A novel probiotic strain exerts therapeutic effects on mouse model of multiple sclerosis by altering the expression of inflammasome and IDO genes and modulation of T helper cytokine profile.

Multiple sclerosis is an inflammatory demyelinating disease that commences to neuronal cell destruction. Recently, a promising evidence of synergic effects of combined supplementation with vitamin D and probiotics in modulating the gut microbiota and metabolome is emerging. Bacillus Coagulans IBRC-M10791 as a novel strain was chosen, prevention and treatment impacts of regular administered were studied in Cuprizone-induced C57bl/6 mouse of demyelination. The mice were divided into six groups and received a daily dose of cuprizone or probiotics. To investigate the effect of probiotic, the IDO-1, CYP27B1, NLRP1, NLRP3, and AIM2 expression were estimated by Real-Time PCR, and IL-4, IL-17, IFN-gamma, and TGF-beta cytokines were measured by ELISA. The results showed that there was significant decrease in IL-17 and IFN-γ and modulatory effects on IL-4 and TGF-ß. On the other hand, we demonstrated that there are significant decrease for expression of IDO-1, CYP27b1, NLRP1, NLRP3 and AIM2 genes in prevention and treatment groups compared to cuprizone group. Also, a significant enhancement in rate of remyelination and alternations proved by LFB staining and Y-Maze test. In conclusion, our study provides insight into how the therapeutic effect of the chosen strain of probiotic was correlated with the modulation of the level of inflammatory and anti-inflammatory cytokines. Further, we demonstrated that the expression of genes related to Tryptophan, Vitamin D and Inflammasome pathways could be affected by B.coagulans. Our study could be beneficial to provide a novel Co-therapeutic strategy for Multiple sclerosis.

Synthesis of pH-sensitive hyaluronic acid nanogels loaded with paclitaxel and interferon gamma: Characterization and effect on the A549 lung carcinoma cell line.

Lung cancer is one of the deadliest cancers in various populations. Apart from the effects that anticancer drugs such as paclitaxel (PTX) have on cancer cells, they also have many side effects on healthy cells. Interferon gamma (IFN-γ) is also one of the cytokines used in the treatment of cancer. Current research is focused on providing new drug carriers to find new therapeutic goals. After synthesis of nanogels and loading of PTX and IFN-γ, the cytotoxicity of these nanogels on A549 and HEK293 healthy cell line was measured by MTT assay and flow cytometry analysis. Finally, the expression of STAT1 gene was investigated using Real time-PCR. The results of MTT assay showed that the survival rate of healthy cells treated with PTX and IFN-γ-loaded nanogels was 2.15 and 2.39 times higher than cancer cells, respectively. The results also showed that the gene expression STAT1 in A549 cells exposed to these nanogels was higher than healthy cells (p < 0.05). Based on flow cytometry results, the death rate of healthy cells treated with the mentioned nanogels was lower than cancer cells (p < 0.05). Therefore, Studies showed that synthesized nanogels have positive effects on cancer cells and also have fewer side effects on healthy cells.

Analysis of inflammasomes and CYP27B1 genes in cuprizone demyelinated C57BL/6 mice and evaluation of Th1 and Th2 patterns after oral administration of Lactobacillus casei strain T2 (IBRC-M10783).

