Comparison of Piperacillin and Tazobactam Pharmacokinetics in Critically Ill Patients with Trauma or with Burn.

Critical illness caused by burn and sepsis is associated with pathophysiologic changes that may result in the alteration of pharmacokinetics (PK) of antibiotics. However, it is unclear if one mechanism of critical illness alters PK more significantly than another. We developed a population PK model for piperacillin and tazobactam (pip-tazo) using data from 19 critically ill patients (14 non-burn trauma and 5 burn) treated in the Military Health System. A two-compartment model best described pip-tazo data. There were no significant differences found in the volume of distribution or clearance of pip-tazo in burn and non-burn patients. Although exploratory in nature, our data suggest that after accounting for creatinine clearance (CrCl), doses would not need to be increased for burn patients compared to trauma patients on consideration of PK alone. However, there is a high reported incidence of augmented renal clearance (ARC) in burn patients and pharmacodynamic (PD) considerations may lead clinicians to choose higher doses. For critically ill patients with normal kidney function, continuous infusions of 13.5-18 g pip-tazo per day are preferable. If ARC is suspected or the most stringent PD targets are desired, then continuous infusions of 31.5 g pip-tazo or higher may be required. This approach may be reasonable provided that therapeutic drug monitoring is enacted to ensure pip-tazo levels are not supra-therapeutic.

Pharmacokinetics of piperacillin and tazobactam in critically Ill patients treated with continuous kidney replacement therapy: A mini-review and population pharmacokinetic analysis.

WHAT IS KNOWN AND OBJECTIVE: Timely and appropriate dosing of antibiotics is essential for the treatment of bacterial sepsis. Critically ill patients treated with continuous kidney replacement therapy (CKRT) often have physiologic derangements that affect pharmacokinetics (PK) of antibiotics and dosing may be challenging. We sought to aggregate previously published piperacillin and tazobactam (pip-tazo) pharmacokinetic data in critically ill patients undergoing CKRT to better understand pharmacokinetics of pip-tazo in this population and better inform dosing. METHODS: The National Library of Medicine Database was searched for original research containing piperacillin or tazobactam clearance (CL) or volume of distribution (V) estimates in patients treated with CKRT. The search yielded 77 articles, of which 26 reported suitable estimates of CL or V. Of the 26 articles, 10 for piperacillin and 8 for tazobactam had complete information suitable for population pharmacokinetic modelling. Also included in the analysis was piperacillin and tazobactam PK data from 4 critically ill patients treated with CKRT in the Military Health System, 2 with burn and 2 without burn. RESULTS AND DISCUSSION: Median and range of literature reported PK parameters for piperacillin (CL 2.76 L/hr, 1.4-7.92 L/hr, V 31.2 L, 16.77-42.27 L) and tazobactam (CL 2.34 L/hr, 0.72-5.2 L/hr, V 36.6 L, 26.2-58.87 L) were highly consistent with population estimates (piperacillin CL 2.7 L/hr, 95%CI 1.99-3.41 L/hr, V 25.83 22.07-29.59 L, tazobactam CL 2.49 L/hr, 95%CI 1.55-3.44, V 30.62 95%CI 23.7-37.54). The proportion of patients meeting pre-defined pharmacodynamic (PD) targets (median 88.7, range 71%-100%) was high despite significant mortality (median 44%, range 35%-60%). High mortality was predicted by baseline severity of illness (median APACHE II score 23, range 21-33.25). Choice of lenient or strict PD targets (ie 100%fT >MIC or 100%fT >4XMIC) had the largest impact on probability of target attainment (PTA), whereas presence or intensity of CKRT had minimal impact on PTA. WHAT IS NEW AND CONCLUSION: Pip-tazo overexposure may be associated with increased mortality, although this is confounded by baseline severity of illness. Achieving adequate pip-tazo exposure is essential; however, risk of harm from overexposure should be considered when choosing a PD target and dose. If lenient PD targets are desired, doses of 2250-3375 mg every 6 h are reasonable for most patients receiving CKRT. However, if a strict PD target is desired, continuous infusion (at least 9000-13500 mg per day) may be required. However, some critically ill CKRT populations may need higher or lower doses and dosing strategies should be tailored to individuals based on all available clinical data including the specific critical care setting.

Meropenem pharmacokinetics in critically ill patients with or without burn treated with or without continuous veno-venous haemofiltration.

INTRODUCTION: Severe burn injury involves widespread skin and tissue damage leading to systemic inflammation, hypermetabolism and multi-organ failure. The hypermetabolic phase of burn injury has been associated with increased systemic antibiotic clearance; however, critical illness in the absence of burn may also induce similar physiologic changes. Continuous renal replacement therapy (CRRT) is often implemented in critically ill patients and may also affect antibiotic clearance. Although the pharmacokinetics (PK) of meropenem has been described in both the burn and non-burn critically ill populations, direct comparative data is lacking. METHODS: For this study, we evaluated PK parameters of meropenem from 23 critically ill patients, burn or non-burn, treated with or without continuous veno-venous haemofiltration (CVVH) to determine the contribution of burn and CVVH to the variability of therapeutic meropenem levels. RESULTS: A two-compartment model best described the data and revealed creatinine clearance (CrCl) and total burn surface area (TBSA) as significant covariates on clearance (CL) and peripheral volume of distribution (Vp), respectively. Of interest, non-burn patients on CVVH displayed an overall lower inherent CL as compared to burn patients on CVVH (6.43 vs. 12.85 L/h). Probability of target attainment (PTA) simulations revealed augmented renal clearance (ARC) may necessitate dose adjustments, but TBSA and CVVH would not. CONCLUSIONS: We recommend a standard dose of 1000 mg every 8 hours; however, if ARC is suspected, or the severity of illness requires a more stringent therapeutic target, we recommend a loading dose of 1000-2000 mg infused over 30 minutes to 1 hour followed by continuous infusion (3000-6000 mg over 24 hours), or intermittent infusion of 2000 mg every 8 hours.

A Review of Genome-Based Precision Medicine Efforts Within the Department of Defense.

INTRODUCTION: Providing patient-specific clinical care is an expanding focus for medical professionals and researchers, more commonly referred to as personalized or precision medicine. The goal of using a patient-centric approach is to provide safer care while also increasing the probability of therapeutic success through careful consideration of the influence of certain extrinsic and intrinsic human factors in developing the patient care plan. Of increasing influence on patient care is the phenotype and genotype information gathered from employing various next-generation sequencing methods. Guided by and partnered with our civilian colleagues, clinical components within the DoD are embracing and advancing genomic medicine in many facets-from the bench to the bedside-and in many therapeutic areas, from Psychiatry to Oncology. In this PubMed-based review, we describe published clinical research and interventions within the DoD using genome-informed data and emphasize precision medicine efforts in earlier stages of development with the potential to revolutionize the approach to therapeutics. MATERIALS AND METHODS: The new PubMed database was searched for articles published between 2015 and 2020 with the following key search terms: precision medicine, genomic, pharmacogenetic, pharmacogenomic, US military, and Department of Defense. RESULTS: Eighty-one articles were retrieved in our initial search. After screening the abstracts for studies that only involved direct testing of (or clinical interaction with) active duty, Reserve, National Guard, or civilian personnel working within the DoD and excluding any epidemiological or microbial isolation studies, seven were included in this review. CONCLUSION: There are several programs and studies within the DoD, which investigate or use gene-based biomarkers or gene variants to deliver more precise clinical assessment and treatment. These genome-based precision medicine efforts aim to optimize the clinical care of DoD beneficiaries, particularly service members in the operational environment.

Evaluation of Pharmacogenomics Testing of Cytochrome P450 Enzymes in the Military Health System From 2015 to 2020.

Pharmacogenomics (PGx) plays a fundamental role in personalized medicine, providing an evidence-based treatment approach centered on the relationship between genomic variations and their effect on drug metabolism. Cytochrome P450 (CYP450) enzymes are responsible for the metabolism of most clinically prescribed drugs and a major source of variability in drug pharmacokinetics and pharmacodynamics. To assess the prevalence of PGx testing within the Military Health System (MHS), testing of specific CYP450 enzymes was evaluated. Data were retrospectively obtained from the Military Health System Management Analysis and Reporting Tool (M2) database. Patient demographics were identified for each test, along with TRICARE status, military treatment facility, clinic, and National Provider Identifier. A total of 929 patients received 1,833 PGx tests, predominantly composed of active duty/guard service members (N = 460; 49.5%), with highest testing rates in the army (51.5%). An even distribution in testing was observed among gender, with the highest rates in Caucasians (41.7%). Of the CYP enzymes assessed, CYP2C19 and CYP2D6 accounted for 87.8% of all PGx CYP testing. The majority of patients were tested in psychiatry clinics (N = 496; 53.4%) and primary care clinics (N = 233; 25.1%), accounting for 56.4% and 24.8% of all tests, respectively. Testing was found to be provider driven, suggesting a lack of a standardized approach to PGx and its application in patient care within the MHS. We initially recommend targeted education and revising testing labels to be more uniform and informative. Long-term recommendations include establishing pharmacy-driven protocols and point-of-care PGx testing to optimize patient outcomes.

