Microorganisms, especially viruses, in the treatment of tumor diseases

Najčešći i uobičajeni pristupi u liječenju tumorskih bolesti uključuju kirurgiju, radioterapiju, kemoterapiju, imunoterapiju, ciljanu terapiju, hormonsku terapiju, transplantaciju matičnih stanica te sveobuhvatno personalizirano liječenje. Izostavlja se gotovo zaboravljeno liječenje primjenom različitih mikroorganizama od kojih su najzastupljeniji virusi. Njihova je neposredna primjena, ako se radi o apatogenim virusima ili njihovim genomskim modifikacijama, sve učestalija i postigla je vrlo visok stupanj primjenjivosti. Ovaj prikaz odnosi se pretežno na mogućnosti primjene mikroorganizama, u manjoj mjeri bakterija, a uglavnom virusa, u liječenju tumorskih bolesti. Neki virusi mogu zaraziti i ubiti tumorske stanice te su poznati kao onkolitički. Neki se od njih nalaze u prirodi, ali su također i modificirani, kako bi se pojačalo njihovo onkolitičko svojstvo, djelujući isključivo na tumorske stanice bez oštećivanja zdravih. U najnovije vrijeme genetički modificirani virusi postigli su stupanj javne upotrebe stavljanjem na tržište te su od FDA-a (Food and Drug Administration) dopušteni za liječenje melanoma (T-VEC)¸ dok se u Kini modificiranim adenovirusom (H101) liječi rak glave i vrata ljudi. Posebna je vrijednost virusa u liječenju tumorskih bolesti, jer se umnažaju u tumorskoj stanici dok je ne razore, tvoreći tako kompleks tumorskog antigena što omogućuje njegovo prepoznavanje od strane imunosnog sustava. Reakcija tog sustava je lokalna ili sustavna, prepoznata danas kao imunoterapija. Uspoređeni s klasičnim oblicima liječenja tumorskih bolesti, primjena onkolitičkih virusa i imunoterapija uspješniji su način s tek blagim nuspojavama. Danas suvereno usmjeruju istraživanja upravo u tom smislu.The most frequent and usual approaches in treating tumor diseases include surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, hormonal therapy, transplantation of stem cells and comprehensively personalized treatment. Treatment using various microorganisms, mainly viruses, has become neglected and almost forgotten. Their direct application, in terms of apathogenic viruses or their genomic modifications, is becoming increasingly common, and has achieved a high level of applicability. This paper relates mainly to the possibility of using microorganisms, mainly viruses and to a lesser extent bacteria, in treating tumor diseases. Some viruses can infect and kill tumor cells, and they are known as oncolytic. Some of them are found in nature, but there are also some that have been modified, in order to enhance their oncolytic potential, acting exclusively on tumor cells without damaging healthy cells. Very recently, genetically modified viruses have achieved a degree of public use by being placed on the market, and have been permitted by the FDA (Food and Drug Administration) for treating melanoma (T-VEC), and in China modified adenovirus (H101) is used in treating cancers of the head and neck in humans. The special value of viruses used for treating tumor diseases is that they multiply inside the tumor cell until they destroy it, thereby creating a complex of tumor antigens, which makes them recognizable by the immune system. The reaction of that system is local or systemic, and this is recognized today as immunotherapy. In comparison with classical forms of treatment for tumor diseases, the use of oncolytic viruses and immunotherapy is more successful, with only mild side-effects. Today, they are dominant in research precisely for that reason

Combined Action of Virus Injection and Local Tumor Irradiation on Tumor Growth in Mice

The dynamics of SCCVII transplantable tumor growth in C3H/H mice was determined after local tumor irradiation and/or virus (NDV LaSota) i.p. injection. The virus applied alone significantly suppressed tumor growth, particularly until the 19th day after tumor transplantation. Local irradiation with 30 Gy resulted in tumor disappearance followed with its regrowth about 15 days later. However, if the virus was injected after the irradiation, there was no tumor growth until the end of the 31 day observation period. It should be noted that virus application prior to local irradiation did not have any additional influence on tumor growth. Thus, the pronounced efficacy of virus applied after tumor irradiation deserves attention. It is possible that the virus injected after irradiation induced a chain of cytokine production joining the action of tumor destruction induced by irradiation. This should be further studied in clarifying the approaches to combined tumor therapy with possible cell-free vaccine production

