Concentrations Of TGF-β1 Research Articles

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.

EIF4A3-induced circle RNA_ 0001860 promotes growth, invasion, and immune evasion of gastric cancer cells through the miR-618/HMGA2 axis.

Gastric cancer is a globally common type of cancer with a dismal prognosis. Circle RNA_ 0001860 (circ_0001860) is dysregulated in several cancers and involved in cancer development. Its involvement in stomach cancer, however, remains unclear. AGS and HGC-27 cells were transfected with shRNA against circ_0001860 to knock down the circ_0001860 level. The function of circ_0001860 in gastric cancer was analyzed by cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, Transwell, ELISA, dual-luciferase reporter, RIP, RNA pull-down, and western blotting. Nude mice were subcutaneously inoculated with AGS cells with lentivirus-packaged sh-circ_0001860 to determine the role of sh-circ_0001860 in gastric cancer by immunohistochemistry assays. circ_0001860 was upregulated in gastric cancer, indicating a poor prognosis for gastric cancer patients. Knockdown of circ_0001860 reduced gastric cancer cells' viability, the percentage of EdU-positive cells, the numbers of invaded cells, and concentrations of IL-10 and TGF-β while fostering the concentrations of IFN-γ and IL-2. circ_0001860 sponged miR-618 to positively modulate the HMGA2 level in gastric cancer cells. The inhibitory role of sh-circ_0001860 on cell growth, invasion, and immune evasion of gastric cancer was abolished with the miR-618 knockdown or the HMGA2 overexpression. Besides, EIF4A3 positively modulated the circ_0001860 level in gastric cancer cells. In vivo, silencing of circ_0001860 reduced tumor size and weight and the expression of HMGA2 and IL-10 but enhanced the level of miR-618 and IFN-γ. Collectively, circle RNA_ 0001860 was induced by EIF4A3 to enhance proliferation, invasion, and immune evasion of gastric cancer through the miR-618/HMGA2 axis.

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.

CYTOKINE-MODULATING INFLUENCE OF HIPEC ON THE INTRAPERITONEAL HOMEOSTASIS FORMATION IN PATIENTS WITH OVARIAN CANCER

Background: Ovarian cancer (OC) has the greatest mortality rate among oncogynecological diseases. Most cases are diagnosed at the peritoneal dissemination stage, resulting in radical treatment. Most cases (75%) are diagnosed in III-IV stages at the stage of peritoneal dissemination, making the radical treatment impossible. Intraoperative hyperthermic intraperitoneal chemoperfusion (HIPEC), which has ceased to be an experimental technique over the past decade, is increasingly used among the methods of combating peritoneal carcinomatosis and demonstrates an increase in recurrence-free and overall survival in advanced stages of OC. Nevertheless, certain pathophysiological aspects of the effect of HIPEC on intra-abdominal homeostasis, and therefore on the further course of the disease, have not been elucidated. However, understanding this effect may be the key to the successful application of HIPEC and predicting its efficacy in each case of OC.&#x0D; Objectives: To access changes in intraperitoneal homeostasis in patients with advanced OC after HIPEC procedure (as a stage of cytoreductive surgery); in particular, to evaluate changes of TNF and TGF-β expression under the influence of HIPEC and systemic chemotherapy in the OC microenvironment.&#x0D; Materials and methods: The study included 33 OC patients treated at Lviv Regional Cancer Centre in 2016–2020. Twelve of them received HIPEC. The primary tumor cultures and primary cultures of peritoneal mesothelium and macrophages were obtained for all patients. The cytokines TNF and TGF-β activity were determined in their conditional media.&#x0D; Results: The decrease of TNF and TGF-β concentrations in the drainage exudate after HIPEC was revealed compared with the same parameters in the ascitic fluid before the operation. One-hour hyperthermia of the primary culture of peritoneal ascites-associated macrophages of recurrent OC patients decreased the TNF level in conditional media. A decline was found in 10 out of 12 cases.&#x0D; Conclusions: HIPEC has a cytokine-modulating effect on the intra-abdominal homeostasis of patients with OC, suppressing TNF expression by peritoneal macrophages. Reduced activity of TNF in supernatants of OC primary tumor cultures was associated with higher sensitivity to chemotherapy.

