Contributions of neuroimmune interactions to chemotherapy-induced peripheral neuropathy development and its prevention/therapy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating sequela that is difficult for both clinicians and cancer patients to manage. Precise mechanisms of CIPN remain elusive and current clinically prescribed therapies for CIPN have limited efficacy. Recent studies have begun investigating the interactions between the peripheral and central nervous systems and the immune system. Understanding these neuroimmune interactions may shift the paradigm of elucidating CIPN mechanisms. Although the contribution of immune cells to CIPN pathogenesis represents a promising area of research, its fully defined mechanisms have not yet been established. Therefore, in this review, we will discuss (i) current shortcoming of CIPN treatments, (ii) the roles of neuroimmune interactions in CIPN development and (iii) potential neuroimmune interaction-targeting treatment strategies for CIPN. Interestingly, monocytes/macrophages in dorsal root ganglia; microglia and astrocytes in spinal cord; mast cells in skin; and Schwann cell near peripheral nerves have been identified as inducers of CIPN behaviors, whereas T cells have been found to contribute to CIPN resolution. Additionally, nerve-resident immune cells have been targeted as prevention and/or therapy for CIPN using traditional herbal medicines, small molecule inhibitors, and intravenous immunoglobulins in a preclinical setting. Overall, unveiling neuroimmune interactions associated with CIPN may ultimately reduce cancer mortality and improve cancer patients' quality of life.

CBD Retailers in NC Promote CBD Online to Treat Pain Violating FDA Rules About Medical Claims and Offer Low-CBD/High-Price Products.

Introduction: Cannabidiol (CBD) products are available nearly nationwide in the US and can coexist with medical or recreational programs. North Carolina (NC) is an example of a state with a program dedicated to integrating hemp cultivation and medicinal CBD exclusively, containing a multitude of retailers selling it as a primary product. The Food and Drug Administration (FDA) mandates that non-FDA approved CBD products cannot be marketed using medical or health-related claims and has sent warning letters to retailers violating these terms. We aim to characterize the online content of the NC CBD market by analyzing retailers' websites to determine whether hemp/CBD shops comply with FDA regulations in terms of medical claims and analyze the claimed CBD content and price of products offered online. Methods: We randomly selected three CBD retailers from the ten most populated cities of NC. We analyzed their website content: product type, medical claims, other disclaimers, price, and CBD content. Results: We found that edible, oral, inhalable, and topical products are offered in similar proportions. Word analysis of product description revealed that "pain" and "pain relief" were the most common medical claim, followed by inflammation and anxiety. Health claims were mostly related to wellbeing. Other attributes indicate that products are associated with pleasant flavors or sensations (ie, cool, lavender, delicious, honey, menthol), which resembles the strategies used for tobacco advertisement. Most products (61%) claimed to contain less than 1000 mg of CBD. The median price of products ranged from $15-30 per 300 mg. We found a positive correlation between CBD content and price. Discussion: Our data demonstrate that the NC online CBD market does not comply with FDA regulations, primarily targets patients with pain, inflammation, or anxiety, and offers products with low CBD concentration and high prices. New policies should limit the access and online promotion of non-pharmaceutical grade CBD products.

Tumor-Derived Lysophosphatidic Acid Blunts Protective Type I Interferon Responses in Ovarian Cancer.

Lysophosphatidic acid (LPA) is a bioactive lipid enriched in the tumor microenvironment of immunosuppressive malignancies such as ovarian cancer. Although LPA enhances the tumorigenic attributes of cancer cells, the immunomodulatory activity of this phospholipid messenger remains largely unexplored. Here, we report that LPA operates as a negative regulator of type I interferon (IFN) responses in ovarian cancer. Ablation of the LPA-generating enzyme autotaxin (ATX) in ovarian cancer cells reprogrammed the tumor immune microenvironment, extended host survival, and improved the effects of therapies that elicit protective responses driven by type I IFN. Mechanistically, LPA sensing by dendritic cells triggered PGE2 biosynthesis that suppressed type I IFN signaling via autocrine EP4 engagement. Moreover, we identified an LPA-controlled, immune-derived gene signature associated with poor responses to combined PARP inhibition and PD-1 blockade in patients with ovarian cancer. Controlling LPA production or sensing in tumors may therefore be useful to improve cancer immunotherapies that rely on robust induction of type I IFN. SIGNIFICANCE: This study uncovers that ATX-LPA is a central immunosuppressive pathway in the ovarian tumor microenvironment. Ablating this axis sensitizes ovarian cancer hosts to various immunotherapies by unleashing protective type I IFN responses. Understanding the immunoregulatory programs induced by LPA could lead to new biomarkers predicting resistance to immunotherapy in patients with cancer. See related commentary by Conejo-Garcia and Curiel, p. 1841. This article is highlighted in the In This Issue feature, p. 1825.

