Concentrations Of EGF Research Articles

CIMAvax-EGF: A New Therapeutic Vaccine for Advanced Non-Small Cell Lung Cancer Patients.

Lung cancer is the common fatal illness with the highest incidence and mortality globally. Epidermal growth factor receptor overexpression by tumor cells is associated with uncontrolled proliferation, angiogenesis, anti-apoptotic signals, metastization, and invasiveness. CIMAvax-EGF vaccine consists of a chemical conjugate of the EGF with the P64 protein derived from the Meningitis B bacteria and Montanide ISA 51, as adjuvant. The vaccine is projected to induce antibodies against EGF that results in EGF withdrawal. CIMAvax-EGF demonstrated to be safe and immunogenic in advanced non-small cell lung cancer (NSCLC) patients. The efficacy study was an open-label, multicentric Phase III clinical trial, which enrolled 405 advanced NSCLC patients. Patients with proven stage IIIB/IV NSCLC, who had completed four to six cycles of chemotherapy (CTP) were randomized to receive CIMAvax-EGF or best supportive care. CIMAvax-EGF resulted in a significantly larger overall survival in patients receiving at least four doses. High EGF concentration at baseline was a good predictive biomarker of the vaccine activity and a poor prognostic biomarker for the non-treated population. The proportion of CD8+CD28− cells, CD4 cells, and the CD4/CD8 ratio after first-line CTP was also associated with CIMAvax-EGF clinical benefit. After completing the Phase III, a Phase IV trial was done where the vaccine was administered in primary care units. Administering the vaccine at primary care institutions granted better access and treatment compliance. Safety was confirmed. Several clinical trials are currently ongoing to validate EGF as a predictive biomarker of CIMAvax-EGF efficacy.

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions.

Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, we identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type- and cell stage-dependent manner by orchestrating AKT and RAF signalling.

Growth Factor Content in Human Sera Affects the Isolation of Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow.

Mesangiogenic Progenitor Cells (MPCs) are human bone marrow-derived multipotent cells, isolated in vitro under selective culture conditions and shown to retain both mesengenic and angiogenic potential. MPCs also co-isolated with multipotent stromal cells (MSCs) when bone marrow primary cultures were set up for clinical applications, using human serum (HS) in place of fetal bovine serum (FBS). MPC culture purity (over 95%) is strictly dependent on HS supplementation with significant batch-to-batch variability. In the present paper we screened different sources of commercially available pooled human AB type serum (PhABS) for their ability to promote MPC production under selective culture conditions. As the majority of “contaminating” cells in MPC cultures were represented by MSC-like cells, we hypothesized a role by differentiating agents present in the sera. Therefore, we tested a number of growth factors (hGF) and found that higher concentrations of FGF-2, EGF, PDGF-AB, and VEGF-A as well as lower concentration of IGF-1 give sub-optimal MPC recovery. Gene expression analysis of hGF receptors was also carried out both in MSCs and MPCs, suggesting that FGF-2, EGF, and PDGF-AB could act promoting MSC proliferation, while VEGF-A contribute to MSC-like cell contamination, triggering MPC differentiation. Here we demonstrated that managing hGF contents, together with applying specific receptors inhibitors (Erlotinib-HCl and Nintedanib), could significantly mitigate the batch-to-batch variability related to serum supplementation. These data represent a fundamental milestone in view of manufacturing MPC-based medicinal products.

