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Major areas of research in the department include:

  • Neurobiology of Pain: Basic mechanisms by which pain signals are initiated, transduced and modulated in sensory pathways
  • Organ Injury and Repair: Basic mechanisms resulting in pulmonary and systemic injury following inhalation of toxic gases and pathogens, ischemia-reperfusion, organ transplantation and trauma.

The division's current projects, organized in alphabetical order by PI:

Saurabh Aggarwal

Free heme impairs bacterial killing by neutrophils in Human Immunodeficiency Virus infection

During the past three decades, a huge number of studies have reported that Human immunodeficiency virus type-1 (HIV-1) destroys the function of different cells in the blood that are involved in fighting bacterial, viral and fungal infections. Polymorphonuclear neutrophils (PMNs) are the most abundant cells in the human blood that play an important role in killing other cells which are infected with HIV-1. PMNs are also the major cells in the blood that fight other bacterial or viral infections and are also involved in healing an injured tissue. However, the PMNs in HIV patients have structural and functional defects. Therefore, these cells are not able to fight HIV-1 or other bacterial infections in HIV patients. The goal of this study is to identify the mechanisms by which PMN function and structure is damaged in HIV patients, and to propose therapy that would improve the function of these cells during HIV-1 infection. We have found that HIV patients have high levels of a highly toxic compound called heme, in their blood. This harmful heme has the ability to destroy the structure and function of several types of cells in the body. Therefore, in this study we will verify if increased heme levels in HIV patients are responsible for damaging the function and structure of PMNs in HIV patients. In our future studies, we will also determine if removing excess heme from the blood of HIV patients can improve the function of PMNs. If our strategy is successful, we would be able reduce HIV-1 infected cells in HIV patients. We would also be able to reduce the chances of HIV patients being able to transmit the disease to other people. In addition, HIV patients will be able to fight better against other bacterial and viral infections, which would overall improve their quality of life.

Shama Ahmad

Targeting cardiopulmonary calpains to mitigate toxicity of halogen gases

Accidental leaks from manufacturing plants are common and large groups of people may be exposed to high halogen (Cl2/Br2) concentrations. Use of halogen gases as chemical weapons is also on the rise. Victims of accidental bromine exposure experience respiratory distress, cardiac arrest and circulatory collapse. Studies evaluating acute and chronic sequelae of Br2 exposure are scant and treatment remains symptomatic as no effective countermeasures exist. Studies in Dr. Ahmad’s lab have established that the heart is severely injured in animals that survive high dose halogen (Cl2/Br2) inhalation. Brominated reactants such as brominated lipids are produced in the lungs upon its inhalation. These reactants reach the heart along with oxygenated blood and inactivate important calcium pumps that regulate the heartbeats. Inactivity of calcium pumps causes calcium accumulation or “calcium overload” in the heart cells. Calcium overload is a serious problem and can lead to sudden cardiac death. Increased calcium also activates destructive proteins, the calpains, that destroy cardiac ultrastructure. The calpain inhibitors based countermeasures can be critical in mitigating acute and chronic effects caused by Br2 inhalation. The outcome from this project will identify an effective antidote for bromine toxicity and enhance readiness for emergencies arising from accidental or intentional exposures.

Aftab Ahmad, Ph.D.

Extracellular RNA as Therapeutic Target after Toxic Chemical Inhalation

The purpose of this U01 is to develop specific medical countermeasures that can be used to rescue victims of civilian disaster, acts of terrorism, and military attack after exposures to toxic inhaled gases. This proposal is aimed at testing certain rescue agents after inhalation of toxic gasses like sulfur mustard.

Exposure to toxic inhaled chemicals like sulfur mustard (SM) can cause injuries to the respiratory system, eyes, skin and also frequently leads to death. The pathogenesis of sulfur mustard-induced injury is incompletely understood and a search for effective treatment regimens has been a challenge. SM-induced toxicities activate several pathways that include the coagulation and inflammatory pathways. Extracellular RNA (exRNA) and hypoxic signaling events drive several factors implicated in these pathways. This proposal focuses on understanding the role of extracellular RNA and associated inflammatory pathways in SM-induced injuries. These studies will be carried out using CEES, a surrogate of sulfur mustard as well as sulfur mustard. We hypothesize that CEES/SM exposures release exRNA that causes activation of the coagulation pathway and also inhibition of HIFs that in turn activate proinflammatory pathways. And that blocking of exRNA and stabilization of HIFs can alleviate toxicity and lung injury. Results of the proposed research will help identify treatment options for sulfur mustard exposures as well as other potentially toxic chemical inhalations associated with increased exRNA and metabolic poisoning.

