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Öğe A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells(Humana Press Inc, 2018) Iz, Sultan Gulce; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, NazliRecent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-alpha), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-alpha mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-alpha mAb production from the humanized anti-TNF-alpha mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-alpha production. The production of anti-TNF-alpha in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-alpha MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.Öğe The biomarker features of miR-145-3p determined via meta-analysis validated by qRT-PCR in metastatic cancer cell lines(Elsevier, 2019) Kumoglu, Gizem Ors; Doskaya, Mert; Iz, Sultan GulceMicroRNAs (miRNAs) play important roles in the cancer biology such as proliferation, differentiation, and apoptosis. The pivotal roles that miRNA expression plays, make them ideal candidates for detection of cancer progression as well as cancer metastasis. Especially for breast, lung and prostate cancer which are originated from soft tissues and prone to metastasis. Thus, the aim of this study is to evaluate the expression level of miR145-3p which is a shared potential biomarker identified by meta-analysis of breast, prostate and lung cancer data sets. Six different data sets representative of three different cancer types were analyzed. These data sets are pooled together to have a master metamiRNA list while getting rid of the platform differentiations between them. As a result, 24 common differentially expressed miRNAs are determined in which miR-145-3p has the topmost rank. To mimic in vivo cancer microenvironment, hypoxia and serum deprivation were used to induce metastasis in breast (MCF-7, MDA-MB-231, MDA-MB-453), prostate (PC3, LNCaP, DU145), lung (A549, NCIH82,) cancer cell lines and noncancerous cell lines of the coresponding tissues (MCF10A, RWPE-1, MRC-5). miR-145-3p expression levels were determined by qRT-PCR. It has been shown that it is down regulated by the induction of metastasis in cancer cell lines while it is up regulated in normal cell lines to suppress the tumor formation. As a conclusion, as representing the same results in three different cancer cell types, miR-145-3p will be a promising biomarker to follow up its expression to detect cancer metastasis.Öğe Co-expression of the Bcl-xL antiapoptotic protein enhances the induction of Th1-like immune responses in mice immunized with DNA vaccines encoding FMDV B and T cell epitopes(Springer, 2013) Iz, Sultan Gulce; Doskaya, Mert; Borrego, Belen; Rodriguez, Fernando; Guruz, Yuksel; Gurhan, Ismet DelilogluFoot-and-mouth disease (FMD) is one of the most devastating animal diseases, affecting all cloven-hoofed domestic and wild animal species. Previous studies from our group using DNA vaccines encoding FMD virus (FMDV) B and T cell epitopes targeted to antigen presenting cells, allowed demonstrating total protection from FMDV homologous challenge in those animals efficiently primed for both humoral and cellular specific responses (Borrego et al. Antivir Res 92:359-363, 2011). In this study, a new DNA vaccine prototype expected to induce stronger and cross-reactive immune responses against FMDV which was designed by making two main modifications: i) adding a new B-cell epitope from the O-serotype to the B and T-cell epitopes from the C-serotype and ii) using a dual promoter plasmid that allowed inserting a new cistron encoding the anti-apoptotic Bcl-xL gene under the control of the internal ribosomal entry site (IRES) of encephalomyocarditis virus aiming to increase and optimize the antigen presentation of the encoded FMDV epitopes after in vivo immunization. In vitro studies showed that Bcl-xL significantly prolonged the survival of DNA transfected cells (p < 0.001). Accordingly, vaccination of Swiss out-bred mice with the dual promoter plasmid increased the total IgG responses induced against each of the FMDV epitopes however no significant differences observed between groups. The humoral immune response was polarized through IgG2a in all vaccination groups (p < 0.05); except peptide T-3A; in correspondence with the Th1-like response observed, a clear bias towards the induction of specific IFN-gamma secreting CD4(+) and CD8(+) T cell responses was also observed, being significantly higher (p < 0.05) in the group of mice immunized with the plasmid co-expressing Bcl-xL and the FMDV B and T cell epitopes.