Methods and protocols for translatable rodent models of postsurgical pain.

Due to the large volume of surgeries and the subsequent incidence of postsurgical pain, it is vital that the underlying mechanisms of postsurgical pain are thoroughly understood. The intensity of acute postsurgical pain is typically dependent on the severity of tissue damage the surgery produces, and the development of chronic pain is more frequent in major surgeries than in minor ones. It is therefore important that postsurgical pain studies are conducted with the differences between major and minor surgeries in mind. To this end, the paw incision and skin muscle incision and retraction models are the focus of this chapter as they feature aspects observed in minor and major surgeries in humans, respectively. Several elements of these models translate to humans with some limitations, as they allow for the measurement of reflexive, spontaneous, and functional pain-like behavior. For these attributes, the SMIR and paw incision surgeries are widely used in postsurgical pain research. Here we layout detailed protocols to instruct experienced as well as inexperienced researchers studying postsurgical pain in rats and mice.

Methods and protocols for chemotherapy-induced peripheral neuropathy (CIPN) mouse models using paclitaxel.

While cancer patients may have chemotherapeutics to thank for being cured of their malignancy, they are often left to suffer a disabling neuropathy induced by that same cancer treatment. This neuropathy, known as chemotherapy-induced peripheral neuropathy, or CIPN, is one of the most debilitating survivorship concerns for patients, with many citing hallmark symptoms of hyperalgesia, allodynia, and numbness, and subsequently reducing their dose or even ceasing treatment altogether. Investigations into this interplay between the antineoplastic activity of chemotherapeutic agents and the preservation of peripheral nerve health are therefore crucial for the development of CIPN treatment and prevention methods. Responding to need, current literature is inundated with varying preclinical models of CIPN. This chapter thus seeks to provide a detailed and reliable methodology for the induction and assessment of CIPN in mice, using a preclinical model that is both reproducible and translatable to several aspects of the clinical phenotype. Specifically, this chapter lays out a model for intermittent low-dose paclitaxel induction of CIPN in C57BL/6J mice, and a testing of this induction via von Frey filament mechanical hypersensitivity assays, a mechanical hyposensitivity (numbness) assay, and a cold-thermal allodynia assay (acetone test). These protocols can easily be adjusted to fit the needs of individual CIPN experiments, as stated throughout the chapter.

IMT504 blocks allodynia in rats with spared nerve injury by promoting the migration of mesenchymal stem cells and by favoring an anti-inflammatory milieu at the injured nerve.

ABSTRACT: IMT504, a noncoding, non-CpG oligodeoxynucleotide, modulates pain-like behavior in rats undergoing peripheral nerve injury, through mechanisms that remain poorly characterized. Here, we chose the spared nerve injury model in rats to analyze the contribution of mesenchymal stem cells (MSCs) in the mechanisms of action of IMT504. We show that a single subcutaneous administration of IMT504 reverses mechanical and cold allodynia for at least 5 weeks posttreatment. This event correlated with long-lasting increases in the percentage of MSCs in peripheral blood and injured sciatic nerves, in a process seemingly influenced by modifications in the CXCL12-CXCR4 axis. Also, injured nerves presented with reduced tumor necrosis factor-α and interleukin-1ß and increased transforming growth factor-ß1 and interleukin-10 protein levels. In vitro analysis of IMT504-pretreated rat or human MSCs revealed internalized oligodeoxynucleotide and confirmed its promigratory effects. Moreover, IMT504-pretreatment induced transcript expression of Tgf-ß1 and Il-10 in MSCs; the increase in Il-10 becoming more robust after exposure to injured nerves. Ex vivo exposure of injured nerves to IMT504-pretreated MSCs confirmed the proinflammatory to anti-inflammatory switch observed in vivo. Interestingly, the sole exposure of injured nerves to IMT504 also resulted in downregulated Tnf-α and Il-1ß transcripts. Altogether, we reveal for the first time a direct association between the antiallodynic actions of IMT504, its promigratory and cytokine secretion modulating effects on MSCs, and further anti-inflammatory actions at injured nerves. The recapitulation of key outcomes in human MSCs supports the translational potential of IMT504 as a novel treatment for neuropathic pain with a unique mechanism of action involving the regulation of neuroimmune interactions.

