koja uzrokuje demijelinizaciju aksona središnjeg živčanog sustava (SŽS). Na razvoj bolesti utječu različiti genetički i okolišni čimbenici, stoga MS spada u multifaktorijalne složene bolesti. Autoreaktivne stanice imunosnog sustava uzrokuju autoimuni odgovor u SŽS-u koji rezultira razaranjem mijelinske ovojnice te je prijenos akcijskog potencijala niz neuron usporen. Uz demijelinizaciju, glavne značajke bolesti su i neurodegeneracija aksona koja nastupa u kasnijim fazama bolesti, glioza te upala u SŽS-u. Simptomi bolesti uključuju gubitak motoričkih, senzoričkih i autonomnih funkcija. Bolest se po intenzitetu simptoma i njihovom razvoju u vremenu dijeli na: klinički izolirani sindrom (CIS), relapsno-remitirajuću multiplu sklerozu (RRMS), primarno-progresivnu multiplu sklerozu (PPMS) i sekundarno-progresivnu multiplu sklerozu (SPMS). RRMS je najčešći oblik MS-a. Za istraživanje složene bolesti poput MS-a potrebni su odgovarajući in vivo animalni modeli. Glavni animalni model istraživanja MS-a je eksperimentalni autoimuni encefalomijelitis (EAE). Kao i MS, karakterizira ga autoimuni odgovor te demijelinizacija i oštećenja aksona u SŽS-u. Najčešće životinje u kojima je induciran EAE jesu miševi i štakori. Postoji nekoliko različitih metoda indukcije EAE-a, no najčešće su aktivna i pasivna imunizacija životinja. Za razliku od CD8+ citotoksičnih T limfocita u MS-u, glavni nositelji patogeneze EAE-a su CD4+ pomagački T limfociti. To čini EAE model vrlo korisnim u istraživanju njihovih efektorskih funkcija te regulacijskih T limfocita. Nadalje, EAE modeli važni su u detekciji potencijalnih antigena koji sudjeluju u autoimunome odgovoru te mehanizmima kojima dolazi do upale i demijelinizacije. Najveći nedostaci EAE-a u istraživanju MS-a su lokalizacija lezija u kralježničnoj moždini, za razliku od dominantnih moždanih lezija u pacijentima koji boluju od MS-a. te nedostatak progresivnosti simptoma. EAE je glavni animalni model MS-a koji je zaslužan za dobivanje velike većina saznanja o patofioziologiji bolesti te razvoju terapije za pacijente koji boluju od MS-a., Multiple sclerosis (MS) is an autoimmune disease, which causes demyelination of axons in central nervous system (CNS). Many different genetic and enviromental factors affect disease develompent, hence MS belongs to multifactorial, complex diseases. Autoreactive immune cells cause autoimmune response in CNS which results in myelin sheath destruction and this slowes down action potential conduction along neurons. Beside demyelination, another elements of MS are neuroaxonal degeneration which occurs in later stages of the disease, gliosis and CNS inflammation. Disease symptoms include loss of motor, sensory and autonomic functions. Disease is divided, by its symptoms intensity and development through the time, to: clinically isolated syndrome (CIS), relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS) and secondary progressive multiple sclerosis (SPMS). RRMS is most common type of MS. For the research of complex diseases, like MS, animal in vivo models are required. The main animal model for MS research is experimental autoimmune encephalomyelitis (EAE). Just like MS, it is characterised by autoimmune response, demyelination and axonal damage in CNS. Most used animals in EAE research are mice and rats. There are several different methods of EAE induction, but the most common are active and passive immunisation of animals. Unlike CD8+ cytotoxic T lymphocytes in MS, main carriers of the EAE pathogenesis are CD4+ T helper lymphocytes. That makes EAE very useful in their effector functions and regulatory T cells research. Furthermore, EAE models are important in detection of potential antigens, which participate in autoimmune response and mechanisms which induce inflammation, and demyelination. Main disadvantages of EAE as a model of MS research are localization of lesions in spinal cord, unlike dominant brain lesions in MS patients, and lack of symptoms progression. EAE is the main animal model of MS which is responisble for the vast majority of pathophysiologic knowledge about the disease and for development of MS therapy.

Jetra je najveći i najsloženiji organ u ljudskom tijelu. Nalazi se u gornjem desnom dijelu trbušne šupljine. Jetra je organ sa velikim brojem vitalno važnih funkcija, te ima ulogu u detoksikaciji organizma. Ljudi su svakodnevno izloženi različitim ksenobioticima, a nalaze se u zraku, tlu, vodi, biljkama i životinjama. Pojam ksenobiotik podrazumijeva svaku tvar koja je „strana“ tijelu. Ksenobiotici su prisutni i u hrani koju svakodnevno konzumiramo u obliku konzervansa. Konzervansi usporavaju biološku i kemijsku razgradnju hrane te tako produžuju rok trajanja. Detoksikacija je proces u kojim se ksenobiotici u organizmu metaboliziraju, te u konačnici izlučuju iz organizma. U tom procesu ključnu ulogu ima jetra. Osnovni princip detoksikacije uključuje reakcije prvog reda i reakcije drugog reda, koje se odvijaju u parenhimu jetre. U reakcije prvog reda pripadaju procesi oksidacije, redukcije i hidrolize. Većinu reakcija prvog reda katalizira enzimski monooksigenazni sustav citokrom P-450. U reakcije drugog reda pripada proces konjugacije, a to je proces u kojem se molekula strana tijelu spaja s endogenim molekulama, s ciljem stjecanja topljivosti u vodi, što naposljetku omogućuje učinkovitije izlučivanje iz organizma. Enzimi reakcija drugog reda kataliziraju dodavanje funkcionalnih skupina kao što su acetat, sulfat ili glukuronat na metabolite ksenobiotika., The liver is the largest and most complex organ in the human body. It is located in the upper right part of the abdominal cavity. The liver is an organ with a number of vitally important functions and it plays a role in the detoxification of the organism. Everyday, humans are exposed to various xenobiotics which are found in the air, soil, water, plants and animals. The term xenobiotic means any substance that is "foreign" to the body. Xenobiotics are also present in the food we consume every day in the form of preservatives. Preservatives slow down the biological and chemical decomposition of food and thus extend its shelf life. Detoxification is a process in which xenobiotics are metabolized in an organism and ultimately excreted from it. The liver plays a key role in this process. The basic principle of detoxification includes Phase I and Phase II reactions which take place in the liver parenchyma. Oxidation, reduction and hydrolysis processes belong to the Phase I reactions. Most Phase I reactions are catalyzed by the cytochrome P-450 enzyme monooxygenase system. Phase II reactions include the conjugation process which is a process in where a molecule foreign to the body combines with endogenous molecules with the aim of acquiring solubility in water, which ultimately enables more efficient excretion from the body. Phase II reaction enzymes catalyze the addition of functional groups such as acetate, sulfate, or glucuronate to xenobiotic metabolites.

