Antibacterial ethylene propylene rubber impregnated with silver nanopowder: AgNP@EPR

Following our interest in reaching for a molded rubber article with possible detergent contact applications, durable silver nanopowder (AgNP) is synthesized by arc discharge, then mixed with varying ratios of ethylene propylene rubber (EPR), affording novel AgNP@EPR nanocomposites. X-ray diffraction (XRD) patterns of AgNP as well as AgNP@EPR show no trace of impurity, while scanning electron microscopy (SEM) indicates an average diameter of 50 nm for the former. Transmission electron microscopy (TEM) images while confirm the SEM results, show quite a few 5 nm AgNP particles lying beside some micro crumbs. Our DC arc discharge technique involves explosion of movable silver anode and static cathode by a current pulse between 5 to 10 A cm-2. A solution blending method is employed for preparation of AgNP@EPR nanocomposites. The AgNP is first dispersed in toluene using an ultrasonic homogenizer, and then thoroughly mixed with EPR in the same solvent whose removal gives nanocomposites of 2, 4, 6 and 8 vol% AgNP in EPR,  showing strong antibacterial activity against both Escherichia coli and Staphylococcus aureus

Evaluation of Immunogenicity of Novel Isoform of EG95 (EG95-5G1) From Echinococcus granulosus in BALB/C Mice

Background: Echinococcosis is a zoonotic parasitic disease of humans and various herbivorous domestic animals transmitted by the contact with domestic and wild carnivores,mainly dogs and foxes. The aim of this study is the production, purification and evaluation immunogenicity of new construction of EG95 protein. Methods: The recombinant plasmid pET32-a+ used for Eg95 expression was constructed with the EG95 gene of Echinococcus granulosus fused with the thioredoxin tag.This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The purification was performed under denaturing conditions in the presence of 8M urea by Ni–NTA column and dialysis. The purified recombinant proteins were confirmed with western blot analysis using polyclonal antiserum. To find out the immunogenicity of the purified protein, the BALB/c mice(10mice/group) were immunized by injecting 20 μg rEG95 protein formulated in Freund’s and alum adjuvant. Results: Immunization of mice with rEG95 using CFA/IFA and alum adjuvant generated high level of total antibody. In proliferation assay, the lymphocytes were able to mount a strong proliferative response with related production of IFN-ɣ, IL-12 and TNF-α but with low secretion of either IL-4 or IL-10. The humoral and cellular immune responses against rEG95 suggested a mixed Th1/Th2 response with high intensity toward Th1. Conclusion: Our findings suggest that new construct of rEG95 formulated with CFA/IFA and alum adjuvant elicited strong cellular and humoral responses supporting further development of this vaccine candidat

The combined use of recombinant and pVAX-omp31 DNA Vaccine for immunological protection against pathogenic Brucellas melitesnsis in an experimental model of BALB/c Mice

Background and Aim: Brucellosis is still an important zoonotic infection and evaluation of immunologic properties of various bacterial antigens along with different vaccination strategies helps in designing efficient vaccines against the disease. The aim of this study is to immunological evaluate the eukaryotic vector pVAX1, carrying the outer membrane protein gene of 31 kDa (Omp31) B.melitensis. Materials and Methods: In this study which was carried out in 2014, whole sequence of omp31 of B. melitensis was inserted between BamHI and XhoI of pVAX1 plasmid vector. Female BALB/c mice aged 6-8 weeks (purchased from Pasteur Institute of Iran) were immunized intra-muscularly with 100μg of the construct, followed by either protein or plasmid boosters separately. The level of IL-4, IL-12, IFN-γ, total serum IgG, and specific IgG1 and IgG2a against recombinant Omp31 were evaluated. Finally the protective immune response following exposure to B. melitensis 16M was evaluated. Results: DNA-vaccine omp31 career with protein reminders Omp31, stimulate higher levels of IFN-γ, IL-12 and IgG2a compared to groups of DNA-vaccine or recombinant protein. Protective immunity was also significantly higher in mice which immunized with DNA vaccine– protein regimen. Conclusions: Mice which immunized with DNA vaccine–protein regimen showed a significantly higher levels of IL-12 and IFN-γ along with serum IgG2a which together imply augmentation of T cell-mediated immune responses against Omp31. The latter was confirmed by significant protective response to B. melitensis 16M challenge

Improvement of Large-scale PRP production by Haemophilus influenzae typeb, using modified CY medium

Background and Objective: Haemophilus influenzae type b (Hib) is a gram negative bacterium and one of the most common causative agents of acute meningitis in infants and less than 5 years old children worldwide. The production of Hib capsular polysaccharide; polyribosyl ribitolphosphate (PRP) is important for the production of conjugate vaccines against Hib infections. The aim of this study is the improvement of Large-scale PRP production by Hib. Materials and Methods: Haemophilus influenzae type b standard strain ATCC10211 was cultivated in 2L fermentors contain 1.5L CY (casaminoacid yeast extract) medium with normal or modified concentrations of glucose, yeast extract, hemin and NAD (nicotinamide adenine dinucleotide). Seed culture of two fermentors was inoculated to 50 L fermentor, separately and range of PRP production and Dry cell weight (DCW) were studied. Results: Cultivation of Hib in 50L fermentor contained modified CY medium with 6gl-1 Glucose, 2.5 gl-1 Yeast extract, 0.03 gl-1 Hemin and 0.015 gl-1 NAD , with controlled pH at 7.3 and 30% Dissolved oxygen tension (DOT) resulted to about 5.1 gl-1 DCW and 1.16 gl-1 PRP , that was significantly higher than normal CY medium. Conclusion: In conclusion, by modification in some medium components of CY medium, control of Dissolved oxygen tension and pH, the Large-scale production of PRP is improved. Improvement of PRP production leads to reduce the final cost of Hib conjugate vaccines

