TGF-β1 Levels Research Articles

Indirubin induces tolerogenic dendritic cells via aryl hydrocarbon receptor activation and ameliorates allergic asthma in a murine model by expanding Foxp3-expressing regulatory T cells

BackgroundAllergic asthma is a chronic bronchial inflammatory disease closely associated with abnormal immune responses of dendritic cells (DCs) and allergen-specific type 2 T helper (Th2) cells. Indirubin (IR), a natural aryl hydrocarbon receptor (AhR) ligand, exerts anti-inflammatory and immunomodulatory properties. PurposeIn this study, we aimed to clarify whether IR exhibits immunomodulatory action on DCs via AhR activation and investigated the antiallergic effects of IR in a mouse model of allergic asthma. MethodsLipopolysaccharide (LPS)-activated bone marrow-derived DCs were treated with IR. Their mRNA expressions, cytokine production, and phenotype patterns were determined by a quantitative real-time PCR, ELISA, flow cytometry, and RNA sequencing. The mixed lymphocyte reaction was utilized to evaluate the regulatory function of IR-treated DCs on T-cell differentiation. Moreover, mice with ovalbumin (OVA)-induced allergic asthma were treated with IR. Thereafter, the airway hyperresponsiveness (AHR), allergen-specific IgE production, cytokine levels, airway inflammation, and T-cell responses were evaluated. ResultsTreatment of LPS-stimulated DCs with 20 μM IR significantly reduced IL-12 and TNF-α production while increasing IL-10 secretion. Meanwhile, these DCs expressed decreased levels of CD80 but increased levels of Jagged 1 surface molecules. However, the effects of IR on DCs were reversed by pretreatment with the AhR antagonist, CH223191. Additionally, the coculture of these tolerogenic-like DCs with allogeneic CD4+T cells promoted the generation of Foxp3+ regulatory T (Treg) cells. A transcriptomic analysis identified several downregulated genes that are involved in regulating cell migration, cytokine secretion, and inflammatory responses in DCs after IR treatment. In an asthmatic murine model, oral administration of 25 mg kg-1 body weight of IR efficiently alleviated the development of AHR, OVA-specific IgE production, and levels of Th2-type cytokines (IL-4, IL-5, and IL-13) and the CCL11 chemokine. IR treatment also attenuated inflammatory cell recruitment and mucus production in the lungs. Notably, an enhanced frequency of Foxp3+ Treg cells and reduced effector T-cell proliferation associated with increased levels of IL-10 and TGF-β were observed in IR-treated mice. ConclusionIR can induce tolerogenic-like BMDCs which promote the differentiation of Treg cells. Importantly, the expansion of Foxp3+ Treg cells contributed to the therapeutic efficacy of IR against allergic asthma.

A CCL2/MCP-1 antagonist attenuates fibrosis of the infrapatellar fat pad in a rat model of arthritis

BackgroundFibrosis of the infrapatellar fat pad (IFP) is a feature of osteoarthritis and contributes substantially to the pain and dysfunction in patients’ joints. However, the underlying mechanisms remain unclear. C-C motif chemokine ligand-2 (CCL2) plays a central role in tissue fibrosis. Thus, we aimed to investigate the role of CCL2 in the development of IFP fibrosis in a rat model of arthritis, hypothesizing that a CCL2 antagonist could mitigate fibrotic progression.MethodsWe induced arthritis in male Wistar rats using intra-articular injections of carrageenan. Furthermore, to evaluate the effects of a CCL2 antagonist on protein expression and collagen deposition in the IFP of the rats, we transferred an N-terminal-truncated CCL2 gene into a rat model via electroporation-mediated intramuscular injection. Macrophage infiltration and collagen deposition in the IFP were analyzed in vivo. Groups were compared using the Mann–Whitney U test and Student’s t-test.ResultsWe identified infiltrating macrophages as well as increases in CCL2 and TGF-β levels as collagen deposition progressed. Gene transfer of the CCL2-antagonist before arthritis induction attenuated collagen deposition remarkably.ConclusionsWe provide initial evidence that anti-CCL2 gene therapy can effectively suppress the development of IFP fibrosis in a rat model. Thus, targeting CCL2 holds promise as a therapeutic strategy for managing tissue fibrosis in osteoarthritis patients.

Knockdown of HM13 Inhibits Metastasis, Proliferation, and M2 Macrophage Polarization of Non-small Cell Lung Cancer Cells by Suppressing the JAK2/STAT3 Signaling Pathway.

