Immunoregulatory Effects of the Active Form of Vitamin D (Calcitriol), Individually and in Combination with Curcumin, on Peripheral Blood Mononuclear Cells (PBMCs) of Multiple Sclerosis (MS) Patients.

OBJECTIVES: Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system. Immune cell subsets, notably T helper (Th) 17 and Th1, exert important roles in MS pathogenesis. Whereas, Treg cells modulate the disease process. Calcitriol, the active form of vitamin D, and curcumin, a bioactive compound derived from turmeric, play immunomodulatory effects relevant to autoimmune disorders, including MS. The objective of this study is to investigate the effects of calcitriol and Curcumin on Peripheral blood mononuclear cells (PBMCs) of individuals with MS. METHODS: PBMCs from twenty MS patients were isolated, cultured, and exposed to 0.004 µg/mL of calcitriol and 10 µg/mL of curcumin. The cells underwent treatment with singular or combined doses of these components to assess potential cumulative or synergistic immunomod-ulatory effects. Following treatment, the expression levels of genes and the cellular population of Treg, Th1 and Th17 were evaluated using Real-time PCR and flow cytometry. RESULTS: Treatment with curcumin and calcitriol led to a significant reduction in the expression levels of inflammatory cytokines and transcription factors related to Th1 and Th17 cells, includ-ing IFN-γ, T-bet, IL-17, and RORC. Furthermore, the frequency of these cells decreased follow-ing treatment. Additionally, curcumin and calcitriol treatment resulted in a significant upregu-lation of the FOXP3 gene expression and an increase in the frequency of Treg cells. CONCLUSION: This study demonstrates that curcumin and calcitriol can effectively modulate the inflammatory processes intrinsic to MS by mitigating the expression of inflammatory cytokines by Th1 and Th17 cells while concurrently enhancing the regulatory role of Treg cells. Moreover, the combined treatment of curcumin and calcitriol did not yield superior outcomes compared to single-dosing strategies.

Alterations in CD4+ T Cell Cytokines Profile in Female Patients with Hashimoto's Thyroiditis Following Vitamin D Supplementation: A Double-blind, Randomized Clinical Trial.

BACKGROUND: Hashimoto's thyroiditis (HT) is an autoimmune disease characterized by the destruction of thyroid cells through immune processes involving T helper (Th)1 cytokines. This clinical trial investigates the impact of vitamin D supplementation on serum cytokine levels and gene expression in CD4+ T cells from HT patients, aiming to understand its effects on Th-1, Th-2, Th-17, and regulatory T (Treg) cell-associated factors. METHODS: Female patients were randomly assigned in a double-blind design to either a vitamin D-supplemented group, which received cholecalciferol [1, 25(OH)2D3] at a dose of 50,000 IU, or the placebo group, which received a weekly placebo for a duration of three months. Serum cytokine levels were assessed using enzyme-linked immunosorbent assay (ELISA), while genes' expression levels were measured using real-time PCR. RESULTS: Serum 25-hydroxyvitamin D and levels exhibited a significant increase following vitamin D supplementation, in comparison to the placebo group. Additionally, the vitamin D supplementation resulted in a significant elevation of serum calcium (Ca) levels compared to baseline. In the vitamin D group, there was a significant decrease in both serum levels and expression of the interleukin (IL)-17 gene when compared to baseline, although no statistical difference was observed between the placebo and vitamin D groups. The gene expression of transforming growth factor-beta (TGFß) was significantly increased in the vitamin D group compared to baseline, with no significant difference between the two study groups. Vitamin D treatment had no effect on serum levels of interferon-gamma (IFNϒ) and IL-4. While the gene expression of IL-4 in the vitamin D group did not exhibit a statistically significant increase, the level of GATA3 transcription factor increased significantly when compared to the placebo group. The expression of IFNϒ and transcription factors, T-bet, RORc, and forkhead box protein 3 (FOXP3) in genes did not show significant changes following vitamin D supplementation. CONCLUSION: The findings suggest that vitamin D supplementation may hold potential benefits for autoimmune diseases, such as HT. However, further longitudinal clinical trials are necessary to gain a more comprehensive understanding of the specific effects of vitamin D on HT. CLINICAL TRIAL REGISTRATION NUMBER: IRCT2016110130644N1.