Öğe Comparison of in vitro cytotoxicity and genotoxicity of MMA-based polymeric materials and various metallic materials(Tubitak Scientific & Technical Research Council Turkey, 2010) Iz, Sultan Gulce; Gurhan, Saime Ismet Deliloglu; Sen, Bilge Hakan; Endogan, Tugba; Hasirci, NesrinAim: To determine the in vitro cytotoxicity and genotoxicity of some polymeric and metallic implant materials used as base materials in dentistry, based on ISO (International Organization for Standardization) and OECD (Organization for Economic Co-Operation and Development) test protocols. Materials and methods: Three different acrylate-based polymeric materials were tested for their in vitro cytotoxicity and genotoxicity (polymethylmethacrylate microspheres [PMMA], a solid cement prepared by mixing PMMA with its monomer methylmethacrylate [PMMA+MMA], a solid cement prepared by mixing PMMA, MMA, and hydroxyapatite [PMMA+MMA+HA], as wells as 4 different metallic materials (titanium [Ti grade 41, nickel alloy 625 [Ni-625], stainless steel alloy 304L [SS-304L], and stainless steel alloy 321 [SS-321]). Cytotoxic effects of the materials were determined using L929 mouse fibroblasts by MTT assay. Cell attachment properties related to the biocompatibility of the materials were analyzed using a scanning electron microscope (SEM). Genotoxicity of the materials was determined with human peripheral lymphocytes via micronucleus assay. Results: The highest compatibility was exhibited by Ti grade 4, followed by Ni-625, SS-304L and, SS-321. Among the polymeric materials, PMMA+MMA+HA had the highest biocompatibility, followed by PMMA+MMA and PMMA. Conclusion: The biocompatibility of the metallic materials was higher than that of the polymeric materials. Ti, the most inert metal, exhibited the highest biocompatibility. The addition of HA reduced the cytotoxic and mutagenic effects of MMA monomer and leachable ingredients.Öğe Development of a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50V and determination of its protective efficacy against acute toxoplasmosis(Bmc, 2020) Sahar, Esra Atalay; Can, Huseyin; Iz, Sultan Gulce; Doskaya, Aysu Degirmenci; Kalantari-Dehaghi, Mina; Deveci, Remziye; Doskaya, MertBackgroundToxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. Toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. A vaccine against T. gondii is required to prevent consequences of the infection. the aim of this study is to generate a multivalent recombinant protein vaccine against T. gondii.Methods49 previously discovered antigenic proteins of T gondii were evaluated by their expression level in E. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. Based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites.ResultsIn mice vaccinated with hexavalent vaccine, strong total IgG (P<0.0001) and IgG2a (P<0.001) responses were induced compared to controls, the ratio of CD4(+) and CD8(+) T lymphocytes secreting IFN-gamma increased, and significantly higher extracellular IFN-gamma secretion was achieved compared to the controls (P<0.001). the survival time of the vaccinated mice increased to 8.382.13days which was significantly higher than controls (P<0.01).Conclusions Altogether, these results show that the hexavalent vaccine which is developed for the first time against T. gondii induced strong and balanced Th1 and Th2 immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model.Öğe Diagnostic Value of a Rec-ELISA Using Toxoplasma gondii Recombinant SporoSAG, BAG1, and GRA1 Proteins in Murine Models Infected Orally with Tissue Cysts and Oocysts(Public Library Science, 2014) Doskaya, Mert; Caner, Ayse; Can, Huseyin; Iz, Sultan Gulce; Gedik, Yaprak; Doskaya, Aysu Degirmenci; Kalantari-Dehaghi, Mina; Guruz, YukselToxoplasma gondii causes congenital toxoplasmosis in newborns resulting with fetal anomalies. Determining the initiation time of infection is very important for pregnant women and current serological assays have drawbacks in distinguishing the recently acute toxoplasmosis. Diagnosis of recently acute infection may be improved by using stage specific antigens in serological assays. In the present study, the diagnostic value of sporozoite specific SporoSAG, bradyzoite specific BAG1 proteins and GRA1 protein expressed by all forms of the parasite have been evaluated ELISA using sera systematically collected from mice administered orally with tissue cyst and oocysts. The anti-SporoSAG IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 1, 10, and 15 (P<0.01). The anti-BAG1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly at days 15, 40, and 120 (P<0.05). The anti-GRA1 IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 2, 10, and 40 (P<0.01). The anti-GRA1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly only at day 40 (P<0.05). The anti-SporoSAG, anti-BAG1, and anti-GRA1 IgG titers of mice showed significant increases at day 40 (P<0.05) and decrement started for only anti-GRA1 IgG at day 120. The presence of anti-SporoSAG IgM and IgG antibodies can be interpreted as recently acute infection between days 10-40 because IgM decreases at day 40. Similarly, presence of anti-BAG1 IgM and absence of IgG can be evaluated as a recently acute infection that occurred 40 days before because IgG peaks at day 40. A peak in anti-GRA1 antibody level at first testing and reduction in consecutive sample can be considered as an infection approximately around day 40 or prior. Overall, recombinant SporoSAG, BAG1 and GRA1 proteins can be accepted as valuable diagnostic markers of recently acute toxoplasmosis.Öğe Discovery of new Toxoplasma gondii antigenic proteins using a high throughput protein microarray approach screening sera of murine model infected orally with oocysts and tissue cysts(Bmc, 2018) Doskaya, Mert; Liang, Li; Jain, Aarti; Can, Huseyin; Iz, Sultan Gulce; Felgner, Philip Louis; Doskaya, Aysu Degirmenci; Davies, David Huw; Guruz, Adnan YukselBackground: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations in immune compromised humans. The parasite has also been recently linked to behavioral diseases in humans and other mammalian hosts. New antigens are being evaluated to develop a diagnostic kit for the diagnosis of acute infection or a protective vaccine. Methods: In this study, we have focused on the discovery of new antigenic proteins from T. gondii genomic data using a high throughput protein microarray screening. To date, microarrays containing > 2870 candidate exon products of T. gondii have been probed with sera collected from patients with toxoplasmosis. Here, the protein microarrays are probed with well-characterized serum samples from animal models administered orally with oocysts or tissue cysts. The aim was to discover the antigens that overlap in the mouse profile with human antibody profiles published previously. For this, a reactive antigen list of 240 antigens recognized by murine IgG and IgM was identified using pooled sera from orally infected mice. Results: Analyses of screening data have identified plenty of antigens and showed strong immunogenicity in both mouse and human antibody profiles. Among them, ROP1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA14, MIC1, MIC2 and MAG1 have shown strong immunogenicity and used as antigen in development of vaccines or serological diagnostic assays in previous studies. Conclusion: In addition to the above findings, ROP6, MIC12, SRS29A and SRS13 have shown strong immunogenicity but have not been tested in development of a diagnostic assay or a vaccine model yet.Öğe Discovery of new Toxoplasma gondii antigenic proteins using a high throughput protein microarray approach screening sera of murine model infected orally with oocysts and tissue cysts (vol 11, 393, 2018)(Bmc, 2024) Doskaya, Mert; Liang, Li; Jain, Aarti; Can, Huseyin; Iz, Sultan Gulce; Felgner, Philip Louis; Doskaya, Aysu Degirmenci[Abstarct Not Available]Öğe The Effect of Ag and Ag plus N Ion Implantation on Cell Attachment Properties(Amer Inst Physics, 2009) Urkac, Emel Sokullu; Oztarhan, Ahmet; Tihminlioglu, Funda; Gurhan, Ismet Deliloglu; Iz, Sultan Gulce; Oks, Efim; Nikolaev, Alexey; Ila, Daryush; McDaniel, FD; Doyle, BLImplanted biomedical prosthetic devices are intended to perform safely, reliably and effectively in the human body thus the materials used for orthopedic devices should have good biocompatibility. Ultra High Molecular Weight Poly Ethylene (UHMWPE) has been commonly used for total hip joint replacement because of its very good properties. In this work, UHMWPE samples were Ag and Ag+N ion implanted by using the Metal-Vapor Vacuum Arc (MEVVA) ion implantation technique. Samples were implanted with a fluency of 1017 ion/cm2 and extraction voltage of 30 kV. Rutherford Backscattering Spectrometry (RBS) was used for surface studies. RBS showed the presence of Ag and N on the surface. Cell attachment properties investigated with model cell lines (L929 mouse fibroblasts) to demonstrate that the effect of Ag and Ag+N ion implantation can favorably influence the surface of UHMWPE for biomedical applications. Scanning electron microscopy (SEM) was used to demonstrate the cell attachment on the surface. Study has shown that Ag+N ion implantation represents more effective cell attachment properties on the UHMWPE surfaces.