Amblyomma americanum tick saliva insulin‐like growth factor binding protein‐related protein 1 binds insulin but not insulin‐like growth factors

Silencing Amblyomma americanum insulin‐like growth factor binding protein‐related protein 1 (AamIGFBP‐rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP‐rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP‐1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP‐rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24–48 h after attachment. Our data suggest that native AamIGFBP‐rP1 is a functional insulin binding protein in that both yeast‐ and insect cell‐expressed rAamIGFBP‐rP1 bound insulin, but not insulin‐like growth factors. When subjected to anti‐blood clotting and platelet aggregation assays, rAamIGFBP‐rP1 did not have any effect. Unlike human IGFBP‐rP1, which is controlled by trypsinization, rAamIGFBP‐rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick‐borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24–48 h of the tick starting to feed makes AamIGFBP‐rP1 an attractive target for antitick vaccine development.     

Vascular defects in gain‐of‐function fps/fes transgenic mice correlate with PDGF‐ and VEGF‐induced activation of mutant Fps/Fes kinase in endothelial cells

Summary. Background: Fps/Fes is a cytoplasmic tyrosine kinase that is abundantly expressed in the myeloid, endothelial, epithelial, neuronal and platelet lineages. Genetic manipulation in mice has uncovered potential roles for this kinase in hematopoiesis, innate immunity, inflammation and angiogenesis. Objective: We have utilized a genetic approach to explore the role of Fps/Fes in angiogenesis. Methods: A hypervascular line of mice generated by expression of a ‘gain‐of‐function’ human fps/fes transgene (fps^MF) encoding a myristoylated variant of Fps (MFps) was used in these studies. The hypervascular phenotype of this line was extensively characterized by intravital microscopy and biochemical approaches. Results: fps^MF mice exhibited 1.6–1.7‐fold increases in vascularity which was attributable to increases in the number of secondary vessels. Vessels were larger, exhibited varicosities and disorganized patterning, and were found to have defects in histamine‐induced permeability. Biochemical characterization of endothelial cell (EC) lines derived from fps^MF mice revealed that MFps was hypersensitive to activation by vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF). Conclusions: MFps mediates enhanced sensitization to VEGF and PDGF signaling in ECs. We propose that this hypersensitization contributes to excessive angiogenic signaling and that this underlies the observed hypervascular phenotype of fps^MF mice. These phenotypes recapitulate important aspects of the vascular defects observed in both VEGF and angiopoietin‐1 transgenic mice. The fps/fes proto‐oncogene product therefore represents a novel player in the regulation of angiogenesis, and the fps^MF line of mice constitutes a unique new murine model for the study of this process.     

MicroRNA miR‐8 regulates multiple growth factor hormones produced from Drosophila fat cells

Metabolic organs such as the liver and adipose tissue produce several peptide hormones that influence metabolic homeostasis. Fat bodies, the Drosophila counterpart of liver and adipose tissues, have been thought to analogously secrete several hormones that affect organismal physiology, but their identity and regulation remain poorly understood. Previous studies have indicated that microRNA miR‐8, functions in the fat body to non‐autonomously regulate organismal growth, suggesting that fat body‐derived humoral factors are regulated by miR‐8. Here, we found that several putative peptide hormones known to have mitogenic effects are regulated by miR‐8 in the fat body. Most members of the imaginal disc growth factors and two members of the adenosine deaminase‐related growth factors are up‐regulated in the absence of miR‐8. Drosophila insulin‐like peptide 6 (Dilp6) and imaginal morphogenesis protein‐late 2 (Imp‐L2), a binding partner of Dilp, are also up‐regulated in the fat body of miR‐8 null mutant larvae. The fat body‐specific reintroduction of miR‐8 into the miR‐8 null mutants revealed six peptides that showed fat‐body organ‐autonomous regulation by miR‐8. Amongst them, only Imp‐L2 was found to be regulated by U‐shaped, the miR‐8 target for body growth. However, a rescue experiment by knockdown of Imp‐L2 indicated that Imp‐L2 alone does not account for miR‐8's control over the insect's growth. Our findings suggest that multiple peptide hormones regulated by miR‐8 in the fat body may collectively contribute to Drosophila growth.     

IGF-1、IGFBP-3及瘦素与巨大儿的相关性

目的探讨胰岛素样生长因子-1（IGF-1）、胰岛素样生长因子结合蛋白-3（IGFBP-3）及瘦素（Leptin）与巨大儿的相关性。方法应用酶联免疫吸附法分别测定巨大儿组（50例）、正常对照组（48例）脐血IGF—1、IGFBP-3和瘦素。结果巨大儿组脐血IGF-1,IGFBP-3及瘦素为（219．8±57．3）μg／L、（2996．1±341．1）μg/L、（65．1±19．2μg／L）,高于对照组的（114．0±31．8）μg/L、（2074．2±175．3）μg／L、（52．2±16．6μg／L）,差异均有统计学意义;巨大儿组脐血IGF-1,IG—FBP-3及瘦素水平与新生儿出生体重呈正相关,相关系数分别为0．63（P〈0．01）,0．59（P〈0．01）,0．58（P〈0．01）。结论胎儿自身分泌的IGF-1、IGFBP-3和瘦素与胎儿生长关系密切,脐血中其水平增高可能是导致巨大儿的重要原因之一。     

