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Objective. After the beginning of the COVID-19 pandemic caused by the SARS-CoV -2 virus, enormous pressure fell on the entire health system. Since there is no adequate cure for this disease, "off-label" use of several drugs (azithromycin, chloroquine, hydroxychloroquine, etc.) was resorted to. The aim of this study was to analyse QTc interval dynamics and its relationship with other factors which could influence outcome in patients with COVID-19. Methods. Study has observational, case-control design with retrospective data collection from medical files of adult patients, with RT-PCR confirmed COVID-19. The cases (n=30) were subjects with fatal outcome and the controls (n=169) were the survivors. The QTc interval was calculated on admission, during and after initial drug treatments with presumed activity against SARS-CoV-2, mostly antimalarials. Primary independent and outcome variables were QTc interval prolongation and all-cause mortality, respectively. Results. Study population included 120 males (60.3%), the mean patients age was 57.3+-15.8 years (+-SD). The most common comorbid illnesses were hypertension (98 patients), pre-existing arrhythmias (32) and diabetes mellitus (29). The most frequently prescribed QTc prolonging drugs were azithromycin (69.8% of patients), chloroquine (50.3%) and hydroxychloroquine (42.7%). Total of 131 patients (65.8%) had QTc interval increase >60 ms from baseline, of whom 5 had QTc prolongation >500 ms (2.5%). De novo ventricular tachyarrhythmias were registered at 14 patients (7%) and 13 (92.8%) of them died. Pre-existing arrhythmias (odds ratio 9.30, 95% confidence interval 2.91-29.73, p480 ms) did not (1.02, 0.22-4.67, p=0.974). Case fatality rate was 15.1%, as 30 patients died during hospitalization. Conclusion. Clinical importance of drug-induced QTc interval prolongation of hospitalized patients with COVID-19 should be considered primarily within the context of other risks, particularly older age, pre-existing cardiovascular disorders and major electrolyte disturbances.

Coronavirus disease (COVID-19), since its appearance, has put a large burden on the global health system which have strived to mitigate the pandemic, but mortality of COVID-19 continues to increase. Many authors have employed machine learning (ML) algorithms in the investigation of COVID-19 in order to identify infected individuals, predict their condition in time, predict the outbreaks and forecast certain numbers. Although there are many studies that examine the application of ML in the diagnosis of prognostic biomarkers and survival prediction several days in advance, there is a limited literature dealing with evidence to label patients in more categories (mild, moderate, severe, etc.) that would help not only to respond in a timely manner to prevent lethal results, but also to minimize the number of patients in hospitals where this is not the case. In this paper we present a methodology for classification of patients into 3 distinct classes of clinical condition (mild, moderate and severe) of COVID-19 disease and prediction of the outcome (change of severity of clinical condition) in advance. The results show that XGBoost classifier achieved average accuracy of 88%. The main advantage of our system is that it is a rule-based algorithm which is easier to implement in a real clinical practice, instead of the use of black box models, which are not appealing for real clinical use.

The use of artificial intelligence, especially machine learning methods in creating models that will be applied in clinical practice has reached its peak with the appearance of the COVID-19 pandemic. This study aims to determine the severity of the clinical condition of COVID-19 patients based on blood marker analysis. The study used data from 60 COVID- 19 patients treated at the Clinical Center Kragujevac. The research methodology includes the selection of the most important laboratory parameters as well as the classification of patients depending on them using methods of supervised learning, regression and classification. With an accuracy of 90%, three parameters were selected that can mostly indicate the severity of the patient's condition, which are: lactate dehydrogenase (LDH), C-reactive protein (CRP), white blood cells (WBC). Laboratory biomarkers such as LDH, CRP and WBC may have an impact on predicting outcomes and help classify patients into an appropriate group based on symptoms.

Introduction/Objective The increasing resistance to macrolides and lincosamides among staphylococci and streptococci is becoming a global problem. The aim of this study was to investigate the prevalence of macrolide-lincosamide-streptogramin (MLS) resistance phenotypes in staphylococcal and streptococcal isolates in southeast Serbia. Methods The MLS phenotypes were determined by the double-disk diffusion method in 2,121 inpatient and outpatient staphylococcal and streptococcal isolates collected during a one-year period at the Center for Microbiology. Results The methicillin-resistant staphylococci isolates were significantly more resistant to penicillin, erythromycin, clindamycin, gentamicin, and ciprofloxacin (100%, 100%, 29.2%, 65.6%, and 53.1%, respectively) than the methicillin-sensitive ones (93.6%, 64.9%, 12%, 28.9%, and 11.7%, respectively). The inducible clindamycin resistance phenotype was dominant in S. aureus and coagulase-negative staphylococci isolates. S. pneumoniae, S. pyogenes, and S. agalactiae isolates showed very high resistance to erythromycin (77.8%, 46.2%, and 32.4%, respectively). All staphylococci and streptococci isolates were sensitive to vancomycin and linezolid, and all beta-hemolytic streptococci isolates to penicillin and ceftriaxone. Conclusion The phenotypic triage of staphylococci is necessary in order to separate inducible resistant and truly clindamycin-sensitive isolates. Macrolides should not be recommended for empirical therapy of streptococcal infections. Penicillins remain the drug of choice for treatment of streptococcal infections in our local area.

Objective. The treatment outcome of chronic HCV infection is influenced by numerous host and viral factors (age, sex, stage of fibrosis, immune response, viral load, genotype, etc.). The aim of this study was to estimate the association between the most important predictive factors and the response to therapy in patients with chronic HCV infection. Methods. Our study included 76 patients with chronic HCV hepatitis, who were treated at Clinic for Infectious Diseases in Kragujevac. The treatment was carried out for 24/48 weeks, depending on the genotype of the virus. Results. HCV infection is more common in men, but we did not confirm the association between the age and gender of patients and the treatment outcome. The most common risk factor in patients with a favorable response (SVR and ETR) was the intravenous use of psychoactive substances, whereas blood transfusion and dialysis were the leading risk factors for patients with unfavorable response (NR/RR). The serum aminotransferase and alkaline phosphatase were lower in the SVR and ETR group of patients. The low level of basal viremia was registered in the group of patients with sustained virological response, while the highest values were recorded in the NR/RR group of patients. The most frequent HCV genotype was genotype 1, presented in a high percentage in both groups, the patients with favorable and the ones with unfavorable responses to the therapy. The presence of genotype 2 was observed only in patients with an unfavorable response to the therapy. The highest percentage of patients with absence of fibrosis was found in the group of patients with a favorable response. Conclusion. The results of our study show that a positive response to treatment was achieved in almost 90 % of patients with chronic HCV infection. Viral load, genotype and stage of fibrosis were associated with treatment outcome in chronic HCV patients. Our study has not confirmed the association between age, sex or biochemical parameters and therapy response.

