Combinational therapy of mesenchymal stem cell-derived extracellular vesicles and azithromycin improves clinical and histopathological recovery in CLP sepsis model.

BACKGROUND: Sepsis is a syndrome that occurs following an infection and marked by severe inflammatory responses, and if not treated in time, it can lead to multi-organ failure syndrome and death. This study examines the effects of a novel combination therapy using azithromycin and mesenchymal stem cell-derived extracellular vesicles (EVs) on a cecal ligation and puncture (CLP) model of sepsis. METHODS: Human Wharton's jelly-mesenchymal stem cells were cultured, characterized, and used to extract EVs. The CLP sepsis model was induced in mice, followed by treatments: saline, AZM, EVs, and combination therapy (A+E). Clinical sepsis scores were recorded 24 h post-treatment. Serum, peritoneal fluid, and organ tissues (kidney, liver, lung) were collected and analyzed for biochemical parameters (AST ALT, and creatinine), inflammatory markers, bacterial load, and histopathological changes. RESULTS: The A+E combined treatment improved the clinical scores of septic mice. The administration of A+E reduced bacterial loads in the peritoneum of septic mice, contributing to effective control of infection. Inflammatory markers of neutrophils-to-lymphocytes ratio (NLR) and TNF-α serum levels were significantly lower in the combinational therapy group, indicating significant anti-inflammatory effect of this combination. Additionally, combination of AZM and EVs alleviated organ damage mainly within liver, kidneys and lungs. Based on histopathological assessments and biochemical parameters, there was diminished tissue damage as well as reduced inflammation, which is correlated with improved functions of these vital organs. CONCLUSION: The combined use of azithromycin and EVs offers a promising therapeutic approach for sepsis by effectively controlling infection and modulating the inflammatory response.

The influence of occupational heat stress on serum inflammatory cytokines among traditional bakery workers in Iran.

Heat exposure exceeding the ISO7243:1989 standard limit can contribute to health problems among employees in a variety of workplaces. Ignoring heat standard requirements in hot working conditions such as bakeries results in physiologic and health problems, as well as an elevated risk of later illnesses. In this analytical case-control study, the serum levels of four inflammatory factors (interleukin-1 beta, interleukin-6, tumor necrosis factor-α, and C-reactive protein) were assessed using an enzyme-linked immunosorbent assay. 105 male artisan bakers (in four job classifications in bakeries and staff) were compared based on demographic characteristics and inflammatory factors. The findings of the study showed correlations between serum interleukin-1ß, interleukin-6, and C-reactive protein levels and thermal exposure in the occupational environment and employment type. Moreover, some differences in serum level of interleukin-1ß and job type were observed. Heat overexposure affected the increase of interleukin-1ß and C-reactive protein secretion. As a result of years of working in high-temperature conditions, inflammation can lead to subsequent diseases in workers. To protect their health from this occupational hazard, additional safeguards are needed. Our recommendations could also be applied to overly hot work environments that may cause heat stress in workers.

Exosomes Derived from Heat-shocked Tumor Cells Promote In vitro Maturation of Bone Marrow-derived Dendritic Cells.

Dendritic cells (DCs), professional antigen-presenting cells that process and deliver antigens using MHC II/I molecules, can be enhanced in numerous ways.  Exosomes derived from heat-shocked tumor cells (HS-TEXs) contain high amounts of heat-shock proteins (HSPs). HSPs, as chaperons, can induce DC maturation. This study aimed to investigate whether HS-TEXs can promote DC maturation. To generate DC, bone marrow-derived cells were treated with Interleukin-4 and GM-CSF. Exosomes were isolated from heat-treated CT-26 cells. The expression level of HSP in exosomes was checked by western blot and the increase in the expression of this protein was observed. Then, HS-TEXs were co-cultured with iDCs to determine DC maturity, and then DCs were co-cultured with lymphocytes to determine DC activity. Our results showed that  DCs treated with HS-TEXs express high levels of molecules involved in DC maturation and function including MHCII, CD40, CD83, and CD86. HS-TEXs caused phenotypic and functional maturation of DCs. In addition, flow cytometric results reflected a higher proliferative response of lymphocytes in the iDC / Tex + HSP group. HS-TEXs could be used as a strategy to improve DC maturation and activation.

