Concentrations Of TGF-beta Research Articles

Mesenchymal stem cell-derived exosomes decrease Hyperplasia in Psoriasis by inducing transforming growth factor β2 (TGF-β2).

Psoriasis, a chronic inflammatory skin disease, is increasingly effectively managed with the targeted immunotherapy; however, long-term immunotherapy carries health risks, and loss of response. Therefore, we need to develop the alternative treatment strategies. Mesenchymal stem/stromal cell (M.S.C.) exosomes stand out for their remarkable immunomodulatory properties, gaining widespread recognition. This study investigated whether M.S.C. exosomes can reduce psoriasis-induced hyperplasia by inducing Transforming Growth Factor beta 2 (TGF-beta2) signaling. Exosomes were isolated from M.S.C.s by ultracentrifugation. Then, scanning electron microscopy was used for the morphology of exosomes. To ascertain the exosome concentration, the Bradford test was used. To ascertain the cellular toxicity of exosomes in Human Umbilical Vein Endothelial Cells ( H.U.V.E.C), an MTT experiment was then conducted. Real-time PCR was used to quantify TGF beta2 expression levels, whereas an ELISA immunosorbent assay was used to determine the protein concentration of TGF beta2. In this study, the exosomes of 15-30nm in size that were uniform, and cup-shaped were isolated. Moreover, the IC50 value for this Treatment was calculated to be 181.750µg/ml. The concentration of TGF-β2 gene in the target cells significantly increased following Treatment with the exosomes. Furthermore, the expression level of the studied gene significantly increased due to the Treatment. Upregulating the expression of TGF-β2 in psoriatic cells via TGF-β2 signaling is one way exosomes can help reduce hyperplasia.

Organ-Dysfunction Markers in Mild-to-Moderate COVID-19 Convalescents.

Background: A coronavirus disease 2019 (COVID-19) outbreak led to a worldwide pandemic. COVID-19 not only caused acute symptoms during the severe phase of the disease, but also induced long-term side effects on the functioning of many organs and systems. Symptoms that were associated with the disease and present at least 3 months after recovery were named long COVID. The aim of this study was to assess if mild-to-moderate COVID-19 may lead to the dysfunction of respiratory, cardiovascular, neural, and renal systems in healthy blood donors who recovered from the disease at least 6 months earlier. Methods: Here, we examined 294 adults among volunteer blood donors divided into convalescents (n = 215) and healthy controls (n = 79). Concentrations of soluble CD163, TGF beta, Lp-PLA2, NCAM-1, S100, NGAL, and creatinine were measured either by ELISA or automated methods. The probability value p < 0.05 was considered as statistically significant. Results: We found significant differences in Lp-PLA2, S100, and NCAM-1 between convalescents and never-infected subjects. Lp-PLA2 and NCAM-1 were lower, and S100 higher, in convalescents than in the control group. Conclusion: Mild-to-moderate COVID-19 convalescents are at a low risk of developing lung fibrosis or chronic kidney disease. However, they should regularly carry out their prophylaxis examinations for early detection of possible negative outcomes of COVID-19.

Pre-Transplant Cytokine Levels as Signatures of Microvascular Inflammation in Kidney Allograft Biopsies.

The presence of microvascular inflammation (MVI) characterized by leukocyte margination in the glomeruli (glomerulitis, Banff score 'g') and peritubular capillaries (peritubular capillaritis, Banff score 'ptc') is a hallmark histological feature of antibody-mediated rejection (AMR), even in the absence of circumferential C4d positivity. In this study, we assessed the efficacy of pre-transplant plasma cytokines as an ancillary screening tool to identify MVI in kidney allograft indication biopsies to facilitate better graft survival. This single-center prospective analytical study comprises 38 kidney transplant recipients whose peripheral blood was collected before transplant and assessed for the plasma cytokine concentrations of FOXP3, IL-6, TGF beta, and IL-17 using enzyme-linked immunosorbent assays (ELISA). Histopathological assessment was done in post-transplant indication biopsies, and Banff scores of 'g+ ptc' were calculated to categorize recipients into three MVI groups. The correlational, regression, and ROC curve analyses were used to assess the association and predictive ability of the cytokines with respect to MVI. In our study cohort, 27 recipients had MVI=0, five had MVI=1, and six had MVI≥2. A significant difference in plasma cytokines was observed between these groups, and we found a strong negative correlation of FOXP3 with MVI, whereas a strong positive correlation of IL-6, TGF beta, and IL-17 was recorded with MVI. We have also assessed the predictive ability of these cytokines, FOXP3, IL-6, TGF-beta, and IL-17, through the ROC curve, which showed an AUC of 0.70, 0.76, 0.84, and 0.72, respectively. Our findings suggest that the pre-transplant levels of cytokines FOXP3, IL-6, TGF-beta, and IL-17 could be measured to identify recipients at risk of post-transplant MVI, which could further serve as an additional tool for effective management of the kidney allograft.

