Concentrations Of TGF-β1 Research Articles

Effects of miR-214 on adenosine A2A receptor and carboxymethyl chitosan nanoparticles on the function of keloid fibroblasts and their mechanisms

This work prepared and investigated the impact of carboxymethyl chitosan nanoparticles (MC-NPs) on the proliferative capability of keloid fibroblasts (KFBs) while analyzing the mechanistic roles of miR-214 and adenosine A2A receptor (A2AR) in fibroblasts within hypertrophic scars. MC-NPs were synthesized through ion cross-linking, were characterized using transmission electron microscopy (TEM) and laser particle size scattering. The influence of MC-NPs on the proliferation capacity of KFBs was assessed using the MTT method. Changes in the expression levels of miR-214 and A2AR in KFBs, normal skin fibroblasts (NFBs), hypertrophic scar tissue, and normal skin tissue were analyzed. KFBs were categorized into anti-miR-214, anti-miR-NC, miR-214 mimics, miR-NC, si-A2AR, si-con, anti-miR-214+ si-con, and anti-miR-214+ si-A2AR groups. Bioinformatics target prediction was conducted to explore the interaction between miR-214 and A2AR. Real-time quantitative PCR and immunoblotting (WB) were employed to detect the expression levels of miR-214, A2AR, apoptotic protein Bax, and TGF-β in different cells. Cell counting kit-8 (CCK8) and flow cytometry were employed to assess cell proliferation activity and apoptosis. The results indicated that MC-NPs exhibited spherical particles with an average diameter of 236.47 ± 4.98 nm. The cell OD value in the MC-NPs group was lower than that in KFBs (P < 0.05). The mRNA levels of miR-214 in KFBs and hypertrophic scar tissue were lower than those in NFBs and normal tissue (P < 0.001), while the mRNA and protein levels of A2AR were significantly elevated (P < 0.05). Compared to the control group and anti-miR-NC, the anti-miR-214 group showed significantly increased cell OD values and Bcl-2 protein expression (P < 0.001), decreased levels of apoptotic gene Bax protein, TGF-β gene mRNA, and protein expression (P < 0.001). Continuous complementary binding sites were identified between miR-214 and A2AR. Compared to the control group, the si-A2AR group exhibited a significant decrease in A2AR gene mRNA and protein expression levels (P < 0.001), reduced cell viability (P < 0.001), increased apoptosis rate (P < 0.001), and a significant elevation in TGF-β protein expression (P < 0.001). miR-214 targetedly regulated the expression of A2AR, inducing changes in TGF-β content, promoting the proliferation of keloid fibroblasts, and inhibiting cell apoptosis.

Coronary artery bypass grafting (CABG) induces pro-inflammatory and immunomodulatory phenotype of platelets in the absence of a pro-aggregatory state

BackgroundCoronary artery bypass grafting (CABG) is considered the choice treatment for patients suffering from coronary artery disease (CAD). In the inflammatory milieu of cardiopulmonary bypass (CPB), systemic inflammatory response syndrome (SIRS) can induce a platelet pro-inflammatory state which could exacerbate post-CABG inflammatory status while affecting hemostatic function in patients. Therefore, focusing on platelets, the study presented here attempted to evaluate the pro-inflammatory and immunomodulatory profile of platelets as well as pro-aggregatory status during CABG. MethodsPlatelets from patients undergoing CABG were subjected to flowcytometry analysis to evaluate P-selectin and CD40L expressions and PAC-1 binding in five intervals of 24 h before surgery, immediately, 2 h, 24 h, and one week after surgery. Moreover, intra-platelet TGF-β1 was also examined with western blotting. ResultsData showed increases of P-selectin and CD40L expressions in patients, with the meaningful loss of platelet contents of TGF-β1 after CABG (p < 0.001), where the changes tended to recover by day 7 of surgery while remaining above baseline (p < 0.001). Meanwhile, no significant change in PAC-1 binding capacity was shown. ConclusionThe study presented here suggests that although the release of pro-inflammatory substances from platelets during CABG supports the post-operative inflammatory state, platelets are not pro-aggregatory enough to enhance thrombotic events after surgery. Whilst these observations could be due to successful medical interventions to optimize hemostasis during and after surgery, post-CABG reversal of anticoagulant by protamine is considered as another factor that may also have contributed to preventing pro-aggregatory but not pro-inflammatory and immunomodulatory functions of platelets.