Abstract 2626A: A lignan compound arctigenin inhibits prostate tumor growth in vitro and in vivo

Abstract Arctigenin is a novel anti-inflammatory lignan derived mainly from the seeds of Arctium lappa, an herb widely used in traditional Chinese medicine to treat inflammation related diseases such as cough, cold and swelling of throat. The present study was designed to investigate whether arctigenin is a potent inhibitor of prostate cancer using both in vitro and xenograft mouse models. The treatments with arctigenin at lower doses (< 2μM) for 48h dose-dependently inhibited the proliferation of two androgen-sensitive prostate cancer cell lines, LNCaP and LAPC-4, by 40-50% compared to control. There was no cytotoxicity observed in normal prostate epithelial PrEC cells with arctigenin doses up to 10μM. An intervention study was then conducted in severe combined immunodeficiency (SCID) mice to confirm the tumor-inhibitory effect of arctigenin in vivo. Male SCID mice (n = 10 per group) were inoculated with 5×10⁁5 LAPC-4 cells subcutaneously. One week later when tumors were palpable the intervention started. Mice were administered with arctigenin at 50mg/kg (LD) or 100mg/kg (HD) or vehicle control daily for 6 weeks by oral gavage. After 4 weeks of intervention the tumor growth was significantly inhibited by both of the arctigenin doses. After 6 weeks of intervention, the tumor volumes were reduced by 48% (LD arctigenin) and 67% (HD arctigenin) compared to control. There was no significant difference in food or water consumption or body weight among groups. An ELISA assay of growth factors demonstrates that arctigenin treatments significantly decreased the concentrations of VEGF, EGF, NGF-β, TNF-α, and FGF-β in tumor tissues compared to control. Western blot analysis revealed a significantly reduced expression of galectin-4 in tumor tissues by arctigenin treatment. Previous studies have shown that strong expression of galectin-4 can be induced in different types of cancer, associated with tumor growth and progression. A microRNA PCR array assay was performed to measure the tumor concentrations of 84 microRNAs involved in prostate cancer development and progression. A panel of microRNAs including miR-126 and miR-135 were identified to be responsive to arctigenin treatment. Further mechanistic investigation using tissue microarray assay is ongoing to measure the protein markers of proliferation and apoptosis in tumor tissues. This study provides a promising non-toxic agent to enhance chemoprevention of prostate cancer. These results warrant future clinical trial studies to confirm the anti-carcinogenic effect of arctigenin in humans. Citation Format: Piwen Wang, Susanne M. Henning, Jaydutt V. Vadgama. A lignan compound arctigenin inhibits prostate tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2626A.

571 - Identification of molecular mechanisms of acquired resistance to trastuzumab in gastric cancer

The HER2 receptor is overexpressed in 7-34% of gastric cancer (GC) tumors, being a potential therapeutic target. Trastuzumab, an anti-HER2 monoclonal antibody, in combination with platinum therapy was approved in 2010 as first-line treatment for advanced GC tumors overexpressing HER2. Despite the clinical benefit of trastuzumab, development of acquired resistance to treatment limits the efficacy of trastuzumab. To study the mechanisms of trastuzumab acquired resistance we generated, upon chronic exposure to trastuzumab, a trastuzumab-resistant cell line derived from the human HER2-positive GC cell NCI-N87, hereafter named NTR2.4. The resistant phenotype of NTR2.4 cell line was associated with an increased basal activation of SRC, MAPK/ERK and PI3K/mTOR HER2 downstream pathways that persisted upon exposure to trastuzumab. Trastuzumab treatment induced a compensatory upregulation of HER3 and an increased expression of the HER family ligands EGF, AREG, HB-EGF, and NRG1 in NTR2.4 compared with the trastuzumab-sensitive NCI-N87 cell line. Ligand stimulation induced resistance to trastuzumab in NCI-N87 cells. These results were replicated in another model of trastuzumab-resistance, OTR6 cell line, suggesting an important role of HER receptor plasticity and HER family ligand in trastuzumab efficacy and resistance. Analysis of clinical samples from HER2-positive GC patients treated with trastuzumab showed higher EGF concentration in serum samples and increased EGF mRNA expression in tumor biopsies at trastuzumab progression compared with samples previous to trastuzumab treatment. Treatment with Pan-HER, a novel antibody mixture against EGFR, HER2, and HER3, was effective in both trastuzumab-sensitive and trastuzumab-resistant cell lines and also in the presence of ligands. Altogether, our results suggested that Pan-HER would be a potential candidate to overcome trastuzumab resistance in advanced GC patients.