Nicorandil and carvedilol mitigates motor deficits in experimental autoimmune encephalomyelitis-induced multiple sclerosis: Role of TLR4/TRAF6/MAPK/NF-κB signalling cascade

Multiple sclerosis (MS) is an inflammatory demyelinating neurodegenerative disease that negatively affects neurotransmission. It can be pathologically mimicked by experimental autoimmune encephalomyelitis (EAE) animal model. ATP-sensitive potassium channels (KATP) plays a crucial role in the control of neuronal damage, however their role in MS are still obscure. Additionally, Carvedilol showed a promising neuroprotective activity against several neurological disorders. Therefore, the present study aimed to investigate the potential neuroprotective effect of KATP channel opener (nicorandil) as well as α and β adrenoceptor antagonist (Carvedilol) against EAE induced neurodegeneration in mice. Mice was treated with nicorandil (6 mg/kg/day; p.o.) and carvedilol (10 mg/kg/day; p.o.) for 14 days. Nicorandil and carvedilol showed improvement in clinical scoring, behaviour and motor coordination as established by histopathological investigation and immunohistochemical detection of MBP. Furthermore, both treatments downregulated the protein expression of TLR4/ MYD88/TRAF6 signalling cascade with downstream inhibition of (pT183/Y185)-JNK/p38 (pT180/Y182)-MAPK axis leading to reduction of neuroinflammatory status, as witnessed by reduction of NF-κB, TNF-α, IL-1β and IL-6 contents. Moreover, nicorandil and carvedilol attenuated oxidative damage by increasing Nrf2 content and SOD activity together with reduction of MDA content. In addition, an immunomodulating effect via inhibiting the gene expression of CD4, TGF-β, and IL-17 as well as TGF-β, IL-17, and IL-23 contents along with anti-apoptotic effect by decreasing Bax protein expression and Caspase-3 content and increasing Bcl-2 protein expression was observed with nicorandil and carvedilol treatments. In conclusion, nicorandil and carvedilol exerted a neuroprotective activity against EAE induced neuronal loss via inhibition of TLR4/MYD88/TRAF6/JNK/p38-MAPK axis besides antioxidant and anti-apoptotic effects.

Elevated CD39+T-Regulatory Cells and Reduced Levels of Adenosine Indicate a Role for Tolerogenic Signals in the Progression from Moderate to Severe COVID-19.

Viral infections trigger inflammation by controlling ATP release. CD39 ectoenzymes hydrolyze ATP/ADP to AMP, which is converted by CD73 into anti-inflammatory adenosine (ADO). ADO is an anti-inflammatory and immunosuppressant molecule which can enhance viral persistence and severity. The CD39-CD73-adenosine axis contributes to the immunosuppressive T-reg microenvironment and may affect COVID-19 disease progression. Here, we investigated the link between CD39 expression, mostly on T-regs, and levels of CD73, adenosine, and adenosine receptors with COVID-19 severity and progression. Our study included 73 hospitalized COVID-19 patients, of which 33 were moderately affected and 40 suffered from severe infection. A flow cytometric analysis was used to analyze the frequency of T-regulatory cells (T-regs), CD39+ T-regs, and CD39+CD4+ T-cells. Plasma concentrations of adenosine, IL-10, and TGF-β were quantified via an ELISA. An RT-qPCR was used to analyze the gene expression of CD73 and adenosine receptors (A1, A2A, A2B, and A3). T-reg cells were higher in COVID-19 patients compared to healthy controls (7.4 ± 0.79 vs. 2.4 ± 0.28; p < 0.0001). Patients also had a higher frequency of the CD39+ T-reg subset. In addition, patients who suffered from a severe form of the disease had higher CD39+ T-regs compared with moderately infected patients. CD39+CD4+ T cells were increased in patients compared to the control group. An analysis of serum adenosine levels showed a marked decrease in their levels in patients, particularly those suffering from severe illness. However, this was paralleled with a marked decline in the expression levels of CD73. IL-10 and TGF-β levels were higher in COVID-19; in addition, their values were also higher in the severe group. In conclusion, there are distinct immunological alterations in CD39+ lymphocyte subsets and a dysregulation in the adenosine signaling pathway in COVID-19 patients which may contribute to immune dysfunction and disease progression. Understanding these immunological alterations in the different immune cell subsets and adenosine signaling provides valuable insights into the pathogenesis of the disease and may contribute to the development of novel therapeutic approaches targeting specific immune mechanisms.

Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-β) signaling drives metastasis and is strongly enhanced during cancer progression. Yet, the use of on-target TGF-β signaling inhibitors in the treatment of cancer patients remains unsuccessful, highlighting a gap in the understanding of TGF-β biology that limits the establishment of efficient anti-metastatic therapies. Here, we show that TGF-β signaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle (sEV) secretion. Demonstrating sEV’s unique role, TGF-β signaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-β. Further, genetic disruption of sEV secretion in highly-metastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-β signaling to nearly-normal levels. Otherwise, TGF-β signaling activity in non-invasive breast cancer cells is inherently low, but can be amplified by sEVs, enabling invasion and metastasis of poorly-metastatic breast cancer cells. Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers, treatment with dimethyl amiloride (DMA) decreases sEV secretion, TGF-β signaling activity, and breast cancer progression in vivo. Targeting both the sEV trafficking and TGF-β signaling by combining DMA and SB431542 at suboptimal doses potentiated this effect, normalizing the TGF-β signaling in primary tumors to potently reduce circulating tumor cells, metastasis, and tumor self-seeding. Collectively, this study establishes sEVs as critical elements in TGF-β biology, demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-β signaling levels in breast cancer cells.

The Influence of Betulin Derivatives EB5 and ECH147 on the Expression of Selected TGFβ Superfamily Genes, TGFβ1, GDF15 and BMP2, in Renal Proximal Tubule Epithelial Cells.

Betulin derivatives are proposed to serve as an alternative to the drugs already established in oncologic treatment. Drug-induced nephrotoxicity leading to acute kidney injury frequently accompanies cancer treatment, and thus there is a need to research the effects of betulin derivatives on renal cells. The objective of our study was to assess the influence of the betulin derivatives 28-propynylobetulin (EB5) and 29-diethoxyphosphoryl-28-propynylobetulin (ECH147) on the expression of TGFβ1, BMP2 and GDF15 in renal proximal tubule epithelial cells (RPTECs) cultured in vitro. The changes in mRNA expression and copy numbers were assessed using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) and the standard curve method, respectively. An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the effect of the betulin derivatives on the protein concentration in the culture media's supernatant. The assessment of the betulin derivatives' influence on gene expression demonstrated that the mRNA level and protein concentration did not always correlate with each other. Each of the tested compounds affected the mRNA expression. The RT-qPCR analyses showed that EB5 and ECH147 induced effects similar to those of betulin or cisplatin and resulted in a decrease in the mRNA copy number of all the analyzed genes. The ELISA demonstrated that EB5 and ECH147 elevated the protein concentration of TGFβ1 and GDF15, while the level of BMP2 decreased. The concentration of the derivatives used in the treatment was crucial, but the effects did not always exhibit a simple linear dose-dependent relationship. Betulin and its derivatives, EB5 and ECH147, influenced the gene expression of TGFβ1, BMP2 and GDF15 in the renal proximal tubule epithelial cells. The observed effects raise the question of whether treatment with these compounds could promote the development of renal fibrosis.

