Glavni urednik

Research programme and results:

Alterations of the DPC4 tumor suppressor gene occurring in sporadic colon adenocarcinoma and renal cell carcinoma of Croatian patients were investigated. The study included a panel of 60 malignant tissues of colon, and 32 tumor specimens diagnosed as renal cell carcinoma. Using three pairs of specific primers for three DPC4 microsatellite repetitive sequences, we investigated the frequency of loss of heterozygosity. In order to increase heterozygosity, all tumor specimens were tested with primers for three polymorphic markers. The presence of the restricted single nucleotide change at specific codon in 2, 8, 10, and 11 exons (which belong to the conserved region of the gene) was examined by specific restriction fragment length polymorphism analysis. Moreover, the investigation was extended to other regions of the DPC4 gene by single strand conformation polymorphism analysis.

Samples used in this study displayed a high frequency of heterozygosity; (97% of colon carcinoma, and 94% of renal carcinoma samples). Out of all tested colon carcinoma samples, even 45% of them exhibited loss of heterozygosity at any investigated marker. On the other hand, merely 13% of informative renal carcinoma samples displayed allelic losses at any marker. We found a mutation in one colon adenocarcinoma sample in exon 11, and the mutation was verified by sequencing. Sequencing demonstrated a novel mutation – a deletion in exon 11 (134-153 del TAGACGAAGTACTTCATACC) of the DPC4 gene in the MH2 domain. No renal cell carcinoma samples were positive for DPC4 mutation.

These data suggest that inactivation of the DPC4 gene contributes to the genesis of colorectal carcinoma (but not renal cell carcinoma) through allelic loss, whereas mutation in the coding region of the gene is infrequently detected.

Oznaka: 0098099

ANTITUMORSKI UČINCI VIRUSA I ONKOLITIČKA VIRUSNA CJEPIVA

ANTITUMOROUS EFFECTS OF VIRUS AND ONCOLYTIC VACCINES
Voditelj/ica projekta: dr. sc. Mislav Jurin

Tel. ++385 1 4680 118   e-mail: [e-mail]

Suradnici na projektu:
Siniša Ivanković, magistar biomed. znanosti, asistent
Mislav Jurin, doktor med. znanosti, redovni profesor, znanstveni savjetnik
Biserka Pokrić, doktorica kem. znanosti, znanstvena savjetnica, (konzultantica)

Tehnički suradnici:
Nevenka Hiršl, samostalna tehničarka

Suradnici iz druge ustanove:
Stanislav Čajavec, doktor biotehnologije, viši znanstveni suradnik, Veterina d.o.o.
Michael Parnham, doktor med. znanosti, redovni profesor, Pliva (konzultant)

Program rada i rezultati na projektu:

Ispitan je učinak virusa Newcastelske bolesti (VNB) soja LaSote na rast fibroblasta L929, te stanica melanoma B16F10, fibrosarkoma FsaR, karcinoma SCCVII u kulturi te učinak različitih načina aplikacije virusa na rast navedenih tumora u miševa. Također je praćen imunološki odgovor organizma s tumorom nakon aplikacije virusa. Rezultati pokusa in vitro pokazali su da postoje diferencijalni mehanizmi citotoksičnosti s obzirom na dozu virusa i vrstu stanica. Virus je djelovao izrazito citotoksično na tumorske stanice, dok je učinak virusa na normalne stanice bio minimalan. Iako nije dovela do potpunog nestanka tumora, trokratna intraperitonealna i intratumorska aplikacija ovog soja VNB signifikantno je reducirala rast implantiranog mišjeg melanoma B16F10, fibrosarkoma FsaR i karcinoma SCCVII. Zabilježen je također i stimulatorni učinak virusa na imunološki sustav miševa s tumorom.

Research programme and results:

The influence of Newcastle disease virus (NDV), strain LaSota, on in vitro growth of L929 (fibroblasts), FsaR (fibrosarcoma), SCCVII (carcinoma) and B16F10 (melanoma) cells was determined. Pronounced cytotoxic action against tumor, but not against normal cells, was noticed. Further, the virus was applied into the mice with growing tumor. Following three injection of virus, either i.p. or in the tumor, significant suppression of tumor growth was noticed but there was no complete tumor regression. A stimulation of immune reaction was noticed in these treated tumorous mice.