Hypoxic Factors in Pulmonary Hypertension

Severe pulmonary hypertension is an irreversible disease that causes morbidity and mortality. The project investigates the role of a hypoxic factor, HIF-2alpha in causing pulmonary hypertension and proposes to block this factor and associated pathway as a therapeutic intervention. Proposed studies could lead to new targets for therapy of pulmonary hypertension and a better understanding of the disease process.

Severe pulmonary hypertension is a progressive and irreversible disease that often leads to mortality due to right heart failure. The pathogenesis of pulmonary hypertension involves proliferation of cells, including endothelial cells (intimal remodeling), in the vessel walls of pulmonary arteries resulting in remodeling and pulmonary hypertension. Several factors implicated in the remodeling process are driven by hypoxia. One mechanism by which cells respond to hypoxia is by stabilization of hypoxia-inducible transcription factors (HIFs), HIF-1alpha and HIF-2alpha. Independent studies suggest that both HIF-1alpha and HIF-2alpha are important mediators in PH. However, recent investigations have reported a marked increase in HIF-2alpha gene locus polymorphism in individuals adapting to high altitudes, suggesting a role of HIF-2alpha in PH. Mechanism by which HIF-2alpha influences pulmonary vascular remodeling and PH, independent of HIF-1alpha is not known. This proposal is focused on understanding the role of HIF-2alpha and its pathways in the pathogenesis of pulmonary hypertension. We have recently identified a new pathway, the HIF-2alpha-A2A receptor pathway, by which HIF-2alpha can promote endothelial proliferation, independent of HIF-1alpha. We hypothesize that hypoxia increases endothelial proliferation and vascular remodeling through a HIF-2alpha and A2A receptor-dependent mechanism and that this is an important step in the pathogenesis of PH. Additionally, we have also identified HIF-1alpha in smooth muscle cell proliferation, independent of HIF-2alpha. The proposed studies are divided into three aims. Aim 1 will elucidate mechanisms by which hypoxia and HIFs influence endothelial growth using cultured primary pulmonary endothelial cells as well as endothelial cells derived from hypertensive rats. The second aim will be used to test the hypothesis that inhibition or knockdown of HIFs, particularly HIF-2alpha will limit vascular remodeling and PA pressures in a rat model of PAH. The third aim will test whether inhibition or knockdown of A2A receptor, a downstream transcriptional target of HIF-2alpha, can mitigate remodeling and PA pressures in a rat model of PAH. The in vivo aims will investigate effects using both a prevention model and a rescue model. Results of the proposed research will help provide insights into hypoxic pathways that influence pulmonary vascular remodeling and offer alternative targets for treatment of PH.

Jennifer DeBerry, Ph.D.

Optogenetic Dissection of the Functional Properties of Bladder Afferent Populations

Interstitial cystitis (IC) and overactive bladder (OAB) are distinguishable, debilitating, chronic urological disorders characterized by bladder hypersensitivity. The symptoms of IC and OAB, which include altered micturition patterns with or without pain, are difficult to manage. The objective of this research is to physiologically dissect the roles of bladder afferent subpopulations in bladder homeostasis and their contributions to pathological sensory changes (e.g., increased urinary frequency, persistent pain) as a first step in development of targeted therapeutic interventions that can be tailored to different bladder pathology depending on the underlying disease mechanism.

Interstitial cystitis (IC) and overactive bladder (OAB) are chronic urological disorders characterized by urinary urgency and increased micturition frequency. A defining characteristic of IC that distinguishes it from OAB is the presence of pelvic/suprapubic pain that worsens as the disease progresses. Because visceral innervation is unique (organs are innervated by two nerves) and noxious visceral stimuli are unlike noxious cutaneous stimuli, mechanisms of bladder hypersensitivity differ from those of cutaneous hyperalgesia. Mechanical and/or chemical hypersensitivity of the bladder largely underlies the symptoms experienced by both IC and OAB patients. Yet, because the defining combination of symptoms (urgency and frequency with or without pain) for each cohort differs, and because symptoms arise from a common global population of bladder afferents, selective changes in transduction must occur either within afferent subpopulations, or among the central pathways engaged by afferent subpopulations, or both. These phenomena are poorly understood. Heterogeneous bladder neuronal subpopulations with lightly myelinated or unmyelinated peripheral fibers (A? - and C-fibers, respectively) convey information about multiple processes, including homeostatic monitoring (metaboreceptors) and detection of potentially damaging stimuli (nociceptors). Some fibers exhibit chemosensitivity, and most respond to mechanical stimuli, although some may acquire mechanosensitivity only during pathological conditions. We propose to use transgenic mouse lines expressing the light-gated ion channel, channelrhodopsin-2 (ChR2), in specific subsets of bladder afferents to address their respective contributions to the cardinal symptoms of IC and OAB. We hypothesize that subpopulations of bladder afferents contribute uniquely to micturition processes and nociception. We will test this hypothesis by studying afferent subpopulation contributions: 1) using an ex vivo preparation to functionally evaluate intrinsic and mechanical, chemical, and light-evoked response characteristics, 2) to the expression of in vivo micturition and nociceptive reflexes, and 3) to the activation of neurons receiving bladder afferent input in distinct regions of the spinal cord.