Multiple sclerosis is characterized by the destruction of myelin in the CNS. Various factors including genetics, epigenetics, and environmental factors are involved in the development of the disease. There is evidence that changes in the gut microbiome profile are associated with immune-related diseases such as MS. Probiotics can alter the composition of the gut microbiota on the mucosal surfaces by differentiating naive T cells into Th1, Th2, Th17, and Treg cells. Female C57BL/6 mice were divided into 6 groups (n = 7): Normal group, cuprizone group (gavage of cuprizone for 4 weeks), Probiotic group (gavage of probiotic for 4 weeks), Treatment1 group (Probiotic for 4 weeks and then cuprizone for 4 weeks), treatment2 group (cuprizone for 4 weeks and then probiotic for 4 weeks) and treatment3 group (cuprizone for 4 weeks and then probiotic for 4 weeks with vitamin D3). Then the expression of NLRP-1, NLRP-3, AIM2, and CYP27B1 genes were evaluated using Real-Time PCR, and serum levels of IFN-γ and IL-4 were also measured by ELISA.The results showed a significant decrease in the expression of inflammasome and CYP27B1 genes in the probiotic-treated groups compared to the cuprizone group. Also, the comparison between probiotic-treated groups and cuprizone group showed a significant decrease in the amount of IFN-γ and IL-4. Due to reduced expression of the inflammasome genes as well as the decrease in IFN-γ levels as an inflammatory cytokine, it appears that L. casei may be effective in the healing process of demyelinated mice.

Effects of Lactobacillus casei Strain T2 (IBRC-M10783) on the Modulation of Th17/Treg and Evaluation of miR-155, miR-25, and IDO-1 Expression in a Cuprizone-Induced C57BL/6 Mouse Model of Demyelination.

Multiple sclerosis (MS) is a complex inflammatory disease in which demyelination occurs in the central nervous system affecting approximately 2.5 million people worldwide. Recent reports have shown that the gut microbiome plays a crucial role in the functioning of the immune system in inflammatory diseases such as MS. In this study, the cuprizone-induced demyelination mouse model was used to investigate the effect of Lactobacillus casei strain T2 (IBRC-M10783) on the alleviation of these mice. Female C57BL/6 mice (8-10 weeks old) were divided into 6 groups: group 1, normal control; group 2, cuprizone control (oral administration of cuprizone 0.2% w/w for 4 weeks); group 3, probiotic control (oral administration of 1 × 109 CFU/ml probiotic for 4 weeks); group 4, treatment 1 (probiotic for 4 weeks then cuprizone for 4 weeks); group 5, treatment 2 (cuprizone for 4 weeks then probiotic for 4 weeks); and group 6, treatment 3 (cuprizone for 4 weeks then probiotic for 4 weeks with vitamin D3 at a dose of 20 IU/day). Then, TGF-ß and IL-17 were measured by ELISA, and the expression of miR-155, miR-25, and IDO-1 was evaluated by real-time PCR. Among the measured microRNAs, the results showed that there was a significant decrease in miR-155 expression between the treatment 1 group and the cuprizone group. In the case of IL-17, the results also showed a significant reduction between the three treatment groups and the cuprizone group. These observations suggest that L. casei can reduce proinflammatory cytokines and reduce demyelinating symptoms in the mouse model.

Using response surface methodology in combination with Plackett-Burman design for optimization of culture media and extracellular expression of Trichoderma reesei synthetic endoglucanase II in Escherichia coli.

Cellulases like endoglucanase II (EGII) from Trichoderma reesei are the industrial enzymes responsible for breakdown of cellulosic materials. Due to its importance for production of eco-friendly commercial products such as alternative biofuels, industrial EGII production and optimization of its production conditions merit consideration. The gene responsible for EGII expression was designed and sub-cloned in to pET26b expression vector and transformed into BL21 (DE3) pLysS cells. Protein expression and purification was followed by a RSM design (20 experiments) to optimize the IPTG Concentration, post induction period and cell density (OD600). Thereafter, another RSM design (20 experiments) was performed to find and optimize the most important permeabilizing factors to achieve higher extracellular EGII expression. The EGII expression levels were assessed by Ghose method. The EGII gene was sub-cloned and protein expression and purification were successfully performed. The RSM experiments indicated that 0.331 mM for IPTG Concentration, 10.89 H for post induction period and 3.41 for cell density (OD600) were the optimum culture. Glycine (0.99%), Triton X-100 (0.73%) and CaCl2 (0.232) have been assigned as the most effective membrane permeabalizing factors. Optimization of culture medium components has led to a 3.06 fold increase in extracellular expression of EGII. RSM is an amenable method to optimize the expression of commercially significant enzymes. Our results indicated that optimization of IPTG concentration, post induction period and cell density along with glycine, Triton X-100 and Ca2+ concentration could lead to more cost effective industrial production of EGII.