An upregulated histocompatibility minor 13 (HM13) has been studied in various tumors, yet the exact mechanism of HM13 in non-small cell lung cancer (NSCLC) is unclear. In view of same, the present study investigates crucial role and action mechanism of HM13 in human NSCLC. HM13 expression was higher in NSCLC tissue and cells through the Western blotting technique along with qRT-PCR. As per data from The Cancer Genome Atlas (TCGA), NSCLC patients having high HM13 expression show lower overall survival. 5-ethynyl-2-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and transwell tests were assessed for NSCLC cell growth, and invasion, and we found that silencing of HM13 inhibited the NSCLC cell proliferation, invasion. Additionally, to investigate the effects of HM13 on THP-1 macrophage polarization, a co-culture model of NSCLC and THP-1 macrophages were used. The CD206 + macrophages were examined using flow cytometry. As the markers of M2 macrophage, the mRNA levels of IL-10 and TGF-β of THP-1 cells were also detected by qRT-PCR. Knockdown of HM13 could inhibit the M2 polarization. Further experiments demonstrated that downregulated HM13 could inhibit the JAK2/STAT3 signaling pathway. RO8191 (activator of JAK/STAT3 pathway) influenced the invasion, proliferation, and expression of JAK2/STAT3 signaling pathway and Epithelial-mesenchymal transition (EMT) markers induced by HM13 silencing. HM13 knockdown also inhibited the tumor growth in vivo by xenograft nude mouse model. By inhibiting JAK2/STAT3 signaling pathway, HM13 knockdown inhibited the NSCLC cell proliferation, metastasis tumor growth, and tumor-associated macrophage M2 polarization. In NSCLC, HM13 could be a therapeutic target to treat the NSCLC.

Cytokines and Pancreatic Ductal Adenocarcinoma: Exploring Their Relationship with Molecular Subtypes and Prognosis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its poor prognosis. The current challenge remains the absence of predictive biomarkers. Cytokines are crucial factors in the pathogenesis and prognosis of PDAC. Furthermore, there is growing interest in differentiating between molecular subtypes of PDAC. The aim of our study is to evaluate the association between the analyzed cytokines and the molecular subtypes of PDAC and to determine their prognostic value. Cytokine levels were measured in 73 patients, and molecular subtypes were analyzed in 34 of these patients. Transforming Growth Factor Beta 2 (TGF-β2) levels were independently associated with the basal-like and null subtypes. In patients with locally advanced and metastatic PDAC, elevated levels of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-9, and IL-15 were associated with a higher risk of progression during first-line treatment, and increased levels of IL-1β, IL-6, IL-8, IL-9, and IL-15 were related to increased mortality. Furthermore, a significant association was observed between higher percentiles of IL-6 and IL-8 and shorter progression-free survival (PFS) during first-line treatment, and between higher percentiles of IL-8 and shorter overall survival (OS). In the multivariate analysis, only elevated levels of IL-8 were independently associated with a higher risk of progression during first-line treatment and mortality. In conclusion, the results of our study suggest that cytokine expression varies according to the molecular subtype of PDAC and that cytokines also play a relevant role in patient prognosis.

The effects of resistance training on cardiovascular factors and anti-inflammation in diabetic rats

Diabetes induces a range of macrovascular and microvascular changes, which lead to significant clinical complications. Although many studies have tried to solve the diabetic problem using drugs, it remains unclear. In this study, we investigated whether resistance exercise affects cardiovascular factors and inflammatory markers in diabetes. The study subjected Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which have genetically induced diabetes mellitus, to a resistance exercise program for 12 weeks and assessed the levels of cardiovascular factors and inflammatory markers using western blotting analysis, ELISA, and immunohistochemistry. During the training period, OLETF + exercise (EX) group exhibited lower body weight and reduced glucose levels when compared with OLETF group. Western blotting analysis, ELISA, and immunohistochemistry revealed that the levels of PAI-1, VACM-1, ICAM-1, E-selectin, TGF-β, CRP, IL-6, and TNF-α were decreased in OLETF + EX group when compared with the OLETF group. Moreover, the anti-inflammatory markers, IL-4 and IL-10, were highly expressed after exercise. Therefore, these results indicate that exercise may influence the regulation of cardiovascular factors and inflammatory markers, as well as help patients with metabolic syndromes regulate inflammation and cardiovascular function.

Oral administration of Lactobacillus delbrueckii UFV-H2b20 protects mice against Aspergillus fumigatus lung infection.

Probiotics provide therapeutic benefits not only in the gut but also other mucosal organs, including the lungs. To evaluate the effects of the probiotic strain L. delbrueckii UFV-H2b20 oral administration in an experimental murine model of A. fumigatus pulmonary infection. BALB/c mice were associated with L. delbrueckii and infected with Aspergillus fumigatus and compared with non-associated group. We investigated survival, respiratory mechanics, histopathology, colony forming units, cytokines in bronchoalveolar lavage, IgA in feces, efferocytosis, production of reactive oxygen species and the cell population in the mesenteric lymph nodes. L. delbrueckii induces tolerogenic dendritic cells, IL-10+macrophages and FoxP3+regulatory T cells in mesenteric lymph nodes and increased IgA levels in feces; after infection with A. fumigatus, increased survival and decreased fungal burden. There was decreased lung vascular permeability without changes in the leukocyte profile. There was enhanced neutrophilic response and increased macrophage efferocytosis. L. delbrueckii-treated mice displayed more of FoxP3+Treg cells, TGF-β and IL-10 levels in lungs, and concomitant decreased IL-1β, IL-17A, and CXCL1 production. Uur results indicate that L. delbrueckii UFV H2b20 ingestion improves immune responses, controlling pulmonary A. fumigatus infection. L. delbrueckii seems to play a role in pathogenesis control by promoting immune regulation.

