The presence of GABA(A)-receptors on astrocytes was studied in explant and primary cultures of rat cerebellum, hippocampus and spinal cord by means of immunohistochemistry. For these studies we have used the monoclonal antibody bd <em>17</em> against the beta2- and beta3-subunits of GABA(A)-receptor. In explant cultures many neurones were intensely stained with the GABA(A)-receptor antibody whereas adjacent astrocytes revealed little or no immunoreactivity. In the far out<em>growth</em> zone of explant culture, however, many immunostained astrocytes were observed. In primary astrocyte cultures, only a few cells were stained by the antibody. Astrocytes which became reactive after producing an artificial scar or after addition of certain compounds such as dibutyryl cyclic AMP, interleukin-6, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and kainic acid, also revealed GABA(A)-receptor immunoreactivity. Furthermore, these astrocytes were intensely stained for glial fibrillary acidic protein and vimentin. From our studies we conclude that only a sub-population of normal astrocytes are immunopositive for the GABA(A)-receptor antibody whereas astrocytes which become reactive following injury of the tissue or after addition of dibutyryl cyclic AMP, the cytokine interleukin-6, <em>fibroblast</em> <em>growth</em> <em>factor</em> or the neurotoxin kainic acid express GABA(A)-sites.

Moderate ethanol consumption demonstrates a protective effect against cardiovascular disease and improves insulin sensitivity, possibly through angiogenesis. We investigated whether 1) ethanol would increase skeletal muscle <em>growth</em> <em>factor</em> gene expression and 2) the effects of ethanol on skeletal muscle <em>growth</em> <em>factor</em> gene expression were independent of exercise-induced <em>growth</em> <em>factor</em> gene expression. Female Wistar rats were used. Four groups (saline + rest; saline + exercise; <em>17</em> mmol/kg ethanol + rest; and <em>17</em> mmol/kg ethanol + exercise) were used to measure the <em>growth</em> <em>factor</em> response to acute exercise and ethanol administration. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF), transforming <em>growth</em> <em>factor</em>-beta(1) (TGF-beta(1)), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), Flt-1, and Flk-1 mRNA were analyzed from the left gastrocnemius by quantitative Northern blot. Ethanol increased VEGF, TGF-beta(1), bFGF, and Flt-1 mRNA at rest and after acute exercise. Ethanol increased resting Flk-1 mRNA. Ethanol increased bFGF mRNA independently of exercise. These findings suggest that 1) ethanol can increase skeletal muscle angiogenic <em>growth</em> <em>factor</em> gene expression and 2) the mechanisms responsible for the ethanol-induced increases in VEGF, TGF-beta(1), and Flt-1 mRNA appear to be different from those responsible for exercise-induced regulation. Therefore, these results provide evidence in adult rat tissue that the protective cardiovascular effects of moderate ethanol consumption may result in part through the increase of angiogenic <em>growth</em> <em>factors</em>.

In two- to five-week tissue cultures of biopsied adult human skeletal muscle, combined addition to the culture medium of insulin, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and epidermal <em>growth</em> <em>factor</em> synergistically increased creatine kinase activity <em>17</em>-fold, increased acetylcholine receptors tenfold, and accelerated muscle differentiation. This study provides the first demonstration of the beneficial influence of these peptides on human muscle. It also establishes a new culture medium, resulting in the following: (1) much better long-term <em>growth</em> and differentiation of biopsied adult human muscle; and (2) by allowing elimination of embryo extract and reduction of serum, an important step toward developing a fully defined medium for culturing biopsied adult human normal and pathologic muscle tissue.

It is known that the extracellular matrix (ECM) is able to signal to cells and thereby direct or modulate the transcription of certain mRNAs. This signaling plays an important role in tumor invasion and metastasis, wound healing, remodeling of the ECM and cell differentiation. There are several mechanisms whereby the ECM signals cells to change their metabolism: (1) receptor molecules binding to specific domains in the ECM, (2) direct phagocytosis of the ECM molecules or domains into the cell, (3) structural changes of the ECM domains. We report the effect of an ECM containing either mutant or normal Fbn1 on the transcription levels of several collagen mRNAs. Tsk/Tsk, Tsk/+ and +/+ mouse embryonic <em>fibroblast</em> cell lines were used. Tsk/Tsk cells produce only mutated fibrillin-1 which arises from mRNA containing an in-frame duplication of exons <em>17</em>-40. To test the effect of the ECM containing mutant Fbn1, cells of the Tsk/Tsk, Tsk/+ and the wild-type (+/+) genotype were each grown on an ECM produced by either Tsk/Tsk, Tsk/+ cells or by wild-type cells (+/+). The embryonic cells were genotyped by Northern analyses for Fbn1 and grown to confluence. The cultures were then harvested and the cells removed, leaving the matrix in the flasks. Matrices produced from Tsk/Tsk, Tsk/+ and from +/+ cells were reseeded with Tsk/Tsk cells, Tsk/+ cells or +/+ cells. The cells were plated at a confluent concentration and incubated on the matrices for 48 h, after which total RNA was harvested and cDNA generated. Real-time PCR using cDNA or Northern analyses using RNA were performed for Fbn1 and Types I, III and V collagens. The PCR and Northern results were normalized using beta-actin and GAPDH, respectively. The Northern analyses showed that the steady state levels of mRNA for Col1a1 were depressed in both Tsk/Tsk and +/+ cells when grown on the matrix produced by Tsk/Tsk cells. Real-time PCR was then performed with primers specific for Col1a2, Col3a1, Col5a1 and Col5a2. The results showed that cells with the Tsk/Tsk, Tsk/+, and +/+ genotype all had lower steady-state levels of the above 4 collagen mRNAs when grown on the matrix produced by homozygous Tsk/Tsk cells or the matrix produced by heterozygous Tsk/+ cells compared with those grown on a matrix produced by +/+ cells. We hypothesize that the mutated Fbn1 molecules with many additional EGF-calcium binding regions and TGF-beta binding domains may (1) change the homeostasis of the ECM by binding additional <em>growth</em> <em>factors</em> and/or (2) present a radically different ECM 3-dimensional architecture. Either or both of these changes could signal the cell to produce less collagen.

