Immunogenicity of glycine nanoparticles containing a chimeric antigen as Brucella vaccine candidate.

PURPOSE: Brucellosis as a worldwide zoonotic illness affect domestic animals and humans doesn't have any vaccine for the prevention of infection in humans yet. The aim of this study was to evaluate the specific immune response following the administration of glycine nanoparticles as adjuvant and delivery system of a chimeric antigen contained trigger factor, Omp31, and Bp26 in murine model. MATERIALS AND METHODS: The chimeric antigen of Brucella was cloned and expressed in Escherichia coli (E. coli) BL21 (DE3). Purification and characterization of recombinant protein was conducted through Ni-NTA (nickel-nitrilotriacetic acid) agarose, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and Western blot. Nanoparticle characteristics including morphology, particle size distribution, zeta potential, protein retention rate, and release rate were measured in vitro. Subsequently, nanoparticle contained antigen was administered to mice and blood sample was taken to measured the antibody level. RESULTS: The protein retention in the nanoparticles was successfully done and the nanoparticle characteristics were appropriate. The average size of glycine particles containing antigen was about 174 nm, and the absorption of protein was approximately 61.27% of the initial value, with a release rate of approximately 70% after 8 hours. Enzyme-linked immunosorbent assay result proved that the immunized sera of mice which were administered with nano-formula contains high levels of antibodies (immunoglobulin G) against recombinant chimeric antigen and also a high level of mucosal antibody (immunoglobulin A) in the oral group, which showed a desirable immunity against Brucella. CONCLUSION: The results showed that chimeric antigen-loaded glycine nanoparticles can act as a vaccine candidate for inducing the cellular and humoral immune response against brucellosis.

Immunologic balance of regulatory T cell/T helper 17 responses in gastrointestinal infectious diseases: Role of miRNAs.

Gastrointestinal Infectious diseases (GIDs) are the second cause of death worldwide. T helper17 cells (Th17) play an important role in GIDs through production of IL-17A, IL-17F, and IL-22 cytokines. Because of their increased activities in GID, Th17 and its inflammatory cytokines can inhibit the progression and eliminate the infection. Actually, although Th17 have the best performance in the acute phase, regulatory T cells (Treg cells) are enhanced in the chronic phase and infection progress through its suppressive function. In addition, Treg cells prevent undesirable inflammatory damages developed by immune system components. On the other hand, miRNAs have important roles in the regulation of immune responses to eliminate bacterial infections and protect host organisms from harmful effects. Actually, miRNAs can reinforce innate and adaptive immunity to remove infections. Of note, miRNAs can develop a regulatory network with the immune system. Additionally, miRNAs can also serve in favor of bacteria to reduce immune responses. Therefore, balance of immune responses in Treg and Th17 cells can influence outcome of many infectious diseases. In conclusion, there is an imbalance in the Treg/Th17 ratio in GIDs; importantly, sets of miRNAs, particularly miR155 and miR146, were determined to be involved clearly in GIDs.

The effect of ethanolic extract of Thymus kotschyanus on cancer cell growth in vitro and depression-like behavior in the mouse.

Cancer and depression are known as two of the most debilitating disease and disorder increasing evidence suggest an urgent need for new therapeutic agents with lower toxicity and high efficacy. Some Thyme species extracts have remarkably been shown to positively affect depression and cancer cells. In the present study, we investigated the effect of Thymus kotschyanus on depression and cancer cells. To this end, in experiment 1, NMRI mice were treated orally with the ethanolic extract of T. kotschyanus (50, 150 and 250 mg/ml) for seven days and then depression-like behavior was measured by Forced Swim Test (FST) and Tail Suspension Test (TST). In experiment 2, the pharmacological effect of the extract on the lung (A549) and cervical (Hela) cancer cell lines was also evaluated by MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) in various concentration_(10, 5, 2.5, 1.25, 0.63, 0.31, 0.15 and 0.08 mg/ml). The results indicated that T. kotschyanus extract treatment (150 and 250 mg/kg) decreased depression-like behavior in the FST and TST tests in adult mice. Moreover, the treatment inhibited cancer cell growth and viability in a dose and time-dependent manner. Collectively these findings suggest that T. kotschyanus have antidepressant and anticancer effects.