The role of angiotensin receptor blocker (losartan) on decreasing fibrotic process of corpora cavernosa in priapism model of wistar rats

Background Priapism induces regulation of Transforming Growth Factor-β1 (TGF-β1) expression and collagen-type-1 deposition. This will replace the normal corpora cavernosa with fibrotic tissue which eventually resulted in erectile dysfunction. It is also known that the fibrosis process of corpora cavernosa is related to Renin-Angiotensin II System (RAS). Angiotensin II receptor blockers (ARB), especially losartan, inhibit the inflammation process and fibrotic tissue formation. This study evaluated the effect of losartan in reducing fibrosis in priapism by evaluating TGF-β1 and collagen-type-1 in cavernous tissue and determined the effect of losartan in preventing fibrosis in priapism model of Wistar rats assessed by the metavir score. Methods A total of eighteen male Wistar rats mean were divided into five groups. For the priapism models, we applied negative pressure on the penis to make an artificial erection to mimic the priapism process. The control groups were observed and the treatment groups were orally given losartan 15 mg/kg/day. Corpora cavernosa was harvested for TGF-β1 and collagen-type-1 measurement using an enzyme-linked immunosorbent assay (ELISA). The fibrotic tissue of each rat was then collected and assessed histopathologically with the metavir scoring system. Results Penile TGF-β1 concentration in the losartan-treated group was not significantly different on day 10 and day 28 of observation (p10=0,30; p28=0,17). Meanwhile, collagen-type-1 concentration was significantly lower compared to control group (p10=0,002; p28=0,01). There was a significant difference in metavir scores in rats that received losartan and those who did not (p&lt;0,05). Conclusion Losartan could suppress the fibrosis process in the priapism model. It could decrease the collagen type 1 deposition during corpora cavernosa tissue regeneration. Based on the metavir score, the group receiving losartan therapy was better than the control group.

The Expression of miR-377-3p in Patients with DKD and the Regulatory Mechanism of miR-377-3p on the Inflammatory Response of HK-2 Cells Through TGF-β.

The purpose of the study was to investigate the expression levels and correlation of inflammatory factors such as miR-377-3p and TGF-β in patients with diabetic kidney disease (DKD), and to investigate the regulatory mechanism of transfection of miR-377-3p on the inflammatory response of HK-2 cell induced by high glucose. According to UACR, patients were divided into normal albuminuria group (Con, n = 29), microalbuminuria group (Micro, n = 31) and macroalbuminuria group (Macro, n = 30), analyzed the correlation and influencing factors between DKD and inflammatory factor. HK-2 cells were randomly divided into four groups: normal control group (NC), high glucose group (HG), miR-377-3p overexpression group (MIN), and miR-377-3p inhibition group (IN). After transfection of miR-377-3p mimics and inhibitors, the contents of TGF-β, IL-6 and IL-18 were detected by RT-PCR and Western blot. The levels of miR-377-3p, TGF-β, IL-6 and IL-18 in both Micro group and Macro group were significantly higher than those in Con group (P < 0.05); Pearson correlation analysis showed that miR-377-3p was positively correlated with UACR, TG, TGF-β, IL-6 and IL-18, and negatively correlated with GFR (P < 0.05). Cell experiment: RT-PCR and Western blot results showed that miR-377-3p, TGF-β, IL-6 and IL-18 in HG group were significantly higher than those in NC group (P < 0.05). After transfection with miR-377-3p inhibitor, the levels of miR-377-3p, TGF-β, IL-6 and IL-18 in IN group were significantly decreased compared with HG group and MIN group. miR-377-3p expression was elevated both in serum of DKD patients and in HK-2 cells with high glucose induced injury, overexpression of miR-377-3p exacerbates the damage to HK-2 cells and promotes the progression of DKD. Silencing miR-377-3p can potentially regulate the levels of inflammatory factors in HK-2 cells by targeting downregulation of TGF-β expression, thereby mitigating the damage to HK-2 cells and delaying the development of diabetic kidney disease.

