Autophagy gene activity may act as a key factor for sensitivity of tumor cells to oncolytic vesicular stomatitis virus

Background: Beclin1 is an important, primary molecule for autophagy

Objectives: It is suggested that the control of the autophagy path increases the sensitivity of tumor cells to VSV

Materials and Methods: In this study, the degree of Beclin1 gene expression in two cell lines, HeLa and A549, has been examined and the percentage of living cells subsequent infection with virus has been evaluated by MTT assay method

Results: The results showed that the degree of Beclin1 gene expression in HeLa cells in comparison with A549 cells has reduced, and the sensitivity of these cells to vesicular stomatits virus [VSV] oncolysis is more than A549

Conclusions: It seems that by using some methods for reducing autophagy, it is possible to make tumor cells more sensitive to virotherapy and even other treatments

Autophagy knock down: as a booster for the replication of viruses in cell culture

Background: Autophagy suppression recently has been known to have a remarkable effect for cellular adjustment and viability in the final stages of cancer. On the other hand, autophagy has the potential effect in preventing many viruses from replication. Beclin1 is the most substantial constituent in autophagy apparatus regulation. This study was intended to investigate the beclin1 siRNA knockdown effect on the extent of activity of the oncolytic vesicular stomatitis virus [VSV] as a model in cell culture

Materials and Methods: In the current study, the cancer cell line, HeLa [cervical squamous cancer cell line ] was infected by VSV, followed by beclin1 siRNA vector transfection. The potential change in the expressions of gene beclin1 in transfected cells, as well as untransfected ones were examined by real time PCR, and also the titer of viruses was compared in cells with and without transfection

Results: The results revealed that the amount of putative gene beclin1 expression in HeLa cells decreased greatly due to siRNA suppressive impact, and also the sensitivity of the cells to VSV oncolytic effect increased upon decrease in beclin1gene expression

Conclusion: It seems that autophagy suppression by using siRNA with VSV is a substantial aid for increase in virus titer in cancer cell lines

Introduction of RNAvirus evolution

Several viruses, in particular RNA viruses, have high mutation rates and relatively short generation times. Particle stability during infection in nature or in laboratory triggers the evolutionary event toward different mechanisms such as genome segmentation, point mutation and recombination. The frequency of mutant genomes increase and modify the previous distribution, which, consequently, lead to emergence of a new infectious particle. Mutation and selection are the most fundamental processes in evolution. High mutation rate of RNA viruses has an important role in viral fitness. Therefore, it increase our understanding about molecular biology of viral infections and their evolution by selection, mutation could reliably determine our ability to challenge destructive viruses. This review focuses on existing impressions of genetic organization and mechanisms of RNA viruses evolution

Efficient lentiviral transduction of adipose tissue-derived mouse mesenchymal stem cells and assessment of their penetration in female mice cervical tumor model

Although the incidence of cervical cancer has reduced during last years, but it causes mortality among women. Many efforts have performed to develop new drugs and strategy for treatment of cervical cancer. Adipose Tissue-Derived mouse Mesenchymal Stem Cells [MSCs] has many advantages which make them a suitable choice as a cell therapeutic agent in cancer treatment. In this study, we aimed to develop an improved protocol for Mouse MSCs transduction as well as assess the homing capacity and incorporation of MSCs in cervical cancer model. MScs were isolated from the mouse adipose tissue and characterized by differentiation and flow cytometry. In our study, lentiviral vector transductions of MSCs performed. Their penetrations were detected in tissue sections after injection of transduced MSCs to female C57BL/6 mice as a cervical cancer model. Results showed that MSCs were efficiently transduced with lentiviral vector resulting in efficient tumor penetration. The results provide evidence that MSCs were able to penetrate into the tumor mass of cervical tumor model and are good vehicles for gene transfer to cervical cancer

[Cellular immune response following neonatal mice immunization with vesicular stomatitis virus]

Infectious microorganisms are major sources of illness and death worldwide, and the leading cause of death in neonates. Effective vaccination of this age group is of particular importance. The lack of a response and greater susceptibility to tolerance are two major features that limit the effectiveness of vaccines in neonates. In this study we compare the cellular immune response generated following antigen injections at different times of life in newborn mice to that of adult mice. Adult and different age neonate mice were vaccinated with vesicular stomatitis virus [VSV]. One week after the last injection, cellular immunity was assayed on spleen cells that targeted EL4 infected cells using lactate dehydrogenase cytotoxicity assay. Antigen injection induced a decreased immune response in newborn mice compared with mice that had been immunized with subsequent injections. In the adult group, due to the evolution of the immune system, we observed a stronger immune response. Immunization of newborn mice may induce a reduced response when compared to adult vaccinations. However this can be corrected by the administration of additional booster doses

[Antigen-specific IL-10 producing CD4[+], CD25[+] regulatory T cells in chronically infected HCV patients]