Hypoxia Preconditioned Serum (HPS) Promotes Proliferation and Chondrogenic Phenotype of Chondrocytes In Vitro.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects remains challenging in terms of maintaining chondrogenic phenotype during in vitro chondrocyte expansion. Growth factor supplementation has been found supportive in improving ACI outcomes by promoting chondrocyte redifferentiation. Here, we analysed the chondrogenic growth factor concentrations in the human blood-derived secretome of Hypoxia Preconditioned Serum (HPS) and assessed the effect of HPS-10% and HPS-40% on human articular chondrocytes from osteoarthritic cartilage at different time points compared to normal fresh serum (NS-10% and NS-40%) and FCS-10% culture conditions. In HPS, the concentrations of TGF-beta1, IGF-1, bFGF, PDGF-BB and G-CSF were found to be higher than in NS. Chondrocyte proliferation was promoted with higher doses of HPS (HPS-40% vs. HPS-10%) and longer stimulation (4 vs. 2 days) compared to FCS-10%. On day 4, immunostaining of the HPS-10%-treated chondrocytes showed increased levels of collagen type II compared to the other conditions. The promotion of the chondrogenic phenotype was validated with quantitative real-time PCR for the expression of collagen type II (COL2A1), collagen type I (COL1A1), SOX9 and matrix metalloproteinase 13 (MMP13). We demonstrated the highest differentiation index (COL2A1/COL1A1) in HPS-10%-treated chondrocytes on day 4. In parallel, the expression of differentiation marker SOX9 was elevated on day 4, with HPS-10% higher than NS-10/40% and FCS-10%. The expression of the cartilage remodelling marker MMP13 was comparable across all culture conditions. These findings implicate the potential of HPS-10% to improve conventional FCS-based ACI culture protocols by promoting the proliferation and chondrogenic phenotype of chondrocytes during in vitro expansion.

Dysfunctions of the C-type natriuretic peptide signaling pathway involved in the development of thoracic aortic aneurysm in Marfan patients

Marfan Syndrome (MFS) is an autosomal dominant inherited connective tissue disorder affecting the cardio-vascular system. Development of thoracic aortic aneurysms (TAA) leading to dissections and ruptures is the primary cause of morbidity and mortality in these patients. No treatment really cures Marfan patients. Interestingly, in humans, mutations in the gene coding for the C-type natriuretic peptide (CNP) or its receptors lead to a “Marfan-like syndrome”. CNP is a local regulator of skeletal growth and of vascular homeostasis, remodeling and angiogenesis. CNP binds to NPR-B or NPR-C receptors, both expressed on endothelial and vascular smooth muscle cells (vSMCs) and modulates the intracellular cGMP and cAMP levels. The aim of this project is to determine whether altered CNP signaling pathway contributes to TAA development and progression to rupture in Marfan patients. Plasma and aorta biopsies were taken from Marfan patients and from Fbn1C1039G/+ mice. The CNP, TGF-beta, cAMP and cGMP concentrations were measured in the plasma. mRNA and protein levels evaluated by qRT-PCR, and Western blot analysis in aortic tissue. Male and female mice studied and alterations of the aorta structure analyzed by ultrasound imaging. Plasma proCNP level in Marfan patients depends on the age and the TAA development. Increased levels found in young patients presenting no or reduced TAA and decreased levels in patients with TAA compared to age and sex-matched non-Marfan patients. CNP and NPR-C decreased at the mRNA and protein levels in the aorta of Marfan patients when compared to the same aorta in non-Marfan patients. In Fbn1+/- mice, aortic structure alterations are detected from 10 weeks in males and 34 weeks in females. CNP and NPR-C mRNA and protein levels drastically decreased in the thoracic aorta in 2–7 week-old male mice and in 34 week-old female mice. In 52-week old Fbn1+/- mice, the plasma cAMP level is increased in males and not in females, whereas the plasma cGMP protein level is increased in females but not in males. CNP injections in 3-week old Fbn1+/- male mice partially prevented the aorta structure alterations. A deficit in CNP protein level or in the expression of the NPR-C receptor contributes to the development and progression of TAA.

Pharmacological Evaluation of Moringa oleifera on Collagen- Induced Arthritis in Rats

Background and Objective: Arthritis associated with oxidative stress and chronic inflammation has been a major health problem among the people worldwide. The present study was aimed to investigate the potential anti-arthritis activity of the methanolic extract of leaves of Moringa oleifera was evaluated on Collagen induced arthritis.&#x0D; Study Design: In-vivo model.&#x0D; Place and Duration of the Study: Department of pharmacology, Karnataka College of pharmacy, Bangalore India, between January to September 2022.&#x0D; Methods: The study was to evaluate anti-arthritic activity of Moringa oleifera (MO) against auto-immune arthritis in Wistar rats, using collagen-induced arthritis (CIA) model. Effect of the methanol extract of Moringa oleifera in inflammatory response during CIA was studied by measuring CRP, different cytokines in serum and assessment of arthritis index, footpad swelling. Level of CRP, LPO, NO and an enzymatic activity of SOD and CAT was determined to assess the effect of the Moringa oleifera extract in neutralizing oxidative stress during CIA. Histology of Synovial membrane experiment has been performed to determine whether Moringa oleifera has any impact on the changes in synovial tissue during CIA.&#x0D; Results: Post oral administration of Moringa oleifera at 250 and 500 mg/kg body weight doses decreased the arthritic index and footpad swelling. Moringa oleifera administration diminished pro-inflammatory cytokines in serum. The concentrations of pro-inflammatory TNFa, TGF-beta, IFNg, and IL-6 were elevated in the serum of the rat challenged with, type II collagen as compared to the std. and MtEMO-fed groups. Treatment with MtEMO in CIA induced rat significantly decreased these cytokines in serum. Decreased the serum level LPO, NO content, and SOD activity along with concomitant rise in CAT with the treatment of MtEMO. In addition decreased CRP in serum was also observed. Serum concentration of CRP, the most common type of acute phase proteins was tested in our experiments and was found to be significantly attenuated in case of CIA rat treated with MtEMO. Protective effect of MtEMO in CIA group is further supported from histopathological studies which showed improvement during bone damage and also shown less inflamed, and lymphocyte accumulation, cartilage damage decreased and less disruption of the synovial lining cell layer.&#x0D; Conclusion: Based on the results it can be concluded that the test drug Moringa oleifera is capable of regulating oxidative stress during CIA and therefore down regulated local and systemic release of pro-inflammatory mediators.