F25P preproinsulin abrogates the secretion of pro-growth factors from EGFRvIII cells and suppresses tumor growth in an EGFRvIII/wt heterogenic model

Extensive heterogeneity is a defining hallmark of glioblastoma multiforme (GBM) at the cellular and molecular levels. EGFRvIII, the most common EGFR mutant, is expressed in 24–67% of cases and strongly indicates a poor survival prognosis. By co-expressing EGFRvIII and EGFRwt, we established an EGFRvIII/wt heterogenic model. Using this approach, we confirmed that a mixture of EGFRvIII and EGFRwt at a certain ratio could clearly enhance tumor growth in vitro and in vivo compared with EGFRwt cells, thereby indicating that EGFRvIII cells promote tumor growth. Furthermore, we demonstrated that the EGFRvIII cells could support the growth of EGFRwt cells by secreting growth factors, thus acting as the principal source for maintaining tumor survival. F25P preproinsulin effectively reduced the concentrations of EGF, VEGF, and MMP-9 in the blood of tumor-bearing mice by competitively inhibiting the endoplasmic reticulum signal peptidase and increased the overall survival in orthotopic models. Taken together, our results provided an effective therapy of F25P preproinsulin in the EGFRvIII/wt heterogenic model.

Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells.

Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration. Here we use a simple modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR. While the threshold is remarkably robust, a more sophisticated model predicted that it could be modulated as a function of EGFR levels on the cell surface. We confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes—at the supraphysiological levels of receptor and/or ligand associated with cancer—uncoupling of the mechanisms leading to signalling through phosphorylation and attenuation through ubiquitination.

Abstract 5345: Enhanced inhibition of PC-3 xenograft prostate tumor growth by combination of green tea and quercetin with docetaxel

Abstract Chemotherapy with docetaxel (Doc) is a standard treatment for metastatic and castration-resistant prostate cancer. However, the development of chemoresistance and its side effects limit the clinical success of Doc. Previously we demonstrated that the combined use of natural products green tea (GT) and quercertin (Q) with Doc synergistically enhanced the anti-proliferative effect in androgen-independent prostate cancer PC-3 cells. The objective of the present study was to confirm the combined effect of GT, Q and Doc in xenograft prostate cancer mouse models and determine underlying mechanisms. Male severe combined immunodeficiency (SCID) mice (n = 10 per group) were inoculated with 5×105 PC-3 cells subcutaneously. When tumors reached a size of about 100mm3 the intervention started. Mice were administered with GT+Q, Doc 5mg/kg (LD), GT+Q+LD Doc, Doc 10mg/kg (HD) or control. The concentration of GT polyphenols in brewed tea administered as drinking water was 0.07% and Q was supplemented in diet at 0.4%. Doc was given through tail vein once a week for 4 weeks. There was a nonsignificant inhibition of tumor growth by GT+Q or LD Doc alone compared to control. However the combination treatment with GT+Q+LD Doc significantly inhibited the tumor growth by 62% compared to LD Doc alone after 7 weeks intervention. The combined effect was comparable to that by HD Doc treatment. Western blot analysis demonstrated a significantly increased ratio of Bax to Bcl-2 protein expression by GT+Q+LD Doc treatment compared to GT+Q or LD Doc. ELISA analysis of blood samples demonstrated that the combination treatment significantly decreased the blood concentrations of several growth factors, including VEGF, EGF, TGF-β, FGF-β, and TNF-α compared to GT+Q or LD Doc alone. A liver toxicity evaluation revealed that the blood alanine aminotransferase (ALT) level was significantly elevated with HD Doc treatment compared to control, while not changed by other groups. Further mechanistic investigations using protein array and tissue microarray assays are ongoing. This study provides a promising regimen by combining GT and Q with Doc to enhance chemotherapeutic effect in advanced prostate cancer in a less toxic manner. The results warrant future clinical trials to confirm the combined effect of the mixture in humans. Citation Format: Piwen Wang, Susanne Henning, David Heber, Jaydutt Vadgama. Enhanced inhibition of PC-3 xenograft prostate tumor growth by combination of green tea and quercetin with docetaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5345. doi:10.1158/1538-7445.AM2015-5345