A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host <em>growth</em> <em>factors</em>. The present study isolated and identified heparin- and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot in these two coagulase-negative staphylococcal (CNS) species. The HBP was shown to be acidic with an approximate pI of 4.6 and a molecular mass around <em>17</em> kDa. The binding of heparin to HBP was inhibited by heparin, fucoidan, pentosan polysulphate and various other sulphated polysaccharides, but not by non-sulphated compounds. However, the purified HBP from both S. epidermidis and S. haemolyticus revealed broad specificity, and also bound bFGF, thrombospondin, von Willebrand <em>factor</em> and, weakly, fibrinogen. The N-terminal sequences of the <em>17</em>-kDa HBP from S. epidermidis and S. haemolyticus showed only limited identity. Comparison of the first 15 amino acid residues derived from either strain with known sequences in the protein databases revealed no close similarities. Taken together, these results suggest that the adhesion of at least some CNS to host sulphated glycosaminoglycans may be mediated by a previously uncharacterised group of surface proteins.

The heparin binding protein <em>17</em>/<em>fibroblast</em> <em>growth</em> <em>factor</em>-binding protein-1 (HBp<em>17</em>/FGFBP-1, GenBank accession no. NP-005121) has been reported to enhance angiogenesis as well as promotes tumor <em>growth</em> in vivo. Furthermore, this molecule was found to be highly expressed in the tissue and cell lines of oral squamous cell carcinoma (OSCC). 1α,25(OH)2D3 is used to study its potential to curb the expression of HBp<em>17</em>/FGFBP-1 in cancer cells. Consequently, we found that HBp<em>17</em>/FGFBP-1 mRNA and protein levels were significantly down-regulated. In this present study, we show that this event takes place via the NF-κB pathway since mRNA and protein levels of this pathway regulator, IκBα, were found to be significantly up-regulated. Furthermore, the promoter activity of HBp<em>17</em>/FGFBP-1 (region between -2<em>17</em> and +61) measured by a luciferase reporter assay was down-regulated following treatment. Silencing of VDR with siRNA showed the effect of 1α,25(OH)2D3 on HBp<em>17</em>/FGFBP-1. Based on these findings, we concluded that 1α,25(OH)2D3 down-regulated HBp<em>17</em>/FGFBP-1 expression via NF-κB. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

BACKGROUND

Venous ulcers are wounds that are thought to occur due to improper functioning of venous valves, usually of the legs. They are the major cause of chronic wounds, occurring in 70% to 90% of chronic wound cases. The treatment of venous ulcers also entails substantial costs. Autologous platelet rich plasma (PRP) is a simple office based procedure which helps in enhancing the wound healing by releasing many growth factors like platelet derived growth factors, fibroblast derived growth factors and epidermal growth factors.

OBJECTIVE

To study the efficacy of autologous platelet rich plasma in the management of chronic venous ulcer.

METHODS

12 patients with 17 venous ulcers were treated with PRP and treatment outcome was measured by percentage of improvement in area and volume of the ulcer.

RESULTS

12 patients with 17 ulcers were treated with PRP. The mean age of the patients was 33.5 years (SD 9.82). 10 were males and 2 were females. The mean duration of the healing of the ulcers was in 5.1 weeks (SD 3.1). The mean percentage improvement in the area and volume of the ulcer was 94.7% (SD 11.12) and 95.6% (SD 10.19) respectively.

CONCLUSIONS

PRP is safe, simple and effective procedure in treating chronic venous ulcers.

