IgG1 and IgG2a Profile of Serum Antibodies to Leishmania major Amastigote in BALB/c and C57BL/6Mice

It is well accepted that in experimental model of Leishmania major infection, BALB/c mice mount a Th2 response and produce IgG1 predominantly whereas C57BL/6mice enhance Th1 response with the biased production of IgG2a antibodies. Therefore, screening for parasite antigens on the basis of reactivity with sera from infected susceptible or resistant mice might be used for the identification of Th1- or Th2-inducing antigens. In this study, the antigenic profile of Leishmania majoramastigote that induce IgG1 or IgG2a isotypes in infected BALB/c or C57BL/6 mice were compared. Western blot analyses revealed that 27, 40, 43, 45 and 114 kDa proteins elicited IgG1 and not IgG2a in both BALB/c and C57BL/6 mice and 55 kDa protein was recognized exclusively by IgG1 of BALB/c mice sera. On the contrary, the bands corresponding to proteins with molecular weights (MW) of 30, 35, 52, 58 and 66 were intensively immunostained with IgG2a from C57BL/6 mice which made them potential candidates for eliciting Th1 response. In conclusion, the results showed that the generation of the immune responses depended on the mouse strain and some leishmanial antigens had an intrinsic potency to elicit Th2 responses

Subcutaneous administration of a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis induces mucosal and systemic immune responses

Objective(s): After decades of containment, pertussis disease, caused by Bordetella pertussis seems to be re-emerging and still remains a major cause of reported vaccine-preventable deaths worldwide. The current licensed whole-cell vaccines display reactogenicity while acellular vaccines are expensive and do not induce Th1-type immune responses that are required for optimum protection against the disease. Thus, there is an urgent need to develop new vaccines and the recombinant technology seems to be the method of choice for this purpose. The present study was an attempt to develop a new, simplified, cost-effective and well-defined vaccine against Bordetella pertussis, with capacity to induce a Th1 response. Materials and Methods: A fusion DNA fragment encoding the N-terminal region of pertussis toxin S1 subunit and filamentous hemagglutinin type 1 immunodominant domain was constructed and the corresponding fusion protein (F1S1) was produced in Escherichia coli. F1S1 in conjunction with imiquimod was administered by subcutaneous (SC) and intranasal (IN) routes to BALB/c mice. Results: This vaccine formulation could elicit high levels of IFN-γ, serum IgG (with higher IgG2a/IgG1 ratio) and lung IgA after the SC and, to a lesser extent, following the IN administration.  Conclusion: Our results indicate that the above-mentioned important proteins of B. pertussis could be successfully produced in E. coli as a single fusion protein. Furthermore, this protein could induce proper systemic and mucosal immune responses after administration via SC or IN routes

Expression of Leishmania major LmSTI1 in Yeast Pichia Pastoris

Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems.Materials and Methods: To express LmSTI1 in the methylotrophic yeast         Pichia pastoris (P. pastoris), the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0.5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L.major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively.Results: The expression level was 0.2% of total soluble proteins.Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization

The role of microbiota and immune system crosstalk in cancer development and therapy

Cancer is a multifactorial disease that is the second leading cause of death after cardiovascular disease in the world. In recent years, microbiota's role in the regulation and homeostasis of the immune system has been considered. Moreover, the immune system can affect the microbiota content. These interactions are critical to the functioning of the immune system. Numerous studies in animal and human models have shown the association of changes in microbiota components with the formation of an inhibitory microenvironment in the tumor and its escape from the immune system. Microbiota also plays a crucial role in the success of various anti-tumor treatments, and its modification leads to success in cancer treatment. The success of anti-tumor therapies that directly target the immune system, such as immune checkpoint blockade and T cell therapy, is also affected by the patient's microbiota composition. It seems that in addition to examining the patient's genetics, precision medicine should pay attention to the patient's microbiota in choosing the appropriate treatment method, and together with usual anti-tumor therapies, microbiota may be modified. This review discusses various aspects of the relationship between microbiota and anti-tumor immunity and its successful treatment

Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: Implication for diagnosis of Kala-azar

Objective(s): Leishmaniasis is endemic in 88 countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran.Materials and Methods: First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c mice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of 10 to 1 in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting.Results: Approximately 25 positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. Results from isotype determination showed the IgG2b sub-class in 6G2FV2 and 8D2FVI6 monoclones. Conclusion: This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar

Adjuvant Effect of Leishmania major Promastigotes on the Immune Response of Mice to Ovalbumin

ABSTRACT The immune responses of mice immunized with ovalbumin (OVA) together with killed L. major (KLM) promastigotes as adjuvant were studied. Three doses (5 × 10 7 , 1 × 10 8 and 2 × 10 8 ) of KLM combined with OVA (100 µg) were injected into the groups of C57BL/6 mice. BCG and complete Freund's adjuvant (CFA) were used as control adjuvants. Lymphocyte proliferation and antibody titers were determined, and IFN-γ and IL-4 were measured in the supernatants of lymph node cell cultures. Results showed that immunization using OVA mixed with KLM enhanced the in vitro proliferative response of T-cells to the antigen and resulted in the production of increased levels of IFN-γ (2800-3700 pg/ml) relative to the mice injected with OVA alone (1750 pg/ml). In the mice receiving OVA + 5 × 10 7 KLM, the production of IL-4 remained lower (18, 20 pg/ml) than OVA alone (105, 109 pg/ml) and almost was similar to that of observed in mice inoculated with OVA + BCG, leading to high IFN-γ/IL-4 ratios. Using higher doses of KLM (1 × 10 8 ), the IL-4 responses were of the same magnitude as or higher than the responses of mice inoculated with OVA + CFA. Antibody titers to OVA were also strongly boosted at the highest KLM dose. These findings indicate that KLM may function as an adjuvant, and its dose plays a role in the eventual outcome of the response. Inoculation of the mice with a low dose of KLM (5 × 10 7 ) tends to promote a Th1-type response. Iran. Biomed. J. 6 (4): 123-128, 200