Role of growth factors (ARTN, GDNF) in TRPA1-dependent mechanisms of chemotherapy-induced peripheral neuropathy

Comprehensive Cancer Center faculty development grant from American Cancer Society Institutional Research Grant

Chemotherapy-induced peripheral neuropathy afflicts ~500,000 cancer patients annually in the United States and is the primary dose-limiting effect of cancer treatment. The implementation of specific mechanism-based therapies such as proteasome inhibitors has failed to significantly reduce the incidence of neuropathy in over 30% of treated individuals. Some chemotherapeutic drugs preferentially damage heavily myelinated (Aβ) large sensory fibers innervating the skin. However many others, including Bortezomib (BTZ), induce neuropathy that is associated with changes in large fibers as well as small (lightly myelinated Aδ and unmyelinated C) sensory fibers that innervate the pelvic viscera. In fact, deviations in both skin and urinary bladder mechanosensation have been reported for BTZ-treated individuals. There is a paucity of information regarding mechanisms underlying chemotherapy-induced peripheral neuropathy with respect to modulation of sensory neurons by growth factors, which can exert complex, tissue-specific effects. A principle mechanism by which artemin (ARTN) and glial cell line-derived neurotrophic factor (GDNF) exert their effects is via modulation of the ion channel, TRPA1, expressed on primary sensory neurons. Thus, the objective of these studies is to determine whether growth factors (ARTN, GDNF) in peripheral tissues mediate chemotherapy-induced peripheral neuropathy through a TRPA1-dependent mechanism. We will use a combination of in vivo and in vitro methods to test the hypotheses that BTZ treatment: 1] alters cutaneous and bladder mechanical sensation in vivo; 2] upregulates skin- and urinary bladder-derived ARTN and GDNF in a tissue- and time-dependent fashion; and 3] upregulates TRPA1 expression and function in vitro in dorsal root ganglion (DRG) neurons innervating the skin and bladder. Mice will be treated for four weeks with BTZ (cumulative dose 4.8 mg/kg) or vehicle. Experimental endpoints will be assessed on days 7, 28, and 42 following the beginning of BTZ (or vehicle) treatment to examine early effects, intermediate effects, and post-treatment sequelae, respectively. Experimental endpoints will include behavioral testing to determine mechanical sensitivity of the skin and bladder, real-time RT-PCR to examine concentration of peripheral growth factor mRNA in skin and bladder, and calcium imaging to assess expression and function of TRPA1 in retrogradely labeled, skin- and bladder-innervating dorsal root ganglion sensory neurons. These studies will provide important preliminary data necessary for the development of a competitive extramural funding application examining a causal link between growth factors and TRPA1 in chemotherapy-induced peripheral neuropathy. There is a high likelihood that experimental findings related to BTZ are relevant to neuropathies induced by multiple other chemotherapeutics that have similar secondary effects (e.g., inflammation and endogenous reactive aldehyde production). In summary, these studies will begin to identify novel, targetable mechanisms underlying chemotherapy-induced peripheral neuropathy (i.e., abnormal sensation and peripheral nerve damage) of the skin and viscera, the latter of which is exceptionally understudied. An improved understanding of these mechanisms will result in increased translation of basic science to clinical practice and ultimately will improve treatment completion rate and quality of life of individuals with cancer.

Jianguo Gu, Ph.D.

Mechanism of Nociception Induced by Innocuous Cold in Trigeminal System

Many clinical conditions including dental procedures, traumatic injury, tumors, and chemotherapy can result in chronic trigeminal nerve injury and degeneration. These often lead to the development of trigeminal neuropathic pain that manifests cold allodynia and hyperalgesia in orofacial regions. The trigeminal neuropathic pain constitutes a huge health problem because of its severity, special location, and resistance to conventional treatment. This project will use advanced neurobiological approaches and animal models to identify molecular and neuronal mechanisms underlying orofacial cold allodynia and hyperalgesia. The accomplishment of the Aims proposed in this project will lead to novel therapeutic targets for treating trigeminal neuropathic pain that manifests cold allodynia and hyperalgesia.