Immunoregulatory Effect of Calcitriol on Experimental Autoimmune Encephalomyelitis (EAE) Mice.

Previous studies noted an imbalance in T helper (Th) 17 and regulatory T cells (Tregs) in experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis animal model. calcitriol, vitamin D's active form, was found to ameliorate EAE symptoms by favoring Tregss over Th17 cells, suggesting immunomodulatory effects. This study aimed to assess calcitriol's impact on EAE manifestations and cytokine profile in mice. In this study, we recruited twenty-eight C57BL/6 mice and divided them into 4 groups: healthy controls, EAE, EAE with calcitriol treatment, and healthy mice with calcitriol treatment. CD4+ T cells were isolated from splenocytes using magnetic-activated cell sorting. Real-time polymerase chain reaction was employed to quantify the genes associated with Th9 cells (i.e., SPI1 encoding PU.1 and IL9 encoding interleukin [IL]-9). Moreover, the levels of IL-17 and transforming growth factor beta (TGF-ß) were evaluated through enzyme-linked immunosorbent assay in the supernatant of CD4+ T cell culture stimulated by anti-CD3 and anti-CD28 antibodies for 72 hours. In the supernatant of CD4+ T cell cultures, the levels of interleukin-17 (IL-17) were significantly increased, while the levels of transforming growth factor beta (TGF-ß) were decreased in the EAE Group compared to the healthy control group. Calcitriol treatment reversed these changes and attenuated EAE symptoms, as confirmed in hematoxylin and eosin, and luxol fast blue stains. Notably, calcitriol increased IL9 gene expression in both EAE and healthy mice.  This study provides further evidence of the anti-inflammatory effects of calcitriol and its role in attenuating EAE.

Secretome-based acellular therapy of bone marrow-derived mesenchymal stem cells in degenerative and immunological disorders: A narrative review.

The bone marrow (BM) plays a pivotal role in homeostasis by supporting hematopoiesis and immune cells' activation, maturation, interaction, and deployment. "BMSC-derived secretome" refers to the complete repertoire of secreted molecules, including nucleic acids, chemokines, growth factors, cytokines, and lipids from BM-derived mesenchymal stem cells (BMSCs). BMSC-derived secretomes are the current molecular platform for acellular therapy. Secretomes are highly manipulable and can be synthesised in vast quantities using commercially accessible cell lines in the laboratory. Secretomes are less likely to elicit an immunological response because they contain fewer surface proteins. Moreover, the delivery of BMSC-derived secretomes has been shown in numerous studies to be an effective, cell-free therapy method for alleviating the symptoms of inflammatory and degenerative diseases. As a result, secretome delivery from BMSCs has the same therapeutic effects as BMSCs transplantation but may have fewer adverse effects. Additionally, BMSCs' secretome has therapeutic promise for organoids and parabiosis studies. This review focuses on recent advances in secretome-based cell-free therapy, including its manipulation, isolation, characterisation, and delivery systems. The diverse bioactive molecules of secretomes that successfully treat inflammatory and degenerative diseases of the musculoskeletal, cardiovascular, nervous, respiratory, reproductive, gastrointestinal, and anti-ageing systems were also examined in this review. However, secretome-based therapy has some unfavourable side effects that may restrict its uses. Some of the adverse effects of this modal therapy were briefly mentioned in this review.

Effect of curcumin on the expression of NOD2 receptor and pro-inflammatory cytokines in fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients.

BACKGROUND: Previous studies has shown that nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) is expressed in Fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients which is stimulated by muramyl dipeptide (MDP) present in the joint environment and induces inflammation via the NF-κB pathway. Also, other studies have shown that curcumin inhibits proliferation, migration, invasion, and Inflammation and on the other hand increases the apoptosis of RA FLSs. In this study, we aim to evaluate the effect of curcumin, a natural anti-inflammatory micronutrient, on the expression of NOD2 and inflammatory cytokines. METHODS: Synovial membranes were collected from ten patients diagnosed with RA and ten individuals with traumatic injuries scheduled for knee surgery. The FLSs were isolated and treated with 40 µM curcumin alone or in combination with 20.3 µM MDP for 24 h. mRNA was extracted, and real-time PCR was performed to quantitatively measure gene expression levels of NOD2, p65, IL-6, TNF-α, and IL-1ß. RESULTS: The study findings indicate that administering MDP alone can significantly increase the mRNA expression levels of IL-6 and IL-1ß in the trauma group and TNF-α in the RA group. Conversely, administering curcumin alone or in combination whit MDP can significantly reduce mRNA expression levels of P65 and IL-6 in FLSs of both groups. Moreover, in FLSs of RA patients, a single curcumin treatment leads to a significant reduction in NOD2 gene expression. CONCLUSION: This study provides preliminary in vitro evidence of the potential benefits of curcumin as a nutritional supplement for RA patients. Despite the limitations of the study being an investigation of the FLSs of RA patients, the results demonstrate that curcumin has an anti-inflammatory effect on NOD2 and NF-κB genes. These findings suggest that curcumin could be a promising approach to relieve symptoms of RA.

