Sultan Gulce-Iz

Background: Toxoplasma 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. Methods: 49 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 50 V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites. Results: In 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-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.001). The survival time of the vaccinated mice increased to 8.38 ± 2.13 days 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.

Background: Serum have been traditionally used to support growth of animal cell cultures. However, the increasing growth of therapeutic biopharmaceuticals market, accelerated the high demand for the serum-free medium (SFM). Objective: The main objective is to design a SFM for a stable rCHO cell line that produces a fully anti-human TNF-α monoclonal antibody (mAb) corresponding to HUMIRA ^® biosimilar. Materials and methods: Design of Experiment (DoE) approaches were used to determine the key factors due to their effect on specific growth rate and mAb production. The production was carried out in T-flasks at different initial cell concentrations and then in Erlenmeyers with the developed SFM. mAb production was compared with commercial SFMs in terms of yield and productivity. Results: Regarding to our findings, when the developed SFM-adapted cells were compared with the cells produced in commercial SFMs, the mAb productivity in developed SFM were higher (1.3–1.6 times) depending on higher mAb concentration and less (3–5 times) cell concentration. Additionally, the produced mAb in the developed SFM provided high conformational similarity with its originator HUMIRA ^® . Conclusion: DoE approaches could be used to reduce cost and time in designing SFM for any commercially important cell line to produce high value biologics.

Breast cancer is one of the most common cancer types among women in which early tumor invasion leads to metastases and death. EpCAM (epithelial cellular adhesion molecule) and HER2 (human epidermal growth factor receptor 2) are two main circulating tumor cell (CTC) subsets in HER2+ breast cancer patients. In this regard, the main aim of this study is to develop and characterize a three-dimensional (3D) breast cancer tumor model composed of CTC subsets to evaluate new therapeutic strategies and drugs. For this reason, EpCAM(+) and HER2(+) sub-populations were isolated from different cell lines to establish 3D tumor model that mimics in situ (in vivo) more closely than two-dimensional (2D) models. EpCAM(+)/HER2(+) cells had a high proliferation rate and low tendency to attach to the surface in comparison with parental MDA-MB-453 cells as CTC subsets. Aggressive breast cancer subpopulations cultured in 3D porous chitosan scaffold had enhanced cell–cell and cell–matrix interactions compared to 2D cultured cells and these 3D models showed more aggressive morphology and behavior, expressed higher levels of pluripotency marker genes, Nanog, Sox2 and Oct4. For the verification of the 3D model, the effects of doxorubicin which is a chemotherapeutic agent used in breast cancer treatment were examined and increased drug resistance was determined in 3D cultures. The 3D tumor model comprising EpCAM(+)/HER2(+) CTC subsets developed in this study has a promising potential to be used for investigation of an aggressive CTC microenvironment in vitro that mimics in vivo characteristics to test new drug candidates against CTCs.

DNA vaccines (nucleic acid vaccines) have been gaining importance as promising therapeutics against infectious diseases, cancer, autoimmune disorders and allergy for the past two decades. However, the immune responses elicited by the DNA vaccines are not at the desired level to stimulate a protective immune response. Thus, studies are focused on the enhancement of DNA vaccine-induced immune response by different approaches. The most common approach is to use biomaterial-based adjuvants for enhanced antigen delivery and uptake by antigen-presenting cells. Some of these adjuvants are alum, saponins, microspheres, nanoparticles, liposomes, polymers, etc. used in vaccine formulations. In addition, molecular adjuvants like cytokines, chemokines and heat shock proteins have been shown to be promising in designing DNA vaccines. In this chapter, molecular adjuvants to improve DNA vaccination-induced immune responses will be summarized with a special focus on Bcl-xL anti-apoptotic protein.

Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. In the laboratory diagnosis of toxoplasmosis, serological tests have importance in detecting antibody response. Traditionally T. gondii tachyzoites grown in vivo are being used as an antigen source in serological assays. Currently, tachyzoites produced in vitro are being tested as an antigen source in order to decrease animal use. Microcarrier technology allowed us to grow anchorage-dependent host cells on microcarrier suspension in short time and approximately 10 times more than traditional flask technique. The ability of T. gondii tachyzoites to grow in host cells adhered to microcarriers has not been analyzed yet. In this study, we aimed to develop a novel in vitro culture method to produce T. gondii tachyzoites abundantly using HeLa cells adhered to Cytodex 1 microcarriers. Initially, the growth of HeLa cells adhered to Cytodex 1 was analyzed using RPMI 1640, DMEM, and EMEM. Next, HeLa cells with a concentration of 1 × 10⁵ cells/ml and 2 × 10⁵cells/ml were adhered to Cytodex 1 and grown in spinner flasks. Then, T. gondii tachyzoites were inoculated with 1:1 and 2:1 cell:tachyzoite ratios to HeLa cells adhered to microcarriers in spinner flaks. During continuous production in spinner flasks, tachyzoites were harvested at the 2nd, 4th, and 7th day of culture and the quality of antigens produced from these tachyzoites were tested in ELISA and Western Blotting using sera of patients with toxoplasmosis. The optimization studies showed that finest HeLa inoculation value was 2 × 10⁵cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 × 10⁷) was 1:1 at the 4th day of inoculation. According to the results of ELISA comparing HeLa cell and mouse derived antigens, the highest correlation with mouse antigen was achieved at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio (P < 0.0001). The sensitivity and specificity ratios of ELISA were 100%. In addition, Western blotting banding patterns of the antigen derived at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio was comparable with mouse derived antigen. Overall, this novel methodology can be an alternative source of antigen in diagnostic assays, decrease animal use for antigen production, and contribute to the solution of ethical and economic problems.

MicroRNAs (miRNAs) are RNA molecules at about 22 nucleotide in length that are non-coding, which regulate gene expression in the post-transcriptional level by performing degradation or blocks translation of the target mRNA. It is known that they play roles in mechanisms such as metabolic regulation, embryogenesis, organogenesis, differentiation and growth control by providing post-transcriptional regulation of gene expression. With these properties, miRNAs play important roles in the regulation of biological processes such as proliferation, differentiation, apoptosis, drug resistance mechanisms in eukaryotic cells. In addition, there are miRNAs that can be used for cancer therapy. Tumor cells and tumor microenvironment have different miRNA expression profiles. Some miRNAs are known to play a role in the onset and progression of the tumor. miRNAs with oncogenic or tumor suppressive activity specific to different cancer types are still being investigated. This review summarizes the role of miRNAs in tumorigenesis, therapeutic strategies in human cancer and current studies.

Benzo[a]pyrene (B[a]P) is a ubiquitous environmental pollutant that reacts with skin and induces intracellular oxidative stress through reactive oxygen species (ROS) accumulation. 'I he antipollution properties of natural extracts, especially including antioxidants, for inhibiting ROS in cells are gaining importance, in addition to the anticancer effects attributed to them. In this study, a commercial functional antipollutant blend of plant extracts consisting of ellagic acid standardized Punica granatum peel extract, Sambucus nigra fruit extract, Prunus cerasus seed extract, and hydrolyzed wheat protein with high antioxidant properties and UV damage-protective properties attributed to each one was investigated. 'the cytoprotective effect of this functional antipollutant blend was determined by ROS assay through reducing the level of intracellular ROS induced by B[a] P as an oxidative stress factor in human neonatal keratinocytes and fibroblast cells. In addition, the cytoprotective effect of the functional antipollutant blend after UVA exposure was also determined. It is shown that the oxidative damage induced by B[a]P and UVA, which are the most abundant factors of chemical and physical pollution, would be prevented by the functional antipollutant blend. 'thus, it can be concluded that this antipollutant functional blend may offer a promising ingredient for the cosmetic industry's skincare products.