Orofacial pain disorders encompass a wide range of conditions including trigeminal neuralgia, reflex sympathetic dystrophy (RSD) of the face, temporomandibular joint disorders, periodontal pain, burning mouth syndrome, dental surgical pain, head and neck cancer pain, pain due to oral infections, and other neuropathic and inflammatory pain conditions. One common symptom in many chronic orofacial pain conditions is cold allodynia/hyperalgesia, the excruciating painful sensation induced by cooling temperatures that would normally produce innocuous or mild painful cooling sensation. Unfortunately, the current clinical treatments are unsatisfactory for this chronic orofacial pain condition. This is largely due to the poor understanding of mechanisms for controlling cold sensitivity in the trigeminal sensory system. We have recently accumulated evidence suggesting that cold sensitivity of nociceptive cold- sensing neurons may largely depend on low-threshold voltage-gated K+ channels (KLT), a subclass of voltage-gated K+ channels that are activated near resting membrane potentials. Furthermore, we found that pharmacologically potentiating KLT channels reversed orofacial cold allodynia/hyperalgesia. In this renewal application, the overall objectives are to study the role of KLT channels in controlling cold sensitivity of nociceptive cold-sensing trigeminal neurons under both physiological and trigeminal neuropathic conditions, and to explore the therapeutic use of KLT channel potentiators for treating orofacial cold allodynia and hyperalgesia. Advanced neurological techniques, including patch-clamp records and calcium imaging together with other approaches, including immunostaining and novel animal models will be used in this project. By accomplishing our goal, we will have identified novel therapeutic targets for treating some intractable orofacial pain conditions.

Cellular and Ion Channel Mechanisms Underlying the Sense of Light Touch in Mammal

The sense of light touch enables tactile discrimination of shapes, texture, vibration and spatial details, and is indispensable in daily life, yet scientific knowledge about how mammals can sense light touch remains lacking at a cellular and molecular level. The expected outcomes of the proposed research will uncover cellular and ion channel mechanisms underlying mechanical transduction for light touch. Because light touch dysfunctions such as the loss of touch sensitivity and the pain induced by light touch are common clinical problems seen under pathological conditions such as diabetes, chemotherapies and other diseases. Understanding cellular and molecular mechanisms of light touch may help us better understand the physiopathology of these sensory dysfunctions, and may further lead to new strategies for preventing or treating these sensory dysfunctions.

The sense of light touch is critically important for daily life but this important sense can be altered to result in sensory dysfunctions such as tactile anesthesia and mechanical allodynia under pathological conditions. How mammals can sense light touch has been one of the biggest mysteries in science. This lack of knowledge prevents development of potentially effective approaches for preventing or treating mechanical sensory dysfunctions. Our long-term-goal is to uncover the cellular and molecular mechanisms underlying the sense of light touch in mammals. As the first stage of our long-term goal, the overall objective of this application is to study mechanisms underlying mechanical transduction of Merkel cell-neurite complex, a sensory structure essential for sensing light touch in mammals. Our central hypothesis is that Merkel cells are mechanical transducer cells that express mechanically activated ion channels (MA) and that activation of these channels triggers Merkel cells to fire action potentials and release excitatory transmitters. This hypothesis is based on our preliminary results obtained by using our recently developed patch-clamp recordings from Merkel cells situated in whisker hair follicles (Merkel cell in situ patch-clamp technique). This innovative technique has, for the first time, led us to successfully record MA currents from Merkel cells. We have further discovered that Merkel cells in situ fire action potentials in response to mechanical stimulation. Our unique expertise of Merkel cell in situ patch-clamp recording technique places us at an advanced position to test the hypothesis with the following specific aims: 1) Elucidate ionic mechanisms of MA currents that excite Merkel cells in situ and characterize Merkel cell MA channel properties; 2) Identity ion channels that encode mechanical activity in Merkel cells; and 3) Delineate the mechanisms underlying the transmission of mechanical activity by Merkel cells. The outcomes of the above investigations will provide scientific knowledge about the sense of light touch at a cellular and molecular level. The study may have clinical implications ranging from sensory dysfunctions seen in diabetes and other disease conditions to Merkel cell malfunctions such as Merkel cell carcinoma.

Kevin Harrod, Ph.D.

Targeting MMP9 to Improve Outcomes in Serious Influenza Infections

Influenza infection is still an important public health concerns despite the availability of vaccines. As influenza viruses undergo high rates of mutation enabling them to evade protection from natural and vaccine-mediated immunity, new drugs are urgently needed for serious influenza infections. Herein a new class of drug will be tested for its efficacy to reduce influenza-associated mortality and morbidity in two animal species to satisfy the FDA "Two Animal Rule".

