Long-Term Effects of Low-Dose Naltrexone on Immunomodulatory Properties of Human Adipose-Derived Mesenchymal Stem Cells.

Background: Low-dose naltrexone (LDN) is involved in the treatment of inflammatory and immune system diseases and can affect immune cells. Mesenchymal stem cells (MSCs) are known for their immunomodulatory effects and the potential for the treatment of certain types of autoimmune diseases. Objective: To investigate the long-term effects of LDN on human adipose-derived mesenchymal stem cells (ASCs) to see how their immunomodulatory properties are affected and also how LDN-treated ASCs interact with other immune cells present in peripheral blood mononuclear cells (PBMCs). Methods: After 14 days of treatment, the ability of LDN-treated ASCs to modulate PBMC proliferation in a two-way mixed lymphocyte reaction (MLR) model was assessed using XTT. The relative expression of IDO, PD-L1, COX-2, HGF genes, and the level of IL-6 and TGF-ß cytokines were measured in IFN-γ stimulated and unstimulated ASCs (treated and not treated cells) using real-time PCR and ELISA respectively. Results: Unstimulated ASCs treated with 10-8 M Naltrexone (10-8 M NTX) showed higher levels of TGF-ß, compared with the controls (P<0.05). Stimulated ASCs treated with 10-6 M NTX showed elevated expression of IDO, PD-L1 genes, and IL-6 level (P<0.05). Conclusion: Our results demonstrated that various LDN concentrations have dissimilar effects on ASCs' immunomodulatory properties. A higher LDN concentration induced an alteration in the immunomodulatory features of ASCs.

Combined effects of pioglitazone and doxorubicin on migration and invasion of MDA-MB-231 breast cancer cells.

BACKGROUND: Despite antitumor properties, chemotherapy medication can create conditions in tumor cells that work in favor of the tumor. Doxorubicin, commonly prescribed chemotherapy agents, can increase the risk of migration and invasion of tumor cells through overexpression of the CXCR4 gene by affecting downstream signaling pathways. The regulatory role of CXCR7 on CXCR4 function has been demonstrated. Therefore, it is hypothesized that combining doxorubicin with another anticancer drug could be a promising approach. METHODS: In this research, we evaluated the anti-invasive property of pioglitazone along with antitumor effects of doxorubicin on MDA-MB-231 breast cancer cell lines. RESULTS: There was no significant difference between two treatment groups in neither the expression nor changes in the expression of CXCR7 and CXCR4 genes (P < 0.05). Pioglitazone-doxorubicin combination reduced cell migration in tumor cells to a significantly higher extent compared to doxorubicin alone (P < 0.05). CONCLUSIONS: Co-administration of pioglitazone and doxorubicin might reduce cell migration in breast cancer tumor cells, and that cell migration function is independent of some specific proteins.

Hydrocortisone Long-term Treatment Effect on Immunomodulatory Properties of Human Adipose-Derived Mesenchymal Stromal/Stem Cells.

Cortisol is secreted in prolonged stress and has therapeutic effects in inflammatory diseases. Considering the immunomodulatory effects of mesenchymal stem cells, here we investigated the effect of hydrocortisone (HC) long-term treatment on immunomodulatory properties of human adipose-derived mesenchymal stromal/stem cells (ASCs). Isolated ASCs from healthy subjects were treated with different HC concentrations for 14 days. The effect of HC-treated ASCs on the proliferative response of peripheral blood mononuclear cells (PBMCs) was evaluated in ASCs/2-way mixed leukocyte reaction coculture using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. HC-treated ASCs were further divided into interferon gamma (IFN-γ) stimulated and unstimulated groups. Transforming growth factor beta 1 (TGF-ß1) and interleukin (IL)-6 levels were measured in culture supernatants by enzyme-linked immunosorbent assay. Relative expression of cyclooxygenase-2 (COX-2), hepatocyte growth factor, indoleamine dioxygenase, and programmed death-ligand 1 genes was assessed by real-time PCR. Levels of TGF-ß1 and COX-2 expression were elevated in unstimulated ASCs, while exposure to high concentration of HC significantly increased TGF-ß1 levels and reduced COX-2 expression. Unstimulated HC-5-µM-treated ASCs increased PBMC proliferation ratio on day 2 of coculture compared to the control group (P = 0.05). In IFN-γ stimulated condition, pretreatment with HC-5 µM resulted in a significantly increased IL-6 and significantly decreased COX-2 expression compared to the HC untreated control group. In conclusion, our results showed various alterations of ASC immunomodulatory related features as a result of long-term exposure of different concentrations of HC. It seems that HC at low concentration pushed the balance toward extended immune response in ASCs, while this observation wasn't persistent in ASCs treated with higher concentrations of HC.

