Evaluation of the immunomodulatory effect of the 14 kDa protein isolated from aged garlic extract on dendritic cells.

Garlic is used all over the world for treatment of different diseases. A wide range of biological activities of garlic has been verified in vitro and in vivo. One of major proteins of garlic which has been isolated and purified is the 14 kDa protein. This protein has been shown to have immunomodulatory effects. In this study, the effect of the 14 kDa protein isolated from aged garlic extract (AGE) was investigated on maturation and immunomodulatory activity of dendritic cells (DC). Proteins were purified from AGE by biochemical method; the semi-purified 14 kDa protein was run on gel filtration Sephadex G50 and its purity was checked by SDS-PAGE. DC were isolated from spleen of BALB/c mice by Nycodenz centrifugation and their adhesiveness to plastic dish. 14 kDa protein from AGE was added to overnight culture of DC medium and the expression percentage of CD40, CD86, and MHC-II was evaluated by flowcytometric analysis. Also, proliferation of T-cells was measured by allogenic mixed lymphocyte reaction (MLR) test. The purified 14 kDa protein isolated from AGE increased the expression of CD40 molecule on DC, but it did not influence CD86 and MHCII molecules. Furthermore, no significant differences were noticed in the pulsed-DC with 14 kDa protein and non-pulsed DC on the MLR.

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

INTRODUCTION: Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. MATERIALS AND METHODS: 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. RESULTS: MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). CONCLUSION: according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

The expression of miR-17 and miR-29a in placenta-derived exosomes in LPS-induced abortion mice model: An experimental study.

BACKGROUND: The expression pattern of microRNAs in placenta-derived exosomes plays a crucial role in the regulation of immune responses and inflammation at the fetal-maternal interface. OBJECTIVE: Considering the immunomodulatory properties of miR-17 and miR-29a, we determined their expression levels in placenta-derived exosomes in a lipopolysaccharide (LPS)-induced abortion mice model. MATERIALS AND METHODS: A total of 14 pregnant BALB/c mice, aged 6-8 wk, were randomly divided into two groups (n = 7/each) on the gestational day 11.5. While the mice in the experimental group were treated with LPS, those in the control group were treated with Phosphate buffered saline; 5 hr after the treatment, the placental cells were isolated and cultured for 48 hr. Then, the cell culture supernatants were collected and used for isolation of exosomes. The isolated exosomes were confirmed by western blot and scanning electron microscopy. The miRNAs were then extracted from exosomes, and cDNA synthesized. The expression levels of miR-17 and miR-29a were evaluated by quantitative real-time PCR analysis. RESULTS: Our results showed that the expression levels of miR-29a in placenta-derived exosomes obtained from the experimental group increased significantly compared to the control group. Also, the expression levels of miR-17 in the placenta-derived exosomes obtained from the experimental group were found to decrease; however, it did not show significant changes compared with the control group (p > 0.05). CONCLUSION: Inflammatory reactions at the fetal-maternal interface can alter miRNAs expression patterns in placenta-derived exosomes, especially miRNAs with immunomodulatory effects such as miR-29a.

Decidual soluble factors, through modulation of dendritic cells functions, determine the immune response patterns at the feto-maternal interface.

Dendritic cells (DCs) can acquire immunogenic or tolerogenic properties depending on intrinsic and tissue environmental factors. We aimed to determine the immunomodulatory effects of decidual soluble factors from abortion- and non-abortion-prone mice on DC functions. The decidual cell supernatants (DS) were obtained from abortion-prone and non-abortion-prone mice. Splenic DCs were treated with DS and conalbumin (as an antigen) and injected into the palms of the mice. After five days, regional lymph node cells were collected and cultured in the presence and absence of conalbumin. The proliferation of lymphocyte cells, the frequency of regulatory T cells (Tregs), and the production of IL-4 and IFN-γ were measured by [(3)H]thymidine incorporation, flow cytometry, and ELISA respectively. Our results indicated that DS from both abortion- and non-abortion-prone mice decreased the ability of DCs to induce lymphocyte proliferation and IFN-γ production, while enhanced their capacity to induce Tregs compared with non-treated DCs. Another important finding was that the immunosuppressive effects of DS from abortion-prone mice on DCs for inducing proliferative responses, developing Tregs, and producing IFN-γ by primed lymphocytes was less than DS from non-abortion-prone mice. We also found that only DS from non-abortion-prone mice could enhance the capacity of DCs to induce IL-4 production by primed lymphocytes. It can be concluded that decidua-secreted factors, by altering DC functions, can determine the pattern of immune responses at the fetomaternal interface and, subsequently, pregnancy outcome.

The immunomodulatory effects of abortion-prone mice decidual and serum soluble factors on macrophages and splenocytes.

OBJECTIVES: To determine the immune status of the abortion mouse model at the feto-maternal interface and at the systemic level simultaneously. STUDY DESIGN: Mid-pregnancy serum and decidual cell supernatants (DS) were obtained from abortion and non-abortion mouse models. The effect of serum and DS on PHA or LPS-induced lymphocyte proliferation was investigated by MTT reduction assay. Treated macrophages and LPS-stimulated macrophages were evaluated for viability and also for nitric oxide (NO) production by Griess reagent. RESULTS: Our results showed that DS from the abortion mouse model significantly decreased LPS-stimulated splenocyte proliferation, and increased proliferation in PHA-stimulated splenocytes, compared with that in the non-abortion mouse model. Proliferation assays for mid-pregnancy serum were the same on LPS- and PHA-stimulated splenocytes. NO production was decreased by non-abortion DS, similar to that observed for serum treatment in LPS-stimulated macrophages in abortion mice. CONCLUSIONS: These findings suggest that in the abortion mouse model, soluble factors within the decidua are more effective than serum soluble factors in altering immune responses that may be involved in the complex process of fetal rejection.