Öğe Genetic Characterization of Toxoplasma gondii Isolates and Toxoplasmosis Seroprevalence in Stray Cats of Izmir, Turkey(Public Library Science, 2014) Can, Huseyin; Doskaya, Mert; Ajzenberg, Daniel; Ozdemir, H. Gokhan; Caner, Ayse; Iz, Sultan Gulce; Doskaya, Aysu Degirmenci; Atalay, Esra; Cetinkaya, Cagdas; Urgen, Saygun; Karacali, Sabire; Un, Cemal; Darde, Marie-Laure; Guruz, YukselCurrently, some Toxoplasma gondii genotypes are being associated with serious clinical presentations. A recent report showing the Africa 1 genotype in two local congenital toxoplasmosis cases acquired in Turkey formed the basis of this study because atypical Africa 1 genotype is most frequently detected in animals and patients from sub-Saharan Africa. Since stray cats are considered as the linkage between wild life and urban life in T. gondii transmission, the present study aimed to isolate and characterize T. gondii strains circulating in stray cats of Izmir (Western Turkey). A secondary objective was to determine toxoplasmosis seroprevalence in this cat population. Tissues obtained from 100 deceased stray cats were bioassayed and isolated strains were genotyped using 15 microsatellite markers. In addition, toxoplasmosis seroprevalence was analyzed in 1121 cat sera collected from several large veterinary clinics in Izmir. Among the 22 isolates, 19 were Type II (86.3%), two were Type III (9%) and one was Africa 1 genotype (4.5%). The overall seropositivity rates in cats were 42-48% and 33.4-34.4% according to IFA and ELISA, respectively. Seroprevalence in deceased cats was significantly higher than in healthy cats (P = 0.0033). Finding both the major clonal Type II lineage together with the Type III lineage also found in Middle East, and an atypical genotype, Africa 1 appears consistent with the specific geographic location of Turkey between three continents and raises the possibility of transportation of these strains between continents through trade routes or long distance migratory birds. In addition, the first large study of toxoplasma seroprevalence in a stray cat population was also reported. The relatively high seropositivity rates and the variety of T. gondii genotypes confirm the local stray cat population as a risk factor for human toxoplasmosis in Izmir.Öğe Polyclonal Antibody Production Against Hapten-Structured KDN Molecule by Using Different Adjuvants Alternative to Freund's Adjuvant(Aves Press Ltd, 2018) Iz, Sultan Gulce; Metiner, Pelin Saglam; Kimiz, Ilgin; Kayali, Caglar; Gurhan, Saime Ismet DelilogluObjective: KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a member of the sialic acid family, is a hapten-structured low-molecular-weight monosaccharide on the cell membrane, which cannot induce immune responses without a carrier protein. Since it is over-expressed on the cancerous cells' membrane, it is thought to be a great target molecule for anti-cancer treatments. The aim of this study is to obtain high titersof the anti-KDN polyclonal antibody response without using any carrier protein against the hapten-structured KDN molecule alternative to Freund's adjuvants. Methods: Montanide (TM) ISA 61 VG, a water-in-oil adjuvant; ISA 201 VG, a water-in-oil-in-water emulsion adjuvant; and IMS 1313 VG NPR, an aqueous-dispersion-based nanoparticle (50-200 nm) microemulsion adjuvant; and Freund's adjuvant were used as anti-KDN antibody response stimulators. FourBALB/c mice were used for each adjuvant group, and immunization was performed at eight different time points. Anti-KDN antibody levels induced after each immunization with different adjuvants were detected with indirect enzyme-linked immunosorbent assay. Results: The adjuvant efficiency of Montanide (TM) ISA 61 VG water in oil adjuvant was 1.4 times higher than in Freund's adjuvant (p<0.0001), with a maximum anti-KDN level on Day 83. Conclusion: It's shown that without any carrier protein conjugation molecules such as hapten-structured KDN, higher amount anti-KDN antibody titres could be obtained by using a more safe and effective Montanide (TM) ISA 61 VG water-in-oil adjuvant as an alternative to Freund's adjuvants. In this regard, it may be possible to produce high-antibody titers without using any carrier molecule, especially when commercial large scale monoclonal antibodies are desired to be produced against haptens as therapeutic approaches.