Health Claims About Cannabidiol Products: A Retrospective Analysis of U.S. Food and Drug Administration Warning Letters from 2015 to 2019.

Background: Cannabidiol (CBD) products are increasingly available to consumers in the United States and are subject to regulation by the U.S. Food and Drug Administration (FDA). CBD products cannot be marketed as unapproved new drugs with claims of therapeutic benefit. In addition, because CBD is the active ingredient in a FDA-approved CBD product, Epidiolex, CBD cannot be marketed as, or in, food products or dietary supplements. The FDA has issued Warning Letters to promote voluntary regulatory compliance. These letters provide insights as to the types of violations for CBD products detected in the U.S. market. Objective: The goal of this retrospective study was to content analyze Warning Letters issued by the FDA to identify illicit marketing of CBD products. Design: Warning Letters issued by the FDA between 2015 and 2019 were content analyzed using a deductive approach. We extracted year of issuance, issuing office, and claim types that are currently prohibited by the FDA, including (i) unapproved new drug, (ii) misbranded drug, (iii) false and/or misleading, (iv) FDA-approved/endorsed, (v) dietary supplement, and (vi) adulterated food product. In addition, we documented the disease or conditions the product claimed to affect, pharmacological effects, and location of violation. Results: Of the 39 Warning Letters issued, 97% were for violations made on company websites and 56% were for social media accounts. Almost all letters (97%) cited violations of marketing CBD as an unapproved new drug. These illicit therapeutic claims were made for >125 unique health problems, including cancer (87.2%), diabetes (71.8%), inflammation (66.7%), pain (66.7%), and arthritis (66.7%). The majority of letters (79.5%) also cited illicit marketing of CBD as a dietary supplement or food product. CBD was promoted as having 16 unique pharmacological effects, including anti-inflammatory (53.8%), anticancer (43.6%), and antipsychotic (30.8%). Conclusions: CBD products have been unlawfully advertised online as unauthorized drugs with health claims that promote therapeutic benefits and as dietary supplements. Efforts are needed to regulate and monitor illicit advertising so consumers are not misled about the risks and benefits of CBD use.

Systemic administration of a ß2-adrenergic receptor agonist reduces mechanical allodynia and suppresses the immune response to surgery in a rat model of persistent post-incisional hypersensitivity.

Beta 2 adrenergic receptor (ß2 AR) activation in the central and peripheral nervous system has been implicated in nociceptive processing in acute and chronic pain settings with anti-inflammatory and anti-allodynic effects of ß2-AR mimetics reported in several pain states. In the current study, we examined the therapeutic efficacy of the ß2-AR agonist clenbuterol in a rat model of persistent postsurgical hypersensitivity induced by disruption of descending noradrenergic signaling in rats with plantar incision. We used growth curve modeling of ipsilateral mechanical paw withdrawal thresholds following incision to examine effects of treatment on postoperative trajectories. Depletion of spinal noradrenergic neurons delayed recovery of hypersensitivity following incision evident as a flattened slope compared to non-depleted rats (-1.8 g/day with 95% CI -2.4 to -1.085, p < 0.0001). Chronic administration of clenbuterol reduced mechanical hypersensitivity evident as a greater initial intercept in noradrenergic depleted (6.2 g with 95% CI 1.6 to 10.8, p = 0.013) and non-depleted rats (5.4 g with 95% CI 1.2 to 9.6, p = 0.018) with plantar incision compared to vehicle treated rats. Despite a persistent reduction in mechanical hypersensitivity, clenbuterol did not alter the slope of recovery when modeled over several days (p = 0.053) or five weeks in depleted rats (p = 0.64). Systemic clenbuterol suppressed the enhanced microglial activation in depleted rats and reduced the density of macrophage at the site of incision. Direct spinal infusion of clenbuterol failed to reduce mechanical hypersensitivity in depleted rats with incision suggesting that beneficial effects of ß2-AR stimulation in this model are largely peripherally mediated. Lastly, we examined ß2-AR distribution in the spinal cord and skin using in-situ hybridization and IHC. These data add to our understanding of the role of ß2-ARs in the nervous system on hypersensitivity after surgical incision and extend previously observed anti-inflammatory actions of ß2-AR agonists to models of surgical injury.

Usefulness of the measurement of neurite outgrowth of primary sensory neurons to study cancer-related painful complications.

