Browsing by Author "Ozbilgin, A"

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    A new experimental in vitro culture medium for cultivation of Leishmania species
    Limoncu, ME; Balcioglu, IC; Yereli, K; Ozbel, Y; Ozbilgin, A
    A new liquid culture medium prepared with chemicals that can be obtained economically and commercially was tested in in vitro cultivation of Leishmania promastigotes to obtain a large number of organisms to use in serological studies, The number of Leishmania infantum and Leishmania tropica promastigotes taken from Novy-MacNeal-Nicolle (NNN) medium reached 1 x 10(7)/ml at the end of the 8th day In our nem medium, though in NNN medium the number of organisms reached only 5 x 10(6)/ml. After 10 subsequent passages, the culture medium prepared was evaluated as being quite inexpensive, simple, and successful compared with other commercially available liquid culture media.
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    Characterization of L. tropica secreted exosomes and development of exosome-based preventive vaccine against L. tropica induced cutaneous leishmaniasis
    Gungor, B; Ayanoglu, IC; Konig, GT; Ozbel, Y; Ozbilgin, A; Girginkardesler, N; Toz, SO; Gursel, I; Gursel, M
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    Comparative proteomic analysis of Leishmania parasites isolated from visceral and cutaneous leishmaniasis patients
    Dinc, M; Yalcin, T; Cavus, I; Ozbilgin, A
    Leishmaniasis is an infectious disease in which different clinical manifestations are classified into three primary forms: visceral, cutaneous and mucocutaneous. These disease forms are associated with parasite species of the protozoan genus Leishmania. For instance, Leishmania infantum and Leishmania tropica are typically linked with visceral (VL) and cutaneous (CL) leishmaniasis, respectively; however, these two species can also cause other form to a lesser extent. What is more alarming is this characteristic, which threatens current medical diagnosis and treatment, is started to be acquired by other species. Our purpose was to address this issue; therefore, gel-based and gel-free proteomic analyses were carried out on the species L. infantum to determine the proteins differentiating between the parasites caused VL and CL. In addition, L. tropica parasites representing the typical cases for CL were included. According to our results, electrophoresis gels of parasites caused to VL were distinguishable regarding the repetitive down-regulation on some specific locations. In addition, a distinct spot of an antioxidant enzyme, superoxide dismutase, was shown up only on the gels of CL samples regardless of the species. In the gel-free approach, 37 proteins that were verified with a second database search using a different search engine, were recognized from the comparison between VL and CL samples. Among them, 31 proteins for the CL group and six proteins for the VL group were determined differentially abundant. Two proteins from the gelbased analysis, pyruvate kinase and succinyl-coA:3-ketoacid-coenzyme A transferase analysis were encountered in the protein list of the CL group.
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    In vivo Antimalarial Activity of Methanol and Water Extracts of Eryngium thorifolium Boiss (Apiaceae Family) against P. berghei in Infected Mice
    Ural, IO; Kayalar, H; Durmuskahya, C; Cavus, I; Ozbilgin, A
    Purpose: To investigate the in vivo antimalarial effect of Eryngium thorifolium, an endemic plant in Turkey. Methods: The methanol and water extracts were prepared and phytochemical analysis conducted on the extracts. Twenty four healthy Balb/c male mice, divided into 4 groups (n = 6), were infected intravenously with Plasmodium berghei and 100 - 250 mg/kg plant extracts administered orally in a single dose per day for 5 days. The untreated group of mice received normal saline solution and chloroquine (standard drug) served as reference drug. Results: The water extract group (250 mg/kg) prolonged the survival of the mice by 6 days compared with the untreated mice while the mice that received choloroquine treatment remained alive at the end of the study of the mice. In the untreated control group, maximum parasitaemia was observed on the 10th day of infection whereas The water extract exhibited some degree of antiplasmodial activity compared to untreated control group. The mice of chloroquine treated group remained alive at the end of the study with 100 % chemosuppression (p < 0.05). In the untreated control group, maximum parasitaemia was observed on the 10th day of infection whereas in the water extract group maximum parasitaemia was attained on the 16th day of infection. The water extract of the plant showed 45.85 % chemosuppression. Phytochemical screening of the water and methanol extracts revealed the presence of flavonoids, terpenoids and tannins. Anthraquinones were positive for water extract. Conclusions: The possible active compounds responsible for the observed chemosupression may be flavonoids, terpeneoids and anthraquinones which are present in the extract. This is the first report on the in vivo antimalarial activity of E. thorifolium.
