Construction of various gamma 34.5 deleted fluorescent-expressing oncolytic herpes simplex type 1 [oHSV] for generation and isolation of HSV-based vectors

Background: Oncolytic herpes simplex virus [oHSV]-based vectors lacking gamma 34.5 gene, are considered as ideal templates to construct efficient vectors for [targeted] cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and gamma 34.5-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors

Methods: Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein [GFP] and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines

Results: We generated three recombinant viruses, HSV-GFP, HSV-GR [Green-Red], and HSV-Red. The HSV-GFP showed two log higher titer [1010 PFU] than wild type [108 PFU]. In contrast, HSV-GR and HSV-Red showed one log lower titer [107 PFU] than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 [P<0.001]. Moreover, HSV-GFP showed higher infection potency [98%] in comparison with HSV-GR [82%]

Conclusion: Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2[nd] and 3[rd] generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells in vitro/in vivo and as oncolytic agents in cancer therapy

enterovirus-like RNA construct for colon cancer suicide gene therapy

In gene therapy, the use of RNA molecules as therapeutic agents has shown advantages over plasmid DNA, including higher levels of safety. However, transient nature of RNA has been a major obstacle in application of RNA in gene therapy. Here, we used the internal ribosomal entry site of encephalomyocarditis virus and the 3' non-translated region of Poliovirus to design an enterovirus-like RNA for the expression of a reporter gene [enhanced green fluorescent protein] and a suicide gene [thymidine kinase of herpes simplex virus]. The expression of these genes was evaluated by flow cytometry and cytotoxicity assay in human colorectal adenocarcinoma cell line [SW480]. We then armed RNA molecules with a target sequence for hsa-miR-143 to regulate their expression by microRNA [miRNA] mimics. The results showed effective expression of both genes by Entrovirus-like RNA constructs. The data also showed that the restoration of hsa-miR-143 expression in SW480 leads to a significant translation repression of the introduced reporter and suicide genes. Collectively, our data suggest the potential use of Entrovirus-like RNA molecules in suicide gene therapy. Additionally, as a consequence of the possible downregulated miRNA expression in cancerous tissues, a decreased expression of gene therapy constructs armed with target sequences for such miRNA in cancer tissue is expected

Homocysteine induces heme oxygenase-1 expression via transcription factor Nrf2 activation in HepG2 cells

Background: elevated level of plasma homocysteine has been related to various diseases. Patients with hyperhomocysteinemia can develop hepatic steatosis and fibrosis. We hypothesized that oxidative stress induced by homocysteine might play an important role in pathogenesis of liver injury. Also, the cellular response designed to combat oxidative stress is primarily controlled by the transcription factor Nrf2, a principal inducer of anti-oxidant and phase II-related genes

Methods: hepG2 cells were treated with homocysteine in different time periods. Glutathione content was measured by flowcytometry. Using electrophoretic mobility shift assay [EMSA] and Western-blotting, anti-oxidant response element [ARE]-binding activity of Nrf2 for heme ocygenase-1 [HO-1] was demonstrated. Real time RT-PCR and Western-blotting were performed to evaluate whether homocysteine was able to induce mRNA and protein expression of HO-1

Results: the role of Nrf2 in cellular response to homocysteine is substantiated by the following observations in HepG2 cells exposed to homocysteine [i] Western-blotting revealed that Nrf2 is strongly stabilized and became detectable in nuclear extracts. [ii] EMSA demonstrated increased binding of Nrf2 to oligomers containing HO-1 promoter-specific ARE-binding site. [iii] Real time RT-PCR and Western-blotting revealed increased mRNA and protein expression of inducible gene HO-1 after treatment with Homocysteine

Conclusion: data presented in the current study provide direct evidence that the immediate cellular response to oxidative stress provoked by homocysteine is orchestrated mainly by the Nrf2-ARE pathway. Therefore, induction of Nrf2-ARE-dependent expression of HO-1 could be a therapeutic option for hepatic cells damage induced by homocysteine. Iran. Biomed. J. 17 [2]: 93-100, 2013

Intrahippocampal injection of 3 alpha diol [a testosterone metabolite] and indomethacin [3 alpha -HSD blocker], impair acquisition of spatial learning and memory in adult male rats