7-DAYS HIGH SALT DIET AFFECTS THE PRODUCTION OF PRO- AND ANTI-INFLAMMATORY CYTOKINES IN YOUNG HEALTHY INDIVIDUALS

Objective: This study aimed to investigate the earliest immunomodulatory effects of NaCl during pre-hypertensive phase of acute salt loading in humans. Thus we have investigated serum levels of systemic pro- and anti-inflammatory cytokines in relation to peripheral Th17 and regulatory T cell abundance following 7-day high-salt (HS) loading in young salt-resistant healthy individuals. Design and method: 30 young healthy individuals adhered to a 7-day low-salt (LS) diet (3.5 g salt/day) followed by a 7-day high-salt (HS) diet (~14.7 g salt/day) protocol. Serum pro- and anti-inflammatory cytokines (INF-gamma, TNF-alpha, IL-6, IL-9, IL-17A, IL-23, IL-10, IL-21, IL-22, TGF-beta1), complement component C3a, as well as frequencies of peripheral blood Th17 and regulatory T cells (Treg), Th17/Treg ratio and the level of enzymes involved in their activation and differentiation (SGK1 and p38/MAP kinase) were measured before and after 7-day HS diet protocol. Results: Short-term HS diet significantly increased serum level of pro-inflammatory cytokines INF-gamma, TNF-alpha, IL-9 and IL-17A level, but also serum concentration of anti-inflammatory cytokines IL-10 and TGF-beta1. In addition, serum INF-gamma and IL-9 levels positively correlated to 24-h urine natriuresis. Relative amount of total and phosphorylated p38/MA kinase remained unchanged, while relative amount of total SGK1 significantly increased following 7-day HS diet compared to LS values. Conclusions: Result of the present study provided evidence of acute salt-loading induced changes in the immune status of young healthy individuals. The increase in relative amount of total SGK1 in peripheral mononuclear cells suggests lymphocyte (re)activation in response to hyperosmotic stress resulting in enhanced production of proinflammatory (IL-17, INF-gamma), but also anti-inflammatory cytokines (IL-10 and TGF-beta1). Still, which effect is prevailing remains elusive and warrants further studies.

BIOMARKERS OF FIBROSIS AND INFLAMMATION IN PATIENTS WITH METABOLIC SYNDROME AND ATRIAL FIBRILLATION: WHAT ARE THE DIFFERENCES IN MOLECULAR MECHANISMS IN HYPERTENSION AND OBESITY?

Objective: To determine the concentration of blood fibrotic and inflammatory biomarkers in patients with atrial fibrillation (AF) associated with metabolic syndrome (MS) components. Design and method: The study included 646 patients aged 35–65 years: patients with AF and MS (n = 142), those with AF and without MS (n = 113), those with MS and without AF (n = 175) and the control group consisted of healthy subjects without cardiovascular and metabolic diseases (n = 107). All participants underwent anthropometric and laboratory investigations. Profibrogenic (aldosterone, galectin-3, TGF-beta1, CTGF) and proinflammatory (CT-1, IL-6) factors were determined in serum and plasma by ELISA. Results: The highest concentrations of fibrotic and inflammatory biomarkers were found in patients with AF in combination with MS. In MS patients without AF, the concentration of aldosterone, galectin-3, TGF-beta1, CTGF, CT-1, and IL-6 was also higher than in healthy subjects. In patients with AF and hypertension (HTN), but without abdominal obesity (AO), higher values of aldosterone (108,1 ± 70,3 pg/ml and 89,3 ± 32,2 pg/ml, p = 0,003) and TGF¬beta1 (3680,1 ± 1863,3 pg/ml and 1968,1 ± 1611,5 pg/ml, p = 0,015) in serum than in AF patients without HTN and without AO. In the group of patients with AF and AO, but without HTN, higher concentrations of IL-6 (2,9 ± 0,7 pg/ml and 1,9 ± 0,6 pg/ml, p = 0,001) and CTGF (162,9 ± 92,2 pg/ml and 116,3 ± 63,4 pg/ml, p = 0,0001). Correlation analysis made it possible to establish a stronger relationship between aldosterone and TGF¬beta1 with systolic blood pressure (r = 0,493, p &lt; 0,0001 and r = 0,530, p &lt; 0,0001), and CT¬1, CTGF and IL¬6 in a greater degree correlated with waist circumference (r = 0,563, p &lt; 0,0001; r = 0,626, p &lt; 0,0001; r = 0,480, p &lt; 0,0001). Conclusions: It can be assumed that hypertension through the aldosterone system and TGF-beta1, as well as abdominal obesity through the cytokine system CT-1 and IL-6 activate various mechanisms and pathways for myocardial emodelling. Integral molecules galectin-3 and CTGF mediate their interactions, in particular in patients with a combination of several MS components and contribute to a higher AF risk.

Early Immunomodulatory Effects of Different Natural Surfactant Preparations in Preterms With Respiratory Distress.