Urinary EGF Receptor Ligand Excretion in Patients with Autosomal Dominant Polycystic Kidney Disease and Response to Tolvaptan.

Recent animal experiments suggest that dysregulation of the EGF receptor pathway plays a role in the pathophysiology of autosomal dominant polycystic kidney disease (ADPKD). Research on EGF receptor ligands in humans with ADPKD is lacking. EGF receptor ligands were measured in patients with ADPKD at baseline and after treatment with a vasopressin V2 receptor antagonist (V2RA) because this information might provide a rationale for future V2RA combination therapy. Blood and urine concentrations of the EGF receptor ligands heparin-binding (HB)-EGF, EGF, and TGF-α were measured by ELISAs in 27 patients with ADPKD who participated in a single-center study investigating a V2RA in 2011-2013 and in 27 controls who were selected from a general population-based observational study. Cyst fluid concentrations were also measured. In patients with ADPKD, ligands were measured at baseline, after 3-week treatment with a V2RA, and 3 weeks after drug withdrawal. The measured GFR (mGFR) was determined by iothalamate infusion, and total kidney volume was measured by magnetic resonance imaging. Urinary HB-EGF excretion and plasma concentration were higher in patients with ADPKD than in controls (median, 1.4 [interquartile range, 1.2-1.9] versus 0.6 [0.4-0.8] µg/24 hours [P<0.001] and 157.9 [83.1-225.9] versus 77.2 [37.2-174.3] pg/ml [P=0.04]). In contrast, urinary EGF excretion and plasma EGF concentration were lower in patients with ADPKD, whereas TGF-α did not differ between patients and controls. Higher HB-EGF excretion was correlated with more severe disease, assessed as lower mGFR (r=-0.39; P=0.05), higher total kidney volume (r=0.39; P=0.05), and higher urinary excretion of albumin and heart-type fatty acid-binding protein, whereas higher EGF excretion and TGF-α excretion were negatively correlated with disease severity. During V2RA treatment, HB-EGF excretion increased (from 1.4 [1.2-1.9] to 2.4 [2.1-3.1] µg/24 hours; P<0.001). In patients with ADPKD, higher urinary HB-EGF excretion is correlated with more severe disease. Whether this association is causal needs to be investigated in intervention studies.

Erlotinib alone or with bevacizumab as first-line therapy in patients with advanced non-squamous non-small-cell lung cancer harboring EGFR mutations (JO25567): an open-label, randomized, multicenter, phase II study.