Vitamins A and D Enhance the Expression of Ror-γ-Targeting miRNAs in a Mouse Model of Multiple Sclerosis.

Autoreactive T cells, particularly those characterized by a Th17 phenotype, exert significant influence on the pathogenesis of multiple sclerosis (MS). The present study aimed to elucidate the impact of individual and combined administration of vitamin A and D on neuroinflammation, and microRNAs (miRNAs) involved in T helper (Th)17 development, utilizing a murine model of experimental autoimmune encephalomyelitis (EAE). EAE was induced in C57BL/6 mice, and 3 days prior to immunization, intraperitoneal injections of vitamins A and D or their combination were administered. Th17 cell percentages were determined in splenocytes utilizing intracellular staining and flow cytometry. Furthermore, the expression of Ror γ-t, miR-98-5p and Let-7a-5p, was measured in both splenocytes and spinal cord tissues using RT-PCR. Treatment with vitamin A and D resulted in a reduction in both disease severity in EAE mice. Treated mice showed a decreased frequency of Th17 cells and lower expression levels of IL17 and Ror γ-t in splenocytes and spinal cord. The spinal cord tissues and splenocytes of mice treated with vitamins A, D, and combined A+D showed a significant upregulation of miR-98-5p and Let-7a-5p compared to the EAE group. Statistical analysis indicated a strong negative correlation between miR-98-5p and Let-7a-5p levels in splenocytes and Ror-t expression. Our findings indicate that the administration of vitamins A and D exerts a suppressive effect on neuroinflammation in EAE that is associated with a reduction in the differentiation of T cells into the Th17 phenotype and is mediated by the upregulation of miR-98-5p and Let-7a-5p, which target the Ror γ-t.

Immunosuppressive Effects of Two Probiotics, Lactobacillus paracasei DSM 13434 and Lactobacillus plantarum DSM 15312, on CD4+ T Cells of Multiple Sclerosis Patients.

T cells play an important role in the development and progression of multiple sclerosis (MS), an autoimmune disease of the central nervous system. In the present study, the immunomodulatory impacts of two Lactobacillus strains, L paracasei DSM 13434 and L plantarum DSM 15312, on the frequency and cytokine production of CD4+ T cells in MS patients were explored. Thirty MS patients were enrolled in this study. The CD4+ T cells were isolated, cultured, and exposed to the media containing cell-free supernatants of L plantarum (group1), L paracasei (group 2), the mixture group of cell-free supernatants of both probiotics (group 3), and vehicle (control) group (group 4). The frequencies of T helper (Th) 1, Th17, Th2, and T regulatory type 1 (Tr1) cells and mean fluorescent intensity (MFI) of the associated cytokines were assessed using flow cytometry. The levels of interleukin 17 (IL-17), transforming growth factor ß (TGF-ß), and interferon-gamma (IFN-γ) cytokines in supernatants of all groups were measured by enzyme-linked immunosorbent assay. The percentage of Th1 cells and the MFI of IFN-γ in Th1 cells (CD4+ IFN-γ+) in all three probiotic treatment groups were significantly decreased compared to the control group. However, no significant changes were observed in the proportion and MFI of Th2, Th17, and Tr1 cells. A significant decrease was observed in IL-17 secretion in the supernatant of cultured CD4+ T cells in all three treatment groups in comparison with control. The levels of TGF-ß and IFN-γ were not significantly different among any of the study groups.  Collectively, cell-free supernatants of the lactobacilli showed an in vitro anti-inflammatory effect. However, further studies are needed to prove the real effects of probiotics on MS.