Prescription Patterns and Relationship to Pharmacogenomics Testing in the Military Health System.

INTRODUCTION: Clinical utilization of pharmacogenomics (PGx) testing is highly institutionally dependent, and little information is known about provider practices of PGx testing in the Military Health System (MHS). In this study, we aimed to characterize Clinical Pharmacogenetics Implementation Consortium (CPIC) actionable prescription (Rx) patterns and their temporal relationship with PGx testing in the MHS. METHODS: Using data from the Military Health System Management Analysis and Reporting Tool (M2) database, this retrospective cohort study included all patients receiving at least one PGx test and at least one CPIC actionable Rx from January 2015 to August 2020 (845 patients, 1,471 PGx, 7,725 index CPIC actionable Rxs). Rx patterns and temporal relationships with PGx testing were characterized via descriptive statistics. Binomial regression was used to determine which patient and provider characteristics were associated with a patient receiving a PGx test within 30 days of an index Rx. RESULTS: Patients had a median of 9 index CPIC actionable Rx's (range 1-26). Pain medications were most commonly prescribed (N = 794, 94% patients with at least 1 Rx). However, pain medication had the lowest Rx-PGx match rate (40%) compared to an average of 62% Rx-PGx match rate for all CPIC drugs. Antidepressants were also commonly prescribed (N = 668, 79.1% patients with at least 1 Rx), and antidepressants had the highest Rx-PGx match rate of 86.7%. A minority of providers (20%, N = 249) ordered the majority of PGx tests (86.1%, N = 1,266) and only 8.3% of PGx tests (N = 398) matched to a CPIC actionable drug within 30 days of the test (defined by Rxs ordered within 30 days before or after the PGx test). However, approximately 39.8% of patients (N = 317) had at least one drug match to a PGx test within 30 days. The largest predictor of whether a patient received a PGx test within 30 days of any index Rx was whether or not a specific psychiatry provider ordered the PGx test (odds ratio; OR 3.7, 95% CI 2.13-6.54, P < 0.001). Neither the CPIC level of evidence nor FDA PGx actionable or informative labels had a significant effect on PGx test timing. CONCLUSIONS: PGx testing was generally limited to high Rx-drug users and was found to be an under-utilized resource. PGx testing did not typically follow CPIC guidelines. Implementing PGx testing protocols, simplifying PGx test-ordering by incorporating at minimum CYP2D6, CYP2C19, and CYP2C9 into PGx-testing panels, and unifying providers' PGx knowledgebase in the MHS are feasible and would improve the clinical utilization of PGx tests in the MHS.

Population Pharmacokinetic Modeling and Simulations of Imipenem in Burn Patients With and Without Continuous Venovenous Hemofiltration in the Military Health System.

Continuous venovenous hemofiltration (CVVH) is a life-sustaining procedure in patients with severe burns and acute kidney injury. Physiologic changes from burn injury and use of CVVH may alter imipenem pharmacokinetics (PK). We aimed to compare imipenem clearance (CL) in burn patients with and without CVVH, determine the effect of burn on imipenem volume of distribution (CVVH, n = 12; no CVVH, n = 11), in combination with previously published models. Model qualification was performed with standard diagnostics and comparing predicted PK parameters/time-concentration profiles with those in the existing literature. Monte Carlo simulations were conducted to evaluate the probability of target attainment. A 2-compartment model best described the data. Utilizing albumin as a covariate on volume parameters and leveraging the clearance model from prior literature, our model predicted imipenem central volume and CL within a 10% margin of error across healthy, renally impaired, and burn populations. We provide direct comparison of imipenem CL in burn patients with and without CVVH. Notably, there was no significant difference. Large imipenem Vd in patients with severe burns is likely explained by increased capillary permeability, for which serum albumin may be a reasonable surrogate. Dosing 500 mg every 6 hours is adequate for burn patients on renally dosed CVVH; however, suspicion of augmented renal clearance or patients placed on CVVH without renal impairment may necessitate dosing of 1000 mg every 6 hours.