BackgroundRespiratory distress syndrome (RDS) is the most common respiratory disease in premature infants. Exogenous natural surfactant preparations are used in the treatment of RDS. In recent years, it has become increasingly evident that surfactant plays an immunoregulatory role.ObjectivesThe aim of this study was to evaluate cytokine and chemokine response following three different regimens of natural surfactant treatment in preterm newborns with RDS.MethodsPremature newborns below 32 weeks of gestation who were intubated for RDS and given early surfactant rescue therapy were included in the study. Newborns were randomly divided into three groups and Beractant 100 mg/kg (B-100), Poractant alfa 100 mg/kg (Pα-100) and Poractant alfa 200 mg/kg (Pα-200) were administered intratracheally. Blood samples and transtracheal aspirates (TA) were collected just before and 4–6 h after the surfactant treatment. Total eosinophil count, inducible T Cell alpha chemoattractant (ITaC), macrophage inflammatory protein 3 beta (MIP3b), interleukins (IL) 5, 8, 9, 10, 13, immunoglobulin E (IgE), interferon gamma (IFN-γ), eotaxin and tumor necrosis factor beta-1 (TGF-β1) were measured from blood and tracheal aspirate samples.ResultsA total of 45 infants, 15 in each group, were included in the study. Mean gestational age, birth weight, antenatal, demographic and clinical characteristics of the study groups were similar. IFNγ concentration and eosinophil counts in TA decreased after surfactant replacement in all groups, especially in the infants treated with Pα-100 and Pα-200. Eotaxin, TGF beta and IL-8 concentrations in TA increased significantly in the infants treated with Pα-100 and Pα-200. IL-9 levels in TA decreased in the B-100 group but increased in the Pα-100 and Pα-200 groups. Blood levels of cytokines and chemokines showed significantly decreased levels of ITaC and MIP3b only in the B-100 group, but no significant change was observed in the Pα-100 and Pα-200 groups.ConclusionIn our study, the different immunomodulatory effects of natural surfactant preparations on newborn lung is proven. We found that Poractant α, one of the natural surfactant preparations, shifted the lung immune system toward TH2.

TMOD-17. A NOVEL GLIOBLASTOMA INVASION MODEL USING HUMAN BRAIN SLICE CULTURES

Abstract OBJECTIVE Glioblastoma (GBM) is among the most common of malignant brain tumours, with a median post-surgical survival of less than one year. Over the past several decades, therapies that appeared promising in mice models have failed during clinical trials due to the differences encountered during translation of research from model organisms to humans. To partially mitigate these difficulties in translation, we present a human cortical organotypic culture based GBM model, which allows us to manipulate individual components of the tumour environment in order to investigate the influence of different cell types in the immunosuppressive tumour microenvironment. METHODS Human neocortical tissue (at least 2 cm away from the tumour core) or entry cortex from epilepsy surgery guided by intraoperative neuro navigation, was cultured for up to 14 days post resection using an optimized medium. The cultured tissue was further injected with patient derived human GBM cells to create an ex vivo human model of glioblastoma model. The role of astrocytes in the tumour microenvironment was studied using microglia loss of function model. RESULTS Our established human neo-cortical slice model can recapitulate an in-vivo characteristics of glioblastoma from functional and imaging aspect. Our data corroborate differences between astrocytes in human and murine models in different reactive states, shows that the glioblastoma microenvironment can be difficult to be accurately modelled using murine models. Results from our human microglia depletion model, provided ample evidence that complex interaction of astrocytes and microglia cells, promotes an immunosuppressive environment in Glioblastoma by releasing high concentration of IL10 and TGFbeta (p&amp;lt; 0.001). CONCLUSION Our model therefore has potential applications to the fields of neuroscience, neuro-oncology, and pharmacotherapy.

What is the role of profibrogenic and proinflammatory factors in developing atrial fibrillation associated with metabolic syndrome components?

Aim. To determine the concentration of blood fibrotic and inflammatory biomarkers in patients with atrial fibrillation (AF) associated with metabolic syndrome (MS) components.Material and methods. The study included 646 patients aged 35-65 years: patients with AF and MS (n=142), those with AF and without MS (n=113), those with MS and without AF (n=175) and the control group consisted of healthy subjects without cardiovascular and metabolic diseases (n=107). All participants underwent anthropometric and laboratory investigations. Profibrogenic (aldosterone, galectin-3, TGF-beta1, CTGF) and proinflammatory (CT-1, IL-6) factors were determined in serum and plasma by ELISA. Statistical analysis was performed using IBM SPSS Statistics software (version 22.0).Results. The highest concentrations of fibrotic and inflammatory biomarkers were found in patients with AF in combination with MS. In MS patients without AF, the concentration of aldosterone, galectin-3, TGF-beta1, CTGF, CT-1, and IL-6 was also higher than in healthy subjects. The levels of aldosterone, CT-1 and IL-6 in patients with AF were higher in the presence of three or more MS components, while the highest values of these parameters were found in patients with five MS components. The lowest concentrations of galectin-3, CTGF, and CT-1 in patients with AF were found in patients without MS components, and in the presence of even 1 MS component, they were significantly higher. Correlation analysis made it possible to establish a stronger relationship between aldosterone and TGF-betal with systolic blood pressure (p=0,493, p&lt;O,O001 and p=0,530, p&lt;O,O001), and CT-1, CTGF and IL-6 in a greater degree correlated with waist circumference (p=0,563, p&lt;0,0001; p=0,626, p&lt;0,0001; p=0,480, p&lt;O,O001). The concentrations of galectin-3 and CTGF were more positively correlated with an increase in the number of MS components. In patients with AF and hypertension (HTN), but without abdominal obesity (AO), higher values of aldosterone (108,1±70,3 pg/ml and 89,3±32,2 pg/ml, p=0,003) and TGF-beta1 (3680,1±1863,3 pg/ml and 1968,1±1611,5 pg/ml, p=0,015) in serum than in AF patients without HTN and without AO. In the group of patients with AF and AO, but without HTN, higher concentrations of IL-6 (2,9±0,7 pg/ml and 1,9±0,6 pg/ml, p=0,001) and CTGF (162,9±92,2 pg/ml and 116,3±63,4 pg/ml, p=0,0001).Conclusion. It can be assumed that hypertension through the aldosterone system and TGF-beta1, as well as abdominal obesity through the cytokine system CT-1 and IL-6 activate various mechanisms and pathways for myocardial remodeling. Integral molecules galectin-3 and CTGF mediate their interactions, in particular in patients with a combination of several MS components and contribute to a higher AF risk.