Comparing Serum Levels of Th17 and Treg Cytokines in Women with Unexplained Recurrent Spontaneous Abortion and Fertile Women.

BACKGROUND: Increased evidences have shown that unexplained recurrent spontaneous abortion (URSA) is associated with inflammatory responses and breakage of immunological autotolerance. Therefore, the balance between Th17 and Treg cells may elucidate the pathophysiology of URSA. OBJECTIVE: To investigate the serum concentration of regulatory and inflammatory cytokines associated with Treg and Th17 in both normal and URSA females. METHODS: Forty-six women with URSA and 28 non-pregnant control women with at least one successful pregnancy were included. Serum was obtained from both groups and stored at -7º C. The serum concentrations of IL-17, IL-21, IL-22, IL-10, and TGF-ß were quantitatively determined by ELISA. RESULTS: The levels of IL-17, IL-21, and IL-22 in sera were significantly higher (P<0.001, P=0.01 and P<0.001, respectively) and TGF-ß serum concentration was significantly lower (P=0.02) in URSA women compared with normal controls. CONCLUSION: Our results suggest that enhancement in Th17-associated cytokine levels and reduction in TGF-ß may be one of the factors involved in URSA.

Green synthesis of silver nanoparticles using Anthemis atropatana extract: characterization and in vitro biological activities.

This study aim to synthesize silver nanoparticles (AgNPs) using Anthemis atropatana extract and to evaluate their chemical characteristics and antimicrobial and cytotoxic effects. The biosynthesis of AgNPs is verified using UV-visible spectrum which showing maximum absorption in 430 nm wavelength. Transmission electron microscope (TEM) and scanning electron microscope (SEM) results revealed that AgNPs has a spherical shape with an average size of 38.89 nanometres. The crystalline structure of green synthesized AgNPs in optimal conditions was confirmed by XRD analysis. The pattern of XRD peaks related to face-centred cubic (fcc) (111), (200), (220), (311) and (222) observed. Also, FTIR results verified the AgNPs synthesis using plant extract. In biological tests, the MTT results indicate the dose dependence of cytotoxic effects of AgNPs on colon cancer cell lines (HT29). The AgNPs had maximum cytotoxicity on HT29 cancer cell line at 100 µg/ml concentration, which were statistically significant comparing control cells (p < .001). Moreover, real time PCR and flow cytometry results confirmed the apoptotic effects of AgNPs. According to the results, it seems that the green synthesis of AgNPs is an eco-friendly and cost effective approach. This research provides insight into the development of new anticancer and antibacterial agents.

The Effect of Licorice Protein Fractions on Proliferation and Apoptosis of Gastrointestinal Cancer Cell Lines.

BACKGROUND: Several methods for the treatment of colon cancer have been introduced, but none of them are safe and effective. We planned to evaluate the inhibitory effect of protein extract of licorice root on HT-29 and CT26 cell lines proliferation and apoptosis. METHODS: Protein extract of licorice root was prepared in phosphate-buffered solution, and SDS-PAGE was used to isolate its fractions. HT-29, CT-26, and HEK293 cell lines were treated with various concentrations of the fractions and full extract of licorice. Cytotoxicity of licorice at various concentrations was assessed using MTT assay. Flow cytometry analysis was applied to evaluate the apoptosis. RESULTS: Our results demonstrated that the concentrations of 5 µg/mL from 25 to 33 kDa fraction and concentration of 8 µg/mL from 62 kDa fraction had a significant inhibitory effect on both cancerous cell lines (P < 0.05), with no significant effect on the noncancerous cell line. The concentrations of 50 and 100 µg/mL from full extracts significantly increased apoptosis in CT26 cells [35.52 ± 7.5 (P = 0.048*) and 47.72 ± 8 (P 0.026*), respectively], but not in HT29 and noncancerous cell lines. CONCLUSIONS: Protein compounds of licorice showed anticancer properties and were able to induce apoptosis in both human colon cancer and mouse colon carcinoma cell lines.