Abnormal outgrowth of sensory nerves is one of the important contributors to pain associated with cancer and its treatments. Primary neuronal cultures derived from dorsal root ganglia (DRG) have been widely used to study pain-associated signal transduction and electrical activity of sensory nerves. However, there are only a few studies using primary DRG neuronal culture to investigate neurite outgrowth alterations due to underlying cancer-related factors and chemotherapeutic agents. In this study, primary DRG sensory neurons derived from mouse, non-human primate, and human were established in serum and growth factor-free conditions. A bovine serum albumin gradient centrifugation method improved the separation of sensory neurons from satellite cells. The purified DRG neurons were able to maintain their heterogeneous subpopulations, and displayed an increase in neurite growth when exposed to cancer-derived conditioned medium, while they showed a reduction in neurite length when treated with a neurotoxic chemotherapeutic agent. Additionally, a semi-automated quantification method was developed to measure neurite length in an accurate and time-efficient manner. Finally, these exogenous factors altered the gene expression patterns of murine primary sensory neurons, which are related to nerve growth, and neuro-inflammatory pain and nociceptor development. Together, the primary DRG neuronal culture in combination with a semi-automated quantification method can be a useful tool for further understanding the impact of exogenous factors on the growth of sensory nerve fibers and gene expression changes in sensory neurons.

CD163 overexpression using a macrophage-directed gene therapy approach improves wound healing in ex vivo and in vivo human skin models.

Large tissue damage or wounds cause serious comorbidities and represent a major burden for patients, families, and health systems. Due to the pivotal role of immune cells in the proper resolution of inflammation and tissue repair, we focus our current study on the interaction of macrophages with skin cells, and specifically on the effects of CD163 gene induction in macrophages in wound healing. We hypothesize that the over-expression of the scavenger receptor gene CD163 in human macrophages would result in a more efficient wound healing process. Using 3D human wounded skin organotypic tissues, we observed that CD163 overexpression in THP-1 and human primary macrophages induced a more efficient re-epithelization when compared to control cells. Using human primary skin cells and an in vitro scratch assay we observed that CD163 overexpression in THP-1 macrophages promoted a more rapid and efficient wound healing process through a unique interaction with fibroblasts. The addition of CD163-blocking antibody, but not isotype control, blocked the efficient wound healing process induced by CD163 overexpression in macrophages. We found that the co-culture of skin cells and CD163 overexpressing macrophages reduced monocyte chemoattractant protein (MCP)-1 and enhanced tumor growth factor (TGF)-α, without altering interleukin (IL)-6 or TGF-ß. Our findings show that CD163 induces a more efficient wound healing and seems to promote a wound milieu with a pro-resolution molecular profile. Our studies set the foundation to study this approach in in vivo clinically relevant settings to test its effects in wound healing processes such as acute major injuries, large surgeries, or chronic ulcers.

Tachykinins modulate nociceptive responsiveness and sensitization: In vivo electrical characterization of primary sensory neurons in tachykinin knockout (Tac1 KO) mice.

Since the failure of specific substance P antagonists to induce analgesia, the role of tachykinins in the development of neuropathic pain states has been discounted. This conclusion was reached without studies on the role of tachykinins in normal patterns of primary afferents response and sensitization or the consequences of their absence on the modulation of primary mechanonociceptive afferents after injury. Nociceptive afferents from animals lacking tachykinins (Tac1 knockout) showed a disrupted pattern of activation to tonic suprathreshold mechanical stimulation. These nociceptors failed to encode the duration and magnitude of natural pronociceptive stimuli or to develop mechanical sensitization as consequence of this stimulation. Moreover, paw edema, hypersensitivity, and weight bearing were also reduced in Tac1 knockout mice 24 h after paw incision surgery. At this time, nociceptive afferents from these animals did not show the normal sensitization to mechanical stimulation or altered membrane electrical hyperexcitability as observed in wild-type animals. These changes occurred despite a similar increase in calcitonin gene-related peptide immunoreactivity in sensory neurons in Tac1 knockout and normal mice. Based on these observations, we conclude that tachykinins are critical modulators of primary nociceptive afferents, with a preeminent role in the electrical control of their excitability with sustained activation or injury.

High glucose induces a priming effect in macrophages and exacerbates the production of pro-inflammatory cytokines after a challenge.