Understanding the cytokine/chemokine networks in CD4(+) and CD8(+) T cells during the acute phase of infection is crucial to design therapies for the control of early human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication. Here, we measured early changes in CD4(+) and CD8(+) T cells in the peripheral blood (PB), bone marrow (BM), and axillary lymph node (ALN) tissue of rhesus macaques infected with SIVMAC251. At 21 days after infection, all tissues showed a statistically significant loss of CD4(+) T cells along with immune activation of CD8(+) T cells in PB and ALN tissue. Twenty-eight different cytokines/chemokines were quantified in either anti-CD3/28 antibody- or staphylococcal enterotoxin B-stimulated single-positive CD4(+) and CD8(+) T cells. PB CD4(+) T cells produced predominantly interleukin-2 (IL-2), whereas CD4(+) and CD8(+) T-cell subsets in tissues produced β-chemokines both before and 21 days after SIV infection. Tissues generally exhibited massive upregulation of many cytokines/chemokines following infection, possibly in an attempt to mitigate the loss of CD4(+) T cells. There was no evidence of a T-helper 1 (TH1)-to-TH2 shift in CD4(+) T cells or a T-cytotoxic 1 (TC1)-to-TC2 cytokine shift in CD8(+) T cells in PB, BM, and ALN T-cell subsets during the acute phase of SIV infection. Despite the upregulation of several important effector cytokines/chemokines (IL-2, IL-12, IL-<em>17</em>, gamma interferon, granulocyte-macrophage colony-stimulating <em>factor</em>) by CD4(+) and CD8(+) T cells, upregulation of β-chemokines (CCL2 and CCL22), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-basic), hepatocyte <em>growth</em> <em>factor</em> (HGF), and migration inhibition <em>factor</em> (MIF) may provide a poor prognosis either by inducing increased virus replication or by other unknown mechanisms. Therefore, drugs targeting β-chemokines (CCL2 and CCL22), FGF-basic, HGF, or MIF might be important for developing effective vaccines and therapeutics against HIV.

OBJECTIVE

Human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection results in early depletion of CD4(+) T cells and dysregulation of protective immune responses. Therefore, understanding the cytokine/chemokine networks in CD4(+) and CD8(+) T cells in different tissues during the acute phase of infection is crucial to the design of therapies for the control of early viral replication. Here, we measured early changes in CD4(+) and CD8(+) T cells in peripheral blood (PB), bone marrow (BM), and axillary lymph node (ALN) tissue of rhesus macaques infected with SIVMAC251. There was no evidence of a T-helper 1 (TH1)-to-TH2 shift in CD4(+) T cells or a T-cytotoxic 1 (TC1)-to-TC2 cytokine shift in CD8(+) T cells in PB, BM, and ALN T-cell subsets during the acute phase of SIV infection. Despite the upregulation of several important effector cytokines/chemokines by CD4(+) and CD8(+) T cells, upregulation of β-chemokines, fibroblast growth factor-basic, hepatocyte growth factor, and migration inhibition factor may provide a poor prognosis.

BACKGROUND

Cardiovascular (CV) risk is high in children with chronic kidney disease (CKD), and further compounded in those who are overweight. Children with CKD have a unique body habitus not accurately assessed by body mass index (BMI). Waist-to-height ratio (WHr), a better predictor of CV risk in populations with short stature, has not been investigated in children with CKD.

METHODS

Analysis of 1723 visits of 593 participants enrolled in the Chronic Kidney Disease in Children (CKiD) study was conducted. CKiD participants had BMI and WHr measured and classified as follows: (1) lean (WHr ≤ 0.49, BMI < 85th percentile); (2) WHr-overweight (WHr>> 0.49, BMI < 85th percentile); (3) BMI-overweight (WHr ≤ 0.49, BMI ≥ 85th percentile); or (4) overweight by both BMI and WHr. Left ventricular mass index (LVMI), fasting lipids, fibroblast growth factor 23 (FGF23), blood pressure, and glucose were measured as markers of CV risk. Linear mixed-effects regression was used to evaluate differences in CV markers between overweight and lean groups.

RESULTS

Participants were 12.2 years old, 60% male, and 17% African-American. Approximately 15% were overweight by WHr but not by BMI. Overweight status by WHr-only or both WHr and BMI was associated with lower high-density lipoprotein (HDL) and higher LVMI, triglycerides, and non-HDL cholesterol compared to lean. CV markers of participants overweight by BMI-only were similar to those of lean children.

CONCLUSIONS

WHr-adiposity is associated with an adverse CV risk profile in children with CKD. A significant proportion of children with central adiposity are missed by BMI. WHr should be utilized as a screening tool for CV risk in this population.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>17</em> (FGF<em>17</em>) is a member of the FGF8 subfamily that appears to be relevant to folliculogenesis and oogenesis, as the prototype member FGF8 is an oocyte-derived protein that signals to cumulus cells. FGF8 has structural and receptor-binding similarities to FGF<em>17</em>, whose expression in the ovary has not been reported. In this study, we demonstrate localization of FGF<em>17</em> protein to the oocyte of preantral follicles, and to the oocyte and granulosa cells of antral follicles. Real-time PCR demonstrated the presence of mRNA in oocytes and, to a lesser extent, in granulosa and theca cells. FGF<em>17</em> mRNA abundance was low in granulosa and theca cells from healthy follicles and increased significantly in atretic follicles. Addition of FSH or IGF-I to granulosa cells in vitro decreased FGF<em>17</em> mRNA abundance, and treatment with FGF<em>17</em> inhibited estradiol and progesterone secretion from granulosa cells in relation to control cultures without these additives. We conclude that FGF<em>17</em> is a potential mediator of granulosa cell differentiation.