An Association between HTRA1 and TGF-β2 in the Vitreous Humor of Patients with Chorioretinal Vascular Diseases.

Background: The aim of this paper was to investigate the protein concentrations of high-temperature requirement A 1 (HTRA1) and transforming growth factor-β (TGF-β) in the vitreous humor of patients with chorioretinal vascular diseases. Methods: This study measured protein concentrations of HTRA1, TGF-β1-3, and vascular endothelial growth factor A (hereinafter called VEGF) in the vitreous humor from seven eyes of patients with chorioretinal vascular diseases (age-related macular degeneration, diabetic macular edema, and retinal vein occlusion) and six control eyes (idiopathic epiretinal membrane and macular hole). We analyzed the mutual relationship among the protein levels. Results: The protein levels of HTRA1 and VEGF were significantly increased in the chorioretinal vascular disease group compared with the control group (1.57 ± 0.79 ×10-9 mol/mL vs. 0.68 ± 0.79 ×10-9 mol/mL, p = 0.039; 3447.00 ± 3423.47 pg/mL vs. 35.33 ± 79.01 pg/mL, p = 0.046, respectively). TGF-β2 levels were not significantly different between groups (2222.71 ± 1151.25 pg/mL for the chorioretinal vascular disease group vs. 1918.83 ± 744.01 pg/mL for the control group, p = 0.62). The concentration of HTRA1 was strongly associated with TGF-β2 levels in the vitreous humor, independent of VEGF (r = 0.80, p = 0.0010). Conclusions: We revealed that vitreous HTRA1 was increased in patients with chorioretinal vascular diseases and strongly correlated with TGF-β2.

Upregulation of Metrnl improves diabetic kidney disease by inhibiting the TGF-β1/Smads signaling pathway: A potential therapeutic target.

This study comprises an investigation of the role of meteorin-like (Metrnl) in an experimental model of diabetic kidney disease (DKD). Twenty-four db/db mice were randomly assigned to one of the following groups: DKD, DKD + Metrnl-/-, and DKD + Metrnl+/+. Plasma Metrnl concentrations were measured using ELISA. Kidney tissues were examined via western blotting, qRT-PCR, and immunohistochemistry to determine the expression levels of inflammatory factors. Electron microscopy was employed to observe stained kidney sections. Compared with the NC group, FBG, BW, and UACR were elevated in the DKD and Metrnl-/- groups, with severe renal pathological injury, decreased serum Metrnl concentration, decreased renal Metrnl expression, and increased expression levels of TNF-α, TGF-β1, TGF-R1, pSmad2, pSmad3, and α-SMA. In contrast, the Metrnl+/+ group showed decreased FBG and UACR, BUN, TC and TG, increased HDL-C and serum Metrnl concentration, increased renal Metrnl expression, and decreased expression of TNF-α, TGF-β1, TGF-R1, pSmad2, pSmad3, and α-SMA, compared to the DKD and Metrnl-/- groups. A Pearson bivariate correlation analysis revealed a negative correlation between UACR and Metrnl, and a positive correlation between UACR and TGF-β1. Upregulation of renal Metrnl expression can improve renal injury by downregulating the expression of molecules in the TGF-β1/Smads signaling pathway in the renal tissues of type 2 diabetic mice; and by reducing the production of fibrotic molecules such as α-SMA.

The TGFβ type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed/refractory multiple myeloma: a phase 1b trial

Functional blockade of the transforming growth factor-beta (TGFβ) signalling pathway improves the efficacy of cytotoxic and immunotherapies. Here, we conducted a phase 1b study (ClinicalTrials.gov., NCT03143985) to determine the primary endpoints of safety, tolerability, and maximal tolerated dose (200 mg twice daily) for the orally-available TGFβ type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed and/or refractory multiple myeloma (RRMM) patients who had received ≥2 lines of chemoimmunotherapy. Secondary endpoints demonstrated sustained clinical responses, favorable pharmacokinetic parameters and a 6-month progression-free survival of 82%. Vactosertib combined with pomalidomide was well-tolerated at all dose levels and displayed a manageable adverse event profile. Exploratory analysis indicated that vactosertib co-treatment with pomalidomide also reduced TGFβ levels in patient bone marrow as well as the level of CD8+ T-cells that expressed the immunoinhibitory marker PD-1. In vitro experiments indicated that vactosertib+pomalidomide co-treatment decreased the viability of MM cell lines and patient tumor cells, and increased CD8+ T-cell cytotoxic activity. Vactosertib is a safe therapeutic that demonstrates tumor-intrinsic activity and can overcome immunosuppressive challenges within the tumor microenvironment to reinvigorate T-cell fitness. Vactosertib offers promise to improve immunotherapeutic responses in heavily-pretreated MM patients refractory to conventional agents.