The JO25567 study was a randomized phase II study to investigate the efficacy of the combination therapy consisted with erlotinib and bevacizumab for non-squamous nonsmall-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) activating mutation (1). The study showed that the progression-free survival (PFS) of the combination therapy group was longer than that of the erlotinib monotherapy group. The median PFS were 16.0 months (95% CI, 13.9-18.1) vs. 9.7 months (95% CI, 5.7-11.1) respectively. The over-all survival (OS) data are immature, but this study suggested a remarkable efficacy of this combination chemotherapy. Angiogenesis is essential for tumor growth, and a high microvessel density has been identified as a worse prognostic and a predictive factor of metastasis. Vascular endothelial growth factor (VEGF) was identified as the most potent and specific angiogenic mitogen for endothelial cells. VEGF expression is regulated by many cytokines and growth factors, for example interleukin-1, interleukin-6, transforming growth factor (TGF), and basic fibroblast growth factor, and so on. EGFR is expressed on vascular endothelial cells, which induce tube formation in response to EGF or TGF-alpha. Signaling through the EGFR axis also plays a role in the regulation of angiogenesis. At the cellular level, activation of EGFR by its ligands such as EGF or TGF-alpha results in increased VEGF expression (2). EGFR-tyrosine kinase inhibitors (EGFRTKIs) inhibit tumor cell growth and blocks synthesis of angiogenic proteins by tumor cells, like VEGF and, TGFalpha. In addition, VEGF production is upregulated by hypoxia via hypoxia-inducible factors (HIFs) in HIH3T3 cells transfected with mutant EGFR constructs. Hypoxia which is common in solid tumors then turns on the signal to initiate angiogenesis. Although the mechanism has not been clearly elucidated, it is suggested that EGFR is associated with enhanced signaling by phosphatidylinositol 3-kinase, which synergizes with hypoxia to regulate VEGF (3). Also, a recent study demonstrated a novel molecular pathway in which hypoxia promotes tumor growth by transcriptional activation of the Met proto oncogene. Through these some pathways, VEGF was regulated in tumors. With regard to prognosis, the role of angiogenesis has been established in the progression of lung cancers. Results from a retrospective study in Japan showed that the serum concentrations of EGF, hepatocyte growth factor (HGF), and VEGF in patients with NSCLC who received EGFR-TKI were significantly higher among patients with progressive disease (PD) than among those with stable disease (SD) or partial response (PR) (4). Increasing HGF and VEGF concentrations were significantly associated with lesser tumor shrinkage, independent of the EGFR mutation status. Furthermore, the higher concentrations of HGF and VEGF were significantly associated with shorter PFS and OS. The study suggested that the serum concentration of VEGF might be an independent prognostic factor in NSCLC. Excessive angiogenesis is also associated with resistance to EGFR-TKI. First-line treatment with an EGFR-TKI of

Effects of Preoperative and Perioperative Administration of Wobenzym Vital on Minimal-Mild Endometriosis

Background Endometriosis is an estrogen-dependent chronic inflammatory disease associated with pelvic pain, inflammation and infertility. Drugs that modulate the release of chemokines and angiogenic and inflammatory molecules are under evaluation. The aim of the present randomized, double-blind, placebo-controlled study was to investigate the effects of Wobenzym Vital, an enzyme-flavonoids combination supplement with antiinflammatory activity, as supportive treatment in patients affected by minimal-moderate endometriosis selected for surgery. Methods Thirty women suspected for endometriosis stage I-II were randomly assigned to receive Wobenzym Vital or placebo for a period of 40-60 days before surgery and 60 days thereafter. Visual analog scale (VAS) scores for pain at enrollment, time of surgery and 7 and 60 days afterwards were evaluated. Blood samples were taken at the same times for the assay of several inflammatory, angiogenic and growth factors and growth factor regulators. Results Treatment with Wobenzym Vital was associated with lower VAS scores before surgery and perioperatively. Surgery further reduced VAS scores in all patients. Patients receiving placebo underwent increases in plasmatic concentrations of sCD40L, EGF, IGFBP-1, IL-6, IL-8, TGF-α and TNF-α in the perioperative period, while this was not noted in patients with active treatment. A trend for lower concentrations of several of the tested analytes in the peritoneal fluid was found in patients treated with Wobenzym Vital. Conclusions In symptomatic patients affected by minimal-mild endometriosis undergoing surgery, preoperative and postoperative treatment with Wobenzym Vital results in reduced pain and lower concentrations of inflammatory and angiogenic factors in peripheral blood and the peritoneal cavity.

Oligomerization–function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor–ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag–probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~10%), whereas ~70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~1nM) was lower than that for half-maximum autophosphorylation (~8nM), which suggested the presence of an inactive dimer binding a single EGF molecule.

Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.

