Plasma Levels Of TGF-β1 Research Articles

FLASH Proton Pencil Beam Scanning Irradiation Minimizes Radiation-Induced Leg Contracture and Skin Toxicity in Mice.

Simple SummaryDose and efficacy of radiation therapy are limited by the toxicity to normal tissue adjacent to the treated tumor region. Recently, ultra-high dose rate radiotherapy (FLASH radiotherapy) has shown beneficial reduction of normal tissue damage while preserving similar tumor efficacy with electron, photon and scattered proton beam irradiation in preclinical models. Proton therapy is increasingly delivered by pencil beam scanning (PBS) technology, and we therefore set out to test PBS FLASH radiotherapy on normal tissue toxicity and tumor control in vivo in mouse using a clinical proton delivery system. This validation of the FLASH normal tissue-sparing hypothesis with a clinical delivery system provides supporting data for PBS FLASH radiotherapy and its potential role in improving radiotherapy outcomes. Ultra-high dose rate radiation has been reported to produce a more favorable toxicity and tumor control profile compared to conventional dose rates that are used for patient treatment. So far, the so-called FLASH effect has been validated for electron, photon and scattered proton beam, but not yet for proton pencil beam scanning (PBS). Because PBS is the state-of-the-art delivery modality for proton therapy and constitutes a wide and growing installation base, we determined the benefit of FLASH PBS on skin and soft tissue toxicity. Using a pencil beam scanning nozzle and the plateau region of a 250 MeV proton beam, a uniform physical dose of 35 Gy (toxicity study) or 15 Gy (tumor control study) was delivered to the right hind leg of mice at various dose rates: Sham, Conventional (Conv, 1 Gy/s), Flash60 (57 Gy/s) and Flash115 (115 Gy/s). Acute radiation effects were quantified by measurements of plasma and skin levels of TGF-β1 and skin toxicity scoring. Delayed irradiation response was defined by hind leg contracture as a surrogate of irradiation-induced skin and soft tissue toxicity and by plasma levels of 13 different cytokines (CXCL1, CXCL10, Eotaxin, IL1-beta, IL-6, MCP-1, Mip1alpha, TNF-alpha, TNF-beta, VEGF, G-CSF, GM-CSF and TGF- β1). Plasma and skin levels of TGF-β1, skin toxicity and leg contracture were all significantly decreased in FLASH compared to Conv groups of mice. FLASH and Conv PBS had similar efficacy with regards to growth control of MOC1 and MOC2 head and neck cancer cells transplanted into syngeneic, immunocompetent mice. These results demonstrate consistent delivery of FLASH PBS radiation from 1 to 115 Gy/s in a clinical gantry. Radiation response following delivery of 35 Gy indicates potential benefits of FLASH versus conventional PBS that are related to skin and soft tissue toxicity.

Association of Transforming Growth Factor β1 Gene Polymorphisms and Inflammatory Factor Levels with Susceptibility to Sepsis.

Objective: To study the association of transforming growth factor β1 (TGF-β1) gene single nucleotide polymorphisms (SNPs) and plasma TGF-β1 levels with susceptibility to sepsis. Methods: The genotypes of the TGF-β1 gene rs1800469, rs1800468, rs1800470, and rs1800471 loci in 285 sepsis patients (119 patients with severe sepsis and 166 patients with mild sepsis) and 285 healthy individuals (control group) were analyzed through Sanger sequencing. Enzyme-linked immunosorbent assay was used to detect the levels of plasma inflammatory factors. Results: The TGF-β1 gene SNP rs1800469 C allele was 0.56 times lower than the T allele in terms of risk of susceptibility to sepsis (95% confidence interval [CI]: 0.43-0.72, p < 0.01). Carriers of the A allele at the rs1800468 locus of the TGF-β1 were 2.82 times more susceptible to sepsis than those with the G allele (95% CI: 1.62-4.91, p < 0.01). The T allele at the rs1800470 locus of TGF-β1 produced a lower risk of sepsis than those with the C allele (odds ratio [OR] = 0.74, 95% CI: 0.57-0.94, p = 0.02). The risk of susceptibility to sepsis in the TGF-β1 rs1800471 locus G allele was 3.54 times higher than that of C allele (95% CI: 2.14-5.86, p < 0.01). The TGF-β1 gene rs1800469 T > C and rs1800470 C > T were associated with mild sepsis, whereas rs1800468 G > A and rs1800471 C > G were associated with severe sepsis (p < 0.01). The TGF-β1 gene rs1800469 T > C and rs1800470 C > T were associated with lower plasma TGF-β1 levels, whereas rs1800468 G > A and rs1800471 C > G were associated with higher TGF-β1 levels (p < 0.05). Conclusion: The alleles T > C of rs1800469 and C > T of rs1800470 of the TGF-β1 gene were associated with lower plasma TGF-β1 levels and a reduced risk of sepsis susceptibility, whereas the alleles rs1800468 G > A and rs1800471 C > G were associated with higher TGF-β1 levels and risk of susceptibility to sepsis.

