Cytokines signatures and susceptibility to cutaneous leishmaniasis in patients from Sistan and Baluchestan province of Iran.

BACKGROUND: Cutaneous leishmaniasis (CL) is a complex, multifactorial disease that results from environmental factors such as parasite polymorphism, phlebotomine vectors, and host genetic factors. Some studies have identified specific genetic factors that may be associated with cutaneous leishmaniasis. The objective of this research was to resolve the association of 8 cytokine polymorphisms, including TNF-α -308 A/G (rs 1800629), TNF-α -238 A/G (rs 361525), TGF-ß1 -509 T/C (rs 1800469), TGF-ß1+ 915 G/C (rs 1800471), IFN-γ -874 T/A (rs 2430561), IFN-γ -179 G/A (rs 2069709), IL-10 -819 C/T (rs 1800871), and IL-10 -592 A/C (rs 1800872) with susceptibility to CL. METHODS: A total of 152 patients with designated CL and 100 healthy controls were selected from those referred to Sistan and Baluchestan hospitals. CL was diagnosed by microscopic examination of Giemsa-stained samples and culture. Leishmania species were identified using ITS2 gene PCR amplification with universal primers. Genetic polymorphism was determined by the ARMS PCR method on extracted genomic DNA of individuals. Eight SNPs cytokines were genotyped. RESULTS: Most of the Genotypic and allelic frequency comparisons between patients with CL and healthy subjects showed no difference, except 3. Individual SNP analysis showed highest association of TGF-ß1 -509 (rs1800469) -CC genotype (P = 0.03, OR = 7.05, 95 % CI = 3.3-15) with 5.7-fold increase, IFN-γ -874 (rs 2430561) -AA genotype (P = 0.04, OR = 4.72, 95 % CI = 1.6-14) with 4.2-fold increase, and IL10 -819 (rs1800871) -CC genotype (P = 0.05, OR = 3.63, 95 % CI = 2.5-5.3) with 1.9-fold increase, with CL. Odds ratios (ORs) and 95 % confidence intervals (CIs) were evaluated to assess the association power. CONCLUSION: Our results conclude that rs1800469 (TGF-ß1), rs2430561 (INF-γ), and rs1800872 (IL10) polymorphisms are associated with CL in southeastern Iranian people.

Activated mesenchymal stem cells increase drug susceptibility of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are major causes of hospital-acquired infections and sepsis. Due to increasing antibiotic resistance, new treatments are needed. Mesenchymal stem cells (MSCs) have antimicrobial effects, which can be enhanced by preconditioning with antibiotics. This study investigated using antibiotics to strengthen MSCs against MRSA and P. aeruginosa. MSCs were preconditioned with linezolid, vancomycin, meropenem, or cephalosporin. Optimal antibiotic concentrations were determined by assessing MSC survival. Antimicrobial effects were measured by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antimicrobial peptide (AMP) gene expression. Optimal antibiotic concentrations for preconditioning MSCs without reducing viability were 1 µg/mL for linezolid, meropenem, and cephalosporin and 2 µg/mL for vancomycin. In MIC assays, MSCs preconditioned with linezolid, vancomycin, meropenem, or cephalosporin inhibited MRSA or P. aeruginosa growth at lower concentrations than non-preconditioned MSCs (p ≤ 0.001). In MBC assays, preconditioned MSCs showed enhanced bacterial clearance compared to non-preconditioned MSCs, especially when linezolid and vancomycin were used against MRSA (p ≤ 0.05). Preconditioned MSCs showed increased expression of genes encoding the antimicrobial peptide genes hepcidin and LL-37 compared to non-preconditioned MSCs. The highest hepcidin expression was seen with linezolid and vancomycin preconditioning (p ≤ 0.001). The highest LL-37 expression was with linezolid preconditioning (p ≤ 0.001). MSCs' preconditioning with linezolid, vancomycin, meropenem, or cephalosporin at optimal concentrations enhances their antimicrobial effects against MRSA and P. aeruginosa without compromising viability. This suggests preconditioned MSCs could be an effective adjuvant treatment for antibiotic-resistant infections. The mechanism may involve upregulation of AMP genes.

Apoptotic effects of mesenchymal stem cells' conditioned medium on colorectal cancer cell lines.