Differential expressions of miR-223, miR-424, miR-145, miR-200c, miR-139 in experimental rat chronic pancreatitis model and their relationship between oxidative stress, endoplasmic reticulum stress, and apoptosis.

Objective(s):This study aimed to research the roles of miR-139, miR-221, miR-200c, miR-145, miR-223, miR-424, and miR-377 in endoplasmic reticulum stress (ERS), oxidative stress (OS), fibrosis, and apoptosis processes in chronic pancreatitis (CP) rat model. Materials and Methods:Fourteen rats were randomized into 2 groups (Group 1, sham group (n=7) and Group 2, CP group (n=7)). TGF-beta and malondialdehyde concentrations were measured in rat blood samples. qRT-PCR was used to investigate the expression levels of 7 miRNAs in the pancreas tissues. The correlations of mRNA undergoing significant changes with inflammation (TNF-α, IL-6), ERS (Ire1-α, Perk), apoptosis (Caspase 3, Bcl-2), OS (Cat, Gpx1), and fibrosis (α-Sma) were investigated.Results:The biochemical results and histopathological scores in Group 1 were statistically significantly high compared with Group 2 (P<0.5). Expression levels of seven miRNAs (miR-200c, miR-145, miR-223, miR-424) were significantly higher, while miR-139 was significantly lower in CP. In our study, we found that miR-200c, miR-145, and miR-139 may contribute to CP progression and cellular processes based on the correlation between ERS, OS, apoptosis, and inflammation with miRNA expression levels. Conclusion:miR-200c, miR-145, miR-139, miR-223, and miR-424 play roles in the CP model. They may be used as candidate biomarkers for the CP process.

Molecular mechanisms of left atrial fibrosis development in patients with atrial fibrillation and metabolic syndrome: what biomarkers should be used in clinical practice?

Aim. To determine the blood concentration of fibrosis biomarkers in patients with atrial fibrillation (AF) in combination with metabolic syndrome (MS) and to analyze the relationship with myocardial fibrosis.Material and methods. This cross-sectional case-control study included 547 patients aged 35 to 65 years: experimental group — patients with MS (n=373), of which 202 patients had AF; comparison group — AF patients without MS (n=110); healthy subjects without cardiovascular diseases and metabolic disorders (n=64). Patients with AF and MS who underwent electroanatomic mapping before pulmonary vein isolation (n=79) were assessed for left atrial (LA) fibrosis severity.Results. It was found that the blood concentration of circulating profibrogenic biomarkers in patients with AF and MS is higher than in patients with AF without MS: aldosterone (135,1 (80,7-224,1) and 90,1 (68,3-120,3) pg/ml, p&lt;0,0001), galectin-3 (10,6 (4,8-15,4) and 5,8 (4,8-8,3) pg/ml, p=0,0001), GDF15 (938,3 (678,3-1352,1) and 671,0 (515,7-879,5) pg/ml, p=0,001), TGF-beta-1 (4421,1 (2513,5-7634,5) and 2630,5 (2020,7-3785,4) pg/ml, p=0,001), CTGF (167,8 (78,9-194,3) and 124,3 (74,4-181,9) pg/ml, p&lt;0,0001), PIIINP (88,5 (58,6120,4) and 58,9 (40,7-86,1) ng/ml, p&lt;0,0001), PINP (3421,4 (1808,1-4321,7) and 2996,1 (2283,8-3894,3) pg/ml, p&lt;0,0001). Patients with paroxysmal AF have higher concentrations of TGF-beta1, CTGF and PINP than patients with persistent and permanent AF. In patients with persistent AF and MS, the concentrations of galectin-3, aldosterone, and PIIINP were higher than in patients with paroxysmal AF, while in patients with permanent AF, they were significantly lower. The plasma concentration of galectin-3 positively correlated with levels of PINP (p=0,465, p&lt;0,0001), PIIINP (p=0,409, p&lt;0,0001), GDF-15 (p=0,369, p&lt;O,O001), CTGF (p=0,405, p&lt;0,0001). According to multivariate regression, of all studied biomarkers, GDF-15 had a greater effect on PIIINP concentration (в=0,234, p=0,038), and galectin-3 — on PINP (в=0,248, p&lt;0,021). Positive correlations of the severity of left atrial fibrosis with the concentration of galectin-3 (p=0,563, p&lt;0,0001), PINP (p=0,620, p&lt;0,0001), TGF-beta-1 (p=0,390, p&lt;0,0001) and CTGF (p=0,551, p&lt;0,0001). According to linear multivariate regression, the most significant effect on LA fibrosis severity among the studied biomarkers is exerted by galectin-3 (в=0,432, p&lt;0,0001), PINP (в=0,343, p=0,001) and PIIINP (в=0,286, p=0,008).Conclusion. An increase in the blood concentration of profibrogenic biomarkers galectin-3, TGF-beta-1, CTGF, PIIINP, and PINP is associated with an increase in LA fibrosis severity and probably has a pathogenetic role in increasing the AF risk in patients with MS.