Vibrio cholerae lipopolysaccharide loaded chitosan nanoparticle could save life by induction of specific immunoglobulin isotype.

The lipopolysaccharide (LPS) of Vibrio cholerae (V. cholerae) plays an important role in cholera disease and the induction of primary protection. In this study, we evaluate mice humoral immune response in intranasal and intraperitoneal administrated V. cholerae LPS. The results showed that the intranasal administration of LPS-chitosan nanoparticle induced the high level of antibodies compared to intraperitoneal injection of antigen without chitosan (P < .001). These results indicated that intranasal and intraperitoneal administration of LPS has been able to induce the high level of antibodies both in the sera and lavage fluid and confirmed our strategy for using intranasal administration of antigen.

In silico designing, cloning, and heterologous expression of novel chimeric human B lymphocyte CD20 extra loop.

Design and production of monoclonal antibody for the diagnosis and immunotherapy of non-Hodgkin lymphoma require a suitable CD20 antigen as an effective immunogen. In this study, a new chimeric human CD20 extra loop (hCD20EXL) protein was designed by bioinformatics tools and was expressed in Escherichia coli BL21 DE3. Amino acid sequences, protein structure, immunogenicity, and other physicochemical property of potential antigens were in silico analyzed. Antigenicity, codon optimization, and other predictions of designed protein were determined by bioinformatics tools. The designed protein was heterologously expressed in E. coli and verified by SDS-PAGE and Western blot. Immunogenicity of this antigen was tested in mice, and reactivity of the antibodies was evaluated using flow cytometry. Experimental analysis confirmed the in silico prediction of the designed chimeric hCD20 in this study. Therefore, based on these results, it is suggested that the new chimeric hCD20 antigen could be an appropriate immunogen for production of monoclonal antibody in immunotherapy purposes.

Production and Characterization of New Anti-Human CD20 Monoclonal Antibody.

The B-cell CD20 antigen is one of the most reliable surface targets in immunotherapy of B lymphoma. In this project, we studied the production and characterization of a new monoclonal antibody against chimeric human CD20 extra loops (hCD20 exl). The results showed that clone C12H, IgG2/k isotype reacted with the antigen in ELISA and immunoblot. The Kd value was found to be 2×10(-9)M and flow cytometry results showed that 99.9% and 99.7% of the Daudi and Raji cells respectively were stained with C12H monoclonal antibody (mab) but not with Jurkat cell lines. It also effectively competed with Rituximab, thus, the staining of the Daudi and Raji cell lines was reduced to 55.9% and 40.5% of cells respectively. Based on the high affinity reaction of C12H mab and appropriate reactivity of C12H mab with the native antigen on the surface of Raji and Daudi cells in flow cytometry, it was concluded that development and evaluation of C12H mab could be a beneficial candidate for further application in genetically engineered monoclonal antibody.

Anti-inflammatory effects of serum isolated from animals on intermittent feeding in C6 glioma cell line.

Multiple sclerosis (MS) is a demyelinating disease of the CNS. Early inflammation leads to later destruction of myelin in MS. Dietary restriction (DR) produces anti-inflammatory and immunomodulatory effects in many species. Based on the reported anti-inflammatory effects of DR, we investigated whether sera collected from rats fed on intermittent feeding (IF, a type of DR) diet could modulate cytokine secretion and matrix metalloproteinase (MMP-2) activity that are involved in MS pathogenesis. Cytokine levels (IL-6 and TGF-ß1) were measured in supernatant from C6 glioma cell line cultures treated with IF and AL fed animals' sera by enzyme-linked immunosorbent assay (ELISA) and MMP-2 activity was detected by gelatin zymography. Our results indicated that sera of animals on IF diet significantly reduced IL-6 (p<0.05) and increased TGF-ß1 (p<0.05) production by C6 glioma cells. A significant decrease (p<0.05) in MMP-2 activity was also found. These results indicate anti-inflammatory and immunomodulatory activity in the sera of animals on IF regimen on cells involved in multiple sclerosis pathogenesis. Further studies on the detection of factors responsible for such activities and their mechanism of action in MS pathogenesis are recommended.