An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

Comparison of EGF and bFGF Expression In Vivo and their Effect In Vitro

Oral mucosal wound healed faster and with minimal scar formation compared with dermal wounds. The present study tried to found useful information in scarless oral wound healing process. This study firstly evaluated the effect of EGF and bFGF on the proliferation and migration of isolated fibroblasts from oral mucosa and skin. Then a liner wound was made in SD rats on transplanted oral mucosa and control skin. In vitro study found that both bFGF and EGF could promote the migration of dermal fibroblasts but not for oral fibroblasts. The oral fibroblasts proliferation was more sensitive to different concentrations of EGF, while bFGF had no significant effect on cell proliferation in this experiment. Our in vivo results demonstrated that oral mucosal wound healed with minimal scar after transplanting to skin and expressed higher EGF. No regular bFGF expression was found either on the transplanted oral mucosal or dermal wound. The results suggested that oral mucosa healed with minimal scar mainly due to its inherent cell phenotypes. The results also suggested the important role of EGF played in scar formation. The ultimate goal of this study is to apply this knowledge to find new ways to reduce scar formation in dermal wound.

CLINICAL CHARACTERISTICS AND DETERMINE THE CONCENTRATIONS OF AUTOBODIES AND CYTOKINES IN PATIENTS WITH RHEUMATOID ARTHRITIS

Background: Rheumatoid arthritis is an autoimmune disease with complex pathophysiological mechanisms, in which cytokines plays an important role. Currently, based on the understanding of the cytokines, the treatment of rheumatoid arthritis with biological agents had changed the course of the disease. Objectives: Study the clinical characteristics and determine the concentrations of autoantibodies (RF, Anti CCP) and cytokines (IL-1α,IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFN-γ, TNF-α, MCP1, EGF) in patients with rheumatoid arthritis, simultaneously, evaluate the correlation between the cytokines concentrations and levels of autoantibodies in patients with rheumatoid arthritis treated at Cho Ray Hospital.Subjects and Methods: Descriptive cross-sectional study conducted in 76 patients with rheumatoid arthritis treated at Cho Ray Hospital form June 2013 to April 2014.Results: - Clinical and laboratory characteristics: The most common clinical manifestations are arthritis (98.7%), symmetry joint damage (96.1%), morning stiffness over 1 hour (90.8%). Subcutaneous nodule is accounted for 3.9%. The most common joints involved in rheumatoid arthritis are wrist (93.4%), MCP and knee (90.8%), PIP (76.3%) and ankle (67.1%), the least common is the hip (14.5%). The percentage of autoantibodies and cytokines: rheumatoid arthritis patients with positive results of anti – CCP is accounted for 86%. rheumatoid arthritis patients with increased IL-1α concentrations is accounted for 40,8%; increased IL-1β concentration in 48.7%; increased IL-2 concentration in 32,9%; increased IL-4 concentration in 86%; increased IL-6 concentration in 100%; increased IL-8 concentration in 39,5%; increased IL-10 concentration in 81,6%; increased VEGF concentration in 51,3%; increased IFN-γ concentration in 67,1%; increased TNF-α concentration in 61,8%; increased MCP1 concentration in 30,3%; increased EGF concentration in 39,43%. The average concentrations of cytolines in rheumatoid arthritis patients is as followings: IL-1α (57.36 ± 196.43), IL-1β (123.77± 532.51), Il-2 (279.93 ± 945.04), IL-4(279.93 ± 945.04), IL-6(91.35 ± 170.52), IL-8(270.84 ± 445.45), IL-10(134.58 ± 496.14), VEGF(638.87 ± 540.18), IFN-γ(136.43 ± 338.68), TNF-α(106.27 ± 265.57), MCP1(292.34 ± 265.52), EGF(152.62 ± 123.64).RF is correlated with IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α. Anti – CCP is correlated with IL-1α, IL-6. Key words: Rheumatoid arthritis, cytokines