Up-regulation of CD200/CD200R1 Immunomodulatory Axis of Allogenic Peripheral Blood Mononuclear Cells in a Co-culture with Adipose-derived Mesenchymal Stem Cells.

Co-inhibitory molecules modulate immune responses. Immunomodulatory properties of mesenchymal stem cells (MSCs) turn them into ideal candidates for cell therapy. This study was designed to investigate the immunomodulatory effect of adipose-derived stem cells (ASCs) on inflammatory environment of a co-culture of allogenic peripheral blood mononuclear cells (PBMCs) in a two-way mixed leukocyte reaction (twMLR) setting. ASCs were co-cultured with allogenic PBMCs in twMLR setting for four days. The proliferation of peripheral blood mononuclear cells (PBMCs), levels of interleukin (IL)-10, and expression of interferon-gamma (IFN-γ), B7-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death-ligand 1 (PD-L1), +, and CD200R1 genes, as well as cell surface expression of CD200 and CD200R1, were measured in twMLR as control, and co-culture groups on days 0, 2 and 4 of the experiment. The proliferation of PBMCs was suppressed on days 2 and 4 of co-culture. The expression of CD200 (p=0.014), CD200R1, CTLA-4, and PD1 genes increased on days 2 and 4 of the co-culture compared to twMLR. CD200 expressing PBMCs decreased by 1.75% on day 2 of the co-culture but increased by 6.23% on day 4 of the co-culture (p=0.013) compared to the same days of twMLR. IL-10 levels increased in the co-culture supernatants on days 2 and 4 compared to twMLR (p<0.05). Our results showed that ASCs upregulate the CD200/CD200R1 axis more than PD-1/PD-L1 and CTLA-4/B7-1 pathways in the twMLR. Also, elevated expression of CD200R1 in the final day of co-culture was similar to PD-1 expression pattern. This finding suggests a role for the CD200/CD200R1 axis in later modulation of the immune response.

MDSCs in pregnancy: Critical players for a balanced immune system at the feto-maternal interface.

Myeloid-derived suppressor cells (MDSCs) have emerged as a new immune regulator at the feto-maternal interface. Although the phenotypes and functions of these cells were primarily studied in pathological conditions such as cancers and infections, new evidence has underscored their beneficial roles in homeostasis and physiological circumstances such as normal pregnancy. In this regard, studies have shown an increased number of MDSCs, particularly granulocytic MDSCs, at the feto-maternal interface. These cells participate in maintaining immunological tolerance between mother and semi-allograft fetus through various mechanisms. They further seem to play critical roles in placentation and fetus growth process. The absence or dysregulation of MDSCs during pregnancy have been reported in several pregnancy complications. These cells are also abundant in the cord blood of neonates so as to balance the immune responses and prevent aggressive inflammatory responses. The current review summarizes and organizes detailed data on MDSCs and their roles during pregnancy.

Impaired immunomodulatory ability of type 2 diabetic adipose-derived mesenchymal stem cells in regulation of inflammatory condition in mixed leukocyte reaction.