AMPK inhibits Smad3‐mediated autoinduction of TGF‐β1 in gastric cancer cells

We have previously shown that adenine monophosphate‐activated protein kinase (AMPK) regulates transforming growth factor β (TGF‐β)‐triggered Smad3 phosphorylation. Here we report that AMPK inhibits TGF‐β1 production. First, metformin reduced mRNA levels of TGF‐β1 in gastric cancer cells, in parallel to the decrease of its protein abundance. The effects were more prominent in the cells containing LKB1, an upstream kinase of AMPK. Second, knockdown of Smad3 by siRNA abrogated the expression of TGF‐β1. Third, metformin suppressed firefly luciferase activity whose transcription was driven by TGF‐β1 promoter. In accordance, deletion of the putative binding site of Smad3 in the TGF‐β1 promoter region severely impaired the promoter activity and response to metformin. Fourth, in support of our in vitro study, clinical treatment of type 2 diabetes with metformin significantly reduced the plasma level of TGF‐β1. Finally, immunohistochemical studies revealed that TGF‐β1 was highly expressed in human gastric cancer tissues as compared with adjacent normal tissues. In contrast, p‐AMPK exhibited opposite changes. Furthermore, the survival rate of gastric cancer patients was positively correlated with p‐AMPK and negative with TGF‐β1. Therefore, our present studies depict a mechanism underlying AMPK suppression of TGF‐β1 autoinduction, which is mediated through inhibition of Smad3 phosphorylation and activation. Collectively, our study sheds a light on the potential usage of AMPK activators in the treatment of TGF‐β1‐mediated gastric cancer progression.

The involvement of neuropilin-1 in primary immune thrombocytopenia

目的探讨神经纤毛蛋白-1（NRP-1）在调节性T细胞（Treg）上的表达与其配体信号素3A（Sema3A）、转化生长因子β1（TGF-β1）以及1型辅助T细胞（Th1）和2型辅助T细胞（Th2）之间平衡的关系。方法纳入2014年3月至2015年5月就诊的62例ITP患者（新诊断ITP 33例、慢性ITP29例），以同期30名健康体检者作为正常对照组。流式细胞术检测Treg细胞NRP-1表达，酶联免疫吸附法（ELISA）检测血浆Sema3A、TGF-β1、IFN-γ和IL-4水平，实时聚合酶链式反应（RT-PCR）检测外周血NRP-1、Sema3A、TGF-β1 mRNA表达水平。分别采用单因素方差和独立样本t检验进行三组间和两组间比较，用Spearman相关系数评估NRP-1、Sema3A、TGF-β1 mRNA表达的相关性。结果与慢性ITP组和正常对照组比较，新诊断ITP组Treg细胞NRP-1表达降低［分别为（0.15±0.03）％、（0.33±0.15）％、（0.46±0.06）％，P<0.01］，血浆Sema3A水平升高［分别为（8.10±1.32）µg/L、（7.41±1.30）µg/L、（2.88±0.82）µg/L，P<0.01］，血浆TGF-β1水平降低［分别为（16.50±3.36）µg/L、（35.17±10.26）µg/L、（41.00±10.02）µg/L，P<0.01］，血浆IFN-γ水平升高［分别为（17.21±2.80）ng/L、（10.23±1.59）ng/L、（8.18±3.27）ng/L，P<0.01］，Th1/Th2（IFN-γ/IL-4）比值升高（分别为1.29±0.30、0.72±0.16、0.61±0.27，P<0.01）。新诊断ITP、慢性ITP组NRP-1、Sema3A mRNA的表达均低于正常对照组（P<0.01）。新诊断ITP组NRP-1 mRNA表达与Sema3A、TGF-β1mRNA表达均呈正相关。结论NRP-1可能参与ITP的发病机制。

TGFB1 polymorphisms and TGF‐β1 plasma levels identify gastric adenocarcinoma patients with lower survival rate and disseminated disease

TGF‐β1 is involved in tumour growth. Four TGFB1 SNPs and TGF‐β1 production by stimulated PBMC were determined in seventy‐eight gastric adenocarcinoma patients. In addition, TGF‐β1 levels were measured in the plasma of further thirty patients. rs1800471‐G/C genotype was prevalent in patients (20.7%) compared to controls (8.4%), as it also was the rs1800468 SNP‐G/A genotype in stage IV patients (20.7%) compared to stage I, II and III patients, combined (10.3%). Conversely, the T/T rs1800469 SNP‐T/T genotype was absent in the former group and present in 19.0% in the latter. Furthermore, the rs1800469‐C/rs1800470‐T (CT) haplotype was found in 15.0% of stage IV patients as compared to 3.0% of the remaining patients (3.0%) and also identifies patients with worse five‐year life expectancy (P = .03). TGF‐β1 synthesis by stimulated PBMCs was significantly lower in patients with the risk SNPs or haplotype, compared to the alternative genotype. Finally, TGF‐β1 plasma levels were lower in patients with worse life expectancy. Analysis of TGFB1 SNPs and measurement of plasma TGF‐β1 levels serves to identify patients at risk of developing a more aggressive disease.