Hippocampus is essentially involved in learning and memory processes, and is known to be a target for androgen actions. The high density of the androgen receptors in hippocampus shows that there must be some relationship between androgens and memory. Androgen effects on spatial memory are complex and contradictory. Some evidence suggests a positive correlation between androgens and spatial memory. While some other reports indicated an impairment effect. The present study was conducted to assess the effect of 3 alpha diol on spatial discrimination of rats. Adult male rats were bilaterally cannulated into CA1 region of hippocampus and then received 3 alpha diol [0.2, 1, 3 and 6 micro g/ 0.5 micro L/side], indomethacin [1.5, 3 and 6 micro g/ 0.5 micro L/side], indomethacin [3 micro g/ 0.5 micro L/side] + 3 alpha diol [1 micro g/ 0.5 micro L/side], 25-35 min before training in Morris Water Maze task. Our results showed that injection of 3 alpha diol [1, 3 and 6 micro g/ 0.5 micro L/side] and indomethacin [3 and 6 micro g/ 0.5 micro L/side] significantly increased the escape latency and traveled distance to find hidden platform. It is concluded that intra CA1 administration of 3 alpha diol and indomethacin could impair spatial learning and memory in acquisition stage. However, intra hippocampal injection of indomethacin plus 3 alpha diol could not change spatial learning and memory impairment effect of indomethacin or 3 alpha diol in Morris Water Maze task

Design of small molecules with HIV fusion inhibitory property based on Gp41 interaction assay

Gp41 of HIV [Human Immunodeficiency Virus] is a protein that mediates fusion between viral and cellular membranes. The agent, T-20, which has been approved for HIV inhibition, can restrain Gp41 function in the fusion process; nevertheless, it has disadvantages like instability, high cost of production and injection form to be delivered twice a day. Several molecules like NB-2 and NB-64 have been discovered that can inhibit HIV infection. These molecules were used as template compounds to design and develop more effective small molecules functioning as HIV-1 fusion inhibitors targeting Gp41. The process included in silico docking protocols using HEX and ArgusLab applications. A multisource database was created, after choosing the best molecules; they were tested in vitro for inhibitory activity by HIV-1 single-cycle model, transfected in HEK cells [293T]. Computational analysis and experimental data were combined to explore molecular properties and the most potent ones were found, with the best suitable criteria for interaction with Gp41. Several examples [DAA-6, DAA-9 and DAA-12] could inhibit infection in vitro as effective as NB-2, NB- 64. Since disadvantages of available fusion inhibitor [T-20], it seems necessary to find similar molecules to be approved and have small size providing suitable bioactivity profile. The molecules explored in this study can be good candidates for further investigations to be used as oral HIV fusion inhibitors in the future

P53 but not cyclin E acts in a negative regulatory loop to control HER-2 expression in MCF-7 breast carcinoma cell line

Cyclin E, HER-2 and p53, are considered as major prognostic markers in breast cancer. As they are related in patho-clinical level, we aimed to check if they have any direct interaction on expression of each other. To study the effect of cyclin E on HER-2 expression, cell lines stably overexpressing cyclin E or its low molecular weight [LMW] isoforms were generated. To understand the results of p53 silencing either alone or in combination with cyclin E overexpression, we created three different p53 stably knocked down cell lines. Protein expression was analyzed by western blot, HER-2 expression in the established cell lines were determined using SYBR green real time PCR and data analyzed by REST software. Results indicate that HER-2 expression is only downregulated following p53 silencing and none of cyclin E isoforms can alter its expression. The presence of cyclin E isoforms in p53 silenced clones also does not altered HER-2 expression. Given the fact that p53 degradation is increased by HER-2 overexpression, these data can draw a regulatory loop in which a non-mutated functional p53 and HER-2 can bidirectionally regulate the expression of these two genes. This study improves our understandings of these pathways and these proteins can be introduced either as a marker or as a target in cancer treatment.

Effect of 3alpha-anderostanediol and indomethacin on acquisition, consolidation and retrieval stage of spatial memory in adult male rats

Testosterone and its metabolites have important roles in learning and memory. The current study has conducted to assess the effect of pre-training, post-training and pre-probe trial intrahippocampal CA1 administration of 3alpha-anderostanediol [one of the metabolites of testosterone] and indomethacin [as 3alpha-hydroxysteroid dehydrogenase enzyme blocker] on acquisition, consolidation and retrieval in Morris water maze [MWM] task. Adult male rats were bilaterally cannulated into CA1 region of hippocampus and then received 3alpha-diol [0.2, 1, 3 and 6 microg/0.5 microl/side], indomethacin [1.5, 3 and 6 microg/0.5 microl/side], indomethacin [3 microg/0.5 microl/side] + 3alpha-diol [1 microg/0.5 microl/side], 25-35 min before training, immediately after training and 25-35 min before probe trial in MWM task. Our results showed that injection of 3alpha-diol and indomethacin significantly increased the escape latency and traveled distance to find hidden platform in acquisition and consolidation stage, but did not have any effect on retrieval of spatial learning as compared with the control group. It is concluded that intra-CAl administration of 3alpha-diol and indomethacin could impair spatial learning and memory in acquisition and consolidation stage. Also, intrahippocampal injection of indomethacin + 3alpha-diol could not change spatial learning and memory impairment effect of indomethacin or 3a-diol in MWM task