Influenza infection remains an enormous public health concern despite the availability of vaccines and worldwide surveillance. The high mutation rates of influenza A viruses (IAVs) enable the viruses to evade natural and vaccine-mediated immunity. Also, current anti-viral therapies do not prevent IAV-related deaths when there is a delay in initiating treatment. Thus, new therapeutic options for treating influenza disease are an immediate public health priority. Our pilot studies identify MMP-9 as an attractive IAV therapeutic target. MMP- 9 is strikingly upregulated in plasma samples from human subjects infected with seasonal and H1N1 IAV and in lungs from mice infected with H1N1 IAV. In mice, Mmp-9 increases IAV-associated mortality and late-stage lung inflammation and late-stage lung viral burdens. We proposed to test the therapeutic efficacy of a "re- purposed" therapeutic candidate (ADZ1236) that selectively and potently inhibits MMP-9 activity in IAV infections in two species in three integrated and highly collaborative aims. Aim 1 will test the therapeutic efficacy of AZD1236 therapy in mice infected with BL2 H1N1 IAV. AZD1236 will be tested in mice alone and in combination with a clinically-used antiviral agent (the neuraminidase inhibitor, oseltamivir). A delayed initiation of treatment approach will be optimized to model "real-world" treatment scenarios for serious IAV infections. Aim 2 will identify the mechanisms by which Mmp-9 promotes (and AZD1236 limits) serious IAV infections in mice. We will study H1N1-infected WT vs. Mmp-9-/- mice and Mmp-9 bone marrow chimeric mice and use in vitro approaches to test our hypothesis that Mmp-9 promotes adverse outcomes in IAV disease by cleaving host or viral proteins. These Mmp-9 substrates will be identified. Aim 3 will test the therapeutic efficacy of AZD1236 in ferrets infected with BL2 H1N1 and the BL3 highly pathogenic avian influenza (HPAI) H5N1 strain (and in H7N9 IAV-infected ferrets if this strain emerges as an epidemic or pandemic strain during the funding period). Our studies will determine whether AZD1236-mediated MMP-9 inhibition has therapeutic efficacy against IAV in both small and large animals sufficient to thereby satisfy the FDA "Two Animal Rule" required to demonstrate efficacy to apply for FDA approval. Successful completion of the work proposed herein may provide a "first in class" therapeutic intervention for serious influenza-mediated lung disease.

Sadis Matalon, Ph.D.

Testing the Efficacy of Hydrogel-based Nanoparticles, Encapsulating Nitrite and Nitric Oxide in the Treatment of Acute Lung Injury and Pulmonary Hypertension (not NIH-funded)

Bromine Inhalation Induced Lung Injury: Novel Mechanisms and Treatment Strategies

World production of Br2 exceeds 300,000 tons per year and accidental spills into the environment during transportation and industrial accidents are common. Br2 inhalation may cause severe injury to the lungs and death from respiratory failure. The purpose of this application is to understand how bromine damages the lungs and find appropriate countermeasures.

Bromine (Br2) is a highly toxic dark-reddish liquid, which evaporates readily to a red vapor with a suffocating odor. World production of Br2 exceeds 300,000 tons per year. Exposure to Br2 causes acute lung injury, death from respiratory failure, and fibrosis. Because of the potential for industrial and transportation accidents to release of large amounts of Br2 in populated areas, Br2 presents a clear and present danger to public health. Few published studies have evaluated the acute and chronic sequelae of Br2 inhalation; treatment remains symptomatic and no effective countermeasures exist. Similar to human pathology, exposure of mice to Br2 causes reactive airway disease syndrome (RADS), increased permeability of the blood gas barrier to plasma proteins, and inflammation followed by sub-epithelial airway fibrosis and significant mortality. The overall purpose of this application is to identify the biochemical and molecular mechanisms responsible for these events and develop appropriate countermeasures. We propose that Br2 and hypobromous acid (HOBr-) interact with and fragment high molecular weight hyaluronan (H-HA), a ubiquitous matrix glycosaminoglycan, to generate highly inflammatory low molecular weight hyaluronan fragments (L-HA). L- HA binds to CD44 and Toll like receptor (TLR)-4, increases intracellular Ca+2 and activates TGF-b1, and RhoA in lung epithelial and airway smooth muscle cells. These events lead to RADS, increased epithelial permeability to plasma proteins, epithelial-mesenchymal cell transition (EMT) of airway cells, sub-epithelial fibrosis, and death from respiratory failure. In addition, we demonstrate for the first time the formation of brominated lipids in the lungs and plasma of mice exposed to Br2. These compounds, formed by the interaction of Br2 with lung plasmalogens, mediate and amplify Br2 lung injury and act as biomarkers of Br2 exposure. Based on solid data we posit that post-Br2 exposure administration of aerosolized YabroÒ (a form of H-HA, currently in clinical trials in Europe for asthma), attenuates lung damage, enhances repair and decreases mortality. Experiments proposed in the first specific aim will assess physiological, biochemical, and morphological changes in mice exposed to Br2 and returned to room air for up to three weeks and test the effectiveness of aerosolized YabroÒ administered post exposure to decrease lung injury and mortality. We will then identify the mechanisms by which Br2 damages rodent and human airway smooth muscle (ASM), bronchial and alveolar type II (ATII) cells. We posit that Br2, brominated lipids, and L-HA increase intracellular Ca2+ and activate RhoA, which lead to increased airway contractility and epithelial permeability. Experiments will: (i) determine membrane potentials by patch clamp; (ii) intracellular Ca+2 by fura-2 fluorescence; (iii) RhoA and ROCK activation; (iv) myosin light chain phosphorylation; and (v) (for epithelial cells) permeability to fluorescent dextrans. Finally, we will isolate mouse tracheal rings at 1, 24, and 72 hr. post Br2 exposure and measure smooth muscle contraction in response to methacholine.