O-030 Colon Epithelial EGFR Inhibits NFκB-Dependent Innate Immune Responses Through CCDC50

Abstract Epidermal growth factor receptor (EGFR) is a key regulator of epithelial proliferation, survival and migration in the colon, where it functions to stimulate epithelial repair following injury. However, recent work by our lab has uncovered a novel role for epithelial EGFR to limit inflammation and subsequent tumorigenesis in mouse colitis models. This anti-inflammatory role for EGFR suggested that inhibition of epithelial innate immune responses might be a key mechanism underlying the therapeutic potential for EGF in IBD. In this study, our aim was to determine how EGFR regulated the epithelial cell response to lipopolysaccharide (LPS), and how a novel target of the EGFR kinase, coiled-coil domain containing 50 (CCDC50), mediates this response. EGFR inhibits colon epithelial responses to LPS by blocking NFκB activation through the adapter protein CCDC50. YAMC mouse colon epithelial (MCE) cells or human HT-29 cells were stimulated with LPS with or without EGF co-treatment. NFκB activity was measured in YAMC cells (NF-YAMC) stably transfected with an NFκB luciferase reporter. IL-8 and CXCL2 mRNA were measured by qPCR, and CXCL2 secretion was measured by ELISA. CCDC50 or TNFAIP3 were knocked down by siRNA with non-targeting siRNA as control. Knockdown was confirmed by qPCR. LPS induced concentration-dependent NFκB activity (EC50 = 40 ng/mL) in NF-YAMC cells, which was inhibited with escalating concentrations of EGF (see figure; IC50 = 0.2 ng/mL EGF, n = 6). This inhibitory effect of EGF was blocked with an EGFR inhibitor, and in MCE cells expressing a dominant-negative EGFR (EGFRwa2). EGF alone did not affect basal NFκB activity. This response was not specific for LPS, as EGF also inhibited NFκB activity induced by L18-MDP (a cell-permeable muramyl dipeptide analog). We next determined that EGF inhibited epithelial chemokine production following LPS stimulation. EGF inhibited LPS-stimulated induction of mRNA for IL-8 (HT-29 cells) and CXCL2 (YAMC and HT-29 cells), and protein for CXCL2 (YAMC cells). Using immunoprecipitation and Western blot, we determined that EGF induced tyrosine phosphorylation of CCDC50 in YAMC cells. Furthermore, siRNA-mediated CCDC50 knockdown blocked the ability of EGF to inhibit LPS-induced NFκB activity and CXCL2 mRNA expression, compared to non-targeting siRNA control. Since CCDC50 has previously been shown to interact with TNFAIP3 (A20), we determined whether CCDC50 required TNFAIP3 to inhibit LPS-induced NFκB activity by siRNA-mediated TNFAIP3 knockdown. However, loss of TNFAIP3 did not prevent the inhibitory effect of EGF, suggesting that the EGFR/CCDC50 mechanism involves an alternate pathway for NFκB inhibition. The inhibition of LPS-induced NFκB signaling by EGFR activation in the colonic epithelium represents a novel mechanism through which EGFR restrains inflammatory responses. EGFR inhibits NFκB through a novel adapter protein, CCDC50, which functions independently of TNFAIP3, and current work is underway to determine this mechanism. This study uncovers a previously underappreciated protective mechanism for EGFR to inhibit innate immune responses in the colon by blunting the response of colon epithelial cells to microbial triggers, and thus limiting subsequent chemokine production and inflammation. Thus, EGFR and CCDC50 are promising targets to modulate mucosal inflammation in IBD.(Equation is included in full-text article.).

Loss of hepatocyte EGFR has no effect alone but exacerbates carbon tetrachloride-induced liver injury and impairs regeneration in hepatocyte Met-deficient mice.