Circulating Endothelin 1 but Not Transforming Growth Factor-β Levels Are Reduced after Pulmonary Endarterectomy in Subjects Affected by Chronic Thromboembolic Pulmonary Hypertension: A Prospective Cohort Study.

Background and objectives: Endothelin-1 (ET-1) and transforming growth factor-β (TGF-β) play a pivotal role in the pathophysiology and vascular remodeling of chronic thromboembolic pulmonary hypertension (CTEPH) which is an under-diagnosed complication of acute pulmonary embolism (PE). Currently, pulmonary endarterectomy (PEA) is still the treatment of choice for selected patients suffering from CTEPH. The aim of this study was to evaluate the preoperative and postoperative circulating levels of ET-1 and TGF-β in subjects affected by CTEPH undergoing successful surgical treatment by PEA. Methods: The data from patients diagnosed with CTEPH who underwent PEA at the Foundation IRCCS Policlinico San Matteo Hospital (Pavia, Italy) were prospectively recorded in the Institutional database. Circulating ET-1 and TGF-β levels were assessed by an ELISA commercial kit before PEA, at 3 months and 1 year after PEA. The demographic data, preoperatory mean pulmonary arterial pressure (mPAP), cardiac output (CO), and pulmonary vascular resistance (PVR) were also recorded. Univariate and multivariate analyses were performed. Results: The analysis included 340 patients with complete ET-1 measurements and 206 patients with complete TGF-β measurements. ET-1 significantly decreased both at 3 months (p < 0.001) and at 1 year (p = 0.009) after PEA. On the other hand, preoperatory TGF-β levels did not significantly change after PEA. Furthermore, ET-1, but not TGF-β, was a good predictor for increased mPAP in multivariate analyses (p < 0.05). Conclusions: ET-1 but not TGF β was significantly modulated by PEA in subjects affected by CTEPH up to 1 year after surgery. The mechanisms leading to prolonged elevated circulating TGF-β levels and their clinical significance have to be further elucidated.

Evaluation of CNPase and TGFβ1/Smad Signalling Pathway Molecule Expression in Sinus Epithelial Tissues of Patients with Chronic Rhinosinusitis with (CRSwNP) and without Nasal Polyps (CRSsNP).

Chronic rhinosinusitis with and without nasal polyps (CRSwNP and CRSsNP, respectively) is a chronic inflammatory disease affecting almost 5 to 12% of the population and exhibiting high recurrence rates after functional endoscopic sinus surgery (FESS). TGFβ1-related pathways contribute to tissue remodelling, which is one of the key aspects of CRS pathogenesis. Additionally, adenosine signalling participates in inflammatory processes, and CNPase was shown to elevate adenosine levels by metabolizing cyclic monophosphates. Thus, the aim of this study was to assess the expression levels of Smad2, pSmad3, TGFβ1, and CNPase protein via immunohistochemistry in sinus epithelial tissues from patients with CRSwNP (n = 20), CRSsNP (n = 23), and non-CRS patients (n = 8). The expression of Smad2, pSmad3, TGFβ1, and CNPase was observed in the sinus epithelium and subepithelial area of all three groups of patients, and their expression correlated with several clinical symptoms of CRS. Smad2 expression was increased in CRSsNP patients compared to CRSwNP patients and controls (p = 0.001 and p < 0.001, respectively), pSmad3 expression was elevated in CRSwNP patients compared to controls (p = 0.007), TGFβ1 expression was elevated in CRSwNP patients compared to controls (p = 0.009), and CNPase was decreased in CRSsNP patients compared to controls (p = 0.03). To the best of our knowledge, we are the first to demonstrate CNPase expression in the upper airway epithelium of CRSwNP, CRSsNP, and non-CRS patients and point out a putative synergy between CNPase and TGFβ1/Smad signalling in CRS pathogenesis that emerges as a novel still undiscovered aspect of CRS pathogenesis; further studies are needed to explore its function in the course of the chronic inflammation of the upper airways.

Effects of Mucuna pruriens (L.) DC. and Levodopa in Improving Parkinson's Disease in Rotenone Intoxicated Mice.

