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Objective: In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed. Materials and Methods: This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26 seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIV-1 Monitor test. Results: The results demonstrated that this technique could detecte up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers (5×102-5×109). Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results (R2= 0.95). Conclusion: On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool of clinical outcome. Indeed, the development and stabilization of HIV-1 RNA assays have given physicians a unique tool for monitoring HIV-1 patients treated with antiviral drugs. In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV-1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test (5×10 9 versus 7.5×10 5). This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis. Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than

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Objective: The global HIV epidemic continues to expand and exceeding previous predictions. An effective vaccine represents the best hope to curtail the HIV epidemic. DNA vaccines induce humoral and cellular responses and mimic live vaccines without their pathogenic potential. The importance of CD8+ CTL responses in controlling HIV and SIV viremia has led to production of a series of vaccine candidates that effectively induce these responses. It is now widely believed that an HIV vaccine strategy must stimulate both a strong humoral (antibody) as well as cell-mediated (CTL) immune response.The p24 and gp41 play many important roles in host-virus interaction and pathogenesis. These proteins are considered as attractive vaccine candidate in which their immunogenecity and immunomodulatory effects have been confirmed. Materials and Methods: In this study, a construct, pcDNA3.1Hygro- (p24-gp41), was evaluated as a DNA vaccine candidate in Balb/C mice for generation of effective cellular immune responses. For immunizing, we used dendrosome, a novel family of vehicles for transfection and therapy. IFN-γ cytokine production and total antibody were detected by ELISA. Lymphoprolifration assay was performed by MTT test. Results: ELISA and MTT assays confirmed that the cited p24-gp41 fusion gene is able to enhance immune responses in mice. Conclusion: The construct that was used in this research can be a good candidate for DNA vaccine against HIV-1, if the future complementary tests demonstrate the same trends of immunogenic responses shown in this study.

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Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

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Objective: The use of bacterial plasmids carrying specific genes of pathogens as genetic vaccines is a relatively new technique for induction of cellular immune responses against microbial pathogens. Mechanisms of production of specific immune responses against these vaccines are not still completely understood. Therefore, it is necessary to examine various routes of inoculation to find the best way of immunization for specific antigens. In this research, intramuscular method of inoculation of influenza vaccine nucleoprotein (NP) encoding vector was compared with that of intra-dermal method. Materials and Methods: In this study, the ability of two different methods of immunization (intramuscular and intra-dermal) in induction of CTL responses as well as their efficiency in clearance of influenza virus from the lung of BALB/c mice was compared. Female BALB/c mice were immunized with influenza virus NP expressing plasmids on days 0, 14 and 28. CTL activity of mice was evaluated by lactate dehydrogenase technique two weeks after the last inoculation. In addition, the mice were challenged by live influenza virus and the viral titer was measured 4 days post-challenge in the lungs of animals. The results of experiments demonstrated that intramuscular immunization of mice induces a stronger CTL response. Mice immunized by intramuscular route also showed a higher ability in virus clearance from the lungs. Conclusion: Results of this study showed that different routes of immunization of influenza NP genetic vaccine induce different levels of cell-mediated immune responses and protection from the live virus.

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Objective: 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. Materials and Methods: 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-γ and IL-4 were also determined with ELISA. Results: 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-γ secretion was dominant. Conclusion: 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.

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This study investigates the factors affecting the commercialization of products in medical biotechnology accelerators, it also demonstrates the priority of these factors. The research is applied and descriptive survey. It has been done in two phases, qualitative and quantitative. Based on the general purpose of this study, to provide a framework for the commercialization of products in medical biotechnology accelerators, we interviewed with informed experts. 62 sub-codes were obtained as effective factors in the commercialization of products in medical biotechnology accelerators, which were classified into 5 main dimensions including individual, organizational, industry and competition, institutional and product. These dimensions are presented as a framework. In the quantitative phase, the main dimensions as well as the sub-themes were ranked in the main dimension. Finally, the priority of the main dimensions was as follows: 1. Individual (personality, behavioral and mental characteristics of the mentor and manager) 2. Industry and competition (interaction with pharmaceutical companies as customers, investors, etc.) 3. Organizational (experience of startup teams, codified strategy, R&D, etc.) 4. Product (value added and high-tech) 5. Institutional (government is pragmatic to the country's slogans, interaction with government managers, etc.).

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Objective: Bone morphogenetic protein-7 (BMP-7) is a multifunctional growth factor predominantly recognized for its osteoinductive properties. Due to the high cost of this protein, the availability of BMP-7 for treatment is limited. The heterologous production of recombinant hBMP-7 has been performed in a number of expression systems. In this study a novel form of BMP-7 was expressed in eukaryotic and prokaryotic hosts. Methods: For expression in the prokaryotic system, the novel protein was secreted to the periplasmic space of Escherichia coli using a pelB signal sequence followed by single-step purification by Ni²⁺-charged column chromatography. In the mammalian cell expression system, we transferred a full-length cDNA encoding precursor of the novel protein to CHO cells then selected stable clones by using the appropriate antibiotic concentration. Expressions in both systems were confirmed by Western blot analysis. Results: The novel recombinant protein was produced as a 36-38 kDa dimer in the CHO cell line and a 16 kDa monomer in the Escherichia coli system. Quantitative analysis according to ELISA showed that the expression levels of the mutant protein in the eukaryotic and prokaryotic expression systems were 40 ng/ml and 135 ng/ml of the culture media, respectively. Conclusion: In this study, the expression level in Escherichia coli was at least three times more than observed in the CHO cells. However, further optimization is required to obtain a dimer protein in Escherichia coli. The results show that periplasmic expression may be suitable for the production of complex proteins such as BMPs.

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Objective: Development of high producing mammalian cell lines is a major bottleneck in manufacturing of recombinant therapeutic proteins. This study examines the effect of using the matrix attachment region from the human interferon beta gene in combination with promoter activation strategy with E1A 13S protein on human tissue plasminogen activator (t-PA) expression in Chinese hamster ovary (CHO) cells. Methods: The matrix attachment region was cloned in 3΄ and 5΄ flanking sides of the t-PA expression cassette in pTPA vector to generate pMTPA. After transfection of the cells with pTPA and pMTPA vectors, stable cell pools were developed and the t-PA expression level determined for each stable cell line. In the next step, E1A 13S expression plasmid was transfected to stable cell pools and t-PA titers were measured after 72 hours. Results: Integration of pTPA and pMTPA vectors in the CHO genome was confirmed by PCR analysis on genomic DNA of stable cell pools. Analysis of the t-PA expression level showed a three-fold enhancement in pMTPA transfected cells compared to pTPA-containing cells. t-PA expression was further enhanced up to 1771 U/ml by transient expression of E1A 13S in pMTPA stable cell pools. Conclusion: These results have shown that incorporation of matrix attachment region in an expression vector in combination with promoter activation can effectively enhance recombinant protein expression levels in CHO cells.