The role(s) of the epidermal growth factor receptor (EGFR) in hepatocytes is unknown. We generated a murine hepatocyte specific-EGFR knockout (KO) model to evaluate how loss of hepatocellular EGFR expression affects processes such as EGF clearance, circulating EGF concentrations, and liver regeneration following 70% resection or CCl4-induced centrilobular injury. We were able to disrupt EGFR expression effectively in hepatocytes and showed that the ability of EGF and heregulin (HRG) to phosphorylate EGFR and ERBB3, respectively, required EGFR. Loss of hepatocellular EGFR impaired clearance of exogenous EGF from the portal circulation but paradoxically resulted in reduced circulating levels of endogenous EGF. This was associated with decreased submandibular salivary gland production of EGF. EGFR disruption did not result in increased expression of other ERBB proteins or Met, except in neonatal mice. Liver regeneration following 70% hepatectomy revealed a mild phenotype, with no change in cyclin D1 expression and slight differences in cyclin A expression compared with controls. Peak 5-bromo-2'-deoxyuridine labeling was shifted from 36 to 48 h. Centrilobular damage and regenerative response induced by carbon tetrachloride (CCl4) were identical in the KO and wild-type mice. In contrast, loss of Met increased CCl4-induced necrosis and delayed regeneration. Although loss of hepatocellular EGFR alone did not have an effect in this model, EGFR-Met double KOs displayed enhanced necrosis and delayed liver regeneration compared with Met KOs alone. This suggests that EGFR and Met may partially compensate for the loss of the other, although other compensatory mechanisms can be envisioned.

Corrigendum to 'Mechanisms of paracrine cardioprotection by cord blood mesenchymal stromal cells'

The authors regret that an error appeared in the above article. When reading the manufacturer’s certificate of the Luminex cytokine/growth factor screening assay, a comma (thousand separator) was mistaken for a decimal separator. Hence, the analyte concentrations given in the article as pg/ml are in fact ng/ml. The paragraph of the subsection ‘Growth factors and cytokines in conditioned medium’ under RESULTS should read: In fibroblast-conditioned medium, the total protein content was 26 ± 10% lower than in CBMSC medium (CBMSC 27 ± 2 vs fibroblasts 20 ± 2 μg/ml, P < 0.05). The concentrations of selected growth factors and cytokines are shown in Fig. 2. The concentrations of VEGF (CBMSC 6.0 ± 0.9; fibroblast 4.9 ± 0.7 ng/ml), EGF (CBMSC 0.83 ± 0.09; fibroblast 0.65 ± 0.06 ng/ml), and Angiopoetin-2 (CBMSC 0.44 ± 0.04; fibroblast 0.49 ± 0.06 ng/ml) were similar, CBMSC medium contained more HGF (CBMSC 0.36 ± 0.05 vs fibroblast 0.04 ± 0.01 ng/ml, P < 0.001), and fibroblast medium more bFGF (CBMSC 0.08 ± 0.02 vs fibroblast 0.59 ± 0.14 ng/ml, P < 0.05) and IL-6 (CBMSC 0.15 ± 0.03 vs fibroblast 5.8 ± 1.0 ng/ml, P < 0.001). IL-10 and TNFα were not detectable in either medium. In addition, the Y-axis in Figure 2 should indicate ng/ml rather than pg/ml.

Growth factor and interleukin concentration in amniotic membrane suspension (AMS) and amniotic tissue homogenates (AMTH)

AbstractApplication of AMS or AMTH eye drops was suggested as a potential non‐surgical treatment alternative of therapy resistant corneal epithelial defects. We determined the concentration of growth factors EGF and FGFb and interleukins IL‐6 and IL‐8 in AMS and AMTH following different preparation methods. In AMTH EGF concentration was about 1.7‐17x and FGFb concentration about 1.2‐8.5x higher than in AMS. With higher EGF and FGFb concentrations and lack of IL‐6 and IL‐8 in AMTH, its use may be more appropriate in treatment of corneal epithelial defects. However, the most effective preparation method and the harvesting timepoint are yet to be determined.