Comparison of Three Different Methods of Transfection for the Production of Recombinant Adenovirus Expressing Human Carcinoembryonic Antigen Gene.

Adenoviral vectors (AdVs) are widely used as a gene delivery vehicle and vaccine design due to their genetic stability, transfer capacity of large genes, production at high titers, and remarkable efficacy of transduction. One of the most important applications of AdVs is in cancer immunotherapy. Tumor-associated antigens are overexpressed in cancer cells; however, they cannot induce immune responses sufficiently. Therefore, the immune system must be stimulated against these antigens to kill the cancer cells. This study described the construction steps of a recombinant AdV expressing human carcinoembryonic antigen (CEA) gene. Furthermore, in order to achieve a high titer of the virus, an efficient transfection was required. Three various transfection reagents were compared to achieve the best method of transfection. Carcinoembryonic antigen was cloned into the pAdV and transfected into the A293 cells using three different reagents, including polyethylenimine (PEI), calcium phosphate, and DMRIE-C. The PEI had the highest transfection efficiency, which was selected for the transfection of the recombinant plasmid. It has low toxicity for cells and is suitable for large-scale transfection. The virus produced in this study can be applied as a vaccine in cancer immunotherapy for stimulating the immune system against CEA-expressing tumors.

[Inhibition of Autophagy by 3-MA Increases Oncolysis Effect of VSV in a Murine Model of Cancer].

Vesicular stomatitis virus has been known as a potent antitumor agent because of its selective replication and lysis of tumor cells and immune-stimulating properties. In response to cellular stress and enhanced metabolism, tumor cells activate autophagy, to provide energy for the cells and preventing tumor destruction. Inhibition of autophagy can increase the therapeutic potential of many antitumor methods. This study aimed to check the efficacy of combined VSV and three-methyl adenine (3-MA) in treating a tumor model in mice. TC-1, a line of C57BL/6 mouse lung cells transformed by HPV-16 E7 and E6 oncoproteins, as well as human Ras, were used for experiments. The viability after treatment with the optimized concentration of 3-MA with or without combination with VSV was assessed by MTT. C57BL/6 male mice were injected with TC-1, and after tumor formation, 3-MA and VSV alone or in combination in two different protocols were injected into tumor mice. Tumor size, tumor-specific CTL response, and apoptosis rate were evaluated. The results showed that 3-MA combined with VSV causes more lethality in tumor cells in vitro. In vivo studies also showed that combined VSV and 3-MA treatment inhibits the progression of TC-1 cancer cells with higher efficiency, especially in daily 3-MA treatment along with four doses of VSV injection with four days' intervals. In addition, the rate of apoptosis and cytotoxic T cells activity in the groups injected with 3-MA and the virus were higher than groups receiving each agent alone. In conclusion, the association of VSV with 3-MA increases its oncolysis activity and subsequently more stimulates the immune system against the tumor. This finding suggests a combinational approach for tumor therapy with therapy. Combining oncolytic VSV with 3-MA as an autophagy inhibitor agent can improve the efficacy of tumor treatment. This combination therapy approach enhances apoptosis in tumors as well as T cell cytotoxicity against tumor cells.

Impact of interplay between autophagy and interferon-alpha in HCV and HCV/HIV infection.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are among the most dangerous pathogens globally. Infection with HCV has been reported in a high percentage of HIV patients. Viruses are obligate intracellular pathogens and their survival is associated with their capability to subvert antiviral defenses of cells and to improve cellular processes required for their replication. The aim of this study was to compare the expression rate of the key gene for autophagy process, Beclin-1, as a cellular response to viral infections, and its effect on IFN-α expression in both HCV and HCV/HIV patient groups. In this study, a total number of 40 samples of peripheral blood mononuclear cells (PBMCs) including 20 HCV and 20 HCV/HIV patients before treatment were evaluated. The HCV viral load in both groups was evaluated by semi quantitative real-time PCR. The level of Beclin-1 and IFN-α gene expression was examined in all samples by semi quantitative real-time PCR assay. The median viral load was 8.3×105 copies/ml in HCV group and 2.1×106 copies/ml in HCV/HIV patients. While the expression level of Beclin-1 gene in HCV group was significantly higher, the level of IFN-α expression was lower compared to the HCV/HIV group (P  Keywords: autophagy; Beclin gene; HCV; HIV; IFN gene.

Combination of virotherapy with VSV and tumor vaccination significantly enhances the efficacy of antitumor therapy.