Parkinson's disease (PD) is the second leading neurodegenerative disease after Alzheimer's disease. Mucuna pruriens (L.) DC. (MP) is a plant that contains Levodopa (L-DOPA) and has been known to improve the symptoms of PD. In this preliminary study, we investigated the anti-parkinsonian potential of MP to compare the effects of L-DOPA. We first developed an in vivo model of the PD in C57BL/6 male mice using rotenone. A total of twelve mice were used for this experiment. Nine mice were injected with rotenone (28 mg/kg) daily for 28 days. The mice experiments were performed to validate the effectiveness of MP to treat PD. Synthetic L-DOPA in a ratio of 1:20 with MP was used as MP contains 5% L-DOPA by weight in it. MP and L-DOPA were injected for 19 days on a daily basis. Cognitive function was evaluated using beam balance and olfactory tests. Serum analysis was performed using serum enzyme-linked immunosorbent assay (ELISA) analysis test. IL-12, IL-6, and TGF-β 1 were evaluated to validate the PD inducement and treatment. The levels of IL-12, IL-6, and TGF-β1 (p < 0.0001) in the PD mice group were significantly higher than those in the control group. The PD mice also showed higher latencies in beam balance and olfactory tests (p < 0.0001) compared to the control group. Both MP and L-DOPA-treated groups showed alleviation in latencies in beam balance and olfactory tests and decreased neuroinflammation in ELISA analysis (p < 0.001). The results treated by MP and L-DOPA showed insignificant differences in their values (p > 0.05). This proved that the MP and L-DOPA had similar effects in improving the symptoms of PD when used in the ratio of 1:20. Furthermore, both MP and L-DOPA reduced the level of IL-6 and TGF-β1 in this study. It may be inferred that a reduction in the level of IL-6 and TGF-β1 eventually leads to a reduction in the Th17 cells. The pathogenic Th17 is thought to be present in virtually all chronic inflammatory disorders. This can be an interesting area of research in further understanding the immunological effect of MP in ameliorating PD symptoms.

Experimental study on H2O2 activation of HSC-T6 and hepatic fibrosis in cholestatic mice by "Yajieshaba"

Ethnopharmacological relevanceYajieshaba (YJSB), approved by the Yunnan Provincial Food and Drug Administration in 2008, are known for their anti-inflammatory, antiviral, and pro-apoptotic properties, effectively treating Hepatic fibrosis (HF). However, its mechanism of action remains unclear. Aim of the studyThe objective of this investigation is to explore how YJSB influences the TGF-β1/Smad signaling pathway as a strategy for reducing HF. MethodsThe establishment of a HF model in mice involved ligation of the common bile duct, followed by administration of YJSB. Body and liver weights were measured, and the liver index calculated. Serum levels of ALT, AST, ALP, TBA, and TBIL were assessed using colorimetric methods. Additionally, liver homogenates were analyzed for PIIINP, Col-IV, LN, HA, and Hyp, as well as TGF-β1 activity, using ELISA. Histological analyses of liver sections, stained with H&E, Ag, and Masson's trichrome, were performed to examine inflammation and the accumulation of collagen and reticular fibers. These studies aimed to elucidate the pharmacodynamic effects of YJSB on HF in mice with bile duct obstruction. The target pathways of YJSB were preliminarily identified through immunofluorescence detection of TGF-β1, P-Smad2L, P-Smad2C, P-Smad3L, P-Smad3C, and Smad4 proteins. In vitro experiments included the induction of hepatic stellate cell (HSC-T6) activation by H2O2. A cell injury model was established for HSC-T6, and the CCK-8 assay was used to determine the optimal YJSB concentration and treatment duration. After pirfenidone (PFD) administration, which inhibits the TGF-β1/Smad pathway, the effects of YJSB on HSC-T6 cell proliferation were observed. ELISA assays quantified Col-III, α-SMA, and Col-I in cell lysates to assess YJSB's impact on collagen synthesis in HSC-T6 cells. Western blot analysis was performed to assess the protein levels within the TGF-β1/Smad signaling cascade. ResultsIn the HF mouse model, administration of YJSB notably augmented the body weight and reduced the liver index. Concurrently, there was an elevation in serum concentrations of ALP, AST, ALT, TBA, and TBIL. Similarly, in the liver homogenates of HF mice, increases were observed in the levels of HA, PIIINP, Col-IV, LN, Hyp, and TGF-β1. Histological assessments using H&E, Ag, and Masson stains indicated a substantial diminution in liver tissue damage. Through immunofluorescence analysis, it was discerned that YJSB modulated the expression of TGF-β1, P-Smad2L, P-Smad2C, and P-Smad3L downwards, while elevating P-Smad3C and Smad4 protein expressions. Additional investigations revealed a significant reduction in α-SMA, Col-I, and Col-III levels in cell culture fluids, suggesting a decrease in collagen synthesis and a protective role against cellular damage. Western blot analyses demonstrated that the TGF-β1/Smad pathway inhibitor, PFD, acted in synergy with YJSB, enhancing its regulatory effects on this pathway, decreasing levels of TGF-β1, P-Smad2L, P-Smad2C, P-Smad3L, and promoting the expression of P-Smad3C. ConclusionsYJSB demonstrates a pharmacodynamic effect against HF, enhancing liver functionality and effectively mitigating the damage associated with bile duct obstruction. The proposed action mechanism of YJSB involves modulation of the TGF-β1/Smad signaling pathway. Research indicates that YJSB might play a role in suppressing the movement, programmed cell death, and activation of HSC-T6, potentially decelerating the advancement of hepatic fibrosis.