Oznaka: 0098101

OKSIDATIVNI STRES I ZLOĆUDNE BOLESTI

OXIDATIVE STRESS AND MALIGNANT DISEASES
Voditelj/ica projekta: dr. sc. Neven Žarković

Tel. ++385 1 1607   e-mail: [e-mail]

Suradnici na projektu:
Suzana Borović, doktor biol. znanosti, znanstvena suradnica
Ana Čipak, dipl. inž. biologije, znanstveni novak
Marija Poljak Blaži, doktor biol. znanosti, znanstvena savjetnica
Neven Žarković, znanstveni savjetnik, voditelj projekta
Morana Živković, dipl. inž. biologije, znanstveni novak

Tehnički suradnici:
Nevenka Hiršl, znanstvena savjetnica, kemijska tehničarka
Tea Vuković, med. lab. ing

Suradnici iz druge ustanove:
Zoran Ćala, asistent, Opća bolnica Sveti Duh
Toni Hanich, asistent, doktor medicine, Opća bolnica Našice
Suzana Kukulj, doktor medicine, Klinika Jordanovac

Rudolf Joerg Schaur, doktor biok. znanosti, KFUni-Graz

Ivo Soldo Soldo, doktor med. znanosti, Opća bolnica Sveti Duh
Igor Stipančić, doktor med. znanosti, docent, Opća bolnica Dubrava


Program rada i rezultati na projektu:

Osnovno područje rada naše skupine temelji se na izučavanju pojavnosti oksidacijskog stresa u in vitro i in vivo uvjetima srodnim patofiziološkim stanjima vezanim uz ljudske bolesti (rak, imunološke i degenerativne poremećaje, traumatske ozljede, i sl.). U istraživanjima težimo ne samo proširenju temeljnih spoznaja o patofiziologiji oksidacijskog stresa, već i razvoju novih analitičkih postupaka, kao i metoda mogućeg nadzora tijeka pojave stresa radi smanjenja neželjenih, patoloških učinaka.

Izučavana je interakcija medijatora oksidacijskog stresa, aldehida 4-hidroksinonenala (HNE), koji nastaje kao produkt lipidne peroksidacije i bioaktivnog peptida BPC 157. Ovaj peptid je zanimljiv zbog svoje ubikvitarne nazočnosti i moguće medicinske primjene (kliničke studije medicinske korisnosti ovog peptida su u tijeku). Utvrđeno je da BPC 157 može smanjiti toksično djelovanje HNEa na stanice kosti i hrskavice, a također i da smanjuje imunosupresivno djelovanje opeklinske ozljede u miševa. Slično tome, uočeno je i imunomodulacijsko djelovanje protu-anemijskog preparata feri sorbitol citratnog kompleksa u eksperimentalnom modelu artritisa. Rad naše skupine omogućio je i objavljivanje revijalnog rada o HNEu kao bioaktivnom medijatoru oksidacijskog stresa u časopisu Molecular Aspects of Medicine koji je pobudio znatan interes stručnjaka u ovom području, pogotovo stoga što je u ovom radu po prvi puta navedena mogućnost da je HNE jedan od najvažnijih čimbenika prijenosa staničnih signala.

Pokazano je imunostimulacijsko djelovanje antianemijskog pripravka feri sorbitol citrata (Jectofer Astra Linz Austrija), za koji smo ranije našli da ima antitumorsko djelovanje in vivo i in vitro. Jektofer u starih miševa uzrokuje porast mase timusa i slezene. Jektofer stimulira antigen prezentirajuće stanice, lučenje TNF alfa i stanični imunološki odgovor. Zanimljivo je da taj spoj željeza ne pojačava simptome antigenom izazvanog artritisa kao što to čine drugi spojeva željeza.

Tijekom 2003. godine publicirali smo i izvorni in vitro model uzgoja tkiva ljudske kosti kojim smo potvrdili nazočnost osteogenih čimbenika u serumu osoba s traumatskim ozljedama središnjeg živčanog sustava koje uzrokuju pojačanu osteogenezu. Ovaj model koristimo i za analizu učinaka medijatora oksidacijskog stresa u regulaciji rasta stanica koštanog podrijetla.