Öğe Semi-IPN Chitosan/PEG Microspheres and Films for Biomedical Applications: Characterization and Sustained Release Optimization(Amer Chemical Soc, 2012) Gunbas, Ismail Dogan; Sezer, Umran Aydemir; Iz, Sultan Gulce; Gurhan, Ismet Deliloglu; Hasirci, NesrinMicro drug carriers are one of the efficient methods for local or systemic cancer treatment. In this study, the aim was to prepare a novel semi-interpenetrated (semi-IPN) micro system by using biocompatible chitosan (CH) and polyethylene glycol (PEG). Various combinations of the systems were prepared and loaded with a model chemotherapeutic drug, methotrexate (MTX), and the effects of composition on the properties and the release behavior of microspheres were examined. Also, the mechanical and thermal properties were examined on film forms of similar compositions. Increase in cross-linking caused a decrease in particle size of CH from 144 to 91 mu m, while the addition of PEG caused an increase up to 163 mu m. Elastic modulus values of the films first increased and then decreased parallel to PEG content. In vitro studies showed faster MTX release from semi-IPN CH-PEG microspheres as compared to pure CH ones. Promising results were obtained in the development of biodegradable drug vehicles.Öğe Semi-IPN chitosan/polyvinylpyrrolidone microspheres and films: sustained release and property optimisation(Informa Healthcare, 2013) Ozerkan, Taylan; Sezer, Umran Aydemir; Gurhan, Ismet Deliloglu; Iz, Sultan Gulce; Hasirci, NesrinA set of chitosan-polyvinylpyrrolidone (CH-PVP) microspheres were prepared as semi-inter penetrating networks (semi-IPN) and loaded with 5-fluorouracil. In vitro release studies showed faster release for semi-IPN microspheres compared to pure CH samples, and the total release was achieved in about 20-30 days, depending on the composition. In vitro cell studies were achieved against human breast adenocarcinoma cell line cells where adsorption of cells on microspheres with a significant decrease in their number was obtained. Meanwhile, the CH-PVP films, which were prepared with the same compositions as in the microspheres, demonstrated an increase in strength from 66 to 118 MPa as the PVP content was decreased. It can be concluded that the prepared CH-PVP semi-IPN microspheres are novel promising carriers compared to pure CH microspheres since it becomes possible to adjust stability and hydrophilicity of the microspheres as well as the release rates of the drugs from the microspheres by changing the ratio of CH/PVP composition.Öğe Semi-IPN chitosan/polyvinylpyrrolidone microspheres and films: sustained release and property optimisation(Informa Healthcare, 2013) Ozerkan, Taylan; Sezer, Umran Aydemir; Gurhan, Ismet Deliloglu; Iz, Sultan Gulce; Hasirci, NesrinA set of chitosan-polyvinylpyrrolidone (CH-PVP) microspheres were prepared as semi-inter penetrating networks (semi-IPN) and loaded with 5-fluorouracil. In vitro release studies showed faster release for semi-IPN microspheres compared to pure CH samples, and the total release was achieved in about 20-30 days, depending on the composition. In vitro cell studies were achieved against human breast adenocarcinoma cell line cells where adsorption of cells on microspheres with a significant decrease in their number was obtained. Meanwhile, the CH-PVP films, which were prepared with the same compositions as in the microspheres, demonstrated an increase in strength from 66 to 118 MPa as the PVP content was decreased. It can be concluded that the prepared CH-PVP semi-IPN microspheres are novel promising carriers compared to pure CH microspheres since it becomes possible to adjust stability and hydrophilicity of the microspheres as well as the release rates of the drugs from the microspheres by changing the ratio of CH/PVP composition.