The Effect of IL-22 and IL-28 in Induction of Type 1 Regulatory T (Tr1) Cells.

Cytokines have been introduced as critical inducers in the development of Th subpopulations.Cytokines like IL-10 are involved in inducing regulatory T cells such as Type 1 regulatory T (Tr1) cells cells. IL-22 is a member of IL-10 family of cytokines, and IL-28A is a member of IFN-γ family. In this study, cord blood mononuclear cells (CBMC) from normal healthy individuals were isolated by Ficoll and then naïve T cells were purified by CD4+CD25+ Regulatory T cell Isolation kit. The effect of these two cytokines on production of IL-5, TGF-ß, IL-10, IL-4 and IFN-γ cytokines from cord blood T cells was investigated to identify Tr1 cells as well as Th1 and Th2 polarization. Flow cytometric analysis showed that IL-28A and IL-22 were not effective in expression of IL-5 and TGF-ß either alone or in synergy, but in view of IL-10, IL-4 and IFN-γ, the results showed that IL-22 increased IL-10 and IL-4 but had a decreasing effect on IFN-γ. The results showed that IL-28A was not effective in increasing or decreasing the level of IL-10, IL-4 and IFN-γ. Therefore, according to these results, IL-22 and IL-28A were not effective in inducing Tr1 cells.

Evaluation of the effect of IL-22 on human cord blood CD4+ T cells.

IL-22 is a member of IL-10 cytokine family which is believed to play an important role in inflammatory responses. IL-22 has similarities with IL-10 including conserved sequences with IL-10. IL-22 receptor is also comprised of two chains known as L-22R1 and L-10R2; supporting the speculation that the two cytokines may have similar effects. The aim of this study was to shed some light on the biological activity of IL-22 upon the cord blood CD4+CD25- T cells. In this research, cord blood T CD4+CD25- cells were cultured in presence of anti CD2/CD3/CD28 coated beads, IL-2 and IL-22 for two weeks at 37 degrees C and 5% CO2. Flow cytometry analysis showed that IL-22 has no effect upon CD25 and Foxp3 expression. Also, the results indicated that IL-22 is not involved in CD4+ T cell proliferation. Moreover, the results of suppression assay did not show any suppression effect on the cultured T cells. Thus, it seems that umbilical cord blood T cells probably do not express IL-22R1 on their surface.

The effect of IL-28A on human cord blood CD4+ T cells.

BACKGROUND AND AIM: The utilization of umbilical cord blood transplantation (UCBT) has been increasing because of the potential advantage of rapid accessibility and the lesser risk of graft-versus-host disease (GVHD), thus allowing less strict HLA matching. IL-28A, also known as IFN-lambda2, has been regarded as a member of a new cytokine family that shares some features with type I interferon (IFN) and was shown to have antiviral activity. The aim of this study was to identify biological activity of IL-28 on cord blood CD4(+) T cells. MATERIALS AND METHODS: In this study, we cultured CD4(+) T cells with IL-28A (20 ng/ml), IL-2 (20 ng/ml) and 5microg/ml MACS Anti-Biotin MACSiBead Particles (bead-to-cell ratio 1:2) for 2 weeks. RESULTS: Flow cytometry analyses showed that IL-28A cannot be effective on CD25 and Foxp3 expression on cord blood CD4(+) T cells, and it is not involved in proliferation of these cells. Treg suppression assay also showed that this cytokine cannot induce production of regulatory T cells. CONCLUSION: We showed that IL-28A is not involved in expression of CD25 and Foxp3 markers and proliferation of CD4(+)CD25(-) T cells, and that our findings also suggest that induction of Foxp3 in T cells activated by anti-CD3/anti-CD28 does not result in the regulatory activity in these cells.