INTRODUCTION: Painful diabetic neuropathy is associated with chronic inflammation, in which macrophages are the key effectors. We utilized an in vitro approach to determine the effects of high glucose on macrophage phenotype. MATERIALS AND METHODS: We exposed human THP-1 macrophages to normal glucose (5 mM) and a clinically relevant high glucose environment (15 mM) and measured the expression and concentration of molecules associated with a diabetic cellular phenotype. RESULTS: We found that THP-1 macrophages in high glucose conditions did not influence the basal expression of cyclooxygenase-2, Toll-like receptor-4, or class A scavenger receptor mRNA, or the concentrations of the cytokines interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and IL-10, but induced a priming effect on tumor necrosis factor (TNF)-α. Then, we stimulated THP-1 macrophages with a strong pro-inflammatory stimulus lipopolysaccharide (LPS; 5 µg/mL). After stimulation with LPS, we observed an exacerbated increase in TNF-α, IL-6, and MCP-1 concentration in the high glucose condition compared to the normal glucose environment. THP-1 macrophages in high glucose conditions developed tolerance to IL-10 anti-inflammatory effects (TNF-α production) when challenged with LPS. CONCLUSION: Our in vitro approach allows the study of macrophages as potential targets for therapeutic purposes since it compares them to primary human macrophages exposed to high glucose and macrophages from patients with diabetes or complications of painful diabetic neuropathy (i.e. ulcers, adipocytes, and pancreas).

Cannabis and Cannabinoids for Chronic Pain.

PURPOSE OF REVIEW: The purpose of this study was to provide the most up-to-date scientific evidence of the potential analgesic effects, or lack thereof, of the marijuana plant (cannabis) or cannabinoids, and of safety or tolerability of their long-term use. RECENT FINDINGS: We found that inhaled (smoked or vaporized) cannabis is consistently effective in reducing chronic non-cancer pain. Oral cannabinoids seem to improve some aspects of chronic pain (sleep and general quality of life), or cancer chronic pain, but they do not seem effective in acute postoperative pain, abdominal chronic pain, or rheumatoid pain. The available literature shows that inhaled cannabis seems to be more tolerable and predictable than oral cannabinoids. Cannabis or cannabinoids are not universally effective for pain. Continued research on cannabis constituents and improving bioavailability for oral cannabinoids is needed. Other aspects of pain management in patients using cannabis require further open discussion: concomitant opioid use, medical vs. recreational cannabis, abuse potential, etc.

Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

M1 macrophages release proinflammatory factors during inflammation. They transit to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1ß, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive and practical approach for inflammatory conditions that could lead to persistent pain, i.e. major surgeries, burns, rheumatoid arthritis, etc.

Mitogen-activated protein kinase phosphatase-3 (MKP-3) in the surgical wound is necessary for the resolution of postoperative pain in mice.

Mitogen-activated protein kinase (MAPK) phosphatase-3 (MKP-3) and its substrates (extracellular signal-regulated kinase [ERK] and p38) play an important role in pathophysiological mechanisms of acute postoperative and chronic neuropathic pain in the spinal cord. This study aimed to understand the role of MKP-3 and its target MAPKs at the site of surgical incision in nociceptive behavior. Wild-type (WT) and MKP-3 knockout (KO) mice underwent unilateral plantar hind paw incision. Mechanical allodynia was assessed by using von Frey filaments. Peripheral ERK-1/2 and p38 phosphorylation were measured by Western blot. Cell infiltration was determined using hematoxylin and eosin histological staining. Peripheral phosphorylated ERK-1/2 (p-ERK-1/2) inhibition was performed in MKP-3 KO mice. In WT mice, mechanical hypersensitivity was observed on postoperative day 1 (0.69±0.17 g baseline vs 0.13±0.08 g day 1), which resolved normally by postoperative day 12 (0.46±0.08 g, N=6). In MKP-3 KO mice, this hypersensitivity persisted at least 12 days after surgery (0.19±0.06 g; N=6). KO mice displayed higher numbers of infiltrating cells (51.4±6 cells/0.1 mm2) than WT mice (8.7±1.2 cells/0.1 mm2) on postoperative day 1 (vs 5-6 cells/0.1 mm2 at baseline) that returned to baseline 12 days after surgery (10-12 cells/0.1 mm2). In WT mice, peripheral p-p38 and p-ERK-1/2 expression increased (5- and 3-fold, respectively) on postoperative days 1 and 5, and returned to basal levels 7-12 days after surgery (N=3 per group). Peripheral p-p38 levels in MKP-3 KO mice followed a similar expression pattern as WT mice. Peripheral p-ERK-1/2 levels in MKP-3 KO mice remained elevated 12 days after surgery (2.5-fold, N=3 per group). Administration of PD98059 (MEK inhibitor, N=8, vehicle N=9) reduced p-ERK-1/2 expression in the incised tissue and blocked hypersensitivity in MKP-3 KO mice (N=6). The findings of this study suggest that MKP-3 is pivotal for normal resolution of acute postoperative allodynia, through the regulation of peripheral p-ERK-1/2.