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    Design, synthesis, and in vitro biological evaluation of novel thiazolopyrimidine derivatives as antileishmanial compounds
    Istanbullu, H; Bayraktar, G; Akbaba, H; Cavus, I; Coban, G; Butuner, BD; Kilimcioglu, AA; Ozbilgin, A; Alptuzun, V; Erciyas, E
    A series of thiazolopyrimidine derivatives was designed and synthesized as aLeishmania majorpteridine reductase 1 (LmPTR1) enzyme inhibitor. TheirLmPTR1 inhibitor activities were evaluated using the enzyme produced byEscherichia coliin a recombinant way. The antileishmanial activity of the selected compounds was tested in vitro againstLeishmaniasp. Additionally, the compounds were evaluated for cytotoxic activity against the murine macrophage cell line RAW 264.7. According to the results, four compounds displayed not only a potent in vitro antileishmanial activity against promastigote forms but also low cytotoxicity. Among them, compoundL16exhibited an antileishmanial activity for both the promastigote and amastigote forms ofL. tropica, with IC(50)values of 7.5 and 2.69 mu M, respectively. In addition, molecular docking studies and molecular dynamics simulations were also carried out in this study. In light of these findings, the compounds provide a new potential scaffold for antileishmanial drug discovery.
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    The Follow-Up of Treatment Process of Malaria By Real-Time Polymerase Chain Reaction: In Vivo Model
    Cavus, I; Ozbilgin, A; Balcioglu, IC
    Microscopic methods are accepted as the gold standard in the diagnosis of malaria and in the follow-up of treatment. However, as the microscopical methods require experienced personnel, it is important to confirm the diagnosis with a different method for accurate diagnosis and treatment follow-up. In our study, we aimed to investigate the utility of the use of real time reverse transcriptase polymerase chain reaction (rRT-PCR), as well as microscopic methods for malaria treatment follow-up. In our study, we formed five groups each consisting of five male Balb/c mice. Each mouse was injected intraperitoneally with 10(7)/ml Plasmodium berghei parasites. After 48 hours following the injection, the mice in the first, second and third groups received 50 mg/kg/day of chloroquine treatment for one, two and three days, respectively. The fourth group was not treated and the fifth group of mice received saline for three days. The parasitemia was monitored for 21 days by blood smears prepared from the end of tail of the mice and searching the presence of the target gene region of the parasite by rRT-PCR. Both the blood smears and rRT-PCR results were positive for groups I, II, IV and V. Both blood smears and rRT-PCR results of mice in groups other than the third group were found to be positive. Blood smears of the mice in third group were found to be positive on the 5th and 7th days of the infection, and the subsequent preparations were evaluated as negative. rRT-PCR results showed positivity on day seven, but no presence of the target gene region of the parasite was detected on the other days. The comparison of microscopy and rRT-PCR methods, had shown parallel results. Apart from the microscopic examination method, it was concluded that the rRT-PCR method is important in the diagnosis of malaria and in the follow-up of the patient during the treatment process, and that different methods that support each other should be used.