Istraživanje je uključilo analizu podataka seroloških nalaza, PCR testova te podataka o cijepljenju 448 pacijenata NZZJZ PGŽ. Izračunate su prosječne vrijednosti titra neutralizirajućih protutijela i komparirane u odnosu na dob, spol i prethodnu izloženost virusnim antigenima; preboljenjem, cijepljenjem ili oboje. Dobivene su više prosječne vrijednosti protutijela u serumu ženskih ispitanika i porast prosječnih vrijednosti protutijela razmjeran s dobi. Također su dobivene preko 7 puta veće prosječne vrijednosti protutijela u serumu cijepljenih u odnosu na necijepljene. Ispitanici koji su cijepljeni barem jednom dozom i uz to su imali preboljenje, imali su 1,5 puta veće prosječne vrijednosti titra od samo cijepljenih. Necijepljeni koji nisu imali preboljenje prije serološkog testa imali su najnižu prosječnu vrijednost protutijela dobivenu u istraživanju. Najviši prosječni titar protutijela dobiven je u ispitanika koji su primili booster dozu Comirnaty cjepiva nakon prethodne primovakcinacije Comirnaty cjepivom. Dobivene su statistički značajne korelacije između titra IgG neutralizirajućih protutijela i vremena proteklog od posljednje doze cjepiva do serološkog nalaza, te vremena proteklog od posljednje doze cjepiva do infekcije. Rađene su i korelacije IgG titra i vremena proteklog između prve i druge doze cjepiva te vremena između primovakcinacije i booster doze. Zaključci koji proizlaze iz navedenih rezultata i analize su: humoralni odgovor pokrenut cjepljenjem značajno je učinkovitiji od humoralnog odgovora pokrenutog preboljenjem, jačina humoralnog imunosnog odgovora razmjerna je broju izloženosti antigenu i važnost primjene booster doze u poticanju humoralnog imunosnog odgovora protiv SARS-CoV-2 virusa je neosporna. Titar protutijela pada tijekom vremena, a što je niži titar manja je imunost protiv SARS-CoV-2. S velikom vjerojatnošću se može zaključiti da je titar protutijela veći što je više vremena proteklo između primitka prve i druge doze cjepiva., This study included the analysis of serological findings, PCR tests and vaccination data of 448 NZZJZ PGŽ patients. Average values of neutralizing antibody titers were calculated and compared in groups of subjects divided by age, sex, and previous exposure to viral antigens that was documented with positive PCR test, vaccination data, or both. Higher mean antibody values in the serum of female subjects and an increase in mean antibody values proportional to age were obtained. Over 7-fold higher mean serum antibodies were found in the serum of vaccinated versus unvaccinated subjects. Subjects who were vaccinated with at least one dose and also had a positive PCR test had 1.5 times higher average titer values than those who were only vaccinated. Unvaccinated individuals who had not recovered prior to the serological test had the lowest average antibody value obtained in the study. The highest mean antibody titer was obtained in subjects who received a booster dose of Comirnaty vaccine after previous primovaccination with Comirnaty vaccine. Statistically significant correlations were obtained between the titer of IgG neutralizing antibodies and the passed time from the last vaccine dose to the serological finding, as the passed time from the last vaccine dose to infection. Correlations were also made between IgG titer and the time passed between the first and second vaccine doses and the time between primovaccination and booster dose. The conclusions derived from the results and the analysis are: the humoral response triggered by vaccination is significantly more effective than the humoral response triggered by the disease and multiple antigen exposures lead to stronger humoral immune response. Also, the role of booster dose in stimulating humoral immune response is undisputable. The antibody titer decreases over time, and the lower the titer, the lower the immunity against SARS-CoV-2. It can also be concluded with high probability that the higher the time passed between the first and second dose of the vaccine, the higher the antibody titer.

U ovome radu navedene su i opisane otrovne biljke i životinje zastupljene u našem geografskom području. Najznačajnije otrovne biljke našeg podneblja su velebilje, kranjski bijeli bun, duhan, bunovina, crna bunika, bijeli kužnjak, crni kukurijek i jesenski mrazovac. Većina otrovnih biljaka pripada porodici pomoćnica (Solanaceae). To su biljke bogate tropanskim alkaloidima koji djeluju kao antikolinergične tvari. Oni imaju antagonističko djelovanje na muskarinske acetilkolinske receptore. S obzirom na široku rasprostranjenost muskarinskih acetilkolinskih receptora u tijelu pri intoksikaciji tim biljkama prisutni su mnogobrojni simptomi. Oni uključuju poremećaje rada srca, inhibiciju ili stimulaciju lučenja pojedinih žlijezda kao i poremećaje intestinalnog motiliteta. Jedan je od najpoznatijih učinaka pojava delirija i halucinacija koji nastaju zbog inhibicije muskarinskih acetilkolinskih receptora u mozgu. Opisane su i otrovne gljive iako one pripadaju zasebnome carstvu. U našem području najznačajnije su zelena pupavka i muhara. Ove gljive pripadaju rodu Amanita koji se smatra najotrovnijim. Za većinu smrtnih slučajeva od posljedica trovanja gljivama odgovorna je zelena pupavka (Amanita phalloides). U gljivama roda Amanita nalazimo dvije grupe toksina. Falotoksini uzrokuju probavne smetnje, a amatoksini inhibiraju RNA polimerazu II i time onemogućuju sintezu vitalno važnih bjelančevina. Životinje čine najveće carstvo. Od životinjskih toksina opisani su toksini zmija, paukova i riba. Većina zmija otrovnica pripada porodici Viperidae. Naše najpoznatije otrovnice su poskok i riđovka. Njihov otrov sadrži hijaluronidaze, koje pojačavaju reapsorpciju ostalih tvari na mjestu ugriza, fosfolipazu A2, koja uzrokuje nekrozu mišićnog tkiva, metaloproteinaze, odgovorne za razgradnju bazalne membrane i proteolitičke enzime, koji oštećuju endotel kapilara i okolno tkivo. Od paukova opisani su crna udovica i smeđi primorski riječni pauk. Otrov pauka smjesa je monoamina, koji djeluju proalgetski, peptida s ekscitatornim učincima koji izazivaju nociceptivni odgovor i enzima, kao što su sfingomijelinaze, fosfolipaza A2 i hijaluronidaze koji pokreću upalnu reakciju. Završni dio rada osvrće se na otrovne ribe. Naše najpoznatije ribe otrovnice su škrpina i morski pauk., In this paper, poisonous plants and animals which live in our geographical area are listed and described. The most significant poisonous plants in our area are belladonna or deadly nightshade (Atropa belladonna), European scopolia or henbane bell (Scopolia carniolica), nicotiana (Nicotiana alata), mandrake (Mandragora officinarum), jimson weed (Datura stramonium), Christmas rose or black hellebore, (Helleborus niger) and autumn crocus or meadow saffron (Colchicum autumnale) The majority of dangerous plants are a part of the family of nightshades (Solanaceae). They are rich with tropane alkaloids, which serve as anticholinergic substance. They have antagonistic effect on muscarinic acetylcholine receptors. Considering the fact that the muscarinic acetylcholine receptors are widespread in the body during the intoxication with these plants, multiple symptoms are present. They include the disturbance in heart rate and rhythm, inhibition or stimulation of secretion of glands and disorders of intestinal motility. One of the most famous effects is the occurrence of delirium and hallucinations due to the inhibition of the muscarinic acetylcholine receptors in the brain. Poisonous mushrooms are described as well, even though they belong to a separate kingdom. In our area, the most significant ones are death cap (Amanita phalloides) and fly agaric (Amanita muscaria). They belong to the Amanita genus, which are considered the most poisonous. In most cases of mushroom poisoning, death cap (Amanita phalloides) is responsible. In Amanita genus, we can find two different toxin groups: phallotoxins, which causes gastrointestinal issues and amatoxins, which inhibit RNA polymerase II and thus disable the synthesis of vitally important proteins. Animals make the biggest kingdom. From animal toxins, snake toxins, spider toxin and fish toxins are described. The majority of poisonous snakes belong to the family Viperidae. Our most famous poisonous snakes are horned viper (Vipera ammodytes) and common European adder (Vipera berus). Their toxin contains hyaluronidase, which increases reabsorption of substances on the bitten area, phospholipases A2, which causes necrosis of muscle tissue, metalloproteinase, responsible for the decomposition of basilar membrane and proteolytic enzymes which damages capillary endothelium and surrounding tissue. From spiders, black widow (Latrodectus tredecimguttatus) and Mediterranean recluse spider (Loxosceles rufescens) are described. Spider poison is a mixture of monoamines which act proalgetically, peptides with excitatory effects which elicit a nociceptive response and initiate an inflammatory reaction from enzymes sphingomyelins, phospholipases A2 and hyaluronidase. Our most famous poisonous fish are scorpionfish (Scorpanea scrofa) and greater weever (Trachinus draco).