Rezime: U ovoj disertaciji sintetisana je serija acilpiruvata u visokim prinosima reakcijom Klajzenove kondenzacije. Acilpiruvati su zatim korišćeni kao prekursori za sintezu tri nove klase jedinjenja pod pretpostavkom da će pokazati bolja biološka svojstva od samih acilpiruvata. Sintetisana je serija od dvadeset i pet novih 1,5- diaril-4-(2-tienilkarbonil)-3-hidroksi-3-pirolin-2-oni. Pored toga, bilo je veoma interesantno ispitati kako koordinacija za neke jone prelaznih metala utiče na aktivnost organskih molekula kao što su acilpiruvati. Zbog toga je sintetisano jedanaest novih metalo-organskih kompleksa sa acilpiruvatima kao ligandima koji sadrže Cu2+ ili Pd2+ kao centralni jon metala. Sva novo-sintetisana jedinjenja su okarakterisana spektroskopskom analizom (UV-Vis, IR, 1H i 13C NMR), masenom spektrometrijom i elementalnom analizom. Rendgenska strukturna analiza je takođe urađena gde je to bilo moguće. Novosintetizovani molekuli testirani su na antioksidativni, antimikrobni i antitumorski potencijal. DPPH test pirolidinona je pokazao da ova jedinjenja poseduju dobru antioksidativnu aktivnost (najniža IC50 vrednost iznosi 166,75 µg/ml). Ovaj test je takođe pokazao da većina acilpiruvata poseduje bolju antioksidativnu aktivnost od odgovarajućih metalo-organskih jedinjenja bakra(II). Ispitivanja antimikrobne aktivnosti su otkrila da pirolidinoni pokazuju značajnu inhibitorsku aktivnost na soju bakterija Staphylococcus aureus u prilično niskim koncentracijama (najniža izmerena MIC vrednost iznosi 0,312 mg/ml), dok je najosetljiviji soj gljivica bio Trichophyton mentagrophytes sa najnižom MIC od 0,625 mg/ml. MIC vrednosti za metalo-organska jedinjenje bakra(II) na testiranim mikroorganizmima bile su u rasponu od 0,048 do 3,125 mg/ml. Najosetljiviji soj bakterija bio je Proteus mirabilis, dok je među gljivicama najosetljiviji soj bio Candida albicans. SUMMARY: In this dissertation a series of acylpyruvates in high yields has been synthesized using the Claisen condensation reaction, which were then used as precursors for the synthesis of three new classes of compounds. Three new classes of compounds were synthesized under the assumption that they would show better biological properties than the acylpyruvates themselves. A series of twenty-five novel and one previously published 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3- pyrrolin-2-ones were synthesized. In addition, it was very interesting to examine how coordination for some metal ion affects the activity of organic molecules such as acylpyruvates, and therefore eleven new metal-organic complexes were synthesized with acylpyruvates as ligands containing Cu2+ or Pd2+ as the central metal ion. All newly synthesized compounds were characterized by spectroscopic analysis (UV-Vis, IR, 1H and 13C NMR), mass spectrometry and elemental analysis. X-ray structural analysis was also performed where possible. The newly synthesized molecules were tested for antioxidant, antimicrobial and antitumor potential. The DPPH test showed that most acylpyruvates showed better antioxidant activity than the corresponding metal-organic copper(II) compounds. MIC values for the metalorganic copper(II) compounds on the tested microorganisms were in range from 0.048 to 3.125 mg/ml. The most sensitive bacteria strain was Proteus mirabilis, while among the fungi the most sensitive strain was Candida albicans. The results of the antitumor activity showed that these compounds showed good cytotoxic activity. All metal-organic copper(II) compounds showed lower IC50 values on the LS174 and A549 cell lines compared to cisplatin. The results of the antitumor potential for palladium(II) metal-organic complexes showed that the three compounds had very good cytotoxic activity on HeLa cells and significant activity on MDA-MB 231 cells. The results obtained by examining the interations between the palladium compound and cisplatin indicate that these compounds may be useful as adjuvants in therapy for the treatment of tumors, reducing the doses of cisplatin and thus the side effects of this drug.

Biljne vrste roda Onosma L se od davnina primenjuju kao prirodne boje. Cvet nekih vrsta se koriste kao povrće u ishrani dok se njihov koren u tradicionalnoj medicini koristi za lečenje rana, opekotina, otežanog disanja, promuklosti, hemoroida, bolova u abdomenu i čireva u želucu. Ipak, kada je reč o vrsti Onosma visianii Clem do sada nisu postojala naučna saznanja o hemijskom sastvu i farmakološkoj aktivnost njenog korena. Prvi zadatak ove disertacije sastojao se u izolovanju bioaktivnih naftohinonskih derivata iz korena biljke O. visianii Clem. Primenom različitih hromatografskih tehnika (stubne hromatografije-silika gel kao adsorbens, preparativne tankoslojne hromatografije-silika gel kao adsorbens, hromatografije na koloni Sephadex-LH20, preparativne tečne hromatografije (HPLC)) optimizovana je efikasna metoda za izolovanje naftohinonskih derivata iz ekstrakta dobijenog hladnom maceracijom primenom rastvaračke smeše petrol etar:metilen hlorid (1:1 v/v). Ovom metodom izolovano je sedam jedinjenja i to deoksišikonin, izobutirilšikonin, αmetilbutirilšikonin, acetilšikonin, β-hidroksiizovalerilšikonin, 5,8-O-dimetil izobutirilšikonin (prvi put identifikovan u prirodnom izvoru) i 5,8-O-dimetil deoksišikonin. Njihova strukturna karakterizacija izvršena je primenom UV-Vis, IR, 1H NMR i 13C NMR spektroskopskih tehnika, kao i upotrebom masene spektrometrije visoke rezolucije. Sledeći zadatak sastojao se u utvrđivanju najpogodnijeg rastvarača za ekstrakciju bioaktivnih komponenata korena biljke O. visianii Clem. Metodom hladne maceracije dobijeno je pet ekstrakata (acetonski, hloroformski, etil acetatni, metanolni i petrol etarski). Upotrebom HPLC analize utvrđeno je da ekstrakti hloroforma, acetona i etil acetata predstavljaju bolje rastvarače za preliminarnu ekstrakciju bioaktivnih derivata naftohinona izobutirilšikonina, α-metilbutirilšikonina i acetilšikonina, dok je za ekstrakciju 5,8-O-dimetil derivata poželjnija upotreba acetona ili hloroforma. U narednom koraku ispitana je i citotoksična aktivnost dobijenih ekstrakata na ćelijama kancera debelog creva i dojke primenom MTT testa i metoda za detekciju apoptoze i analize ćelijskog ciklusa. Dobijeni rezultati su pokazali značajno bolju aktivnost ekstrakata acetona, hloroforma i etil acetata pri čemu je citotoksični efekat ekstrakata u direktnoj korelaciji sa količinom individualnih komponenti naftohinonskih pigmenata. Ispitana je i biološka aktivnost izolovanih jedinjenja, najpre upotrebom MTT testa, kao i metodama za detekciju apoptoze i analize ćelijskog ciklusa na ćelijama kancera debelog creva i dojke. Naftohinonski derivati α-metilbutirilšikonin, acetilšikonin i β-hidroksiizovalerilšikonin pokazali su najjači citotoksični efekat pa je dalje ispitan i njihov efekat na markere oksidativnog stresa. Tretman ovim jedinjenjima izazvao je poremećaj oksidativne homeostaze ćelija raka debelog creva i dojke, povećavajući nivo superoksid anjon radikala, nitrita, oksidovanog i redukovanog glutationa. U sledećem koraku je ispitana i mogućnost interakcija ova tri naftohinona sa molekulima DNK i HSA. Rezultati dobijeni primenom UV-Vis i FL spektroskopskih tehnika kao i molekulske doking simulacije jasno ukazuju da se testirana jedinjenja vezuju u oblasti malog žleba molekula DNK kao i da imaju mogućnost da se čvrsto vežu za HSA, i sasvim bezbedno transportuju do ciljnih ćelija. Takođe, ispitana je i antibakterijska aktivnost svih izolovanih naftohinona. Dobijeni rezultati su pokazali da izolovani naftohinoni pokazuju dobru antibakterijsku aktivnost kako prema gram pozitivnim, tako i prema gram negativnim bakterijskim sojevima, pri čemu su α-metilbutirilšikonin i acetilšikonin pokazali najznačajniju aktivnost. Naftohinoni su po prirodi lipofilna jedinjenja na čiju stabilnost utiče i dejstvo kiseonika iz vazduha kao i temperatura ali i UV zračenje. U cilju povećanja bioraspoloživosti i farmaceutske efikasnosti naftohinona iskorišćena je mogućnost njihove enkapsulacije unutar hidrofobne šupljine molekula β-CD. Rezultati dobijeni primenom UV-Vis, IR i 1H NMR spektroskopije, kao i rendgenske difraktometrije praha (XPRD) i skenirajuće elektronske mikroskopije (SEM) potvrdili su da je dobijen binarni sistem acetilšikonin/β-CD. Test fazne rastvorljivosti pokazao je da je dobijen inkluzioni sistem AL-tipa, kao i da je odnos substrat/ligand 1:1 M/M. Kako bi potvrdili da li je enkapsulacijom povećana biološka aktivnost povećanjem bioraspoloživosti enkapsuliranog naftonihona, ispitano je citotoksično dejstvo enkapsuliranog i slobodonog acetilšikonina na ćelijama kancera debelog creva i dojke primenom MTT testa, klonogenog testa kao i metodama za detekciju apoptoze i analize ćelijskog ciklusa. Takođe, ispitan je i njihov efekat na produkciju intracelularnih reaktivnih kiseoničnih vrsta (ROS), kao i na ekspresiju ključnih apoptotskih proteina, i na inhibiciju autofagije. Dobijeni rezultati su ukazali da formiranje binarnog sistema značajno utiče na poboljšanje aktivnosti naftohinonskih derivata. Since ancient times, plant species of the genus Onosma L have been used as natural colorаnts. Flowers of some species are used as a vegetable in nutrition, while their roots are used in traditional medicine in the treatment of wounds, burns, shortness of breath, hoarseness, hemorrhoids, abdominal pain and gastric ulcers. Nevertheless, for the plant species of Onosma visianii Clem, so far there has been no scientific knowledge of its root chemical composition and pharmacological activity. The first task of this dissertation was isolation of bioactive naphthoquinone derivatives from the roots of the plant O. visianii Clem. Using different chromatographic techniques (column chromatography-silica gel as adsorbent, preparative thin layer chromatography-silica gel as adsorbent, column chromatography Sephadex-LH20, preparative liquid chromatography (HPLC)), we optimized an efficient method for isolation of naphthoquinones from the extract obtained by cold maceration using a solvent mixture of petroleum ether:methylene chloride (1:1 v/v). Using this