Oncolytic virotherapy offers the potential to treat tumors both as a single agent and in combination with conventional therapies such as chemotherapy and immunological therapy. Here, we describe an effective treatment regimen which combines virotherapy with immunotherapy. IFN-α and co-stimulator IL-2 along with tumor cell lysate vaccination with intratumoral administration of oncolytic vesicular stomatitis virus (VSV) resulted in regression of established TC1 papilloma tumor model in C57BL/6 mice. The remarkable results especially in the group receiving tumor vaccination and virotherapy together (TC1-VSV) were obtained. Combination therapy synergistically enhanced CTL activity against tumor cells and reduced tumor size, although significant reduction in tumor size was observed in both groups receiving VSV or tumor vaccine alone. The presented data suggest that the effectiveness of virotherapy is enhanced when combined with immunotherapy by priming specific CD8 T cells against tumor antigens through tumor vaccination and boosting by exposure of antigens upon virus infection. Keywords: virotherapy; VSV; tumor vaccine; immunotherapy; IFN-α; IL-2.

[3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways].

Dikkoppf-1 (DKK1) is an antagonist of the canonical Wnt signaling pathway. The importance of DKK1 as a diagnostic and therapeutic agent in a wide range of diseases along with its significance in a variety of biological processes accentuate the necessity to decipher its 3D structure that would pave the way towards the development of relevant selective inhibitors. A DKK1 structure model predicted by the Robetta server with structural refinements including a 10 ns molecular dynamics run was subjected to functional and docking analyses. We hypothesize that the N-terminal region of the DKK1 molecule could be functionally important for both canonical and noncanonical Wnt pathways. Moreover, it seems that DKK1 could be involved in interactions with the Frizzled receptors, leading to the activation of the Planar Cell Polarity (PCP) pathway (activation of Jun N-terminal kinase (JNK) Pathway) and Wnt/Ca^(2+) pathway (activation of CamKII).

[Antitumor effect of mIFN-λ3 in C57BL/6 mice model for papilloma tumors].

Although several years have passed since the determination of the human papilloma virus (HPV) as the causative agent for cervical cancer, a definitive treatment has not yet been found. Interferon-alpha (IFN-α) immunotherapy is one of the promising methods for tumor treatment, although numerous side effects were observed in clinical trials. Recently, a new type of interferon, lambda-interferon (IFN-λ), has been discovered with fewer side effects than IFN-α since its receptor repertoire is limited. IFN-λ has a series of activities including antiviral, anti-proliferative and anti-tumor actions. In the present study, the effects of IFN-α and IFN-λ on the TC1 papilloma tumor model in C57BL/6 mice were evaluated. TC1 cells were injected into the mice subcutaneously. Upon tumor formation, murine IFN, mIFN-α and mIFN-λ, expression plasmids were injected intratumorally in combination or alone. The survival time and tumor size as well as apoptosis in tumors and NK cytoxicity were measured after three injections. As compared with the control group, the remarkable results especially in the group which received mIFN-α and mIFN-λ together were obtained for all of the measured parameters. Although IFN-λ is a new member of the interferon family and its properties should be studied in detail, the data obtained suggests that the use of IFN-λ especially in combination with IFN-α could be considered as an effective strategy for papilloma cervical cancer immunotherapy.

Immunogenicity and protective effect of a DNA construct encoding certain neutralizing epitopes of herpes simplex virus type-1 glycoprotein B.

Much attention is presently focused on the vaccination with certain epitopes of an antigen. To further study the ability of neutralizing epitopes mapped in the first 1515 nucleotides of glycoprotein B of herpes simplex virus type-1 (gB-1) to induce neutralizing antibodies, a DNA immunization approach was employed. Vaccination of mice with a plasmid expressing the neutralizing epitopes induced humoral immune responses, although the antibody titre was significantly lower than that of antibodies induced by the full-length gB-1 gene. Furthermore, the plasmid DNA could not protect the mice against HSV-1 lethal challenge, but could significantly prolong the survival time compared to mock-vaccinated group.

Prevalence of HI antibody titer against rubella virus to determine the effect of mass vaccination in Tehran.

BACKGROUND: Rubella is an infectious viral disease, has a worldwide distribution and is normally a mild childhood disease. Infection during early pregnancy may cause fetal death or congenital rubella syndrome. The highest risk of CRS is found in countries with high susceptibility rates among women of childbearing age. In many developed and some developing countries, large-scale rubella vaccination during the past decade has drastically reduced or practically eliminated rubella and CRS. Mass vaccination campaigns and Expanded Program of Immunization (EPI) have increased vaccine coverage in the world with a substantial impact on the reduction of rubella infections, such as CRS. OBJECTIVE: The present study was preformed to evaluate the immune status against rubella before and after the mass campaign vaccination on 22 December 2003. STUDY DESIGN: A total of 320 samples were collected from the healthy subjects before and after the vaccination and 80 paired sera were collected and tested for the presence of rubella antibody using HI test. RESULTS AND CONCLUSIONS: Based on the results, 98.1% of the population has gained anti-rubella antibody, compared with 92.2% before the vaccination. The data revealed that 98.75% of the paired subjects had rubella antibody after mass vaccination which is statistically significant.