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    Leishmania kinetoplast DNA contributes to parasite burden in infected macrophages: Critical role of the cGAS-STING-TBK1 signaling pathway in macrophage parasitemia
    Yilmaz, IC; Dunuroglu, E; Ayanoglu, IC; Ipekoglu, EM; Yildirim, M; Girginkardesler, N; Ozbel, Y; Toz, S; Ozbilgin, A; Aykut, G; Gursel, I; Gursel, M
    Leishmania parasites harbor a unique network of circular DNA known as kinetoplast DNA (kDNA). The role of kDNA in leishmania infections is poorly understood. Herein, we show that kDNA delivery to the cytosol of Leishmania major infected THP-1 macrophages provoked increased parasite loads when compared to untreated cells, hinting at the involvement of cytosolic DNA sensors in facilitating parasite evasion from the immune system. Parasite proliferation was significantly hindered in cGAS- STING- and TBK-1 knockout THP-1 macrophages when compared to wild type cells. Nanostring nCounter gene expression analysis on L. major infected wild type versus knockout cells revealed that some of the most upregulated genes including, Granulysin (GNLY), Chitotriosidase-1 (CHIT1), Sialomucin core protein 24 (CD164), SLAM Family Member 7 (SLAMF7), insulin-like growth factor receptor 2 (IGF2R) and apolipoprotein E (APOE) were identical in infected cGAS and TBK1 knockout cells, implying their involvement in parasite control. Amlexanox treatment (a TBK1 inhibitor) of L. major infected wild type cells inhibited both the percentage and the parasite load of infected THP-1 cells and delayed footpad swelling in parasite infected mice. Collectively, these results suggest that leishmania parasites might hijack the cGAS-STING-TBK1 signaling pathway to their own advantage and the TBK1 inhibitor amlexanox could be of interest as a candidate drug in treatment of cutaneous leishmaniasis.
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    Cutaneous leishmaniasis caused by Leishmania infantum in Turkey: reports of two cases diagnosed with genotyping and protein fingerprinting
    Culha, G; Akyar, I; Zeyrek, FY; Gündüz, C; Kurt, Ö; Ostan, I; Töz, S; Kocagoz, T; Ozbel, Y; Ozbilgin, A
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    Design, synthesis, in vitro- In vivo biological evaluation of novel thiazolopyrimidine compounds as antileishmanial agent with PTR1 inhibition
    Istanbullu, H; Bayraktar, G; Karakaya, G; Akbaba, H; Perk, NE; Cavus, I; Podlipnik, C; Yereli, K; Ozbilgin, A; Butuner, BD; Alptuzun, V
    The leishmaniasis are a group of vector-borne diseases caused by a protozoan parasite from the genus Leish-mania. In this study, a series of thiazolopyrimidine derivatives were designed and synthesized as novel anti-leishmanial agents with LmPTR1 inhibitory activity. The final compounds were evaluated for their in vitro antipromastigote activity, LmPTR1 and hDHFR enzyme inhibitory activities, and cytotoxicity on RAW264.7 and L929 cell lines. Based on the bioactivity results, three compounds, namely L24f, L24h and L25c, were selected for evaluation of their in vivo efficacy on CL and VL models in BALB/c mice. Among them, two promising compounds, L24h and L25c, showed in vitro antipromastigote activity against L. tropica with the IC50 values of 0.04 mu g/ml and 6.68 mu g/ml; against L. infantum with the IC50 values of 0.042 mu g/ml and 6.77 mu g/ml, respec-tively. Moreover, the title compounds were found to have low in vitro cytotoxicity on L929 and RAW264.7 cell lines with the IC50 14.08 mu g/ml and 21.03 mu g/ml, and IC50 15.02 mu g/ml and 8.75 mu g/ml, respectively. LmPTR1 enzyme inhibitory activity of these compounds was determined as 257.40 mu g/ml and 59.12 mu g/ml and their selectivity index (SI) over hDHFR was reported as 42.62 and 7.02, respectively. In vivo studies presented that L24h and L25c have a significant antileishmanial activity against footpad lesion development of CL and at weight measurement of VL group in comparison to the reference compound, Glucantime (R). Also, docking studies were carried out with selected compounds and other potential Leishmania targets to detect the putative targets of the title compounds. Taken together, all these findings provide an important novel lead structure for the anti-leishmanial drug development.