VEGFA contributes to tumor property of glioblastoma cells by promoting differentiation of myeloid-derived suppressor cells

BackgroundGlioblastoma (GBM) is a malignant astrocytic tumor and its progression involves the regulation of vascular endothelial growth factor-A (VEGFA). However, the mechanism of VEGFA in regulating GBM progression remains unclear.MethodsVEGFA mRNA expression was analyzed by quantitative real-time polymerase chain reaction. Protein expression of VEGFA, cluster of differentiation 9 (CD9), CD81, and transforming growth factor-β1 (TGF-β1) was detected by western blotting assay. Flow cytometry assay was conducted to assess cell proliferation, cell apoptosis and myeloid-derived suppressor cell (MDSC) differentiation. TUNEL cell apoptosis detection kit was utilized to analyze cell apoptosis of tumors. Angiogenic capacity was investigated by tube formation assay. Transwell assay was used to assess cell migration and invasion. The effect of VEGFA on tumor formation was determined by a xenograft mouse model assay. Immunohistochemistry assay was used to analyze positive expression rate of VEGFA in tumor tissues. TGF-β1 level was detected by enzyme-linked immunosorbent assay.ResultsVEGFA expression was upregulated in GBM tissues, GBM cells, and exosomes from GBM patients and GBM cells. VEGFA silencing led to decreased cell proliferation, tube formation, migration and invasion and increased cell apoptosis. Moreover, VEGFA knockdown also delayed tumor formation. VEGFA promoted MDSC differentiation and TGF-β1 secretion by MDSCs by being packaged into exosomes. In addition, TGF-β1 knockdown displayed similar effects with VEGFA silencing on GBM cell phenotypes, and MDSCs attenuated VEGFA knockdown-induced effects by secreting TGF-β1 in A172 and U251 cells.ConclusionVEGFA contributed to tumor property of GBM cells by promoting MDSC differentiation and TGF-β1 secretion by MDSCs, providing potential targets for GBM treatment.

Endogenous PGD2 acting on DP2 receptor counter regulates Schistosoma mansoni infection-driven hepatic granulomatous fibrosis.

Identifying new molecular therapies targeted at the severe hepatic fibrosis associated with the granulomatous immune response to Schistosoma mansoni infection is essential to reduce fibrosis-related morbidity/mortality in schistosomiasis. In vitro cell activation studies suggested the lipid molecule prostaglandin D2 (PGD2) as a potential pro-fibrotic candidate in schistosomal context, although corroboratory in vivo evidence is still lacking. Here, to investigate the role of PGD2 and its cognate receptor DP2 in vivo, impairment of PGD2 synthesis by HQL-79 (an inhibitor of the H-PGD synthase) or DP2 receptor inhibition by CAY10471 (a selective DP2 antagonist) were used against the fibrotic response of hepatic eosinophilic granulomas of S. mansoni infection in mice. Although studies have postulated PGD2 as a fibrogenic molecule, HQL-79 and CAY10471 amplified, rather than attenuated, the fibrotic response within schistosome hepatic granulomas. Both pharmacological strategies increased hepatic deposition of collagen fibers - an unexpected outcome accompanied by further elevation of hepatic levels of the pro-fibrotic cytokines TGF-β and IL-13 in infected animals. In contrast, infection-induced enhanced LTC4 synthesis in the schistosomal liver was reduced after HQL-79 and CAY10471 treatments, and therefore, inversely correlated with collagen production in granulomatous livers. Like PGD2-directed maneuvers, antagonism of cysteinyl leukotriene receptors CysLT1 by MK571 also promoted enhancement of TGF-β and IL-13, indicating a key down-regulatory role for endogenous LTC4 in schistosomiasis-induced liver fibrosis. An ample body of data supports the role of S. mansoni-driven DP2-mediated activation of eosinophils as the source of LTC4 during infection, including: (i) HQL-79 and CAY10471 impaired systemic eosinophilia, drastically decreasing eosinophils within peritoneum and hepatic granulomas of infected animals in parallel to a reduction in cysteinyl leukotrienes levels; (ii) peritoneal eosinophils were identified as the only cells producing LTC4 in PGD2-mediated S. mansoni-induced infection; (iii) the magnitude of hepatic granulomatous eosinophilia positively correlates with S. mansoni-elicited hepatic content of cysteinyl leukotrienes, and (iv) isolated eosinophils from S. mansoni-induced hepatic granuloma synthesize LTC4 in vitro in a PGD2/DP2 dependent manner. So, our findings uncover that granulomatous stellate cells-derived PGD2 by activating DP2 receptors on eosinophils does stimulate production of anti-fibrogenic cysLTs, which endogenously down-regulates the hepatic fibrogenic process of S. mansoni granulomatous reaction - an in vivo protective function which demands caution in the future therapeutic attempts in targeting PGD2/DP2 in schistosomiasis.