[Study on co-administration of GM-CSF, IL-15 on the enhancement of the immunogenicity of HIV-1 P24-Nef expression vector in BALB/c mouse model]

Today, AIDS is considered as a global problem and many efforts to generate an effective vaccine against this disease have been made, but remain inconclusive. DNA vaccines are a member of the new generation of vaccines that can efficiently stimulate the immune system. However, recent findings indicate low immunogenicity for these vaccines and it is believed that these types of vaccines require strategies that could infer more immunogenicity. The employment of adjuvants could be considered as one of the most important methods involved. In this study, a DNA vaccine candidate for HIV P24-Nef is constructed and then using genetic adjuvants IL-15 and GM-CSF, cellular immune responses have been studied. In this study the gene structure of HIV P24-Nef in eukaryotic expression vector was constructed and expression vectors of IL-15 and GM-CSF were used as adjuvants. After inoculation of the candidate vaccine to BALB/c mice, cytokine patterns, lymphocytes proliferation and cytotoxicity were analyzed. Our findings indicate that candidate vaccine significantly stimulated cellular immune responses. The usage of IL-15 and GM-CSF as DNA adjuvants together and separately with candidate vaccine has strengthened cellular immune responses significantly. Co-administration of DNA adjuvants significantly increased cellular immune responses when the ratio of the vaccine dose was more than the adjuvants. The sequences that we selected as candidate vaccine demonstrated good immunogenicity in mouse model and co-administration of IL-15 and GM-CSF DNA adjuvants increased cellular immune response to DNA vaccine construct

[Evaluation of immune responses from immunization of HIV-1 P24-Nef fusion peptide vaccine with monophosphoryl lipid A [MPL] in BALB/c mice]

Several vaccines against HIV have been investigated but none has been approved as an effective HIV vaccine. An approach that could induce stronger immune response against the pathogen is utilizing a multi-epitopic vaccine. This strategy was used in the design of several vaccines and resulted in improved immune responses. In this study a multi-epitopic fusion peptide including parts of HIV-1 Nef and P24 as a vaccine candidate was injected into mice and immune humoral responses measured with total antibody and IgG sub-classes using ELISA. Also measurement of cellular immune responses through evaluation of spleen cells proliferation response using MTT and cytotoxicity by LDH were performed. Finally, the cytokine pattern of IFN-gamma and IL-4 were also determined with ELISA. The results indicate that candidate vaccine stimulated mouse splenic lymphocyte proliferation response and also induced strong cytotoxicity responses. Analysis of humoral immune response has shown that the candidate vaccine has induced specific antibody production mainly of the IgG2a sub-class. Also cytokine pattern evaluation has shown that IFN-gamma secretion was dominant. The use of immunogen and conserved epitopes from P24 and Nef induced strong humoral and cellular immune responses and this construct could be candidate for further studies in animal models

Immunogenicity of a new HIV-1 DNA Construct in a BALB/c mouse model

Cell mediated immunity, especially cytotoxic T cell responses against HIV-1 infection, plays a critical role in controlling viral replication and disease progression. DNA vaccine is a novel technology which is known to stimulate strong cellular immune responses. Many DNA vaccines have been tested for HIV infection but there is still no effective vaccine against this infection. Construction of a vaccine consisting of multiple conserved and immunogenic epitopes may increase vaccine efficacy. In the present study a DNA vaccine candidate constructed from HIV-1 P24-Nef was evaluated and cellular immune responses were assessed in murine BALB/c model. HIV-1 P24-Nef gene was cloned in PCDNA3.1 expression vector. Mice were immunized with DNA construct and IL-4 and IFN-gamma evaluation was per-formed using ELISPOT. Cytotoxicity response was evaluated with Granzyme B ELIS-POT assay and lymphocyte proliferation was evaluated with LTT assay. Analysis of immune responses showed that, compared to control groups, the candidate vaccine induced production of higher levels of both IL-4 and IFN-gamma [p<0.05]. Cytotoxicity and lymphocyte proliferation responses of mice vaccinated with the candidate vaccine were significantly increased compared to control groups [p<0.05]. HIV-1 P24-Nef DNA construct displayed strong immunogenicity in a murine model