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    Circular RNAs as a new perspective in the diagnosis and mechanism of Leishmania infections
    Alizadeh, H; Muftuoglu, C; Omondi, ZN; Mert, U; Asadi, M; Ozbilgin, A; Caner, A
    Leishmaniasis is a neglected infectious disease that affects millions of people worldwide. Visceral leishmaniasis (VL) caused by Leishmania infantum and cutaneous leishmaniasis (CL) caused by L. major/ L. tropica are the main clinical forms of this disease, which are life-threatening if not diagnosed and treated properly. Considering the problems in sampling and laboratory diagnosis of leishmaniasis, new molecular markers such as circular RNAs (circRNAs) are needed. circRNAs, a novel class of RNAs, have been one of the most promising targets for the diagnosis and prognosis of diseases. Although the therapeutic and diagnostic role of circRNAs in many diseases and some parasitic diseases are known, not much research has been done in the field of leishmaniasis. We determined the gene expressions of circRNAs in human leukemia monocytic (THP-1) cells after infection with Leishmania. For this, the human cell line THP-1 was differentiated into macrophages by Phorbol 12-myristate 13acetate (PMA) treatment. Differentiated THP-1 cells were infected with L. infantum and L. tropica promastigotes. After 24 hours, expression levels of circRNAs were determined by RT-qPCR technique. Also, the microRNAs associated with differentially expressed circRNAs were investigated. Then, the molecular pathways associated with expressed circRNAs were obtained by GO and Reactome. The results showed that five circRNAs were differentially expressed in THP1 macrophages infected with L. infantum and L. tropica. These findings suggest that some circRNAs may be potential biomarkers for diagnosis in Leishmania-infected patients. The enrichment analysis revealed that differentially expressed circRNAs are mainly involved in the regulation of protein stability, RNA catabolic process, and P53/PTK6 signaling mechanism. This is the first study to report an overview of Leishmania-induced circRNAs, which can be potential biomarker candidate for diagnosis especially at species level. Notably, expression of some circRNAs in supernatant of Leishmania infected macrophages suggests that these genes are available in body fluids, therefore, can easily be accessed from the patient without invasive methods especially during treatment monitoring.
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    Genotyping of Giardia lamblia in a Cohort of Turkish Patients: A Search for a Relationship between Symptoms and Genotypes
    Balcioglu, C; Kurt, O; Sevil, N; Dagci, H; Tetik, A; Ergunay, K; Yereli, K; Ozbilgin, A; Turgay, N; Toz, SO
    Recent surveys investigating the molecular biology of Giardia lamblia revealed two distinct assemblages with different clinical outcomes. However, there is not a universal compromise about the clinical effects of each assemblage, warranting further studies. Here, we report the results of the first analyses of the assemblages of G. lamblia in Manisa province located in western Turkey, together with their relationships with the symptoms and DNA sequence analyses of the PCR products. DNA samples were isolated from the stools of 63 patients infected with G. lamblia and 54 DNA samples, amplified successfully with PCR, were digested with the enzyme Xho I for RFLP. Thirty-eight of 54 samples (70.4%) were found to be in Assemblage A, while the remaining 16 samples (29.6%) were found to be in Assemblage B. The number of female patients was found significantly higher in Assemblage B (P=0.18). There was a statistically significant relationship between the occurrence of both abdominal pain and diarrhea and Assemblage B (chi-square, 10.52; P<0.05). No other statistically significant relationship was detected between the assemblages and neither with the symptoms nor with the age groups of the patients. The comparison of the DNA sequences of the PCR products from two assemblage B (one subtype B1 and one B) and one assemblage A samples both with each other and with other DNA sequences in the NCBI website by multialignment analyses, revealed specific regions for assemblages B (B1-B) and A on tpi gene region. Further studies with more patients are required to assess these initial results. Now, our aim is to design a probe for tpi gene region to set up a real-time PCR assay that is easier to conduct and requiring shorter time for the analyses.