Multipotent Mesenchymal stem cells (MSCs) have vigorous immunomodulatory activity, apoptotic effects, and the capacity to migrate to inflammatory and tumor sites. This study focuses on the apoptotic effects of MSCs conditioned medium (CM) on colorectal cancer cell lines. MSCs were preconditioned with lipopolysaccharide (LPS) to induce apoptosis in colorectal cancer cells. The conditioned medium (LPS-CM) from the preconditioned cells was isolated and used to treat colorectal cancer cells (HT29 and SW48). The survival and proliferation of cancer cells were assessed using Trypan blue staining and MTT assay. The apoptosis rate was evaluated through flow cytometry analysis and caspase-3 activity. Additionally, Real-Time PCR was used to measure the mRNA level of apoptotic and anti-apoptotic factors, including bcl2, bax, and p53 genes. The results showed that LPS-CM significantly increased (p < 0.001) the percentage of apoptosis in the SW48 and HT29 cell lines. Caspase-3 activity significantly increased (p < 0.001) in these cell lines after treatment with LPS-CM. The mRNA level of bcl2 was significantly decreased (p < 0.001), while bax and p53 genes were significantly overexpressed (p < 0.001) in the LPS-CM treated cell lines. Notably, the mRNA level of bcl2 and bax genes was significantly altered at a higher concentration of LPS-CM. In conclusion, the conditioned medium from LPS-preconditioned MSCs can effectively induce apoptosis in colorectal cancer cells. This finding suggests that LPS-CM could be a potential strategy for inhibiting the proliferation and progression of colorectal cancer cells.

Evaluation of antioxidant and antibacterial effects of lactobacilli metabolites- preconditioned bone marrow mesenchymal stem cells in skin lesions amelioration.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) therapeutically intensify wound-healing. Meanwhile, probiotics are capable to inhibit pathogenic bacteria by secreting antimicrobial and antioxidant agents. They ameliorate more the wounds, by MSCs' synergistic effect. METHODS AND RESULTS: This way, MSCs exposed to probiotics metabolites (the extracts of Lactobacillus plantarum and Lactobacillus casei), become more viable, and the optimum doses of probiotics metabolites were determined by Trypan blue and MTT assays. Then, preconditioned MSCs (pre-MSCs) faced again the oxidative stress (100 µM H2O2), and their survival rate was assessed by MTT assay. Conditioned medium (CM) prepared from pre-MSCs and the expression rate of antioxidant proteins evaluated by Real-time PCR. The antimicrobial activity of the samples was evaluated using the MIC method. Then mice wound models were treated with the mixture of a basal ointment and each CM of pre-MSCs. The proliferation and viability of MSCs improved by the probiotic's metabolites preconditioning (P ≤ 0.05). pre-MSCs exhibited significant up-regulated in antioxidant genes expression (P ≤ 0.001). Also, probiotics metabolites presented a significant free radical scavenging effect (P ≤ 0.001). CMs of pre-MSCs showed considerably increased antibacterial activity (P ≤ 0.05). Experimental groups compared with the controls revealed that wound contraction was more rapid in groups that received CMs of LP-MSCs, LC-MSCs, MSCs, and then finally mixed probiotics metabolites, respectively. CONCLUSIONS: These results provide a novel insight into the probiotics extract effect on the improvement of MSCs' potential for wound healing procedures.

Anti-apoptotic effect of Nisin as a prebiotic on human mesenchymal stem cells in harsh condition.

Mesenchymal stem cells (MSCs) are progenitor cells of connective tissue with the ability of proliferation, self-renewal, and multilineage differentiation that make it a promising source with an enormous potential to be utilized for tissue repairing and vehicles of cell-based gene therapy. The low survival rate of MSCs following transplantation is their drawback. Preconditioning with some factors is a novel and effective strategy, improving the survival of the cells by protecting them from harmful conditions and result in the good recovery of injured tissues. Nisin is a prebiotic with antimicrobial activity. This manuscript aimed to evaluate the effect of Nisin preconditioning of MSCs on in vitro cell viability. MSCs were cultured and preconditioned with Nisin in different concentrations. Then, they are separately exposed to H2O2 and serum deprivation. Cell survival and cell apoptosis were evaluated by MTT assay and Real-time PCR, respectively. Furthermore, Annexin-PI staining and caspase activity was performed to visualize apoptotic cells. MSC-Nisin viability and proliferation significantly increased when exposed to H2O2 and serum deprivation, compared to that of MSCs. About 250 and 500 IU/mL of Nisin donate a significant anti-apoptotic impact to MSCs. Our data suggest that preconditioning with Nisin has been improved cell viability and the anti-apoptotic capacity of MSCs. However, the mechanism related to the protective properties of preconditioning and using this strategy in stem cell therapy requires more research.

Laboratory methods to decipher epigenetic signatures: a comparative review.