Bromine Inhalation Induced Lung Injury: Novel Mechanisms and Treatment Strategies

Between 1940 and 2007, the accidental release of large amounts of Cl2 in 30 large cities world-wide (such as the train derailment in Graniteville, South Carolina(3), the industrial accident in a chemical plant near Apex, NC (described in the local press), the malfunction of Cl2 delivery systems to a water park near Sacramento, CA (ibid) and the deliberate release of Cl2 during acts of terrorisms by insurgents in the Iraq conflict (9), caused significant mortality and morbidity to humans and animals. In addition to these public disasters, from 2000-2004, there were about 6,000 calls for Cl2 related injuries to US poison control centers for Cl2 related injuries each year. For these reasons, Cl2 is specifically mentioned in the PAR-10-180 as an “agent of interest.”

Currently there are no specific treatments for humans or animals exposed to Cl2. “The purpose of the U01 research is to foster and support the development of FDA-approved therapeutics that can be used effectively to treat individuals during a chemical emergency”.

CIALIS® Reverses Halogen Induced Injury to Pregnant Animals and Their Offspring (Co-PI: Tamas Jilling)

We have discovered that pregnant mice are highly susceptible to bromine. Tadalafil, an FDA approved agent, reverses injury to pregnant mice and offsprings when administered after Br2 exposure. We will investigate the mechanisms involved and identify the optimum way of delivering tadalafil.

The halogen bromine (Br2) is used as water disinfectant, for bleaching fibers, for manufacturing antiepileptic drugs, dyestuffs, flame-retardants, insecticides, drilling fluids, and gasoline additives. When inhaled, it causes exposure-level-dependent acute and chronic pulmonary and systemic injuries ranging from mild eye and airway irritation, to significant damage to the cardiopulmonary system and other organs, which can lead to death. Survivors may develop reactive airway disease syndrome, pulmonary fibrosis as well as restrictive and obstructive pulmonary diseases. Presently, there are no studies evaluating acute and chronic sequelae of Br2 inhalation in pregnant rodent and non-rodent models, even though US census bureau data predicts two of every 100 people in the US being pregnant. Exposure of pregnant mice at gestational day 15 (E15) to Br2 (600 ppm for 30 min.) results in 75% mortality over four days, in contrast to 25% mortality in males or non-pregnant females (p<0001). When delivered at E19, fetuses of surviving Br2-exposed mice exhibit severe fetal growth restriction (FGR) and fetal demise (FD). Placentas are poorly developed and express increased levels of short-FMS-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic mediator and biomarker of both preeclampsia and pulmonary hypertension. When born naturally, none of the fetuses survive. Oral administration of an FDA- approved type 5 cyclic nucleotide-specific phosphodiesterase inhibitor (PDE5i; tadalafil) to the dams post- exposure, dramatically improved maternal survival, fetal growth restriction and neonatal survival. We hypothesize that brominated intermediates, formed by the reaction of Br2 and HOBr with plasmalogens cause injury to the endothelium and the placenta, inducing the release of vasoconstrictor and anti-angiogenic mediators which in turn mediate pulmonary vasoconstriction, increased pulmonary artery pressure and right ventricular dysfunction. Tadalafil restores pulmonary and uterine vasodilation, preserves heart function and improves uterine/placental blood supply resulting in maternal and fetal survival. We will test these proposed mechanisms and we will perform the necessary efficacy studies to identify the optimum therapeutic regimen of tadalafil to decrease maternal morbidity and mortality, improve fetal growth restriction and increase fetal survival when administered orally post exposure. Specific Aim #1. To test the hypothesis that exposure of pregnant mice to Br2 at E15 causes extensive pulmonary injury as well as systemic endothelial injury, placental injury, pulmonary hypertension, right heart failure resulting in maternal mortality, fetal growth restriction and fetal demise/stillbirth. Specific Aim #2: To identify the sequence of events and mechanisms involved in the development of maternal vasoconstriction, pulmonary hypertension and right heart failure. Specific Aim #3. To investigate the efficacy of post halogen exposure administration of tadalafil to decrease maternal and fetal death and morbidity and to develop a rabbit (non-rodent) model of Br2 toxicity in pregnancy.