The immunomodulatory effects of all-trans retinoic acid and docosahexaenoic acid combination treatment on the expression of IL-2, IL-4, T-bet, and GATA3 genes in PBMCs of multiple sclerosis patients.

OBJECTIVES: Multiple sclerosis (MS) is a potentially disabling autoimmune disease of the central nervous system. Neither the pathogenesis nor the effectiveness of treatment of MS has been fully understood. This in vitro trial evaluated the beneficial immunomodulatory effects of single and combined treatments of all-trans retinoic acid (ATRA) and docosahexaenoic acid (DHA) on the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting MS (RRMS) patients who were receiving interferon beta (IFN-ß). METHODS: The PBMCs of 15 RRMS patients were isolated, cultured, and treated with single and combined treatments of ATRA and DHA. The expressions of IL-2, IL-4, T-bet, and GATA3 genes were evaluated using real-time PCR. RESULTS: The results showed that a single treatment of ATRA could significantly suppress the gene expression of the pro-inflammatory cytokine, IL-2 (P < 0.05), and related transcription factor, T-bet (P < 0.001). The gene expression level of the anti-inflammatory cytokine, IL-4, and its transcription factor, GATA3, were not significantly changed. The expression of IL-2 and T-bet genes was significantly decreased in combination treatments of ATRA and DHA (P < 0.001). Significant suppression of IL-2 and T-bet (P < 0.001) was observed in ATRA and DHA combination therapy with half doses of their single treatment, which suggested a synergistic effect of these components. DISCUSSION: Co-administration of vitamin A and DHA, an omega-3 fatty acid derivative, may exert a synergistic effect in modulating the immune system in MS patients; however, more studies are needed to evaluate the exact effects and mechanism of their actions on the immune cells.

Effect of curcumin on the expression of NOD2 receptor and pro-inflammatory cytokines in fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients

Abstract Background Previous studies has shown that nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) is expressed in Fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients which is stimulated by muramyl dipeptide (MDP) present in the joint environment and induces inflammation via the NF-κB pathway. Also, other studies have shown that curcumin inhibits proliferation, migration, invasion, and Inflammation and on the other hand increases the apoptosis of RA FLSs. In this study, we aim to evaluate the effect of curcumin, a natural antiinflammatory micronutrient, on the expression of NOD2 and inflammatory cytokines. Methods Synovial membranes were collected from ten patients diagnosed with RA and ten individuals with traumatic injuries scheduled for knee surgery. The FLSs were isolated and treated with 40 μM curcumin alone or in combination with 20.3 μM MDP for 24 h. mRNA was extracted, and real-time PCR was performed to quantitatively measure gene expression levels of NOD2, p65, IL-6, TNF-α, and IL-1β. Results The study findings indicate that administering MDP alone can significantly increase the mRNA expression levels of IL-6 and IL-1β in the trauma group and TNF-α in the RA group. Conversely, administering curcumin alone or in combination whit MDP can significantly reduce mRNA expression levels of P65 and IL-6 in FLSs of both groups. Moreover, in FLSs of RA patients, a single curcumin treatment leads to a significant reduction in NOD2 gene expression. Conclusion This study provides preliminary in vitro evidence of the potential benefits of curcumin as a nutritional supplement for RA patients. Despite the limitations of the study being an investigation of the FLSs of RA patients, the results demonstrate that curcumin has an anti-inflammatory effect on NOD2 and NF-κB genes. These findings suggest that curcumin could be a promising approach to relieve symptoms of RA.

The Impact of Vitamin D Supplementation on the IFNγ-IP10 Axis in Women with Hashimoto's Thyroiditis Treated with Levothyroxine: A Double-blind Randomized Placebo-controlled Trial.