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    Antileishmanial Activity of Selected Turkish Medicinal Plants
    Ozbilgin, A; Durmuskahya, C; Kayalar, H; Ertabaklar, H; Gunduz, C; Ural, IO; Zeyrek, F; Kurt, O; Cavus, I; Balcioglu, C; Toz, SO; Ozbel, Y
    Purpose: To determine the in vitro and in vivo anti-leishmanial activities of extracts obtained from Centaurea calolepis, Phlomis lycia, Eryngium thorifolium, Origanum sipyleum and Galium incanum ssp. centrale. Methods: To estimate the cytotoxicity of plant extracts, WST-1 assay was used. Parasite inhibition in the presence of plant extracts (25 - 500 mu g/ml) in comparision with control group and reference group (glucantime, 25 mu g/ml) at 12 - 72 h were determined in vitro on L. tropica promastigotes. The in vivo leishmanicidal activity of the extracts was evaluated against L. tropica-infected mice with glucantime as reference drug. Results: The chloroform extract of Galium incanum ssp. centrale showed the highest cytotoxicity with IC50 value of 0.0316 +/- 0.005 mu g/ml. In vitro parasite inhibition by the plant extracts ranged between 16.7 +/- 0.01 % and 100 +/- 0.00 % at 25 mu g/ml concentration. The methanol extract of Eryngium thorifolium possessed the highest activity on promastigotes of L. tropica with 100 % inhibition at 25 mu g/ml. The water and chloroform extracts of C. calolepis and water and methanol extracts of E. thorifolium at a dose of 100 mg/kg reduced parasitaemia in L. tropica infected mice. Conclusion: Parasite viability results suggest that the methanol extract of Eryngium thorifolium, regarded as non-cytotoxic, is a promising candidate drug for treating L. tropica infection.
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    Origanum Sipyleum Methanol Extract in Combination with Ponatinib Shows Synergistic anti-Leukemic Activities on Chronic Myeloid Leukemia Cells
    Kayabasi, C; Susluer, SY; Okcanoglu, TB; Yelken, BO; Mutlu, Z; Bagca, BG; Kurt, CC; Saydam, G; Durmuskahya, C; Kayalar, H; Ozbilgin, A; Avci, CB; Gunduz, C
    Origanum sipyleum is used in folk medicine due to its anti-inflammatory, antimicrobial, and antioxidant properties. Ponatinib, an effective tyrosine kinase inhibitor in the treatment of chronic myeloid leukemia (CML), has severe side effects. Thus, we aimed to determine a novel herbal combination therapy that might not only increase the anti-leukemic efficacy but also reduce the dose of ponatinib in targeting CML cells. Origanum sipyleum was extracted with methanol (OSM), and secondary metabolites were determined by phytochemical screening tests. The cytotoxic effects of OSM on K562 cells were measured by WST-1 assay. Median-effect equation was used to analyze the combination of ponatinib and OSM (p-OSM). Apoptosis, proliferation, and cell-cycle were investigated by flow-cytometry. Cell-cycle-related gene expressions were evaluated by qRT-PCR. OSM that contains terpenoids, flavonoids, tannins, and anthracenes exhibited cytotoxic effects on K562 cells. The median-effect of p-OSM was found as synergistic; OSM reduced the ponatinib dose similar to 5-fold. p-OSM elevated the apoptotic and anti-proliferative activity of ponatinib. Consistently, p-OSM blocked cell-cycle progression in G(0)/G(1), S phases accompanied by regulations in TGFB2, ATR, PP2A, p18, CCND1, CCND2, and CCNA1 expressions. OSM enhanced the anti-leukemic activity of ponatinib synergistically via inducing apoptosis, suppressing proliferation, and cell-cycle. As a result, OSM might offer a potential strategy for treating patients with CML.