Timothy Ness, M.D., Ph.D.

A Phase 4 Randomized, Active-Comparator Controlled Clinical Trial to Study the Safety of Sugammadex (MK-8616) for the Reversal of Neuromuscular Blockade Indiced by Either Rocuronium Bromide or Vecuronium Bromide in American Society of Anesthesiologists (ASA) Class 3 or 4 Subjects (Not NIH-Funded)

Quantitative Studies of Urinary Bladder Sensation

The proposed studies examining acute stress-related effects in rodent model systems will give insight related to the effect of an acute exacerbating factor related to bladder pain. Neural pathways and mechanisms related to an acute stressor - footshock - will be defined. Novel therapeutics related to GABAB-receptor mechanisms will be explored. An improved understanding of sensory processing related to IC and of urinary bladder sensory pathways and their modulation by acute stress and pharmacological manipulations will result in an increased translation of basic science to therapeutics for bladder pain.

Acute and chronic pains originating from the urinary bladder are common clinical entities affecting more than 50% of females at some time in their lives. In an attempt to understand urinary bladder hypersensitivity in a translational manner, this ongoing research project has used rodents to define basic neurophysiological elements of bladder sensation at spinal and supraspinal levels. Using urinary bladder distension (UBD)-evoked reflexes and spinal/supraspinal neuronal responses as experimental endpoints, clinically-relevant models of bladder hypersensitivity have been developed. Whereas the effect of inflammation as an exacerbator of pain has been investigated, the effects of acute stress have not. The present set of studies seeks to reverse the deficit of knowledge that exists in relation to the mechanisms of acute stress as an exacerbator of urinary bladder pain by performing systematic experimental investigations in our translational model. Further, it seeks to explore novel therapeutics in the form of GABAB-receptor based mechanisms. Three Specific Aims are proposed: Specific Aim #1: To quantitatively characterize effects of serotonergic receptor antagonists on reflex and neuronal responses to UBD in rats which experienced neonatal bladder inflammation (NBI) and subsequently received acute footshock (aFS). Specific Aim #2: To quantitatively characterize using immunohistochemical and neurochemical measures, serotonergic agonist and receptor content in the spinal cord of rats which experienced NBI subsequently subjected to aFS. Specific Aim #3: To quantitatively characterize central nervous system effects of GABAB receptor agonists and antagonists on reflex and neuronal responses to UBD in rats which experienced NBI in the presence of exacerbating factors (inflammation, aFS). These studies will expand upon preliminary studies and will determine quantitatively the effects of the classic stressor, intermittent nonpainful footshock, on reflex (visceromotor) and spinal dorsal horn neuronal responses to UBD. Effects of pharmacological manipulations related to pain facilitation will be assessed in animals which experienced neonatal bladder inflammation and controls. The proposed studies examining acute stress-related effects in rodent model systems will give insight related to the effect of an acute exacerbating factor related to bladder pain and will explore novel therapeutics. An improved understanding of sensory processing related to IC and of urinary bladder sensory pathways and their modulation by acute stress will result in an increased translation of basic science to therapeutics for bladder pain.

Multidisciplinar Approach to Pelvic Pain I & II

This is a local project investigating potential phenotypes within the interstitial cystitis population using psychophysical and imaging methods. It is part of a multicenter, multidisciplinary investigation performing deep phenotyping of subjects with the disorder of interstitial cystitis.

Jean-Francois Pittet, M.D.

Regulation of phosphodiesterases and cAMP signaling during the host-pathogen interaction in the pulmonary endothelium