Ugljikov tetraklorid (CCl4) liposolubilni je organski spoj koji se nalazi u sastavu različitih lakova, insekticida, gumenih voskova, a može se pronaći i u otpadnim vodama u koje dospijeva prilikom proizvodnje željeza, čelika, industrijskih boja te rafiniranja nafte. Sustavna toksičnost ovog spoja poznata je dugo vremena. Svrha ovoga rada bila istražiti učinke akutne intoksikacije ugljikovim tetrakloridom na mozak eksperimentalnih životinja. U tu svrhu 10 mužjaka laboratorijskih miševa jednokratno je intraperitonealno injicirano sa 100 μL 10 % (v/v) CCl4 otopljenog u suncokretovom ulju. Grupa od 5 intoksiciranih životinja neurološki je testirana jedan dan nakon intoksikacije, a druga grupa sastavljena od jednakog broja životinja nakon tri dana. Pet životinja injiciranih samo sa suncokretovim uljem poslužilo je kao kontrolna skupina. Prikupljeni uzorci moždanog tkiva obojani su standardnom histološkom tehnikom pomoću hemalauna i eozina (HE) te imunohistokemijskim bojanjem uporabom protutijela protiv mikroglijalnog biljega IBA-1 te protiv mijelinskog proteolipidnog proteina (PLP). Neurološki status miševa ispitao se standardnim brzim neurološkim testom i vrednovao bodovanjem. U miševa žrtvovanih već jedan dan nakon intoksikacije u mozgu su pronađeni znakovi neuronskog oštećenja i degeneracije, infiltracija upalnim stanicama i demijelinizacijske lezije. Tri dana nakon intoksikacije spomenute promjene postale su opsežnije, prisutne u dubljim regijama mozga, a zabilježena je i periventrikularna hemoragija sa intersticijskim edemom mozga. Intoksicirani miševi pokazali su neurološke poremećaje u smislu slabosti stražnjih nožica te poremećaj koordinacije ravnoteže. Ovim radom jasno je pokazan akutni neurotoksični učinak jednokratne aplikacije ugljikova tetraklorida, no potrebna su daljnja istraživanja kako bi se podrobnije rasvijetlili mehanizmi nastanka opisanih neuropatoloških promjena. Carbon tetrachloride (CCl4) is a liposolubile organic compound being a constituent part of various varnishes, insecticides, rubber waxes, but can also be found in waste water from the production of iron, steel, industrial paints and oil refining. The purpose of this study was to investigate the effects of the acute carbon tetrachloride intoxication on the brain of experimental animals. For this purpose, 10 male laboratory mice were injected intraperitoneally with the single dose of 100 μL of 10 % (v/v) CCl4 dissolved in the sunflower oil. A group of 5 intoxicated animals was neurologically tested and sacrificed one day after the intoxication, and another group composed of an equal number of animals underwent the same procedures, three days after intoxication protocol. Five animals injected only with sunflower oil served as a control group. Collected brain tissue samples were stained by standard histological tehnique, using hemalaun and eosin (HE) and by immunohistochemical staining using antibodies against the microglial marker IBA-1 and against myelin proteolipid protein (PLP). The neurological status of the mice was examined by a standard rapis neurological test and evaluated by scoring. Brain tissues of mice sacrificed one day after the intoxication showed signs of neuronal damage and degeneration, inflammatory infiltration and demyelinating lesions. Three days after intoxication, the mentioned changes became more extensive and present in deeper regions of the brain. Periventricular hemorrhage and interstitial brain edema were also recorded in these animals. Intoxicated mice showed neurological disorders in terms of hind leg weeknes, as well as coordination and balance disorders. The present study clearly demostrated the acute neurotoxic effects of a single-dose carbon tetrachloride application, but further research is needed to clarify the mechanisms of the neuropathological changes described here.

Cilj: Cilj ovog rada bio je procijeniti izražaj, tkivnu raspodjelu, odnos i moguće interakcije između metalotioneina (MT) i megalina (LRP-2) u različitim patohistološkim stupnjevima karcinoma pločastih stanica usne šupljine (OSCC) i potencijalno malignim oralnim poremećajima (PMOP- oralna leukoplakija (OL) i oralni lihen planus (OLP)), te procijeniti njihov odnos prema proliferacijskim biljezima i biljezima apoptoze. Materijali i metode: Ova retrospektivna studija uključivala je arhivirane uzorke tkiva 122 pacijenata i kontrolnih ispitanika. Izražaj proteina ispitan je imunohistokemijom i imunofluorescencijom, a kvantifikacija obojenja procijenjena je korištenjem Image J softvera. Interakcija proteina u tumorskom tkivu testirana je i vizualizirana PLA metodom (prema engl. Proximity Ligation Assay). Mann-Whitney i Kruskal-Wallis testovi korišteni su za utvrđivanje značaja razlike između svake skupine, dok je Pearsonov koeficijent korelacije proveden kako bi se testirala korelacija. Rezultati: Izražaj oba proteina značajno se razlikovao između svake skupine koja pokazuje isti obrazac postupnog povećanja od oralnog lihen planusa do slabo diferenciranog OSCC-a. Štoviše, utvrđeno je da MT i megalin suizražavaju i međusobno djeluju u tumorskom tkivu, a njihov izražaj pozitivno korelira unutar cjelokupne ispitivane skupine. Nalazi istaknutog izražaja megalin a u jezgri i kromosomima sugeriraju da prolazi reguliranu intramembransku proteolizu (RIP) nakon vezanja s MT-a, što ukazuje na njegovu sposobnost izravnog utjecaja na genski izražaj i staničnu diobu u tumorskom tkivu. Zaključak: Dobiveni podaci ukazuju na potencijalnu interakciju MT-megalina u onkogenezi. Objectives: The aim of this study was to assess the expression, tissue distribution, relationship and possible interactions between metallothioneins (MTs) and megalin (LRP-2) in different grades of oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMD, oral leukoplakia (Ol) and oral lichen planus (OLP)), as well as to determine their relationship to proliferative markers and markers of apoptosis. Material and Methods: This retrospective study included archived samples of 114 patients and control subjects. Protein expression was examined by immunohistochemistry and immunofluorescence, and staining quantification was performed by ImageJ software. Protein interaction in cancer tissue was tested and visualized by proximity ligation assay. MannWhitney and Kruskal-Wallis tests were used to determine the significance of differences between each group, whereas Pearson correlation coefficient was performed to test correlation. Results: Expression of both proteins differed significantly between each group showing the same pattern of gradual increasing from oral lichen planus to poorly differentiated OSCC. Moreover, MTs and megalin were found to co-express and interact in cancer tissue, and their expression positively correlated within the overall study group. Findings of prominent nuclear and chromosomal megalin expression suggest that it undergoes regulated intramembrane proteolysis upon MTs binding, indicating its ability to directly affect gene expression and cellular division in cancer tissue. Conclusion: Data obtained point to the onco-driving potential of MTs-megalin interaction.

Cilj istraživanja: Cilj istraživanja je bio utvrditi dolaze li do pojačanog izražaja HSP60, TLR2 i TLR4 kod ljudi s patološki uvećanom lijevom srčanom klijetkom i na taj način doprinijeti rasvjetljavanju molekulskih zbivanja tijekom hipertrofije srčanog mišića te inicijalnih procesa uključenih u patogenezu njenih posljedica. Takvo bi istraživanje dokazalo da je stečena hipertrofija lijeve klijetke čovjeka posredovana humoralnom imunosti, što se do sada samo pretpostavljalo, budući da su dosadašnje studije na tu temu rađene isključivo na životinjskim modelima. Materijali i metode: U tu svrhu izvršene su analize imunohistokemijskog izražaja na uzorcima tkiva srčanog mišića dobivenima tijekom obdukcija ljudi u kojih je hipertrofija lijeve klijetke bila jedini patološki nalaz i kod kojih je kao neposredni uzrok smrti određena iznenadna srčana smrt. Uzorci iz kontrolne skupine dobiveni su od ispitanika kod kojih histološki nije pronađena srčana patologija, a koji su umrli nasilnom smrću (nesretni slučaj ili samoubojstvo). Za ispitivanje translokacije HSP60 korištena je dvostruka imunoflorescencija, dok je interakcija HSP60 i TLR2/4 dokazivana testom neposrednog vezivanja (PLA). Rezultati: Rezultati su pokazali značajno veći izražaj HSP60, TLR2 i TLR4 u hipertrofičnom srčanom mišiću u usporedbi s normalnim. Nadalje, u hipertrofičnim smo kardiomiocitima pronašli membransku translokaciju HSP60 i znakove interakcija HSP60/TLR. Korelacijska je analiza pokazala vrlo značajne međukorelacije između kvantificiranih vrijednosti intenziteta imunohistokemijskog bojenja HSP60, TLR2 i TLR4 kod ispitanika s hipertrofijom lijeve klijetke, dok u kontrolnoj skupini rezultati nisu pokazali značajne korelacije. Rezultati dobiveni PLA analizom jasno su pokazali da se HSP60 u hipertrofičnom srčanom mišiću veže za TLR2 i TLR4. Zaključci: Između sekundarno hipertrofiranog i normalnoga srčanog mišića lijeve srčane klijetke čovjeka postoje razlike u izražaju HSP60, TLR2 i TLR4. Osim toga, dobiveni podatci ukazuju na važnu potpornu ulogu HSP60 u adaptivnom rastu kardiomiocita, dok istodobna indukcija TLR2 i TLR4 predlaže interakcije HSP60-TLR kao rana molekulska zbivanja kod sekundarne hipertrofije lijeve klijetke. Objectives: The study aimed to determine whether HSP60, TLR2 and TLR4 are overexpressed in people with pathologically enlarged left ventricles and thus contribute to the elucidation of molecular events during myocardial hypertrophy and the initial processes involved in its pathogenesis. Such research would prove that acquired left ventricular hypertrophy is mediated by humoral immunity, which has so far only been hypothesized, given that previous studies on this topic have been done exclusively on animal models. Material and methods: For this reason, immunohistochemical expression analyzes were performed on myocardial tissue samples acquired during autopsies of people in whom left ventricular hypertrophy was the only pathological finding and in whom sudden cardiac death was determined as the immediate cause of death. Samples from the control group were obtained from subjects who were not found to have cardiac pathology and who died a violent death (accident or suicide). Double immunofluorescence was used to examine the HSP60 translocation, while the interaction of HSP60 and TLR2/4 was demonstrated by a direct ligation test (PLA). Results: The results showed significantly higher expression of HSP60, TLR2, and TLR4 in the hypertrophic myocardium compared to the normal myocardium. Moreover, we found HSP60/TLR interactions in hypertrophic cardiomyocytes. Correlation analysis showed very significant intercorrelations between quantified values of HSP60, TLR2, and TLR4 immunohistochemical intensities in subjects with left ventricular hypertrophy while in the control group the results showed no significant correlations. The results obtained by PLA analysis clearly showed that HSP60 in hypertrophic myocardium binds to TLR2 and TLR4. Conclusion: There are differences in the expression of HSP60, TLR2, and TLR4 between the secondary hypertrophied myocardium and the normal left ventricular myocardium. In addition, the data obtained indicate an important supporting role of HSP60 in adaptive cardiomyocyte growth, while concomitant induction of TLR2 nad TLR4 suggests HSP-TLR interactions as early molecular events in secondary left ventricular hypertrophy.