Vitamin D Supplementation: Effect on Cytokine Profile in Multiple Sclerosis

Vitamin D is known for its role in modulating calcium and phosphate homeostasis and is implicated both in bone mineralization and immune system regulation. The immune-modulatory role of vitamin D and its impact on multiple sclerosis (MS) courses are still debated. The aim of this review was to check the effect of vitamin D supplementation on cytokine profile regulation in people with MS. A significant increase in serum concentrations of interleukin (IL)-10 and Transforming growth factor (TGF)-β1 after vitamin D supplementation was demonstrated in most studies, with some of them reporting a reduction in disability scores after vitamin D supplementation and an inverse correlation between IL-10 levels and disability. The effect of vitamin D on the serum levels of IL-17 and IL-6 was controversial; different results across studies could be explained by a variability in the treatment duration, route, and frequency of administration, as well as the dosage of vitamin D supplementation, responses to vitamin D treatment and the serum levels reached with supplementation, including the methods used for cytokine analysis and the different cell types investigated, the MS phenotype, the disease phase (active vs. non-active) and duration, and concomitant treatment with disease-modifying therapies. Nevertheless, the significant increase in the serum concentrations of IL-10 and TGF-β1, demonstrated in most studies, suggests an anti-inflammatory effect of vitamin D supplementation.

Expression of MicroRNA-3162-3p in Different Clinical Stages of Children with Primary Immune Thrombocytopenia and Its Significance

To explore the expression of microRNA-3162-3p in different clinical stages of childhood primary immune thrombocytopenia (ITP) and its significance. Ninety-six children with ITP were enrolled and divided into new diagnosis group (n=40), persistent group (n=30) and chronic group (n=26) according to the course of disease. 80 healthy children were selected as the control group. Peripheral blood mononuclear cells (PBMNC) of ITP children and healthy children were isolated and cultured, and the expression of microRNA-3162-3p in PBMNC of subjects was detected by real-time fluorescence quantitative PCR. The contents of IL-17, IL-23, IL-10 and TGF-β in PBMNC of subjects were determined by ELISA. The correlation between microRNA-3162-3p and platelet count, IL-17, IL-23, IL-10 and TGF-β was analyzed. Compared with the control group, the expression of microRNA-3162-3p and IL-10 in PBMNC and platelet count of ITP children were significantly decreased（P < 0.05）, while IL-17, IL-23 and TGF-β were significantly increased (P < 0.05). With the prolongation of the disease course, the expressions of microRNA-3162-3p and IL-10 in PBMNC and platelet count were significantly decreased（P < 0.05）, while the expressions of IL-17, IL-23 and TGF-β were significantly increased (P < 0.05). The expression of microRNA-3162-3p in PBMNC was positively correlated with platelet count and IL-10 (r =0.716, 0.667), and negatively correlated with IL-17, IL-23, and TGF-β (r =-0.540, -0.641, -0.560). MicroRNA-3162-3p expression is significantly reduced in PBMNC of children with ITP, and is involved in the regulation of Th17/Treg imbalance, which can be used as a potential therapeutic target of ITP.

The evaluation of PD-1 and Tim-3 expression besides their related miRNAs in PBMCs of women with recurrent pregnancy loss

Recurrent pregnancy loss (RPL) is a multifactorial disorder, associated with immunologic abnormalities. During pregnancy, the maternal immune system uses different tolerance mechanisms to deal with a semi-allogenic fetus. The expression of immune checkpoints and their related miRNAs in immune cells can ensure pregnancy at the feto-maternal interface by modulating immune responses. This study aims to evaluate the expression of the immune checkpoint molecules PD-1 and Tim-3 on circulating T cells by flow cytometry, that of mir-138 and mir-155 in PBMCs by Real-time PCR, and the concentrations of TGF-β and IP-10 in the sera of women suffering from RPL as well as of gestational age-matched healthy pregnant women by ELISA. The percentage of PD-1 or Tim-3 expressing CD8+ T cells was significantly lower in RPL patients compared to the controls, while there was no significant difference in Tim-3 expression of CD4+ T cells between the two groups. The mRNA of both the PD-1 and Tim-3 genes were downregulated in PBMCs of RPL patients compared to controls, however, the difference was not statistically significant for Tim-3. The concentration of TGF-β was significantly lower and that of IP-10 was significantly higher in the sera of RPL patients than in those of the controls. The relative expression of mir-138 and miR-155 were significantly lower, in PBMCs of RPL patients than in those of healthy pregnant women. These data confirm that by affecting cytokine production, immune checkpoints, and microRNAs play a role in establishing the appropriate local immune environment for successful pregnancy. The wider analysis of immune checkpoints may also yield new biomarkers for the diagnosis and prevention of RPL.