Deficiency of protease-activated receptor (PAR) 1 and PAR2 exacerbates collagen-induced arthritis in mice via differing mechanisms

Abstract Objectives Protease-activated receptor (PAR) 1 and PAR2 have been implicated in RA, however their exact role is unclear. Here, we detailed the mechanistic impact of these receptors on the onset and development of inflammatory arthritis in murine CIA and antigen-induced arthritis (AIA) models. Methods CIA or AIA was induced in PAR1 or PAR2 gene knockout (KO) and matched wild type mice. The onset and development of arthritis was monitored clinically and histologically. Immune cells, cytokines and MMPs were detected by ELISA, zymography, flow cytometry, western blot or immunohistochemistry. Results In CIA, PAR1KO and PAR2KO exacerbated arthritis, in opposition to their effects in AIA. These deficient mice had high plasma levels of IL-17, IFN-γ, TGF-β1 and MMP-13, and lower levels of TNF-α; T cells and B cells were higher in both KO spleen and thymus, and myeloid-derived suppressor cells were lower only in PAR1KO spleen, when compared with wild type cells. Th1, Th2 and Th17 cells were lower in PAR1KO spleens cells, whereas Th1 and Th2 cells were lower and Th17 cells higher in both KO thymus cells, when compared with wild type cells. PAR1KO synovial fibroblasts proliferated faster and produced the most abundant MMP-9 amongst three type cells in the control, lipopolysaccharides or TNF stimulated conditions. Conclusion This is the first study demonstrated that deficiency of PAR1 or PAR2 aggravates inflammatory arthritis in CIA. Furthermore, the protective functions of PAR1 and PAR2 in CIA likely occur via differing mechanisms involving immune cell differentiation and cytokines/MMPs.

Megakaryocyte-Derived TGF-β1 Negatively Regulates Megakaryopoiesis in Mice

Megakaryocytes (MK) and platelets contain a high concentration of transforming growth factor β1 (TGFβ1). Mice with conditional deletion of Tgfb1 in megakaryocytes (PF4Cre/Tgfb1flox/flox) resulted in &amp;gt;90% reduction of TGFβ1 in platelets and 50% reduction of TGFβ1 in plasma. TGFβ1 has been shown to play an inhibitory role in megakaryopoiesis in vitro, and inhibiting TGFβ1 increased megakaryopoiesis in vivo. However, the source of TGFβ1 in megakaryopoiesis is unknown. In this study, we tested whether megakaryocyte-derived TGFβ1 contributes to megakaryopoiesis in bone marrow (BM) by comparing three groups of mice: PF4Cre/Tgfb1flox/flox, littermate control Tgfb1flox/flox, and WTC57Bl/6 mice. Bones (femurs) from these mice (n=12) (age 15-30 weeks, males 60% and females 40%) were harvested, fixed, decalcified, sectioned, and H&amp;E stained. Whole stained BM areas of the sectioned femurs were scanned with an Aperio slide scanner to quantify the number of megakaryocytes and the demarcation membrane system (DMS) and ploidy (nucleus size) of the megakaryocytes were quantified by manually counting megakaryocytes and tracing their DMS and nucleus. The percentage of MK among total BM cells was calculated by dividing total numbers of BM cells in the total area of a BM section with the number of MK in the section. Freshly isolated BM cells were cultured in vitro in culture medium (DMEM+10%FBS) in the presence of thrombopoietin (TPO, 100 ng/ml) with and without TGFβ1 (20 ng/ml) or with a neutralized antibody against the active form of TGFβ1 (AF-101; 2 ug/ml). TGFβ1 and TPO levels in plasma, BM exudates, and cells were measured by ELISA. PF4Cre/Tgfb1flox/floxmice had &amp;gt;50% reduction in TGFβ1 levels in BM cells and exudates (TGFβ1 levels in BM exudates were 1.4 ± 0.033 ng in WT and 0.68 ± 0.065 ng in PF4CreTgfb1flox/floxmice, p&amp;lt;0.01; and in BM cells 50 ± 9 ng/ml in WT and 22 ± 4.2 ng/ml in PF4CreTgfb1flox/flox; p&amp;lt;0.001). MK numbers were ~25% higher in PF4Cre/Tgfb1flox/floxmice (n=6) compared to combined littermate controls (n=3) and WT (n=3) (MK was 0.30 ± 0.02% in PF4Cre/Tgfb1flox/flox and 0.23 ± 0.16% in combined controls; p&amp;lt;0.001 (n=6), whereas blood platelet counts were only marginally higher in PF4Cre/Tgfb1flox/flox (1114 ± 300) vs. controls (806 ± 116; p&amp;lt;0.05). There was a ~2-fold higher plasma TPO levels in PF4CreTgfb1flox/floxmice vs. WT (p=0.04, n=4). Increased DMS and nucleus areas in MK have been shown to correlate with proplatelets formation and platelet production. However, DMS and nuclear areas remained unchanged between genotypes [(DMS area was 197 ± 46 in PF4CreTgfb1flox/flox and 228 ± 50 um2 in combined WT and littermate controls (p=0.3), and nucleus size was 154 ± 23 in PF4CreTgfb1flox/flox and 160 ± 33 um2 in controls (p=0.7)], indicating that the role of TGFβ1 is limited to megakaryopoiesis. To test whether the in vivo phenotype was recapitulated, we cultured whole BM isolated from WT and PF4Cre/Tgfb1flox/flox mice, which showed a ~2.5-fold increase in MK numbers vs. WT when cultured for 5 days in TPO-supplemented medium. The addition of recombinant TGFβ1 in culture medium inhibited MK numbers, and a neutralizing antibody against TGFβ1 resulted in increased MK numbers. We conclude that MK-derived TGFβ1 negatively regulates megakaryopoiesis in mice. Further investigation is needed to determine the mechanism by which TGFβ1 regulates TPO-induced megakaryopoiesis. Our study may be important in megakaryocyte generation in vitro and may have important implications in vivo under normal and stress-inducing conditions where variable megakaryopoiesis is observed, such as essential thrombocythemia and primary myelofibrosis. Disclosures No relevant conflicts of interest to declare.