We previously detected immunoreactive <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) in maternal and fetal circulations. Here, we determined whether the amounts of FGF-2 in term maternal serum, cord serum, and amniotic fluid were altered in pregnancies complicated by diabetes, as these are associated with a higher incidence of fetal macrosomia and increased placental size. Serum and amniotic fluid were collected at term from normal pregnancies (n = <em>17</em>), women with pregestational insulin-dependent diabetes (n = 37; group A), patients with previously undiagnosed diabetes (n = 32; group B), women with gestational diabetes (n = 85; group C), and women with a milder form of glucose intolerance in pregnancy (n = 16; group D). Mean newborn weight and length, and placental weight did not significantly differ between normal and diabetic pregnancies, although the placental weight tended to be higher in the latter. However, 24% of the infants in group A and 19% in group B had a birth weight in excess of the 90th percentile. Levels of insulin in cord serum and amniotic fluid in groups A and B were significantly elevated compared to control values. FGF-2 was extracted from serum and amniotic fluid by heparin-Sepharose affinity chromatography and subjected to Western blot analysis or quantified by specific RIA. Western blot analysis of maternal serum, cord serum, and amniotic fluid from diabetic pregnant patients revealed, in each case, a single immunoreactive FGF-2 species of 18 kilodaltons; this was absent from nonpregnancy serum. In normal term pregnancies, the mean immunoreactive FGF-2 level in cord serum was 119 +/- 28 pmol/L, and that in amniotic fluid was 91 +/- 35 pmol/L. Values were significantly increased (2- to 4-fold) in both cord serum and amniotic fluid for all groups of diabetic patients. The mean FGF-2 level in normal term maternal serum was 104 +/- 24 pmol/L, and this was significantly increased in diabetic patients in groups B and C. The amount of FGF-2 in maternal serum showed a positive correlation with newborn weight and length, and placental weight (P < 0.05 or better, by Spearman rank correlation), and significant positive correlations also existed between the amounts of FGF-2 in cord serum and newborn or placental weight. The results suggest that the FGF-2 levels in maternal serum, cord serum, and amniotic fluid at term are elevated in pregnancies complicated by diabetes, and that the amounts of FGF-2 in maternal serum and cord serum are correlated with fetal and placental size.

BACKGROUND

Whether the type of dietary fat could alter cardiometabolic responses to a hypercaloric diet is unknown. In addition, subclinical cardiometabolic consequences of moderate weight gain require further study.

RESULTS

In a 7-week, double-blind, parallel-group, randomized controlled trial, 39 healthy, lean individuals (mean age of 27±4) consumed muffins (51% of energy [%E] from fat and 44%E refined carbohydrates) providing 750 kcal/day added to their habitual diets. All muffins had identical contents, except for type of fat; sunflower oil rich in polyunsaturated fatty acids (PUFA diet) or palm oil rich in saturated fatty acids (SFA diet). Despite comparable weight gain in the 2 groups, total: high-density lipoprotein (HDL) cholesterol, low-density lipoprotein:HDL cholesterol, and apolipoprotein B:AI ratios decreased during the PUFA versus the SFA diet (-0.37±0.59 versus +0.07±0.29, -0.31±0.49 versus +0.05±0.28, and -0.07±0.11 versus +0.01±0.07, P=0.003, P=0.007, and P=0.01 for between-group differences), whereas no significant differences were observed for other cardiometabolic risk markers. In the whole group (ie, independently of fat type), body weight increased (+2.2%, P<0.001) together with increased plasma proinsulin (+21%, P=0.007), insulin (+<em>17</em>%, P=0.003), proprotein convertase subtilisin/kexin type 9, (+9%, P=0.008) <em>fibroblast</em> <em>growth</em> <em>factor</em>-21 (+31%, P=0.04), endothelial markers vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin (+9, +5, and +10%, respectively, P<0.01 for all), whereas nonesterified fatty acids decreased (-28%, P=0.001).

CONCLUSIONS

Excess energy from PUFA versus SFA reduces atherogenic lipoproteins. Modest weight gain in young individuals induces hyperproinsulinemia and increases biomarkers of endothelial dysfunction, effects that may be partly outweighed by the lipid-lowering effects of PUFA.

BACKGROUND

http://ClinicalTrials.gov. Unique identifier: NCT01427140.

OBJECTIVE

To correlate the intraoperative concentrations of 20 synovial fluid biomarkers with preoperative symptoms, intraoperative findings, and postoperative outcomes in patients undergoing knee arthroscopy, with comparisons made to samples obtained from asymptomatic knees.

METHODS

Synovial fluid samples were obtained from 81 patients undergoing knee arthroscopy meeting the inclusion criteria, which included 70 samples from operative knees and 32 samples from contralateral knees. Preoperatively, baseline data obtained from clinical questionnaires including a visual analog scale (VAS) score, the Lysholm score, and the Knee Injury and Osteoarthritis Outcome Score-Physical Function Short Form were recorded. Synovial fluid was collected from both the operative knee and asymptomatic contralateral knee. Synovial fluid was stored with a protease inhibitor at -80°C until analysis. Intraoperative findings, procedures performed, and International Cartilage Repair Society (ICRS) cartilage status scores in all operative knees were documented. The concentrations of the following 20 biomarkers were measured using a multiplex magnetic bead immunoassay: matrix metalloproteinase (MMP) 3; MMP-13; tissue inhibitor of metalloproteinase (TIMP) 1; TIMP-2; TIMP-3; TIMP-4; <em>fibroblast</em> <em>growth</em> <em>factor</em> 2; eotaxin; interferon γ; interleukin (IL) 10; platelet-derived <em>growth</em> <em>factor</em> BB; IL-1 receptor antagonist; IL-1β; IL-6; monocyte chemotactic protein 1 (MCP-1); macrophage inflammatory protein 1α; macrophage inflammatory protein 1β; RANTES (regulated upon activation, normal T cell expressed and secreted); tumor necrosis <em>factor</em> α; and vascular endothelial <em>growth</em> <em>factor</em>. Clinical outcome scores were obtained in 83% of patients at a mean of <em>17</em> months' follow-up postoperatively. Analysis of variance and Pearson correlation analysis were performed to determine statistical significance between preoperative data, intraoperative findings, postoperative outcomes, and synovial fluid biomarker concentrations compared with asymptomatic contralateral knees.