Ganoderma lucidum induces the expression of CD40/CD86 on peripheral blood monocytes.

BACKGROUND: The major immuno-modulating effects of Ganoderma lucidum include mitogenicity and activation of immune effector cells such as T cells, macrophages and natural killer cells resulting in the production of cytokines. OBJECTIVE: The purpose of this study was to evaluate the expression of CD40 and CD80 by G. lucidum-treated human peripheral blood mononuclear cells. METHODS: Monocytes were isolated and incubated at 37 C and 5% CO2 for 24 h and 48 h in the presence or absence of different concentrations of G. lucidum. Cells were then incubated with labelled monoclonal antibodies against CD14, CD40 and B7-1(CD80) molecules utilizing standard protocols, and analyzed by flow cytometry. RESULTS: The results showed that incubation of monocytes with G. lucidum led to marked enhancement of CD40 and B7-1 expression in a dose- and time- dependent manner (p<0.001). G. lucidum was more effective in enhancing the expression of CD80 and CD40 molecules of cells obtained from females than male donors (p<0.001). CONCLUSION: G. lucidum enhanced the expression of CD40 and CD80 molecules on peripheral blood monocytic cells derived from both sexes in a dose-dependent manner, with a preferential higher effect on cells obtained from female donors.

Effects of T-2 toxin on cytokine production by mice peritoneal macrophages and lymph node T-cells.

BACKGROUND: T-2 toxin is a mycotoxin of type A trichothecenes produced by several fungal genera such as Fusarium species. Mycotoxins can affect both cell mediated and humoral immune compartments. OBJECTIVE: The purpose of this study was to investigate the effect of T-2 toxin on cytokine production by mouse peritoneal macrophages and lymph node T cells. METHODS: Mouse peritoneal macrophages and lymph node T cells were isolated and treated with different concentrations of T-2 toxin and incubated at 370C and 5% CO2 in air for 48 hours. Cell free media were removed and used for cytokine assay by an ELISA method. RESULTS: T-2 toxin significantly reduced IL-1beta release in a concentration dependent manner (p<0.005, p<0.001). Interleukin-12 and TNF-alpha production were significantly increased in response to 0.001ng/ml, 0.01ng/ml and 0.1ng/ml of T-2 toxin (p<0.001). However, T-2 toxin at higher concentrations ranging from 1ng/ml to 100ng/ml, reduced both IL-12 (p<0.001) and TNF-alpha production (p<0.005, p<0.05). The effects of T-2 toxin on lymph node T cells showed that IL-4 and IL-10 release was decreased in a concentration dependent manner (all with p<0.01). T-2 toxin at concentrations between 1ng/ml and 100ng/ml reduced the release of both IL-2 and IFN-gamma (p<0.05, p<0.001). CONCLUSION: The results suggest that T-2 toxin at low concentrations can highly induce secretion of IL-12, TNF-alpha, IFN-gamma and IL-2 and it may be used as a positive immunomodulator in the human model.

Effect of Ganoderma lucidum on cytokine release by peritoneal macrophages.