Human corneal fibroblast migration and extracellular matrix synthesis during stromal repair: Role played by platelet‐derived growth factor‐BB,basic fibroblast growth factor,and transforming growth factor‐β1

The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5β1‐integrin and syndecan‐4. Using in vitro HCFs, a mechanical wound model was developed to study the influence of the GFs basic fibroblast GF (bFGF), platelet‐derived GF (PDGF‐BB) and transforming GF‐β1 (TGFβ1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFβ1 at different time points during the wound closure. The HCF response to PDGF‐BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing nonfibrotic healing. bFGF stimulated nonfibrotic ECM production and limited the migration process. Finally, TGFβ1 induced expression of the fibrotic markers collagen type III and α5β1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process. Copyright © 2016 John Wiley & Sons, Ltd.     

IGF-1, IGFBP-3, VEGF and MMP-9 levels and their potential relationship with renal functions in patients with compensatory renal growth

BACKGROUND: Mechanisms of compensatory renal growth (CRG) still remain a mystery. Various growth factors, including growth hormone, insulin-like growth factor-1 (IGF-1) have been implicated in different forms of CRG. AIMS: To investigate the serum levels of IGF-1, vascular endothelial growth factor (VEGF - role in vascular remodelling), matrix metalloproteinase-9 (MMP-9 - essential for normal nephrogenesis) and correlation of renal function in patients with unilateral nephrectomized, agenesis and hypoplasic kidney. METHODS: Thirty patients were included in this study. In group I, there were 10 patients with unilateral nephrectomy, while in group II, there were 10 patients with unilateral agenesis. As for group III, there were 10 patients with unilateral hypoplastic kidney. The serum levels of IGF-1, IGF-binding protein-3 (IGFBP-3), VEGF and MMP-9 were studied in all the cases. Clearance of creatinin (Ccr) and protein excretion were examined in the 24 h urine. CRG was determined with ultrasonography and scintigraphy. Twenty-six control subjects were also studied. RESULTS: The levels of IGF-1, IGFBP-3, VEGF and MMP-9 were significantly higher in patients than in the control subjects (P < 0.001). Ccr and protein excretion levels were different in study groups than in those of the control group (P < 0.01). There were positive correlations between the serum levels of IGF-1 with IGFBP-3; IGF-1 with MMP-9; IGFBP-3 with MMP-9 (r = 0.825, P = 0.0001; P < 0.001 r = 0.611; P < 0.001 r = 0.585, respectively). There were negative correlations between GFR and the serum levels of IGF-1, IGFBP-3 and MMP-9 (P < 0.01 r = -0.708; P = 0.002 r = -0.803; P < 0.05 r = -0.442, respectively). Furthermore, there were positive correlations between proteinuria and the serum levels of IGF-1, IGFBP-3 and MMP-9 (P = 0.039 r = 0.600; P < 0.05 r = 0.456; P < 0.05 r = 0.424). CONCLUSIONS: Increased IGF-1, IGFBP-3, VEGF and MMP-9 were observed in CRG in the follow-up period. IGF-1 and MMP-9 seemed to have increased in patients with CRG in defiance of the development of fibrosis. Moreover, IGF-1 and MMP-9 seem to be associated with reduced renal function and proteinuria.     

Exenatide upregulates gene expression of glucagon‐like peptide‐1 receptor and nerve growth factor in streptozotocin/nicotinamide‐induced diabetic mice

Glucagon‐like peptide‐1 (GLP‐1) is an incretin hormone that has modulating effects on insulin release. GLP‐1 and receptors for GLP‐1 are widely expressed throughout the body including the brain. The expression of GLP‐1 receptors is very specific to large neurons in hippocampus, neocortex, and cerebellum. GLP‐1 receptor stimulation enhances glucose‐dependent insulin secretion and lowers blood glucose in type 2 diabetes mellitus. Studies on adipobiology of neurotrophins have focused on nerve growth factor (NGF) as an example of adipose‐derived neurotrophins. Compromised trophic factor signaling may underlie neurodegenerative diseases ranging from Alzheimer's disease to diabetic neuropathies. Exenatide, a potent and selective agonist for the GLP‐1 receptor, is currently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to assess the effect of chronic exenatide treatment on the hippocampal gene expression levels of GLP‐1 receptor and NGF in diabetic mice. The effects of chronic exenatide treatment (0.1 μg/kg, s.c., twice daily for 2 weeks) on GLP‐1 receptor and NGF gene expression levels in the hippocampus of streptozotocin/nicotinamide (STZ–NA)‐induced diabetic mice were assessed by quantitative real‐time polymerase chain reaction (RT‐PCR). The results of this study revealed that hippocampal gene expression of GLP‐1 receptor and NGF were downregulated in diabetic mice. Importantly, a significant increase in the gene expression level of GLP‐1 receptor and NGF was determined after 2 weeks of exenatide administration. Increased gene expression level of GLP‐1 receptor and NGF may underlie the beneficial action of exenatide in STZ/NA‐induced diabetes.     

