Current State of the Art in DNA Vaccine Delivery and Molecular Adjuvants: Bcl-xL Anti-Apoptotic Protein as a Molecular Adjuvant

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

Conducting Informal Discovery of a Party\u27s Former Employees: Legal and Ethical Concerns and Constraints

This Article identifies and critiques existing sources of confusion in the law and proposes revised and alternative discovery procedures to provide equal access to information possessed by ex-employees, while simultaneously safeguarding the integrity of that information. Its primary emphasis is on federal jurisprudence, although important points of consensus and departure between state and federal law are noted, as appropriate. Part I explains the issues that arise in informal discovery, and the difficulties with clearly resolving those issues given the conflicting state of the law. Part II discusses application of the attorney-client privilege to communications between corporate counsel and former employees, concluding that the privilege should not shield the content of such communications from discovery by opposing counsel. Because the attorney-client privilege issue and the debate over ex parte contact both turn on whether a former employee is a party to the litigation, an exposition of the ethical concerns of ex parte contact follows in Part III, which concludes that ex parte contact by opposing counsel should be allowed. Part IV examines the applicability of the work product doctrine in shielding from discovery certain tangible materials related to the interview. This Article advocates absolute immunity from discovery for attorney notes and memoranda, and for collections of documents selected by counsel for discussion with former employees; limited discovery of signed witness statements generated pursuant to the interview; and fairly broad discovery of the content of counsel\u27s questions and statements during the interview. The proposed discovery guidelines in the Conclusion are offered with the ambitious, if not elusive, goal of reconciling the competing interests and policies that rouse litigants to battle when informal discovery is conducted of former employees, while simultaneously giving due regard to the pragmatic impact that new or revised rules may have on the litigants and the adversarial process. The overriding objective is to provide equal access by all parties to the information possessed by former employees, while at the same time providing mechanisms to deter, and if necessary to reveal, inappropriate manipulation of these potential witnesses by counsel

The biomarker features of miR-145-3p determined via meta-analysis validated by qRT-PCR in metastatic cancer cell lines

WOS: 000483423500041PubMed ID: 31195093MicroRNAs (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

Therapeutic microRNAs in human cancer

WOS: 000458237600034PubMed ID: 30600466MicroRNAs (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

Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis

WOS: 000314273600006Background: 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.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [110O809]; Ege University, Science and Technology CenterEge University [2011BIL020]This work was partly funded by Scientific and Technological Research Council of Turkey (TUBITAK) by grant number 110O809 and Ege University, Science and Technology Center (2011BIL020)

Monoclonal antibodies in cancer immunotherapy

WOS: 000452543200139PubMed ID: 30311129Nowadays, in cancer treatments, immunotherapy which can be classified as a cancer type specific therapy is more popular than non-specific therapy methods such as surgery, radiotherapy and chemotherapy. The main aim of immunotherapy is to enable patients' immune system to target cancer cells and destroy them. The mainly used treatment methods in cancer immunotherapy are cancer vaccines, adoptive cell therapy, cytokines and monoclonal antibodies. In this review, we discuss the immunotherapy approaches, especially monoclonal antibodies which are mostly used in cancer immunotherapy in clinical applications

Cytotoxic responses of carnosic acid and doxorubicin on breast cancer cells in butterfly-shaped microchips in comparison to 2D and 3D culture

WOS: 000398104900012PubMed ID: 28191587Two dimensional (2D) cell culture systems lack the ability to mimic in vivo conditions resulting in limitations for preclinical cell-based drug and toxicity screening assays and modelling tumor biology. Alternatively, 3D cell culture systems mimic the specificity of native tissue with better physiological integrity. In this regard, microfluidic chips have gained wide applicability for in vitro 3D cancer cell studies. The aim of this research was to develop a 3D biomimetic model comprising culture of breast cancer cells in butterfly-shaped microchip to determine the cytotoxicity of carnosic acid and doxorubicin on both estrogen dependent (MCF-7) and independent (MDAMB231) breast cancer cells along with healthy mammary epithelial cells (MCF-10A) in 2D, 3D Matrigel (TM) and butterfly-shaped microchip environment. According to the developed mimetic model, carnosic acid exhibited a higher cytotoxicity towards MDAMB 231, while doxorubicin was more effective against MCF-7. Although the cell viabilities were higher in comparison to 2D and 3D cell culture systems, the responses of the investigated molecules were different in the microchips based on the molecular weight and structural complexity indicating the importance of biomimicry in a physiologically relevant matrix

Development and verification of a three-dimensional (3D) breast cancer tumor model composed of circulating tumor cell (CTC) subsets

Inevi, Muge Anil/0000-0003-2854-3472; Gulce Iz, Sultan/0000-0002-9275-6272; saglam metiner, pelin/0000-0002-5726-1928WOS: 000489549500001PubMed: 31583566Breast 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.Ege UniversityEge University [15-MUH-038]; [TUBITAK-1919B011503631]This project was supported by the research fund as Ege University Scientific Research Project (Project Number: 15-MUH-038) and by TUBITAK-1919B011503631 under the supervision of Sultan GULCE-IZ. the authors would like to thank Dr Aylin SENDEMIR-URKMEZ for providing the chitosan scaffolds