A Rediscovered Immunomodulatory Action of Cancer Chemotherapy to Pilot the Cytokine Network in an Antitumor Way

The recent proposal of cancer immunotherapy with anti-checkpoint inhibitor monoclonal antibodies, as well as the previous immunotherapy with IL-2, would require an interpretation of cancer chemotherapy not only in terms of therapeutic strategy carried out to destroy cancer cells, but also as an approach potentially able to influence and modulate the cytokine network in an attempt to correct cancer-related cytokine alterations responsible for tumor progression itself. This statement is justified by the fact that the efficacy of cancer immunotherapy is depending at least in part on the cytokine secretions of patients. Despite the great number of cancer-related cytokine alterations, the main alterations provided by a physiopathological and prognostic significance would consisting of low blood levels of IL-2 and IL-12 in association with abnormally high concentrations of IL-1, IL-6, TNF-alpha, IL-10 and TGF-beta. The effects of chemotherapy on cytokine secretions have appeared to depend on the type of agent, as well as on its dosage and schedule of administrarion. At present, the main effects of chemotherapy provided by a potential clinical application would be represented by the stimulatory action of adriamycin on IL-2 secretion, the inhibitory effect of cisplatin on IL-6 secretion, the stimulatory effect of gemcitabine on IL-12 production, and the inhibitory action of both cyclophosphamide and gemcitabine on regulatory T cell system. Then, the future chemo-immunotherapeuticregimenswouldhavenot to be elaborated not only on the bases of empiristic criteria and on the cytotoxic effects of che various chemotherapeutic agents, but also by taking into consideration their specific activity on the secretion of those cytokines, whose anomalous production has to be corrected and neutralized.

AUTOLOGOUS MONOCYTE-MEDIATED HUMAN CD4+TREG EXPANSION

CD4+Foxp3+regulatory T cells (Tregs) implies huge importance in regulating immune activation and autologous self-tolerance. In transplantation, the concern of immunosuppressants could be reduced by using adoptive autologous Tregs. However, the limitation of current Tregs expansion protocols are the small number of expanded cells. In this study, our lab managed to expand human Tregs ex-vivo using autologous monocytes cells which could dramatically improve the expansion rate. Here we evaluated the possible therapeutic application of consistently ex-vivo expanded human CD4+CD25+FoxP3+T cells from gated CD4+CD25hiCD127- population. Ex-vivo enriched CD4+CD25+CD127-T cells were successfully and consistently expanded to 5,000-8,800 times from the initial culture for 10 days with optimized culture conditions. A high concentration of IL-2(>500units) and TGF-beta together with anti-CD3 and anti-CD28 stimulation were essential to the massive ex-vivo expansion. Expanded Tregs showed 99% of FoxP3 expression and expressed high level of CD25, CD95, CD47 as well as maintain the suppressive capacity. Thus, we proved that ex-vivo expanded CD4+Treg retain regulatory activity and could match the requirement for cells therapy.

A new DNA vaccine expressing HspX-PPE44-EsxV fusion antigens of Mycobacterium tuberculosis induced strong immune responses.

Objective(s):Infection with tuberculosis (TB) is regarded as a major health issue. Due to the emergence of antibiotic resistance during TB treatment, prevention via vaccination is one of the most effective ways of controlling the infection. DNA vaccines are developed at a greater pace due to their ability in generating a long-lasting immune response, higher safety compared to the live vaccines, and relatively lower cost of production. In the present study, we evaluated a new DNA vaccine encoding the fusion HspX-PPE44-EsxV antigens, separately, and in combination with Bacillus Calmette–Guérin (BCG) administration, in a prime-boost method in mice.Materials and Methods:A novel DNA vaccine encoding HspX-PPE44-EsxV fusion antigen of Mycobacterium tuberculosis was constructed, and RT-PCR and Western blot analysis were performed to verify the expression of the antigen. Female BALB/c mice were divided into five groups (PBS, BCG, pcDNA3.1 (+) vector, pDNA/HspX-PPE44-EsxV vaccine, and the BCG-prime boost groups). In order to evaluate the immunogenicity of the recombinant vector, BALB/c mice were injected with 100 μg of pDNA at 2-week intervals. Then, cytokine assay was conducted using eBioscience ELISA kits (Ebioscience, AUT) according to manufacturers’ instructions to evaluate the concentrations of IL-4, IL-12, TGF-β, and IFN-γ.Results:The concentrations of INF-γ, IL-12, and TGF-beta were significantly increased compared to the control groups (P<0.001). INF-γ and IL-12 production were increased significantly in pDNA/HspX-PPE44-EsxV+BCG group compared to pDNA/HspX-PPE44-EsxV group (P<0.001).Conclusion:This study showed that the present DNA vaccine could induce a high level of specific cytokines in mice. It was also shown that using this DNA vaccine in a BCG prime-boost protocol can produce significant amounts of IFN-γ, IL-12, and TGF-β.