metode we isolated seven compounds: deoxyshikonin, isobutyrylshikonin, αmethylbutyrylshikonin, acetylshikonin, β-hydroxyisovalerylshikonin, 5,8-O-dimethyl isobutyrylshikonin (first time identified in a natural source) and 5,8-O-dimethyl deoxyshikonin. Тheir structural characterization was performed by UV-Vis, IR, 1H NMR and 13C NMR spectroscopic techniques, as well as using high resolution mass spectrometry. The next task was to determine the most suitable solvent for the extraction of the bioactive components from the roots of the O. visianii Clem. Five extracts (acetone, chloroform, ethyl acetate, methanol and petroleum ether) were obtained using cold maceration method. Performed HPLC analysis showed that chloroform, acetone, and ethyl acetate are better solvents for the preliminary extraction of isobutyrylshikonin, α-methylbutyrylshikonin and acetylshikonin, while the use of chloroform and acetone as solvents is preferable for extraction of 5,8-O-dimethyl derivatives. The cytotoxic activity of the obtained extracts was examined on colon and breast cancer cells using the MTT assay, methods for the detection of apoptosis and cell cycle analysis. Obtained results showed significantly better activity of acetone, chloroform and ethyl acetate extracts, suggesting that cytotoxic effect of the extracts are in direct correlation with the amount of individual components of the naphthoquinone pigments. Next, we examined the biological activity of the seven isolated naphthoquinone compounds. Initially we studied their cytotoxic effects on colon and breast cancer cells using the MTT assay, methods for the detection of apoptosis as well as cell cycle analysis. The naphthoquinone derivatives α-methylbutyrylshikonin, acetylshikonin and βhydroxyisovalerylshikonin showed the strongest cytotoxic activity so we additionally examined their effect on the oxidative stress markers. The results showed that treatment with these compounds caused a disruption of the oxidative homeostasis of colon and breast cancer cells, increasing the levels of superoxide anion radical, nitrite, oxidized and reduced glutathione. The following step was to examine the possibility of interactions of these three naphthoquinones with DNA and HSA molecules. The results using UV-Vis and FL spectroscopic techniques as well as molecular docking simulations clearly indicate that the tested compounds can bind in the region of the small groove of DNA molecule, and that can bind strongly to HSA and be transported safely to the target cells. Additionally, we studied antibacterial activity of seven isolated naphthoquinones. Obtained results showed that all isolated naphthoquinones exhibited potent antibacterial activity against both gram-positive and gram-negative bacterial strains, with αmethylbutyrylshikonin and acetylshikonin showing the most significant activity against all tested bacterial strains. Naphthoquinone are known as lipophilic compounds whose stability is affected by the action of oxygen from the air as well as temperature and UV radiation. In order to increase bioavailability and pharmaceutical efficiency of naphthoquinones, the possibility of their encapsulation within the hydrophobic cavity of the β-CD molecule was exploited. The results of UV-Vis, IR and 1H NMR spectroscopy, as well as X-ray powder diffractometry (XPRD) and scanning electron microscopy (SEM) showed that the acetylshikonin/β-CD binary system was obtained. The phase solubility test showed that the AL-type inclusion system was obtained, and that the substrate/ligand ratio was 1:1 M/M. In order to confirm whether encapsulation increased biological activity, the cytotoxic effect of encapsulated and free acetylshikonin on colon and breast cancer cells was examined using MTT assay, clonogenic assay as well as apoptosis detection methods and cell cycle analysis. Also, the effect on the production of intracellular reactive oxygen species (ROS) were examined, as well as their effect on expression of key apoptotic proteins, and the inhibition of autophagy. The obtained results indicate that the formation of a binary system significantly influences the enhancement of the activity of naphthoquinone derivatives. The results obtained represent a significant contribution to the pharmacological and phytochemical description of the Onosma visianii Clem plant and indicate the possible use of isolated naphthoquinone derivatives in the development of new antibacterial formulations, as well as in the development of new strategies against colon and breast cancer.Since ancient times, plant species of the genus Onosma L have been used as natural colorаnts. Flowers of some species are used as a vegetable in nutrition, while their roots are used in traditional medicine in the treatment of wounds, burns, shortness of breath, hoarseness, hemorrhoids, abdominal pain and gastric ulcers. Nevertheless, for the plant species of Onosma visianii Clem, so far there has been no scientific knowledge of its root chemical composition and pharmacological activity. The first task of this dissertation was isolation of bioactive naphthoquinone derivatives from the roots of the plant O. visianii Clem. Using different chromatographic techniques (column chromatography-silica gel as adsorbent, preparative thin layer chromatography-silica gel as adsorbent, column chromatography Sephadex-LH20, preparative liquid chromatography (HPLC)), we optimized an efficient method for isolation of naphthoquinones from the extract obtained by cold maceration using a solvent mixture of petroleum ether:methylene chloride (1:1 v/v). Using this metode we isolated seven compounds: deoxyshikonin, isobutyrylshikonin, αmethylbutyrylshikonin, acetylshikonin, β-hydroxyisovalerylshikonin, 5,8-O-dimethyl isobutyrylshikonin (first time identified in a natural source) and 5,8-O-dimethyl deoxyshikonin. Тheir structural characterization was performed by UV-Vis, IR, 1H NMR and 13C NMR spectroscopic techniques, as well as using high resolution mass spectrometry. The next task was to determine the most suitable solvent for the extraction of the bioactive components from the roots of the O. visianii Clem. Five extracts (acetone, chloroform, ethyl acetate, methanol and petroleum ether) were obtained using cold maceration method. Performed HPLC analysis showed that chloroform, acetone, and ethyl acetate are better solvents for the preliminary extraction of isobutyrylshikonin, α-methylbutyrylshikonin and acetylshikonin, while the use of chloroform and acetone as solvents is preferable for extraction of 5,8-O-dimethyl derivatives. The cytotoxic activity of the obtained extracts was examined on colon and breast cancer cells using the MTT assay, methods for the detection of apoptosis and cell cycle analysis. Obtained results showed significantly better activity of acetone, chloroform and ethyl acetate extracts, suggesting that cytotoxic effect of the extracts are in direct correlation with the amount of individual components of the naphthoquinone pigments. Next, we examined the biological activity of the seven isolated naphthoquinone compounds. Initially we studied their cytotoxic effects on colon and breast cancer cells using the MTT assay, methods for the detection of apoptosis as well as cell cycle analysis. The naphthoquinone derivatives α-methylbutyrylshikonin, acetylshikonin and βhydroxyisovalerylshikonin showed the strongest cytotoxic activity so we additionally examined their effect on the oxidative stress markers. The results showed that treatment with these compounds caused a disruption of the oxidative homeostasis of colon and breast cancer cells, increasing the levels of superoxide anion radical, nitrite, oxidized and reduced glutathione. The following step was to examine the possibility of interactions of these three naphthoquinones with DNA and HSA molecules. The results using UV-Vis and FL spectroscopic techniques as well as molecular docking simulations clearly indicate that the tested compounds can bind in the region of the small groove of DNA molecule, and that can bind strongly to HSA and be transported safely to the target cells. Additionally, we studied antibacterial activity of seven isolated naphthoquinones. Obtained results showed that all isolated naphthoquinones exhibited potent antibacterial activity against both gram-positive and gram-negative bacterial strains, with αmethylbutyrylshikonin and acetylshikonin showing the most significant activity against all tested bacterial strains. Naphthoquinone are known as lipophilic compounds whose stability is affected by the action of oxygen from the air as well as temperature and UV radiation. In order to increase bioavailability and pharmaceutical efficiency of naphthoquinones, the possibility of their encapsulation within the hydrophobic cavity of the β-CD molecule was exploited. The results of UV-Vis, IR and 1H NMR spectroscopy, as well as X-ray powder diffractometry (XPRD) and scanning electron microscopy (SEM) showed that the acetylshikonin/β-CD binary system was obtained. The phase solubility test showed that the AL-type inclusion system was obtained, and that the substrate/ligand ratio was 1:1 M/M. In order to confirm whether encapsulation increased biological activity, the cytotoxic effect of encapsulated and free acetylshikonin on colon and breast cancer cells was examined using MTT assay, clonogenic assay as well as apoptosis detection methods and cell cycle analysis. Also, the effect on the production of intracellular reactive oxygen species (ROS) were examined, as well as their effect on expression of key apoptotic proteins, and the inhibition of autophagy. The obtained results indicate that the formation of a binary system significantly influences the enhancement of the activity of naphthoquinone derivatives. The results obtained represent a significant contribution to the pharmacological and phytochemical description of the Onosma visianii Clem plant and indicate the possible use of isolated naphthoquinone derivatives in the development of new antibacterial formulations, as well as in the development of new strategies against colon and breast cancer.