Öğe Seroprevalence of Leishmania infection and molecular detection of Leishmania tropica and Leishmania infanturn in stray cats of Izmir, Turkey(Academic Press Inc Elsevier Science, 2016) Can, Huseyin; Doskaya, Mert; Ozdemir, H. Gokhan; Sahar, Esra Atalay; Karakavuk, Muhammet; Pektas, Bayram; Karakus, Mehmet; Toz, Seray; Caner, Ayse; Doskaya, Aysu Degirmenci; Iz, Sultan Gulce; Ozbel, Yusuf; Guruz, YukselLeishmaniasis caused by more than 20 species of genus Leishmania is transmitted by the bite of infected phlebotomine sand flies. The studies on Leishmania infection in cats is very few in Turkey and therefore we aimed to screen stray cats living in city of Izmir located in western Turkey using nested PCR targeting kinetoplast DNA and serological techniques (ELISA and IFA). Leishmania DNA positive samples were also studied by ITS1 real time PCR. Whole blood and serum samples were obtained from stray cats (n: 1101) living in different counties of Izmir. In serological assays, a serum sample was considered positive in 1:40 dilution in IFA and for ELISA a serum sample was accepted positive when the absorbance value (AV) exceeded the mean AV + Standard Deviation (SD) of the negative control serum samples. According to the results, the seropositivity rates were 10.8% (119/1101) and 15.2% (167/1101) by in house ELISA and IFA, respectively. Among serology coherent samples, the seropositivity rate was 11.1% (116/1047) as detected by both assays after discordant samples (n: 54) were discarded. Of the 1101 stray cats, six (0.54%) were positive by nested PCR while only one of these six samples was positive by ITS1 real time PCR. During PCR, three controls designated as Leishmania infantum, Leishmania tropica, and Leishmania major were used for species identification. According to nested PCR results, L. tropica was identified in two cats (no.76 and 95). In another cat (no. 269), there were two bands in which one of them was well matched with L infantum and the other band had similar to 850 bp size which does not match with any controls. Remaining three cats (no. 86, 514, and 622) also had the similar to 850 bp atypical band size. ITS1 real time PCR detected L tropica in only one cat (no. 622) which showed an atypical band size in nested PCR. These results indicated that three cats with only one atypical band (no. 86, 514, and 622) and the cat with mixed infection (no. 269) were infected with L tropica. Altogether, L tropica was detected in all six DNA positive cats and L infantum was detected in one cat with mixed infection. In conclusion, although the reservoir role of cats in nature is still unclear the high seroprevalence rate against Leishmania parasites and detecting parasite DNA in stray cats in Izmir indicates that the stray cats are frequently bitten by infected sand flies. Further research activities are required to reveal the frequency of leishmaniasis in cats in different regions of Turkey where Leishmania species are endemic. (C) 2016 Elsevier Inc. All rights reserved.Öğe Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis(Univ Catolica De Valparaiso, 2012) Iz, Sultan Gulce; Calimlioglu, Beste; Gurhan, Saime Ismet DelilogluBackground: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate the anti-apoptotic efficacy of Bcl-xL expressing dual promoter plasmid system as a candidate to be used for recombinant protein production and DNA vaccination approaches. For this purpose, Bcl-xL anti-apoptotic protein gene was inserted in a dual expressing vector system in frame with EGFP (enhanced green fluorescence protein) after IRES (internal ribosomal site). The plasmid has a multiple cloning site after CMV (cytomegalovirus promoter) left empty to be inserted a biopharmaceutical protein gene region or DNA vaccine antigens. Results: In order to determine the anti-apoptotic efficacy of Bcl-xL inserted dual expressing vector, BHK-21 cells were transfected both with this plasmid and empty vector as control. Apoptosis was stimulated by several apoptosis inducing agents and serum deprivation in the transfected cells for 48 hrs. Cells expressing Bcl-xL protein in frame with EGFP were determined by flow cytometry as an indicator of cell viability. Additionally, apoptosis were determined by intracellular cleaved Casp 3 staining in Bcl-xL expressing EGFP positive cells. The dual expression plasmid bearing Bcl-xL anti-apoptotic protein prolonged the cell survival rate and protected cells from apoptosis upon apoptosis induction by doxorubicin and camptothecin in which the anti-apoptotic efficacies are inhibited through over expressing of Bcl-xL. pIRES2EGFP/Bcl-xL transfected cell ratio was significantly higher compared to empty vector transfected cells (P < 0.001). In contrast, apoptotic cell ratio was significantly lower in pIRES2EGFP/Bcl-xL transfected cell population compared to empty vector transfected cells (P < 0.001). Conclusion: In conclusion, it was shown that in vitro transient expression of Bcl-xL efficiently inhibited apoptosis induced by serum deprivation, doxorubicin and camptothecin. Thus, the dual expression plasmid bearing Bcl-xL anti-apoptotic protein could be a good candidate for recombinant protein production and DNA vaccination applications.