In vitro antifungal effect of phenylboronic and boric acid on Alternaria alternata

The ascomycete fungus Alternaria alternata causes early blight, one of economically the most important tomato diseases. Due to frequent use of fungicides, A. alternata has developed resistance with negative economic and environmental consequences. Research of new ways to control fungal pathogens has turned its eye to environmentally friendly chemicals with low toxicity such as boronic acids. The aim of our study was therefore to test the antifungal effects of phenylboronic and boric acid in vitro on A. alternata. We isolated the pathogen from a symptomatic tomato plant and determined the minimum inhibitory concentration of phenylboronic and boric acid on A. alternata mycelial growth using the poisoned food technique. The antifungal effect was tested on a wide range of phenylboronic and boric acid concentrations (from 0.04 % to 0.3 %) applied separately to agar with mycelial disc of the pathogen. After five days of incubation, phenylboronic acid at low concentration (0.05 %) completely inhibited mycelial growth. Boric acid, in turn, did not significantly slow down mycelial growth but did reduce sporulation and confirmed its fungistatic effect. Our findings point to the potential use of phenylboronic acid to control phytopathogenic fungi. This is, to our knowledge, the first report on its antifungal effect on an agriculturally important pathogen in vitro. Moreover, since A. alternata is also a human pathogen, these results may have clinical ramifications

The Effect of Nonaromatic Naphthalane on Mice Oral Planocellular Carcinoma - a Pilot Study

Nearomatski visokosteranski naftalan (NAVS) posebna je frakcija hrvatske nafte, iznimno bogata steranima, iz koje je odstranjen aromatski sastav. Steranima se pripisuje bioaktivnost slična steroidnim hormonima, modulatorima tumorskoga rasta. Potaknuti rezultatima prijašnjih in vitro i in vivo studija o inhibitornom efektu NAVS-a na rast planocelularnog karcinoma, proveli smo studiju o njegovu učinku na oralni planocelularni karcinom (OPCC) u miševa. Ciljevi: 1) ispitati valjanost jednostavnog eksperimentalnog modela OPCC-a; 2) ispitati mogući antiproliferativni učinak NAVS-a na postavljenome modelu praćenjem tumorskoga rasta; 3) ispitati antineoangiogeni učinak NAVS-a kao objašnjenje mogućeg antiproliferativnog učinka, te za tu prigodu procijeniti mogućnost križne reaktivnosti anti-humanih imunohistokemijskih markera s mišjim tkivom. Suspenzija od 100 μl s 105 SCC VII stanica inokulirana je intraoralnim putem pod bukalnu sluznicu u 48 singeničnih C3H miševa. Sedam dana nakon inokulacije životinje su podijeljene u 6 jednakih skupina, te se miševima, ovisno o skupini, intratumorski injiciralo po 100 μl sljedećih tvari: parafinsko ulje (PO) kao negativna kontrola, NAVS (u jednoj skupini 7 dana, a u drugoj skupini 14 dana nakon inokulacije tumora), 1, 25 dihidroksiergotamin (1,25-D3) kao pozitivna kontrola, te kombinacije NAVS s 1,25-D3 i PO s 1,25-D3. Rast tumora praćen je tjednim mjerenjem s pomoću kalipera. Životinje su žrtvovane 1., 2., 3. i 4. tjedna nakon aplikacije ispitivanoga sredstva. Histološki pripravci eksplantiranih tumora bojeni su hematoksilin-eozinom te imunohistokemijski s anti-CD34 protutijelima radi procjene tumorske neoangiogeneze. U usporedbi s PO skupinom, tumorski rast i angiogeneza bili su sniženi u 1,25-D3 i NAVS skupinama. NAVS vjerojatno smanjuje rast OPCC-a inhibicijom vaskularne proliferacije potrebne za tumorski rast.Nonaromatic naphthalane (NAVS) is a specific fraction of Croatian oil, extremely rich in steranes from which the aromatic system is removed. Steranes are attributed with bioactivity similar to steroid hormones, modulators of tumour growth. Encouraged by the results of earlier in vitro and in vivo studies on the inhibitory effect of NAVS on the growth of planocellular carcinoma, we carried out a study on its effect on oral planocellular carcinoma (OPCC) in mice. Aims: 1) To test the validity of the simple experimental model OPCC, 2) to test possible antiproliferative effect of NAVS on the above model by monitoring tumour growth, 3) to test the antineoangiogenic effect of NAVS to explain the possible antiproliferative effect, and to estimate the possibility of crisis reactivity of anti-human immunohistochemical markers with mice tissue. A suspension of 100 μl s 105 SCC VII cell was inoculated intraorally under the buccal mucous membrane in 48 syngeneic C3H mice. Seven days after inoculation the animals were divided in six equal groups and the mice, depending on the group, were intratumorously injected with 100 μl of the following substances: paraffin oil (PO) as a negative control, NAVS (in one group 7 days, and in the second group 14 days, after inoculation of the tumour), 1.25 dihydroxyergotamine (1.25-D3) as a positive control, and a combination of NAVS with 1.25 D3 and PO with 1.25- D3. Tumour growth was monitored weekly by measuring with callipers. The animals were sacrificed 1, 2, 3 and 4 week after application of the tested substance. Histological specimens of explanted tumours were stained with hematoxylin-eozine, and immunohistochemically with anti-CD34 antibodies for estimation of tumour neoangiogenesis. Compared with the PO group, tumour growth and angiogenesis were decreased in the 1.25-D3 and NAVS groups. NAVS probably reduced growth by OPCC inhibition of vascular proliferation, needed for tumour growth