Uvod/cilj U poslednjoj deceniji zabeleženo je povećanje rezistencije Grampozitivnih koka na antibiotike iz grupe makrolida, linkozamida i streptogramina A/B (MLSa/b). Složeni mehanizmi rezistencije na MLS antibiotike uključuju: modifikaciju ribozoma, aktivno izbacivanje antibiotika i modifikaciju antibiotika. Porodica gena odgovornih za kodiranje enzima koji vrše metilaciju ribozoma je označena skraćenicom erm (od engl. erythromycin resistance methylase) i dovode do pojave unakrsne rezistencije na makrolide, linkozamide i streptogramine grupe B (MLSb). Profil rezistencije poznat kao MLSb fenotip može biti: inducibilan (iMLSb) i konstitutivan (cMLSb). Sojevi sa erm posredovanom eritromicin rezistencijom mogu se pokazati osetljivim na klindamicin u rutinskom disk difuzionom testiranju osetljivosti. Ciljevi Utvrditi učestalost fenotipova i gena rezistencije na MLS antibiotike kod Gram-pozitivnih koka na području jugoistočne Srbije. Metode MLS fenotipovi rezistencije: konstitutivni (cMLSb), inducibilni (iMLSb), M/MSb i LSa/b, bili su utvrđeni dupli-disk difuzionom metodom (D-testom). Ispitano je 2366 kliničkih izolata Gram-pozitivnih koka, sakupljenih u toku jednogodišnjeg perioda u Centru za mikrobiologiju Zavoda za javno zdravlje Vranje. Geni koji kodiraju MLSb rezistenciju (ermA, ermB, ermC, msrA, msrB, mefA/E, lnuA, lnuB, lsaA i lsaC) su identifikovani korišćenjem multipleks PCR-a. Rezultati Najveću učestalost rezistencije na eritromicin imali su izolati meticilin-rezistentnih koagulaza-negativnih stafilokoka (MRCNS, 91,5%), zatim meticilin-rezistentnih S. aureus (MRSA, 86,4%) i enterokoka (80,8%). Niža, ali još uvek visoka stopa rezistencije na eritromicin je bila prisutna i kod drugih izolata Gram-pozitivnih koka. Nasuprot makrolidima, izuzev za izolate Enterococcus spp. (96,7%), Streptococcus pneumoniae (35,9%) i MRSA (28,4%), drugi izolati Grampozitivnih koka su pokazali značajno nižu rezistenciju na klindamicin. Otkrili smo da je iMLSb fenotip najučestaliji kod stafilokoka, kod enterokoka i bakterija S. agalactiae i S. pnemoniae bio je dominantan cMLSb fenotip, dok je M/MSb fenotip bio najčešće prisutan kod bakterija S. pyogenes. Od ukupnog broja izolata četiri soja suV pokazala novi "keyhole" fenotip. Najčešći pojedinačni geni i njihove kombinacije detektovane kod stafilokoka su bile msrA/B, ermC i ermS+msrA/B, dok su kod enterokoka i ß-hemolitičkih streptokoka bile lsaA, ermB i ermB+lsaA. Zaključak Utvrdili smo visoku stopu rezistencije na eritromicin kod Grampozitivnih koka. Meticilin-rezistentne stafilokoke su bile značajno rezistentnije na MLS antibiotike u odnosu na meticilin-senzitivne stafilokoke. Najčešće identifikovani gen MLS rezistencije među stafilokokama bio je ermC, dok je među enterokokama i ß-hemolitičkim streptokokama bio ermB gen. Utvrđena je jasna razlika u obrascu MLS osetljivosti među stafilokokama i enterokokama/streptokokama i potvrđena je neophodnost uvođenja D testa u rutinskom testiranju antimikrobne osetljivosti kod izolata stafilokoka. Introduction The increase of resistance to macrolide, lincosamide, and streptogramin a/b antibiotics (MLSa/b) among Gram-positive cocci has been reported in the last decade. The complex mechanisms of resistance to MLS antibiotics include: modification of ribosome, active efflux of antibiotics and antibiotic inactivation. The gene family called erm (erythromycin resistance methylase) that encoding enzymes responsible for ribosome methylation leads to cross- resistance to macrolides, lincosamides and streptogramin b (MLSb resistance phenotype). The antimicrobial resistance profile known as MLSb resistance phenotype can be either inducible (iMLSb) and constitutive (cMLSb). Macrolide resistant strains carrying erm gene can show false sensitivity to clindamycin, using routine antimicrobial susceptibility test. Objective This study investigated the prevalence of MLS resistance phenotypes and genes in Gram-positive cocci in region of the Southeast Serbia. Methods MLS resistance phenotypes: constitutive (cMLSb), inducible (iMLSb), M/MSb, and LSa/b, were determined by the double-disk diffusion method. We have analyzed 2366 clinical isolates of Gram-positive cocci, collected during 1-year period at the Center for Microbiology of the Public Health Institute in Vranje, Serbia. The MLS resistance genes (ermA, ermB, ermC, msrA, msrB, mefA/E, lnuA, lnuB, lsaA, and lsaC) were identified by multiplex PCR. Results The highest frequencies of isolates resistant to erythromycin were found among methicillin-resistant coagulase-negative staphylococci (MRCNS, 91.5%), methicillin-resistant S. aureus (MRSA, 86.4%) and Enterococcus strains (80.8%). Lower, but still high resistance rate to erythromycin was found among other Gram-positive cocci. Contrary to macrolides, except for Enterococcus spp. (96.7%), S. pneumoniae (35.9%), and MRSA (28.4%), other Gram-positive cocci showed significantly lower resistance to clindamycin. We have found that iMLSb is the most prevalent phenotype in staphylococci, the cMLSb is predominantly in enterococci, S. agalactiae, and S. pnemoniae, whereas the M/MSb phenotype is the most frequent in S. pyogenes. LSa/b phenotype was the most common in enterococci and beta haemolytic streptococci, but nearly or totally absented among staphylococci, S. pneumoniae, and S. pyogenes. The novel keyhole zone phenomenon was detected in four out of total number of clinical isolates. The most prevalent MLS resistance genes and their combinations detectedVII in staphylococci were msrA/B, ermC and ermС+msrA/B, whereas lsaA, ermB и ermB+lsaA were predominantly found in enterococci and beta haemolytic streptococci. Conclusion Generally, high rates of erythromycin resistance in Gram-positive cocci was found. The methicillin-resistant staphylococci were significantly more resistant to MLS antibiotics than methicillin-sensitive staphylococci. The most frequent MLS resistance gen among staphylococci isolates was ermC, whereas the ermB gen was the most prevalent in enterococci and ß-haemolytic streptococci. We have also discovered a clear differences of MLS resistance pattern in staphylococci and enterococci/streptococci, and confirmed the necessity of routine D-testing of all clinical isolates of staphylococci.

Autofagija, proces programirane ćelijske razgradnje unutarćelijskog sadržaja, je uključena u regulaciju preživljavanja i smrti ćelija kancera. U ovoj doktorskoj disertaciji je po prvi put ispitivana sposobnost antileukemijskih lekova citarabina i idarubicina da indukuju autofagiju u različitim humanim leukemijskim ćelijskim linijama i mononuklearnim ćelijama periferne krvi (MNPK) pacijenata obolelih od leukemije in vitro. Takođe, ispitivani su unutarćelijski mehanizmi odgovorni za indukciju autofagije, kao i uloga autofagije u citotoksičnosti ovih lekova. Vijabilitet REH, HL-60, K562 leukemijskih ćelijskih linija, MNPK pacijenata obolelih od leukemije i MNPK zdravih kontrola je određivan merenjem aktivnosti kisele fosfataze i mitohondrijalnih dehidrogenaza. Protočna citofluorimetrija je korišćena za detekciju apoptoze i zakišeljavanja citoplazme. Indukcija autofagije je ispitivana fluorescentnom mikroskopijom (detekcija unutarćelijskih kiselih vezikula obojenih akridin oranžom), transmisionom elektronskom mikroskopijom (posmatranje autofagnih vezikula) i imunoblot analizom (konverzija LC3-I u LC3-II, degradacija SQSTM1/p62). Aktivacija signalnih puteva koji učestvuju u regulaciji autofagije je analizirana imunoblot metodom. Uloga autofagije u citotoksičnosti citarabina i idarubicina je ispitivana primenom farmakološke inhibicije bafilomicinom A1, hlorokinom, vortmaninom i amonijum hloridom, kao i genetske inaktivacije ekspresije beklina-1, LC3β i SQSTM1 transfekcijom odgovarajućim malim interferirajućim RNK. Citarabin i idarubicin su izazvali povećanje unutarćelijske kiselosti i pojavu autofagnih vezikula sa delimično razgrađenim ćelijskim sadržajem u leukemijskim ćelijskim linijama. Antileukemijski lekovi su stimulisali razgradnju supstrata autofagije SQSTM1 i povećali konverziju LC3-I u LC3-II formu asociranu autofagozomima u odsustvu ili prisustvu inhibitora proteolize, ukazujući tako na povećanje autofagnog fluksa. Oba leka su smanjila fosforilaciju mTOR kinaze, glavnog negativnog regulatora autofagije i njegovog supstrata p70S6 kinaze, dok je tretman mTOR aktivatorom leucinom sprečio indukciju autofagije. Idarubicin je suprimirao aktivnost mTOR aktivatora... Autophagy, a process of programmed cellular self-digestion, has been implicated in regulation of cancer cell survival and death. The present study investigated for the first time the ability of antileukemic drugs cytarabine and idarubicin to induce autophagy in different human leukemic cell lines and peripheral blood mononuclear cells (PBMC) from leukemia patients in vitro. Intracellular mechanisms responsible for the induction of autophagy, as well as the role of autophagy in cytotoxicity of these drugs were also investigated. Cell viability of REH, HL-60, K562 leukemic cell lines, and PBMC from leukemic patients and healthy controls was determined by measuring the acid phosphatase and mitochondrial succinate dehydrogenase activity. Flow cytometry was used for the detection of apoptosis and intracellular acidification. Autophagy induction was assessed by fluorescent microscopy (detection of acridine orange stained intracellular acidic vesicles), by transmission electron microscopy (observation of autophagic vacuoles), as well as by immunoblot analysis of LC3 conversion and SQSTM1/p62 proteolysis. Activation of autophagy-regulating signaling pathways was analyzed by immunoblotting. Pharmacological inhibition of autophagy with bafilomycin A1, chloroquine, wortmannin, and NH4Cl or RNA interference-mediated knockdown of beclin-1, LC3β and SQSTM1 were used to determine the role of the autophagy in cytotoxicity of antileukemic drugs. Cytarabine and idarubicin induced an increase in intracellular acidification and appearance of autophagic vesicles with partially digested cellular components in leukemic cell lines. Antileukemic drugs stimulated the degradation of autophagic target SQSTM1 and enhanced the conversion of LC3-I to autophagosome-associated LC3-II in the absence or presence of proteolysis inhibitors, thus indicating the increase in autophagic flux...

Uvod: Glukokortikoidni hormoni (GH) su mimo svog anti-inflamatornog dejstva i jedni od najpoznatijih inhibitora zarastanja rana. Nedavno je pokazano da farnezil pirofosfat (FPP), ključni međuprodukt mevalonatskog puta sinteze holesterola i farnezilacije proteina može delovati i kao agonista za nekoliko nuklearnih hormonskih receptora uključujući glukokortikodni receptor (GR). Interesantno je da statini, lekovi koji se koriste u terapiji ateroskleroze, svoje dejstvo ostvaruju upravo inhibirajući ovaj sintetski put. Međutim, statini imaju i dodatne plejotropne efekte, koji se ostvaruju nezavisno od sniženja koncentracije holesterola u plazmi i značajno doprinose njihovom korisnom dejstvu u različitim bolestima. Jedan od dobro dokumentovanih plejotropnih efekata je poboljšana reparacija tkiva uključujući i zarastanje rana. Zanimljivo je da se upravo inhibicija farnezilacije signalnih molekula, usled sniženja nivo ćelijskog FPP-a, smatrala do sada ključnim mehanizmom kojim statini ostvaruju ove svoje dodatne efekte. Međutim otkriće da FPP može delovati kao ligand za GR otvara vrata za drugačiju interpretaciju mehanizma dejstva plejotropnih efekata statina i novu primenu ovih lekova. Ciljevi: U ovoj disertaciji dokazivano je postojanje novog mehanizma dejstva statina, kojim oni mogu pospešiti zarastanje rana. Ovaj efekat se zasniva na sniženju koncentracije endogenog FPP-a u ćelijama kože, putem inhibicije HMG-CoA reduktaze Osnovna hipoteza je da, s obzirom da statini svoje dejstvo ostvaruju upravo inhibirajući ovaj sintetski put, modifikuju FPP-GR signalizaciju i tako učestvuju u stimulaciji zarastanje rana. Prvi cilj istraživanja je bio aktivacije glukokortikoidnog signalnog puta i stimulacije transkripcije gena. Potom je ispitatano da li povećanje nivoa endogenog FPP-a putem inhibicije enzima skvalen sintetaze i farnezil transferaze ima isti efekat na aktivaciju glukokortikoidnog receptora kao i dodavanje egzogenog FPP-a... Introduction: Glucocorticoid hormones, in addition to their anti-immflamatory effect, are well known inhibitors of wound healing. Recent studies have shown that farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway of the cholesterol synthesis and protein farnesylation, can act as an agonist for several nuclear hormone receptors including glucocorticoid receptor (GR). Interestingly, mechanism of action of statins, drugs used in therapy of atherosclerosis, is based on inhibition of mevalonath pathway. However, statins might exert also additional pleiotropic, noncholesterol lowering effects which significantly contribute to their therapeutic action. One of the well known pleiotropic effects of the statins is stimulation of tissue repair, including wound healing. Interestingly, mechanism of pleiotropic effects is traditionally explained by statin mediated inhibition of the farensylation of signaling molecules. By inhibiting the enzime HMG-CoA reductase, statins prevent the synthesis of substrate for reaction, farnesylpyrophosphate. Given that is recently shown that FPP can act as a ligand for GR, it is not surprising that statins might have additional mechanisms of action beyond inhibition of the farnesylation. Better understanding of this mechanism may contribute to novel therapeutic roles for this drugs. Objectives: In this study we are exploring new mechanism of action of statins that can contribute to improvement of wound healing. This effect is based on decreasing the levels of endogenus FPP in keratinocytes by inhibition of the HMG-CoA reductase. We postulate that statins, acting as an inhibitors of mevalonate pathway, stimulate wound healing through modulation of FPP-GR signal. The first step in in our study is to prove that the exogenous FPP, acting as an agonist for GR, can activate the glucocorticoid signaling pathway and regulate the transcription of the target genes. Second, will explore the effects of ZGA and B581, which both can increase the level of endogenous FPP, on activation of glucocorticoid receptors. ZGA and B581 acomplish their effects through inhibition of squalene synthetase and farnesyl transferase respectively...

Multipla skleroza (MS) je hronična, zapaljenska, demijelinizaciona i neurodegenerativna bolest centralnog nervnog sistema (CNS). Smatra se da je MS autoimunska bolest kod koje autoreaktivni pomoćnički T-limfociti (Th) specifični za antigene mijelina pokreću proces oštećenja direktno i aktivacijom drugih imunskih i neimunskih ćelija. Njihovo dejstvo bar delom antagonizuju regulatorni T-limfociti (Treg). Prva linija terapije za najčešću formu MS, relapsno-remitentnu (RR), je interferon-β koji svoje efekte ostvaruje dejstvom na ekspresiju različitih gena. Problem koji se nameće u lečenju ovim lekom je relativno visoka učestalost slabog terapijskog odgovora. U ovoj doktorskoj disertaciji prospektivno je praćena relativna genska ekspresija iRNK u mononuklearnim ćelijama periferne krvi kod bolesnika sa RRMS. Mereni su markeri biološkog odgovora na IFN-β, marker T-limfocita, Th1/17 − polarišući citokini, njihovi receptori, transkripcioni faktori Th1/17 i Treg, efektorski citokinii endogeni IFN-β pre početka terapije sa IFN-β1b i nakon 6, 12, 24 i 36 meseci trajanja terapije. Pored toga, praćena je kinetika koncentracije odabranih proinflamatornih citokina u plazmi. Analiziran je potencijal ovih medijatora kao biomarkera kliničkog odgovora na terapiju. Tokom IFN-β terapije su se značajno menjali nivoi ekspresije svih ispitivanih gena, i to na isti način kod pacijenata koji dobro odgovaraju na terapiju i kod pacijenata koji suboptimalno odgovaraju na terapiju. IFN-β terapija nije uticala na koncentracije merenih proinflamatornih citokina u plazmi bolesnika sa RRMS. Nije postojala povezanost nivoa RNK, kao ni koncentracije citokina u plazmi sa kliničkim i demografskim parametrima obolelih. Međutim, kada su podaci analizirani u zavisnosti od pojave relapsa, nađena je povezanost niže ekspresije β2 subjedinice receptora za IL-12 (IL-12Rβ2) sa višim rizikom za pojavu relapsa. Relativna genska ekspresija IL-12Rβ2 ima potencijal kao biomarker odgovora na terapiju sa IFN-β1b. Dalja istraživanja koja bi uključila veći broj bolesnika sa RRMS su potrebna kako bi se ispitala ova pretpostavka i procenila primenjivost korišćenja relativne genske ekspresije IL-12Rβ2 kao biomarkera odgovora na terapiju sa IFN-β1b...

Uvod. Langerhansove ćelije (LĆ) predstavljaju subpopulaciju dendritskih ćelija (DĆ) koja ima ulogu u započinjanju imunskog odgovora na antigene koji su dospeli u epidermis ili sluzokože. DĆ vezuju molekulske obrasce patogena (engl. Pathogen-associated molecular patterns, PAMP) i oštećenih ćelija (engl. Damage-associated molecular patterns, DAMP) pomoću membranskih i citosolnih receptora za prepoznavanje molekulskih obrazaca (engl. Pattern recognition receptors, PRR). Među PRR su najviše proučavani receptori slični Tollu (engl. Toll-like receptors, TLR) i lektinski receptori C-tipa (engl. C-type lectin receptors, CLR). Uspostavljanje kontakta između DĆ i PAMP i DAMP je ključni događaj kojim se povezuju urođena i stečena imunost, što za posledicu ima eradikaciju antigena i nastanak imunološke memorije ili izazivanje periferne tolerancije. Nakon internalizacije i obrade PAMP i DAMP u proinflamacijskom okruženju, DĆ započinju migraciju u T-zavisne zone regionalnih limfnih čvorova u toku koje fenotipski i funkcionalno sazrevaju. Aktivacija CD40 receptora na DĆ nakon vezivanja liganda ovog molekula (CD40L) ispoljenog na površini aktivisanih T-limfocita dovodi do okončanja maturacije DĆ. Tada one postaju potpuno efikasne antigen-prezentujuće ćelije sposobne da polarizuju imunski odgovor CD4+ T-limfocita u Th1 smeru i da indukuju citotoksičnu efektorsku funkciju CD8+ T-limfocita. Konvencionalne DĆ, sa napred navedenim funkcionalnim karakteristikama, su našle primenu u prevenciji i terapiji infektivnih bolesti i maligniteta u obliku DĆ vakcina. Iako je za in vivo ili in vitro sazrevanje DĆ često dovoljna stimulacija samo jednim agonistom TLR ili CLR, fenotipski stabilniji i funkcionalno potentniji imunski odgovor u eksperimentalnim i kliničkim ispitivanjima se najčešće dobija tek kombinovanom primenom njihovih analoga, što je posledica specifične integracije različitih, međusobno nezavisnih, unutarćelijskih signala. Nakon nedavne potvrde da LĆ imaju sposobnost unakrsne prezentacije antigena stvorili su se uslovi da i one postanu kandidati za DĆ vakcine. Do sada nije bilo studija koje su ispitivale načine modulacije fenotipskih i funkcionalnih karakteristika LĆ koji mogu imati potencijalnu ulogu u razvoju maturacijskih protokola neophodnih za terapijsku primenu ovih ćelija... Introduction. Langerhans cells (LCs) represent a subpopulation of dendritic cells (DCs) whose function is to initiate immune responses against the antigens from epidermis and mucoses. DCs bind pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) via membrane and cytosolic pattern recognition receptors (PRRs). The most frequently studied PRRs are Toll-like receptors (TLRs) and C-type lectine receptors (CLRs). Similarly to conventional DCs, the contact established between LC’s PRRs and PAMPs and DAMPs is the key event that links innate and adaptive immunity and leads to eradication of the antigen, initiation of the peripheral tolerance and introduction of the immunological memory. After LCs internalise and process PAMPs and DAMPs in a proinflammatory environment, they start to migrate toward T-dependent regions of the regional lymph nodes and, at the same time, become phenotipically and functionally mature. The activation of the CD40 molecule on LCs with a CD40 ligand (CD40L) expressed on the surface of activated T-lymphocytes brings the maturation of LCs to its end, when they become fully efficient antigen-presenting cells, capable of inducing the Th1 polarisation of CD4+ T-lymphocytes and citotoxic effector functions of CD8+ T-lymphocytes. Conventional DCs with the aforementioned functional characteristics are used in the prevention and therapy of infectious or malignant diseases as DC-vaccines. Although DCs require stimulation with a single TLR or CLR agonist to become mature in vitro and in vivo conditions, phenotipically stable and functionally more potent immune response of these cells in experimental and clinical studies is usually achieved only after combined treatment with analoges of these receptors because of the specific integration of the various, mutually independent, intracellular signals. LCs became candidates for DC-vaccines after recent confirmation that they are capable to cross-present antigens. Until now, there were not any studies that examined the means to modulate phenotypic and functional characteristics of LCs that could have a potential role in the development of maturational protocols necessary for therapeutic application of these cells...

Akutna HCV infekcija u većini slučajeva (>80%) protiče kao oligosimptomatska ili asimptomatska bolest. Nakon akutne faze, spontana rezolucija HCV infekcije događa se u samo 20 % obolelih, dok kod najvećeg broja inficiranih virus perzistira i bolest prelazi u hroničnu fazu. Pretpostavlja sa da oko 180 miliona ljudi širom sveta, ima hroničnu HCV infekciju. Patogeneza HCV infekcije je rezultat multiplih interakcija između imunskog sistema domaćina i virusnih faktora. Snažan HCV specifičan CD4+ i CD8+ T ćelijski odgovor usmeren na multiple HCV epitope u nekim slučajevima omogućava spontanu eliminaciju virusa u akutnoj fazi. Međutim, HCV virus poseduje različite mehanizme kojima, tokom rane faze akutne infekcije, može da izbegne odbrambene snage domaćina, što omogućava dalju progresiju bolesti u hroničnu fazu. Supresija koju izazivaju Treg indukovane HCV virusom može da snizi imunski odgovor T ćelija u ranim fazama infekcije i tako onemogući otklanjanje virusa, ali se time može smanjiti i zapaljenski infiltrat u jetri i sprečiti masivno oštećenje tkiva. Cilj ove studije je da se utvrdi prisustvo i odredi apsolutni i relativni broj CD4+, CD4+/Foxp3+ (Treg), CD8+ i CD20+ limfocita u jetri i perifernoj krvi pacijenata sa hroničnom HCV infekcijom, ispita citokinski profil imunskog odgovora u hroničnoj HCV infekciji kao i postojanje eventualne povezanosti ovih parametara, virusne replikacije i nivoa oštećenja jetrinog parenhima. Rezultati naše studije ukazuju da su pacijenti sa hroničnom HCV infekcijomom češće muškog pola, da je najčešći put infekcije IVUPAS, a najčešći genotip virusa genotip 1. Povoljan odgovor na terapiju je zabeležen kod tri četvrtine ispitanika. Metode logističke regresije su pokazale da su starost ispitanika i genotip značajni prediktivni faktori za nastanak fibroze, dok se kao najznačajniji prediktivni faktori povoljnog odgovora na terapiju izdvajaju starost ispitanika, put infekcije i stadijum fibroze. Ispitivanje citokinskog profila imunskog odgovora u hroničnoj HCV infekciji je pokazalo da su ovi pacijenti heterogena grupa, tako da kod pacijenata bez fibroze dominiraju proinflamatorni citokini, pri čemu je uočena tendencija smanjenja nivoa svih citokina ka višim stadijumima fibroze. tako da su kod pacijenata sa F3 stadijumom fibroze vrednosti svih citokina, osim IL-4, niže su od vrednosti u kontrolnoj grupi, što ukazuje na preovlađivanje Th2 odgovora u višim stadijumima fibroze. Po završetku terapije nivoi svih citokina, i proinflamatornih i antiinflamatornih, su niži u odnosu na vrednosti pre terapije. Rezultati protočne citometrije su pokazali da u odnosu na kontrolu pacijenti imaju značajno veći broj CD3+, kao i subpopulacija CD4+, CD8+ i Foxp3+ T limfocita u perifernoj krvi, ali statistički značajna razlika u indeksu CD4/CD8 nije pokazana. U perifernoj krvi pacijenata broj aktiviranih plazmocitoidnih dendritskih ćelija značajno je veći od broja aktiviranih mijeloidnih dendritskih ćelija, a iako statističku razliku nismo utvrdili, uočeno je da je broj aktiviranih mijeloidnih DC veći u nižim stadijumima fibroze, a broj aktiviranih plazmocitoidnih DC u višim stadijumima fibroze. Fenotipizacijom mononuklearnih leukocita u jetri pokazano je da pacijenti sa nepovoljnim odgovorom na terapiju imaju značajno niži broj CD4+ i Foxp3+ ćelija, ali viši broj CD8+ ćelija u odnosu na pacijente sa povoljnim odgovorom. Kao rezultat toga, CD4/CD8 indeks pacijenata sa nepovoljnim odgovorom je gotovo tri puta niži u odnosu na pacijente sa povoljnim odgovorom na terapiju. Sa druge strane, pacijenti sa višim stadijumima fibroze imaju značajno veći broj CD4+, Foxp3+, CD8+ i CD20+ ćelija, ali niži broj DC-SIGN+ ćelija u odnosu na pacijente bez fibroze. Iako je frekvencija i CD4+ i CD8+ ćelija veća kod pacijenata sa uznapredovalom fibrozom, indeks CD4/CD8 pozitivnih ćelija kod ovih pacijenata je više od dva puta niži u odnosu na pacijente bez fibroze. Uočene razlike u frekvenciji pojedinih subpopulacija limfocita u tkivu jetre pacijenata u različitim stadijumima bolesti, ukazuju da analiza imunofenotipskih parametara u jetri, u odnosu na perifernu krv, bolje odslikava fine mehanizme imunskog odgovora na HCV virus. Metodama korelacije utvrđeno je da postoji statistički visoko značajna korelacija između broja T limfocita u infiltratima jetre i serumskog nivoa AST i ALT. Interesantno je da je korelacija serumskih koncentracija AST u pozitivnoj korelaciji sa brojem CD4+ limfocita, dok je ta korelacija negativnog predznaka u slučaju Foxp3+ Treg. Sumarno, rezultati ove opsežne studije su pokazali da su pacijenti sa hroničnom HCV infekcijom heterogena grupa, te da se pravi uvid u imunopatogenetska dešavanja tokom hronične HCV infekcije može dobiti jedino uporednom analizom pacijenata u različitim stadijumima bolesti. Shodno tome, pokazano je da je imunski odgovor kod ovih pacijenata polarizovan u odnosu na stadijum bolesti. Kod pacijenata u nižim stadijumima bolesti dominira Th1 imunski odgovor koji, iako praćen umerenom intrahepatičnom ifiltracijom efektorskih T limfocita, ne dovodi do oštećenja jetrinog parenhima. Nasuprot tome, kod pacijenata sa visokim stadijumom fibroze dominira neadekvatan Th2 odgovor, koji praćen izrazito snažnom intrahepatičkom infiltracijom efektorskih limfocita uz izrazitu dominaciju CD8+ limfocita, dovodi do masivnog oštećenja jetrinog parenhima. Naši rezultati ukazuju da regulatorni mehanizmi uključeni u ovu polarizaciju mogu biti dvojaki. Sa jedne strane, pacijenti u nižim stadijumima bolesti imaju veći broj aktiviranih mDC, kako u perifernoj krvi, tako i u inflamiranom tkivu jetre, dok pacijenti sa izraženom fibrozom pokazuju dominaciju pDC. Pored toga, pacijenti sa izraženom fibrozom imaju znatno veći broj intrahepatičnih Treg, pri čemu negativna korelacija između broja Treg u jetri i serumskog nivoa AST i ALT, kao i pozitivna korelacija između broja CD4 i serumskog nivoa AST mogu biti odraz pokušaja imunskog sistema da ograniči intenzivan, ali ne i efikasan T ćelijski imunski odgovor. In up to 80% of patients acute form of hepatitis C is asymptomatic. After the acute phase, the spontaneous resolution of HCV infection occurs in only 20% of cases, while most of infected patients develop a chronic desease. It is assumed that about 180 million people worldwide, have chronic HCV infection. The pathogenesis of HCV infection is the result of multiple interactions between the host's immune system and viral factors. Strong HCV-specific CD4+ and CD8+ T cell response directed to multiple epitopes of HCV in some cases eliminate the virus spontaneously in the acute phase. However, HCV has mechanisms to escape humoral and cellular response, which allows progression of the disease to the chronic phase. A subset of regulatory T cells contributes to failure of T cell-mediate immunity in the early stages of infection and thus prevent the removal of the virus, but also can reduce the inflammatory infiltrate in the liver and help prevent massive tissue damage. The aim of this study was to determine the presence and absolute and relative number of CD4+, CD4+/Foxp3+ (Treg), CD8+ and CD20+ cells in the liver and peripheral blood of patients with chronic HCV infection, to assess sera cytokine profile and to find possible correlations of these parameters, viral replication and liver histopathology. The results of our study showed that chronically infected patients were mostly males, IVUPAS was most frequent route of infection and genotype 1 was dominant. About 77% of patients achieved sustained virologic response. The logistic regression showed that an age of the patients and genotype are significant predictive factors for the development of fibrosis, while the most important predictors of a favorable response to therapy are age of the patient, route of infection and the stage of fibrosis. Cytokine profiling has shown predominance of inflammatory cytokines in patients without fibrosis and tendency of decrease in cytokine levels with progression of liver injury. In F3 stage patients values of all cytokines except IL- 4 were lower than in control group, indicating predominance of Th2 response in the higher stages of fibrosis. After the treatment, levels of both proinflammatory and antiinflammatory cytokines decreased. Results of flow cytometry showed that, in comparison to control, patients had significantly higher number of peripheral blood CD3+, as well as CD4+, CD8+ and Foxp3+ T cell subpopulations, but the difference in CD4/CD8 index was not statistically significant. Furthermore, the number of activated pDCs was significantly higher than the number of activated mDCs. In addition, we have found higher number of activated mDCs in the lower stages of fibrosis, and activated pDCs in the higher stages of fibrosis, although these differences were not statistically significant. In the liver of patients with poor response to therapy we have noted significantly lower number of CD4+ and Foxp3+, but higher number of CD8+ cells, as compared to responders. As a result, the CD4/CD8 index was almost three times lower in patients with poor response to therapy. On the other hand, patients with higher stages of fibrosis, have a significantly higher number of CD4+, Foxp3+, CD8+ and CD20+ cells, but a lower number of DC-SIGN+ cells as compared to patients without fibrosis. Although the frequency of CD4+ and CD8+ cells increased in patients with advanced fibrosis, CD4/CD8 index was more than two times lower than in patients without liver fibrosis. These facts indicate that immunophenotypic analysis of liver reflects more certain the mechanisms of immune response to HCV virus. Statistically significant correlation between the number of T cell in liver infiltrates and serum levels of AST and ALT was found. Interestingly, serum concentrations of AST were positively correlated with the number of CD4+ lymphocytes, whereas negatively correlated with number of Foxp3+ Treg. In summary, the results of these extensive studies have shown that patients with chronic HCV infection represent a heterogeneous group, and that the real insight into immunopathogenetic events during chronic HCV infection may be provided only by comparative analysis of patients in different stages of the disease. Accordingly, it has been shown that the immune response is polarized in relation to the stage of fibrosis. In patients with lower stages of fibrosis Th1 response is dominant and, although accompanied by moderate intrahepatic effector T lymphocytes infiltration, does not lead to damage of liver parenchyma. In contrast, in patients with high-stage fibrosis inappropriate Th2 response dominates, in concomitance with strong intrahepatic infiltration of effector cells and distinct dominance of CD8+ lymphocytes, resulting in massive damage of liver parenchyma. Our results indicate dual regulatory mechanisms involved in this polarization. On the one hand, the patients with lower stages of fibrosis have a higher number of activated mDC, both in the peripheral blood and in inflamed liver tissue, while patients with marked fibrosis showed dominance of pDC. In addition, patients with marked fibrosis have a significantly larger number of intrahepatic Treg, wherein a negative correlation between the number of Treg in the liver and serum levels of AST and ALT, and the positive correlation between the number of CD4 and serum levels of AST may be a reflection of the immune system attempts to limit intense, but inefficient T cell immune response.