RESULTS

Analysis was performed on 70 operative and 32 contralateral samples. There were strong positive correlations between ICRS score and age, symptom duration, VAS score, and Knee Injury and Osteoarthritis Outcome Score-Physical Function Short Form. A strong positive correlation was found between MCP-1 and IL-6 concentrations, intraoperative ICRS score, and continued pain at the time of final follow-up. MCP-1 and IL-6 were the strongest predictors of severe cartilage lesions, whereas IL-1 receptor antagonist was inversely related. MMP-3 levels were consistently elevated in all operative samples and directly correlated to increased preoperative VAS scores. RANTES, vascular endothelial growth factor, and platelet-derived growth factor BB were the strongest predictors of postoperative improvement at final follow-up regardless of injury and cartilage status.

CONCLUSIONS

Synovial fluid biomarkers have the capacity to reflect the intra-articular environment before surgery and potentially predict postoperative clinical outcomes. Recognition of key molecular players may yield future therapeutic targets, and large clinical trials exploring these discoveries are anticipated.

METHODS

Level III, therapeutic case-control study.

Macroautophagy/autophagy is one of the major responses to stress in eukaryotic cells and is implicated in several pathological conditions such as infections, neurodegenerative diseases and cancer. Interestingly, cancer cells take full advantage of autophagy both to support tumor growth in adverse microenvironments and to oppose damages induced by anti-neoplastic therapies. Importantly, different human oncogenes are able to modulate this survival mechanism to support the transformation process, ultimately leading to 'autophagy addiction'. Still, oncogenic signaling events, impinging on the control of autophagy, are poorly characterized, limiting our possibilities to take advantage of these mechanisms for therapeutic purposes. Here, we screened a library of activated kinases for their ability to stimulate autophagy. By this approach, we identified novel potential regulators of the autophagic process and, among them, the IKBKE oncogene. Specifically, we demonstrate that this oncoprotein is able to stimulate autophagy when overexpressed, an event frequently found in breast tumors, and that its activity is strictly required for breast cancer cells to support the autophagic process. Interestingly, different oncogenic pathways typically involved in breast cancer, namely ERBB2 and PI3K-AKT-MTOR, also rely on IKBKE to control this process. Ultimately, we show that IKBKE-dependent autophagy is necessary for breast cancer cell proliferation, suggesting an important supporting role for this oncogene and autophagy in these tumors.

BACKGROUND

AAK1: AP2 associated kinase 1; AMPK: 5'-prime-AMP-activated protein kinase; AKT1: AKT serine/threonine kinase 1; BAF: bafilomycin A1; CA: constitutively activated; CDK17: cyclin dependent kinase 17; CDK18: cyclin dependent kinase 18; CHUK: conserved helix-loop-helix ubiquitous kinase; EGF: epidermal growth factor; ERBB2: erb-b2 receptor tyrosine kinase 2; FGF: fibroblast growth factor; FM: full medium; GALK2: galactokinase 2; IKBKB: inhibitor of nuclear factor kappa B kinase subunit beta; IKBKE: inhibitor of nuclear factor kappa B kinase subunit epsilon; IKK: IκB kinase complex; KD: kinase dead; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK1: mitogen-activated protein kinase 1; MAPK15: mitogen-activated protein kinase 15; MTORC1: mammalian target of rapamycin kinase complex 1; myr: myristoylation/myristoylated; NFKBIA: NFKB inhibitor alpha; PDGF: platelet derived growth factor; PFKL: phosphofructokinase, liver type; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKCD: protein kinase C delta; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TNBC: triple-negative breast cancer; TSC2: TSC complex subunit 2; WB: western blot; WT: wild-type.

Obesity and its associated metabolic disorders are related to the onset of fatty liver and the balance of white adipose tissue (WAT) and brown adipose tissue (BAT). We hypothesized that metformin, an effective pharmacological treatment for type 2 diabetes, would inhibit white adipogenesis, fatty liver, and metabolic dysfunction. Metformin was treated daily for 14 weeks in a high-fat dieting C57BL/6J mice. Serum biomarkers were analyzed and protein level was assessed using confocal staining or flow cytometry. The development of lipid drops in the liver cells and white adipocyte was measured using hematoxylin and eosin or Oil Red O stains. Gene expressions were analyzed with quantitative real-time PCR. Metformin treatment decreased the body weight and improved the metabolic profile of obese mice. In obese mice, metformin also induced the expression of BAT-related markers and increased <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 21 expression in the liver and in white adipocyte. Metformin suppressed white adipocyte differentiation via induction of FGF21. Metformin improves Treg/Th<em>17</em> balance in CD4+ T cells in mice with high-fat diet-induced obesity. Metformin also improves glucose metabolism and metabolic disorder. Interleukin-<em>17</em> deficiency also decreases inflammation in mice. Therefore, metformin may be therapeutically useful for the treatment of obesity and metabolic dysfunction.