Evaluation of a nanotechnology-based approach to induce gene-expression in human THP-1 macrophages under inflammatory conditions.

Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5µg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes.

Mitogen-activated protein kinase (MAPK) phosphatase-3 (MKP-3) displays a p-JNK-MAPK substrate preference in astrocytes in vitro.

Mitogen-activated protein kinases (MAPKs) play critical roles in the central nervous system immune responses through glial function, which are regulated with relative selectivity (or preference) by MAPK phosphatases (MKP). Phosphorylated extracellular signal-regulated protein kinase (p-ERK) is preferentially dephosphorylated by MKP-3, which display little activity over p-p38 and p-c-Jun NH2-terminal kinases (p-JNK). It has been proposed that these substrate preferences may vary depending on tissue or functional cellular processes. Since astrocytes display a prominent activity of JNK>ERK under stressed or reactive phenotype, we hypothesize that MKP-3 possess a similar or differential substrate preference in astrocytes for JNK and ERK (ERK=JNK or JNK>ERK). We generated transient expression of MKP-3 by transfecting a specific cDNA in primary rat neonatal brain cortex astrocytes. Cells were stimulated with lipopolysaccharide (LPS), and MAPKs and downstream pro-inflammatory products were measured by Western blot and ELISA analyses. MKP-3 expression in primary astrocytes reduced LPS-induced p-ERK and p-p38 by ∼50%, and p-JNK by ∼75%, and moderately reduced nitrite oxide (NO), while completely blocked Interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). We confirmed MKP-3 specific activity by developing a BV-2 microglia cell line stably overexpressing MKP-3 and using a specific siRNA against MKP-3. Our data demonstrate MKP-3 has differential substrate preference in astrocytes compared to other cells types, since it preferentially dephosphorylated p-JNK over p-ERK. Our results indicate also that astrocytic immune functions can be modulated by MKP-3 induction, a strategy that could be beneficial in neurological conditions in which astrocytes play a pathophysiological role, i.e. persistent pain.

Evidence for a role of endocannabinoids, astrocytes and p38 phosphorylation in the resolution of postoperative pain.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Alpha2-adrenoceptor stimulation transforms immune responses in neuritis and blocks neuritis-induced pain.

Neuropathic pain may be primarily driven by immune responses in peripheral nerves. Peripherally released catecholamines may exacerbate neuropathic pain and also modulate immune responses in a complex and sometimes opposing manner by actions on multiple adrenoceptor subtypes. We showed previously that injection of the alpha2-adrenoceptor agonist clonidine at the site of peripheral nerve injury reduces pain behavior and local tissue pro-inflammatory cytokine content in rats. The current study used a model of acute inflammatory neuritis to test the efficacy and mechanisms of action of alpha2-adrenoceptor stimulation to reduce pain. Zymosan, injected on the sciatic nerve, caused hypersensitivity to mechanical stimuli ipsilateral to injection and contralaterally, so-called mirror image pain. Ipsilateral hypersensitivity was inhibited dose-dependently by perineural injection of clonidine. Zymosan increased leukocyte number at the site of injection 3 d later as well as their content of interleukin 1alpha (IL-1alpha), IL-1beta, and IL-6. Perineural clonidine prevented both the increase in leukocyte number and cytokine expression induced by zymosan. Additionally, clonidine reduced the capacity of leukocytes to express pro-inflammatory cytokines as assessed by treatment of cells ex vivo with lipopolysaccharide, whereas no repression of IL-10 production occurred. Clonidine reduced the number of macrophages and lymphocytes as well as their expression of tumor necrosis factor alpha. All of the effects of clonidine were prevented by coadministration of an alpha2A-adrenoceptor-preferring antagonist. These results suggest that alpha2-adrenoceptor stimulation transforms cytokine gene expression, especially in macrophages and lymphocytes from a pro- to an anti-inflammatory profile in the setting of neuritis, likely relieving neuritis-induced pain by this mechanism.