Characterization of platelet rich plasma in feline immunodeficiency virus-infected cats: Cell, and PDGF-BB and TGF-ß1 growth factor analysis

Autologous platelet-rich plasma (PRP) contains growth factors (GFs) that modulate the expression of inflammatory cells; thus, these products could be considered a good strategy to favor tissue regeneration in feline immunodeficiency (FIV) positive cats. However, there is no scientific documentation on obtaining PRP in FIV-positive cats.Authors hypothesized that PRP can be obtained in FIV cats following the PRGF®-Endoret® methodology. The objectives of this study were to compare the platelet, erythrocyte, and leukocyte concentration between whole blood (WB) and the PRP; and determine the concentration of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor β1 (TGF-β1) in FIV-positive cats. Sixteen adults FIV-positive asymptomatic cats were included in the study. WB samples were drawn and the PRP was obtained by centrifugation at 265g for 10 min. Erythrocyte and leukocyte, platelets, and mean platelet volume (MPV) were determined both in WB and in PRP. PDGF-BB and TGF-β1 concentrations were additionally determined in PRP.Platelet concentration increased 1.1 times in PRP fraction compared to WB, but no significant differences were reported. MPV was statistically higher in WB than in PRP (p = 0.001). Erythrocytes and leukocytes counts were decreased by 99% and 92%, respectively in the PRP fraction (p < 0.001). Regarding TGF-ß1, a higher concentration was shown in the PRP (p < 0.02). Although the product obtained could not be classified as PRP according to the PRGF®-Endoret® methodology, based on the drastic reduction of RBC and WBC, the PLT concentrate is of high purity.

Tanshinone IIA alleviates bleomycin-induced pulmonary fibrosis by inhibiting Zbtb16

Pulmonary fibrosis is a complex disease that can occur in a variety of clinical settings. The Zinc Finger and BTB Domain Containing 16 (Zbtb16) is a transcription factor and has not been studied in pulmonary fibrosis. Lung tissues from rats which were treated with bleomycin and Tanshinone IIA (Tan IIA) were collected for mRNA sequencing. Zbtb16, a differentially expressed gene, was screened. Using adeno-associated virus to knock down Zbtb16 in rats, it was found that the lung index and the content of hydroxyproline in lung tissue were decreased. HE and Masson staining revealed that pathological symptoms of lung histopathology were relieved after Zbtb16 knockdown. Protein expressions of α-SMA, Collagen I and Fibronectin were significantly decreased after Zbtb16 knockdown in vivo and in vitro. Meanwhile, the protein content of TGF-β1 and the phosphorylation of Smad2/3 were inhibited by Zbtb16 knockdown. Conversely, under the treatment of Tan IIA and TGF-β1, overexpression of Zbtb16 improved cell viability, increased the expression of fibrosis-related proteins, and promoted the phosphorylation of Smad 2/3. All above demonstrates that Zbtb16 inhibition ameliorates pulmonary fibrosis and suppresses the TGF-β/Smad pathway. Furthermore, Zbtb16 mediates the inhibitory process of Tan IIA on pulmonary fibrosis. This study provides a novel candidate therapeutic target for pulmonary fibrosis.

Characterization of the immunosuppressive environment induced by larval Echinococcus granulosus during chronic experimental infection.