Endometriosis in the Mouse: Challenges and Progress Toward a 'Best Fit' Murine Model.

Endometriosis is a prevalent gynecologic condition associated with pelvic pain and infertility characterized by the implantation and growth of endometrial tissue displaced into the pelvis via retrograde menstruation. The mouse is a molecularly well-annotated and cost-efficient species for modeling human disease in the therapeutic discovery pipeline. However, as a non-menstrual species with a closed tubo-ovarian junction, the mouse poses inherent challenges as a preclinical model for endometriosis research. Over the past three decades, numerous murine models of endometriosis have been described with varying degrees of fidelity in recapitulating the essential pathophysiologic features of the human disease. We conducted a search of the peer-reviewed literature to identify publications describing preclinical research using a murine model of endometriosis. Each model was reviewed according to a panel of ideal model parameters founded on the current understanding of endometriosis pathophysiology. Evaluated parameters included method of transplantation, cycle phase and type of tissue transplanted, recipient immune/ovarian status, iterative schedule of transplantation, and option for longitudinal lesion assessment. Though challenges remain, more recent models have incorporated innovative technical approaches such as in vivo fluorescence imaging and novel hormonal preparations to overcome the unique challenges posed by murine anatomy and physiology. These models offer significant advantages in lesion development and readout toward a high-fidelity mouse model for translational research in endometriosis.

Risk of type 2 diabetes mellitus by antimuscarinic agents among adult females receiving care in the military health system.

PURPOSE: To explore patterns of antimuscarinic medication as a risk factor for type 2 diabetes mellitus (T2DM). METHODS: This is a retrospective cohort study of females 18 years or older within the Military Health System from 2006 to 2016. Administrative and claims data were used to select patients who initiated therapy with tolterodine, fesoterodine, oxybutynin, darifenacin, solifenacin, or trospium. Patients with no documented history of T2DM were followed for the occurrence of T2DM, the end of the study or loss of eligibility. Rates of T2DM were calculated for the overall population, by duration of therapy and by individual drugs. Crude and adjusted Cox proportional hazards were calculated to assess differences by duration of use and specific muscarinic antagonist. RESULTS: Over 2.6 million antimuscarinic prescriptions were dispensed to 241 829 females (mean age/SD, 62 ± 18 years). Patients exposed to M3 selective antagonists had highest risk of developing T2DM compared to those exposed to nonselective antagonists. Using oxybutynin, a nonselective antagonist as a comparator, adjusted rate ratios of T2DM were 57% (HR 1.57, 95%CI 1.48-1.67) and 29% (HR 1.29, 95%CI 1.24-1.35) significantly higher for darifenacin and solifenacin, respectively (both M3 selective). CONCLUSIONS: We found exposure to M3 selective antagonists darifenacin and solifenacin had the highest risk of developing T2DM compared to nonselective antagonist oxybutynin. This is supported by well described physiologic mechanisms and may allow for more informed prescribing decisions, particularly if minimizing risk of T2DM is a priority.

The utilization of an ocular wound chamber on corneal epithelial wounds.

PURPOSE: Currently available ocular moisture chambers are not adequate to manage the treatment of periocular burns, corneal injuries, and infection. The purpose of these studies was to demonstrate that a flexible, semi-transparent ocular wound chamber device adapted from technology currently used on dermal wounds is safe for use on corneal epithelial injuries. MATERIALS AND METHODS: A depilatory cream (Nair™, 30 seconds) was utilized to remove the excess hair surrounding the left eyes of anesthetized Institute Armand Frappier (IAF) hairless, female guinea pigs (Crl:HA-Hrhr). A 4 mm corneal epithelium defect was created using a corneal rust ring remover (Algerbrush®II). Epithelial defects were either left untreated or the eyes were fitted with an ocular wound chamber and 0.5 mL of hydroxypropyl methylcellulose (HPMC) gel (GenTeal®) or HPMC liquid (GenTeal®) was injected into each chamber (N=5 per group). At 0, 24, 48, and 72 hours fluorescein and optical coherence tomography imaging was collected and the intraocular pressure (IOP) was measured. H&E staining was performed on corneal and eyelid skin samples and evaluated by a veterinary pathologist. RESULTS: Corneal epithelial wounds demonstrated 100% closure rates when left untreated or treated with an ocular wound chamber containing HPMC gel at 72 hours while wounds treated with an ocular wound chamber containing HPMC liquid were 98% healed. No significant differences were found in corneal thickness and wound healing, IOP, or eyelid skin pathology in any treatment group when compared to controls. CONCLUSIONS: This study indicates that adapted wound chamber technology can be safely used on sterile, corneal epithelial wounds without adverse effects on periocular or ocular tissue when filled with a liquid or gel.