Synergistic effects of mesenchymal stem cell-derived extracellular vesicles and dexamethasone on macrophage polarization under inflammatory conditions.

The undesirable inflammation and the excessive M1 macrophage activity may lead to inflammatory diseases. Corticosteroids and stem cell therapy are used in clinical practice to promote anti-inflammatory responses. However, this protocol has limitations and is associated with numerous side effects. In this study, the synergistic anti-inflammatory effects of dexamethasone (Dex) and mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) were evaluated to enhance the polarization of M1 inflammatory macrophages into the anti-inflammatory (M2) phenotype. Hence, we designed different combinations of Dex and EVs using three methods, including EVs isolated from Dex-preconditioned MSCs (Pre-Dex-EVs), EVs loaded with Dex (L-Dex-EVs), and EVs and Dex co-administration (Dex + EVs). All designed EVs had a significant effect on reducing the expression of M1-related genes (iNOS, Stat1, and IRF5), cytokines (IL6 and TNF-a), and CD markers (CD86) in lipopolysaccharide-stimulated macrophages. On the other hand, these combinations promoted the expression of alternative-activated M2-related genes (Arg-1, Stat6, and IRF4), cytokine (IL10), and CD markers (CD206).The combination of Dex and MSC-EVs enhances the effectiveness of both and synergistically promotes the conversion of inflammatory macrophages into an anti-inflammatory state.

Cancer-associated mesenchymal stem/stromal cells: role in progression and potential targets for therapeutic approaches.

Malignancies contain a relatively small number of Mesenchymal stem/stromal cells (MSCs), constituting a crucial tumor microenvironment (TME) component. These cells comprise approximately 0.01-5% of the total TME cell population. MSC differentiation potential and their interaction with the tumor environment enable these cells to affect tumor cells' growth, immune evasion, metastasis, drug resistance, and angiogenesis. This type of MSC, known as cancer-associated mesenchymal stem/stromal cells (CA-MSCs (interacts with tumor/non-tumor cells in the TME and affects their function by producing cytokines, chemokines, and various growth factors to facilitate tumor cell migration, survival, proliferation, and tumor progression. Considering that the effect of different cells on each other in the TME is a multi-faceted relationship, it is essential to discover the role of these relationships for targeting in tumor therapy. Due to the immunomodulatory role and the tissue repair characteristic of MSCs, these cells can help tumor growth from different aspects. CA-MSCs indirectly suppress antitumor immune response through several mechanisms, including decreasing dendritic cells (DCs) antigen presentation potential, disrupting natural killer (NK) cell differentiation, inducing immunoinhibitory subsets like tumor-associated macrophages (TAMs) and Treg cells, and immune checkpoint expression to reduce effector T cell antitumor responses. Therefore, if these cells can be targeted for treatment so that their population decreases, we can hope for the treatment and improvement of the tumor conditions. Also, various studies show that CA-MSCs in the TME can affect other vital aspects of a tumor, including cell proliferation, drug resistance, angiogenesis, and tumor cell invasion and metastasis. In this review article, we will discuss in detail some of the mechanisms by which CA-MSCs suppress the innate and adaptive immune systems and other mechanisms related to tumor progression.

Lanthanide complexes facilitate wound healing by promoting fibroblast viability, migration and M2 macrophage polarization.