Tijekom 2003. u našoj je skupini izrađen i diplomski rad pod nazivom “Dokazivanje antitumorskog učinka neutrofila” (student Morana Živković), kojim smo uveli izvorni model za izučavanje uloge neutrofila u oksidacijskom stresu te njihove antitumorske aktivnosti (istraživanja su provedena u suradnji s kolegama iz Graza). Istraživanje pojavnosti oksidacijskog stresa na subcelularnoj razini u granulocitima provedeno je izvornom metodom imunoelektronske mikroskopije i kemiluminescencijom.

Naposljetku, 2003. godine smo, zajedno s kolegama iz Austrije i Rumunjske, objavili rezultate prve kliničke studije protutumorskog djelovanja biljnog lijeka Isorel, koji je zahvaljujući našim istraživanjima prepoznat kao jedan od pet fitomedicinski učinkovitih lijekova.

Research programme and results:

Major research topic is the evaluation of oxidative stress under experimental conditions in vitro and in vivo that resemble clinical disorders based on oxidative stress (cancer, immune disorders and degeneration, traumatic injuries, etc.). Our research tends not only to study the basic aspects of pathophysiology of oxidative stress but also to develop novel analytical methods and treatments for the control of oxidative stress that would attenuate its harmful, pathological consequences.

Interaction between the mediator of oxidative stress, the aldehyde 4-hydroxynonenal (HNE), generated as a product of lipid peroxidation, and the bioactive peptide BPC 157 was studied. This peptide is attractive for its ubiquitary presence and possible medical application (clinical studies on its medical efficiency are under progress). It was observed that BPC 157 can attenuate toxic effects of HNE on the bone and cartilage cells, as well as it can decrease immunosuppressive effects of burn injury in mice.

Similarly, immune modulating effects were noticed for the antianemic ferric-sorbitol-citrate complex drug in experimental model of arthritis.

The work of our team lead to the review article on HNE as bioactive marker of oxidative stress, published in the Molecular Aspects of Medicine, which raised significant interest among researchers in the field, in particular because, in this review - for the first time, HNE was considered as one of most important cellular signaling factors.

We have shown that ferric-sorbitol citrate (Jectofer, Astra, Linz, Austria), which has anti-tumor activities in vivo and in vitro, also stimulates immune reaction. Enlargement of thymus end spleen were noted in old normal mice treated with Jectofer. This iron compound also stimulates TNF-alfa production, antigen presenting cell activity and cellular immune response in normal mice. However, Jektofer did not intensify antigen-induced arthritis in mice, while other ferric compounds aggravated arthritis.

In 2003, we published genuine model for in vitro tissue cultures of the human bone in which we confirmed the presence of osteogenic factors in the sera of patients with traumatic brain injury that might cause enhanced osteogenesis. This model is used nowadays to analyze effects of the mediators of oxidative stress in the growth control of the bone cells.

During the 2003, a graduate (diploma) thesis was completed under the title Investigation of Anti-tumor Activity of Neutrophils (Morana Živković), where we introduced a genuine model for research on oxidative stress and antitumor activity of neutrophils (research was carried in collaboration with the colleagues from Graz). The research on subcellular appearance of oxidative stress in granulocytes was evaluated by the genuine immuno-electron microscopy method and by chemiluminescence.

Finally, in 2003., we published, together with the colleagues from Austria and Romania, the results of the first clinical trial on the plant extract Isorel, which is, due to our research, considered among five efficient phytotherapeutic drugs of this kind.

Oznaka: 0098102

MOLEKULARNI MEHANIZMI IMUNOSUPRESIJE

MOLECULAR MECHANISMS OF IMMUNOSUPPRESSION
Voditelj/ica projekta: dr. sc. Renata Novak Kujundžić

Tel. ++385 1 4560949   e-mail: [e-mail]

Suradnici na projektu:
Katja Ester, dipl. inž. molekularne biologije, znanstvena novakinja

Suradnici iz druge ustanove:
William Lauman Ragland, doktor med. znanosti, znanstveni savjetnik, University of Georgia, College of Veterinary Medicine, Athens, Georgia, SAD
Vladimir Savić, doktor biol. znanosti, znanstveni suradnik, Veterinarski institut RH, Centar za peradarstvo