Angiotensin Converting Enzyme Inhibitor Reduces Radiation Induced Fibrosis in Peri-Capsular Implant

Radiotherapy after implant-based breast reconstruction can cause fibrosis and contracture in the capsule around the implant that may lead to deterioration of the cosmetic result, pain and tenderness and the need for a second surgery. Angiotensin converting enzyme (ACE) inhibitors reduce fibrosis by suppressing angiotensin-II synthesis and TGF-β pathway. In our study, the efficacy ACE inhibitor (Ramipril), on radiation induced capsular fibrosis was evaluated. Thirty Wistar rats in 5 groups were used. Group 1: implant; Group 2: implant + radiation; Group 3: ramipril + implant; Group 4: ramipril + implant + radiation; Group 5: sham. Ramipril treatment was started 5 days before surgery and continued for 12 weeks after surgery. A mini silicone implant was placed in the back of the rats. Single fraction of 21.5 Gy radiation was applied. Tissues were examined histologically and immunohistochemically (TGF- β1, MMP-2, TIMP-2 expression). The alteration of plasma TGF-β1 levels were examined before and after the experiment. After applying implant or implant + radiation, capsular thickness, percentage of fibrotic area, tissue and plasma TGF-β1 levels significantly increased and MMP-2/TIMP-2 ratio significantly decreased compared to sham group. In ramipril treated groups, the decrease in capsular thickness, fibrosis, TGF-β1 positivity, and increase in MMP-2 /TIMP-2 ratio was found to be significant. In ramipril + implant + radiation group, the alteration values of TGF-β1 dramatically decreased. Our results show that Ramipril reduces fibrosis in the capsule around the implant on histological, immunohistochemical and biochemical analysis. The results of our study may be important for the design of the clinical trials required to investigate the effective and safe doses of Ramipril, which is an inexpensive and easily tolerated drug, on human.Abstract 2049; Table 1The comparison of histological, immunohistochemical and biochemical Results of implant, implant + radiation and ramipril + implant + radiation groupsImplantp value*Implant + RadiationRamipril + Implant + Radiationp value**Capsular thickness (μm)163±35<0.003193±45115±25<0.001Fibrotic area (%)52±9<0.00164±1243±11.5<0.001TGF-β1 (%)28±5<0.00842±823±5<0.001MMP-2 (%)18±5117±4.518±51TIMP-2 (%)22±50.72325±419±5<0.001MMP-2/TIMP-2 ratio (%)0.85±0.30.4730.7±0.21.05±0.5<0.002After the experiment TGF-β1 levels (pg/ml)66±16179.5±2647±70.095Plasma TGF-β1 alteration value (pg/ml)29±15141±246±90.037* p value is statistical difference of between implant and implant + radiation groups ** p value is statistical difference of between implant + radiation and ramipril + implant + radiation groups Open table in a new tab

The role of thrombospondin-1 in the pathogenesis of antiphospholipid syndrome

ObjectiveAntiphospholipid syndrome (APS) is an acquired thrombophilia characterized by recurrent thrombosis and/or pregnancy morbidity, in the presence of antibodies to β2 glycoprotein-I (β2GPI), prothrombin or Lupus anticoagulant (LA). Anti-β2GPI antibodies recognize complexes of β2GPI dimers with CXCL4 chemokine and activate platelets. Thrombospondin 1 (TSP-1) is secreted by platelets and exhibits prothrombotic and proinflammatory properties. Therefore, we investigated its implication in APS. MethodsPlasma from APS patients (n = 100), Systemic Lupus Erythematosus (SLE) (n = 27) and healthy donors (HD) (n = 50) was analyzed for TSP-1, IL-1β, IL-17A and free active TGF-β1 by ELISA. Human Umbilical Vein Endothelial Cells (HUVECs) and HD monocytes were treated with total HD-IgG or anti-β2GPI, β2GPI and CXCL4 and CD4+ T-cells were stimulated by monocyte supernatants. TSP-1, IL-1β, IL-17A TGF-β1 levels were quantified by ELISA and Real-Time PCR. ResultsHigher plasma levels of TSP-1 and TGF-β1, which positively correlated each other, were observed in APS but not HDs or SLE patients. Patients with arterial thrombotic events or those undergoing a clinical event had the highest TSP-1 levels. These patients also had detectable IL-1β, IL-17A in their plasma. HD-derived monocytes and HUVECs stimulated with anti-β2GPI-IgG-β2GPI-CXCL4 secreted the highest TSP-1 and IL-1β levels. Supernatants from anti-β2GPI-β2GPI-CXCL4 treated monocytes induced IL-17A expression from CD4+ T-cells. Transcript levels followed a similar pattern. ConclusionsTSP-1 is probably implicated in the pathogenesis of APS. In vitro cell treatments along with high TSP-1 levels in plasma of APS patients suggest that high TSP-1 levels could mark a prothrombotic state and an underlying inflammatory process.