Pharmacological Study of Guben Yifei-Tang on a Rat Model of Pneumoconiosis

Background Diffuse pulmonary fibrosis is a progressively worsening lung disease that poses a serious threat to human health. Current treatment options are limited and generally ineffective. Objectives This study aimed to investigate the therapeutic effects of GubenYifei-Tang (GB) in a rat model of pneumoconiosis. Materials and Methods We established a chronic pneumoconiosis model in rats through intratracheal injection of silica dust. Subsequently, the rats were randomly assigned to various groups, including the model group (NC group), Hanfangji Masu tablet group (TET group), high-dose GB group (GB-H group), low-dose GB group (GB-L group), and a blank control group comprising 12 normal rats (NC group). We assessed blood biochemical, vascular endothelial function, immunoinflammatory factors, and oxidative stress indices in four rats from each group at the 1st, 3rd, and 6th months of administration. Results Throughout the 6-month administration period, rats in the GB-L and GB-H groups displayed no significant abnormalities in their general condition or body weight. Both high and low doses of GB significantly reduced the respiratory rate and increased the respiratory amplitude of the pneumoconiosis model rats ( p &lt; 0.05). Additionally, they significantly decreased the mean pulmonary arterial pressure of these rats ( p &lt; 0.05). Moreover, we observed noteworthy improvements in blood gas indices and hypoxemia symptoms, including increased pH, decreased arterial partial pressure of carbon dioxide, increased arterial oxygen partial pressure, and arterial oxygen saturation. Compared to the NC group, the model group rats exhibited significantly elevated concentrations of serum transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and interleukin-1 ( p &lt; 0.01). High-dose GB significantly reduced serum levels of TGF-β, TNF-α, and interleukin-10 in rats, while low-dose GB partially improved serum inflammatory factor levels. Additionally, the lung mass index, malondialdehyde (MDA) levels, and hydroxyproline levels were significantly increased in the model group rats compared to the NC group ( p &lt; 0.01), accompanied by decreased superoxide dismutase (SOD) levels. Both high and low doses of GB demonstrated substantial improvements in lung mass index, MDA, SOD, and hydroxyproline levels in rats ( p &lt; 0.05). Conclusion In the rat model of pneumoconiosis, GB exerts a positive therapeutic impact on lung tissue lesions and respiratory function, with its effectiveness being both time- and dose-dependent.

TGFβ in malignant canine mammary tumors: relation with angiogenesis, immunologic markers and prognostic role

Transforming growth factor-β (TGFβ) and FoxP3 regulatory T cells (Treg) are involved in human breast carcinogenesis. This topic is not well documented in canine mammary tumors (CMT). In this work, the tumoral TGFβ expression was assessed by immunohistochemistry in 67 malignant CMT and its correlation to previously determined FoxP3, VEGF, and CD31 markers and other clinicopathologic parameters was evaluated. The high levels of TGFβ were statistically significantly associated with skin ulceration, tumor necrosis, high histological grade of malignancy (HGM), presence of neoplastic intravascular emboli and presence of lymph node metastases. The observed levels of TGFβ were positively correlated with intratumoral FoxP3 (strong correlation), VEGF (weak correlation) and CD31 (moderate correlation). Tumors that presented a concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31 markers were statistically significantly associated with parameters of tumor malignancy (high HGM, presence of vascular emboli and nodal metastasis). Additionally, shorter overall survival (OS) time was statistically significantly associated with tumors with an abundant TGFβ expression and with concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31. The presence of lymph node metastasis increased 11 times the risk of disease-related death, arising as an independent predictor of poor prognosis in the multivariable analysis. In conclusion, TGFβ and Treg cells seem involved in tumor progression emerging as potential therapeutic targets for future immunotherapy studies.

Relation of T Cell Profile with Vitamin D Receptor and Vitamin D-Binding Protein Gene Polymorphisms in Atopy.