NF-α, IL6, IL1-α, IL-10 and TGF-β1 Gene Expression in Iranian Acute Myeloid Leukemia Patients

Acute myeloid leukemia is a heterogeneous clonal disorder of blood-producing cells that are among the most common malignant disorders in adults. The purpose of this study was to examine the gene expression of TNF-α, IL6, IL1-α, IL-10, and TGF-β1 at diagnosis in acute myeloid leukemia. The current study included 50 patients with acute myeloid leukemia and 50 control subjects. ELISA was used to determine the serum concentrations of TNF-α, IL6, IL1-α, IL-10, and TGF-β1. After cDNA synthesis and RNA extraction, the expression of TNF-α, IL6, IL1-α, IL-10, and TGF-β1 genes was determined using real-time PCR (ΔΔCT computational). Statistical analysis was conducted with Statistical Package for the Social Sciences 19, and P&lt;0.05 was deemed statistically significant. The mean serum levels of TNF-α, IL6, IL1-α, IL-10, and TGF-β1 were lower in patients with acute myeloid leukemia than in the control group. In addition, cytokine mRNA expression in peripheral blood mononuclear cells was significantly lower in AML patients compared to the control group (p&lt;0.0001). Changes in TNF-α, IL6, IL1-α, IL-10, and TGF-β1 were associated with acute myeloid leukemia, according to the findings. The current study supports the use of cytokines as diagnostic biomarkers for acute myeloid leukemia.

Integrin αV mediated activation of myofibroblast via mechanoparacrine of transforming growth factor β1 in promoting fibrous scar formation after myocardial infarction

BackgroundMyocardial infarction (MI) dramatically changes the mechanical stress, which is intensified by the fibrotic remodeling. Integrins, especially the αV subunit, mediate mechanical signal and mechanoparacrine of transforming growth factor β1 (TGF-β1) in various organ fibrosis by activating CFs into myofibroblasts (MFBs). We investigated a possible role of integrin αV mediated mechanoparacrine of TGF-β1 in MFBs activation for fibrous reparation in mice with MI. MethodsHeart samples from MI, sham, or MI plus cilengitide (14 mg/kg, specific integrin αV inhibitor) treated mice, underwent functional and morphological assessments by echocardiography, and histochemistry on 7, 14 and 28 days post-surgery. The mechanical and ultrastructural changes of the fibrous scar were further evaluated by atomic mechanics microscope (AFM), immunofluorescence, second harmonic generation (SHG) imaging, polarized light and scanning electron microscope, respectively. Hydroxyproline assay was used for total collagen content, and western blot for protein expression profile examination. Fibroblast bioactivities, including cell shape, number, Smad2/3 signal and expression of extracellular matrix (ECM) related proteins, were further evaluated by microscopic observation and immunofluorescence in polyacrylamide (PA) hydrogel with adjustable stiffness, which was re-explored in fibroblast cultured on stiff matrix after silencing of integrin αV. The content of total and free TGF-β1 was tested by enzyme-linked immunosorbent assay (ELISA) in both infarcted tissue and cell samples. ResultIncreased stiffness with heterogeneity synchronized with integrin αV and alpha smooth muscle actin (α-SMA) positive MFBs accumulation in those less mature fibrous areas. Cilengitide abruptly reduced collagen content and disrupted collagen alignment, which also decreased TGF-β1 bioavailability, Smad2/3 phosphorylation, and α-SMA expression in the fibrous area. Accordingly, fibroblast on stiff but not soft matrix exhibited obvious MFB phenotype, as evidenced by enlarged cell, hyperproliferation, well-developed α-SMA fibers, and elevated ECM related proteins, while silencing of integrin αV almost abolished this switch via attenuating paracrine of TGF-β1 and nuclear translocation of Smad2/3. ConclusionThis study illustrated that increased tissue stiffness activates CFs into MFBs by integrin αV mediated mechanoparacrine of TGF-β1, especially in immature scar area, which ultimately promotes fibrous scar maturation.

Leucine-Rich Repeat Containing 15-Mediated Cell Adhesion Is Essential for Integrin Signaling in TGF-β1-Induced PDL Fibroblastic Differentiation.