A tridentate ligand LH3 ((2-hydroxy-3-methoxybenzylidene)-2-(hydroxyimino)propanehydrazide) comprising o-vanillin, hydrazone and oxime donor groups has been employed to prepare a series of tetranuclear Ln(III) complexes. The reaction of ligand LH3 with Ln(NO3)3 [Ln = Sm, Eu, Gd, Tb, Dy, Ho, Er] in MeOH yielded Ln4(LH)6(MeOH)2 (Ln = Sm(1), Eu(2), Gd(3), Tb(4), Ho (6) and Er (7))] whereas the corresponding reaction with Dy(NO3)3 afforded Dy4(LH)4(LH2)2(OH)2 (5). All complexes were characterized by various analytical techniques including single crystal X-ray diffraction, IR spectroscopy, UV-Vis spectroscopy, and elemental analysis. To investigate the potential of these lanthanide complexes for wound healing applications, their effects on fibroblast viability, migration, and M2 macrophage polarization were evaluated. The cytotoxicity assessment revealed that complexes 2(Eu), 4(Tb), 5(Dy), and 7(Er) significantly enhanced fibroblast viability compared to the negative control (NC). In vitro wound healing assay demonstrated that complexes 2(Eu) and 7(Eu) substantially promoted fibroblast migration compared to the NC. Moreover, complex 2(Eu) exhibited significant anti-inflammatory effects by reducing the phagocytic ability of lipopolysaccharide (LPS)-stimulated macrophage cells and attenuating nitric oxide (NO) production. In conclusion, among the series of complexes tested, complex 2(Eu) displayed the most potent anti-inflammatory effect on macrophage cells, while simultaneously promoting fibroblast viability and migration. This unique combination of properties renders complex 2 (Eu) highly promising for wound healing applications.

Oleuropein reduces LPS-induced inflammation via stimulating M2 macrophage polarization.

Oleuropein (OLEU) is the most prevalent phenolic component in olive varieties, and it has been considered for its powerful antioxidant properties in therapeutic applications. OLEU has anti-inflammatory properties and performs this property by suppressing inflammatory cells' function and reducing oxidative stress caused by various factors. This study investigated the ability of OLEU to polarize LPS-stimulated murine macrophage (MQ) cell RAW 264.7 into M1/M2 macrophages. As a first step, the cytotoxicity effects of OLEU were evaluated on LPS-stimulated RAW 264.7 cells using the thiazolyl blue (MTT) colorimetric test. Then, cytokines production, gene expression (Real-Time PCR), and functions (Nitrite oxide assay and phagocytosis assay) of OLEU-treated LPS-stimulated RAW 264.7 cells were evaluated. Our findings demonstrated that OLEU could reduce nitrite oxide (NO) production in LPS-stimulated RAW 264.7 cells by downregulating the inducible nitric oxide synthase gene expression. Furthermore, OLEU therapy decreases the expression of M1-associated pro-inflammatory cytokines production (IL-12, IFN-γ, and TNF-α) and genes expression (iNOS, TNF-α) while increasing the M2-associated anti-inflammatory gene expression and cytokines production (IL-10, and TGF-ß). Based on the result, OLEU may be considered a potential therapeutic approach for inflammatory diseases due to its possible effects on oxidative stress-related factors, cytokine expression and production, and phagocytosis.

Role of microbiota short-chain fatty acids in the pathogenesis of autoimmune diseases.

There is emerging evidence that microbiota and its metabolites play an important role in helath and diseases. In this regard, gut microbiota has been found as a crucial component that influences immune responses as well as immune-related disorders such as autoimmune diseases. Gut bacterial dysbiosis has been shown to cause disease and altered microbiota metabolite synthesis, leading to immunological and metabolic dysregulation. Of note, microbiota in the gut produce short-chain fatty acids (SCFAs) such as acetate, butyrate, and propionate, and remodeling in these microbiota metabolites has been linked to the pathophysiology of a number of autoimmune disorders such as type 1 diabetes, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, celiac disease, and systemic lupus erythematosus. In this review, we will address the most recent findings from the most noteworthy studies investigating the impact of microbiota SCFAs on various autoimmune diseases.

The potential application of encapsulated exosomes: A new approach to increase exosomes therapeutic efficacy.

Cell therapy is one of the methods that have shown promising results in treating diseases in recent decades. However, the use of different types of cells comes with limitations. The application of immune cells in cell therapy can lead to cytokine storms and inappropriate responses to self-antigens. Also, the use of stem cells has the potential to create tumors. Also, cells may not migrate to the injury site after intravenous injection. Therefore, using exosomes from different cells as therapeutic candidates were proposed. Due to their small size and favorable characteristics, such as biocompatibility and immunocompatibility, the easy storage and isolation, exosomes have attracted much attention. They are used in treating many diseases, including cardiovascular diseases, orthopedic diseases, autoimmune diseases, and cancer. However, the results of various studies have shown that the therapeutic efficiency of exosomes (Exo) can be increased by loading different drugs and microRNAs inside them (encapsulated exosomes). Therefore, analyzing studies investigating encapsulated exosomes' therapeutic ability is critical. In this study, we have examined the studies related to the use of encapsulated exosomes in treating diseases such as cancer and infectious diseases and their use in regenerative medicine. Compared to intact exosomes, the results show that the application of encapsulated exosomes has a higher therapeutic ability. Therefore it is suggested to use this method depending on the treatment type to increase the treatment's efficiency.

