Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine

PURPOSE: FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. MATERIALS AND METHODS: In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. RESULTS: The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-gamma and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. CONCLUSION: In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

HESA-A exerts its cytoprotective effects through scavenging of free radicals: an in vitro study

Natural medicines have been recently considered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect. Chinese hamster ovary [CHO] and human embryonic kidney [HEK293T] cells were treated with different concentrations of HESA-A and H2O2 followed by cell proliferation assays. The antioxidant effect of the HESA-A preparations was evaluated by an antioxidant assay kit. The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H2O2 were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H2O2, respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Antioxidant assay revealed that HESA-A scavenges free radicals. The findings indicate that HESA-A had cytoprotective effects in vitro, and that such an effect might be due to antioxidant properties

Down-regulation of metallothionein 1 and 2 after exposure to electromagnetic field in mouse testis

It is proved that testis is sensitive to electromagnetic field [EMF] and its damage results in infertility. Exposure to EMF induces reactive oxygen species production and affects on anti-oxidants defense mechanisms. Metallothionein [MT] is a name for a group of low molecular weight [6-7 kDa], sulfhydryl rich proteins. Expression of MT1 and MT2 genes in testis tissue after EMF exposure was aimed in this study. Male BALB/c mice [8 weeks old] were exposed to 3 MT EMF for 8 weeks, 4 hours/day. After 8 weeks, the mice were sacrificed and the testis tissue was removed. The testis pieces were stained with hematoxylin and eosin and analyzed under an optical microscope. Assessment of MT1 and MT2 genes and also protein expression was performed by real-time PCR and Western-blot, respectively. In light microscopic observation, the number of primary spermatocytes was increased significantly in EMF group [P<0.01]. In addition, in interstitial space, the number of leydig cells was increased significantly in EMF group [P<0.01] and basement membrane thickness was increased as well. MT1 and MT2 genes were down-regulated significantly in testis tissue of mice exposed to EMF both in mRNA and protein level compared to control. It is clear that MT is mediated in testis development and spermatogenesis. Down-regulation of MT1 and MT2 after EMF in mouse testis might be followed by some consequences that result in infertility

Genetically engineered mesenchymal stem cells stably expressing green fluorescent protein

Mesenchymal stem cells [MSCs] are nonhematopoietic stromal cells that are capable of differentiating into and contribute to the regeneration of mesenchymal tissues. Human mesenchymal stem cells [liMSCs] are ideal targets in cell transplantation and tissue engineering. Enhanced green fluorescent protein [EGFP] has been an important reporter gene for gene therapy. The aim of this study was establishment of MSCs expressing GFP. MSCs were isolated and characterized by Immunophenotyping. The pEGFP-Nl plasmid was extracted from previously transformed Escherichia, coli cells and transfected into MSCs using FuGENE HD transfection reagent. Stable cells were established in the presence of geneticin. Expression of GFP was detected by RT-PCR, western blot analysis and immunoflorecent microscope. MSCs were successfully isolated and characterized. The MSCs transfected with the pEGFP-Nl plasmid expressed GFP both in mRNA and protein levels while cells transfected with empty vector did not. The results suggested that this engineered cell line will be used in the future studies and can easily be traced in vivo

High frequency electromagnetic field induces lipocalin 2 expression in mouse liver

Neutrophil gelatinase-associated lipocalin [NGAL/Lcn2], comprise a group of small extracellular proteins with a common beta-sheet-dominated 3-dimensional structure. In the past, it was assumed that the predominant role of lipocalin was acting as transport proteins. Recently it has been found that oxidative stress induces Lcn2 expression. It has been also proved that electromagnetic field [EMF] produces reactive oxygen species [ROS] in different tissues. Expression of Lcn2 following exposure to electromagnetic field has been investigated in this study. Balb/c mice [8 weeks old] were exposed to 3 mT, 50 HZ EMF for 2 months, 4 hr/day. Afterwards, the mice were sacrificed by cervical dislocation and livers were removed. The liver specimens were stained with Haematoxylin-Eosin [H and E] and analyzed under an optical microscope. Total RNA was extracted from liver and reverse transcription was performed by SuperScript III reverse transcriptase with 1 micro g of total RNA. Assessment of Lcn2 expression was performed by semiquantitative and real time-PCR. The light microscopic studies revealed that the number of lymphocyte cells was increased compared to control and dilation of sinosoids was observed in the liver. Lcn2 was up-regulated in the mice exposed to EMF both in mRNA and protein levels. To the extent of our knowledge, this is the first report dealing with up-regulation of Lcn2 in liver after exposure to EMF. The up-regulation might be a compensatory response that involves cell defense pathways and protective effects against ROS. However, further and complementary studies are required in this regards

Effects of leukemia inhibitory factor on gp130 expression and rate of metaphase II development during in vitro maturation of mouse oocyte

Leukemia inhibitory factor [LIF] is a 45-56 kDa glycoprotein that has an important role in proliferation and embryo implantation. Its effect on oocyte maturation and how to exert the function remained to be elucidated. Immature mice superovulated with human menopausal gonadotropin and germinal vesicle [GV] oocytes were obtained from ovary 48 hours after. GV oocytes were cultured in tissue culture medium 199 with 0, 100, 500 and 1,000 U/ml LIF. Cumulus expansion and in vitro maturation [IVM] rate were assessed after culture. For reverse transcriptase PCR, total RNA from GV and metaphase II [Mil] oocytes were extracted by Trizol reagent. The quantity and quality of RNA were determined by spectrophotometry and electrophoresis, respectively. Reverse transcription was performed by Super Script III reverse transcriptase with 1 micro g of total RNA followed by DNase I treatment and heat inactivation. Expression of gp130 was determined by RT-PCR. Our results showed that cumulus expansion was improved with 1,000 U/ml in culture medium compared to others. GV breakdown and Mil rate in groups with LIF were higher than control group and were dose dependant. In 1,000 U/ml, LIF rate of Mil was significantly higher than control group [P<0.05]. Our results also showed that gp130 is expressed neither in GV nor in Mil oocytes during IVM of mouse oocytes. gp130 is expressed in human oocyte but not in mouse. Our results suggest that in mouse, LIF could affect the oocyte via another receptor or via cumulus cells; however, further studies are warranted