Passive Immunity with Recombinant Anti-Pseudomonas Aeruginosa Type B Fagellin Antibody in a Burned Mouse Model

Abstract Background: Pathogenic Pseudomonas aeruginosa strains produce a polar flagellum that required for motility, adhesion, invasion and secretion of virulence factors. The aim of this study was to evaluate the effect of prevention and treatment with anti-recombinant type B flagellin antibody in a burned mouse model. Materials and Methods: One hundred twenty six mice were divided into 16 groups injected with different regimen of anti-recombinant type B flagellin antibody. Infections were caused by sub-dermal injection of P. aeruginosa PAO1 and PAK strains at the burn site. Groups were monitored for mortality for one week. Additionally, functional activity of this antibody was assessed by motility inhibition and opsonophagocytosis assays. Results: Immunotherapy with rabbit antisera substantially increased (85.71%) survival rate of mice challenged with a homologous strain PAO1 compared with the control PBS. The mortality rate in mice infected by the heterologous strain PAK was only 28.57%. This antibody increased phagocytosis killing of the homologous strain but it only had a slight effect on the heterologous strain. Conclusion: Passive immunotherapy protected burned mice challenged with the homologous strain but showed lower efficacy against the heterologous strain

The Assessment of Cytokine and Antibody Responses to Recombinant 31kDa Brucella Cell-Surface Protein in Brucella Abortus Infected Mouse Model

Background & Objective: One of the most common diseases between zoonosis - especially in developing countries – is brucellosis. Identification of Brucella cell antigen combinations in terms of the amount and type of immune response in infected hosts, are important in vaccine design. 31kDa Brucella cell surface protein (BCSP31) is shared among all Brucellae. We aimed to define specific immune responses to BCSP31 which are elicited during infection of murine host with B. abortus. Surface protein of 31 kDa (BCSP31) is present in all strains, and here, the host immune response during murine infection with Brucella abortus which are formed in proportion to the proteins are studied. Materials & Methods: Recombinant BCSP31 (rBCSP31) of B. abortus was produced and purified. One group of BALB/c mice were infected with pathogenic B. abortus 544 and maintained for 60 days; a no-infected group of mice also was considered. Specific serum antibodies directed to rBCSP31 was evaluated through ELISA. Splenocytes of mice were cultured and stimulated with rBCSP31; thereafter, IL-4, IL-12 and IFN-γ cytokine responses of spleen lymphocytes were assessed. Results: We detected a remarkable IgG titer along with IgG1 and IgG2a specific to recombinant BCSP31 in serum samples from infected mice. Significant titers of IgG, IgG1 and IgG2a antibodies than BCSP31 in the the serum of mice infected with the virulent strain were observed. The Evaluation of Splenocytes responses to rBCSP31 also showed a significant IL-12 and IFN-γ production along with remarkable IL-4 production in infected mice. All responses were significantly different from that of non-infected mice (p<0.05). Conclusion: These findings suggest that specific humoral and cell-mediated responses to BCSP31 is formed during murine host infection with B. abortus. Based on these findings, rBCSP31 can be used in further design of immunogenic strategies for vaccination against brucellosis

Cloning and expression of Brucella outer membrane protein 36kDa (OMP2b) in E. coli

Background & Objective: Brucellosis is an important zoonotic disease of economic significance. Brucella species are gram-negative, facultative intracellular bacteria, and are capable of replicating in the phagosomes of macrophages. They cause infection in several animal species and humans. Prevention of new diseases and diagnosis of cases infected with the organism are both essential for eradication of the disease. Characterization and evaluation of different antigens of Brucella cells has a key role in progression of prevention and diagnosis programs. Here, we report the production and purification of recombinant 31kDa outer membrane protein Brucella abortus (Omp2b). Materials & Methods: Brucella abortus 36kDa outer membrane protein gene was amplified with PrimeSTAR® HS DNA polymerase, cloned in pJET1.2. The target gene was subcloned in pET28a (+). Recombinant pET28a vectors were transformed into E coli BL21 (DE3). Expression of recombinant protein was induced with 1mM IPTG. Proteins were absorbed to Ni-NTA agarose resins and Recombinant proteins were eluted with 250mM imidazol. Imidazol removed by dialysis. Proteins were assayed by Western-blotting and rOmp2b was probed by Brucella rabbit anti serum. Result: Appearance of a golden brown band at the site of reaction, in Western blotting confirmed successfully clone and expression. We purified Omp2b by affinity chromatography and this method prepared refolds proteins on the column. Conclusion: Omp2b were successfully cloned, expressed and purified. The recombinant proteins were recognized by polyclonal antiserum which suggests the accuracy of procedure