Vitamin D je liposolubilan spoj čija je osnovna uloga održavanje homeostaze kalcijevih iona u organizmu. No, receptor vitamina D i enzim 1α-hidroksilaza prisutni su u mnogim tkivima što čini temelj pretpostavke, koja je in vitro i in vivo modelima i potvrđena, kako uz osnovnu postoje i druge uloge vitamina D. Prema dostupnoj literaturi jedna od njih je i antitumorska aktivnost. Cilj ovoga rada bio je istražiti postoji li povezanost i koji je oblik povezanosti između koncentracije cirkulirajućeg 25(OH)D i incidencije karcinoma bubrega. Istraživanje je obuhvatilo 227 osoba, od čega je njih 144 dijagnosticirano s karcinomom bubrega, a 83 sudionika su činila kontrolnu skupinu. Ispitanicima je imunoenzimskim testom određena serumska koncentracija 25(OH)D, a anketnim upitnikom su prikupljeni podaci koji su kasnije korišteni u regresijskim modelima prilikom statističke obrade podataka. Statističkom obradom podataka pronađen je nelinearan odnos u obliku U-krivulje između cirkulirajućeg 25(OH)D i incidencije karcinoma bubrega pri čemu je porast koncentracije do 75 nmol/L obrnuto proporcionalan s incidencijom karcinoma bubrega, a isto se ne može reći za vrijednosti više od navedene u odnosu na skupinu s vrijednostima nižim od 30 nmol/L. Rezultati prilagođenih modela za određene parametre nisu toliko uvjerljivi. Stratifikacijom podataka pokazalo se kako niske vrijednosti 25(OH)D značajno povećavaju rizik kod muškaraca. Istraživanje je pokazalo važan odnos između koncentracije cirkulirajućeg 25(OH)D i karcinoma bubrega koji se može opisati U-krivuljom premda vrijednost dobivenih rezultata svakako treba provjeriti kliničkim studijama., Vitamin D is a liposoluble compound whose primary role is maintaining the homeostasis of calcium levels in the body. However, vitamin D receptor and enzyme 1α-hydroxylase are present in many tissues, which forms the basis of the assumption, which is confirmed in in vitro and in vivo models, of additional roles of vitamin D. According to the available literature one of them is antitumor activity. The aim of this study was to determine the association and shape of that association between circulating 25(OH)D levels and the incidence of kidney cancer. 227 people were included in the study, 144 were diagnosed with kidney cancer, and remaining 83 participants formed the control group. Serum 25(OH)D was measured with an enzyme immunoassay, while other data used in regression models during statistical analysis were collected with the questionnaire. Statistical analysis revealed a non-linear association in the form of a U-shaped curve between circulating 25(OH)D levels and the incidence of kidney cancer. Incidence is reversely proportional to increasing 25(OH)D levels till the value of 75 nmol/L compared to the group with levels lower than 30 nmol/l, where there was no difference in incidence between the groups with highest and lowest levels of 25(OH)D. Results of adjusted models for certain parameters are not as conclusive. Stratification of the data showed that low values 25(OH)D significantly increased the risk in men. Research has shown an important association in form of U-shaped curve between circulating 25(OH)D and kidney cancer, although the value of the results obtained should certainly be explored by clinical trials.

Vitamin D se smatra prohormonom čiji aktivni oblik koji nastaje u koži ljudi djelovanjem ultraljubičastog zračenja tipa B djeluje kao hormon, odnosno kao signalna molekula koja, vežući se na unutarstanične receptore za vitamin D koji se nalaze u gotovo svim tkivima u tijelu, utječe na različite fiziološke funkcije. Glavninu svojih funkcija obavlja povećavajući intestinalnu apsorpciju i renalnu reapsorpciju kalcija i fosfata sudjelujući na taj način u održavanju njihove homeostaze. U brojnim studijama je pokazano da vitamin D ima protektivni učinak u razvoju određenih malignih tumora, uključujući i maligne tumore mokraćnog mjehura koji su deveti najčešći malignitet u svijetu dok se u Hrvatskoj u muškaraca nalaze na 5. mjestu. Sudionici ove studije su podijeljeni u dvije grupe; jedna se sastojala od 236 pacijenata oboljelih od karcinoma mokraćnog mjehura, dok je druga grupa bila kontrolna grupa od 83 sudionika koji su povezani s prvom grupom na osnovi dobi, a regrutirani su kao urološki pacijenti koji ne boluju od karcinoma mokraćnog mjehura. Svi sudionici su ispunili upitnik koji se sastojao od 4 dijela s otvorenim i zatvorenim pitanjima te su dali svoj uzorak krvi. Cilj ovog rada bio je dokazati povezanost serumske razine 25-hidroksi vitamina D i karcinoma mokraćnog mjehura u sjevernom priobalju Hrvatske. Ovom studijom pronađena je korelacija između visoke serumske razine vitamina D i nižeg rizika razvoja karcinoma mokraćnog mjehura u grupi pacijenata koji obitavaju u sjevernom priobalju Hrvatske, Vitamin D is considered a prohormone whose active form, which is synthesized in the skin by ultraviolet light type B, acts as a hormone – a signal molecule that affects different physiological functions by binding to vitamin D receptors in different body tissues. Most of its functions are done through mediating an increase in intestinal apsorption and renal reapsorption of calcium and phosphates. It has been shown in many studies that vitamin D has a certain protective effect when it comes to development of some malignant tumours, including malignant tumours of the bladder which are the ninth most common malignancy in the world and fifth in Croatia's male population. The participants of this study were divided into two groups; one consisted of 236 patients with bladder carcinoma while the other group consisted of 83 participants which were connected with the first group based on age and were recruited as urologic patients which did not have bladder carcinoma. All participants have filled a questionnaire which consisted of 4 parts with open-ended and close-ended questions and they all gave their blood sample for analysis. The aim of this study was to find a connection between serum level of 25-hydroxy vitamin D and bladder carcinoma in the northern coastal region of Croatia. With this study a correlation between high serum level of vitamin D and lower level of risk of development of bladder carcinoma has been found among a group of patients in the northern coastal region of Croatia.