BACKGROUND: The water-soluble extract of Ganoderma lucidum (Reishi) has been used as an immunomodulator to stimulate spleen cells proliferation and cytokine expression. OBJECTIVES: To investigate the effect of Ganoderma lucidum (G. lucidum) on cytokine production by mice peritoneal macrophages. METHODS: Mice peritoneal macrophages were prepared by intra-peritoneal injection of 5 ml cold PBS. Peritoneal macrophages were plated out at 1X10(6) cell/well in 1ml RPMI 1640 medium supplemented with 10%FCS, 50 microg streptomycin and 50U penicillin. Cells were incubated in the presence or absence of different concentrations of G. lucidum at 37 degrees C and 5% CO2 for 48 hours. Cell free medium was removed and used for cytokine assay by ELISA method (Bender med system). RESULTS: The results showed no significant differences in cell viability at concentrations ranged from 0-40 microg/ml compared with control group. G. lucidum enhanced IL-1beta, TNF-alpha and NO production in a concentration dependent manner. However, it is not clear if the enhancement of NO release is due to direct effect of G. lucidum on NO synthesis or by indirect endogenous modulation via cytokines. IL-12 release by peritoneal macrophages was also increased in response to different concentrations of G. lucidum, but maximum enhancement was induced in response to 5 microg/ml of G. lucidum (P<0.001). CONCLUSION: Our results indicate that G. lucidum at concentrations used has a positive effect on cytokine release and NO production by peritoneal macrophages. Therefore, it is concluded that G. lucidum at moderate concentrations improves macrophage function through cytokine and NO release.

Androgen binding site in J111 cell line.

BACKGROUND: The finding of sex steroid receptor protein in non classical reproductive tissues suggested the possibility that sex steroids may have a relevance to the immune system. MATERIAL/METHODS: The J111 cells were maintained in RPMI 1640 complete medium at 37 degrees C in 5% CO2 in air. Cells were resuspended at 1x10(6) cells in 0.2 ml complete medium in 1.5 ml eppendorf tubes. A single saturating concentration 1x10(-9) M of [3H]5alpha-DHT was added to the cells suspension. Unlabelled steroids (5alpha-DHT, 17-beta estradiol, or the synthetic glucocorticoid triamcinolone acetonid) were added over the range 1x10(-8) to 1x10(-9) M. Duplicate tubes were incubated at 37 degrees C for 1h. For autoradiography, the supernatant was discarded and the pellet resuspended in 0.2 ml medium. For binding assay, Labeled cells were separated from unbound steroid by immunomagnetic bead using anti-CD68 antibody. RESULTS: In autoradiography, a population of approximately 96% of J111 cells that contain receptors for androgen has been demonstrated. The results of immunomagnetic showed that binding identified in the J111 cells was modest selective towards androgenic compounds. Schatchard analysis of data showed the KD value of 2.5x10(-9) M and the number of receptor in each cell was found to be 257+/-1. Little competition was seen from 17 beta estradiol or the synthetic glucocorticoid triamcinolone acetonid. CONCLUSIONS: These data indicate that androgen binding in J111 cells is of modest affinity and specific, due to the inability of 100-fold molar excess of estradiol to displace bound [3H]-5alphaDHT.

Macrophage may responses to androgen via its receptor.

BACKGROUND: Sex hormones have profound effects on immune responses and may influence the disease which caused by intracellular parasite(Leishmania) and bacterial (tuberculosis)and also autoimmune disease such as rheumatoid arthritis (RA). It has also been demonstrated that 5alpha-Dihydrotestosterone (5alpha-DHT) modulate nitric oxide and cytokine release by macrophages. These effects seem to be exerted by specific receptors for androgen in macrophages. MATERIAL/METHODS: Protein secretion: The effect of 5alpha-DHT on protein secretion by peritoneal macrophages of NZB\BALBc mice was investigated using radiolabelled protein secretion following SDS-PAGE and Fluorography. Binding assay: Androgen binding was also investigated using an autoradiography method. Peritoneal macrophages were treated with [3H]- 5alpha-DHT and incubated for 2 h before smearing on to microscope slides. Slides were air dried, dipped in Kodak NTB photographic emulsion, sealed in light proof boxes and left at 4 degrees C for 6 weeks. RESULTS: The results showed that protein secretion by macrophages changed under 5alpha-DHT treatment. Analysis of the data according to quantitation of [(3)H]-5alphaDHT binding receptors in fixed-slide mounted cells, identified a high specific androgen binding at physiological concentration. The receptors had a relatively high affinity for the 5alpha-DHT, So that binding affinity was not inhibited in the presence of 100-fold excess of non labelled 17-beta Estradiol. CONCLUSIONS: These results suggest that the immunosuppressive action exerted by androgen is at least partially achieved through a direct influence on macrophages.