Atopic diseases, including atopic dermatitis (AD) and allergic asthma (AA), are characterized by complex immune responses involving various T cells subsets and their cytokine profiles. It is assumed that single nucleotide polymorphisms (SNPs) in the Vitamin D receptor (VDR) gene and the Vitamin D-binding protein (GC) gene are related to the action of Vitamin D and, consequently, play a role in regulating the immune response. However, there is not enough data to unequivocally support the hypothesis about the relationship between T cells profile and VDR or GC SNPs. Two hundred sixty-six subjects (aged > 18 years) were involved in the study: 100 patients with mild or moderate AD, 85 patients with mild or moderate AA, and 81 healthy individuals. Blood cell counts were determined by standard methods. Flow cytometric analysis was used to evaluate CD4+ T-helper (Th) cell subtypes: Th2, Th1, Th17, and T regulatory (Treg) cells in peripheral blood. Measurements of cytokines, total immunoglobulin E (IgE), and Vitamin D levels in serum were evaluated by ELISA. Significantly higher levels of Th1, Th2, and Th17 cells, along with lower levels of Tregs, were found in patients with atopic diseases compared to healthy individuals. Additionally, higher serum levels of interleukin (IL) 5, IL-17A, and transforming growth factor-β1 (TGF-β1), as well as lower levels of IL-10, were observed in patients with atopic diseases than in control. The study established associations between VDR SNPs and immune profiles: the AA genotype of rs731236 was associated with increased Th2 and Th17 cells and a higher Th1/Th2 ratio; the GG genotype of rs731236 was linked to decreased serum IL-10 and TGF-β1 levels; and the TT genotype of rs11168293 was associated with increased IL-10 levels. Additionally, the GG genotype of GC gene SNP rs4588 was associated with reduced Th2 and Th17 lymphocytes, while the TT genotype of rs4588 was linked to decreased IL-10 levels. Furthermore, the CC genotype of rs7041 was associated with higher levels of Th2, Th17, IL-10, and IL-35, as well as reduced levels of TGF-β1, while the GG genotype of rs3733359 was associated with reduced IL-10 levels. In conclusion, our study demonstrates that the Vitamin D receptor gene single nucleotide polymorphisms rs731236 and rs11168293, along with polymorphisms in the Vitamin D-binding protein gene (rs4588, rs7041, rs3733359), are significantly associated with variations in T cell profiles in atopy. These variations may play a crucial role in promoting inflammation and provide insight into the genetic factors contributing to the pathogenesis of atopy.

Effect of photodynamic therapy on peripheral immune system for unresectable cholangiocarcinoma

BackgroundPhotodynamic therapy (PDT) has been emerging as a promising treatment for unresectable cholangiocarcinoma (CCA). A number of experiments have demonstrated that PDT could enhance antitumor immunity significantly. However, the impact of PDT on peripheral immune system for unresectable CCA remains unclear. MethodsIn a clinical trial comparing the perioperative and long-term outcomes of PDT+stent treatment and stent alone treatment for unresectable CCA, we tested the levels of lymphocytes (CD4+ T cells, CD8+ T cells, NK cells, B cells and Treg cells) and immune-related cytokines (IL-4, IL-6, IL-10, TNF-α, TGF-β, perforin, GM-CSF and IFN-γ) in peripheral blood before and after PDT+stent treatment or stent alone treatment and analyzed the influence of PDT on peripheral immune system for unresectable CCA. ResultsBefore treatment, the levels of all the immune cells and immune-related cytokines did not show significant differences between the PDT+stent group and stent alone group. The ratio of CD8+ T cells increased significantly after PDT treatment, but other kinds of lymphocytes did not show significant difference. Increased level of IL-6 and decreased level of perforin and TGF-β after PDT treatment were demonstrated, whereas no significant changes were found for other immune-related cytokines. ConclusionPDT altered the levels of immune cells and immune-related cytokines in the peripheral blood of unresectable CCA patients, potentially correlating with the therapeutic efficacy of PDT in unresectable CCA treatment. Future studies could delve deeper into this aspect to explore how PDT can be more effectively utilized in the management of unresectable CCA.

Enhanced wound healing activity of naturally derived Lagenaria siceraria seed oil binary nanoethosomal gel: formulation, characterization, in vitro/in vivo efficiency

BackgroundThe present study aims to enhance the wound healing potential of the seed oil (SO) of Lagenaria siceraria (Egyptian cultivar) via the preparation of SO-loaded binary nanoethosomal (SO-BNE) gels. SO-BNEs were prepared using 23 factorial design, characterized for vesicle size, zeta potential, polydispersity index, linoleic and oleic acid EE% for ensuring improved skin permeability. The L. siceraria SO, optimized SO-BNE gels (0.5% and 5%) and Mebo® were topically applied in full-thickness wounded rat model twice daily for 10 days.ResultsIn the SO-BNE gel groups, the normal appearance of the skin architecture and structure of the dermis was revealed. In addition, the levels of NRF2, TGF-β1 and FOXO1, collagen type I, SMA-α and MIP2 were significantly elevated. The wound healing potential of SO-BNE gels was proposed to be via suppression of oxidative stress and stimulation of skin regeneration biomarkers. Furthermore, the SO screening through GC/MS unveiled high percentages of unsaturated fatty acids. SO was also found to be nontoxic to human skin fibroblast cells; enhanced viability and migration rates at concentration of 50 g/mL by 99.76% and 75.9%, respectively.ConclusionThese findings demonstrate that the Lagenaria siceraria SO-loaded BNE gels represent a promising delivery for wound healing with enhanced release and bioavailability.