The ancient Chinese formula Longdan Xiegan Tang improves antipsychotic-induced hyperprolactinemia by repairing the hypothalamic and pituitary TGF-β1 signaling in rats

Ethnopharmacological relevanceAntipsychotics often induce hyperprolactinemia. The transforming growth factor (TGF)-beta1 signaling in the pituitary and hypothalamus inhibits prolactin synthesis and secretion, and its impairment is implicated in neuropsychiatric disorders. Longdan Xiegan Tang (LXT) alone or together with antipsychotics have been used to treat various neuropsychiatric diseases and hyperprolactinemia-associated disorders. Aim of the studyTo investigate the effect of LXT on hyperprolactinemia and involvement of the TGF-beta1 signaling. Materials and methodsMale rats were co-administered with olanzapine (5 mg/kg) and LXT extract (50 and 500 mg/kg) (p.o., × 8 weeks). Plasma concentrations of prolactin and TGF-beta1 were determined by ELISA. Protein expression was analyzed by Western blot. ResultsTreatment of rats with LXT extract suppressed olanzapine-induced increase in plasma prolactin concentration and overexpression of pituitary and hypothalamic prolactin protein. Importantly, LXT restored olanzapine-induced decrease in protein expression of the key components of the TGF-beta1 signaling, TGF-beta1, type II TGF-beta receptor, type I TGF-beta receptor and phosphorylated SMAD3 in the pituitary and hypothalamus. Further, it antagonized downregulation of pituitary and hypothalamic dopamine D2 receptor (D2R) protein level, and inhibited pituitary estrogen receptor (ER) alpha and ERbeta protein expression. ConclusionsThe present results suggest that LXT ameliorates antipsychotic-induced hyperprolactinemia in rats by repairing the pituitary and hypothalamic TGF-beta1 signaling possibly via D2R, ERs or/and other pathways. Our findings may also provide scientific elucidation for use of the ancient Chinese formula to treat the impaired TGF-beta1 signaling-associated neuropsychiatric disorders.

Proportion and role of CD45+ erythroid progenitor cells in patients with tongue cancer metastasis

Objective: To investigate the proportion and role of CD45+ erythroid progenitor cells (EPC) in patients with tongue cancer metastasis. Methods: The initial treatment of tongue cancer patients (n=40) from January 2017 to June 2018 in He'nan Provincial People's Hospital was included in this study. According to the presence or absence of lymph node metastasis, they were divided into tumor group (no lymph node metastasis was found in imaging and pathology) and metastasis group (both imaging and pathology confirmed lymph node metastasis). The expression of Ki-67 was detected by immunohistochemistry and the proportion of CD45+CD71+TER119+EPC was detected by flow cytometry. EPC was sorted by flow cytometry, interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were detected by enzyme-linked immunosorbent assay (ELISA), and reactive oxygen species (ROS) was detected by flow cytometry. Transwell was used for tumor invasion test; methyl thiazolyltetrazolium (MTT) assay was used to detect proliferation level. Results: There were 20 cases in the tumor group and metastasis group. There was no significant difference between the two groups in terms of age, sex, time of onset and size of tumors. Flow cytometry showed that the ratio of CD45+EPC in peripheral blood of tumor group and metastasis group was (1.2±0.2)% and (3.1±0.2)% (t=7.823, P<0.001). Correlation analysis showed that the ratio of CD45+EPC was positively correlated with the proliferation index of Ki-67 cells (r=0.592, P=0.006). The results of flow cytometry showed that the mean fluorescence intensity (MFI) of ROS in EPC was 102.1±22.9 in tumor group and 530.0±67.2 in metastasis group (t=6.025,P<0.001). The results of ELISA showed that the mass concentrations of IL-10 and TGF-β in EPC supernatant of tumor group were (10.8±1.6) and (3.2±0.8) μg/L, respectively. The mass concentrations of IL-10 and TGF-beta in EPC supernatant of metastasis group were (26.9±3.7) and (6.4±0.9) μg/L, respectively (t=3.956, P=0.003; t=2.595, P=0.027). Transwell results showed that the proportion of invasive cells in the CD45+EPC group [(40.3±4.4)%] was higher than that in the control group [(17.5±2.2)%] (t=4.607, P=0.001). MTT proliferation experiment showed that the proliferation rate of the CD45+EPC group [(52.0±3.3)%] was higher than that of the control group [(30.5±1.9)%] (t=5.656, P<0.001). Conclusions: The proportion of CD45+EPC in patients with tongue cancer metastasis is significantly increased. CD45+EPC can promote the proliferation and metastasis of tongue cancer by secreting immunosuppressive molecules and ROS.

