Evaluation of Heating and Irradiation Methods for Production of Purified Protein Derivative (PPD) of Mycobacterium Tuberculosis.

Tuberculin skin test, also known as the tuberculin or purified protein derivative (PPD) test, is an extensively applied diagnostic test for the detection of primary infection with Mycobacterium tuberculosis (Mtb). The production of PPD is accompanied by some difficulties that require a series of modifications in the production and purification processes. The present study aimed to determine the facilitation level of the manufacturing process by modifying evaluation methods for the production of PPD tuberculin. Mtb strains were cultured in Lowenstein-Jensen media, and the cultured strains were inoculated into the Dorset-Henley liquid medium by the biphasic medium of potato-Dorset-Henley. After incubation, flasks containing cultured strain were selected for bacterial inactivation, and the optimal gamma radiation dose(s) was determined. Tuberculoproteins were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentration was determined using the Bradford and Kjeldahl protein assay methods. Finally, the lymphocyte transformation test and potency test were performed. Based on the results, the Dorset-Henley liquid medium is suitable for the massive growth of the bacterium. The transferal of Mtb from solid to liquid medium was directly carried out without intermediate culture. It was found that during tuberculoprotein production, heating at 100°C for 3 h would be safe for killing mycobacterium. Furthermore, the simultaneous use of heating and gamma irradiation (8 kGgy) killed all of the mycobacteria, while doses of 1, 1.5, and 7 kGy decreased a significant number of bacterial cells. The results also indicated that the concentration of tuberculoprotein extracted by TCA precipitation method was higher than that obtained by AS precipitation. The tuberculoproteins which were produced by these two methods in the lymphocyte transformation test were not significantly different in terms of potency (P>0.05). Moreover, due to the high volume of produced protein, the protein measurement was more efficiently carried out by the Kjeldahl method, compared to the Bradford method. Finally, the results of the present study demonstrated that in addition to the novel approach of gamma irradiation, optimum methods are efficient and applicable in the production of PPD tuberculin.

Enhancement of Th1 immune response against Leishmania cysteine peptidase A, B by PLGA nanoparticle.

Recombinant cysteine peptidase vaccine can induce protective immunity against cutaneous leishmaniasis. However, the antigenic diversity and variable immunogenicity prevents them from being approved for general vaccination. Different approaches like adjuvant application and antigen delivery systems are studied to increase their efficacy. Nanoparticles can both stimulated antigen uptakes and affect direction of immune response. In this study the effect of PLGA nanoparticles were considered to enhance the immune response against recombinant CPA (CPA) and CPB (CPB). For this purpose, L. major CPA and CPB were prepared. PLGA were conjugated to the proteins using Aldehyde/Hydrazine Reaction. Conjugation efficacy and created nanoparticle morphology were determined by FTIR and SEM methods, respectively. BALB/c mice were received intraperitoneally three boosts of 7 µg/mouse of each antigen alone (CPA/CPB/CPA + CPB) or as PLGA conjugated form in different Study groups, at 3 weeks interval. After vaccination, mice were challenged with 106L. major, subcutaneously. Time course study of lesion development demonstrated nanoparticle efficacy in parasite dissemination control that confirmed by spleen parasite burden assay. Significant induction of nitric oxide production by peritoneal macrophages and increase in splenocyte IFN-γ production showed the protective effect of PLGA-CPA/CPB vaccination in comparison to CPA and CPB alone. Current study demonstrated that the conjugation of the antigen with the PLGA can activate immune responses against L. major. However, further study is necessary to assess the long-term effect and other aspects of immune response.

Elevated levels of IL-2 and IL-21 produced by CD4+ T cells in inflammatory bowel disease.

CD4+ T cells are considered as a subset of cells that play a pivotal role in the development of inflammatory bowel disease (IBD). The aim of this study was to assess the levels of interleukin (IL)-2, IL-21 and their receptors produced by CD4+ T cells in patients with inflammatory bowel disease. Thirty-two patients with ulcerative colitis (UC) and mean age of 37.93±10.37 years, as well as 22 patients with Crohn’s disease (CD) and mean age of 37.04±10.44 years, were studied. The healthy controls (HC) included 31 subjects with a mean age of 36.7±10.48 years. Peripheral blood mononuclear cells (PBMCs) were isolated from all the participants. The CD4+ T cells were isolated and the expression of IL-2 and IL-21 and also their receptors were examined by flow cytometry. The level of IL-2+ cells was significantly increased in UC patients compared with HC (40.71±6.04 vs 37.24±6.54, respectively, p=0.04). The level of IL-21+ cells was also significantly elevated in CD patients compared with HC (4.44±1 vs 3.83±0.74, respectively, p=0.02). Furthermore, we found a significant positive correlation between clinical activity index (CAI) and IL-21+ cells. According to the results, we hypothesize that the elevated level of IL-2+ and IL-21+ T cells and a positive correlation between IL-21+ cells with CAI in UC patients may contribute to the pathogenesis of disease. Moreover, the assessment of cells producing such cytokines constitutes a potential diagnostic and therapeutic strategy for IBD.

Immune response to recombinant Leishmania infantum lipophosphoglycan 3 plus CpG oligodeoxynucleotides in BALB/c mice.

Development of a protective antileishmanial vaccine is an urgent priority for successful control of different forms of leishmaniasis. The potential of a recombinant lipophosphoglycan 3 (rLPG3) expressed by Leishmania tarentolae was evaluated in combination with CpG oligodeoxynucleotides (CpG-ODN) as a Th1-promoting adjuvant against Leishmania infantum infection in BALB/c mice. First, mice were immunized subcutaneously with rLPG3 either alone or in combination with CpG-ODN. Next, the immunogenic and protective efficacies of this vaccine were analysed in immunized mice. It was observed that coadministration of rLPG3 with CpG-ODN led to enhance in a Th1 response to rLPG3 induced by itself as the IFN-γ production was promoted in association with the predominant presence of IgG2a antibodies in the sera. However, immunization with rLPG3 plus CpG-ODN induced partial protection against infectious challenge in BALB/c mice. Taken together, further studies are required to improve the protective efficacy using either more potent immune enhancers or vaccination strategies.

Proliferation responses in preimmunized mice lymphocytes by Bordetella pertussis cell wall components.

Bordetella pertussis infects the respiratory tract of the human host and causes whooping cough in children. The nature of immunity against Bordetella pertussis infection and disease is poorly understood. The aim of this study was to investigate cell mediated immunity in mice immunized with outer membrane component of cell wall, of B. Pertussis. A group of mice were immunized with outer membrane complex (OMC) and killed whole cell (WCV) of B. pertussis, with an interval of 2 weeks. During a period of 7 weeks following the immunization, lymphocytes were isolated from lymph nodes of immunized mice. The in vitro proliferative response of isolated lymphocyte to stimulation with 20 ig of 30 and 69 kDa outer membrane protein (OMP) were measured as parameters for cell mediated immunity (CMI). The data were expressed as mean count per minute (CPM)x103 after subtraction of the CPM of unstimulated control cultures. Lymphoblastogenic response was observed in immunized mice with WCV and OMC. At 30 days of post immunization a significant increase in response to 30 and 69 kDa OMP was observed, a small decrease in the response was evident against P30 and P69 at 60 and 120 days of post immunization, but the response was still higher than what was observed in control mice. Current findings indicate strongly the potential of outer membrane protein component of B. perlussis in proliferating lymphocytes in the mice.