Program rada i rezultati na projektu:

Interferon alfa i gama djeluju protuvirusno direktno - inhibicijom replikacije virusa ili indirektno - aktivacijom efektorskih stanica. Istraživali smo sposobnost pilića da proizvode interferon tijekom infekcije virusom Marekove bolesti (MDV) uz pomoć testa kompetitivne hibridizacije za određivanje količine mRNA za pileći interferon alfa i gama. Pokusi su rađeni na liniji pilića B13/B13, genetski sklonoj razvoju tumora uslijed infekcije virusom Marekove bolesti i na liniji B21/B21, koja je rezistentna na nastanak tumora. Istraživan je učinak onkogenog RB-1B soja MDV-a i cijepnog soja herpesvirusa purana (HVT) na transkripciju gena za interferon alfa i gama.

Onkogeni MDV je imao negativan učinak na transkripciju gena za interferon alfa i gama prvog i sedmog dana nakon inokulacije u pilića B13/B13 linije, ali samo sedmog dana po inokulaciji u pilića B21/B21 linije. Suprotno ovom nalazu, neonkogeni HVT nije utjecao na transkripciju gena za interferon alfa i gama prvog dana nakon inokulacije, a sedmog dana nakon inokulacije pilića linije B21/B21 supresivni učinak HVT-a na transkripciju gena za interferon alfa i gama je bio blaži od učinka MDV-a. Navedeni rezultati upućuju na to da sposobnost MDV-a da onemogući proizvodnju mRNA za interferon, mjereno u krvi, već prvog dana nakon inokulacije pilića linije B13/B13 ne samo da dovodi do imunosupresije, nego može biti povezan i sa onkogenošću virusa.

Research programme and results:

Interferon alpha and gamma display antiviral activity either directly through inhibition of viral replication or indirectly through activation of effector cells. We evaluated the ability of chickens to produce interferon during infection with Marek's disease virus (MDV) using competitive hybridization for measuring the abundance of mRNA for chicken interferon alpha and gamma. Chicken line B13/B13, genetically susceptible to tumor development during infection with Marek's disease virus, and chicken line B21/B21, genetically resistant to tumor development, were used throughout the experiments. The effects of oncogenic strain of MDV and vaccinal strain of turkey herpes virus (HVT) on transcription of interferon alpha and gamma genes were studied. Oncogenic strain of MDV had negative effect on transcription of genes for interferon alpha and gamma on the first and seventh day post infection in B13/B13 chickens, while it had negative effect only the seventh day in B21/B21 chickens. Vaccinal strain had no influence on transcription of interferon genes the first day post infection, and on the seventh day it had milder negative effect in B21/B21 chickens compared to oncogenic MDV. Our results imply that the ability of oncogenic strain of MDV to block transcription of interferon genes, measured in blood, on the first day post infection of B13/B13 chickens not only causes immunosuppression but also may be related to the oncogenicity of the virus.

Oznaka: 0098103

NEUROTRANSMITORI U STRESU I REGULACIJA GABA RECEPTORA IN VITRO

NEUROTRANSMITTERS IN STRESS AND REGULATION OF GABA RECEPTORS IN VITRO
Voditelj/ica projekta: dr. sc. Danka Peričić

Tel. ++385 1 4561-126   e-mail: [e-mail]

Suradnici na projektu:
Milica Bjegović, doktorica med. znanosti, znanstvena suradnica
Maja Jazvinšćak Jembrek, magistrica biol. znanosti, asistent, znanstvena novakinja
Danka Peričić, doktorica med. znanosti, znanstvena savjetnica
Ivana Rajčan, znanstvena novakinja, doktorica medicine
Dubravka Švob Štrac, dipl. inž. molekularne biologije, mlađi asistent, znanstvena novakinja
Suradnici iz druge ustanove:
Branimir Živković, doktor med. znanosti, znanstveni savjetnik, (konzultant), Medicinski fakultet Sveučilišta u Zagrebu

Program rada i rezultati na projektu:

Cilj projekta jest bolje upoznavanje mehanizama koji nakon kronične primjene lijekova dovode do adaptivnih promjena GABA-A receptora. U tu svrhu, kao model nam je poslužila kultura embrionalnih stanica bubrega čovjeka (HEK 293) koje stabilno eksprimiraju alpha1 beta2 gama2s GABA-A receptore, što je najčešći tip GABA-A receptora u mozgu. Kronično izlaganje HEK 293 stanica GABA-i i muscimolu, ali ne i diazepamu, povećalo je broj GABA-A receptora. Kako se čini, do porasta dolazi zbog povećane sinteze, a ne smanjene razgradnje proteina. Za razliku od kronične primjene diazepama, koja dovodi do kidanja funkcionalnih veza između veznog mjesta za GABA-u i onog za benzodiazepine ("uncoupling"), kronična primjena GABA-e ne izaziva te promjene. Daljnja istraživanja trebaju utvrditi relevantnost ovih podataka za objašnjenje fenomena tolerancije i ovisnosti koji se javlja nakon duže primjene benzodiazepina i drugih pozitivnih modulatora GABA-A receptora.

Cilj našeg projekta jest također rasvjetljavanje mehanizama odgovornih za antikonvulzivno djelovanje stresa. Ranije smo pokazali da su alfa2-adrenoceptori važni za spomenuto djelovanje. Sada smo pokazali da stres plivanjem inhibira mehanizmom koji ne ovisi o alpha2-adrenoceptorima, ponašanje ovisno o 5-HT2A receptorima, što znači da su učinci stresa na konvulzivni prag i na ponašanje ovisno o 5-HT2A receptorima izazvani različitim mehanizmima.

Research programme and results:

The aim of our project is to better understand the mechanisms that underlie adaptive changes in GABA-A receptors, following their prolonged exposure to drugs. Stably transfected human embryonic kidney (HEK) 293 cells were used as a model to study the effects of prolonged drug exposure on the recombinant alpha1 beta2 gamma2s GABA-A receptors, the most common type of GABA-A receptors found in the brain. Chronic exposure of HEK 293 cells to GABA and muscimol, but not to diazepam, enhanced the number of GABA-A receptors. This increase appears to be due to increased synthesis rather than decreased degradation of receptor proteins. Unlike chronic diazepam, which produced functional uncoupling of GABA and benzodiazepine binding sites in the absence of GABA and neuronal elements, chronic GABA failed to produce allosteric uncoupling. Further studies are needed to determine the relevance of these results to the phenomena of tolerance and dependence produced by prolonged treatment with benzodiazepines and other positive modulators of GABA-A receptors.

The aim of our study was also to elucidate the mechanism of the anticonvulsant effect of stress. The involvement of alpha2-adrenoceptors has been proposed. We demonstrated that swim stress inhibits by an alpha2-adrenoceptor unrelated mechanism, 5-HT2A receptor-mediated head twitch behaviour in mice, suggesting that this effect and the swim stress-induced anticonvulsant effect are produced by two separate and independent mechanisms.

Oznaka: 0098104

MOLEKULARNO-GENETIČKI I PROGNOSTIČKI ČIMBENICI U NASTANKU RAKA VRATA MATERNICE

GENETIC AND MOLECULAR PROGNOSTIC FACTORS OF CERVICAL CANCEROGENESIS
Voditelj/ica projekta: dr. sc. Magdalena Grce

Tel. ++385 1 4561110   e-mail: [e-mail]

Suradnici na projektu:
Koraljka Husnjak, magistrica biol. znanosti, asistent, znanstveni novak
Mihaela Matovina, magistrica biol. znanosti, znanstveni novak

Tehnički suradnici:
Jasminka Golubić, med. lab. inž.

Suradnici iz druge ustanove:
Ivan Fistonić, doktor med. znanosti, doc., KB Merkur, Zagreb
Petar Klarić, doktor med. znanosti, doc., KB Sestre milosrdnice, Zagreb

Program rada i rezultati na projektu:

Cilj projekta je praćenje prognostičkih čimbenika uključenih u nastanak i progresiju raka vrata maternice. U tu svrhu praćena je prisutnost ključnih čimbenika rizika, poglavito genitalna infekcija humanim papilomavirusima (HPV) u žena. Budući da je infekcija HPV-om nužna, ali ne i dovoljna za razvoj raka vrata maternice, razmatrali smo, također, i ulogu ostalih spolno prenosivih infekcija (STI) kao npr. herpesvirusa, klamidija, mikoplazme i ureaplazme. Osim toga, postavili smo in vitro model istraživanja protuvirusnog učinka raznih substanci, te ispitali 1,2,5-oksadiazinske derivate.