Human periodontal ligament cells (hPDLCs) cultured from periodontal ligament (PDL) tissue contain postnatal stem cells that can be differentiated into PDL fibroblasts. We obtained PDL fibroblasts from hPDLCs by treatment with low concentrations of TGF-β1. Since the extracellular matrix and cell surface molecules play an important role in differentiation, we had previously developed a series of monoclonal antibodies against PDL fibroblast-specific cell surface molecules. One of these, the anti-PDL51 antibody, recognized a protein that was significantly upregulated in TGF-β1-induced PDL fibroblasts and highly accumulated in the PDL region of the tooth root. Mass spectrometry revealed that the antigen recognized by the anti-PDL51 antibody was leucine-rich repeat containing 15 (LRRC15), and this antibody specifically recognized the extracellular glycosylated moiety of LRRC15. Experiments presented here show that as fibroblastic differentiation progresses, increased amounts of LRRC15 localized at the cell surface and membrane. Inhibition of LRRC15 by siRNA-mediated depletion and by antibody blocking resulted in downregulation of the representative PDL fibroblastic markers. Moreover, following LRRC15 inhibition, the directed and elongated cell phenotypes disappeared, and the long processes of the end of the cell body were no longer found. Through a specific interaction between integrin β1 and LRRC15, the focal adhesion kinase signaling pathway was activated in PDL fibroblasts. Furthermore, it was shown that increased LRRC15 was important for the activation of the integrin-mediated cell adhesion signal pathway for regulation of cellular functions, including fibroblastic differentiation, proliferation, and cell migration arising from the expression of PDL-related genes in TGF-β1-induced PDL fibroblastic differentiation.

SiRNA-Mediated B7H7 Knockdown in Gastric Cancer Lysate-Loaded Dendritic Cells Amplifies Expansion and Cytokine Secretion of Autologous T Cells.

Gastric cancer, ranked as the fifth most common cancer worldwide, presents multiple treatment challenges. These obstacles often arise due to cancer stem cells, which are associated with recurrence, metastasis, and drug resistance. While dendritic cell (DC)-based immunotherapy has shown promise as a therapeutic strategy, its efficacy can be limited by the tumor microenvironment and certain inhibitory immune checkpoint molecules, such as B7H7. SiRNA-medicated knockdown of B7H7 in tumor cell lysate-pulsed DCs can increase cytokine secretion and autologous T lymphocyte expansion. This study aimed to evaluate the impact of B7H7 suppression in gastric cancer cell lysate-pulsed DCs on the stimulatory potential of autologous CD3+ T lymphocytes. Peripheral blood mononuclear cells (PBMCs) were isolated and monocytes were obtained; then, they were differentiated to immature DCs (iDCs) by GM-CSF and IL-4. Tumor cell lysates from human gastric cancer cell lines were harvested, and iDCs were transformed into mature DCs (mDCs) by stimulating iDCs with tumor cell lysate and lipopolysaccharide. B7H7-siRNA was delivered into mDCs using electroporation, and gene silencing efficiency was assessed. The phenotypic characteristics of iDCs, mDCs, and B7H7-silenced mDCs were evaluated using specific surface markers, an inverted light microscope, and flow cytometry. CD3+ T cells were isolated via magnetically activated cell sorting. They were labeled with CFSE dye and co-cultured with mDCs and B7H7-silenced mDCs to evaluate their ability to induce T-cell proliferation. T-cell proliferation was assessed using flow cytometry. The concentration of TGF-β, IL-4, and IFN-γ secreted from CD3+ T cells in the co-cultured supernatant was evaluated to investigate the cytokine secretory activity of the cells. Transfection of B7H7 siRNA into mDCs was performed in optimal conditions, and the siRNA transfection effectively reduced B7H7 mRNA expression in a dose-dependent manner. SiRNA-mediated B7H7 knockdown in mDCs enhanced maturation and activation of the DCs, as demonstrated by an increased surface expression of CD11c, CD86, and CD40. Co-culture experiments revealed that B7H7-silenced mDCs had more capacity to induce T cell proliferation compared to non-transfected mDCs. The cytokine production patterns of T cells were also altered. Upon examining the levels of TGF-β, IL-4, and IFN-γ released by CD3+ T cells in the co-culture supernatant, we found that silencing B7H7 in mDCs resulted in a rise in IL-4 secretion and a reduction in TGF-β levels compared to mDCs that were not transfected. The study found that suppressing B7H7 expression in DCs significantly enhances their maturation and stimulatory activity when exposed to gastric cancer cell lysate. These B7H7-silenced DCs can substantially increase cytokine production and promote co-cultured T-cell expansion. Consequently, inhibiting B7H7 in DCs may offer a practical strategy to enhance the ability of DCs to initiate T lymphocyte responses and improve the effectiveness of DC-based cell therapy for cancer patients.