Kost je složeno, organizirano tkivo koje se sastoji od mineralne i organske tvari. To je i izrazito dinamično tkivo, što se očituje neprestanom pregradnjom. Osteoblasti su koštane stanice koje stvaraju novu kost, dok su osteoklasti koštane stanice odgovorne za razgradnju već stvorene kosti. Ključnu ulogu u regulaciji koštane pregradnje ima OPG/RANKL/RANK sustav, koji se sastoji od glikoproteinske molekule RANKL, njezinog srodnog receptora RANK te „lažnog“ receptora OPG. Renalna osteodistrofija je naziv za različita patofiziološka stanja koja pogađaju koštani sustav bolesnika s kroničnim bubrežnim zatajivanjem. Glavni patofiziološki mehanizmi koji su uključeni u pojavu renalne osteodistrofije su pojava hiperfosfatemije, hipokalcijemije, uz poremećaj sinteze vitamina D. Prema rezultatima ovog istraživanja, osobe na hemodijalizi imaju značajno više razine AP, OPG, sRANKL i iPTH, u odnosu na osobe s transplantiranim bubregom (Tx12) i na osobe bez bubrežne bolesti. Hemodijalizirani ispitanici i primatelji bubrežnog transplantata na terapiji vitaminom D imaju značajno više razine OPG-a, uz značajno niže razine sRANKL-a. Ovi rezultati govore u prilog povoljnog učinka vitamina D na očuvanje koštane mase u ispitanika na HD i primatelja bubrežnog transplantata. Omjer razina serumskih vrijednosti RANKL/OPG, mogao bi bolje korelirati s PTH vrijednostima, nego li zasebno mjerenje tih čimbenika, ali ostaje nejasno zašto je omjer RANKL/OPG najniži u umjereno povišenim vrijednostima iPTH pa je potrebno je provesti dodatna istraživanja, kako bismo dobili odgovor na ovo pitanje, Bone is a complex, organized tissue that consists of both, mineral and organic substances. It is also a highly dynamic tissue that is manifested by continuous bone remodeling. Osteoblasts are bone cells that create new bone, while osteoclastic bone cells are responsible for the resorption of the already created bone. OPG/RANKL/RANK system plays a key role in bone remodeling regulation. This system is consisting of the RANKL glycoprotein molecule, its related RANK receptor, and OPG as the "decoy receptor''. Renal osteodystrophy is a name for various pathophysiological conditions affecting the bone of patients with chronic renal failure. The main pathophysiological mechanisms present in renal osteodystrophy are hyperphosphatemia, hypocalciemia, with vitamin D synthesis disorders. According to the results of this study, patients on hemodialysis had significantly higher levels of AP, OPG, sRANKL and iPTH compared to transplanted kidney recipients (Tx12) and those with no renal disease. Patients on hemodialysis and transplanted kidney recipients on vitamin D therapy had significantly higher levels of OPG, with significantly lower sRANKL levels. These results suggest the beneficial effect of vitamin D on bone mass loss prevention in patients on hemodialysis and in kidney transplant recipients. The RANKL/OPG serum level ratio could be better correlated with iPTH values than a separate measurement of these factors, but it remains unclear why the RANKL/OPG ratio is low in moderately elevated iPTH values and further research is needed in order to get the answer to this question.

Uvod: Depresija je čest psihijatrijski poremećaj kojeg karakteriziraju ponovljene epizode depresivnog raspoloženja, ali i različiti somatski simptomi, kao što su metabolički simptomi koji mogu rezultirati razvojem metaboličkog sindroma. Etiologija depresije još nije potpuno poznata. Smatra se da su geni uključeni u kodiranje proteina važnih za regulaciju i funkciju serotonergičkog (5-HT) sustava, kao što je gen za serotoninski transporter (5HTT) i njegov funkcionalni polimorfizam 5-HTTLPR (polimorfna regija vezana za gen serotoninskog transportera), povezani s depresijom. Cilj istraživanja: Cilj istraživanja bio je utvrditi postoji li povezanost 5-HTTLPR i metaboličkog sindroma i/ili pojedinih sastavnica metaboličkog sindroma u depresivnih bolesnika. Naime, očekivana je veća zastupljenost SS genotipa, odnosno S alela obzirom na 5-HTTLPR kod bolesnika s metaboličkim sindromom prema onima koji nisu razvili metabolički sindrom. Ispitanici i metode: Istraživanje je uključilo 202 depresivna bolesnika sa i 204 depresivna bolesnika bez metaboličkog sindroma. Dijagnoza depresije postavljena je prema kriterijima MKB-10, a metaboličkog sindroma prema kriterijima SZO. Komponente metaboličkog sindroma određene su rutinskim laboratorijskim metodama. Genotipizacija je provedena lančanom reakcijom polimeraze. Rezultati su obrađeni pomoću Kruskal-Wallisove analize rangova i Dunnovog testa, ili Mann Whitneyevog testa, Spearmanovog koeficijenta korelacije i višestruke linearne regresije. Rezultati: Nisu utvrđene značajne razlike u učestalosti 5-HTTLPR genotipova ili alela između depresivnih bolesnika sa i bez metaboličkog sindroma. Međutim, nosioci LL genotipa imali su značajno viši dijastolički tlak i opseg struka od nosioca drugih genotipova. Druge komponente metaboličkog sindroma (sistolički tlak, koncentracije triglicerida, glukoze i HDL kolesterola, izlučivanje albumina) nisu bile povezane s polimorfizmom 5-HTTLPR u depresivnih bolesnika. Zaključak: 5-HTTLPR polimorfizam nije značajno povezan s metaboličkim sindromom u depresivnih bolesnika. Utvrđena je značajna povezanost 5-HTTLPR i sastavnica metaboličkog sindroma (dijastoličkog tlaka i opsega struka) upućujući da je LL genotip obzirom na 5-HTTLPR rizičan za viši dijastolički tlak i veći opseg struka. Ti podatci upućuju da 5-HTTLPR polimorfizam ima određenu ulogu u razvoju pojedinih komponenti metaboličkog sindroma u depresivnih bolesnika Introduction: Depression is frequent psychiatric disorder characterized by the reccurent episodes of depressed mood, but also with different somatic symptoms such as metabolic symtpoms that might result in development of metabolic syndrome. Etiology of depression is not completely known. It is assumed that genes coding for proteins important for regulation and fuction of serotonin (5-HT) system, such as a gene for serotonin transporter (5HTT), and its functional polymorphism 5-HTTLPR (serotonin-transporter-linked polymorphic region), are associated with depression. Aim of study: Study aimed to determine association between 5-HTTLPR and metabolic syndrome and/or components of metabolic syndrome in depressed patients. Namely, we expected that patients with metabolic syndrome will be more frequently carriers of the 5- HTTLPR SS genotype or S allele compared to depressed patients without metabolic syndrome. Subjects and methods: The study included 202 depressed patients with and 204 depresssed patients without metabolic syndrome. Diagnosis of depression was made according to the ICD-10 criteria, and metabolic syndrome according to the criteria of the WHO, and use of routine laboratory and clinical measurements. Genotyping was determined using PCR. Results were analyzed with Kruskal-Wallis ANOVA and Dunn test, Mann Whitney test, Spearman's coefficient of correlation and multiple linear reggression. Results: 5-HTTLPR polymorphism was not significatly associated with metabolic syndrome in depressed patients. However, carriers of the LL genotype had significantly higher diastolic blood pressure and weist circumference than other genotype carriers. Other components of the metabolic syndrome (sistolic blood pressure, triglycerides, glucose, HDL cholesterol concentrations and albumines) were not significantly associated with 5-HTTLPR polymorphism in deppressed patients. Conclusion: 5-HTTLPR polymorphism is not significantly associated with metabolic syndrome in depressed patients. Significant association between 5-HTTLPR and components of metabolic syndrome (diastolic blood pressure and weist circumference) was found, suggesting that LL genotpye of the 5-HTTLPR is a risk genotipe for higher diastolic blood pressure and increased weist circumference. These data suggest that 5-HTTLPR polymorphism has particular role in development of specific components of metabolic syndrome in depressed patients.