Research programme and results:

The proposed research involves the investigation of risk and prognostic factors of cervical cancerogenesis. Thus, the most important risk factor human papillomavirus (HPV) was investigated in female genital specimens. As the HPV infection is necessary but not sufficient, other sexually transmitted infections (STI), such as herpesviruses, chlamydia, micoplasma and ureaplasma were investigated. Beside that, an antiviral testing model was established and 1,2,5-oksadiazine derivates were tested.

Oznaka: 0098106

ULOGA GENA FHIT U NASTANKU NEUROENDOKRINIH TUMORA

THE ROLE OF FHIT GENE IN NEUROENDOCRINE TUMORS
Voditelj/ica projekta: dr. sc. Šimun Križanac

Tel. ++385 1 4561 114   e-mail: [e-mail]

Suradnici na projektu:
Sanja Kapitanović, doktorica med. znanosti, viša znanstvena suradnica, (konzultantica)
Krešimir Pavelić, doktor med. znanosti, znanstveni savjetnik, (konzultant)


Tehnički suradnici:
Jelena Barač
Ozrenka Poljak

Suradnici iz druge ustanove:
Zlata Ivanovi-Herceg, doktorica med. znanosti, izvanredna profesorica, Klinika za plućne bolesti Jordanovac, Zagreb
Jerolim Karadža, doktor med. znanosti, Klinika za plućne bolesti Jordanovac, Zagreb
Smiljka Lambaša, magistrica med. znanosti, Klinička bolnica Dubrava, Zagreb
Ljubomir Pavelić, doktor med. znanosti, izvanredni profesor, Klinika za plućne bolesti Jordanovac, Zagreb (konzultant)
Fadila Pavičić, doktor med. znanosti, redovni profesor, Klinika za plućne bolesti Jordanovac, Zagreb (konzultantica)
Miroslav Samaržija, doktor med. znanosti, znanstveni suradnik, Klinika za plućne bolesti Jordanovac, Zagreb (konzultant)

Program rada i rezultati na projektu:

Osnovni cilj projekta je rasvijetliti molekularne osnove nastanka neuroendokrinih tumora s posebnim naglaskom na gen FHIT. Unutar gena FHIT nalazi se izuzetno lomljivo mjesto, FRA3B, zbog kojeg je gen podložan brojnim mutacijama. Točkaste mutacije gena FHIT relativno su rijetke, međutim, homozigotna delecija eksona ili insercija intronskih sekvenci, nastanak aberantnih transkripata, kao i nemogućnost detekcije transkripta, relativno su česte u različitim primarnim tumorima čovjeka npr. tumorima pluća, želuca, glave i vrata. Izgleda da se radi o stečenim mutacijama jer, npr., izlaganje karcinogenima može potaknuti početnu zloćudnu preobrazbu (stvaranje preneoplastičnih lezija) u različitim slojevima epitela dušnika i to upravo kreiranjem različitih promjena gena FHIT.

Research programme and results:

The general goal of the project is to elucidate molecular pathophisiology of neuroendocrine tumors. Specific goal is to determine the role of FHIT gene inactivation in development of tumors using molecular genetic methods: reverse transcription of FHIT mRNA, PCR, DNA-amplification, DNA-sequencing, allelic loss detection by microsatellite polymorphism analyses, cell proliferation assay. The genomic structure of FHIT overlaps with the FRA3B fragile site and coincides with a genomic region that is known to be frequently involved in allelic loss, genetic rearrangement, and cytogenetic abnormality in solid tumors. Although point mutations within the FHIT gene have rarely been reported, genomic alterations, such as homozygous deletions of exons or insertions of intronic sequences and aberrant transcripts of the FHIT gene, as well as the lack of detectable FHIT protein, have all been frequently observed in lung and head and neck cancers. There is a possibility that a constitutional alteration within the FHIT gene could be associated with a predisposition to develop both lung and renal cancer or other types of multiple primary tumors.

Oznaka: 0098107