Hashimoto's thyroiditis (HT) results from chemoattraction of inflammatory cells toward the thyroid gland by inducing the production of interferon-gamma (IFNγ)-induced protein 10 (IP10) by T helper (Th) 1 cells. Vitamin D may suppress the IFNγ-IP10 axis, but this new function of vitamin D has not yet been investigated in HT patients. In an intervention and control group, patients received 50000 IU cholecalciferol or placebo every week for three months, respectively. The CD4+ T cells of 40 patients were isolated, and the mRNA expression levels of vitamin D receptor (VDR), peroxisome proliferator-activated receptors (PPAR)-α, and PPAR-γ genes were determined by real-time PCR. ELISA method was used to determine serum levels of vitamin D, tumor necrosis factor-alpha (TNF-α), IFN-γ, and IP10. Vitamin D levels in the intervention group were significantly higher than in the placebo group after supplementation. PPAR-α and PPAR-γ gene expression levels did not differ significantly between the two groups. The serum levels of IP10, IFNγ, and TNF-α decreased significantly in the vitamin D group, as well as in the placebo group.  During this study, vitamin D levels significantly increased in the intervention group and inflammatory factors decreased. Based on the similar results obtained in the placebo group, further studies with larger sample sizes and longer intervention times are recommended.

Influence of Vitamins A and D on the Expression of MicroRNA27-3p Isoforms and GATA3 in Experimental Autoimmune Encephalomyelitis.

Vitamins A, D, and microRNAs contribute to T cell differentiation into TH2 phenotypes. We investigated the molecular mechanisms and effects of vitamin A and D on the expression of GATA3 and miR-27-3p isoforms in experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein, mixed with Complete Freund's Adjuvant, together with injection of pertussis toxin. Treatments began one day before immunization with (200 µg and 100 ng of vitamin A and vitamin D per mouse, respectively, and vitamin A+D (100 µg+50 ng) per mouse. Expression levels of GATA3 and miR­27­3p isoforms were measured in the CNS and splenocytes by real-time RT-PCR. The expression level of GATA3 in the mice spinal cords and splenocytes was increased in the vitamin A and A+D-treated EAE mice at 24 h and 48 h after restimulation by 10 µg and 40 µg of myelin oligodendrocyte glycoprotein. Vitamins A and D and their combination upregulated the miR-27-3p isoforms compared with EAE mice with no treatments. We also demonstrated that miR-273p isoform expression was altered in splenocytes of vitamin-treated EAE mice. The results showed a positive correlation between splenocyte GATA3 levels and miR-27-3p isoform expression. The protective impacts of vitamins A and D in EAE mice may be mediated by the upregulation of GATA3. However, it is not specified whether suppression of GATA3-targeting miRNAs of the miR-27-3p family is involved in this effect. These results do not rule out the possibility that miR-27-3p isoforms might have beneficial effects by targeting other transcripts, such as GluA2 and NR2B.

Genome Analysis of an Enterococcal Prophage, Entfac.MY.

Background: Bacteriophages are bacterial parasites. Unlike lytic bacteriophages, lysogenic bacteriophages do not multiply immediately after entering the host cells and may integrate their genomes into the bacterial genomes as prophages. Prophages can include various phenotypic and genotypic effects on the host bacteria. Enterococcus spp. are Gram-positive bacteria that cause infections in humans and animals. In recent decades, these bacteria have become resistant to various antimicrobials, including vancomycin. The aim of this study was to analyze genome of an enterococcal prophage. Methods: In this study, Enterococcus faecium EntfacYE was isolated from biological samples and its genome was analyzed using next-generation sequencing method. Results: Overall, 254 prophage genes were identified in the bacterial genome. The prophage included 39 housekeeping, 41 replication and regulation, 80 structural and packaging, and 48 lysis genes. Moreover, 46 genes with unknown functions were identified. All genes were annotated in DNA Data Bank of Japan. Conclusion: In general, most prophage genes were linked to packaging and structure (31.5%) gene group. However, genes with unknown functions included a high proportion (18.11%), which indicated necessity of further analyses. Genomic analysis of the prophages can be effective in better understanding of their roles in development of bacterial resistance to antibiotics. Moreover, identification and study of prophages can help researchers develop genetic engineering tools and novel infection therapies.

The epidemiology and etiology of onychomycosis in 2 laboratory centers affiliated to Tehran university of medical sciences during 2019-2020.