Uloga regulatornih T limfocita u imunosnoj kontroli MC-2 fibrosarkoma

The role of T regulatory lymphocytes (Treg) particularly in cancer is well known. The goal of the present study was to determine the contribution of these lymphocytes in the regulation of anti-tumor immunity of CBA/HZgr mice against MC-2 fibrosarcoma (4th generation of methylcholanthrene induced tumor). The levels of T lymphocytes (CD4+, CD8+ and CD4+CD25+) were determined 8 and 20 days after tumor transplantation. Further, the role of CD4+CD25+ (Tregs) in tumor-host interaction was evaluated in vitro and in vivo by using specific monoclonal antibodies. We found that splenocytes of both control and Treg depleted tumor bearing mice strongly but differently inhibited growth of tumor cells in vitro. While splenocytes of untreated mice exhibited significant decrease of this activity (from 74.4% to 62.6% and 32.95%), the splenocytes of Treg depleted mice showed increase of this activity (from 79.5% to 84.3% and 86.2%) from day 6 to day 13 and day 21 after tumor grafting, respectively. Further, upon i.v. injecting specific monoclonal anti-Treg antibody tumor immediately prior to tumor cell intracutaneous transplantation, the tumor was rejected after initial growth. In treated mice, the incidence of Treg cells was very low initially, reaching normal values two weeks later. These animals were shown to be resistant to tumor transplantation four months later.Uloga T regulatornih limfocita dobro je poznata, osobito kod tumora. Cilj ovoga istraživanja bio je utvrditi doprinos ovih limfocita u regulaciji antitumorske imunosti CBA/HZgr miševa protiv MC-2 fibrosarkoma (4. generacija metilkolantrenom izazvanog tumora). Razine T limfocita (CD4+, CD8+ and CD4+CD25+) određivale su se 8 i 20 dana nakon transplantacije tumora. Nadalje, uloga CD4+CD25+ T limfocita (Treg) u interakciji tumora i domaćina procijenjena je in vitro i in vivo primjenom specifičnih monoklonskih protutijela. Utvrdili smo da splenociti kako kontrolnih miševa tako i miševa s tumorom bez Treg snažno, ali različito suzbijaju rast tumorskih stanica in vitro. Dok su splenociti netretiranih miševa pokazivali značajno smanjenje ove aktivnosti (sa 74,4% na 62,6% i 32,95%), splenociti miševa bez Treg pokazivali su porast ove aktivnosti (sa 79,5% na 84,3% i 86,2%) od 6. do 13. i 21. dana nakon transplantacije tumora. Uz to, nakon i.v. injektiranja specifičnog monoklonskog anti-Treg tumorskog protutijela neposredno prije intrakutane transplantacije tumorskih stanica, nakon početkog rasta nastupilo je odbacivanje tumora. Kod tretiranih miševa incidencija Treg stanica bila u početku vrlo niska i dostigla je normalne vrijednosti dva tjedna kasnije. Nakon četiri mjeseca pokazalo se da su ove životinje otporne na transplantaciju tumora