The Effects of Mesenchymal Stem Cells on Antimelanoma Immunity Depend on the Timing of Their Administration.

There is still a lively debate about whether mesenchymal stem cells (MSCs) promote or suppress antitumor immune response. Although several possible explanations have been proposed, including different numbers of injected and engrafted MSCs, heterogeneity in phenotype, and function of tumor cells, the exact molecular mechanisms responsible for opposite effects of MSCs in modulation of antitumor immunity are still unknown. Herewith, we used a B16F10 murine melanoma model to investigate whether timing of MSC administration in tumor-bearing mice was crucially important for their effects on antitumor immunity. MSCs, intravenously injected 24 h after melanoma induction (B16F10+MSC1d-treated mice), significantly enhanced natural killer (NK) and T cell-driven antitumor immunity, suppressed tumor growth, and improved survival of melanoma-bearing animals. Significantly higher plasma levels of antitumorigenic cytokines (TNF-α and IFN-γ), remarkably lower plasma levels of immunosuppressive cytokines (TGF-β and IL-10), and a significantly higher number of tumor-infiltrating, IFN-γ-producing, FasL- and granzyme B-expressing NK cells, IL-17-producing CD4+Th17 cells, IFN-γ- and TNF-α-producing CD4+Th1 cells, and CD8+cytotoxic T lymphocytes (CTLs) were observed in B16F10+MSC1d-treated mice. On the contrary, MSCs, injected 14 days after melanoma induction (B16F10+MSC14d-treated mice), promoted tumor growth by suppressing antigen-presenting properties of tumor-infiltrating dendritic cells (DCs) and macrophages and by reducing tumoricidal capacity of NK cells and T lymphocytes. Significantly higher plasma levels of TGF-β and IL-10, remarkably lower plasma levels of TNF-α and IFN-γ, and significantly reduced number of tumor-infiltrating, I-A-expressing, and IL-12-producing macrophages, CD80- and I-A-expressing DCs, granzyme B-expressing CTLs and NK cells, IFN-γ- and IL-17-producing CTLs, CD4+Th1, and Th17 cells were observed in B16F10+MSC14d-treated animals. In summing up, the timing of MSC administration into the tumor microenvironment was crucially important for MSC-dependent modulation of antimelanoma immunity. MSCs transplanted during the initial phase of melanoma growth exerted tumor-suppressive effect, while MSCs injected during the progressive stage of melanoma development suppressed antitumor immunity and enhanced tumor expansion.

Sodium copper chlorophyllin attenuates adenine-induced chronic kidney disease via suppression of TGF-beta and inflammatory cytokines.

The present study was designed to evaluate the effect of sodium copper chlorophyllin (SCC) in adenine-induced chronic kidney disease (CKD). CKD was induced in male Wistar rats by feeding 0.3% w/w adenine diet for 28 days. After induction, animals were treated with sodium copper chlorophyllin at dose 2.7, 5.4, and 10.8 mg/kg for the next 28 days. The biochemical and urines parameters like creatinine, blood urea nitrogen (BUN), albumin, total protein creatinine clearance, urea clearance, and glomerular filtration rate were assessed on days 0, 14, and 28. Plasma TGF-β1, COX-2, and IL-6 levels were assessed. Various oxidative stress parameters and TGF-β1 expression were determined in the kidney. Histopathology of the kidney was studied with different stains. Sodium copper chlorophyllin-treated animals showed a significant reduction in urine output and relative kidney weight. The treatment with sodium copper chlorophyllin significantly improved kidney function by normalizing biochemical and urine parameters. Treatment with SCC significantly reduced circulatory inflammatory mediators-TGF-β1, COX-2, and IL-6. Additionally, the treatment also significantly reduced oxidative stress and TGF-β1 expression in kidney tissues. Histopathology studies showed inhibition in the kidney damage due to the treatment of SCC. The sodium copper chlorophyllin treatment attenuated adenine-induced chronic kidney disease in rats.