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease associated with high morbidity/mortality and limited treatment options. A breakdown of pulmonary endothelial barrier function, leading to edema and impaired lung function, is a hallmark of ARDS. It is well established that stimulation of cAMP synthesis, such as with β-adrenoceptor agonists, enhances endothelial barrier function and is protective in preclinical models of sterile lung injury such as upon LPS administration. However, clinical trials probing the utility of β-agonists in ARDS have failed and the reasons remain unclear. Cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, play a critical role in the regulation of cellular cAMP levels, the subcellular compartmentalization of these signals, and hence endothelial cell functions. Our preliminary data indicate that a single Type-4 PDE isoform, PDE4D, contributes the predominant portion of cAMP-hydrolytic capacity in the pulmonary endothelium and is tightly regulated under physiologic conditions. P.aeruginosa (PA) is a common cause of nosocomial pneumonia that can progress to sepsis and ARDS. We observed that during the host-pathogen interaction, distinct PA virulence factors induce PDE4D activation, resulting in a suppression and dysregulation of endothelial cAMP signals. Specifically, the bacterial exotoxin cyclase ExoY promotes a PKA-mediated phosphorylation and activation of PDE4D that alters endothelial cAMP signaling. In addition, PA infection can induce a PKA-independent, but type-3 secretion system- and flagellin-dependent PDE4D regulation that results in activation and subcellular relocalization of the enzyme. With this proposal, we will define the pathways by which PA virulence factors alter PDE4D functions and its contribution to endothelial barrier disruption and lung injury. We will test the idea that PA-induced PDE4D activation correlates with health outcomes in patients with PA-associated ARDS, and that, conversely, inhibition of PDE4 is endothelial barrier protective. We will test the idea that aberrant PDE4 activation limits the therapeutic efficacy of β-agonists in settings of PA-lung infection as well as other causes of ARDS. The PDE4 family comprises four genes and non-selective PDE4 inhibitors have established therapeutic effects in preclinical models of ARDS, but also induce side effects, such as emesis and nausea, that limit their clinical utility. Given the unique and non-overlapping physiological and pathophysiological roles of each PDE4 isoform, targeting individual PDE4 proteins can serve to dissect the therapeutically beneficial from the side effects of the PAN-PDE4 inhibitors available to date. To this end, we will determine whether selective ablation of PDE4D is protective in ex vivo and in vivo models of PA-lung injury, paving the way for development of PDE4D-selective inhibitors as ARDS therapeutics with an improved safety profile compared to the non- selective PDE4 inhibitors available to date.

Nosocomial Pneumonias Impair Cognitive Function

Patients in intensive care units are at high risk for long-term health threats including cognitive impairment. The correlation was only recently revealed after large-scale follow-up cognitive assessments on intensive patient survivors after their discharge from the hospital. There are testimonials, reviews and calls-to-action on many critical care websites and in journal issues over the last 2-3 years on this public health crisis. However, the causative mechanisms that lead to abrupt cognitive impairment are unclear—they are not attributable to age, gender, relative brain hypoxia, anesthetics and sedatives.

Nosocomial bacteria such as Pseudomonas aeruginosa impair endothelial cell function during the course of infection that culminates in acute lung injury. Recent studies implicate that after P. aeruginosa infection, endothelial cells produces and releases cytotoxic amyloids that are transmissible among cells. Having access to the endothelium of whole blood circulation, these cytotoxic amyloids likely propagate. In fact, the amyloids are detectable in the blood and cerebrospinal fluid of nosocomial pneumonia patients in intensive care units. Further, when applied to brain slices, the cytotoxic amyloids derived from endothelium impair neural activity and synaptic information transfer.

This project takes advantage of vertically integrated approaches, ranging from the use of different bacteria stains and cultured cells, to in vitro brain slice recordings and in vivo animal behavior studies. The studies are designs to test the hypothesis that nosocomial pneumonias induce amyloid protein accumulation in CSF, resulting in LTP suppression and learning deficit. Altogether, studies systematically examine a possible lung-brain axis, where a primary lung infection induces production of cytotoxic amyloids that spread to the cerebrospinal fluid and contribute to cognitive impairment. This work addresses a novel mechanism underlying the end organ dysfunction that is evident during, and in the aftermath of, critical illness. Mechanistic insight into endothelium-derived cytotoxicity to brain function will reveal novel therapeutic approaches to preventcognitive decline.

Mark F. Powell, M.D.

A Randomized Controlled Trial of Regional versus General Anesthesia for Promoting Independence After Hip Fracture (REGAIN Trial)

Ursula Wesselmann, M.D., Ph.D.

Racial Disparities: Pain in Women Treated for Breast Cancer

Due to advances in breast cancer diagnosis and treatment, there has been a dramatic increase in the breast cancer survivor population. Chronic pain during and after breast cancer treatment is a major clinical problem affecting 25 - 60% of breast cancer survivors, and striking racial health disparities in pain and pain-related disability have been well documented with African-American women experiencing a disproportionate burden, however, the mechanisms underlying these disparities remain poorly understood. The goal of this project is to identify pathophysiological mechanisms and psychosocial factors contributing to racial group differences in pain in African- American and non-Hispanic White women treated for breast cancer, and the results of this study should have a major impact on racial pain disparities in the clinical setting by developing methods to identify patients at-risk and to target those patients for early and tailored interventions.