The larval stage of Echinococcus granulosus causes the chronic infection known as cystic echinococcosis, deploying strong inhibitory mechanisms on host immune responses. Using experimental intraperitoneal infection in C57BL/6 mice, we carried out an in-depth analysis of the local changes in macrophage populations associated with chronic infection. In addition, we analyzed T cells and relevant soluble mediators. Infected animals showed an increase in local cell numbers, mostly accounted for by eosinophils, T cells, and macrophages. Within macrophage populations, the largest increases in cell numbers corresponded to resident large peritoneal macrophages (LPM). Monocyte recruitment appeared to be active, as judged by the increased number of monocytes and cells in the process of differentiation towards LPM, including small (SPM) and converting peritoneal macrophages (CPM). In contrast, we found no evidence of macrophage proliferation. Infection induced the expression of M2 markers in SPM, CPM, and LPM. It also enhanced the expression of the co-inhibitor PD-L1 in LPM, SPM, and CPM and induced the co-inhibitor PD-L2 in SPM and CPM. Therefore, local macrophages acquire M2-like phenotypes with probable suppressive capacities. Regarding T cells, infection induced an increase in the percentage of CD4+ cells that are PD-1+, which represent a potential target of suppression by PD-L1+/PD-L2+ macrophages. In possible agreement, CD4+ T cells from infected animals showed blunted proliferative responses to in vitro stimulation with anti-CD3. Further evidence of immune suppression in the parasite vicinity arose from the observation of an expansion in FoxP3+ CD4+ regulatory T cells and increases in the local concentrations of the anti-inflammatory cytokines TGF-β and IL-1Ra.

Kinetics of neutrophil extracellular traps and cytokines in oral mucositis and Candida infection.

This study investigated the concentrations of neutrophil extracellular traps (NET) and salivary cytokines (IL-1β, IL-6, IL-8/CXCL8, TNF, and TGF-β1) in patients undergoing chemotherapy and their associations with oral mucositis (OM) and Candida infection. This prospective longitudinal study performed at a Brazilian service included 60 adults diagnosed with hematolymphoid diseases. Saliva samples were collected on days D0, D3, D10, and D15. Cytokines were analyzed by ELISA and NET formation by identification of the myeloperoxidase-DNA complex. Oral Candida spp. was cultured. OM occurred in 43.3% of patients and oral candidiasis in 20%. However, 66% of individuals had positive cultures for C. albicans. Higher concentrations of IL-6, IL-8/CXCL8, and TNF and lower concentrations of TGF-β1 were observed in patients with OM. C. albicans infection contributed to the increase in IL-8/CXCL8, TGF-β1, and TNF. Individuals with OM or with oral candidiasis had significant reductions in NET formation. In contrast, individuals with C. albicans and with concomitant C. albicans and OM exhibited higher NET formation. The kinetics of cytokine levels and NET formation in chemotherapy-induced OM appears to be altered by Candida infection, even in the absence of clinical signs of oral candidiasis.

SMAD7 expression in CAR-T cells improves persistence and safety for solid tumors.

Despite the tremendous progress of chimeric antigen receptor T (CAR-T) cell therapy in hematological malignancies, their application in solid tumors has been limited largely due to T-cell exhaustion in the tumor microenvironment (TME) and systemic toxicity caused by excessive cytokine release. As a key regulator of the immunosuppressive TME, TGF-β promotes cytokine synthesis via the NF-κB pathway. Here, we coexpressed SMAD7, a suppressor of TGF-β signaling, with a HER2-targeted CAR in engineered T cells. These novel CAR-T cells displayed high cytolytic efficacy and were resistant to TGF-β-triggered exhaustion, which enabled sustained tumoricidal capacity after continuous antigen exposure. Moreover, SMAD7 substantially reduced the production of inflammatory cytokines by antigen-primed CAR-T cells. Mechanistically, SMAD7 downregulated TGF-β receptor I and abrogated the interplay between the TGF-β and NF-κB pathways in CAR-T cells. As a result, these CAR-T cells persistently inhibited tumor growth and promoted the survival of tumor-challenged mice regardless of the hostile tumor microenvironment caused by a high concentration of TGF-β. SMAD7 coexpression also enhanced CAR-T-cell infiltration and persistent activation in patient-derived tumor organoids. Therefore, our study demonstrated the feasibility of SMAD7 coexpression as a novel approach to improve the efficacy and safety of CAR-T-cell therapy for solid tumors.

NR4A1 PROMOTES OSTEOGENIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS AND IMPROVES INFLAMMATION-INHIBITED BONE REGENERATION

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases.