Enhanced Integrin Activation of PLD2-Deficient Platelets Accelerates Inflammation after Myocardial Infarction

Background: Phospholipase (PL)D1 is crucial for integrin αIIbβ3 activation of platelets in arterial thrombosis and TNF-α-mediated inflammation and TGF-β-mediated collagen scar formation after myocardial infarction (MI) in mice. Enzymatic activity of PLD is not responsible for PLD-mediated TNF-α signaling and myocardial healing. The impact of PLD2 in ischemia reperfusion injury is unknown. Methods: PLD2-deficient mice underwent myocardial ischemia and reperfusion (I/R). Results: Enhanced integrin αIIbβ3 activation of platelets resulted in elevated interleukin (IL)-6 release from endothelial cells in vitro and enhanced IL-6 plasma levels after MI in PLD2-deficient mice. This was accompanied by enhanced migration of inflammatory cells into the infarct border zone and reduced TGF-β plasma levels after 72 h that might account for enhanced inflammation in PLD2-deficient mice. In contrast to PLD1, TNF-α signaling, infarct size and cardiac function 24 h after I/R were not altered when PLD2 was deleted. Furthermore, TGF-β plasma levels, scar formation and heart function were comparable between PLD2-deficient and control mice 21 days post MI. Conclusions: The present study contributes to our understanding about the role of PLD isoforms and altered platelet signaling in the process of myocardial I/R injury.

The impact of vitamin D3 intake on inflammatory markers in multiple sclerosis patients and their first-degree relatives.

Background & aimsIn our previous study, a Seesaw model was proposed for the fluctuation of crucial anti- (IL-10) and pro-inflammatory (Il-6 & IL-17A) cytokines through vitamin D3. In this paper, however, it is intended to extend the mentioned model by assessing the expression mRNA levels of IL-27 and TGF-β1 as well as the changes of plasma levels of IL-27, TGF-β1, IL-17A, IL-10, and IL-6 after treatment by vitamin D3.MethodVenous blood samples were drawn from Healthy Participants (HP, n = 25) and First-Degree Relative Participants (FDRP, n = 25) as control groups and Multiple Sclerosis Participants (MSP, n = 25) before and after eight weeks of supplementation with 50000 IU vitamin D3. The mRNA expression and plasma concentrations were gauged by using Real-Time PCR and ELISA assay, respectively.ResultsThe mRNA surfaces of IL-27, as well as TGF-β1, were up-regulated. However, the plasma levels of TGF-β1, IL-17A, and IL-6 were significantly different among the three groups. In addition, the plasma levels of IL-27, TGF-β1, IL-10, IL-17A, and IL-6 significantly changed following the administration of vitamin D3.ConclusionThe findings of this paper illustrate that anti-inflammatory cytokines could have a key role in immunomodulatory functions due to their anti-inflammatory functions. To conclude, this might contribute to preventing the pathophysiological process of MS. Also, the proposed model could be used as a preventive way on disposed people to multiple sclerosis, particularly in first degree relatives of these patients.

Clinical features and monocyte/macrophage subsets characterization in granulomatous vs non-granulomatous Crohn’s disease

Aims: Granuloma, mainly composed of macrophages, is a histological feature of Crohn’s disease (CD). However, its significance in CD has not been investigated adequately. Our study aims to address this issue by comparing the clinical manifestations and monocyte/macrophage subtypes between granulomatous and non-granulomatous CD.Materials and methods: Demographics, symptoms, endoscopic manifestations, histopathological features, and Montreal classification of patients with and without granulomas were compared. Flow cytometry was used to determine the phagocytosis and subsets of monocytes. ELISA was used to measure the plasma levels of TNF-α, IL-6, IL-1β, IL-10, CCL22, and TGF-β1. Immunohistochemistry was performed to quantify the expression of CD68, CD163 and iNOS.Results: Of the222 CD patients enrolled, granulomas were detected in 90. Compared with non-granulomatous CD patients, those with granulomas had younger age, increased rates of diarrhea and perianal complications, along with higher endoscopic score. Intestinal stenosis and crypt abscess were more frequently observed in granulomatous CD patients. A defective phagocytosis of monocytes was observed in granulomatous CD patients. Meanwhile, higher percentages of intermediate and non-classic monocytes, with a lower percentage of classic monocyte were found in them. Besides, they had higher levels of TGF-β1 and IL-10, a lower level of TNF-α, an increased ratio of CD163+/CD68+cells, and a decreased ratio of iNOS+/CD68+ cells.Conclusions: Granulomatous CD patients exhibited different manifestations compared with their non-granulomatous counterparts. More aggressive therapy may be needed in granulomatous CD patients. Furthermore, the heterogeneity of monocyte/macrophage subsets and altered plasma cytokine may underlie the difference between those two groups.

Transforming growth factor beta 1 levels in the blood of pediatric liver recipients: Clinical and biochemical correlations.