Background and Objectives: Onychomycosis is caused by dermatophyte species, non-dermatophyte moulds (NDMs), and accounts for roughly 50% of all nail diseases. As the prevalence of onychomycosis is increasing, new epidemiologic documents may help with treatment and prevention. The present investigation aims to determine the epidemiological profile of onychomycosis in 2 mycology laboratories. Materials and Methods: A cross-sectional study conducted during eight months (2019-2020) on 169 patients with positive nail mycology tests referred to two mycological laboratory centers affiliated with Tehran University of Medical Science. The nail clippings were examined by direct smear and culture. Also, molecular assays were performed if needed. Results: 10% of nail lesions referred to Razi Hospital (RH), and 30% of nail lesions referred to TUMS mycology laboratory were positive. Middle age (40-60) suffer more from onychomycosis. Aspergillus flavus, Trichophyton mentagrophytes, and Candida albicans were the most common etiologic agents in each of the three main classes of fungi causing onychomycosis. Females were more infected. NDMs were the predominant etiologic agents, and toenails were the most common site of onychomycosis. Conclusion: The pattern of etiologic agents and clinical signs of onychomycosis differs according to geographical region and age, so repeated epidemiological surveys of onychomycosis seem to be fundamental.

Genome Analysis of the Enterococcus faecium Entfac.YE Prophage.

Background: Bacteriophages are viruses that infect bacteria. Bacteriophages are widely distributed in various environments. The prevalence of bacteriophages in water sources, especially wastewaters, is naturally high. These viruses affect evolution of most bacterial species. Bacteriophages are able to integrate their genomes into the chromosomes of their hosts as prophages and hence transfer resistance genes to the bacterial genomes. Enterococci are commensal bacteria that show high resistance to common antibiotics. For example, prevalence of vancomycin-resistant enterococci has increased within the last decades. Methods: Enterococcal isolates were isolated from clinical samples and morphological, phenotypical, biochemical, and molecular methods were used to identify and confirm their identity. Bacteriophages extracted from water sources were then applied to isolated Enterococcus faecium (E. faecium). In the next step, the bacterial genome was completely sequenced and the existing prophage genome in the bacterial genome was analyzed. Results: In this study, E. faecium EntfacYE was isolated from a clinical sample. The EntfacYE genome was analyzed and 88 prophage genes were identified. The prophage content included four housekeeping genes, 29 genes in the group of genes related to replication and regulation, 25 genes in the group of genes related to structure and packaging, and four genes belonging to the group of genes associated with lysis. Moreover, 26 genes were identified with unknown functions. Conclusion: In conclusion, genome analysis of prophages can lead to a better understanding of their roles in the rapid evolution of bacteria.

Mass spectrometry in research laboratories and clinical diagnostic: a new era in medical mycology.

Diagnosis by clinical mycology laboratory plays a critical role in patient care by providing definitive knowledge of the cause of infection and antimicrobial susceptibility data to physicians. Rapid diagnostic methods are likely to improve patient. Aggressive resuscitation bundles, adequate source control, and appropriate antibiotic therapy are cornerstones for success in the treatment of patients. Routine methods for identifying clinical specimen fungal pathogen are based on the cultivation on different media with the subsequent examination of its phenotypic characteristics comprising a combination of microscopic and colony morphologies. As some fungi cannot be readily identified using these methods, molecular diagnostic methods may be required. These methods are fast, but it can cost a lot. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs. It can be considered an alternative for conventional biochemical and molecular identification systems in a microbiological laboratory. The reliability and accuracy of this method have been scrutinized in many surveys and have been compared with several methods including sequencing and molecular methods. According to these findings, the reliability and accuracy of this method are very high and can be trusted. With all the benefits of this technique, the libraries of MALDI-TOF MS need to be strengthened to enhance its performance. This review provides an overview of the most recent research literature that has investigated the applications and usage of MT-MS to the identification of microorganisms, mycotoxins, antifungal susceptibility examination, and mycobiome research.