Background: the mechanism behind the apparent lack of effective antiviral immune response in patients with chronic hepatitis C virus [HCV] infection is poorly understood. Although multiple levels of abnormalities have been identified in innate and adaptive immunity, it is postulated that production of specific cytokines such as IL-10 may contribute to the induction and maintenance of HCV persistence. Production of IL-10 by CD4[+], CD25[+], and IL-10 [+] regulatory T cells with regulatory capacity [Tregs] appears to be one of the viral mechanisms that alter the antiviral immune response. As the first report, that attempts to mimic physiological forces that can occur during HCV infection, in this study we evaluate the ability of HCV-core antigens in increasing the frequency of CD4[+],CD25[+],IL-10[+] regulatory T cells

Materials and Methods: we analyzed peripheral blood mononuclear cells [PBMCs] from chronic HCV-infected patients [n=19] and normal controls [n=6] to determine the effect of the HCV-core antigen in the frequency of HCV-specific IL-10 production. PBMCs of different groups were isolated, cultured and stimulated with core antigen. Then, an in-house triple-stain flow cytometric method was used to investigate the frequency of CD4[+],CD25[+],IL-10 producing cells

Results: following incubation of PBMCs with HCV-core antigen, a population of CD4[+],CD25[+],IL-10[+] cells [regulatory T cells] increased. However we observed no increase in Tregs in the negative controls

Conclusion: the study supports the view that specific CD4[+],CD25[+],IL-10[+] T cells may be implicated in host immune tolerance during an HCV infection. It is likely that HCV vaccine candidates avoid epitopes that lead to strong IL-10 production

endogenous immune adjuvant released by necrotic cells for enhancement of DNA vaccine potency

Improving vaccine potency in the induction of a strong cell-mediated cytotoxicity can enhance the efficacy of vaccines. Necrotic cells and the supernatant of necrotic tumor cells are attractive adjuvants, on account of their ability to recruit antigen-presenting cells to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells. To evaluate the utility of supernatant of necrotic tumor cells as a DNA vaccine adjuvant in a murine model. The supernatant of EL4 necrotic cells was co-administered with a DNA vaccine expressing the glycoprotein B of Herpes simplex virus-1 as an antigen model under the control of Cytomegalovirus promoter. C57BL/6 mice were vaccinated three times at two weeks intervals with glycoprotein B DNA vaccine and supernatant of necrotic EL4 cells. Five days after the last immunization, cell cytotoxicity, IFN-gamma and IL-4 were evaluated. The obtained data showed that the production of IFN-gamma from the splenocytes after antigenic stimulation in the presence of the supernatant of necrotic EL4 cells was significantly higher than the other groups [p<0.002]. The flow cytometry results showed a significant increase in the apoptosis/necrosis of EL4 cells in the mice immunized with DNA vaccine and supernatant of necrotic EL4 cells comparing to the other groups [p<0.001]. The supernatant of necrotic cells contains adjuvant properties that can be considered as a candidate for tumor vaccination

Investigation in vitro expression of catSper sub fragment followed by production of polyclonal antibody: potential candidate for the next generation of non hormonal contraceptive

CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper [1-4] ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive. In this experimental study, a small extracellular fragment of Cat-Sper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1] pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2] The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay [ELISA] and western-blot, and 3] The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test. The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with [p< 0.05] unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility. CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive

Effect of LIGHT adjuvant on kinetics of T-cell responses induced by HSV-1 DNA Immunization

Studies on efficacy of various vaccines that prevent or reduce the primary and recurrent HSV-1 infection have demonstrated the importance of cellular immunity for protection against the infection. We previously used DNA vaccination to induce cellular immunity against HSV-1 infection in mice. The aim of our study was to evaluate the effect of LIGHT; a member of TNF super family, on the kinetic of CTL response induced by HSV-1 glycoprotein B based DNA vaccine. Using a granzyme B ELISA for detection and analysis of CD8+ T cells, CTL activity was determined in the spleen of BALB/c mice at various time points after primary and booster dose of vaccination. The kinetics of CTL response to primary and secondary HSV-1 infection and DNA vaccination were compared to those induced by DNA vaccination in combination with LIGHT adjuvant in the present study. In primary and secondary immunization, the CTL activity in the HSV injected group peaked 7 days and 12 hours post immunization, respectively. After 5 days, LIGHT could neither accelerate the CTL response compared to DNA vaccination alone nor could enhance the CTL activity in the primary and the first peak of memory response, the amount of granzyme B induced by the LIGHT containing vaccine was significantly higher than that induced by the vaccine without the adjuvant. Although LIGHT enhances the cellular response in the booster dose of vaccination, it does not accelerate the CTL response

[Evaluation of intramuscular versus intra-dermal inoculation of bacterial plasmid encoding nucleoprotein of influenza virus]

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. 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 postchallenge 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. 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

[In vitro expansion of antigen-specific natural regulatory T cells in hepatitis C virus infection mediated by Core antigen]

Hepatitis C virus [HCV] is one of the most relevant persistent infections afflicting the human population. Control of viral replication in HCV infection has been associated with the cellular component of the host immune response. Several mechanisms have been proposed to explain this abnormal immune response, among them an altered activity of regulatory T cells [Tregs] being the most recently postulated. As the first report, in the present study the ability of HCV-core antigen in increase the frequency of natural Tregs [nTregs] in the mixed population of PBMCs was evaluated. Peripheral blood mononuclear cells [PBMCs] from chronic HCV infected patients [n=19] and normal controls [n=6] were analyzed to study the effect of HCV-core antigen in frequency of HCV specific nTregs. For this, PBMCs of different groups were isolated, cultured and stimulated with core antigen. Then an in-house triple-stain flowcytometric method was used to investigate the frequency of nTregs. The results showed that, following incubation with HCV-core Ag, a population of nTregs was increased but, in negative controls the number of nTregs did not increase. The present data supporting the idea that nTregs are able to respond specifically to HCV antigen suggests that Tregs could contribute to an inadequate response against the HCV infection, leading to chronic infection and supports the view that specific natural Tregs may be implicated in host immune tolerance during HCV infection. It is reasonable that HCV vaccine candidates avoid epitopes that lead to strong nTregs stimulation

Expansion of CD4+CD25+FoxP3+ regulatory T cells in chronic hepatitis C virus infection

Regulatory T cells [Tregs] have been involved in impaired immunity and may have a pivotal role in persistence of viral infections. To develop a simple and reliable in-house three color flow cytometery of peripheral blood to understand the role of HCV infection in the increase of Tregs. The level of naturally occurring CD4+ CD25+ FoxP3+ regulatory T cells [nTregs] in 20 chronically infected with hepatitis C virus [HCV] patients was compared to those of 15 healthy individuals by flowcytometry. In a different approach we performed permeabilization and intracellular staining before surface staining which allows the preservation of the surface molecules in the combined detection process and results in the normal frequency of nTregs in blood. Using the optimized method, it was shown that a significantly higher proportion of nTregs in the total CD4+ T cell population was seen in the peripheral blood of chronic HCV patients [0.83 +/- 0.21%, p=0.05] as compared to controls [0.26 +/- 0.1, p=0.05]. Conclusions: In accordance with other studies, we showed that HCV infection induces a dramatic increase in Tregs, which might contribute to the immune response failure during HCV infection

Kinetics of primary and memory cytotoxin B lymphocyte responses to herpes simplex virus 1 infection: granzyme mediated CTL activity

Herpes simplex virus type 1 is one of the most common viruses among human population. Studies demonstrate the essential role of cell mediated immunity, especially CD8+ T cells, in prevention and clearance of HSV1. It is of great importance to improve our knowledge about the kinetics of CTL responses to primary and secondary HSV-1 infection. Using a sensitive technique for detection and analysis of CD8+ T cells, granzyme B ELISA, the CTL activity in the spleens of Balb/c mice at various time points after intraperitoneal administration of HSV1 [strain KOS] in primary and secondary infections were determined. During acute HSV-1 infection, virus specific cytotoxic T cells were detected at day 5 post virus inoculation and peaked at day 7. Six hours after secondary infection the activity of memory CD8+ T cells was detected and peaked at 12 hours post infection. The peak of CTL activity was found to be day 7 post infection in primary HSV-1 infections which decreased with time. In secondary infections, the activity of CTLs reached the highest level at 12 hours post infection

effect of herpes simplex virus virion host shutoff gene- a new suicide gene- on tumor cells

The herpes simplex virus [HSV] UL41 gene product, virion host shutoff [Vhs] protein, mediates the rapid degradation of both viral and cellular mRNA. This ability suggests that Vhs protein can be used as a suicide gene in cancer gene therapy applications. The recent reports have shown that the degradation of cellular mRNA during herpes simplex infection is selective. RNA containing AU-rich elements [ARE] in their 3' untranslated ends are the targets for the Vhs protein. RNA that are not subject to Vhs protein-dependent degradation are up-regulated during HSV infection. ARE are frequently found in mRNA that encode proto-oncogenes, nuclear transcription factors, and cytokines. In many human cancers, the AU-rich stretch of proto-oncogenes and regulatory genes has impaired. To investigate whether Vhs protein might be useful for inhibition of tumor cell proliferation, a eukaryotic expression vector containing Vhs protein gene was constructed. Cell degradation and RNA content of HeLa and MRC-5 tumor cells after transfection with the constructed vector were studied. The results showed a strong inhibitory activity in proliferation of transfected tumor cells and a sharp decrease in their RNA content. These data suggest that Vhs protein can be considered as a candidate for suicide cancer gene therapy