Assessing angiogenesis factors as prognostic biomarkers in breast cancer patients and their association with clinicopathological factors

Introduction Angiogenesis is fundamental for tumor growth and metastasis across many solid malignancies. Considerable interest has focused on the molecular regulation of tumor angiogenesis as a means to predict disease outcomes and guide therapeutic decisions. Methods In the present study, we investigated the prognostic value of transforming growth factor beta (TGF-β), epidermal growth factor (EGF), fibroblast growth factor (FGF), delta-like ligand 4 (DLL4), and vascular endothelial growth factor (VEGF) in the serum of 120 women diagnosed with breast cancer using ELISA as well as examined their associations with clinical parameters and the outcome of the disease. Results Our results demonstrated that the serum concentration of TGF-β and EGF were remarkably higher in patients with higher tumor size, end stages of the disease, and positive lymph node involvement compared to patients with lower tumor size, early stages of the disease, and negative lymph node involvement. In addition, we found a significant correlation between the serum concentration of VEGF and the level of EGF, FGF, and DLL4 in patients with breast cancer. Furthermore, both univariate and multivariate analyses showed that TGF-β and EGF can be used as end-stage predictors. Discussion/Conclusion Based on our findings, increasing the level of angiogenesis factors is significantly associated with higher tumor size and late stages of the disease in patients with breast cancer. Moreover, measuring the level of angiogenesis factors could lead to better prediction of disease outcomes and choosing the best treatments for patients.

Enhancement of the immunogenicity of a Mycobacterium tuberculosis fusion protein using ISCOMATRIX and PLUSCOM nano-adjuvants as prophylactic vaccine after nasal administration in mice.

Tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (M. tuberculosis), remains a health problem worldwide and this infection has the highest mortality rate among bacterial infections. Current studies suggest that intranasal administration of new TB vaccines could enhance the immunogenicity of M. tuberculosis antigens. Hence, we aim to evaluate the protective efficacy and immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA through intranasal administration in a mice model. In the present study, the recombinant fusion protein was expressed in Escherichia coli and purified and used to prepare different nanoparticle formulations in combination with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA. Mice were intranasally vaccinated with each formulation three times at an interval of 2 weeks. Three weeks after the final vaccination, IFN-γ, IL-4. IL-17, and TGF-β concentrations in the supernatant of cultured splenocytes of vaccinated mice as well as serum titers of IgG1 and IgG2a and sIgA titers in nasal lavage were determined. According to obtained results, intranasally vaccinated mice with formulations containing ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA could effectively induce IFN-γ and sIgA responses. Moreover, both HspX/EsxS/ISCOMATRIX/MPLA and HspX/EsxS/PLUSCOM/MPLA and their BCG booster formulation could strongly stimulate the immune system and enhance the immunogenicity of M. tuberculosis antigens. The results demonstrate the potential of HspX/EsxS-fused protein in combination with ISCOMATRIX, PLUSCOM, and MPLA after nasal administration in enhancing the immune response against M. tuberculosis antigens. Both nanoparticles were good adjuvants in order to promote the immunogenicity of TB-fused antigens. So, nasal immunization with these formulations, could induce immune responses and be considered a new TB vaccine or a BCG booster.

Effects of adipose-derived mesenchymal stem cell conditioned medium on human tenocytes exposed to high glucose.

Diabetic tendinopathy is a common invalidating and challenging disease that may be treated using stem cells. However, the effects of adipose-derived mesenchymal stem cell conditioned medium (ASC-CM) in diabetic tendinopathy have never been explored. The present study evaluated the effects of ASC-CM on morphology, cell viability, structure, and scratch wound closure of human tenocytes (HTNC) exposed to high glucose (HG). Experimental study. HTNC were exposed to HG (25 mM) for 7, 14 and 21 days with or without ASC-CM for the last 24 h. CM was collected from 4 × 105 ASCs, centrifuged for 10 min at 200 g and sterilized with 0.22 μm syringe filter. At 7 days, HG-HTNC had decreased cell viability [72 ± 2%, p < 0.01 versus normal glucose (NG)] compared to NG-HTNC (90 ± 5%). A further decrement was detected after 14 and 21 days (60 ± 4% and 60 ± 5%, both, p < 0.01 versus NG and p < 0.01 versus HG7). While NG-HTNC evidenced a normal fibroblast cell-like elongated morphology, HG-HTNC showed increased cell roundness. In contrast, HG-HTNC exposed to ASC-CM showed a significant increase in cell viability, an improved cell morphology and higher scratch wound closure at all HG time points. Moreover, the exposure to ASC-CM significantly increased thrombospondin 1 and transforming growth factor beta 1 (TGF-β1) content in HG-HTNC. The TGF-β1 elevation was paralleled by higher Collagen I and Vascular Endothelial Growth Factor in HG-HTNC. ASC-CM may restore the natural morphology, cell viability and structure of HTNC, promoting their scratch wound closure through TGF-β1 increase.