The fatty acid-binding protein (FABP) decreases the clinical signs and modulates immune responses in a mouse model of experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: An increasing body of studies has shown that Fasciola hepatica can affect immune responses. This study explored whether the fatty acid-binding protein (FABP) of F. hepatica can modulate the immune system in a mouse model of experimental autoimmune encephalomyelitis (EAE). METHODS: EAE-induced C57BL/6 mice were treated with vehicle, F. hepatica total extract (TE) or FABP. The clinical signs, body weights, and the expression of IFN-γ, T-bet, IL-4, GATA3, IL-17, RORγ, TGF-ß, FOXP3, IL-10, TNF-α genes and proteins were determined in the isolated CD4+ splenocytes. Besides, the percentage of Treg cells and degree of demyelination were evaluated. RESULTS: We found that TE and FABP treatments decreased the clinical scores, lymphocyte infiltration rate, and demyelinated plaques in EAE mice. The expressions of IL-4 and GATA3 were increased, whereas IL-17 and TNF-α were down-regulated. FABP did not affect the expression of IFN-γ, RORγ, IL-10, and TGF-ß genes or proteins but reduced the expression of T-bet. TE administration did not affect the expression of IL-10 and the Tbet genes, and increased the expression levels of IFN-γ and FOXP3 in CD4+ lymphocytes. Both FABP and TE treatment did not affect the Treg cell percentage. CONCLUSION: This study indicates that F. hepatica FABP and TE can suppress the inflammatory responses in EAE-induced mice and shift the immune system toward Th2 responses. However, FABP exerts stronger anti-inflammatory effects and seems to be more effective than TE for EAE treatment.

The Frequency of CD4+ T Cells in Women with Hashimoto's Thyroiditis.

BACKGROUND: Hashimoto's thyroiditis (HT) is the most prevalent autoimmune disease, and there is no definitive treatment available for this disease. To find the appropriate therapeutic approach, it is necessary to determine the mechanism of this disease. To achieve this purpose, the frequency of CD4+ T cells was evaluated in patients with HT and compared with healthy individuals. METHODS: Twenty-six female patients with HT, aged 20 - 45 years, enrolled in this study. Based on the level of thyroglobulin antibody (anti-TG) and anti-thyroid peroxidase antibody (anti-TPO) in serum of patients with HT, they were divided into two groups. The serum level of anti-TPO was above 100 IU/mL in the group 1 (n = 13), whereas the serum levels of both anti-TPO and anti-TG were above 100 IU/mL in the group 2 (n = 13). Eleven healthy women were considered control group, or group 3. Using flow cytometry, the frequency of T helper (Th)1, Th2, Th17, T regulatory type 1 (Tr1), and LT CD4+IL-4+IL-17+ cells and mean fluorescent intensity (MFI) of their related cytokines were evaluated. RESULTS: The frequency of Th2 cells in the groups 1 (anti-TPO > 100) and 2 (anti-TPO > 100 and anti-TG > 100) were more than control group. Only the difference between groups 3 (healthy control) and 2 was significant (P = 0.022). The frequency of LT CD4+IL-4+IL-17+ cells in the group 1 was significantly more than group 3 (P = 0.027); However, the difference between group 2 and 3 was not significant (P = 0.126). The expression of interferon-gamma (IFN-γ) in the group 2 (P = 0.001) and group 1 (P = 0.001) was significantly higher than group 3. The frequency of Th17, Th1, and Tr1 cells and MFI of IL-17 and IL-10 were not significantly different between the study groups. CONCLUSIONS: In the present study, no significant differences were observed in the frequency of Th17 and Tr1 cells and in MFI of IL-17 and IL-10 in comparison to healthy individuals. Therefore, trying to make a change in the population of these cells probably does not have a significant therapeutic effect. Since Th2 cells and the expression of IFN-γ increased in women with HT, reducing the frequency of Th2 cells or the expression of IFN-γ may be effective in controlling the disease progression. It may be helpful for these patients to prevent the progression of the disease.

The effect of calcitriol and all-trans retinoic acid on T-bet, IFN-γ, GATA3 and IL-4 genes expression in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease which affects the central nervous system (CNS). In the present study, the in vivo effects of ATRA, calcitriol, and their combinations on the expression of murine CD4+ T cell cytokines and their specific transcription factors in experimental autoimmune encephalomyelitis (EAE)-induced mice were explored. Thirty-two EAE induced inbred C57BL/6 female mice with an age ranged from 8 to 10 weeks were divided into four categories in a random manner. The first, second, and third groups received ATRA, calcitriol, ATRA+ calcitriol, respectively, and the fourth group received vehicle. The treatment started on the day prior to immunization and through the IP injections every other days for 21 days. The dosages of administration for calcitriol, ATRA, and calcitriol+ ATRA were 100 ng, 250 µg, and 50ng + 125 µg, respectively per mouse. An equal volume of excipient was administered for the vehicle group. T-bet, IFN-γ, GATA-3, and IL-4 genes expression were assessed in the splenocytes of EAE -induced mice. The expression of T-bet and IFN-γ genes in the splenocytes of ATRA, calcitriol and combination- treated mice were significantly reduced compared to vehicle group (p < 0.05). A significant decrease in T-bet expression was observed in the combination-treated group compared to the ATRA-treated group (p < 0.05). The expression of GATA3 and IL-4 genes was significantly increased in the ATRA-, calcitriol-, and combination-treated mice when compared with the control group (p < 0.05). Furthermore, the effect of calcitriol alone and in combination with ATRA was more considerable than that of ATRA alone. The nutraceutical approaches may be promising in the prevention and/or treatment of MS.