OBJECTIVE

We evaluated the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the healing of large traumatic tympanic membrane perforations (TMPs).

METHODS

Prospective clinical study.

METHODS

Tertiary university hospital.

METHODS

A randomized, prospective analysis was performed between June 2013 and August 2014 on the treatment of traumatic TMPs larger than 25% of the TM. Closure rate, closure time, hearing gain, and rate of otorrhea were compared between EGF and bFGF groups, as well as to an observation-only group.

RESULTS

Final analysis was performed on 86 patients at 3 months. The closure rates of perforation in the EGF, bFGF, and observation groups were 86.2%, 89.3%, and 72.4%, respectively. The closure rates in the EGF and bFGF groups were 14% to 17% higher than in the observation group, although the difference was not statistically significant for the total closure rate among the three groups (P = 0.200). The average closure time was significantly longer (P < 0.01) in the observation group than in the EGF and bFGF groups. However, the closure times in the EGF and bFGF groups were not significantly different (P = 0.92). In addition, differences in purulent otorrhea rates among the groups were not statistically significant (P = 0.82).

CONCLUSIONS

Both EGF and bFGF can accelerate the closure of human large traumatic TMPs. The healing outcomes among the two growth factors were not significantly different.

Interstitial lung <em>fibroblast</em> activation coupled with extracellular matrix production is a pathological signature of idiopathic pulmonary fibrosis (IPF), and is governed by transforming <em>growth</em> <em>factor</em> (TGF)-β/Smad signalling. We sought to define the role of heat shock protein (HSP)90 in profibrotic responses in IPF and to determine the therapeutic effects of HSP90 inhibition in a murine model of pulmonary fibrosis.We investigated the effects of HSP90 inhibition in vitro by applying <em>17</em>-AAG (<em>17</em>-allylamino-<em>17</em>-demethoxygeldanamycin) to lung <em>fibroblasts</em> and A549 cells and in vivo by administering <em>17</em>-DMAG (<em>17</em>-dimethylaminoethylamino-<em>17</em>-demethoxygeldanamycin) to mice with bleomycin-induced pulmonary fibrosis.HSP90 expression was increased in (myo)<em>fibroblasts</em> from fibrotic human and mouse lungs compared with controls. <em>17</em>-AAG inhibited TGF-β1-induced extracellular matrix production and transdifferentiation of lung <em>fibroblasts</em> and epithelial-mesenchymal transition of A549 cells. The antifibrotic effects were associated with TGF-β receptor disruption and inhibition of Smad2/3 activation. Co-immunoprecipitation revealed that HSP90β interacted with TGF-β receptor II and stabilised TGF-β receptors. Furthermore, <em>17</em>-DMAG improved lung function and decreased fibrosis and matrix metalloproteinase activity in the lungs of bleomycin-challenged mice.In conclusion, this is the first study to demonstrate that HSP90 inhibition blocks pulmonary <em>fibroblast</em> activation and ameliorates bleomycin-induced pulmonary fibrosis in mice.

OBJECTIVE

To investigate whether protease-activated receptor 1 (PAR-1) and/or PAR-2 promotes the invasiveness/proliferation of synovial fibroblasts (SFs) and to determine the signaling mechanisms of these pathways.

METHODS

SFs were isolated from the synovial tissue of patients with rheumatoid arthritis (RA), patients with osteoarthritis (OA), and PAR-1- or PAR-2-knockout (KO) mice. Expression of PAR-1 and PAR-2 was detected by immunofluorescence and Western blotting. The invasion and proliferation of SFs were measured by invasion assay and MTT assay, respectively. Matrix metalloproteinase 2 (MMP-2) and MMP-9 were detected by zymography, and cytokines were measured by enzyme-linked immunosorbent assay.

RESULTS

PAR-1 and PAR-2 were colocalized with SFs in RA and OA synovium and, to a considerably lesser extent, in normal synovium. Inhibition of PAR-2 by small interfering RNA (siRNA) inhibited RASF invasion and proliferation, whereas blocking of PAR-1 by siRNA had the reverse effects. SFs from PAR-2-KO mice exhibited slower rates of proliferation and invasion. SFs from PAR-1-KO mice produced less MMP-2 and, in response to tumor necrosis factor α (TNFα) stimulation, had increased MMP-9 secretion when compared to SFs from wild-type and PAR-2-KO mice. Inhibition of PAR-1, but not PAR-2, stimulated the secretion of interleukin-17 (IL-17) and TNFα by RASFs. Furthermore, PAR-1 and PAR-2 had opposing effects on the activation of ERK, p38, and NF-κB.

CONCLUSIONS

Activation of PAR-1 stimulates MMP-2 secretion, inhibits RASF growth and invasion, and decreases production of IL-17 and TNFα by RASFs, whereas activation of PAR-2 stimulates RASF growth and invasion and increases production of TNFα. Thus, although PAR-1 and PAR-2 are coexpressed by RASFs, PAR-2 alone appears to be responsible for the aggressive properties of RASFs and is likely to contribute to the pathologic progression of RA.