Regulation of the Th17/Treg balance by human umbilical cord mesenchymal stem cell-derived exosomes protects against acute experimental colitis.

Increasing evidence suggests that mesenchymal stem cells (MSCs) have immunosuppressive properties mediated by MSC-derived small extracellular vesicles (sEV). Exosomes are small extracellular vesicles that contain components that regulate immune cell function. We investigated the immunomodulatory effects of MSC-derived Exosome (MSC-Exo) on the severity of colitis using the dextran sulfate sodium (DSS)-induced colitis model. Exosomes were administrated intraperitoneally. Daily changes in body weight, stool consistency, and bleeding were assessed to determine the impact of MSC-Exos on colitis. Several measurements were taken, including the colon weight, length, and histological analysis of the colon tissues. The percentage of regulatory T cells and IL-10, TGF-ß, IL-17, TNF-α, and IFN-γ levels were calculated in the mesenteric lymph node (MLN) and spleen. The results showed MSC-Exos improved clinical manifestations of colitis. Colon macroscopic and histological observations also showed improvement in tissue destruction. The results illustrated that MSC-Exos might attenuate colitis by regulating Treg/Th17 balance, increasing anti-inflammatory, and decreasing pro-inflammatory cytokines expression. As a result, MSC-Exos could be used as an immunomodulatory approach to treating bowel inflammation.

Agent-based Modeling of Tumor and Immune System Interactions in Combinational Therapy with Low-dose 5-fluorouracil and Dendritic Cell Vaccine in Melanoma B16F10.

This study is designed to present an agent-based model (ABM) to simulate the interactions between tumor cells and the immune system in the melanoma model. The Myeloid-derived Suppressor Cells (MDSCs) and dendritic cells (DCs) are considered in this model as immunosuppressive and antigen-presenting agents respectively. The animal experiment was performed on 68 B16F10 melanoma tumor-bearing C57BL/6 female mice to collect dynamic data for ABM implementation and validation. Animals were divided into 4 groups; group 1 was control (no treatment) while groups 2 and 3 were treated with DC vaccine and low-dose 5- fluorouracil (5-FU) respectively and group 4 was treated with both DC Vaccine and low-dose of 5-FU. The tumor growth rate, number of MDSC, and presence of CD8+/CD107a+ T cells in the tumor microenvironment were evaluated in each group. Firstly, the tumor cells, the effector immune cells, DCs, and the MDSCs have been considered as the agents of the ABM model and their interaction methods have been extracted from the literature and implemented in the model. Then, the model parameters were estimated by the dynamic data collected from animal experiments.  To validate the ABM model, the simulation results were compared with the real data. The results show that the dynamics of the model agents can mimic the relations among considered immune system components to an emergent outcome compatible with real data. The simplicity of the proposed model can help to understand the results of the combinational therapy and make this model a useful tool for studying different scenarios and assessing the combinational results. Determining the role of each component helps to find critical times during tumor progression and change the tumor and immune system balance in favor of the immune system.

Dose-dependent effects of oleuropein administration on regulatory T-cells in patients with rheumatoid arthritis: An in vitro approach.