Repeat low-level blast exposure increases transient receptor potential vanilloid 1 (TRPV1) and endothelin-1 (ET-1) expression in the trigeminal ganglion.

Blast-associated sensory and cognitive trauma sustained by military service members is an area of extensively studied research. Recent studies in our laboratory have revealed that low-level blast exposure increased expression of transient receptor potential vanilloid 1 (TRPV1) and endothelin-1 (ET-1), proteins well characterized for their role in mediating pain transmission, in the cornea. Determining the functional consequences of these alterations in protein expression is critical to understanding blast-related sensory trauma. Thus, the purpose of this study was to examine TRPV1 and ET-1 expression in ocular associated sensory tissues following primary and tertiary blast. A rodent model of blast injury was used in which anesthetized animals, unrestrained or restrained, received a single or repeat blast (73.8 ± 5.5 kPa) from a compressed air shock tube once or daily for five consecutive days, respectively. Behavioral and functional analyses were conducted to assess blast effects on nocifensive behavior and TRPV1 activity. Immunohistochemistry and Western Blot were also performed with trigeminal ganglia (TG) to determine TRPV1, ET-1 and glial fibrillary associated protein (GFAP) expression following blast. Increased TRPV1, ET-1 and GFAP were detected in the TG of animals exposed to repeat blast. Increased nocifensive responses were also observed in animals exposed to repeat, tertiary blast as compared to single blast and control. Moreover, decreased TRPV1 desensitization was observed in TG neurons exposed to repeat blast. Repeat, tertiary blast resulted in increased TRPV1, ET-1 and GFAP expression in the TG, enhanced nociception and decreased TRPV1 desensitization.

Polarized Secretion of Matrix Metalloproteinases and Their Inhibitors by Retinal Pigment Epithelium Derived from Induced Pluripotent Stem Cells During Wound Healing.

PURPOSE: To characterize the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) by induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. We hypothesize that iPS-RPE secretes mediators of tissue remodeling such as MMPs and TIMPs to promote migration and proliferation of cells during wound healing. METHODS: iPS-RPE was grown on transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Conditioned media were collected from the apical and basolateral sides of the transwells every 72 h for 12 days. The media were analyzed by multiplex ELISA assays to detect secreted MMPs and TIMPs. Activity assays were performed to detect the active form of MMP-2 in conditioned media. RESULTS: MMP-2 and TIMP-1, -2, -3, and -4 were detected in conditioned media from iPS-RPE. The proteins were found to be secreted in a polarized manner. The apical secretion and activation of MMP-2 was elevated from days 3 to 12 after wounding. TIMP-1, -2, -3, and -4 were detected in conditioned media from both the apical and basolateral sides of wounded cells. Apical secretion of all 4 TIMPs increased within 3 days after wounding. CONCLUSIONS: These results indicate that iPS-RPE secretes MMP-2 and all 4 TIMPs in a polarized manner. After wounding, apical secretion of MMP-2 was higher compared to control. Apical secretion of all 4 TIMPs increased compared to control, while only TIMP-1 showed increased basolateral secretion compared to control.

Secretion Profile of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium During Wound Healing.

PURPOSE: The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing. METHODS: iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67, ß-catenin, e-cadherin, n-cadherin, and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction, cell proliferation, and migration were measured. RESULTS: iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of ß-catenin to the nucleus, cadherin switch, and expression of S100A4. GRO, GM-CSF, MCP-1, IL-6, and IL-8 were secreted by both the control and the wounded cell cultures. VEGF, FGF-2, and TGFß expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction, as well as proliferation and migration of ARPE 19 cells. CONCLUSIONS: These results indicate that after the monolayer is wounded, iPS-RPE secretes proteins into the culture medium that promote increased proliferation, contraction, and migration.