BACKGROUND

An exploratory translational analysis was conducted as part of a phase II study of dovitinib to assess the relevance of soluble serum proteins and circulating tumor (ct) DNA (ctDNA) as biomarkers in patients with tyrosine kinase inhibitor (TKI)-refractory gastrointestinal stromal tumors (GISTs).

METHODS

Predose serum samples were collected from 30 patients on day 1 of cycle 1 and cycle 2. Serum levels of angiogenesis-related proteins were assessed by enzyme-linked immunosorbent assay, and Beads, emulsions, amplification, and magnetics (BEAMing) assays were carried out to detect mutations in serum ctDNA.

RESULTS

Dovitinib increased vascular endothelial <em>growth</em> <em>factor</em> (VEGF)165 (1.26-fold, P = 0.006), VEGF-A (1.27-fold, P = 0.004), placental <em>growth</em> <em>factor</em> (6.0-fold, P = 0.002), <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (1.45-fold, P = 0.02), and interleukin 8 (1.75-fold, P = 0.04) levels, and decreased soluble vascular endothelial <em>growth</em> <em>factor</em> receptor (sVEGFR)-2 levels (0.8-fold, P = 0.001). The changes in sVEGFR-2 were significantly associated with metabolic response determined by positron emission tomography (P = 0.02) and progression-free survival (PFS; P = 0.02). Secondary kinase mutations were identified in the ctDNA of 11 patients (41%), and these patients all had mutations involving KIT exon <em>17</em>. Patients with secondary KIT mutations had significantly worse overall survival {median, 5.5 months [95% confidence interval (CI) 3.8-7.2 months]} than those with no detectable secondary mutations [9.8 months (95% CI 9.6-10.0 months); hazard ratio = 2.7 (95% CI 1.0-7.3); P = 0.047].

CONCLUSIONS

Changes in sVEGFR-2 levels were associated with dovitinib-mediated antitumor activity. Genotyping of serum ctDNA with BEAMing is useful for the identification of resistant mutations potentially associated with poor prognosis in patients with GISTs.

To study the tear cytokine and the conjunctival and oral mucosal marker profile in chronic ocular Stevens-Johnson syndrome (SJS) and their alteration following mucous membrane grafting (MMG) for lid margin keratinisation (LMK).

In a 1-year prospective study, SJS cases (n=25) and age-matched/sex-matched healthy controls (n=25) were recruited. Tear specimen (Schirmer's strip), conjunctival and oral mucosal imprints were collected from controls and SJS cases pre-MMG and post-MMG (at first follow-up, n=<em>17</em>). Tear cytokines were profiled using 27-bioplex array. Transforming <em>growth</em> <em>factor</em>-beta (TGF-β)-mediated extracellular matrix changes in conjunctival and oral mucosal cells were analysed by gene expression studies. 30 RESULTS: Tear cytokine profiling of chronic SJS cases at pre-MMG stage revealed significant upregulation of cytokines granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF), interleukin (IL)-8, IL-1β, monocyte chemoattractant protein-1, IL-15, IL-2, IL-<em>17</em>A and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) with downregulation of IP-10 (interferon gamma-induced protein 10), tumour necrosis <em>factor</em>-α, interferon-γ, IL-10, vascular endothelial <em>growth</em> <em>factor</em>, regulated upon activation normal T-cell expressed and secreted (RANTES), IL-7, IL-12p70 and IL-13, with maximal increase in GM-CSF and maximal downregulation of IP-10, respectively. Of these, IL-2, IL-15, bFGF and IL-<em>17</em>A showed significant correlation with disease severity, pre-MMG. Conjunctival cells pre-MMG showed increase in TGF-β1, TGF-βRII, connective tissue <em>growth</em> <em>factor</em> and collagen-III gene expression by 10, 67, <em>17</em>3 and 184 folds, respectively, which dropped to 1.3, 11, 13.5 and 19 folds correspondingly, post-MMG. However, their expressions in oral mucosa were negligible.

A proinflammatory, profibrotic, antiapoptotic ocular surface milieu characterises chronic ocular SJS. IP-10, an antifibrotic cytokine was noted to be maximally downregulated, unlike in other forms of chronic dry eye disease. The alterations in the ocular surface are seen to reverse largely with MMG for LMK.

The protein kinase AKT1 (v-akt murine thymoma viral oncogene homolog 1), also referred to as protein kinase B (PKB), is an essential mediator of the phosphatidylinositol 3-kinase signaling pathway. Elevated activity of AKT1 is common in human cancer. Localization at the plasma membrane, leading to enhanced phosphorylation and activation of AKT1, is an important <em>factor</em> determining the oncogenicity of this kinase. Although the phosphatidylinositol 3-kinase signaling pathway is frequently upregulated in cancer, cancer-specific mutations in AKT1 are not common. Recently, such a mutation has been identified in breast, colon and ovarian cancers. The mutation is located in the pleckstrin homology (PH) domain of AKT1 and results in a glutamic acid to lysine substitution at residue <em>17</em>. The resultant change in the conformation of the PH domain facilitates membrane binding of the mutant protein. Here we show that exchange of the PH domain leading to preferential binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) over phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) constitutively activates AKT1. AKT1 with this altered PIP affinity induces oncogenic transformation in cultures of chicken embryo <em>fibroblasts</em> and causes neoplastic <em>growth</em> and angiogenesis in the chorioallantoic membrane of the chicken embryo. Gain-of-function mutants of AKT1 may not be affected by PI3K inhibitors that are currently in development. Therefore, AKT1 remains a distinct and important cancer target.

