Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse

Objective: Polytope DNA vaccines, capable of focusing the cytotoxic T lymphocyte (CTL)response on critical epitopes, represent a promising approach in HCV immunotherapy. Nevertheless,due to controversial rules governing epitope processing and the low level expression/immunogenicity of recombinant polytope peptides, designing and primary expression/immunogenicity analysis of these vaccine types should be the first consideration prior tocostly transgenic animal studies.Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected anddesigned in three appropriate sequential tandems based on epitope and proteasomal cleavagepredictions. The related nucleotide sequences were synthesized using SOEing PCRmethod and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatitis B surfaceantigen (HBsAg-S) gene. Following the preparation of polyclonal anti-sera, expression/secretion of polytopes was evaluated in Cos-7 cells by using immunofluorescence, Westernblot,dot blot, ELISA and RT-PCR techniques. The immunogenicity of the plasmids was alsoassessed through the delayed-type hypersensitivity (DTH) assay in BALB/c mice.Results: Due to in silico designs and optimizations, the polytope products of constructedplasmids were efficiently detected in vitro through common techniques and HBsAg-S-basedparticles were shown to be secreted into the culture media (up to 30%). Moreover, all plasmidswere able to efficiently induce a positive DTH response while HBsAg-S fusion constructsindicated a significant immunopotential effect towards the incorporated mouse epitopes.Conclusion: Designed polytope constructs of this study are efficiently expressed and processed.They have the required initial potency for further immunogenicity analysis in transgenicmice

Number and subtypes of natural killer cells in patients with allergic rhinitis in comparison to healthy subjects

"n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Allergic rhinitis is a common disorder with great morbidity. Its prevalence has increased during recent years, therefore attracting attentions to its mechanisms. Type 2 cytokines play a major role in allergies. It has been proposed that Natural killer (NK) cells may be able to produce type 2 cytokines. This study was done to evaluate NK cells number and subtypes in patients with allergic rhinitis, comparing healthy subjects."n"nMethods: In a case control study, patients with allergic rhinitis were compared to healthy non-atopic subjects. Allergic rhinitis was diagnosed according to ARIA guidelines. NK cells quantity was studied by staining of peripheral blood mono nuclear cells with anti-CD16-FITC and anti-CD56-PE and evaluated by two color flowcytometry. Intracellular cytokines were evaluated by tri-color flowcytometry. NK cells were separated by magnetic beads, and cultured for 72 hours. Secretion of IL-4, IL-5, IL-10, IL-13, and IFN-γ was measured by ELISA, in stimulated and unstimulated conditions."n"nResults: Patients had more CD16+ CD56+ NK cells than control group. IL-4+ NK cells were significantly higher in patients (p<0.001), but the number of IFN-γ+ NK cells was not different. Cytokine secretion of NK cells was similar in case and control groups. Although IL-13 level after stimulation seemed higher in patients, the difference was not significant."n"nConclusion: NK cells number is increased in patients with allergic rhinitis and a considerable number of them produce IL-4