Decreased circulating transforming growth factor-beta (TGF-β) and kidney TGF-β immunoreactivity predict renal disease in cats with naturally occurring chronic kidney disease.

The aim of the present study was to compare the circulating transforming growth factor-beta (TGF-β) of clinically normal age-matched and naturally occurring chronic kidney disease (CKD) cats and to determine the correlation between the TGF-β expression and histopathological changes in cats with CKD. A total of 11 clinically normal age-matched and 27 cats with naturally occurring CKD were included in this study. Circulating TGF-β was quantified by immunoassays. Kaplan-Meier analysis was used to calculate the association between survival time and the concentration of circulating TGF-β. A general linear model was used to compare the circulating TGF-β between groups. Immunohistochemical analyses revealed TGF-β expression in renal tissues from cats with CKD that died during the study (n = 7) and in available archived renal tissue specimens taken at necropsy from cats that had previous CKD with renal lesions (n = 10). Correlations of the TGF-β expression and clinical parameters (n = 7) and histopathological changes (n = 17) were analysed using Spearman's rank correlation. The median survival time of cats with a lower concentration of circulating TGF-β was shorter than that of cats with a higher concentration. The area under the curve of circulating TGF-β for predicting CKD was 0.781, indicating good differentiation. The study indicated a significant difference in circulating TGF-β concentrations between clinically normal cats and those with CKD and demonstrated that TGF-β expression is correlated with tubular atrophy. The study findings suggest that decreased serum TGF-β and tubular atrophy with TGF-β immunoreactivity may be significant in cats with CKD.

Industrial Mass Production of Platelet Dry Powder

The goal of this paper is to examine the use of pig blood in the industrial mass production of platelet dry powder and to transform platelet dry powder into a low-cost and mass-produced material. Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) contain multiple types of growth factors (GFs) and can be widely used in medical applications. However, neither can be mass-produced, due to the complexity of the PRP preparation process and the lack of anticoagulants in the PRF preparation process, increasing the risk of coagulation during mass production. Another obstacle is the insufficient supply of autologous PRP and autologous PRF. In this study, platelet dry powder was mass-produced from pig blood through the indirect addition of calcium chloride solution. Furthermore, the results showed that different concentrations and percentages of calcium chloride solution had significant effects on concentrations of TGF-β1 and PDGF-BB in the platelet dry powder. The platelet dry powder mass-produced from pig blood demonstrated high concentrations of GFs and long-term shelf stability, increasing the supply to industries that use it in product development.

Electroacupuncture improves cognitive dysfunction in rats with Alzheimer's disease by regulating microglial cells.