Contraceptive and molecular function of a novel recombinant vaccine based human leukemia inhibitory factor on Balb/c mice: An experimental in vivo study.

The functional competence of leukemia inhibitory factor (LIF), as immunocontraceptive vaccine in mice, was investigated. Balb/c mice were divided into two groups of vaccinated and controls. The recombinant human LIF (rhLIF) protein and phosphate buffer saline was emulsified with Freund's adjuvant and injected into vaccinated and control groups, respectively. Theinhibition of implantation was evaluated in mice uterine. The concentration of secreted interferon-γ (IFN-γ) and interleukin (IL)-4 were measured in cultured splenocyte of mice stimulated by rhLIF. The expressions of immune responsive gene 1 (IRG-1), cochlin (COCH), amphiregulin(Ar), and heparin-binding EGF-like growth factor (HB-EGF) genes were determined. Mice were assessed for inhibition of fertility after delivery, reversibility of immune response against rhLIF, and survival rate. Active immunization of mice with rhLIF resulted in reduction of the implantation and fertility rate up to 80.49% and 75%, respectively. All mice produced a high titer of anti-rhLIF antibodies in serums and vaginal fluids washes after 16 weeks; however, these antibodies were cleared from vaginal fluid washes after six months. A significant down-regulation in mRNA levels of IRG-1, Ar and HB-EGF was observed in vaccinated group compared to controls; however, no significant change in the expression profile of cochlin gene was detected. The results showed that rhLIF prevented pregnancy in a high percentage of female mice. Although the immunization of female Balb/c mice with rhLIF inhibited fertility and expression of genes associated with this molecule, further studies are needed to support this protein as a suitable candidate for contraceptive vaccine in mammals.

Production and characterization of recombinant human leukemia inhibitory factor and evaluation of anti-fertility effects of rabbit anti-rhLIF in Balb/c mice.

Human leukemia inhibitory factor (hLIF) is a cytokine of interleukin-6 family. This study aimed to evaluate the recombinant production rate of active hLIF by different vector-host systems under various conditions. Moreover, a rabbit polyclonal antibody (pAb) against recombinant hLIF (rhLIF) was produced and its anti-fertility effects were explored in Balb/c mice. Four different constructs including pET22b/hLIF, pET28b/hLIF, pET32b/hLIF and pColdI/hLIF were designed and transformed into BL21-(DE3), Rosetta-(DE3), Origami-(DE3) and Shuffle T7-(DE3) host cells. The expression level and proliferative effect of rhLIF were measured by SDS-PAGE and MTT assays, respectively. Rabbit pAb to rhLIF was produced and characterized using enzyme-linked immunosorbent assay and western blot techniques. The Balb/c mice were divided into two intervention and control groups. Then, they were intraperitoneally injected by purified rabbit anti-rhLIF and non-immunized rabbit pAb, respectively. After sacrifice on day 7, the number of implantation sites was counted. The rhLIF was successfully expressed by pET32b/hLIF and pColdI/hLIF vectors in all hosts with no significant difference in the rate of their expression. The rhLIF was purified and checked for activity. The results showed that it is functionally active and the produced anti-rhLIF pAb could specifically bind to commercial rhLIF. Passive immunization results showed that anti-rhLIF antibody completely inhibited fertility in all injected Balb/c mice compared to controls. Although previous studies showed expression of rhLIF using various methods, using different vector-host systems ensures us of successful biological active expression of it. The pAb against rhLIF could be a powerful tool for inducing in vivo infertility.