Far from just a poke : Common painful needle procedures and the development of needle fear

Background: Vaccine injections are the most common painful needle procedure experienced throughout the lifespan. Many strategies are available to mitigate this pain; however, they are uncommonly utilized, leading to unnecessary pain and suffering. Some individuals develop a high level of fear and subsequent needle procedures are associated with significant distress. Objective: The present work is part of an update and expansion of a 2009 knowledge synthesis to include the management of vaccinerelated pain across the lifespan and the treatment of individuals with high levels of needle fear. This article will provide a conceptual foundation for understanding: (a) painful procedures and their role in the development and maintenance of high levels of fear; (b) treatment strategies for preventing or reducing the experience of pain and the development of fear; and (c) interventions for mitigating high levels of fear once they are established. Results: First, the general definitions, lifespan development and functionality, needle procedure-related considerations, and assessment of the following constructs are provided: pain, fear, anxiety, phobia, distress, and vasovagal syncope. Second, the importance of unmitigated pain from needle procedures is highlighted from a developmental perspective. Third, the prevalence, course, etiology, and consequences of high levels of needle fear are described. Finally, the management of needle-related pain and fear are outlined to provide an introduction to the series of systematic reviews in this issue. Discussion: Through the body of work in this supplement, the authors aim to provide guidance in how to treat vaccination-related pain and its sequelae, including high levels of needle fear

Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDIα showed two potential protein kinase A (PKA) phosphorylation motifs, Ser174 and Thr182. Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDIα-GST fusion protein and PKA motif-containing GDIα peptide at Ser174, but not Thr182. Transfection of COS-7 cells with mutated full-length GDIα at Ser174 to Ala174 (GDIα-Ser174A) abrogated the ability of cAMP to phosphorylate GDIα. However, mutation of Thr182 to Ala182 (GDIα-Thr182A) did not abrogate, and cAMP increased phosphorylation of GDIα to a similar extent as wild-type GDIα transfectants. The mutant GDIα-Ser174A, but not GDIα-Thr182A, was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDIα-Ser174A was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIα binding with RhoA, suggesting that phosphorylation of Ser174 regulated preformed GDIα-RhoA complexes. The findings provide strong support that the selective phosphorylation at Ser174 by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury

MicroRNA-146 Inhibits Thrombin-Induced NF-jB Activation and Subsequent Inflammatory Responses in Human Retinal Endothelial Cells

PURPOSE. Nuclear factor-jB (NF-jB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-jB through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-jB activation and retinal endothelial functions. METHODS. Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays. RESULTS. We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-jB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-jB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-jB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-jB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs. CONCLUSIONS. We uncovered a novel negative feedback regulatory mechanism on thrombininduced GPCR-mediated NF-jB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-jB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-jB activation pathways and related inflammatory processes in DR. Keywords: microRNA, diabetic retinopathy, retinal endothelial cells, NF-kB activation, thrombin D iabetic retinopathy (DR) is the leading cause of blindness in people between ages of 25 and 74 in the industrialized world. 1 Nearly all individuals who have had type I diabetes for more than 15 years develop DR. Approximately 50% to 80% of type II diabetic patients also develop retinopathy after 20 years of diabetes. 2 Although progress has been made, there is still no preventive treatment. MicroRNAs (miRNAs) are small, noncoding RNAs, and represent a newly recognized, important level of geneexpression regulation. 3,4 However, the roles of miRNAs in DR are still largely unknown. Previously, to identify miRNAs involved in DR, we performed miRNA-expression profiling and established miRNA transcriptomes of the retina and retinal endothelial cells (RECs) of normal control and streptozotocin (STZ)-induced diabetic rats 3 months after the onset of diabetes. 5 Among inflammation-related miRNAs, we showed that miR-146 was upregulated in RECs of diabetic rats