Evaluation of the Immune Checkpoints, TIM-3 and PD-1, as well as Anti-Inflammatory Cytokines IL-10, and TGF-β along with Diseases Activity in Chronic Spontaneous Urticaria.

Chronic spontaneous urticaria (CSU) is a skin disease caused by mast cells that produce inflammatory mediators. Immune checkpoint receptors such as program death-1 (PD-1) and T-cell immunoglobulin and mucin domain 3 (TIM-3) are essential for the pathophysiology of many autoimmune and allergic diseases. The aim of this study was to investigate the expression of PD-1 and TIM-3 in CSU patients and their relationship to the anti-inflammatory cytokines (TGF-β and IL-10). In the current study, peripheral blood mononuclear cells (PBMCs) from CSU patients and healthy individuals were used and the Urticaria Activity Score 7 (UAS7) was used to assess disease severity. TaqMan-based RT-PCR was used to assess the expression of TIM-3 and PD-1 as well as the anti-inflammatory cytokines transforming growth factor-β (TGF-β) and IL-10. The protein concentrations of TGF-β and IL-10 were also measured by ELISA. The relationship between the expression of TIM-3 and PD-1 as well as TGF- β and IL-10 and the severity of the disease was investigated. The results showed that PD-1 mRNA expression was significantly increased in CSU patients (P<0.0001), while TGF- β and IL-10 levels were higher in CSU patients, but this difference was not significant (p=0.638, p= 0.798). The increase in protein level of IL-10 was significant (P<0.0001). There was also a positive correlation between the expression of PD-1 and TGF- β molecules and disease activity (P=0.0043, P=0.0018). In conclusion, the study found that the immune system expresses inhibitory molecules and anti-inflammatory cytokines to control disease severity. The higher expression of PD-1 molecules and IL-10 is associated with disease severity, suggesting that the immune system is trying to control inflammation and reduce disease severity.

Inhibition of TGFβ1 activation prevents radiation-induced lung fibrosis.

Radiotherapy is the main treatment modality for thoracic tumours, but it may induce pulmonary fibrosis. Currently, the pathogenesis of radiation-induced pulmonary fibrosis (RIPF) is unclear, and effective treatments are lacking. Transforming growth factor beta 1 (TGFβ1) plays a central role in RIPF. We found that activated TGFβ1 had better performance for radiation pneumonitis (RP) risk prediction by detecting activated and total TGFβ1 levels in patient serum. αv integrin plays key roles in TGFβ1 activation, but the role of αv integrin-mediated TGFβ1 activation in RIPF is unclear. Here, we investigated the role of αv integrin-mediated TGFβ1 activation in RIPF and the application of the integrin antagonist cilengitide to prevent RIPF. ItgavloxP/loxP ;Pdgfrb-Cre mice were generated by conditionally knocking out Itgav in myofibroblasts, and wild-type mice were treated with cilengitide or placebo. All mice received 16Gy of radiation or underwent a sham radiation procedure. Lung fibrosis was measured by a modified Ashcroft score and microcomputed tomography (CT). An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum TGFβ1 concentration, and total Smad2/3 and p-Smad2/3 levels were determined via Western blotting. Conditional Itgav knockout significantly attenuated RIPF (p<.01). Hounsfield units (HUs) in the lungs were reduced in the knockout mice compared with the control mice (p<.001). Conditional Itgav knockout decreased active TGFβ1 secretion and inhibited fibroblast p-Smad2/3 expression. Exogenous active TGFβ1, but not latent TGFβ1, reversed these reductions. Furthermore, cilengitide treatment elicited similar results and prevented RIPF. The present study revealed that conditional Itgav knockout and cilengitide treatment both significantly attenuated RIPF in mice by inhibiting αv integrin-mediated TGFβ1 activation. Activated TGFβ1 has a superior capacity in predicting radiation pneumonitis (RP) risk and plays a vital role in the development of radiation-induced pulmonary fibrosis (RIPF). Conditional knock out Itgav in myofibroblasts prevented mice from developing RIPF. Cilengitide alleviated the development of RIPF by inhibiting αv integrin-mediated TGFβ1 activation and may be used in targeted approaches for preventing RIPF.