BACKGROUND

Dental pulp inflammation and repair are closely related. Osteocalcin (OCN), a glycoprotein present in dentin matrix, is expressed by odontoblasts. Although OCN is considered a reparative molecule inside the dental pulp, it is not clear if it is involved in pulpal inflammation. The objective of this study was to localize OCN in reversible and irreversible pulpitis and to describe its possible function in inflammation.

METHODS

Pulp tissues in the form of reversible and irreversible pulpitis were collected from the endodontic clinic. Those from impacted teeth were used as controls. Immunohistochemistry was used to localize OCN. Samples were analyzed for OCN and inflammatory mediator expression using multiplex assay.

RESULTS

OCN in inflamed tissues was localized in cells and matrix around calcification areas and in cells around blood vessels but not in normal tissues. The plex assay (Bio-Plex 200, Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) showed OCN expression in reversible pulpitis significantly higher than in irreversible pulpitis, and both were significantly higher than in the controls. A panel of inflammatory mediators showed an increase in reversible and irreversible pulpitis. Another panel was decreased in both stages compared with the controls. OCN expression in reversible pulpitis was positively correlated to the expression of vascular endothelial <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, macrophage inflammatory protein-1β, monocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin (IL)-<em>17</em>, and soluble IL-2 receptor α and negatively correlated to that of IL-1α, IL-1β, IL-8, granulocyte macrophage colony-stimulating <em>factor</em>, and macrophage inflammatory protein-1α.

CONCLUSIONS

Profound understanding of the pulp inflammatory process would lead to new molecular treatment strategies. Our data indicate that OCN expression in reversible pulpitis is associated with angiogenic markers, suggesting its potential use in regenerative treatment.

BACKGROUND

Although airway remodeling is a central feature of COPD, the mechanisms underlying its development have not been fully elucidated. The goal of this study was to determine whether histone deacetylase (HDAC) 2 protects against cigarette smoke (CS)-induced airway remodeling through IL-17A-dependent mechanisms.

METHODS

Sputum samples and lung tissue specimens were obtained from control subjects and patients with COPD. The relationships between HDAC2, IL-17A, and airway remodeling were investigated. The effect of HDAC2 on IL-17A-mediated airway remodeling was assessed by using in vivo models of COPD induced by CS and in vitro culture of human bronchial epithelial cells and primary human fibroblasts exposed to CS extract, IL-17A, or both.

RESULTS

HDAC2 and IL-17A expression in the sputum cells and lung tissue samples of patients with COPD were associated with bronchial wall thickening and collagen deposition. Il-17a deficiency (Il-17a-/-) resulted in attenuation of, whereas Hdac2 deficiency (Hdac2+/-) exacerbated, CS-induced airway remodeling in mice. IL-17A deletion also attenuated airway remodeling in CS-exposed Hdac2+/- mice. HDAC2 regulated IL-17A production partially through modulation of CD4+ T cells during T helper 17 cell differentiation and retinoid-related orphan nuclear receptor γt in airway epithelial cells. In vitro, IL-17A deficiency attenuated CS-induced mouse fibroblast activation from Hdac2+/- mice. IL-17A-induced primary human fibroblast activation was at least partially mediated by autocrine production of transforming growth factor beta 1.

CONCLUSIONS

These findings suggest that activation of HDAC2 and/or inhibition of IL-17A production could prevent the development of airway remodeling by suppressing airway inflammation and modulating fibroblast activation in COPD.

In this short communication we are providing insight about the regulatory role of the phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) kinase system in psoriatic disease. This is an upcoming active research field in respect to elucidating the inflammatory and proliferative cascades of psoriatic disease. To provide a new dimension to the understandings of the molecular principles of the pathogenesis of autoimmune diseases, we hypothesized that (i) dysregulation of cytokines and <em>growth</em> <em>factors</em> in autoimmune diseases activate the mTOR signaling system and (ii) the activated mTOR kinase system is a key regulator of the inflammatory/proliferative cascades of the disease process. In support of this hypothesis we have earlier reported that <em>growth</em> <em>factors</em> (nerve <em>growth</em> <em>factor</em> (NGF) and platelet-derived <em>growth</em> <em>factor</em> (PDGF)) and relevant cytokines (interleukin (IL)-<em>17</em>, IL-22) known to be critical for psoriasis, psoriatic arthritis, and rheumatoid arthritis activate the mTOR signaling system. Here, we are providing our latest observations that the mTOR signaling proteins are upregulated in psoriatic skin and further we observed that proliferation of keratinocytes (KC) and synovial cells (synovial <em>fibroblasts</em> (FLS)) of psoriatic arthritis are dependent on the PI3K-AKT-mTOR kinase system. To our knowledge, we are the first to explore whether a double kinase inhibitor of mTOR signal proteins has a therapeutic potential for psoriatic disease. Here we will be sharing our views, our research work in this field and as well we will provide evidences how a double kinase inhibitor of mTOR signal proteins can be an effective therapeutic agent for psoriatic disease.