3123 TGFbeta, Early Cytokine Dysregulation, and Airway Smooth Muscle Dysfunction in Cystic Fibrosis

OBJECTIVES/SPECIFIC AIMS: This study aims to first describe the unique cytokine profile and TGFbeta levels of young children with CF, then understand the pathologic effects of TGFbeta on lung function in a CF animal model. These powerful translational studies linking observations in clinical disease with a transgenic mouse model allow us a unique opportunity to investigate the role of TGFbeta in early CF lung disease. METHODS/STUDY POPULATION: Cytokine levels (TGFbeta, TNFalpha, IL-8, IL-6, HNE, and IL-1beta) in bronchoalveolar lavage fluid (BALF) from CF patients (n = 15) and non-CF control patients (n = 21) under 6 years old were determined by ELISA and Luminex assay. Tracheotomized patients without significant underlying lung disease were chosen as non-CF inflamed control patients, as they had similar levels of neutrophilic inflammation and infection as CF patients. The percentage of BAL neutrophils (% PMNs) in each sample was assessed. The relationships between cytokines were analyzed using linear regression and principal components analysis. In animal studies, CF and non-CF mice (n = 4-5 per group) were treated with intratracheal adenoviral TGFbeta1 vector, an empty vector control, or PBS. After one week, animals were collected; lung function, response to the bronchoconstrictor methacholine, and rescue with albuterol were measured utilizing a FlexiVent machine. Lungs were collected for histology. Immunohistochemistry for alpha-SMA was performed and pictures of all cross-sectional airways were obtained. Burden of ASM was assessed by dividing the square root of alpha-SMA stained airway smooth muscle by the basement membrane perimeter length of each airway. RESULTS/ANTICIPATED RESULTS: Patient characteristics of CF and non-CF inflamed control patients were similar in terms of age (3.6 yrs vs 3.3 yrs respectively, p = 0.49), positive BAL culture (13% vs 14%, p = 0.94), and % PMNs (65% vs 56%, p = 0.64). Despite these similarities, TGFbeta levels were 2-fold higher in CF versus non-CF BAL (p = 0.034). Analysis of BAL cytokines from both patient groups showed that three principal components describe 86% of total variance across the cytokine variables. These components represent different contributions from the cytokines, with TGFbeta, IL6, and % PMNs comprising one component of similarly regulated inflammatory markers. These components can distinguish CF versus non-CF patients with 77% accuracy (area under the curve: 0.77). TGFbeta concentrations were uniquely associated with increased IL-6 in CF samples (r = 0.74; p = 0.0015) but did not demonstrate association with other cytokines. After TGFbeta exposure, CF mice demonstrated greater abnormalities in airway resistance than non-CF mice, with heightened response to methacholine. Importantly, this increase in airway obstruction in CF mice was reversible with albuterol treatment, indicating airway smooth muscle dysfunction as a principal driver of lung function abnormalities. Furthermore, TGFbeta induced an increased ASM burden on lung histology in both CF and non-CF mice (p&lt;0.05). IL-6 levels in the BAL of CF mice showed greater increases after TGFbeta treatment compared to non-CF mice (p&lt;0.05). Empty vector control treatment did not cause lung pathology. DISCUSSION/SIGNIFICANCE OF IMPACT: Young children with CF have a unique pattern of pulmonary inflammation compared to inflamed non-CF control patients. In CF, TGFbeta pulmonary levels are uniquely associated with IL-6, a driver of ASM dysfunction in other pulmonary diseases. We followed up this clinical observation study by investigating the effect of TGFbeta on pulmonary disease in a mouse model. CF mice demonstrate increased pulmonary IL-6, airway obstruction, and ASM dysfunction after TGFbeta exposure. This study provides evidence that TGFbeta is associated with a distinct cytokine pattern that may promote ASM dysfunction in early CF lung disease. Understanding the mechanism of early CF pathophysiology will be critical in developing targeted therapeutics that can prevent early lung damage.

Are epicardial adipose tissue and transforming growth factor beta1 risk factors of atrial fibrillation in patients with metabolic syndrome?

Objective. To determine the relationship between the thickness of epicardial adipose tissue (EAT) and the serum concentration of transforming growth factor beta1 (TGF-beta1) with the severity of left atrial myocardial fibrosis in patients with metabolic syndrome (MS) and to define the role of these factors in atrial fibrillation (AF) development.Design and methods. The study included 258 patients: patients with MS without AF (n = 57), patients with MS and AF (n = 83), patients with AF without MS (n = 36), and otherwise healthy subjects without cardiovascular disease and metabolic disorders (n = 82). Serum level of TGF-beta1 was assessed by ELISA. Epicardial adipose tissue thickness (EAT) was measured by ultrasound on the Vivid 7 apparatus (General Electric, USA). To assess fibrosis we constructed anatomical and amplitude maps of the left atrium (LA) using the non-fluoroscopic electro-anatomical mapping system CARTO3 (Biosense Webster, USA)Results. EAT in patients with MS and AF is higher than in patients with MS without AF (4,7 ± 1,9 and 4,2 ± 1,6 mm, respectively, p = 0,023), higher than in patients with AF without MS (4,7 ± 1,9 and 4,3 ± 1,7 mm, p = 0,01) and significantly higher than in healthy subjects (4,7 ± 1,9 and 2,3 ± 0,9 mm, respectively, p &lt; 0,001). The EAT positively correlated with the percentage of fibrosis of LA estimated by the mapping method (r = 0,549, p &lt; 0,0001). The serum concentration of TGF-beta1 in patients with AF and MS was 6700,2 [2588,4, 17500,3] pg/ml. It was 4,7 times higher than in healthy subjects (p &lt; 0,0001), 2,6 times higher than in patients with MS without AF and higher than in patients with AF without MS. Positive correlations were found between TGF-beta1 and LA volume (r = 0,203, p = 0,03). Binomial regression analysis showed that the probability of AF presence in patients with MS was higher when traditional predictors of this arrhythmia were present — the volume of the LA (odds ratio (OR) = 1,092, 95% confidence interval (CI) 1,026–1,162, p = 0,005), systolic blood pressure (OR = 1,093, 95% CI 1,021–1,169, p = 0,01), as well as EAT (OR = 2,21, 95% CI 1,111–4,386, p = 0,024) and TGF-beta1 (OR = 1,01, 95% CI 1,006–1,015, p = 0,002).Conclusions.Thickness of epicardial adipose tissue and transforming growth factor beta1 are associated with AF also in MS. Probably, epicardial fat and transforming growth factor beta1 stimulate atrial myocardial fibrosis which is the risk factor of AF.