3'-terminated Overhangs Regulate DNA Double-Strand Break Processing in Escherichia coli

Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in Escherichia coli To study DSB processing in vivo, we induced DSBs into the E. coli chromosome by gamma irradiation and measured chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with ssDNA. RecA decreased DNA degradation in wild- type, recB and recD strains, indicating that it is a general phenomenon in E. coli On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos). 3'-5' ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding 3' tail from degradation by 3'-5' ssExos. Since 3'-tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3' tails emanating from DSBs, along with 3' tail's massive length, or prevention of their degradation by inactivation of 3'-5' ssExos. Thus, we conclude that 3' overhangs are crucial in controlling the extent of DSB processing in E. coli This study suggests a regulatory mechanism for DSB processing in E. coli, wherein 3' tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends

Control of early blight fungus (Alternaria alternata) in tomato by boric and phenylboronic acid

Finding a suitable alternative to the small pool of existing antifungal agents is a vital task in contemporary agriculture. Therefore, intensive research has been conducted globally to uncover environmentally friendly and efficient agents that can suppress pathogens resistant to the currently used antimycotics. Here, we tested the activity of boric acid (BA) and its derivative phenylboronic acid (PBA) in controlling the early blight symptoms in tomato plants infected with pathogenic fungus Alternaria alternata. By following the appearance and intensity of the lesions on leaves of the tested plants, as well as by measuring four selected physiological factors that reflect plant health, we have shown that both BA and PBA act prophylactically on fungal infection. They did it by reducing the amount and severity of early blight symptoms, as well as by preventing deterioration of the physiological traits, occurring upon fungal inoculation. Phenylboronic acid was more efficient in suppressing the impact of A. alternata infection. Therefore, we conclude that BA, and even more so PBA, may be used as agents for controlling early blight on tomato plants, as they are both quite effective and environmentally friendly

ZADOVOLJSTVO UČENIKA, RODITELJA I NASTAVNIKA ŠKOLOM U SVJETLU RASTEREĆENJA UČENIKA I NASTAVNOGA PROCESA U SREDNJOJ ŠKOLI (pregled rezultata istraživanja)

Ovo istraživanje i usporedba mišljenja učenika, roditelja i nastavnika ide u prilog potrebi rasterećenja učenika u školi, a upućuje i na mjere i postupke koje bi trebalo poduzeti u smislu kvalitetnijega i potpunijega rasterećenja učenika i nastavnoga procesa u cijelosti. Cilj je povećanje zadovoljstva radom kod nastavnika, pozitivniji stav učenika prema školi, kao i veće zadovoljstvo školom i povećano uključivanje roditelja.This research and comparison of the attitudes of students, parents and teachers greatly supports the need to relieve students from excess duties in schools and points at measures and procedures that ought to be taken with the purpose of relieving students and the teaching process as a whole as efficiently and fully as possible. The aim of this research is the increase of teachers’ satisfaction with their work, achievement of positive attitude of students to school in general and their satisfaction with it, as well as an increased involvement of their parents