Establishment of hormone reference intervals for infants born < 30 weeks' gestation

Objective

Preterm infants, especially those born very preterm (< 32 weeks' gestation), suffer a number of morbidities. Immaturity of the endocrine system and its potential impact on morbidity is the subject of numerous studies. Hormone concentrations are sometimes measured in very preterm infants, however there are little normative data available to be able to interpret the results. The aim of this study was to describe age appropriate hormone reference intervals for babies born less than 30 weeks' gestation.

Study design

Samples were collected at 1, 4, 7, 14, 21, 28 and 42 days after birth from babies born 23–29 weeks' gestation. The serum was analyzed for seven hormones by automated chemiluminescent immunoassay (Siemens Immulite 2000). Results from the 107 infants who survived beyond 40 weeks' corrected gestational age were included in the data analysis.

Results

Cortisol, dehydroepiandrosterone sulfate, growth hormone and progesterone levels were highest during the first seven days with levels up to 10,801 nmol/L; 26.6 μmol/L; 343 mU/L; and > 63.6 nmol/L respectively. Free thyroxine levels were as low as < 2.6 pmol/L for the first 28 days with the nadir at 7 days. Estradiol levels ranged from < 73 to 1626 pmol/L over the six weeks. Reference intervals for IGF-1 could not be established as the levels were below the analyzer's sensitivity. There were no differences in reference intervals between male and female infants.

Conclusions

We describe gestation appropriate reference intervals for six hormones measured in babies born < 30 weeks' gestation. Utilization of these reference intervals permits the correct and timely interpretation of results to the clinician.     

胰岛素样生长因子-I及其受体在糖尿病足溃疡的表达及意义

目的探讨胰岛素样生长因子-I（Insulin—likegrowthfactor-I,IGF-I）与其受体两亚基（α,B）在非糖尿病正常皮肤和单纯糖尿病患者皮肤,以及糖尿病足患者足溃疡创面中的分布和表达特征,旨在阐明其表达水平变化与糖尿病足溃疡创面的内在联系。方法按照病例入选标准收集30例糖尿病足患者,23例单纯糖尿病患者,以及25例非糖尿病正常人,取足部皮肤组织标本,经固定脱水,石蜡包埋,切片后备用。采用免疫组织化学SP法检测所有取材组织中IGF-I、IGF-IRα和IGF-IRB蛋白定位及含量。在400倍光镜下观察免疫组化结果。结果在非糖尿病正常皮肤和单纯糖尿病患者皮肤,以及糖尿病足溃疡创面皮肤中IGF—I、IGF—I Rα和IGF—IRβ都有阳性表达,但变化规律不尽相同。IGF—I的阳性细胞率单纯糖尿病组较正常对照组表达减弱（P〉0．05）,糖尿病足溃疡组较单纯糖尿病组表达进一步减弱（P〉0．05）。IGF—IRα和IGF—IRβ的阳性细胞率单纯糖尿病组较对照组表达减弱（P〉0．05）,糖尿病足溃疡组较单纯糖尿病组表达进一步减弱（P〉0．05）。结论糖尿病患者创面IGF-I的缺乏或不足可能与糖尿病所引发的内皮细胞损害有关,与糖尿病足部溃疡经久不愈有直接关系。创面IGF-IRα和IGF—IRβ表达减少,和生物活性的降低,影响细胞因子引起的信号传递和核内目的基因表达,导致创面修复失控形成慢性难愈性溃疡。     

The influence of sustained dual‐factor presentation on the expansion and differentiation of neural progenitors in affinity‐binding alginate scaffolds

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co‐transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate–sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor‐2 (bFGF) were affinity‐bound to alginate–sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium‐crosslinked alginate. NPCs isolated from 18 day‐old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10–14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ. Copyright © 2013 John Wiley & Sons, Ltd.     

Staphylococcus aureus‐induced complement activation promotes tissue factor‐mediated coagulation

Essentials

• Complement, Toll‐like receptors and coagulation cross‐talk in the process of thromboinflammation.
• This is explored in a unique human whole‐blood model of S. aureus bacteremia.
• Coagulation is here shown as a downstream event of C5a‐induced tissue factor (TF) production.
• Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation.

Summary

Background

There is extensive cross‐talk between the complement system, the Toll‐like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression.

Objectives

To study the relative roles of complement, TLRs and TF in Staphylococcus aureus‐induced coagulation.

Methods

Lepirudin‐anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF_{1 + 2}) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively.

Results

All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF_{1 + 2}, and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF_{1 + 2} levels to baseline levels.

Conclusions

S. aureus‐induced coagulation in human whole blood was mainly attributable to C5a‐induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus‐induced coagulation.