Trichostatin A Inhibits Retinal Pigmented Epithelium Activation in an In Vitro Model of Proliferative Vitreoretinopathy.

PURPOSE: Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however, the mechanisms leading to enhanced RPE proliferation, migration, and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease. METHODS: ARPE-19 cells, primary cultures of porcine RPE, and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGFß2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor, trichostatin A (TSA; 0.1 µM), were assessed for contraction and migration through collagen contraction and scratch assays, respectively. Western blotting and immunofluorescence analysis were performed to assess α-smooth muscle actin (α-SMA) and ß-catenin expression after TGFß2 treatment alone or in combination with TSA. RESULTS: TGFß2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 µM). In agreement with these data, immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased α-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGFß2-mediated iPS-RPE cell migration. CONCLUSIONS: Our findings indicate a role of acetylation in RPE activation. Specifically, the HDAC inhibitor TSA decreased RPE cell proliferation and TGFß2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR.

Low-Level Blast Exposure Increases Transient Receptor Potential Vanilloid 1 (TRPV1) Expression in the Rat Cornea.

BACKGROUND: Blast-related ocular injuries sustained by military personnel have led to rigorous efforts to elucidate the effects of blast exposure on neurosensory function. Recent studies have provided some insight into cognitive and visual deficits sustained following blast exposure; however, limited data are available on the effects of blast on pain and inflammatory processes. Investigation of these secondary effects of blast exposure is necessary to fully comprehend the complex pathophysiology of blast-related injuries. The overall purpose of this study is to determine the effects of single and repeated blast exposure on pain and inflammatory mediators in ocular tissues. METHODS: A compressed air shock tube was used to deliver a single or repeated blast (68.0 ± 2.7 kPa) to anesthetized rats daily for 5 days. Immunohistochemistry was performed on ocular tissues to determine the expression of the transient receptor potential vanilloid 1 (TRPV1) channel, calcitonin gene-related peptide (CGRP), substance P (SP), and endothelin-1 (ET-1) following single and repeated blast exposure. Neutrophil infiltration and myeloperoxidase (MPO) expression were also assessed in blast tissues via immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis, respectively. RESULTS: TRPV1 expression was increased in rat corneas exposed to both single and repeated blast. Increased secretion of CGRP, SP, and ET-1 was also detected in rat corneas as compared to control. Moreover, repeated blast exposure resulted in neutrophil infiltration in the cornea and stromal layer as compared to control animals. CONCLUSION: Single and repeated blast exposure resulted in increased expression of TRPV1, CGRP, SP, and ET-1 as well as neutrophil infiltration. Collectively, these findings provide novel insight into the activation of pain and inflammation signaling mediators following blast exposure.

Persistent Nociception Triggered by Nerve Growth Factor (NGF) Is Mediated by TRPV1 and Oxidative Mechanisms.

Nerve growth factor (NGF) is elevated in certain chronic pain conditions and is a sufficient stimulus to cause lasting pain in humans, but the actual mechanisms underlying the persistent effects of NGF remain incompletely understood. We developed a rat model of NGF-induced persistent thermal hyperalgesia and mechanical allodynia to determine the role of transient receptor potential vanilloid 1 (TRPV1) and oxidative mechanisms in the persistent effects of NGF. Persistent thermal hypersensitivity and mechanical allodynia require de novo protein translation and are mediated by TRPV1 and oxidative mechanisms. By comparing effects after systemic (subcutaneous), spinal (intrathecal) or hindpaw (intraplantar) injections of test compounds, we determined that TRPV1 and oxidation mediate persistent thermal hypersensitivity via peripheral and spinal sites of action and mechanical allodynia via only a spinal site of action. Therefore, NGF-evoked thermal and mechanical allodynia are mediated by spatially distinct mechanisms. NGF treatment evoked sustained increases in peripheral and central TRPV1 activity, as demonstrated by increased capsaicin-evoked nocifensive responses, increased calcitonin gene-related peptide release from hindpaw skin biopsies, and increased capsaicin-evoked inward current and membrane expression of TRPV1 protein in dorsal root ganglia neurons. Finally, we showed that NGF treatment increased concentrations of linoleic and arachidonic-acid-derived oxidized TRPV1 agonists in spinal cord and skin biopsies. Furthermore, increases in oxidized TRPV1-active lipids were reduced by peripheral and spinal injections of compounds that completely blocked persistent nociception. Collectively, these data indicate that NGF evokes a persistent nociceptive state mediated by increased TRPV1 activity and oxidative mechanisms, including increased production of oxidized lipid TRPV1 agonists.

