Sultan Gülçe İz

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.

Toxoplasma 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.

Genetic Characterization of Toxoplasma gondii Isolates and Toxoplasmosis Seroprevalence in Stray Cats of İzmir, Turkey

Currently, 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 İzmir (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 İzmir. 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 İzmir.

Biocompatibility of glass ionomer cements with and without chlorhexidine.

Objective: The aim of the present study is to evaluate the biocompatibility of glass ionomer cements (GICs) with and without chlorhexidine (CHX) as well as coated with varnish or not using in vitro cytotoxicity test. Materials and Methods: Biocompatibility of Fuji IX, Fuji IX with varnish, Fuji IX with 1% CHX diacetate and Fuji IX with 1% CHX diacetate with varnish was determined with in vitro cytotoxicity assay by using L929 mouse connective tissue fibroblasts. After 72 h, cell viabilities were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to determine the effects of the cements on the mitochondrial function and microscopic images were taken by scanning electron microscopy. Results: Statistical analysis was performed by one-way analysis of variance followed by the Bonferroni post-hoc test at a significance level of P < 0.05. 72 h after treatment, there were statistically significant differences between Fuji IX and Fuji IX-CHX (P < 0.001). In addition, the reduction of the cytotoxicity by coating the GICs with varnish was indicative and increased the cell viability ratio (P < 0.001). Conclusions: Fuji IX coated with varnish was found to be the most biocompatible one among others. Thus adding CHX significantly reduced the cell viability, it is assumed that, due to the leakage of CHX and the other components of the GICs to the cell culture medium, the cell viabilities were decreased, so it is highly recommended to use varnish not only to reduce the water loss from the GICs, but also to reduce the cytotoxicity of the GICs.

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.

Foot-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 T3A; in correspondence with the Th1-like response observed, a clear bias towards the induction of specific IFN-γ 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.

Semi-IPN chitosan/polyvinylpyrrolidone microspheres and films: sustained release and property optimisation.

Abstract A 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.

Cryopreservation of Toxoplasma gondii tachyzoites and tissue cysts

Toxoplasma gondii tachyzoites and tissue cysts are largely used for developing diagnostic assays, vaccines and in drug research as well as biochemical and molecular structure studies. Continuous passaging of tachyzoites or tissue cysts in animal models encounter ethical and economical problems and it is a time consuming procedure. Cryopreservation of tachyzoites and tissue cysts and revitalization of cryopreserved samples whenever needed, can decrease the economical loss, ethical problems and labour. In the present article, production of tachyzoites and tissue cysts in mice, preparation of samples for cryopreservation, cryopreservation of tachyzoites and tissue cysts, defrosting of cryopreserved samples and reinoculation to mice have been described in detail.

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

BackgroundToxoplasma 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.MethodsIn 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.ResultsAnalyses 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.ConclusionIn 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.

The Effect of Ag and Ag+N Ion Implantation on Cell Attachment Properties

Implanted 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 show...