TGF-β1 is a cytokine with profibrogenic and immunosuppressive activities, which suggest the clinical significance of TGF-β1 for the assessment of graft function after LT. We analyzed the dynamics of TGF-β1 levels in the blood after LDLT in 135 pediatric liver recipients and examined the relationship between the cytokine levels and the laboratory and clinical variables. We found that TGF-β1 levels in the blood of patients with ESLD were lower than that in healthy children of the same age, P=.001. Moreover, blood levels of TGF-β1 were associated with liver disease etiology (r=.23) and hepatic fibrosis severity (r=.33). Before LDLT, TGF-β1 levels were significantly higher in children with good outcomes than in recipients who developed graft dysfunction early in the post-transplant period, P=.047. One month after LDLT, TGF-β1 levels in blood plasma increased in pediatric recipients, P=.002. Cytokine levels were significantly correlated with gender (r=.21) and HLA (r=-.24) mismatches, as well as with TAC dosage (r=-.32) later in the post-transplant period. One year after LDLT, TGF-β1 plasma levels were higher (P=.01) than those before LDLT and did not correlate with most of the investigated biochemical and clinical variables. Conclusion: Blood levels of TGF-β1 are associated with hepatic fibrosis severity, graft dysfunction development, and TAC dosage and can be regarded as a potential prognostic biomarker for the assessment of graft function and the optimization of immunosuppressant dosage in pediatric recipients after LDLT.

LncRNA CASC11 was downregulated in coronary artery disease and inhibits transforming growth factor-β1.

ObjectiveTo investigate the involvement of long non-coding RNA (lncRNA) Cancer Susceptibility 11 (CASC11) in patients with coronary artery disease (CAD).MethodsThis case–control study enrolled patients with CAD and age- and sex-matched healthy control subjects. The plasma levels of lncRNA CASC11 and transforming growth factor-beta 1 (TGF-β1) were measured. Diagnostic values of lncRNA CASC11 and TGF-β1 for CAD were determined using receiver operating characteristic curve analysis. Correlations between plasma levels of lncRNA CASC11 and TGF-β1 were analysed using linear regression.ResultsThe study enrolled 82 patients with CAD and 82 healthy controls. Plasma levels of lncRNA CASC11 were downregulated in patients with CAD, while plasma TGF-β1 levels were upregulated in patients with CAD, compared with healthy controls. Plasma levels of lncRNA CASC11 and TGF-β1 distinguished patients with CAD from healthy controls and were inversely correlated in both groups. LncRNA CASC11 over-expression mediated the downregulation of TGF-β1 in human primary coronary artery endothelial cells, while TGF-β1 over-expression showed no significant effects on lncRNA CASC11 levels. An 8-year follow-up study showed that low lncRNA CASC11 levels were closely correlated with a higher mortality rate in patients with CAD.ConclusionLncRNA CASC11 is downregulated in CAD and inhibits TGF-β1.

Short-term prognostic effects of circulating regulatory T-Cell suppressive function and vascular endothelial growth factor level in patients with non-small cell lung cancer and obstructive sleep apnea

ObjectiveTo determine if suppressive function of regulatory T-cells (Tregs) and vascular endothelial cell growth factor (VEGF) levels are closely associated with prognosis of patients with non-small cell lung cancer (NSCLC) and obstructive sleep apnea (OSA). MethodsPeripheral blood from 20 OSA patients, 44 newly diagnosed NSCLC patients with (n = 22) and without (n = 22) OSA was collected. Forkhead box protien 3 plus (Foxp3+) and CTLA-4+ Tregs ratio were analyzed with flow cytometry. Levels of VEGF, IL-10 and TGF-β1 were analyzed with enzyme-linked immuno sorbent assay. NSCLC patients with and without OSA were followed up for two years. Optimal cutoff values were determined by receiver operating characteristic curves. Survival analysis were performed using the Kaplan–Meier test. ResultsNSCLC patients with OSA showed higher Foxp3+Tregs ratio, higher plasma VEGF and TGF-β1 levels when compared with NSCLC patients without OSA (P < 0.05). In NSCLC patients with OSA or not, subjects with higher Foxp3+Treg ratio, higher TGF-β1 and VEGF levels tended to have poor mean survival time and two-year overall survival (OS, Foxp3+Treg: 636.7 vs. 704.8 days, 59.0% vs. 82.6%, P = 0.125; TGF-β1: 637.8 vs. 698.4 days, 57.0% vs. 84.4%, P = 0.054; VEGF: 642.9 vs. 677.5 days, 48.6% vs. 81.3%, P = 0.074). Multivariate Cox regression adjusted for disease stage and receipt of systemic treatments, confirmed the links between high VEGF level and worse OS (HR: 1.003; 95% CI: 1.001–1.005; P = 0.021). ConclusionsOSA may up-regulate the expression of circulating TGF-β1, VEGF and Foxp3+Tregs expression in NSCLC patients. Elevated VEGF level is closely associated with worse short-term survival in NSCLC patients with OSA or not.

Nilotinib Treatment of Patients Affected by Chronic Graft-versus-Host Disease Reduces Collagen Production and Skin Fibrosis by Downmodulating the TGF-β and p-SMAD Pathway.