Epigenetics refers to nucleotide sequence-independent events, and heritable changes, including DNA methylation and histone modification (as the two main processes), contributing to the phenotypic features of the cell. Both genetics and epigenetics contribute to determining the outcome of regulatory gene expression systems. Indeed, the flexibility of epigenetic effects and stability of genetic coding lead to gene regulation complexity in response signals. Since some epigenetic changes are significant in abnormalities such as cancers and neurodegenerative diseases, the initial changes, dynamic and reversible properties, and diagnostic potential of epigenomic phenomena are subject to epigenome-wide association studies (EWAS) for therapeutic aims. Based on recent studies, methodological developments are necessary to improve epigenetic research. As a result, several methods have been developed to explore epigenetic alterations at low, medium, and high scales, focusing on DNA methylation and histone modification detection. In this research field, bisulfite-, enzyme sensitivity- and antibody specificity-based techniques are used for DNA methylation, whereas histone modifications are gained based on antibody recognition. This review provides a mechanism-based understanding and comparative overview of the most common techniques for detecting the status of epigenetic effects, including DNA methylation and histone modifications, for applicable approaches from low- to high-throughput scales.

A revealing review of mesenchymal stem cells therapy, clinical perspectives and Modification strategies.

Multipotent mesenchymal stem cells (MSCs) have been considerably inspected as effective tool for cell-based therapy of inflammatory, immune-mediated, and degenerative diseases, attributed to their immunomodulatory, immunosuppressive, and regenerative potentials. In the present review, we focus on recent research findings of the clinical applications and therapeutic potential of this cell type, MSCs' mechanisms of therapy, strategies to improve their therapeutic potentials such as manipulations and preconditioning, and potential/unexpected risks which should be considered as a prerequisite step before clinical use. The potential risks would probably include undesirable immune responses, tumor formation and the transmission of incidental agents. Then, we also review some of the milestones in the field, briefly discuss challenges and highlight the new guideline suggested for future directions and perspectives.

Mesenchymal stem cells preconditioned by staphylococcal enterotoxin B enhance survival and bacterial clearance in murine sepsis model.

Sepsis, a health-threatening progressive infectious disease, is the major cause of morbidity and mortality worldwide. Cell therapy using mesenchymal stromal cells (MSCs) is an innovative strategy with excessive therapeutic potential in the treatment of sepsis. Staphylococcal enterotoxin B (SEB) preconditioning aims to prolong the interval of survival of transplanted MSCs which induces the production of cytoprotective agents, anti-apoptotic and anti-inflammatory factors. The MSCs were preconditioned with an optimum dose of SEB (470 µmol/L). The expression levels of apoptosis genes and antibacterial activity of MSC and SEB-MSC and their conditioned medium (CM), as well as cell survival, were studied in vitro in an oxidative stress and serum deprivation condition. Following treatment of the septic mice with MSCs and SEB-MSCs, pro/anti-inflammatory cytokines, hematological factors, bacterial clearance and animal survival were assessed. The apoptotic and pro-inflammatory cytokine's genes expression was down-regulated while antibacterial peptides and anti-inflammatory cytokines were up-regulated in SEB-MSC-treated mice. The animal survival rates were improved; bacterial clearance was enhanced in the peritoneal fluids, blood and organs; aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were reduced in blood, compared with saline and MSCs alone. This research concludes that transplantation of SEB-MSCs presents improved therapeutic effects on a live bacterial model of sepsis.

Antimicrobial effects of mesenchymal stem cells primed by modified LPS on bacterial clearance in sepsis.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs)-based regenerative therapy is now considered as an alternative approach to revive infectious diseases, including sepsis. Nevertheless, the efficiency of MSC application is limited by the poor survival rate of engrafted MSCs. Hence, preconditioning was established as a strategy to increase the cells' efficiency. METHODS: MSCs were preconditioned with 1 µg/ml of three different lipopolysaccharides (LPSs) of Pseudomonas (Pse-LPS), Acinetobacter (Ac-LPS), and Acinetobacter inactivated lipid A by PagL (Ac-LPS-PagL). Then, preconditioned MSCs were exposed to oxidative stress and serum deprivation followed by evaluation of the antibacterial activity, survival, and apoptosis of MSCs. Then, the murine sepsis model treated with 100 µl phosphate-buffered saline (control group, sepsis group), 100 µl of 1 × 10 6 wild MSCs (MSC group), and three remained groups received 100 µl of 1 × 10 6 LPS-preconditioned MSCs (Pse-LPS-MSCs group: LPS purified from Pseudomonas, or Ac-LPS-MSCs group: LPS purified from Acinetobacter, and Ac-PagL-LPS-MSCs group: detoxified LPS Pagl). RESULTS: After 4 days, LPS-preconditioned MSC transplantation modulated the immune response and reduced inflammation in septic mice. Apoptosis of Pse-LPS/Ac-LPS-preconditioned-MSCs was obviously reduced in vitro, and the survival rate of engrafted mice was evidently elevated in Pse-LPS-MSCs and Ac-LPS-MSCs groups compared with other three groups. CONCLUSION: LPS preconditioning provides an innovative strategy for evolving functional and biological properties of MSCs and ameliorates the survival rate of the mouse model of sepsis after MSC transplantation, protects cells from apoptosis and organ damages, and evaluates therapeutic properties, including immunemodulatory.