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    Assessment of in vivo antimalarial activities of some selected medicinal plants from Turkey
    Ozbilgin, A; Durmuskahya, C; Kayalar, H; Ostan, I
    Resistant infections lead to increased necessity of searching novel drugs and drug combinations. The purpose of this paper was to investigate antimalarial properties of some selected medicinal plants that have been traditionally used in Turkey for antipyretic and analgesic purposes. Lavandula stoecheas subsp. cariensis, Phlomis nissolii, Phlomis bourgaei, Phlomis leucophracta, Centaurea hierapolitana, Centaurea polyclada, Centaurea lydia, Scrophularia cryptophila, Scrophularia depauperata, Scrophularia floribunda, Rubia davisiana, and Alkanna tinctoria subsp. subleiocarpa were investigated for their in vivo antimalarial activities in mice infected with Plasmodium yoelii. Two hundred fifty to 500 mg/kg doses of plant extracts were given to mice as a single daily dose for 4 days. P. nissolii water extract, C. lydia chloroform extract, S. cryptophila ethanol extract, and C. polyclada methanol extract showed antimalarial activity with reducing parasitaemia. The chemotherapeutic effects of plant extracts ranged between 13.5 % and 66.91 %. The chemosuppressions exerted by combined plant extracts of P. nissolii, S. cryptophila, and C. lydia with C. polyclada methanol extract were detected as 51.25 %, 57.33 %, and 58.33 %, respectively. Investigation of cytotoxic activities against brine shrimps revealed that methanol extract of C. polycada, chloroform extract of C. lydia, and ethanol extract of S. cryptophila showed cytotoxic activities, while water extract of P. nissolii was not active against brine shrimps.
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    Detection of anti-leishmanial effect of the Lucilia sericata larval secretions in vitro and in vivo on Leishmania tropica: First work
    Polat, E; Cakan, H; Aslan, M; Sirekbasan, S; Kutlubay, Z; Ipek, T; Ozbilgin, A
    It is known that some of the enzymes and substances secreted by 2nd and 3rd stages of the Lucilia sericata larvae to have bacteriostatic and bactericidal effects. From this point of view, we investigated the anti-leishmanial effect of larval secretions of the L. sericata on the Leishmania tropica both in vitro and in vivo conditions. In vitro: It was observed that promastigotes of L. tropica had undergone lyzis within 1 min in the larval secretions of L. sericata. However, larval secretion was ineffective on the promastigotes within Novy-Mac-Neal-Nicolle (NNN) cultures and RPMI 1640 medium. In vivo: Seven groups of male Balb/C mice (6 study groups and 1 control group), each composed of eight weeks old 10 mice were formed. L. tropica promastigotes were injected subcutaneosly to the soles of the SG mice' feet. In study groups, cutaneous lesions were developed Limoncu et al., 1997 in 2 (20%) and 1 (10%) of the SG-1 and SG-2, respectively after 15 days. There were L. tropica in the smears prepared from the lesions and L tropica was observed in the cultures. Cutaneous lesions were not developed in 8 (80%), 9 (90%) and 10(100%) of the SG-I, SG-II and SG-III, respectively. There were no cutaneous lesions developed in the soles of the feet. There were no L. tropica in the smears prepared from the infected soles of the feet neither L. tropica was observed in the cultures. Larval secretions were given into the cutaneous lesions to the feet soles of the SG-IV, V and VI mice after 6 months. No healing was observed in the cutaneos lesions of 4 (40%), 5 (50%) and 1(10%) of SG-IV, SG-V and SG-VI, after 6 months, respectively. There were L tropica in the smears prepared from the lesions and L. tropica was observed in the cultures. On the other hand, the lesions of 6 (60%), 5 and 9(90%) of SG-IV, SG-V and SG-VI were diminished in size and disappeared completely after 6 months. There were L. tropica observed in the smears prepared from the infected soles of the feet and no growth was observed in the cultures. In the smears prepared from the cutaneous lesions developed in the soles of the feet of the control group mice. L. tropica was visualized and observed in the cultures. A statistical significant difference was observed between study groups and control group (p < 0.001). In our study we demonstrated for the first time that the secretions of the 2nd and 3rd stages sterile and pure larvae of L. sericata had effects on promastigotes of L. tropica in in vitro and very effective on amastigote forms in in vivo conditions. (c) 2012 Elsevier Inc. All rights reserved.