Activation of mu opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1) via ß-arrestin-2-mediated cross-talk.

The transient receptor potential family V1 channel (TRPV1) is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C). Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that ß-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of ß-arrestin2 sequestration by G-protein coupled receptors (GPCRs) on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in ß-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates ß-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven ß-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.

Phosphorylation regulates TRPV1 association with ß-arrestin-2.

Post-translational modifications in TRPV1 (transient receptor potential vanilloid 1) play a critical role in channel activity. Phosphorylation of serine/threonine residues within the N- and C-termini of TRPV1 are implicated in receptor sensitization and activation. Conversely, TRPV1 desensitization occurs via a calcium-dependent mechanism and leads to receptor de-phosphorylation. Importantly, we recently demonstrated that TRPV1 association with ß-arrestin-2 is critical to receptor desensitization via its ability to scaffold the phosphodiesterase PDE4D5 to the receptor, regulating TRPV1 phosphorylation. In the present study, we demonstrate that phosphorylation of TRPV1 and ß-arrestin-2 regulates this association at the membrane. Under serum-free media conditions, we observed a significant decrease in TRPV1 and ß-arrestin-2 association in transfected CHO (Chinese-hamster ovary) cells. Pharmacological activation of the kinases PKA (protein kinase A) and PKC (protein kinase C) led to a robust increase in TRPV1 and ß-arrestin-2 association, whereas inhibition of PKA and PKC decreased association. Previously, we identified potential PKA residues (Ser(116), Thr(370)) in the N-terminus of TRPV1 modulated by ß-arrestin-2. In the present study we reveal that the phosphorylation status of Thr(370) dictates the ß-arrestin-2 and TRPV1 association. Furthermore, we demonstrate that CK2 (casein kinase 2)-mediated phosphorylation of ß-arrestin-2 at Thr(382) is critical for its association with TRPV1. Taken together, the findings of the present study suggest that phosphorylation controls the association of TRPV1 with ß-arrestin-2.

ß-Arrestin-2 desensitizes the transient receptor potential vanilloid 1 (TRPV1) channel.

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel activated by multiple stimuli and is implicated in a variety of pain disorders. Dynamic sensitization of TRPV1 activity by A-kinase anchoring protein 150 demonstrates a critical role for scaffolding proteins in nociception, yet few studies have investigated scaffolding proteins capable of mediating receptor desensitization. In this study, we identify ß-arrestin-2 as a scaffolding protein that regulates TRPV1 receptor activity. We report ß-arrestin-2 association with TRPV1 in multiple cell models. Moreover, siRNA-mediated knockdown of ß-arrestin-2 in primary cultures resulted in a significant increase in both initial and repeated responses to capsaicin. Electrophysiological analysis further revealed significant deficits in TRPV1 desensitization in primary cultures from ß-arrestin-2 knock-out mice compared with wild type. In addition, we found that ß-arrestin-2 scaffolding of phosphodiesterase PDE4D5 to the plasma membrane was required for TRPV1 desensitization. Importantly, inhibition of PDE4D5 activity reversed ß-arrestin-2 desensitization of TRPV1. Together, these results identify a new endogenous scaffolding mechanism that regulates TRPV1 ligand binding and activation.

Regulation of ROS production and vascular function by carbon monoxide.

Carbon monoxide (CO) is a gaseous molecule produced from heme by heme oxygenase (HO). CO interacts with reduced iron of heme-containing proteins, leading to its involvement in various cellular events via its production of mitochondrial reactive oxygen species (ROS). CO-mediated ROS production initiates intracellular signal events, which regulate the expression of adaptive genes implicated in oxidative stress and functions as signaling molecule for promoting vascular functions, including angiogenesis and mitochondrial biogenesis. Therefore, CO generated either by exogenous delivery or by HO activity can be fundamentally involved in regulating mitochondria-mediated redox cascades for adaptive gene expression and improving blood circulation (i.e., O(2) delivery) via neovascularization, leading to the regulation of mitochondrial energy metabolism. This paper will highlight the biological effects of CO on ROS generation and cellular redox changes involved in mitochondrial metabolism and angiogenesis. Moreover, cellular mechanisms by which CO is exploited for disease prevention and therapeutic applications will also be discussed.