Impact of gut microbiota on immune system

The commensal microflora collection known as microbiota has an essential role in maintaining the host's physiological homeostasis. The microbiota has a vital role in induction and regulation of local and systemic immune responses. On the other hand, the immune system involves maintaining microbiota compositions. Optimal microbiota-immune system cross-talk is essential for protective responses to pathogens and immune tolerance to self and harmless environmental antigens. Any change in this symbiotic relationship may cause susceptibility to diseases. The association of various cancers and autoimmune diseases with microbiota has been proven. Here we review the interaction of immune responses to gut microbiota, focusing on innate and adaptive immune system and disease susceptibility

Anti-mitogenic and apoptotic effects of 5-HT1B receptor antagonist on HT29 colorectal cancer cell line

Purpose: There is lack of evidence about impact of 5-HT receptors on colorectal cancers. The current study was designed to investigate the role of serotonin and its receptors in colorectal cancer cell line and tissues. Methods: In cell cultures, we investigated the effects of 5-HT and 5-HT agonists and antagonists on proliferation of HT29 cells. We also tested apoptosis for the receptor antagonists. The expression of 5-HT1 receptor subtypes was examined by immunohistochemistry and western blotting. Results: Our data indicated that 5-HT receptor was fully expressed in HT29 cell line and tumor tissues. MTT proliferation assay also revealed that serotonin and CP93129 dihydrochloride, a selective 5-HT receptor agonist, stimulated growth of HT29 cells. Further, SB224289 hydrochloride (that is a selective 5-HT receptor antagonist) had anti-proliferative and apoptotic effects on HT29 cells. Conclusions: The findings of this study provide evidence for the potential role of 5-HT receptor in colorectal cancer. Further investigation is required to explore the effect of receptor antagonists on the prevention, prognosis and treatment of patients with colorectal cancer

Tumor suppressive function of microRNA-192 in acute lymphoblastic leukemia

Non-coding RNAs play a critical role in gene regulation in cancer cells. Reduced expression of microRNA-192 (miR-192) has been detected in many cancers. In this study, we investigated the role of miR-192 in cell proliferation and cell cycle control in NALM-6 cell line, a model of acute lymphoblastic leukemia (ALL). Cell cycle analysis by DNA content using propidium iodide staining and cell apoptosis analysis using Annexin V assay were carried out. Cell proliferation changes were monitored using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, the relative changes in P53, BAX, CASP3, and BCL-2 gene expression were determined by quantitative reverse transcription PCR. Overexpression of miR-192 resulted in cell proliferation arrest in ALL cells. After 72 and 96 hours of transduction, apoptosis was significantly increased in the cells transduced with miR-192-overexpressing virus compared with control cells. The expression of P53, BAX, and CASP3 increased after 48 hours of transduction in miR-192-overexpressing cells, but no change was observed in BCL-2 expression. The G0/S and G1/S ratio changed to 7.5 and 4.5, respectively, in the cells overexpressing miR-192 compared with controls. The results of our study suggest, for the first time, tumor suppressive effects of miR-192 in ALL cells

Combinatorial delivery of antigen and TLR agonists via PLGA nanoparticles modulates Leishmania major-infected-macrophages activation

Appropriate activation of macrophages is critical for the elimination of Leishmania parasites, which resides in this cell. Some species of Leishmania (L.) fails to stimulate macrophages and establish a chronic infection. To overcome this suppression and induce an innate immune response, the effect of PLGA-encapsulated soluble antigens of Leishmania (SLA) along with agonists of TLR1/2 (Pam3CSK4) and TLR7/8 (R848) nanoparticles (NPs) on activation of L. major-infected-macrophages were investigated and were compared with those of soluble formulations. SLA and R848 were encapsulated into the PLGA, while Pam3CSK4 adsorbed onto the surface of nanoparticles. The kinetics of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and iNOS genes expression were investigated by qPCR over 72 h. The parasite load was also quantified by qPCR.The results indicated that engulfment of L. major promastigotes does not induce any pro-inflammatory cytokines expression by macrophages; however, the infected-cells are capable of responding to the TLRs agonists, and a lesser extent, to the SLA stimulation. Encapsulation resulted in increased strength of the IL-1β, IL-6, TNF-α, and increased and prolonged time of iNOS expression. Also, encapsulation showed the leishmanicidal activity by decreasing parasite load in treated NPs formulations. Among the different combinations of the components, the triple (SLA-R848-Pam3CSK4) forms promoted the highest activation of macrophages, followed by dual SLA-Pam3CSK4 and SLA-R848 NPs.In conclusion, the findings of this study indicate that the addition of SLA in combination with TLR1/2 and TLR7/8 agonists either in NPs or in soluble forms overcome the suppression of L. major-infected macrophages. Moreover, encapsulation increases the strength and duration of the cytokines and iNOS expression, in parallel with decreasing parasite load, suggesting a longer availability or delivery of the NPs into the macrophages. These findings highlight the advantages of particulate therapeutic vaccine formulations