Multiple sclerosis (MS) is an autoimmune disorder that targets myelin sheaths in the central nervous system. It is a chronic disorder and although the axonal damage is initially reversible, at later stages it eventually results in neuroaxonal loss. It has a wide range of symptoms including sensory, autonomic nervous system, motoric and cognitive problems. It affects over 2.5 million people in the world, and starts in most patients at 20 to 40 years of age. MS is a multifactorial disease, with both genetic and environmental factors playing a role in its pathogenesis. Based on its clinical course it is categorized into four types: relapse-remitting, secondary progressive, primary progressive, and progressive relapsing MS. There is still no definite cure for MS, and all current therapies work solely by alleviating symptoms and the severity of the disease. The immune system plays a key role in MS. Autoreactive CD4+ T cells in the periphery are accepted as the most likely cause of the disease, although the triggering mechanisms for their activation are still unclear. Microglia and macrophages have both pro- and antiinflammatory effects in MS, whereas the natural killer cells have been shown to have a regulatory role and could suppress the autoreactive CD4+ T cells. B cells have been extensively studied in MS in recent years, and were found to play an important role in its pathogenesis and course, thus presenting an attractive therapeutic target. Oligoclonal immunoglobulin bands in the cerebrospinal fluid are produced by B cells and are found in over 95% of MS patients. B cells also act as antigen-presenting cells to T cells, and secrete cytokines which can be either pro-inflammatory or anti-inflammatory. In this thesis we will elaborate on the pathogenesis of MS and review MS therapies, all of which target the immune system. Specifically, we will list and compare the traditional therapies such as interferon-β and glatiramer acetate, which are still the first-line therapy for MS, with second- and third-line therapies that include various B cell-targeting approaches. With a higher efficacy than older therapies and a smaller risk of serious side effects, B cell-focused therapies are proving to be the future of MS treatment, Multipla skleroza (MS) je kronični autoimuni poremećaj koji pogađa mijelinske ovojnice u središnjem živčanom sustavu. U ranijim stadijima aksonalna šteta je reverzibilna, ali u kasnijima dolazi do neuroaksonalnog gubitka. Ima širok spektar simptoma uključujući probleme u senzornom, autonomnom živčanom i motoričkom sustavu, kao i kognitivne poteškoće. Više od 2,5 milijuna ljudi u svijetu boluje od ove bolesti, a u većine pacijenata započinje u dobi od 20 do 40 godina. MS je multifaktorijalna bolest u čijoj patogenezi ulogu imaju i okolišni i genetski faktori. Na temelju kliničkog tijeka, MS je kategorizirana u četiri tipa: relapsno-remitirajuća, sekundarno progresivna, primarno progresivna i progresivno relapsirajuća. Još uvijek ne postoji definitivni lijek za MS i sve trenutno dostupne terapije samo umanjuju simptome. Imuni sustav igra ključnu ulogu u ovoj bolesti. Autoreaktivne CD4+ T stanice na periferiji su prihvaćene kao najvjerojatniji uzrok bolesti, iako mehanizmi njihove aktivacije još uvijek nisu jasni. Mikroglija i makrofagi imaju i proupalne i protuupalne učinke u MS-u. NK stanice imaju regulatornu ulogu i mogle bi utišati autoreaktivne CD4+ T stanice. Posljednih godina B stanice su opsežno istraživane i pokazalo se da igraju bitnu ulogu u patogenezi i tijeku MS-a te ih to čini dobrom terapijskom metom. B stanice proizvode oligoklonalne vrpce imuoglobulina koje se nalaze u preko 95% pacijenata. B stanice su također antigen-prezentirajuće stanice T stanicama, te proizvode citokine koji imaju ili proupalni ili protuupalni učinak. U ovom završnom radu bit će pojašnjena patogeneza MS-a i opisane terapije za MS. Sve terapije za MS djeluju na imunosni sustav. Tradicionalne terapije poput glatiramer acetata i interferona-β usporedit će se s novijim terapijama koje na neki način djeluju na B stanice. Zbog veće učinkovitosti od starijih terapija i manjeg rizika od ozbiljnih nuspojava, terapije fokusirane na B stanice su se pokazale kao budućnost tretiranja MS-a.

Ishemijsko-reperfuzijska ozljeda jedan je od najvažnijih uzroka oslabljene funkcije transplantiranog bubrega. Dosad su identificirani mnogi čimbenici, koji mogu povećati vjerojatnost njezina nastanka, a jedan od najvažnijih je način skladištenja bubrega, odnosno duljina trajanja hladne ishemije. Osnovni uzrok oštećenja tkiva u hladnoj ishemiji je nedostatak kisika, koji dovodi do smanjenja koncentracije adenozin trifosfata (engl. adenosine triphosphate, ATP-a) te slabljenja aktivnosti Na/K-ATP-aze (Na/K crpke). Puno kompleksniji i teži oblik oštećenja tkiva nastaje ponovnom uspostavom cirkulacije, odnosno reperfuzijom, prilikom koje dolazi do pojačanog ulaska kisika u stanice te stvaranja velike količine slobodnih kisikovih radikala. Njihovo nakupljanje unutar tkiva uzrokuje oštećenja DNA, proteina i lipida stanične membrane. Ljudski organizam djelomično je zaštićen od djelovanja slobodnih kisikovih radikala pomoću vlastitih antioksidativnih enzimatskih sustava, poput superoksid dismutaze (SOD), glutation peroksidaze (engl. gluthatione peroxidase, GSH-Px) te katalaze. Kako bi se spriječio nastanak navedenih oštećenja, u kliničkoj se praksi za vrijeme trajanja ishemije bubrega koriste različite otopine za njegovo čuvanje. Cilj ovog rada bio je ispitati da li primjena koštanog morfogenetskog proetina 7 (engl. bone morphogenetic protein 7, BMP-7) smanjuje oštećenje tkiva bubrega tijekom hladne i tople ishemije uspješnije od UW otopine te da li je dodavanjem BMP-7 u UW otopinu oštećenje tkiva manje nego kada se tkivo ispire samo s UW otopinom. Rezultati ovog istraživanja pokazali su kako primjena rhBMP-7 tijekom hladne i tople ishemije, uspješnije čuva tkivo bubrega od oksidativnog oštećenja proteina i lipida te NA+/K+ ATP-aze u odnosu na UW otopinu, dok istovremeno ne postoji značajnija razlika u aktivnosti antioksidativnih enzima, GSH-Px te SOD., Ischemic-reperfusion injury is important cause of transplanted kidney decreased function. So far many risk factors have been identified, which may increase the probability of its occurrence, the most important one is duration of the cold ischemia. The main cause of tissue damage in cold ischemia is lack of oxygen, which leads to a decreased concentration of adenosine triphosphate (ATP) and decrease in Na/K-ATPase activity. More complex and severe form of tissue damage is caused by re-establishment of circulation, known as reperfusion, which leads to increased oxygen level in the cells and creation of reactive oxygen species (ROS). ROS cause damage of the DNA, proteins, and lipids of cell membrane. Human body is partially protected from effects of ROS by its own antioxidant enzymatic systems, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase. In order to prevent occurrence of these impairments, different organ preservation solutions can be used during cold ischemia period. Aim of this study was to examine whether the application of bone morphogenetic protein 7 (BMP-7) reduces kidney damage during cold and warm ischemia more successfully than UW solution, and whether the adding of BMP-7 into UW solution will prevent tissue damage more efficiently than UW solution itself. The results of this study demonstrated that rhBMP-7, compared with UW solution, better preserves kidney tissue after cold and warm ischemia, decreasing the levels of LPO, PCC and preserving the damaging of NA+/K+ ATP-ase. However, there was no significant difference in antioxidative enzymes activity levels between these two groups.