Human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium.

To investigate human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium (CM). A human corneal epithelial cell line (HCE-T), human corneal fibroblasts (HCF) and keratoconus fibroblasts (KC-HCF) have been used. Twenty-four hours after RB-PDT (0.001% RB concentration, 565 nm wavelength illumination, 0.17 J/cm2 fluence) cell migration rate using scratch assay and growth factor concentrations in the cell culture supernatant using ELISA have been determined. In addition, the effect of CM has been observed. RB-PDT significantly reduced migration rate in all cell types, compared to controls (p≤0.02). Migration rate of HCE-T cultures without RB-PDT (untreated) was significantly higher using HCF CM after RB-PDT, than using HCF CM without RB-PDT (p<0.01). Similarly, untreated HCF displayed a significantly increased migration rate with HCE-T CM after RB-PDT, compared to HCE-T CM without treatment (p<0.01). Furthermore, illumination alone and RB-PDT significantly decreased keratinocyte growth factor (KGF) concentration in HCF and KC-HCF supernatant, and RB-PDT significantly decreased soluble N-Cadherin (SN-Cad) concentration in HCF supernatant, compared to controls (p<0.01 for all). In HCE-T CM, RB-PDT increased hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGFb) concentration (p≤0.02), while decreasing transforming growth factor β (TGF-β) concentration (p<0.01). FGFb concentration increased (p<0.0001) and TGF-β concentration decreased (p<0.0001) in HCF CM, by RB-PDT. Epidermal growth factor (EGF), HGF, and TGF-β concentration decreased (p≤0.03) and FGFb concentration increased (p<0.01) in KC-HCF CM, using RB-PDT. HCE-T, HCF and KC-HCF migration rate is reduced 24 hours after RB-PDT. In contrast, HCE-T migration is enhanced using HCF CM after RB-PDT, and HCF migration rate is increased through HCE-T CM following RB-PDT. Modulation of EGF, KGF, HGF, FGFb, TGF-β and N-Cadherin secretion through RB-PDT may play an important role in corneal wound healing.

PRODUCTION OF GROWTH FACTORS AND DESQUAMATION OF ENDOTHELIOCYTES IN THE HEART IN ISCHEMIC CARDIOMYOPATHY

Highlights Dysregulation of angiogenesis may be the pathogenetic factor of ischemic cardiomyopathy (ICMP). Aim. To determine the content of growth factors and desquamated endothelial cells (DEC) in the blood from the coronary sinus and ulnar vein in association with the number of progenitor endothelial cells (PEC) in the blood from the ulnar vein in patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Methods. The study included 30 patients with ICMР and 22 patients with CHD, and 18 healthy donors. The content of DEC (CD45–CD146+) was determined in blood from the cubital vein (peripheral) and coronary sinus, and the content of DEC (CD14+CD34+VEGFR2+) was determined in peripheral blood by flow cytometry (antibodies “BD Biosciences”, USA). The concentrations of VEGF-A, VEGF-B, PDGF, SDF-1, SCF, FGF-1, TGF-β1 in blood plasma from both locations were evaluated by multiplex analysis (set “Cloud-Clone Corp.”, USA).Results. The content of DEC in peripheral blood was elevated in patients with CHD of both groups, and in patients with ICMP in sinus blood was higher than in peripheral. At the same time, in patients with CHD without cardiomyopathy, an excess of PEC and SDF-1 in the blood from the ulnar vein was established in combination with an increase in the concentration of PDGF and a decrease in the content of VEGF-B in the blood from the coronary sinus relative to the parameters of systemic blood flow. In patients with ICMP, these changes were not detected, but there was an increase in the concentration of TGF-β1 in sinus blood compared with peripheral blood. Regardless of the presence of ICMP, the concentration of SCF, FGF-1, VEGF-A in the blood from the ulnar vein corresponded to the norm and that in sinus blood; the content of VEGF-A in the coronary bloodstream exceeded its systemic level.Conclusion. In patients with ICMP, desquamation of the coronary vascular endothelium is enhanced against the background of violations of its repair processes due to insufficient (relative to CHD without cardiomyopathy) mobilization of PEC from the bone marrow due to the absence of an excess of SDF-1 in the blood and their insufficient homing into the myocardium due to weak PDGF production in the heart.