The present study was conducted to investigate cellular and molecular features of chronic graft-versus-host disease fibroblasts (GVHD-Fbs) and to assess the effectiveness of nilotinib as a fibrosis modulator. Growth kinetics, phenotype, and differentiation of cultured skin biopsy-derived GVHD-Fbs were compared with normal fibroblasts from both a dermal cell line (n-Fbs) and healthy individuals undergoing cosmetic surgery (n-skin-Fbs). Collagen genes (COL1α1/COL1α2) and p-SMAD2 expression were assessed by real-time PCR and immunofluorescence. The in vivo effects of nilotinib on chronic GVHD (cGVHD)-affected skin were investigated by immunohistochemistry; the relationship to TGF-β plasma levels was assessed. Although the morphology, phenotype, and differentiation of cultured GVHD-Fbs were comparable to normal fibroblasts, growth was slower and senescence was reached earlier. The expression of COL1α1 and COL1α2 mRNAs was respectively 4 and 1.6 times higher in cGVHD-Fbs (P = .02); the addition of TGF-β increased n-Fbs, but not GVHD-Fbs, collagen gene expression. Compared with the baseline, the addition of 1 μM nilotinib induced 86.5% and 49% reduction in COL1α1 and COL1α2 expression in cultured GVHD-Fbs, respectively (P< .01). In vivo immunohistochemistry analysis of skin biopsy specimens from patients with cGVHD showed strong baseline staining for COL1α1 and COL1α2, which decreased sharply after 180 days of nilotinib; immunofluorescence revealed TGF-β inhibition and p-Smad2 reduction at the intracellular level. Of note, nilotinib treatment was associated with normalization of TGF-β levels both in culture supernatants and in plasma. In general, the data show that cGVHD fibroblasts promote fibrosis through abnormal collagen production induced by hyperactive TGF-β signaling. TGF-β inhibition at the intracellular and systemic level represents an essential antifibrotic mechanism of nilotinib in a clinical setting.

Changes in the Th9 cell population and related cytokines in the peripheral blood of infants with recurrent wheezing.

IntroductionT helper type 9 (Th9) cells have been shown to play a key role in initiating allergic reactions and promoting airway inflammation. However, to the best of our knowledge, their role has not been analyzed in infants with recurrent wheezing.Material and methodsWe performed a case-control study including 34 infants with recurrent wheezing and the same number of healthy infants as controls; all subjects were aged 1- to 3-years-old. The Th9 cell populations in the peripheral blood of these subjects were analyzed using flow cytometry, along with the assessment of Th9- and Th2-related plasma cytokine levels, including interleukin (IL)-1β, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, and IL-33, and transforming growth factor β1 (TGF-β1) using a Luminex 200 immunoassay.ResultsOur results indicatedthat infants with recurrent wheezing had higher percentages of Th9 cells (median, 0.69%; range, 0.46-1.08%) as compared to healthy infants (median, 0.25%, range, 0.13-0.36%; p < 0.05). In addition, infants with recurrent wheezing also exhibited higher plasma levels of cytokines IL-4, IL-9, IL-10, IL-33, and TGF-β1. Furthermore, the percentage of Th9 cells was positively correlated with the levels of IL-4 (r = 0.408, p < 0.05) and IL-9 (r = 0.644, p < 0.05) in the peripheral blood of wheezing infants.ConclusionsOur findings suggest that the percentage of Th9 cells is increased in infants with recurrent wheezing; thus, Th9 cells may play an important role in the pathogenesis of recurrent wheezing.

Long Noncoding RNA loc285194 Expression in Human Papillomavirus-Positive and -Negative Cervical Squamous Cell Carcinoma, C33A, and SiHa Cells and Transforming Growth Factor-β1.

BackgroundThis study aimed to investigate the expression of long noncoding RNA (lncRNA) loc285194 in cervical squamous cell carcinoma (CSCC) biopsies that were positive and negative for human papillomavirus (HPV) and in human CSCC cell lines SiHa and C33A and to investigate the overexpression of lncRNA loc285194.Material/MethodsCervical biopsy tissue and plasma samples from 66 patients with histologically confirmed CSCC, that were HPV16-positive (N=22), HPV18-positive (N=27), and HPV-negative (N=17), and healthy controls (N=20) and human CSCC cell lines SiHa (HPV16-positive) and C33A (HPV-negative) were studied. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure the expression of lncRNA loc285194 in cervical biopsies and plasma. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to measure levels of transforming growth factor-β1 (TGF-β1). A lncRNA loc285194 expression vector was constructed and transfected into SiHa and C33A cells that underwent a transwell assay for cell migration.ResultsExpression of lncRNA loc285194 was downregulated in HPV-positive and HPV-negative tissue samples and plasma from patients with CSCC and distinguished between patients and healthy controls. Plasma levels of loc285194 and TGF-β1 were significantly correlated with the presence of CSCC. In SiHa and C33A cells, TGF-β1 expression was downregulated, and cell migration was inhibited following lncRNA loc285194 overexpression. Although lncRNA loc285194 expression was not affected by TGF-β1 treatment, its effects on cell migration were reduced by TGF-β1.ConclusionsThe expression of lncRNA loc285194 inhibited the migration of CSCC cells in vitro through the inactivation of TGF-β1.