Cilj istraživanja ovog doktorskog rada je ispitati izražaj citotoksične molekule granulizina na razini bjelančevine i glasničke ribonukleinske kiseline (gRNK) na spoju majčinih i embrijskih tkiva u anembrijskoj trudnoći i zadržanom pobačaju te rezultate usporediti s decidualnim tkivom elektivno prekinute normalne trudnoće prvog tromjesečja. Materijali i metode: Uzorke bazalne decidue normalne i anembrijske trudnoće, te zadržanog pobačaja dobivali smo s Klinike za ginekologiju i porodništvo, KBC Rijeka. Od imunoloških metoda koristili smo imunohistologijsko obilježavanje antigena u decidualnom tkivu uklopljenom u parafin (citokeratin, granulizin, IL-15, Apaf-1, NF-B, CD56) uz kompjutersko kvantificiranje broja obilježenih stanica/mm2 tkiva i semikvantitativnu analizu jačine obilježavanja (H score). Višestrukom imunofluorescencijom obilježavali smo citokeratin i CD56, IL-15, granulizin, Apaf-1 ili NF-B u decidualnom tkivu uz očitavanje na fluorescentnom mikroskopu i obilježavali smo CD3/CD56 i NKp46, NKp44, CD94, NKG2A ili NKG2D u suspenziji DMS dobivenih enzimatskom razgradnjom protočnom citometrijom. Metodom lančane reakcije polimeraze u stvarnom vremenu (RT-qPCR) određivali smo izražaj gena za granulizin i perforin. Rezultati: Udio CD3ˉCD56⁺ stanica NK u suspenziji DMS, apsolutni broj CD56⁺ stanica i IL-15⁺ stanica/mm2 decidualnog tkiva bio je manji u zadržanom pobačaju Učestalost NKG2A, NKG2D i NKp46 biljega na CD3ˉCD56⁺ stanicama NK u suspenziji DMS iz zadržanog pobačaja je bila statistički značajno manja u odnosu na normalnu trudnoću. U anembrijskoj trudnoći udio stanica NK se nije mijenjao. Učestalost NKG2C, CD94 i NKP44 nije se značajnije mijenjala. Glasnička RNK za granulizin je četverostruko manja u DMS zadržanog pobačaja, a četverostruko veća u DMS anembrijske trudnoće, dok je gRNK za perforin povećana u objema. U normalnoj trudnoći dominiralo je citoplazmatsko obilježavanje granulizina, dok se u patološkim trudnoćama granulizin nalazio u jezgrama stanica. Apsolutni broj NF-B⁺ stanica/mm2 decidualnog tkiva je najveći u anembrijskoj trudnoći a najniža učestalost NF-B⁺ stanica je utvrđena u resicama trofoblasta zadržanog pobačaja. Zaključak: Slabije učinkovito ubijanje trofoblasta granulizinom može biti uključeno u sporije odbacivanje trofoblasta u anembrijskoj trudnoći i zadržanom pobačaju, zbog smanjene dopreme granulizina u jezgre trofoblasta u odnosu na normalnu trudnoću., The research objective of this doctoral thesis was to examine the expression of the cytotoxic molecule of granulysin at the level of protein and messenger ribonucleic acid (mRNA) at the interface of maternal and embryonic tissues in anembryonic pregnancy and missed abortion, and to compare the results with decidual tissue from an electively terminated normal first trimester pregnancy. Materials and methods: Basal decidua samples from a normal and an anembryonic pregnancy as well as a missed abortion were obtained from the Obstetrics and Gynecology Clinic of the Clinical Hospital Center Rijeka Of immunoassays, we used immunohistochemical staining of antigens in decidual tissue embedded in paraffin (cytokeratin, granulysin, IL-15, Apaf-1, NF-B, CD56) with computer quantification of the number of stained cells/mm2 of tissue and semiquantitative analysis of the intensity of staining (H score). We performed multiple immunofluorescence labelling of cytokeratin and CD56, IL-15, granulysin, Apaf-1 or in decidual tissue with fluorescent microscope reading, as well as the staining of CD3/CD56 and NKp46, NKp44, CD94, NKG2A or NKG2D in the suspension of DMCs obtained by enzymatic degradation using flow cytometry. Using the real-time polymerase chain reaction method (RT-qPCR), we determined the gene expression for granulysin and perforin. Results: The content of CD3-CD56⁺ NK cells in the DMC suspension, the absolute number of CD56⁺ cells and IL-15⁺ cells/mm2 of decidual tissue was lower in the missed abortion. The frequency of NKG2A, NKG2D and NKp46 markers on CD3ˉCD56⁺ NK cells in the DMC suspension from the missed abortion was statistically significantly lower than in the normal pregnancy. In anembryonic pregnancy proportion of NK cells did not change. The frequency of NKG2C, CD94 and NKP44 did not differ significantly. The messenger RNA for granulysin was four times lower in the DMC from the missed abortion, and four times higher in the DMC from the anembryonic pregnancy, while the mRNA for perforin was increased in both. In the normal pregnancy, cytoplasmic staining of granulysin was predominant, while, in the pathological pregnancies, granulysin was present in cell nuclei. The absolute number of NF-B⁺ cells/mm2 of decidual tissue was the highest in the anembryonic pregnancy. The lowest frequency of NF-B⁺ cells was determined in the trophoblast villi of the missed abortion. Conclusion: A less efficient killing of trophoblasts with granulysin may be involved in a slower rejection of trophoblasts in anembryonic pregnancy and missed abortion, because of a decreased supply of granulysin to trophoblast nuclei compared to normal pregnancy.

Cilj istraživanja: Implantacija razvojno kompetentnog embrija u receptivan uterus je ključan proces tijekom uspostavljanja trudnoće u sisavaca. Prema današnjim saznanjima smatra se da je nepravilna implantacija uzrok 75 % neuspjelih trudnoća. Aktivnost steroidnih hormona, estrogena (E) i progesterona (P), preko njihovih jezgrinih receptora je ključna za proces implantacije. Mehanizam djelovanja E-a, a pogotovo P-a tijekom rane trudnoće još nije dovoljno razjašnjen. Glavni cilj ovog istraživanja bio je utvrditi ulogu progesteronskog receptora (PR) u mehanizmu implantacije blastociste in vivo. Ispitala sam: 1. vremenski i prostorni odnos između PR-a i njime reguliranih gena: Hand2 i Cox2 te ER i FGF9 u uterusu miša tijekom peri-implantacijskog razdoblja, 2. prostorne promjene izražaja PR-om reguliranih gena tijekom implantacije, 3. promjene izražaja Hand2, Cox2, FGF9 i PR gena u implantacijskom mjestu u odnosu na ne-implantacijsko mjesto i 4. regulaciju izražaja Hand2 i FGF9 s P-om i E-om u ovarijektomiziranim mišicama. Materijali i metode: Kao model za istraživanje mehanizama uključenih u trudnoću koristila sam BALB/c miševe. Metodom imunofluorescencije na rezovima tkiva odredila sam prostorni i vremenski raspored PR-a, ERα, COX2, HAND2 i FGF9, kao i njihov međusobni odnos tijekom rane trudnoće. Metodu lančane reakcije polimeraze u stvarnom vremenu (RT-PCR) koristila sam za određivanje izražaja Hand2 i FGF9 gena tijekom rane trudnoće. Razliku izražaja P-om reguliranih gena na implantacijskom u odnosu na ne-implantacijsko mjesto sam odredila RT-PCR metodom. Promjene izražaja bjelančevina Hand2 i FGF9 tijekom rane trudnoće odredila sam western blot metodom. Da bi odredila regulaciju izražaja Hand2 i FGF9 u uterusu koristila sam ovarijektomizirane WT mišice tretirane steroidnim hormonima. Ulogu PR izoformi u izražaju Hand2 u uterusu odredila sam koristeći ovarijektomizirane PRAKO i PRKO mišice tretirane egzogenim E-om i P-om. Rezultati: Tijekom peri-implantacijskog razdoblja dinamika izražaja i odnos PR-a i njime reguliranih gena, Hand2, COX2, ERα i FGF9 mijenja se tijekom procesa rane trudnoće u miševa i strogo je stanično specifičan. Transkripcijska aktivnost PR-a je neophodna za izražaj Hand2 i Cox2 u specifičnoj populaciji stromalnih stanica na implantacijskom mjestu. Na implantacijskom mjestu izražaj Hand2 i COX2 je značajno povećan u usporedbi s ne-implantacijskim mjestom. Izražaj Hand2 u mjestu je utišana. Ovim istraživanjem je po prvi puta pokazano da je FGF9 važan čimbenik u uspostavljanju komunikacije između kompetentne blastociste i receptivnog uterusa. Zaključak: Precizno usklađeni mehanizmi koje reguliraju P i E, preko PR-a i ERα su ključni za stvaranje pogodnog okoliša za implantaciju embrija. Transkripcijski čimbenici, PR-a i Hand2 zajedno potiču diferencijaciju stromalnih stanica na mjestu implantacije. Ta populacija stanica je mjesto biosinteze prostaglandina. Prostaglandini su medijatori angiogeneze i decidualizacije u uterusu. Čimbenik rasta, FGF9 je važan za proces implantacije i uspostavljanje trudnoće u miša., Aim of the study: Implantation of a developmentally competent embryo in a receptive uterus is an essential process during the establishment of pregnancy in mammals. Based on the current knowledge, it is assumed that improper implantation is the cause of 75% of failed pregnancies. Synchronized activity of steroid hormones estrogen (E) and progesterone (P) through their nuclear receptors is the key for successful pregnancy. Mechanisms of E and particularly P-regulated pathways during implantation still need to be clarified. The aim of this research was to determine progesterone receptor’s (PR) role in the establishment of pregnancy in vivo using a mouse model organism. I investigated: 1. spatiotemporal relationship between PR and its targets: Hand2, COX2, ER and FGF9 during the periimplantation period; 2. spatial changes in the expression of PR-regulated genes during implantation; 3. changes in expression of Hand2, COX2, FGF9 and PR genes at the implantation site in relation to the non-implantation site and 4. steroid hormone regulation of Hand2 and FGF9 expression. Material and methods: For these studies BALB/c mice were used. Immunofluorescence analyses of uterus tissue obtained from pregnant mice were used to study the spatiotemporal relationship between PR and PR-regulated genes during peri-implantation. Changes in the expression of PR regulated genes at the implantation site in relation to the non-implantation site were analyzed by using immunofluorescence on uterus tissue, as well as using quantitative real time-PCR (RT-PCR). Differences in Hand2 and FGF9 gene and protein expression during periimplantation were determined by using RT-PCR as well as by western blot. To study the regulation of Hand2 and FGF9 gene expression by steroid hormones in the uterus, ovariectomized WT mice were hormonally treated. The role of PR isoforms in Hand2 expression was studied by using E and P treated ovariectomized PRAKO and PRKO mice. Results: During peri-implantation period the expression of PR and PR regulated genes Hand2, COX2, ERα and FGF9 dynamically changes in the uterus and is highly cell specific. Transcriptional activity of PR is necessary for the expression of Hand2 and COX2 in the specific population of the stromal cells at the implantation site. At the implantation site the expression of Hand2 and Cox2 was significantly higher in comparison to the non-implantation site. The expression of Hand2 is regulated by the PR-A isoform in endometrium. Transcriptional activity of ERα is downregulated at the implantation site. For the first time, this study showed that FGF9 is an important factor for the establishment of communication between a competent blastocyst and a receptive uterus. Conclusion: Balance in P and E regulated activity, throughout their cognate receptors is crucial to establish a suitable environment for embryo implantation in mice. The interaction of transcription factors PR and Hand2 is necessary for differentiation of specific stromal cells at the site of implantation. In these cells PR and Hand2 stimulate production of prostaglandins, which are the key mediators of angiogenesis and decidualization in the uterus. Growth factor FGF9 is an important factor for implantation and the establishment of pregnancy in the mice.