Upotreba biljaka u lečenju i ishrani stara je koliko i sama ID istorija ljudskog roda. Veština lečenjem biljem razvijala se kod svih naroda sveta i sačuvala se manje ili više sve do današnjih dana uz tendeciju porasta primene fitoterapije u ekonomski razvijenim zemljama poslednjih godina. Naučni interes za lekovitost belog luka počinje u drugoj polovini XIX veka kada je Luj Paster utvrdio da ova biljka sprečava rast i razmnožavanje određenih sojeva bakterijsih vrsta i od tada je predmet stalnih naučnih istraživanja. U ovom radu ispitivana je antimikrobna aktivnost vodenog ekstrakta belog luka gajenog sa područja Šumadije, glavnog aktivnog principa alicina kao i nekih organosumpornih jedinjenja konstituenasa biljke Alium Sativum L.,rastvornih u vodi derivata cisteina, na gram-negativne bakterije Eserihia coli ATCC 10536, Pseudomonas aeruginosa ATCC 9027 i gram-pozitivne bakterije Staphylococcus aureus ATCC 43300, Bacillus subtilis ATCC 11774. Merenja i kvantifikovanje antimikrobnog dejstva su bila određena korišćenjem površinskog disk difuzionog metoda na Mueller Hinton agarnoj podlozi uz primenu aplikativnog softvera Motic images plus 2.0 ML na čistim bakterijskim kulturama i referentnom supstancom antibiotikom gentamicinom korišćenog u svim eksperimentima kao kontrola. Očekivanu antimikrobnu aktivnost je ispoljio ekstrakt belog luka i najači aktivni princip alicin, ali je 112 istovremeno i konstituens N-acetil-L-cistein takođe ispoljio efekat na sve u radu ispitivane bakterijske sojeve. N-acetil-L- cistein je veći antimikrobni efekat pokazao na ispitivane gram- pozitivne nego na gram-negativne bakterijske vrste. Pretpostavka za dobijeni antimikrobni efekat kod gram negativnih bakterija bi bila da tiolna grupa reaguje prvo sa lipopolisaharidnim slojem pa tek nakon toga sa kapsulnim polisaharidima poznatih kao glikokaliks, odnosno da razlike u strukturi ćelijskog zida direktno utiču na dobijene vrednosti zone inhibicije. Dobijeni rezultati naše studije upravo bi mogli da potvrde ovu pretpostavku gde je minimalna inhibitorna aktivnost bila znatno niža od vrednosti dobijenih za gram-negativne bakterije. Određivanje prečnika zone inhibicije, minimalne inhibitorne koncetracije, preciznije određivanje antibakterijskog efekta ekstrakta belog luka kao i neposredno utvrđivanje da i konstituens N-acetil-L-cistein pripada grupi aktivnih principa koji su prisutni u belom luku bi mogli da doprinesu znatno boljem razumevanju obima antimikrobnog dejstva belog luka. Činjenica da pored alicina i N-acetil-L-cistein poseduje direkni antimikrobni potencijal na ispitivane bakterijske vrste u studiji bi mogla biti osnov za izradu novih lekovitih antimikrobnih preparata kao i za proširenje indikacija postojećih lekova. Poznato je da se N-acetil-L-cistein sada već koristi kao mukolitik, a u ovom radu je pokazano da njegovo direktno antimikrobno dejstvo može uticati na potencijalno korenite promene u načinu primene terapije kod pacijenata sa respiratornim infekcijama, u cilju unapređenja terapijske efikasnosti i bezbednosti. The use of plants in medical treatment and nutrition is as old as AB human history itself. The skills for treatment using plants have been developed in all nations of the world and have been maintained more or less up to the present day. Developed countries show the tendency to increase use of phototherapy in recent years. Scientific interest in the curative properties of garlic begins in the second half of the nineteenth century, when Louis Pasteur found out that this herb inhibits the growth and reproduction of certain strains of bacteria. The garlic has been subject to ongoing scientific research since then. This study examined the antimicrobial activity of aqueous extract of garlic cultivated in Šumadija region and its main active principle of allicin and some organosulfur compound constituents of plants Alium sativum L., soluble in water derivatives of cysteine on gram- negative bacteria Eserihia coli ATCC 10536, Pseudomonas aeruginosa ATCC 9027 and gram- positive bacteria Staphylococcus aureus ATCC 43300, Bacillus subtilis ATCC 11774. Measurement and quantification of antimicrobial activity were determined using surface disk diffusion method on Mueller Hinton agar substrate with the use of Motic Images Plus 2.0 ML software on pure bacterial cultures. The 115 antibiotic gentamicin is reference substance and is used in all experiments as a control. The garlic extract and its strongest active constituent of allicin has showed the expected antimicrobial activity, but also the constituent N-acetyl-L-cysteine has showed the effect on all bacterial strains used for this study. N-acetyl-L-cysteine demonstrated greater antimicrobial effect on gram-positive bacteria tested than gram- negative bacteria. The presumption of resulting antimicrobial effect of gram negative bacteria would be that the thiol group reacts first with lipopolysaccharide layer and only then with the capsule polysaccharides known as glycocalyx and differences in the structure of the cell wall directly affect the resulting value of the inhibition zone. The results of our study would be able to confirm this hypothesis where the minimum inhibitory activity was significantly lower than the values obtained for gram-negative bacteria. Determining the diameter of the inhibition zone, minimum inhibitory concentration, or more precisely determine the antibacterial effect of garlic extract and the identification and assessment of the N-acetyl-L cysteine as a group of active substances which are present in garlic could contribute significantly to a better understanding of the scope of antimicrobial activity of garlic. The fact that N-acetyl-cysteine beside allicin has direct antimicrobial potential on bacterial species examined in the study could be the basis for the development of new therapeutic antimicrobial agents. This also can be used to expand the indications of existing drugs. It is known that N-acetyl-L-cysteine is now used as mucolytic. This work shows that its direct antimicrobial activity may potentially cause the fundamental changes in therapy with respiratory infections patients, to improve therapeutic efficacy and security.