INTRODUCTION: Rheumatoid arthritis (RA) is an autoimmune disease that is identified with chronic inflammation and progressive destruction of the joints. The defective activity of regulatory T cells (Tregs) plays a crucial role in RA development. Oleuropein (OLEU) is the most common polyphenolic compound in olive leaf extracts with numerous pharmacological activities. In this study, the potential effects of OLEU in shifting CD4+ T cells toward Tregs are evaluated in patients with RA. METHODS: 32 healthy controls (HC) and 45 RA patients were included in two groups. The immunoturbidometric technique was used to measure serum levels of c-reactive protein (CRP) and rheumatoid factor (RF). Isolated CD4+ T cells from peripheral blood mononuclear cells (PBMCs) of HC and RA patients were cultured with appropriate concentrations of OLEU. The cytotoxicity effects of OLEU were determined using the MTT assay at 24, 48, and 72 h. The percentage of CD4+CD25 + FoxP3 regulatory T lymphocytes (Tregs) and the expressions of IL-10 and TGF-ß were evaluated by flow cytometry and immunoassay techniques after treatment of cells with different concentrations of OLEU for 24 h. The serum levels of RF and CRP in patients with RA were 11.8 ± 5.32 IU/ml and 6.36 ± 5.82 mg/l, respectively. RESULTS: OLEU had a dose-dependent effect on the CD4+ T cells via increasing the frequency of CD4+CD25 + FoxP3 Tregs (p = 0.0001). Moreover, it induced the production of IL-10 (p = 0.0001) and TGF-ß (p < 0.01) in both HC and RA patients. CONCLUSION: The findings of this study suggest that OLEU may have immunomodulatory effects by inducing Tregs, and it might help in developing a novel nutrition strategy for management of autoimmune diseases such as RA.

Optimized Dose of Dendritic Cell-based Vaccination in Experimental Model of Tumor Using Artificial Neural Network.

Previous studies have demonstrated that maturation of dendritic cells (DCs) by pathogenic components through pathogen-associated molecular patterns (PAMPs) such as Listeria monocytogenes lysate (LML) or CpG DNA can improve cancer vaccination in experimental models. In this study, a mathematical model based on an artificial neural network (ANN) was used to predict several patterns and dosage of matured DC administration for improved vaccination. The ANN model predicted that repeated co-injection of tumor antigen (TA)-loaded DCs matured with CpG (CpG-DC) and LML (List-DC) results in improved antitumor immune response as well as a reduction of immunosuppression in the tumor microenvironment. In the present study, we evaluated the ANN prediction accuracy about DC-based cancer vaccines pattern in the treatment of Wehi164 fibrosarcoma cancer-bearing mice. Our results showed that the administration of the DC vaccine according to ANN predicted pattern, leads to a decrease in the rate of tumor growth and size and augments CTL effector function. Furthermore, gene expression analysis confirmed an augmented immune response in the tumor microenvironment. Experimentations justified the validity of the ANN model forecast in the tumor growth and novel optimal dosage that led to more effective treatment.

CD73 specific siRNA loaded chitosan lactate nanoparticles potentiate the antitumor effect of a dendritic cell vaccine in 4T1 breast cancer bearing mice.

The efficacy of conventional anti-tumor immunotherapeutic approaches is markedly affected by the immunosuppressive microenvironment of tumor. Since adenosine is one of the main orchestra leaders in immunosuppression symphony of tumor, targeting its producing molecules such as CD73 can help to achieve a better clinical outcome following conventional cancer immunotherapeutic approaches. In the present study, we evaluated the efficacy of CD73-specific siRNA-loaded chitosan-lactate nanoparticles (ChLa NPs) in combination with tumor lysate pulsed dendritic cells (DCs) vaccine in treatment of 4T1 (murine derived) breast cancer bearing mice. Our results showed that intravenous administration of CD73-specific siRNA-loaded NPs led to reduced expression of CD73 in tumor cells which was associated with decreased tumor growth and metastasis, and improved mice survival. Furthermore, we found that the mechanism by which combination therapy inhibits tumor growth is in part related to downregulation of regulatory T (Treg), myeloid derived suppressor cells (MDSCs), and tumor associated macrophages, an augmented CTL effector function, improved proliferation status of T cells, increased production of inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17 and reduced levels of IL-10. Moreover, this treatment protocol attenuated the expression and activities of matrix metalloproteinases (MMPs) 2 and 9 which could be associated to the prevention of lung metastasis. In conclusion, our findings indicate that the use of CD73-specific siRNA-loaded NPs provides an immune potentiating function, thereby improves the efficacy of DC based cancer immunotherapy.