Cilj istraživanja: Cilj istraživanja je bio utvrditi postoje li razlike u konstitutivnom i inducibilnom izražaju stresnih bjelančevina metalotioneina I+II (MT I+II) i bjelančevine toplinskog šoka glikoproteina 96 (Gp96) između štakora DA i AO soja tijekom razvoja eksperimentalnog autoimunosnog encefalomijelitisa. Pošto su citokini IL-6 i TGF-β (od engl. transforming growth factor β), kao i transkripcijski čimbenik NF-kB (od. engl. nuclear factor kappa-light-chain-enhancer of activated B cells), važni za odvijanje patogeneze multiple skleroze i EAE-a, istovremeno su se ispitivale pridružene razlike njihovog izražaja u spomenutim tkivima. Budući da je izražaj MT I+II usko vezan uz tkivne metale, ispitivale su se razlike u tkivnim sadržajima iona cinka i bakra. Materijali i metode: Istraživanja provedena su na mužjacima štakora AO i DA soja, starosti 2-3 mjeseca. Uzorci za analizu uključivali su tkivo jetre, velikog i malog mozga i zasebno hipokampusa iz intaktnih neimuniziranih životinja te imuniziranih životinja žrtvovanih u predkliničkoj fazi bolesti (7. dan) i trenutku najintenzivnije kliničke slike (12. dan). Promjene izražaja bjelančevina utvrđene su imunohistokemijskim bojanjem na parafinskim tkivnim rezovima te Western blot analizom, uporabom odgovarajućih protutijela. Indukcija MT-I gena utvrđena je metodom reverzne transkripcije u realnom vremenu. Koncentracije tkivnih metala određene su spektrofotometrijski, a tkivni sadržaj i distribucija slobodnih cinkovih iona prikazani bojanjem na parafinskim tkivnim rezovima uporabom specifičnog fluorescentnog kelatora cinkovih iona Zinquina. Dodatna obrada rezultata je uključivala kvantifikaciju inteziteta imunohistokemijskoh bojanja, inteziteta bojanja Zinquinom te intenziteta signala bendova dobivenih Western blotom u odgovarajućim softverskim programima. Rezultati: Rezultati su pokazali veći konstitutivni izražaj MT I+II u hipokampusu, malom mozgu i jetri štakora AO soja u odnosu na DA soj. Izražaj MT I+II nije se znatno mijenjao nakon imunizacijskog protokola za indukciju EAE-a. U štakora DA soja je utvrđena znatna indukcija MT I+II u istim tkivima nakon imunizacije. Transkripcijska aktivnost MT-I gena ponašala se prema identičnom obrascu u oba soja. Prateći porast izražaja MT I+II nakon indukcije EAE-a, u DA soja povećala se i ukupna koncentracija tkivnog cinka u navedenim tkivima, posebice u jetri, gdje je utvrđen i porast bakra, dok se količina slobodnih cinkovih iona u mozgu i jetri štakora DA soja nije znatno mijenjala nakon imunizacijskog protokola. U AO soja sadržaj slobodnih cinkovih iona porastao je nakon imunizacije u hipokampusu i jetri, dok se ta razina značajno smanjila u malome mozgu. Izražaji citokina TGF-β i IL-6 ponašali su se prema obrascu izražaja MT I+II u oba soja. Utvrđen je viši konstitutivni izražaj transkripcijskog čimbenika NF-kB u jetri štakora AO soja, u odnosu na DA soj, koji je značajno snižen nakon imunizacije u štakora AO soja. U mozgu intaktnih štakora oba soja nije utvrđena znatna razlika tog čimbenika, te je on ostao gotovo nepromijenjen nakon imunizacije u štakora AO soja, a u hipokampusu DA soja se znatno povisio. Izražaj bjelančevine toplinskog šoka Gp96 pokazao je sličnost s izražajima MT I+II i citokina u štakora AO soja, dok se u DA soja izražaj istoga smanjio u dentatnom girusu hipokampusa nakon imunizacije. Zaključci: Između štakora DA i AO soja postoje razlike u inducibilnosti i konstitutivnom izražaju stresnih bjelančevina MT I+II i Gp96, što doprinosi različitoj sklonosti razvoju EAE-a. Osim toga, različitoj sklonosti, barem djelomično, doprinose i različiti izražaji citokina TGF-β i IL-6, kao i transkripcijskog čimbenika NF-kB u mozgu i jetri, molekula s dobro poznatom ulogom u upalnim i imunosnim procesima. Objectives: The aim of this study was to determine differences of constitutive and inducibile expression of stress proteins metallothioneins I+II (MT I+II) and heat shock protein glycoprotein 96 (Gp 96) between DA and AO rat strains which were subjected to immune protocol for induction of experimental autoimmune encephalomyelitis (EAE). Concomitantly, the difference of expression of cytokines IL-6 and TGF-β as well as transcription factor NF-kB was examined, since it is known that they have important role in pathogenesis of multiple sclerosis and EAE. Considering that MT I+II are closely related to tissue metals, concentrations of Zn and Cu ions were additionally assessed in the studied tissues. Materials and methods: The study was performed on male AO and DA rat strains, aged 2-3 months. The samples included hepatic, cerebral, cerebellar and hippocampal tissue from intact (nonimmunized) and immunized animals sacrificed in preclinical (7th day) phase of disease as well as in the moment of the most intensive clinical symptoms (12th day). Protein expression was determined by immunohistochemistry on paraffin-embedded tissue sections and by Western blot analysis using antibodies of interest. The gene expression of MT-Ia was analyzed by absolute quantitative real-time polymerase chain reaction. Tissue metal concentrations were determined spectrometricaly, while tissue content and distribution of free zinc ions was determined using ion chelator Zinquin. The intensity of immunohistochemical and Zinquin staining and Western blot bends was quantficated using computer software. The results showed higher constitutive hippocampal, cerebellar and hepatic expression of MT I+II proteins in AO rats in comparison to DA rats. Expression of MT I+II did not change significantly after immunization protocol for EAE induction in AO rats. In DA rats, high induction of MT I+II proteins was found after immunization in all analyzed tissues. Transcriptional activity of MT-Ia gene correlated with protein expression in both rat strains. Concurrently with MT I+II expression in DA rats after EAE induction, totally tissue zinc concentration has also increased, especially in liver, where increase of total copper concentration has also been observed. Furthermore, brain and hepatic level of free zinc ion did not change significantly after immunization. Hippocampal and hepatic level of free zinc ions increased, but cerebellar level decreased after immunization in AO rats. TGF-β and IL-6 expression correlated with MT I+II expression in both rat strains. Furthermore, higher constitutive hepatic NF-kB expression was found in AO rats in comparison to DA rats, and expression decreased significantly after immunization. No differences of NF-kB expression was found between brain tissues of both intact rat strains, but after immunization this transcription factor was significantly upregulated in hippocampus of DA rats, while in AO strain remained at a basal level. Expression of heat shock protein Gp96 showed similarity with MT I+II and cytokines expressions in AO rats, but this expression decreased in hippocampal dentate gyrus in DA rats after immunization. Conclusions: There is a different constitutive and inducible expression of stress proteins MT I+II and Gp96 between AO and DA rats, suggesting their role in different susceptibility to immune mediated encephalomyelitis. Moreover, different sensitivity could also be, at least partly, attributed to different expression of TGF, IL-6 and NF-kB, molecules with well-known role in the inflammatory and immune processes.