To observe the effect of electroacupuncture (EA) on microglia (MG), Janus kinase-2 (JAK2) and signal transducer and activator of transcription-3 (STAT3) in hippocampal CA1 region of Alzheimer's di-sease (AD) rats, so as to explore its mechanisms in the treatment of AD. Thirty-six male SD rats were randomly divided into sham operation, model and EA groups, with 12 rats in each group. The AD rat model was established by intraperitoneal injection of D-galactose combined with intrahippocampal injection of aggregated Aβ25-35. The rats in the EA group were given EA (2 Hz/20 Hz, 2 mA) at "Baihui"(GV20) and"Shenting"(GV24) for 30 min, once daily, 6 days a week for 4 weeks. Morris water maze test was used to detect the learning and memory ability and spatial exploration ability of rats. HE staining was used to observe the pathological changes of hippocampus. The ultrastructure of hippocampal neurons was observed by transmission electron microscopy. The positive expression of MG marker io-nized calcium adaptor protein (Iba-1) in hippocampus was observed by immunofluorescence staining. The expression levels of serum inflammatory factor interferon-γ (IFN-γ) and transforming growth factor beta 1 (TGF-β1) were detected by ELISA. The mRNA expression levels of JAK2, STAT3, inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) in hippocampal CA1 region were detected by real-time quantitative PCR. The protein and phosphorylation levels of JAK2 and STAT3 in hippocampal CA1 region were detected by Western blot. Compared with the sham operation group, the escape latency of the model group was significantly prolonged (P<0.01), and the number of crossing the original platform was significantly reduced (P<0.01), the positive expression of Iba-1 in CA1 region, the content of serum IFN-γ, the relative mRNA expressions of JAK2, STAT3 and iNOS, and the protein and phosphorylation levels of JAK2 and STAT3 were significantly increased (P<0.01), while the content of serum TGF-β1 and the relative expression of Arg-1 mRNA were significantly decreased (P<0.01). Compared with the model group, the escape latency of rats in the EA group was significantly shortened (P<0.01), the number of crossing the original platform was significantly increased (P<0.01), the positive expression of Iba1, the content of serum IFN-γ, the mRNA expressions of JAK2, STAT3 and iNOS, and the protein and phosphorylation levels of JAK2 and STAT3 were significantly decreased (P<0.05, P<0.01), while the content of serum TGF-β1 and the expression of Arg-1 mRNA were significantly increased (P<0.01). Moreover, pathological and ultrastructural observation showed a reduction in the number of hippocampal neurons, changement of nuclear morphology, dilation of intercellular space, and decreased number of mitochondria in the model group；these situations were relatively milder in the EA group. EA can improve the learning and memory function of AD rats, which may be associated with its functions in decreasing MG activities, and inhibiting the JAK2 / STAT3 signaling pathway in the hippocampus.

Euphorbia factor L1 inhibited transport channel and energy metabolism in human colon adenocarcinoma cell line Caco-2

Euphorbia factor L1 (EFL1) is a kind of lathyrane-type diterpenoid and is isolated from the medical herb Euphorbia lathyris L. (Euphorbiaceae); it has been reported with the toxicity that causes intestinal irritation, but the underlying mechanisms are still obscure. The objective of this study was to assess the EFL1-induced intestinal cytotoxicity in human colon adenocarcinoma Caco-2 cells. The Caco-2 cells were treated with EFL1, and the intracellular calcium ion concentration, mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP), adenosine 5′-triphosphate (ATP) content, ATPase activities, TGF-β1 concentration, and transepithelial electrical resistance (TEER) were detected. The interaction between EFL1 and the tight junction proteins Occludin, Claudin-4, Tricellulin, ZO-1, JAM-1, and E-cadherin was simulated by molecular docking. The expression of proteins involved in the energy metabolism, the ion transporters and aquaporins, the tight junction, and the F-actin cytoskeleton were detected by Western blotting and cell immunofluorescence. As a result, EFL1 decreased the intracellular Ca2+, MMP, mPTP, ATP content, and ATPase activities in the Caco-2 cells. The AMPK/SIRT1/PGC-1α signaling pathway, which regulates the energy metabolism, was inhibited. The ion transporters NEH and CFTR, as well as the aquaporins in the Caco-2 cells, were decreased. The tight junction proteins were down-regulated, and the integrity of the intestinal barrier was injured; TGF-β1 was compensatively increased; so, the intestinal permeability was increased and was characterized by decreased TEER. The morphology of the F-actin cytoskeleton was destroyed. These findings indicated that EFL1 caused cytotoxicity in the human intestinal Caco-2 cells through mitochondrial damage, inhibition of the energy metabolism, and suppression of the ion and water molecule transporters, as well as the down-regulation tight junction and cytoskeleton protiens.