The immunomodulatory properties of type 2 diabetic patients' adipose-derived mesenchymal stem cells (D-ASCs) has not been extensively studied. In this study, we compared the immunomodulatory properties of D-ASCs and non-diabetic subjects mesenchymal stem cells (ND-ASCs) in co-culture with mixed leukocyte reaction (MLR). ASCs were isolated from adipose tissue samples of type 2 diabetic and non-diabetic subjects (age: 40-55). D-ASCs and ND-ASCs were co-cultured with two-way MLR. Peripheral blood mononuclear cells (PBMCs) proliferation ratio, protein levels of IFN-γ and IL-10, mRNA expression of COX-2, TNF-α, TGF-ß1 and IL-6 genes in MLR, D-ASCs and ND-ASCs co-cultures were assessed using XTT, ELISA and Real-time qRT-PCR, respectively. PBMCs proliferation on days 2 and 4 of D-ASCs co-culture was higher than ND-ASCs co-culture of the same days (p < 0.001). IFN-γ level decreased on day 4 compared to day 2 of ND-ASCs co-culture, but its level had not changed in D-ASCs co-culture. COX-2 expression on days 2 and 4 of D-ASCs co-culture was lower than ND-ASCs co-culture of the same days (p < 0.05). The expression of TNF-α and IL-6 on days 2 and 4 of D-ASCs co-culture were higher than ND-ASCs co-culture of the same days (p < 0.001). TGF-ß1 on day 4 of ND-ASCs co-culture showed a slightly higher expression than D-ASCs co-culture of the same day. Lower suppression of PBMCs proliferation, declined expression of anti-inflammatory and upregulated expression of pro-inflammatory factors in D-ASCs co-culture, indicated an impairment of these cells in modulation of the inflammatory condition.

Effect of gestational age on migration ability of the human umbilical cord vein mesenchymal stem cells.

PURPOSE: Migration ability of mesenchymal stem cells (MSCs) towards chemotactic mediators is a determinant factor in cell therapy. MSCs derived from different sources show different properties. Here we compared the migration ability of the term and the pre-term human umbilical cord vein MSCs (hUCV-MSCs). MATERIALS/METHODS: MSCs were isolated from term and pre-term umbilical cord vein, and cultured to passage 3-4. Migration rate of both groups was assessed in the presence of 10% FBS using chemotaxis assay. Surface expression of CXCR4 was measured by flow cytometery. The relative gene expression of CXCR4, IGF1-R, PDGFRα, MMP-2, MMP-9, MT1-MMP and TIMP-2 were evaluated using real time PCR. RESULTS: The isolation rate of the pre-term hUCV-MSCs was higher than the term hUCV-MSCs. Phenotype characteristics and differentiation ability of the term and pre-term hUCV-MSCs were not different. The migration rate of the pre-term hUCV-MSCs was more than the term hUCV-MSCs. Gene and surface expressions of the CXCR4 were both significantly higher in the pre-term hUCV-MSCs (P≤0.05). The mRNA levels of PDGFRα, MMP-2, MMP-9, MT1-MMP and TIMP-2 showed no significant difference between the two groups. CONCLUSION: Our results showed that the gestational age can affect the migration ability of the hUCV-MSCs.

Immunomodulatory properties of umbilical cord vein mesenchymal stromal cells influenced by gestational age and in vitro expansion.

In vivo and in vitro aging of the mesenchymal stromal cells (MSCs) can affects their properties. We investigated the immunomodulatory properties of the term and preterm human umbilical cord vein MSCs (UCV-MSCs) at the passages (P) 2 and 5. Term and preterm UCV-MSCs at P2 and 5 were co-cultured with two-way mixed lymphocyte reaction. Proliferation, IFN-γ and IL-10 protein levels, mRNA levels of the COX-2, TGF-ß1, TNF-α, IL-4 and FoxP3 were assessed. The term UCV-MSCs and P5 of the term and preterm UCV-MSCs had stronger inhibitory effects on cell proliferation than the preterm UCV-MSC and P2, respectively (P = 0.001). In supernatants of the co-cultures, IFN-γ was higher in the term UCV-MSC than the preterm UCV-MSC, while IL-10 was higher in the preterm UCV-MSCs than the term UCV-MSCs. Also in the co-cultures, COX-2 expression in the term UCV-MSCs and P2 was higher than the preterm UCV-MSCs and P5, respectively and TGF-ß1 expression in the term UCV-MSCs was higher than preterm. Conclusively it appears that the term UCV-MSCs, and P5 of the term and preterm UCV-MSCs showed a higher immunomodulatory ability than the preterm UCV-MSCs and P2, respectively.