Mitogen Activated Protein Kinase Phosphatase-1 Prevents the Development of Tactile Sensitivity In a Rodent Model of Neuropathic Pain

Neuropathic pain due to nerve injury is one of the most difficult types of pain to treat. Following peripheral nerve injury, neuronal and glial plastic changes contribute to central sensitization and perpetuation of mechanical hypersensitivity in rodents. The mitogen activated protein kinase (MAPK) family is pivotal in this spinal cord plasticity. MAPK phosphatases (MKPs) limit inflammatory processes by dephosphorylating MAPKs. For example, MKP-1 preferentially dephosphorylates p-p38. Since spinal p-p38 is pivotal for the development of chronic hypersensitivity in rodent models of pain, and p-p38 inhibitors have shown clinical potential in acute and chronic pain patients, we hypothesize that induction of spinal MKP-1 will prevent the development of peripheral nerve-injury-induced hypersensitivity and p-p38 overexpression. We cloned rat spinal cord MKP-1 and optimize MKP-1 cDNA in vitro using transfections to BV-2 cells. We observed that in vitro overexpression of MKP-1 blocked lipopolysaccharide-induced phosphorylation of p38 (and other MAPKs) as well as release of pro-algesic effectors (i.e., cytokines, chemokines, nitric oxide). Using this cDNA MKP-1 and a non-viral, in vivo nanoparticle transfection approach, we found that spinal cord overexpression of MKP-1 prevented development of peripheral nerve-injury-induced tactile hypersensitivity and reduced pro-inflammatory cytokines and chemokines and the phosphorylated form of p38

Evidence for a Role of Endocannabinoids, Astrocytes and p38 Phosphorylation in the Resolution of Postoperative Pain

An alarming portion of patients develop persistent or chronic pain following surgical procedures, but the mechanisms underlying the transition from acute to chronic pain states are not fully understood. In general, endocannabinoids (ECBs) inhibit nociceptive processing by stimulating cannabinoid receptors type 1 (CB(1)) and type 2 (CB(2)). We have previously shown that intrathecal administration of a CB(2) receptor agonist reverses both surgical incision-induced behavioral hypersensitivity and associated over-expression of spinal glial markers. We therefore hypothesized that endocannabinoid signaling promotes the resolution of acute postoperative pain by modulating pro-inflammatory signaling in spinal cord glial cells.To test this hypothesis, rats receiving paw incision surgery were used as a model of acute postoperative pain that spontaneously resolves. We first characterized the concentration of ECBs and localization of CB(1) and CB(2) receptors in the spinal cord following paw incision. We then administered concomitant CB(1) and CB(2) receptor antagonists/inverse agonists (AM281 and AM630, 1 mg x kg(-1) each, i.p.) during the acute phase of paw incision-induced mechanical allodynia and evaluated the expression of glial cell markers and phosphorylated p38 (a MAPK associated with inflammation) in the lumbar dorsal horn. Dual blockade of CB(1) and CB(2) receptor signaling prevented the resolution of postoperative allodynia and resulted in persistent over-expression of spinal Glial Fibrillary Acidic Protein (GFAP, an astrocytic marker) and phospho-p38 in astrocytes. We provide evidence for the functional significance of these astrocytic changes by demonstrating that intrathecal administration of propentofylline (50 microg, i.t.) attenuated both persistent behavioral hypersensitivity and over-expression of GFAP and phospho-p38 in antagonist-treated animals.Our results demonstrate that endocannabinoid signaling via CB(1) and CB(2) receptors is necessary for the resolution of paw incision-induced behavioral hypersensitivity and for the limitation of pro-inflammatory signaling in astrocytes following surgical insult. Our findings suggest that therapeutic strategies designed to enhance endocannabinoid signaling may prevent patients from developing persistent or chronic pain states following surgery

Cannabinoid receptor type 2 activation induces a microglial anti-inflammatory phenotype and reduces migration via MKP induction and ERK dephosphorylation

<p>Abstract</p> <p>Background</p> <p>Cannabinoid receptor type 2 (CBR2) inhibits microglial reactivity through a molecular mechanism yet to be elucidated. We hypothesized that CBR2 activation induces an anti-inflammatory phenotype in microglia by inhibiting extracellular signal-regulated kinase (ERK) pathway, via mitogen-activated protein kinase-phosphatase (MKP) induction. MKPs regulate mitogen activated protein kinases, but their role in the modulation of microglial phenotype is not fully understood.</p> <p>Results</p> <p>JWH015 (a CBR2 agonist) increased MKP-1 and MKP-3 expression, which in turn reduced p-ERK1/2 in LPS-stimulated primary microglia. These effects resulted in a significant reduction of tumor necrosis factor-α (TNF) expression and microglial migration. We confirmed the causative link of these findings by using MKP inhibitors. We found that the selective inhibition of MKP-1 by Ro-31-8220 and PSI2106, did not affect p-ERK expression in LPS+JWH015-treated microglia. However, the inhibition of both MKP-1 and MKP-3 by triptolide induced an increase in p-ERK expression and in microglial migration using LPS+JWH015-treated microglia.</p> <p>Conclusion</p> <p>Our results uncover a cellular microglial pathway triggered by CBR2 activation. These data suggest that the reduction of pro-inflammatory factors and microglial migration via MKP-3 induction is part of the mechanism of action of CBR2 agonists. These findings may have clinical implications for further drug development.</p

Sindbad: A new operational service for a safer leisure and boating navigation

The SINDBAD- Leisure and Boating Safety Navigation - project goal is the development of an advanced operational service to support navigation in a specific area. The first prototype covers the Ligurian Sea (a very busy touristic area in the North Mediterranean Sea) It develops an ICT Service Infrastructure to provide innovative intelligent automation functions and to develop customized services, accessible by your mobile device, for conducting a boat and avoiding any kind of risk ensuring the best degree of comfort

Multicentric validation of proteomic biomarkers in urine specific for diabetic nephropathy

Background: Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration &gt;= 5 years, cases of DN were defined as albuminuria &gt;300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82). Methodology/Principal Findings: Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In &lt;10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patient's subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients. Conclusions: These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin

Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations.

A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA