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The antibacterial role of exogenous nitric oxide gas Miller, Christopher C. 2004

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T H E A N T I B A C T E R I A L R O L E O F E X O G E N O U S N I T R I C O X I D E G A S by C H R I S T O P H E R C. M I L L E R RRT, Northern Alberta Institute of Technology, 1982 B.A., Ottawa University, 1991 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Experimental Medicine) /Accept this tresis as conforming to the required standard, T H E U N I V E R S I T Y O F BRITISH C O L U M B I A November 2004 © Christopher Clayton Miller, 2004 ABSTRACT Experimental evidence indicates that nitric oxide (NO) has a role in the host defense mechanism, and may have antibacterial characteristics. The goal of this thesis is to explore the feasibility of exogenous or gaseous N O (gNO) as a potential therapeutic antibacterial agent by confirming that gNO is bacteriocidal to bacteria and to elucidate the mechanism by which it is bacteriocidal. A successful bacteriocidal effect is defined as a decrease in bacteria of greater than 3 loglO cfu/ml. In order to test the bacteriocidal or bacteriostatic effects of gNO, we built and validated a closed in vitro environment for delivery of a variety of gases including NO. Under controlled in vitro conditions, we determined the time required to effectively induce a bacteriocidal effect with 200 parts per million gNO on a representative collection of Gram-positive and Gram-negative strains of bacteria associated with clinical infection. Further, Candida albicans, Methicillin Resistant Staphylococcus aureus (MRSA), a particularly resistant strain of Pseudomonas aeruginosa from a cystic fibrosis patient, Group B Streptococcus, and Mycobacterium smegmatis were also included to determine if yeasts, a multi-drug resistant strain of bacteria and actinomycetes had a similar response. These bacteria represented a variety of bacterial pathogens that contribute to both respiratory and wound infections. A series of in vitro studies was performed using wild-type M. smegmatis, isogenic mycothiol (MSH) mutants, and complemented mutants to show that MSH was essential for the protection of M. smegmatis against oxidative and nitrosative stressors. The results showed that 200 ppm gNO was completely bacteriocidal for all bacteria tested. Without exception, every bacterial strain challenged with 200 ppm gNO had at least a 3 loglO reduction in cfu/ml. The bacterial death curve was characterized by a latent period when it appeared that the bacteria were unaffected. This latent period was followed by an abrupt death of all cells. Mycobacteria typically had a longer latent period compared to other organisms, suggesting that mycobacteria have a highly active mechanism that ii protects the cell from gNO cytotoxicity. Eukaryotic cell and Gram negative bacteria synthesize glutathione (GSH), which detoxifies N O and reactive oxygen species. Some bacteria that do not make GSH produce other low-molecular weight thiols to protect themselves from oxidative damage. M smegmatis, which does not make GSH, produces MSH, which may act analogously to GSH in protecting the cell from N O and other electirophilic molecules. Mycothiol was shown to protect M. smegmatis against gNO damage and, to achieve this, an intact MSH biosynthesis pathway was required. The studies described here also determined that continuous exposure to a high level of gNO (200 ppm) was not required for the observed bacteriocidal effect. A cyclic regimen of 160 ppm gNO for 30 minutes, followed by 3.5 hours of 20 ppm gNO also resulted in a complete bacteriocidal effect. These studies begin to lay the foundation for potential use of gNO as a therapeutic agent against bacterial infections. This thesis will advance the current body of knowledge and confirm that N O is a cytocidal, non-organism specific, broad-spectrum antibacterial agent. Data from these studies will add insight into the detoxification pathways that drug-resistant bacteria use to fight antibiotic therapies. This new validated exposure chamber methodology may provide opportunities for further research on agents that block these pathways. Ultimately, the knowledge derived from this thesis might provide a rationale for further exploration of gNO as a non-organism specific, non-antibiotic based, treatment for pulmonary and wound infections. ni T A B L E OF C O N T E N T S Abstract ii Table of contents iv List of tables viii List of figures ix Abbreviations xi Acknowledgements xiv 1.0 INTRODUCTION TO T H E ANTIBACTERIAL R O L E OF NITRIC O X I D E 1 1.1 NITRIC O X I D E 1 1.2 CHEMICAL REACTIVITY A N D TOXICITY OF N I T R O G E N SPECIES 2 1.3 GASEOUS NITRIC O X I D E IN A PHYSIOLOGICAL C O N T E X T 6 1.4 NITRIC O X I D E AS A SECOND MESSANGER 8 1.5 T H E R O L E OF N O IN HOST D E F E N S E 9 1.6 ANTIBACTERIAL PROPERTIES OF N O 20 1.7 USE OF NITRIC O X I D E IN A CLINICAL SETTING 22 1.8 D E F I N I N G T H E P R O B L E M 23 2.0 W O R K I N G HYPOTHESIS & SPECIFIC AIMS 25 2.1 HYPOTHESIS 25 2.2 SPECIFIC AIMS 25 2.3 SUMMARY 26 3.0 E X P E R I M E N T A L 27 3.1 MATERIALS 27 iv 3.2 SPECIALIZED EQUIPMENT - IN VITRO C E L L EXPOSURE D E V I C E 28 3.3 METEIODS 31 3.3.1 Method validation and Safety 31 3.3.1.1 Cell Culture Preparation for method validation and safety experiments 31 3.3.1.2 Skin Cell Culture — pilot study for growth on solid media 32 3.3.1.3 Exposure Conditions 32 3.3.1.4 Analysis and Measurements — Cell Viability 33 3.3.1.5 Analysis and Measurements — Nitric Oxide and pH Analysis 33 3.3.1.6 Characterizing kinetics of gNO delivery from the the air to liquid interphase 34 3.3.2 Measuring Bacteriostatic Action of Gaseous Nitric Oxide on S. aureus and P. aeruginosa 34 3.3.3 Measuring Bacteriocidal Action of Gaseous Nitric Oxide on a variety of bacterial pathogens 36 3.3.3.1 Intermittent exposure 37 3.3.3.2 N O z exposure 38 3.3.4 Measuring the Protective Effect of Mycothiol Against Nitric Oxide Damage in Mycobacteria 38 3.3.4.1 Strains and Microbiology Techniques 38 3.3.4.2 Mycothiol (MSH) Measurement 39 3.3.5 Statistics . 40 RESULTS 42 4.1 V A L I D A T I N G T H E STABILITY OF IN VITRO D E L I V E R Y SYSTEM 42 4.1.1 Deterrruning the kinetics of Nitric Oxide Uptake 45 4.1.2 Cell Growth and Viability 47 4.1.2.1 Bacterial Growth in the in vitro cell exposure device 47 v 4.1.2.2 Skin Cell Growth in the in vitro cell exposure device 49 4.2 BACTERIOSTATIC A C T I O N OF GASEOUS NITRIC O X I D E O N S. aureus AND P.aeruginosa 53 4.2.1 Determining the effect of gNO on P. aeruginosa growth 53 4.2.2 Determining the effect of gNO on S. aureus growth 54 4.2.3 Time exposure study 56 4.3 BACTERIOCIDAL A C T I O N OF GASEOUS NITRIC O X I D E O N A VARIETY OF P A T H O G E N I C BACTERIA 57 4.3.1 Exposing bacteria to 200 ppm gNO 58 4.3.2 Exposing bacteria to intermittent 160 ppm gNO 66 4.3.3 Results from N 0 2 Exposure 72 4.4 MYCOTHIOL PROTECTS MYCOBACTERIA AGAINSTNITRIC O X I D E D A M A G E 73 5.0 DISCUSSION 80 5.1 V A L I D A T I O N OF A N IN VITRO SYSTEM TO EXPOSE MICROORGANISMS T O G N O 80 5.2 D E T E R M I N I N G T H E BACTERIOSTATIC A C T I O N OF GASEOUS NITRIC O X I D E O N S. aureus A N D P.aeruginosa 85 5.3 BACTERIOCIDAL A C T I O N OF GASEOUS NITRIC O X I D E O N A VARIETY OF P A T H O G E N I C BACTERIA 88 5.4 U N D E R S T A N D I N G D E F E N S E MECHANISMS B Y WHICH E U K A R Y O T E S A N D M A M M A L S PROTECT THEMSELVES AGAINST NITRIC O X I D E D A M A G E 93 5.5 D E T E R M I N I N G T H E M E C H A N I S M OF A C T I O N B Y WHICH BACTERIA ATTEMPT TO PROTECT THEMSELVES F R O M G N O C E L L D E A T H 95 5.6 M O V I N G TO T H E FUTURE - T H E POTENTIAL USE OF G N O AS A THERAPEUTIC A G E N T 99 6.0 FUTURE W O R K 102 6.1 M E T H O D O L O G Y FOR G N O ANALYSIS A N D STUDIES O N ANTIBACTERIAL ACTIVITY OF gNO 103 v i 6.2 IN VIVO STUDIES 105 7.0 REFERENCES 108 Appendix 1 128 Appendix 2 168 LIST O F T A B L E S Table Title Page Table 1 Calculation of nitre oxide conversion to nitrogen dioxide 4 Table 2 Calculated uptake rate of gNO into liquid media 46 Table 3 Survival curve analysis for microorganisms exposed to 200 65 ppm until L D 1 0 0 is achieved Table 4 Survival curve analysis for microorganisms exposed 68 intermittendy to 160 ppm gNO for 30 rninutes Table 5 Summary of survival curves for M. smegmatis studies 74 Table 6 Mycothiol measurement tables 80 vm LIST O F FIGURES Figure Title Page 1 Relative size of N O molecule to bacterial cell wall 9 2 Potential N O damage with the cell cytoplasm 11 3 Glutathione detoxification pathway — normal or initial phase 13 4 Potential exogenous gNO cytotoxic pathway — initial phase 14 5 Potential exogenous gNO cytotoxic pathway — final phase 15 6 Mycothiol structure 18 7 Mycothiol biosynthesis pathway 19 8 Cell exposure device 29 9 Precision gas manifold for mixing 31 10 Determining reproducibility of gNO delivery system 43 a) gNO concentration b) N 0 2 concentration c) 0 2 partial pressure d) Temperature 11 N O gas delivery rate in various culture mediums 45 12 Variation of the pH levels in saline 47 13 Bacteial cell viability inside the gNO chamber 48 a) E. coli b) S. aureus c) P. aeruginosa 14 Viability of human fibroblast culture incubated in the gNO chamber 51 a) Fibroblast cell proliferation b) Fibroblast cell attachment c) Fibroblast morphology 15 P. aeruginosa dosage curve on solid media 54 16 S. aureus dosage curve on solid media 55 17 S. aureus time exposure to 160 ppm gNO 56 ix 18 Survival curves for microorganisms exposed to 200 ppm gNO 58-62 19 Survival curve analysis 63 20 Survival curves after intermittent dosing 70-71 21 Survival curves for N O z exposure 72-73 22 Wild Type vs Mycothiol deficient survival curve 75 23 Comparison of survival curves for strains of M. smegmatis 76 24 Mycothiol assay HPLC chromatograph 78 25 gNO resulted in a time-dependent decrease in Mycothiol 78 26 Two potential cellular MSH detoxification pathways for N O 98 X LIST O F ABBREVIATIONS Abbreviation Definition ATCC American Type Culture Collection Cfu Colony forming units CINRG Clinical inhaled nitric oxide research group c o 2 Carbon dioxide D M E M Dulbecco's modified Eagle's medium EDRF Endothelium-derived relaxing factor E D T A Etnylenediaminetetraacetic acid E M Electron microscopy FBS Fetal bovine serum GISA Glycopeptide intermediate S. aureus gNO Gaseous nitric oxide, as a potential drug GS F D H Glutathione-dependent formaldehyde dehydrogenase GSH Reduced glutathione GSNO S-nitrosoglutathione HEP A filter High Efficiency Particulate Accumulator filter H N 0 3 Nitric acid H O N O Nitrous acid HPLC High-pressure liquid chromatography ICU Intensive care unit L D Lethal dose L - N M M A NG-monomethyl-L-arginine L P M Litres per minute LPS Lipopolysaccharide LP, Latent period of time mBBr Monobromonimane MB 7H9 Middlebrook 7H9 Mca Mycothiol S-conjugate amidase XI MetHb Methemoglobin MRSA Methicillin Resistant Staphylococcus aureus MSH Mycothiol MSSM Mycothiol disulfide N A D P H Nicotinamide adenine dinucleotide phosphate NINOS Neonatal inhaled nitric oxide study N 2 0 2 Nitrous oxide N 2 0 3 Dinitrogen trioxide NOS Niric Oxide Synthase N O Nitric oxide N O z Nitrogen dioxide N02~ Nitrite N 0 3 or eNOS An isoform of NOS endothelial type 3 N03" Nitrate NOS1 or nNOS An isoform of NOS enzyme or type 1 NOS2 or iNOS An isoform of NOS enzyme or type 2 N O x Oxides of nitrogen NP Nosocomial pneumonia 0 2 Elemental oxygen O A D C Oleic acid-albumin-dextrose-catalase ONOO" Peroxynitrite OSHA Occupational Safety and Health Adrrrinistration Ppm Parts per rnillion R H Relative humidity RNIs Reactive nitrogen intermediates ROIs Reactive oxygen intermediates RPM Revolutions per minute SNO S-niliosothiol TSA Tryptic soy agar xi i M l Microlitre VRSA Vancomycin- resistant S. aureus x i i i ACKNOWLEDGMENTS In 1991, when nitric oxide was only known as an infamous deadly gas, I had a poignant nocturnal epiphany and thus my nitric oxide journey began No scientific endeavor can be accomplished without the assistance and support of many people. As such, I wish to thank my supervisor Dr. Jeremy Road for believing in me when I first came to him with the idea for a thesis in nitric oxide back in 1991 and then having the patience to watch as I struggled through the process. I also wish to thank Dr. Norman Wong for his extreme patience by allowing me to have an extension when I had to start all over again in 1999. I also wish to thank my supervisory committee Drs. Yossi Av-Gay, Diane Roscoe and Grant Stiver for their constructive input and instruction. A special thanks to Dr. Yossi Av-Gay for guiding me at a critical time and for allowing me to call his laboratory my home for two years. In Dr. Av-Gay's laboratory I would like to thank Dr. Mamta Rawat for her insights and mycobacteria colonies and laboratory assistance from Ray Chow, Mary Ko, Dr. Neora Pick, Negra Nazemi and my friend and colleague Bevin McMullin. This project would not have been possible without collaboration from the University of Alberta under the direction of Drs. Aziz Ghahary, Nick Nation and David Neil. I would like to thank Dr. Richard Long from the University of Alberta who has inspired me to continue to search for answers related to the treatment of tuberculosis with gaseous nitric oxide. I also wish to thank all of my friends at PulmoNOx Medical Inc. with special thanks to Abdi Ghaffari, Al i Ardakani, Robert E. Lee, Rob Rolfson and Mark Rirnkus who helped direcdy with this project. I wish to extend my appreciation to Doug Hole and his father J.F. Hole for financial assistance that has made it possible for me to pursue my nitric oxide dream. I want to express my thanks to my very dear colleague Bruce Murray for always being at my side through this journey and for Alex Stenzler who has joined this crazy nitric oxide journey with us. Thanks to my family and mother who have always believed in me. Finally, I wish to thank Minna, my eternal wife and friend for her patience, faith and unwavering love. I would like to dedicate this thesis to my late father, Victor B. Miller. x i v 1.0 INTRODUCTION TO T H E ANTIBACTERIAL ROLE OF NITRIC OXIDE 1.1 NITRIC OXIDE Nitric oxide (NO) is a small, unstable, colorless, highly diffusible gas with a density about the same as air (1.04). Composed of a single atom of oxygen combined with nitrogen, as recently as two decades ago, N O was recognized only as either an air polluting toxic molecule found in cigarette smoke and smog or as a noxious gas causing Silo Filler's disease . Ironically, in the late 1980's, N O was discovered to be the smallest, lightest, and the first gas molecule known to act as a biological messenger in mammals, which eventually lead to the Nobel prize in medicine in 1998 (Raju, 2000). Following this discovery, the number of scientific studies on N O soared from a few hundred to tens of thousands by the late 1990's (Culotta and Koshland 1992, Web Search on Medline: 60,000 articles), and today there is an entire periodical dedicated to this molecule (Nitric Oxide — Biology and Chemisty, © Elsevier). N O is now recognized for the vital regulatory and physiological functions it fulfills in every major system of the human body (Raju, 2000), including the vasodilation of smooth muscle (Papapetropoulos et ai, 1997) and blood pressure regulation (Kelm, 2003), neurotransmission (Yun et ai, 1996), inhibition of platelet adhesion (Riddell and Owen, 1999), wound healing (Witte and Barbul, 2002), and nonspecific immune response to infection (Sasaki et ai, 1998). As evidence of its importance, a number of disorders have been associated with altered levels of N O or abherent expression of nitric oxide synthases (NOS) (Bruch-Gerharz et al, 1998). The role of N O in the immune response has led to to the hypothesis that this molecule may have antibacterial properties. This thesis explores and examines the potential use of gNO for antibacterial therapy, specifically for bacteria that are involved in respiratory tract and wound infections. 1 1.2 C H E M I C A L R E A C T I V I T Y A N D T O X I C I T Y O F N I T R O G E N SPECIES N O is a highly reactive gas. In the presence of oxygen, N O gas combines with molecular oxygen to form a red-brown gas called nitrogen dioxide (NOJ. When exposed to NO, N 0 2 reacts readily to produce dinitrogen trioxide (N 20 3), which is soluble in water, forming either nitrous (HONO) or nitric acid (HNO s) (Falke et al, 1991). N 0 2 is a highly reactive gas and is a toxic irritant to the human lung, with concentrations of 2-5 parts per million (ppm) for a 4-hour period causing mild inflammation (Austin, 1967; Clutton-Brock, 1967; Greenbaum et ai, 1967; Devlin et al, 1990; Sackheim and Lehman, 1990; Gaston et al, 1992). In animal models, exposure to high levels of mixed oxides of nitrogen (NOx) greater than 500 ppm causes considerable immediate damage to the alveolar epithelium, followed by a six-hour latent period that may culminate in the development of pulmonary edema (Brown et al, 1983). A review of the risk of human exposure to N O z in the form of environmental pollutants from combustion engines warns of toxic effects through increased susceptibility to infection and increased airway deficits (Samet and Utell, 1990). Some of these findings are consistent with the clinical symptoms and postmortem examination of a patient death in Bristol, England in 1966, which was initially attributed to the presence of a contaminating gas, nitric oxide, delivered along with nitrous oxide (NzO) anesthesia (Clutton-Brock, 1967). The reaction of N O with oxygen to form poisonous compounds is dependent on the concentration of oxygen and the square of the concentration of N O (Falke et al, 1991). Conversion of N O to N 0 2 in the presence of oxygen is represented by the following equation: 2NO + 02^^2N02 Eq (1) The rate of N O z formation is: Eq(2) or Eq(3) Integrating Eq (3) gives: 2 - { [NO\2d[NO}= k3 J [02\lt Eq(4) At high 0 2 concentrations relative to the concentration of N O , the 0 2 concentration will be essentially constant. Therefore, Eq (4) becomes: J [NO\2 d[NO] = k3 [02) | dt or [NO] [NO\ k,[02} Eq(5) In order to estimate the time for conversion of N O to N 0 2 we know that the rate constant k 3 is found to have values in the range of: 2 x 10~38 to 27.8 x 10~38 cm6/molecule2 sec Since there are 6.02 x 1023 molecules / mole and each mole occupies 22.4 liters, we have 2.6875 x 1023 molecules per cubic centimeter. Assuming a rate constant of: 2 x 10~38 cm6/molecule2 sec, the time to convert 50% of 160 ppm N O in 21% oxygen can be obtained from Eq (5) as follows: 1 1 = 2 x l 0 " 3 8 x.21 x2.6875xlO 1 9 xT 80 x 10"° x 2.6875 x 1019 160 x 10"6 x 2.6875 x 1019 Solving for T: T = ~ — = 2060.3 sec = 34.3 min 2x l0" 3 8 x.21 x2.6875xlO 1 9 x 160xlO"6 x2.6875xlO 1 9 If k3 is 27.8 x 10 3 8 cmf>/molecule2 sec, T becomes 148.22 sec or 2.47 minutes (Beckman, 1996; Francoe et al., 1998). Also, the 50% conversion times for 200 ppm N O in 21% oxygen are 1648.3 sec or 27.5 min and 118.6 sec or 1.98 min corresponding to reaction rates of 2 x 10"38 3 cm6/molecule2 sec and 27.8 x 10 3 8 cm6/molecule2 sec respectively. Practically speaking, this means that a 50% conversion of N O to N 0 2 at 10 ppm concentration in room air (20°C) takes 7 hours. At 100 ppm it would take 40 minutes, 160 ppm 34 minutes and at 200 ppm 27 minutes (Table 1). This may explain why no adverse effects have been observed in recent short-term exposure of laboratory animals to concentrations as high as 200 ppm N O (Stavert and Lehnert, 1990; Frostell et aL, 1991; FDA-FOI, 2000). NO Concentration(ppm) Oxygen Concentration(%) NO Half LM e (sec) k3=2.0x1038 k3=27.8x1038 160 21 2060.32 148.22 200 21 1648.25 118.58 160 100 432.67 31.13 200 100 346.13 24.90 Table 1. Calculation of nitric oxide conversion to nitrogen dioxide. Values of 200 and 160 ppm in the presense of 21% and 100% oxygen. In the human pulmonary hypertensive model, similarly encouraging findings have been reported while breatiiing an air/NO (40 ppm) mixture. (Blomqvist et aL, 1991) The United States Occupational Safety and Health Administration (OSHA) has established the time-weighted-average N O exposure value at 25 ppm (MMWR, 1998). In the mid nineties, despite these encouraging preliminary findings, a number of important toxicological studies remained to be done prior to general approval and acceptance of N O as a a selective pulmonary vasodilator (gNO). N O is active in the body for less than 15 seconds as once it interfaces with a red-blood cell, it forms methemoglobin (MetHb). Methemoglobin bonds with oxygen binding sites on the hemoglobin; high levels of MetHb (>5%) can interfere with oxygen delivery. The reported half-life of MetHb is approximately one hour. As a result, while low doses of inhaled nitric oxide are safely used clinically (typically 1-40 ppm), there is a concern that using inhaled concentrations above 80 ppm may be unsafe due to the formation MetHb (Wang and Deen, 2003). 4 By itself, gNO in low concentrations may not be as toxic as other oxides of nitrogen, as previously thought. Indeed, at low concentrations, gNO reacts very slowly with oxygen to form these toxic products (Falke et al., 1991). This observation provides encouraging data for exploring the use of gNO safely as an antibacterial therapeutic, assuming tissue susceptibility is less than bacterial to gNO. 5 1.3 GASEOUS NITRIC O X I D E IN A P H Y S I O L O G I C A L C O N T E X T The identification of N O as an important biological messenger molecule originated in vascular smooth-muscle research during the mid seventies. The walls of arteries are lined with endothelial cells lying adjacent to smooth-muscle cells (Williams et al, 1989), and in 1975, Moncada and co-workers discovered these endothelial cells to be necessary for vascular smooth-muscle relaxation (Moncada et al, 1976). In 1980, Furchgott and Zawadzki found that the cellular depolarizing action of acetylcholine stimulated the release of a powerful smooth-muscle relaxing agent in the endothelial cell. They named this substance endothelium-derived relaxing factor (EDRF) (Furchgott and Zawadzki, 1980). They further concluded that the relaxation of arterial smooth muscle is dependent upon an intact endothelium to produce EDRF (Furchgott and Zawadzki, 1980). The endothelium also produces potent vasoconstrictors like endothelin (Yanagisawa et al, 1988). Dinh-Xuan and Higenbottam have suggested that vascular muscle tone is dynamic and represents a balance between "vasorelaxing" and "vasoconstacting" influences (Dinh-Xuan and Higenbottam, 1989). A wide range of endothelium-dependent drugs have since been used in the clinical setting as vasodilators. However, only recendy has their vasodilating action been linked to EDRF. Throughout the mid-1980s, N O was reported to either be EDRF or a major component of EDRF (Griffith et al, 1984; Ignarro et al, 1987; Palmer et al, 1987). Bioassay studies demonstrated that EDRF and N O were both highly diffusible gases with very short half-lives in aqueous buffered solutions (Griffith et al, 1984; Cocks et al, 1985; Furchgott, 1984), and two research groups simultaneously proposed that EDRF and N O gas were the same (Ignarro et al, 1987; Furchgott, 1988). It is still debated whether EDRF and N O are identical active species, or if EDRF is a NO-producing compound such as nitiosothiol (Gaston et al, 1994). Gaseous N O is now recognized as a major vasodilator originating from the endothelium (Ignarro etal, 1987; Furchgott, 1984; Furchgott, 1988; Cremona etal, 1991; Vane etal, 1990), and the premise that inhaled gNO can function as a selective pulmonary vasodilator is based upon this fact. 6 It was not until 1992 that N O could be measured in biological tissues using a porphyrinic-based microsensor (Malinski and Taha, 1992). Once N O could be measured, N O was shown to be present in a wide variety of cells throughout the body, including endothelial cells (Papapetropoulos et al, 1997), macrophages (MacMicking et al, 1997), neutrophils (Yui et al, 1991), fibroblasts (Nathan, 1992), and keratinocytes (Heck et al, 1992), as well as tissues including the brain, arteries, veins, immune system, liver, pancreas, uterus, peripheral nerves, and lungs (Moncada et al, 1991). Under normal physiological conditions, production of N O occurs continuously during oxygen-based metabolism. It is produced from L-arginine by three different isoforms of nitric oxide syntases (NOS). The NOS catalyzes a reaction of L-arginine with 2 0 2 and nicotinamide adenine dinucleotide phosphate (NADPH) to form L-citrulline, NADP+, 2 H 2 0 and N O . Neuronal nitric oxide syntase (nNOS or NOS1) and endothelial nitric oxide syntase (eNOS or NOS3) are constitutive calcium-calmodulin-dependent enzymes, producing N O in the nanomolar range. Inducible NOS (NOS2) is mostiy synthesized in response to inflammatory mediators (bacteria, lipopolysaccharides, cytokines) and releases N O in micromolar concentrations (Wang and Deen, 2003). Although N O concentrations vary from tissue to tissue - depending on expression of NOS, strength and duration of stimulus, as well as cell type - studies show that under normal physiological conditions this value lies between 1 — 4000 nM (Fang, 1999). As examples, N O concentrations in the range of 0.9 - 2.7 \iM are reported in ventricular myocardium (Pinsky et al, 1997), 0.27 - 4.3 piM in human skin (Clough, 1999), 1.4 — 7.8 [iM witxiin the lumen of gastrointestinal tract under normal conditions (Lijima and Henry, 2002) and peak at 50 p:M in aorta walls (Brovkovych et al, 1997). The balance of available N O within the host cell is critical for maintaining physiologic homeostasis. Pathophysiology of a number of disorders have been associated with altered level of N O or expression of its enzymes, NOS (Bruch-Gerharz et al, 1998). Too little available N O and functional processes are disrupted, as observed in the pulmonary system when lack of N O causes vasoconstriction, resulting in pulmonary hypertension (Frostell et al, 1991). On the other hand, too much N O released within the body, as occurs during septic shock, can cause profound systemic hypotension (Symeonides et al, 1999). 7 1.4 N I T R I C O X I D E AS A S E C O N D M E S S E N G E R In a biological system, a second messenger is a low-weight diffusible molecule used to relay a signal witliin a cell. Second messengers are typically synthesized by specific enzymatic reactions, usually as a result of an external signal that was received by a transmembrane receptor and pre-processed by other membrane-associated proteins. As an uncharged, relatively hydrophobic, short-lived nonelectrolyte, N O diffuses readily through lipid membranes, enabling it to play an important role as a second messenger in biological systems. N O is uncharged and almost 9 times more soluble in hydrophobic solvents than in aqueous solution (Shaw and Vosper, 1977), and as the smallest known signaling molecule with a radius of about 3-4 Angstroms, it can pass easily though biological membranes. Figure 1 depicts the relative size of the nitric oxide molecule (3-5 Angstroms) to the cell wall thickness (20 nanometers). In order to help visualize the comparative size of a nitric oxide molecule passing through the gaps in a phospholipid bilayer in a cell wall, it would be analogous to a car driving through a five lane tunnel. Thus the N O molecule can easily diffuse across cell membranes. N O reacts readily with oxygen species (oxygen, superoxide, etc.) and transition metals (iron, copper, etc.) and as a consequence, possesses a finite and short half-life in the range of 5 — 15 seconds (Lancaster, 1997). This short half-life makes N O ideal as a second messenger, 8 Figure 1. Relative size of N O molecule to the bacterial cell wall. The nitric oxide molecule is 3-4 Angstroms in diameter (depicted as blue bubbles). The cell wall is approximately 10 nanometers thick. Nitric oxide molecules diffuse passively through the cell wall unhindered because of their small size and lipophilic nature. indeed, N O is now known to be involved in at least 30 important messenger mechanisms in the human body (Gow et al, 1999; Raju, 2000). It has been reported that N O plays a key role as a messenger molecule within many biological systems (Moncada et al, 1991). With its short half-life, N O can potentially diffuse to a radius of 150 - 300 um in human tissue. This means that N O delivered exogenously has a limited volume of influence and potentially little generalized systemic effect. 1.5 T H E R O L E O F NO IN HOST DEFENSE Once it was established that N O was a prevalent messenger molecule throughout the body, the role of N O in the host defense system was evaluated. Evidence that nitric oxide may play a role in the host defense mechanism was provided by the fact that the inhibition of NOS by specific inhibitors, exacerbated infection. NOS inhibitors are relatively selective, nontoxic L-arginine analogs which inhibit the NOS enzymes and can be administered to cultured cells, experimental animals, and people. In vitro studies of phagocytotic cells and a variety of 9 microbial targets have demonstrated that cytokine-inducible microbiostatic or microbial activity is L-arginine dependent and inhibitable by competitive NOS inhibitors such as N G -monomethyl-L-arginine (L-NMMA) (Fang, 1997). L - N M M A is a relatively non-selective inhibitor of all NOS isoforms. Malawista demonstrated a role of reactive nitrogen intermediates (RNIs) in killing of staphylococci by showing that killing was inhibited by L-N M M A (Malawista etal. 1992). NOS inhibitors have been shown to worsen the course of diseases caused by an impressive array of phyla—viruses, bacteria, fungi, protozoa, and helminthes (MacMicking et al, 1997; Fang, 1997). In both animal models and humans, NOS2 or iNOS can be up-regulated by cytokines and bacterial products like lipopolysaccaride and lipoteichoic acid which upregulate the body's response to infection (Hibbs et ai, 1988; MacMicking et al, 1997). Indeed, it has been demonstrated that NOS is upregulated and N O is produced at the site of infection (Stenger et al, 1996; Nicholson et ai, 1996). Systemic nitrite and nitrate (end products of N O metabolism) levels are often elevated during infection (Ochoa et al, 1991). It has been reported that NOS expression is associated with good clinical outcomes in individuals infected with malaria (Anstay et al, 1996). Studies point toward the fact that bacteria such as staphylococci seem to be susceptible to NO-mediated killing prior to or following phagocytosis of organisms by the immune system (Kharitonov et al., 1994a). It has been shown that during the host's response to systemic infections, N O production by the endothelium and macrophage increases dramatically (Cotran 1999; Bogdan et al, 2000). Billiar and Hasan have used a rodent model to confirm that cytokines induce NOS along with the up-regulation of macrophage cytotoxicity against pathogens such as protozoa, viruses, fungi, and bacteria (Billiar etal., 1990; Hansan et al., 1993). Arginine, an N O precursor, can stimulate antibacterial components of the immune system (Nirgiotis et aL, 1991). Arginine promotes the synthesis of N O that is believed to improve resistance to infection (Gianotti et al, 1993). Arginine supplementation in a diet after burn injury has been shown to decrease mortality rate by improving host immunity and bacteriocidal mechanisms via the arginine-nitric oxide pathway. Arginine supplementation also produced a major reduction in the incidence of microbial translocation (Gennari and 10 Alexander, 1997; Horton et al, 1998). Taken together this information strongly supports a role for N O in the host defense mechanism. Although initially controversial, it is now well established that human macrophages, the large phagocytic cells in the immune system, induce iNOS in order to generate N O as a primary mechanism of killing foreign bacteria (Hibbs et al, 1992; MacMicking et al, 1997). In vitro studies have shown that oxides of nitrogen inhibit growth or kill a number of fungi, parasites, helminths, protozoa, yeast, mycobacteria and bacteria (Moncada et al, 1991; De Groote and Fang, 1995; Liaudet et al, 2000; Bogdan et al, 2000). N O may even play a role in killing tumor cells and in halting viral replication (Karupiah et al, 1993). N O may inflict lethal nitrosative damage directly or through reactive nitrogen intermediates (RNIs) on crucial cellular enzymes. RNIs can nitrosylate cysteine sulfhydyls and heme prosthetic groups, disrupt iron-sulfur clusters and inactivate tyrosyl radicals (Stamler, 1994). N O can also initiate oxidative damage caused by reactive oxygen intermediates (ROIs) when N O reacts with superoxide or peroxide to form peroxynitrite (St. John, et al, 2001; Pacelli, et al, 1995). Peroxynitrite can nitrate tyrosine residues (Beckman and Koppenol, 1996), or oxidize cysteines (Bryk, et al, 2000) and methionines (Pryor and Squadrito, 1995; Vogt, 1995), to cause oxidative D N A damage (St. John etal, 2001; Park and Imlay, 2003) Figure.2. NO cell wall 1 r N itrosation R N I / N O nitrosylate cysteine sulfhydyls nitrosylate heme prosthetic groups disrupt iron-sulfur clusters inactivate tyrosyl radicals e + N O r 0 N 0 0 ' / peroxynitrite nitrate tyrosine residues oxidize cysteines oxidize methionines Figure 2. Potential NO damage within the cell cytoplasm. Potential interactions between NO resulting in nitrosative action as reactive nitrogen intermediates (RNIs) or oxidative action by autooxidating with super oxide to create peroxynitrite (ONOO). Potential reactive targets are listed 11 Bacterial death is thought to be attributed to N O crossing the bacterial cell membrane, binding with iron containing enzymes associate with the cell's mitochondria and then mterrupting cellular respiration causing apoptosis (Tamir et al, 1993). However, the exact mechanism of how N O exerts this cytocidal effect is unknown. The literature describes four primary routes of N O reactivity within the cell (Gow et al, 1999). These involve reactions with reduced thiols, transition metals (especially iron complexes), superoxides and auto-oxidation to higher oxides of nitrogen, like peroxynitrite. WitJiin the host, under normal physiological conditions, homeostaric N O levels are maintained, tolerated and N O even participates as a key messenger in many biological activities. (Moncada et ai, 1991; De Groote et al., 1995). The host cells are not immune to the high levels of N O released, and they have evolved multiple defenses against nitrosative injury whereas most bacteria have not. The human host cell protective strategies are discussed below. Within the cell, N O is primarily attracted to and most likely to react preferentially with thiols to form S-nitrosothiol (SNO). SNO is thought to be the active form of N O in the cytosol. SNO is inactivated by a low molecular weight thiol called glutathione. Glutathione is the predominant low-molecular-weight thiol that is present in eukaryotes and Gram-negative bacteria (Meister, 1988). GSH mediates many diverse activities, some of which are to provide the cell with reducing properties, function in catalysis, metabolism, transport, and synthesis of proteins and nucleic acids. GSH is essential for mitochondrial function and serves as a storage and transport form of cysteine (Martensson et al., 1990). The reduced form of glutathione, referred to as GSH, plays a critical role in the detoxification of N O and other potentially reactive oxygen species like hydrogen peroxide and peroxynitrite (Anderson and Meister, 1983; reviewed by Dolphin et al, 1989). GSH reacts readily with N O because of its predisposition to react with the GSH to form S-nittosoglutathione (GSNO). GSNO is the major N O scavenger within biological systems and prevents N O building to levels that cause unwanted nitrosative reactions. Most N O is bound in the GSNO reservoir and N O and/or SNO levels are regulated by glutathione dependent formaldehyde dehydrogenase or GS-FDH. (Liu et al, 2001). The GSH dependent detoxification pathway uses enzymes called glutathion-S-tranferases (GST). GST's conjugate GSH to xenobiotics through the sulphur group on the 12 GSH to form an S-conjugate. These S-conjugates are then eliminated via the host excretion system as mercapturic acid derivatives. Based on the literature described above, we suggest that the destructive pathway, which is non-specific to both bacteria and host cells, for gNO would occur as follows. First all available GSH would react with the influx of gNO to protect the cell by creating the innate GSNO. GSH is a key electron donor for reducing proteins and free radicals (Dolphin et ai, 1989). This defensive scavenging process would continue until all GSH was consumed (Figure 3). Figure 3. Glutathione Detoxification Pathway - normal or initial phase Reduced Glutathione ( G S H ) ; S-nitrosothiol (SNO) ; S-nitrosoglutathione ( G S N O ) ; Glutathione-dependent formaldehyde dehydrogenase ( G S - F D H ) ; Glutathione (GSH) is replaced by Mycoth io l ( M S H ) i n some bacteria. The G S H pool protects the bacteria from the attack by N O and H 20 2. N O and H 2 O z are two endogenous mechanisms of pathogen control, N O being released by macrophages and neutrophils. However, once GSH was depleted, the cell would be highly vulnerable. During the initial phase, high concentrations of gNO would react with non-heme irons in transition metals 13 found in many key enzymes. One such enzyme is aconitase and is central to the mitochondrial respiration cycle. These enzymes are critical for the mitochondrial electron transport chain for cellular respiration (Karupiah and Harris, 1995). The bonding by N O with the aconitase enzyme would result in nearly complete cessation of oxygen consumption by the bacteria and would either result immediately in cell death or a temporary transition to anaerobic metabolism (Figure 4). Oxygen now abundantly available, would transition into reactive oxygen species like superoxide and react with available gNO, to create deadly reactive nitrogen intermediates like peroxynitrite. Without the GSH available, hydrogen peroxide is also left unchecked to target the D N A of the bacteria. With gNO binding the iron in the mitochondria there would be a mobilization of metal ions associated with the mitochondria that have multiple destructive pathways to destroy D N A by dearnination (Figure 5). Mitochondrial Death H 2 0 2 NO O , NO NO [SNO] [SNO] Figure 4. Potential exogenous g N O cytotoxic pathway — Initial phase Exogenous N O overwhelms the G S H pool and then locks up the iron based enzymes causing 0 2 consumption cessation and mitochondrial death. It also eliminates the bacterial protection from H 2 0 2 and metal ions. In 1991, Wink et al. demonstrated the ability of N O to deaminate DNA. Bacterial mutants deficient in D N A repair mechanism exhibit an increased sensitivity to NO-donor compounds. De Groote et al. reported using D N A repair-deficient mutant mice and showed 14 that D N A might be an important target of N O host-pathogen interaction (De Groote et aL, 1995). Thus it would only be a matter of time when irreversible cytotoxicity is achieved (Shiloh et aL, 1999). One might extrapolate that the viability of a cell is direcdy related to how much N O it is exposed to and the availability of GSH to detoxify it. In summary, the overall consumption of gNO is first with primary reactions of normal physiology and then secondary reactions called "nitrosative stress" that may be hazardous to the cell integrity. Each of these pathways induces enzymatic protein modifications resulting in altered function. Consequently, depending on conditions and cell type, this will determine whether gNO reactivity is cytoprotective or cytotoxic. Figure 5. Potential exogenous gNO cytotoxic pathway - Final Phase. Free oxygen and hydrogen peroxide molecules produce reactive oxygen species that damage bacterial D N A by dearnination. Iron ions from metal mobilization also contribute to D N A damage. Bacteria have more non-heme iron proteins than mammalian cells, offering more targets for N O . The closer proximity of aconitase (therefore Fe-ions) to D N A in bacteria may explain the difference in response to oxidative stress between mammalian and bacterial cells and affords a unique means to combat pathogens with minimal damage to host cells. 15 N O is an endogenous molecule produced in high levels and delivered as a defensive mechanism by host defense cells, yet these same host defense cells are required to recognize differences between host cell and the pathogenic cells so that the same molecule kills one, the bacteria, but not the other, the host cell. Normal mammalian cells have fewer non-heme iron enzymes than bacteria and therefore present fewer targets for N O attachment. More importandy, since eukaryotic cells are segregated by a membrane and their D N A is bound to histones while bacterial chromosomes are associate with basic proteins, the aconitase enzymes in bacteria are much closer spatially to their D N A than in normal mammalian cells. Host cell lines may also have higher levels of GSH than the pathogenic bacterial cells. The combination of these differences may account for the ability of N O to be cytotoxic to pathogens while not harming normal host cells. Mycobacterium tuberculosis infects about 1.75 billion people with 8 million new cases and 3 million deaths annually (WHO, 2000). Mycobacteria primarily reside in the macrophage, the core of the host's antibacterial defense arsenal. Macrophages erigulf and phagocytose bacteria. Activation of the macrophage with the cytokine interferon — gamma makes the milieu even harsher with acidification to lower the pH, fusing the phagosome with lysosomes and stimulating the production of NO. Mycobacteria are able to inhibit the phagosome-lysosome fusion, ameliorate acidification and inhibit maturation (Sturgill-Koszycki et a l , 1994). Witfiin the activated phagosome, iNOS or NOS2 is upregulated to produce high levels of NO. It has been hypothesized that the mycobacteria that survive N O damage may be able to switch from active cell division to a state of nonreplicating persistence that helps to explain the latency in tuberculosis disease (Schnappinger et a l , 2003; Voskuil et a l , 2003). N O is a potent antibacterial micromolecule that is produced by mammalian cells that can kill tuberculosis in vitro with a concentration potency exceeding that of most antituberculosis drugs (Long et a l , 1999; Nathan and Ehrt, 2004). Despite this inhospitable environment that typically results in bacterial cell death, some mycobacteria are able to replicate and persist. Understanding how mycobacteria are able to cope in such a hostile environment has remained elusive. Studying the mechanisms by which mycobacteria avoid being completely eliminated widiin the harsh environment of the macrophage may explain their unique growth characteristics within the host and provide new drug targets for the prevention or treatment of tuberculosis. 16 There was a time when it was thought that the only way that mycobacteria could protect themselves from oxidative and nitrosative damage was to reduce the two primary precursors of ROIs and RNIs, these being the amount of superoxide and NO. The main pathways described for bacteria to reduce the cystolic levels of superoxide was through the action of superoxide dismutase (Piddington et a l , 2001) and to decrease N O levels, was through oxididation by flavohemoglobins into nitrates (N03~) (Poole et a l , 2000). However, recent research has provided some insight into several new mechanisms that mycobacteria utilize to defend against oxidative and nitrosative stressors. One such alternative mechanism relies on bacterial peroxiredoxins that can catabolize peroxynitrite fast enough to protect cellular targets (Bryk, et a l , 2000). The genome sequencing project of M. tuberculosis found that it encodes for a number of thioredoxins and thioredoxin reductases (Heym et a/.,1997). Peroxiredoxin AhpC and AhpD proteins from M. tuberculosis have been expressed, purified and characterized and both are peroxiredoxin alkyl hydroperoxide reductases (Hillas et a l , 2000). Studies show that these members of the peroxiredoxin family of non-heme peroxidases, protect the cell against oxidative and nitrosative injury (Storz, et a l , 1989; Chen, et a l , 1998). Two more proteins Lpd and SucB have been shown to be central to this pathway. The AhpC system consists of four proteins and relies on the reduction of AhpC by the thiol transferase AhpD (Bryk, et a l , 2002). The endogenous electron donor for the catalytic activity of this system to reduce proteins and scavenge free radicals is probably a low molecular-weight thiol called mycothiol (MSH). The second novel protection mechanism against oxidative and nitrosative stressors involves the newly discovered proteasomes. Proteasomes are organelles within the cell that are responsible for degrading proteins that have been irreversibly oxidized (Davies, 2001). Proteasomes are essential in eukaryotes and are well characterized (Kisselev and Goldberg, 2001). They are composed of a regulatory particle with a ubiquitin site that targets proteins for destruction on a ring—like structure called the core. Proteins destined for destruction are bound, unfolded, cleaved into peptides about 8 amino acids long and disgorged into the cytosol. Little is known about prokaryote proteasomes and they have no known function in bacteria. The only eubacteria known to contain proteasomes are actinomycetes such as mycobacteria. Recently it has been shown that the mycobacterial proteasome is required for 17 resistance to N O (Grune, et al, 1998). Darwin and colleagues screened 1,100 M. tuberculosis transposon mutants for hypersensitivity to acidified nitrite (Darwin et aL, 2003). Of 12 mutants that had increased sensitivity to RNIs, five had insertions in two genes encoding putative components of the proteasome. A series of in vitro studies using wild-type, the proteasome mutants and complementation of the mutant strains showed that the proteasome was essential and nonredundant for protection of M. tuberculosis and Gram positive bacteria to oxidative and nitrosative stress. They concluded that one of the functions of the mycobacterial proteasome is to protect it against oxidative and nitrosative stress. It is speculated that the mechanism of protection involves the degradation of proteins that are irreversibly oxidized, nitrated or nitrosated. Inhibition of the proteasome might be useful to sensitize mycobacteria to the immune system, either alone or in combination with other therapies. Mycobacteria and Streptomycetes have the highest reported levels of MSH (Newton et al, 1996). The chemical structure of MSH is L-cysteine, D-glucosamine, and lD-myo-inositol and is shown in Figure. 6 (Sakuda, et al, 1994; Spies and Steenkamp, 1994; Newton et al, 1995). Analagous to GSH, it is proposed that MSH functions as a Figure 6. Mycothiol (MSH) structure, common name and abbreviations for mycothiol; 1D-/n/oinosityl 2- (N-acetylcysteinyl)amido- 2 -deoxy- a - D - glucopyranoside. storage reservoir for the amino acid cysteine (Newton et aL, 1995), maintains a homeostatic redox environment by reducing oxidized molecules and free radicals that are a natural byproduct of aerobic metabolism and reacts with toxic substances, functioning as a detoxification agent (Newton et al, 2000a, Rawat et al, 2002). Deace ty l a se L igase Acety l t ransferase - A' Mycothiol G l c N A c - l n s — G l c N - l n s — ^ ^ » • C y s - G l c N - l n s — A c C y s - G l c N - l n s A c O H A T P A M P + ppi A c e t y l - C o A C o A C y s mshX mshB mshC mshD 18 Figure 7. Mycothiol biosynthesis pathway. Ac, acetyl-CoA, coenzyme A The pathway for biosynthesis of MSH (Figure 7) has been characterized over a period of five years and is summarized in a review by Newton and Fahey (2002). The characterization of chemical and transposon mutants defective in the MSH biosynthesis pathway has provided insights into the role of MSH in protection against ROIs (Anderberg et a l , 1998; Newton et a l , 2000a; Rawat et a l , 2002; Newton et a l , 2003). For instance, MSH deficient mutants are hypersensitive to alkylating agents, free radicals and several antibiotics (Rawat et a l , 2002). Studies have shown that when the M. smegmatis mutant was blocked in the early step of MSH synthesis encoded by the mshA gene, it could not tolerate even 0.8 mM of hydrogen peroxide while the wild-type strain could grow in the presence of up to 12 mM hydrogen peroxide (Newton et ai, 2003). The importance of this pathway is shown by the fact that a single enzyme called MSH-S-conjugate amidase detoxifies several toxins by producing a mercapturic acid waste derivative from an S-conjugate from MSH and the toxin (Newton et a l , 2000a). The exact mechanism and specificity of the MSH dependent detoxification pathway are still unknown. However, disruption of this MSH dependent detoxification make mycobacteria more vulnerable to oxidative damage caused by free radicals like gNO (Rawat et ai, 2004, in press J.Bact). gNO is a potent free radical (Gaston et ai, 1994). 1.6 A N T I B A C T E R I A L PROPERTIES O F N O Over fifty years ago, oxides of nitrogen where shown to prevent spoilage of meats (Tarr, 1941). The bacteriostatic effect was attributed to N O being released from sodium nitrite in an acidic environment (Shank et al., 1962). An extensive literature survey was conducted in order to examine the in vitro role of N O or oxides of nitrogen as an antibacterial (Appendix 1). These studies reported in vitro methods for exposing cellular systems to N O include the release of N O from NO-donor compounds, stimulation of N O producing enzymes (nitric oxide synthases), and the use of NO-saturated solutions (Maragos et a l , 1991; Ashok et a l , 1995; Thomas et a l , 2002). In most cases the 19 delivery of N O is mdirect, without using a pure gas form, which may introduce extraneous variables and not represent the true effect of exogenous gaseous N O (gNO) within the testing model. The majority of research in the literature investigating the role of gNO on biological systems has utilized N O donors (Kavdia et ai, 1998). However, experimental designs that use N O -donor compounds, the solution pH, light, and aqueous medium, can all affect the rate of N O production (Feelisch and Stamler, 1996) making the actual levels of N O or even N O species that interact with the microorganisms difficult to determine. With the intracellular stimulation of N O synthase (NOS), other species may form or exist within the cell cytoplasm. In most cases, N O release rate is not constant, difficult to maintain in long exposures, and the toxicity of donor compounds following release of N O are difficult to predict (Feelisch and Stamler, 1996). N O produced by stimulation of NOS enzymes can react intracellularly with other nitrogen species generated or existing within the cytoplasm, such as superoxide, to form other potentially damaging species like peroxynitrite (Kavdia et al, 1998). This effect may considerably reduce the availability or mask the true effects of N O on the cells being studied. In the application of NO-saturated solutions to cell cultures, there is an inability to maintain or deliver consistent levels of N O (Kavdia et al, 1998). These factors all contribute to the variability and perhaps incorrect conclusions regarding the direct effect of gNO as an antibacterial agent. As an alternative to the above-described methods that may have resulted in experimental ambiguities, Kavdia et al. (1998), have indicated that delivering a constant rate of exogenous N O gas could be an ideal experimental methodology. Studies have shown that constant delivery of N O gas above liquid surfaces will lead to a steady-state N O concentration in the solution, even in the presence of other species that are reacting with the nitric oxide Tamir et al, 1993). Work by Tamir's group illustrated that by permeating exogenous N O through a membrane, a constant formation of nitrogen dioxide (NO^ in the presence of oxygen (Oj) can be achieved. These observations supported the reports that followed indicating that an N O gas concentration can achieve a steady state in the treated solution (Kavdia et a l , 1998). Although the system used by Tamir was designed for exposure of bacterial cells to exogenous N O for growth modeling, the system does not support the optimal growth environment 20 required for other cells such as human cell cultures nor does it provide a method for the control arm other than conventional incubation. Further, these systems are cumbersome to set up and have not been validated against conventional incubators. Without a comparison of the methodology to a conventional incubator, it is possible that the actual device itself may in fact be inhibiting microbial growth rather than an effect of gNO. There are two delivery systems that have been described in the literature that more closely address the needed methodology (Hoehn et al, 1998; Long et al, 1999). These devices were used to expose various pathogens such as S. aureus, Staphylococcus epidermidis, E. coli, P. aeruginosa, and Mycobacterium tuberculosis to exogenous N O gas (Hoehn et al, 1998; Long et al, 1999). In the Hoehn experiment, agar plates were placed inside an airtight container (gas pack system) and connected directiy to a N O tank (Hoehn et. al, 1998). A detailed description was not available in the report. Long's device, conceptually closer to the device presented in this study and very well described, used an airtight Plexiglas chamber inside a heated biological safety cabinet. A baffle box mixed 5% C O z in air and N O gas before delivering it to the chamber (Long et. al, 1999). Both of these devices came short of providing an optimal growth environment that takes into account the humidity factor, flow rate, nitrogen dioxide buildup, and suitable oxygen levels. In addition, with primary focus on bacterial growth, these devices do not support favorable test conditions for human cell cultures. Both the Hoehn and Long devices take advantage of pressurized cylinders to drive and control the flow of gas through the system. This arrangement may have led to inconsistent flow rates during long exposure periods, as flows increase as pressure in gas cylinders decrease (even in presence of a regulator). Additionally, in the absence of a constant negative pressure, a reliable and accurate flow rate would be difficult to achieve in the previously described systems. The systems described had large volumes and N O z levels would be difficult to keep consistent for prolonged periods of time. Both research teams measured gases with an analyzer. However, N 0 2 in these devices was measured prior to entering the device rather than inside the device just above the Petri dishes. Thus, it is possible that uncontrolled variables may have caused cell death rather than the gNO. Although their methodologies were good first attempts to look at the direct effects of gNO, these systems could be improved upon to control for more external variables. Finally a system would need to be compared to a conventional incubator in order to ensure that the results would be directly due to gNO. 21 Pneumonia induced in animal models treated with inhaled gNO at 40 ppm and 10 ppm are shown to reduce bacterial load (Webert et ai, 2000; Jean et ai, 2002). They found that N O resulted in a statistically significant reduction in bacterial load as well as significant reductions in myeloperoxidase levels as a marker of inflammation. They concluded that further research was warranted to address the potential clinical use of inhaled N O for pneumonia. These findings suggest that there are worthwhile questions to be answered regarding the use of gNO as an antibacterial agent in the human airways. 1.7 U S E O F NITRIC O X I D E IN A C L I N I C A L S E T T I N G As N O was initially classified as a toxic substance, a lot of ground work was required before N O could be safely studied and used in the medical field as a potential drug. The author participated with the respiratory community to establish safe delivery systems and procedures for gNO. (Millet and Miller, 1992; Miller, 1994; Miller, 2003). Currentiy exogenous gaseous gNO is used clinically as an inhaled selective pulmonary vasodilator to treat persistent pulmonary hypertension of the newborn (PPHN) and to improve oxygenation due to ventilation-perfusion mismatching in adult respiratory distress syndrome (Hurford, 2002). gNO has been approved as a drug to treat P P H N in the USA, based on its role as a significant messenger molecule in the cardiovascular system (Miller, 2003). Two recent trials in hypoxic newborns demonstrated statistically significant reduction in the need for Extra Corporeal Membrane Oxygenation in these eligible patients (NINOS, 1997; Clark et ai, 2000). These two trials were submitted as part of a New Drug Admission submission by INO Therapeutics for FDA approval for sale of inhaled Nitric Oxide Gas (INOmax). That approval was issued in early 2000. No adverse effects were associated with either of these trials. The CINRG trial (one of these two trials) also reported a statistically significant reduction in chronic lung disease in those infants treated with gNO, again supporting its potential as an anti-inflammatory drug. The use of gNO has proven to be safe and effective as an inhaled drug. During inhalation therapy, over the last decade, many thousands of infants have received gNO to reverse P P H N 22 without adverse side effects. During this therapy NO, N 0 2 and the percentage of methemoglobin is monitored to ensure safe delivery. Exploring the use of gNO as an antibacterial agent would require monitoring of these critical parameters. Because gNO is already used within humans, performing research to ascertain its potential for use as an antibacterial in respiratory infections and for infectious wounds is promising from a safety and toxicology point of view. Positive results in this area would give promise, in an era of increasing drug resistant bacteria, for a novel approach to treat diseases such as cystic fibrosis, tuberculosis and non-healing wounds. 1.8 D E F I N I N G T H E P R O B L E M After reviewing the literature, it was apparent that a significant role for N O in inhalational, topical applications and diagnostic use may exist (Gianetti et al, 2002; Weller et ai, 1998; de Arruda-Chaves etal., 2002). In order to study the efficacy and safety of direct application of the N O molecule, a methodology is needed that emulates, as close as possible, the clinical milieu. One of the major obstacles in N O research is the lack of validated and accurate in vitro models to study the direct effects of the exogenous gaseous molecule. To the best of our knowledge, a validated device and methodology for N O in vitro studies does not exist. In order to do further research into the interaction of gNO with both potentially pathogenic bacteria and cultured human cells a new system that would enable the delivery of a constant N O concentration would be required. The system needed would be similar to the device described by Long but would address its inadequacies from an experimental and practical standpoint. Despite the promising reports on antibacterial and immunoregulatory effects of N O , there have been only a few attempts to study the direct bacteriocidal effect of gNO on drug resistant bacteria and human cells. More specifically, no one to date has systematically studied the dose and time effects of gNO on pathogens directly responsible for causing either respiratory tract infections or surface wound infections in the human host. Nor has 23 anyone attempted to propose and test a methodology to safely and effectively deliver gNO once the targeted pathogens have been identified. Further, there is a limited body of knowledge attempting to explain the mechanism of how gNO may act as an antibacterial agent. The following thesis attempts to address some of these issues and expand the knowledge base in this area. 24 2.0 W O R K I N G H Y P O T H E S I S & S P E C I F I C A I M S The introduction provides a background review of gNO chemistry, physiology, biologicial roles, antibacterial properties, and clinical applications. In an era when multi-drug resistant bacteria are on an increase, there is a need to explore novel approaches to antibacterial therapy that are non-organism specific and non-antibiotic based. Experimental evidence indicates that N O has a role in the host defense mechanism, and may have antibacterial characteristics. The goal of this thesis is to explore the feasibility of exogenous or gaseous N O (gNO) as a potential therapeutic antibacterial agent by confirming that gNO is bacteriocidal to bacteria that are involved in respiratory tract and wound infections and to elucidate the mechanism by which it is bacteriocidal. 2.1 H Y P O T H E S I S Gaseous N O is bacteriocidal to bacteria that are involved in diseases of the respiratory tract and wound infections. 2.2 S P E C I F I C A I M S The specific aims of this project are to: 1 . Validate an in vitro system to expose cells to gNO. 2. Determine the lethality of gNO on a variety of bacteria associated with nosocomial acquired respiratory tract and/or dermal wound infections. 3. Determine the mechanism of action by which bacteria attempt to protect themselves from gNO cell death and test this by: I. Demonstrating that Mycobacteria smegmatis are less susceptible to gNO damage because they have an exceptional thiol, mycothiol (MSH), which maintains the redox balance in the cell, and protects the cell from nitrosative and or oxidative stress. 25 II. Demonstrating that M. smegmatis exposed to gNO will utilize its MSH to protect against N O and that MSH levels will be depleted in the process. III. Demonstrating that MSH-deficient M. smegmatis mutants are more susceptible to gNO than the wild-type strain. 2.3 SUMMARY This thesis will advance the current body of knowledge and confirm that N O is indeed a cytocidal, non-specific broad-spectrum antibacterial agent. Data from this study will add insight into the detoxification pathways that bacteria utilize to fight antibiotic therapies. The new validated methodology used may provide opportunities for further research on agents that block these pathways. Ultimately, the knowledge derived from this thesis might provide a rationale for further exploration of the use of gNO for the treatment of pulmonary and skin/soft tissue infections. 26 3.0 E X P E R I M E N T A L 3.1 M A T E R I A L S Gases were supplied in pressurized cylinders. These included 800 ppm medical grade N O , balance nitrogen (ViaNOx-H, SensorMedics Corporation, Yorba Linda, CA, USA), medical air, oxygen and carbon dioxide (Praxair, Mississauga, O N , Canada) that were delivered at a constant pressure (50 psig) using appropriate standard Compressed Gas Association (CGA) approved gas regulator. Bacterial Cell Cultures from JB. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and S. aureus (ATCC 25923) strains were purchased from the American Type Culture Collection (ATCC) or provided by several laboratories in Vancouver General Hospital, a large metropolitan teaching hospital (British Columbia, Canada). The main laboratory provided Methicillin Resistant S. aureus (MRSA) and C. albicans. An extremely resistant strain of P. aeruginosa, isolated from a cystic fibrosis patient, was donated by Dr. Speert's cystic fibrosis research laboratory. M. smegmatis strains were generously provided by Dr. Mamta Rawat in Dr. Yossi Av-Gay's infectious disease laboratory. Dermal skin fibroblast cells were obtained by normal skin punch biopsies obtained from adult patients undergoing elective reconstructive surgery. Patient sputum samples were obtained from the multidisciplinary intensive care unit (ICU) of the Vancouver General Hospital after institutional review board approval. The attending physician identified patients as being suspect for nosocomial pneumonia (NP), and informed consent was sought from the patient or their surrogate. Petri dishes (100x15mm, 8-758-03) were obtained from Fisher Scientific (Canada). 6-Well Cell Culture Cluster plates (3516) were purchased from Corning (NY, USA). The 75-cm2 cell culture flasks were from Fisher Scientific (USA). 27 3.2 SPECIALIZED E Q U I P M E N T - in vitro C E L L E X P O S U R E D E V I C E In order to test the bacteriocidal or bacteriostatic effects of gNO, we have built a closed in vitro environment for delivery of a variety of gases mcluding nitric oxide (Miller et al, in press) The delivery system consisted of two cylindrical Plexiglas exposure chambers with separate gas entry ports and a common exit port (Figure 8). An airtight Plexiglas "heating jacket" that created a thermally isolated environment surrounded these exposure chambers. Enclosed in the jacket was an electrical heater unit controlled by an internal thermostat (Invensys Appliances Control, Carol Stream, Illinois, USA), providing stable temperatures inside the chamber. An electrical fan (12 V Touch Safe Car Fan, Canadian Tire Corporation, Ltd, Canada) was placed inside the jacket to circulate the heated air thereby allowing steady state temperatures to be reached rapidly. A vent, covered by High Efficiency Particulate Accumulator (HEPA) Filter (Sentry Air Systems, Houston, TX) to prevent contamination, was placed on the sidewall to deliver room air and avoid rainout inside the insulation jacket. A hygrometer (VWR Canlab, Mississauga, ON) was used at the exhaust port to measure relative humidity of the gas mixture exiting the cell exposure chambers. Separate sample lines from each of the two exposure chambers provided samples of the gas mixtures to a nitric oxide/ nitrogen dioxide/oxygen electrochemical analyzer (AeroNOx, Pulmonox Medical Inc, Tofield, AB, Canada) to detect the exact composition of each gas in the mixture. The two sample lines were adjusted to be approximately 1 cm above the level of the media in each of the two exposure chambers. The AeroNOx analyzer was calibrated at least every tHrd experiment with National Institute of Standards and Technology (NISI) referenced nitrogen dioxide (11.9 ppm N O J and N O (78.2 ppm) calibration gas (Pulmonox Medical Inc., Tofield, A B , Canada) based on the manufacturer's guidelines and specifications. The overall apparatus dimensions were 84 x 66 x 44 cm and was placed either on a countertop in a Level II biological safety room or under a biological cabinet for the duration of the studies (Figure 8). 28 Hvarometer Humidifier HeDa Filter Vacuum pump B Figure 8. Cell Exposure Device A: Schematic representation of the gNO exposure chamber. The gas enters from bottom left of the diagram using stainless steel quick connects. Flow for each gas can be adjusted using valves and 2 digital flowmeters. The system has the capacity to mix and deliver five different gases simultaneously. The gas mixture is then passed through special filters to prevent possible contamination ofthe system (black arrows). A flow rate of 2 L P M or 10.0 L P M was used in all experiments. B: Insulating jacket surrounding the exposure chambers. Glove port in front of the jacket allows access to the chamber without danger of cross contamination (Miller et al., in press) 29 and carbon dioxide (Praxair, Mississauga, O N , Canada). Using an appropriate Compressed Gas Association (CGA) approved gas regulator, the gases were delivered to the system at a constant pressure (50 psig). Gases were then mixed together at pre-calculated flows in a dilution manifold (Figure 1A) to produce the desired concentrations and which included digital TSI mass flowmeters (TSI Inc., Shoreview, M N , USA) to accurately meter the gas flow. The gas manifold allowed for up to 5 different gas mixtures and at various flow rates. Two identical channels in the manifold provided for two different sources of gas mixtures for exposing samples in a control chamber and exposing samples in a test treatment chamber. The gases mixed in the manifold were delivered through two separate 22 millimeter (inside diameter) corrugated respiratory tubes to two humidifiers (MR850, Fisher&Paykel Healthcare, CA, USA). The humidifiers were set to humidify gases to greater than 60% relative humidity (RH%) using sterile water (Baxter Corporation, Clintec Nutrition Division, Canada) at a temperature of 40.0°C in order to achieve 37° C in the exposure chambers. Both heated and humidified gas mixtures (treatment and control mixes independently) were delivered to each exposure chamber at a constant flow rate of 2 Liters per minute (LPM) for all the initial bacteriostatic studies and then adjusted to 2 L P M for the rest of the studies. Each of the exposure chambers could contain five standard 100 x 15 mm Petri dishes or three six-well plates or four 25-cm2 cell culture flasks. A vacuum pump was placed at the exhaust port to create negative pressure throughout the system and prevent possible back-flow of gas, and ensure a one-way gas flow. The exhaust gas mixture was filtered through a double layered H E P A filter to avoid any potential contamination of exhaust air. The exhaust gas mixtures, comprised mainly of N O , N O z , O z , and C 0 2 , were safely vented to a Class II biosafety cabinet through a vinyl duct. Data was collected over a 72 hour period to evaluate and to establish reliable steady-state environmental conditions inside the exposure chamber. N O distribution, N O z , 0 2 levels, and chamber temperature and relative humidity were monitored to establish a validated 72 hour continuous exposure system. 30 3.3 M E T H O D S 3.3.1 Method validation and Safety 3.3.1.1 Cell Culture Preparation for method validation and safety experiments Single colonies of E. coli, P. aeruginosa and S. aureus were inoculated into brain heart infusion media and grown overnight (Dalynn Biologicals, Calgary, AB, Canada). On the day of the experiment, dilutions were performed using normal saline to adjust the suspension to approximately 2.5 x 108 colony forming units per rnl(cfu/rnl) by visual comparison with the appropriate McFarland standard (# 0.5). Colony counts were performed as a control as per standard laboratory protocol. An estimated but consistent quantity of actively growing bacteria from each strain was plated on tryptic soy agar (TSA) (Dalynn Biologicals, Calgary, AB, Canada) following dilution by McFarland nephelometry. In brief, a l-in-10 dilution was further diluted to an estimated 105 cfu/ml and a 0.1 ml inoculum of this suspension was spread on TSA plate (Hendrickson and Krenz, 1991). Four control plates were placed inside 31 a conventional incubator (UltraTech WJ301D, Baxter, USA) at 37°C for 24 hours in each experiment. The treated plates (n=8) were placed inside the exposure chamber, with four plates in each of the two chambers, at 37°C for 24 hours. 3.3.1.2 Skin Cell Culture - Pilot study for growth on solid media Dermal skin fibroblast cells were obtained by normal skin punch biopsies obtained from adult patients undergoing elective reconstructive surgery. Tissue samples were collected individually and washed three times in sterile Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, New York) supplemented with antibiotic-antimycotic preparation (lOOug/ml penicillin, 100 u.g/ml streptomycin, 0.25 u.g/ml amphotericin B) (Gibco). Specimens were dissected free of fat and minced into small pieces less than 0.5 mm in diameter, washed six times with D M E M , and distributed into 25 cm flasks, 3 pieces per flask. Four milliliters of D M E M supplemented with 10% fetal bovine serum (FBS) (Gibco) and antibiotic-antimycotic was added to each dish. These dishes were incubated in a commercially available cell culture incubator adjusted to 37°C in a humidified atmosphere of 5% carbon dioxide (COj) in humidified air. Medium was replaced weekly, and by 4 weeks, fibroblasts covered more than 50% of the growth surface. At this time the fibroblasts were released from the dishes by brief (less than 5 minutes) digestion with 0.25% trypsin and subsequently seeded into 75-cm2 culture flasks (Corning, NY) in DMEM-10% FBS and then further incubated. Upon reaching confluence, the cells were released by trypsinization, split for subculture at a ratio of 1:5, and reseeded into 75-cm2 flasks. Strains of dermal fibroblasts at passages 3-7 were used in this study. 3.3.1.3 Exposure Conditions In order to test the efficacy of the gNO exposure chamber, the growth and viability of various bacterial strains as well as human skin fibroblast cultures were compared with cells incubated in a conventional incubation chamber (Forma Scientific, Marietta, Ohio, USA). Cells were exposed to medical air (in absence of gNO) continuously at 10.0 L P M up to 24 hours for bacteria and 96 hours for fibroblast cells. For skin fibroblasts, 5.0% C 0 2 was also added to the gas mixture in each of the control and treatment chambers. 32 3.3.1.4 Analysis and Measurements - Cell Viability For bacterial cells, visual count of cfu from each plate was used as a measure of cell growth. Cell counts were expressed as a relative percentage of cfu in treated groups in comparison to the control. For dermal fibroblasts, a proliferation assay was performed on cells seeded onto 25 cm 2 flasks and compared with control. In brief, fibroblasts were detached by 5 minutes treatment with 0.1% trypsin (Life technologies Inc., Gaithersburg, MD) and 0.02% ethylenediaminetetraacetic acid (EDTA) (Sigma, St. Louis, MO) in PBS (pH 7.4), re-suspended in 1 ml of D M E M with 10% FBS (GIBCO, Grand Island, NY) and counted in a hemocytometer by an inverted microscope. The plating efficiency of fibroblast cultures incubated in the gNO chamber was calculated by ratio of cell count on day 4 to original number of cells plated. To further validate optimal conditions for growing skin cells and their viability, 1-hour culture plate attachment efficiencies of treated fibroblasts were compared with control cells, immediately following 24 hour incubation in the gNO chamber and the conventional incubator. Fibroblasts were sub-cultured into 25 cm2 flasks and trypsinized for count after one hour of incubation in a conventional incubator. Morphology, confluency, and contamination of cells were also evaluated microscopically. 3.3.1.5 Analysis and Measurements - Nitric Oxide and p H Analysis Continuous readings of N O , N 0 2 , and 0 2 concentrations in the gas mixtures entering the exposure chambers were obtained using a calibrated AeroNOx electrochemical N O , N O z , and 0 2 analyzer (Pulmonox Medical Inc:, To field, AB). The uptake of N O into cell culture media was monitored by measuring the stable end products of N O , namely nitrite (N0 2 ) and nitrate (N03~) at various time points of exposure using the Griess reaction (Green, 1982). Following conversion of nitrates to nitrites and addition of Griess reagent, absorbance of samples were measured at 540 nm using a spectrophotometer (Helios Gamma & Delta, Unicam UV-Visible Spectroscopy, Cambridge, UK). Following exposure to N O , the pH level was measured at 0, 24, and 48 hours in sample aliquots taken from the cell culture medium using a digital pH-meter (Corning pH meter 240, Corning Life Sciences, USA). 33 3.3.1.6 Characterizing kinetics of gNO delivery from the air to liquid interphase In order to characterize kinetics of gNO delivery from the air to liquid interphase, variations in cumulative nitrite + nitrate (stable end product of NO) content were measured in exposed solutions (Wang and Deen, 2003). Saline, broth, D M E M , and Middlebrook 7H9 mediums were continuously exposed to 20 and 200 ppm N O for 48 hours, under identical conditions to determine how much gNO would transfer into each type of liquid media that was to be used for the studies. Samples were collected at various time points and analyzed for nitrite and nitrate levels using Griess reaction to analyze the amount of N O in a liquid. 3.3.2 Measuring Bacteriostatic Action of Gaseous Nitric Oxide on S. aureus and P. aeruginosa Strains of each species, P. aeruginosa and S. aureus, were prepared one day before each experiment according to the following procedure. ATCC samples were prepared aseptically by adding 0.3 to 0.4 ml TSB liquid medium to the freeze-dried material with a sterile pipette and vortexed. The mixture was then transferred to a test tube containing 5 to 6 ml of TSB medium. A few drops of inoculate were pipetted onto a tryptic soy agar plate (TSA). The plate was placed in a conventional incubator at 37°C for 18-24 hours and then observed for acceptable bacterial colony growth. On the day of each experiment, 3 to 5 isolated colonies from the freshly cultured TSA plate were selected. Using a sterile glass rod, colonies were transferred and suspended in 5 ml of tryptic soy broth (TSB) solution and labeled. The inoculum was mixed well and then the rarbidity was visually compared against a number 0.5 McFarland standard, which approximated a cell count of 2.5 x 108 cfu/ml. If the turbidity exceeded that of the McFarland standard, the inoculum was then diluted with sterile broth or saline. Inoculums were placed in a conventional incubator for 20 minutes to acclimatize. A dilution of 0.1 ml inocula of S. aureus and P. aeruginosa cells were then aseptically pipetted onto clear TSA plates. The 0.1 ml sample was dropped onto the center surface of the agar 34 plate and was then rapidly spread using a sterile (alcohol and flame) culture-spreader metal loop. The plates were maintained in an upright position until the inoculum was absorbed by the agar (approximately 10 minutes). A number of preliminary serial dilution studies had been done during previous studies as described in Section 3.3.1.1, in order to determine the optimal dilution to achieve approximately 30-200 colonies per plate. This was done using a micropipetter and aseptically transferring 0.1 ml of freshly prepared inoculum into a labeled 0.9 ml test tube containing 0.85% saline solution. Then 0.1 ml was transferred from the latter test tube (diluted suspension) into a second 0.9 ml of saline solution. This procedure was repeated until the desired dilution level was reached. The inoculated plates were then transferred and placed in an inverted position inside the exposure chamber. Eight identical plates were prepared for each study. Four plates were placed in each arm or chamber of the in vitro cell exposure chamber. Control plates were exposed to 2 L P M medical air at 37.0°C and with a relative humidity (RH) of approximately 60% for period of 24 hours. The total gas flow was adjusted downward and the R H maintained from those values reported in section 3.3.1.3. This procedure was done because it was noted in the pilot trials that the edges of the agar were drying out and cracking when exposure was greater than about 16 hours. N 0 2 was monitored to keep the level as low as possible. Plates in the treated groups were exposed to identical conditions with the exception that gNO was added. Concentrations of gNO of 50, 80, 120, and 160 ppm were individually evaluated. Following the incubation period, a visual count of cfus was obtained. Plates were then transferred to a conventional incubator and grown for an additional 24 hours to ascertain any difference in colony size and number. It was assumed that each colony-forming unit originated from O N E single bacterial cell. The difference in cfus between control and gNO exposed plates were used to evaluate the bacteriostatic effect of nitric oxide. The difference between colony count and colony diameter after the additional incubation for 24 hours in the conventional incubator was to suggest bacteriocidal effect. 35 A further set of studies were done with a concentration dose of 160 ppm gNO for time intervals of 0, 4, 8, 12, 16 and 20 hours. Only S. aureus was used for this preliminary study. The potential for an inhalation administration in humans to reduce P. aeruginosa bioload seemed impractical due to the potential toxicity sequalae if the optimal dose exceeded 120 ppm gNO. We therefore used S. aureus only for this study since it is the most prevalent skin pathogen and we suspected that topical application of gNO would be less susceptible to the toxic effects of gNO. Colony counts were obtained from all treated plates and compared to control plates at various time endpoints. These studies were repeated three times. 3.3.3 Measuring Bacteriocidal Action of Gaseous Nitric Oxide on a variety of bacterial pathogens Preliminary studies described above, ascertained that the effective dosage range for a greater than 90% reduction in cfu for both S. aureus and P. aeruginosa was between 160 -200 ppm of gNO. Based on this data, 200 ppm gNO were utilized for this portion of the study. The clinical bacteria portion of the study was performed at Vancouver General Hospital (Vancouver, Canada), after instimtional review board approval. Patients in the intensive care unit (ICU), who developed pneumonia, were recruited to donate a sputum sample. From this sample, the causative organism was isolated and cultured in the main hospital microbiology laboratory. These isolates were then exposed to 200 ppm gNO using the same in vitro system that was described and validated in Section 3.2, to determine if gNO had either a bacteriocidal, bacteriostatic, or no effect on these bacteria. Bacteriocidal effect was defined for these studies as a reduction by 3 Log 1 0 cfu/mL (Stiver, 2002). Additional bacterial samples were obtained from the sputum samples collected from patients in the 24 bed multidisciplinary ICU. Sputum samples were collected in a sterile container via endotracheal tube aspiration during a routine endotracheal tube suctioning procedure, the samples were labeled and then sent to the hospital microbiology lab where they were cultured and the suspect bacteria identified as per institutional standard microbiology procedures. The bacteria collected for this study were S. aureus, Serratia marcescens, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Enterobacter aerogenes and Acinetobacter baumanii. Each of 36 the bacteria that were obtained from the ventilated patients was found to be resistant to at least one antibiotic. Once the organism was identified, it was isolated and grown according to standard operating procedures of the certified laboratory. From these cultures, a 0.5 McFarland standard with 108 cfu/ml was prepared and further diluted 1:1000 with sterile saline to 105 cfu/ml to a volume of 20 ml. The concentration of 105 cfu was chosen as that is an accepted threshold for determining infection (Bowler, 2003). Three milliliter aliquots were then pipetted into each well of a six-well cell culture cluster flat bottom with lid plate (Corning 3516, Corning, NY). Triplicate wells were prepared for each bacteria, for both the treatment and control. Two sets of organisms per six-well plate were prepared. When the temperature and gNO concentration were in a steady state, the control plates were placed in the control arm and the treatment plates were placed in treatment arm. To measure the effect of gNO, each plate was sampled at 0, 1, 2, 3, 4, 5, 6, 8, 10, 12 and 24 hours. Additional time points at every 30 minute intervals were done to obtain more robust data for intitial S. aureus and P. aeruginosa studies. Samples were taken in three volumes 0.1ml, 0.01ml and 0.001ml and were plated onto blood agar plates (Columbia agar with 5% sheep's blood, P M L Microbiologicals, Willsonville, Oregon). The blood agar plates were then placed in a conventional incubator (35°C) for 24 hours. After 24 hours, the blood agar plates were removed from the incubator and the colony forming units were visually counted and reported independendy by a senior microbiology technologist blinded to the exposure. 3.3.3.1 Intermittent exposure To simulate the minimal exposure of the lungs to a safe level of gNO that might have the equivalent bacteriocidal effectiveness, an mtermittent exposure protocol was developed. Instead of continuous exposure to 200 ppm gNO, the S. aureus P. aeruginosa, E. coli, Group B Streptoccci, and an extremely resistant strain of P. aeruginosa, isolated from a cystic fibrosis patient were exposed to 160 ppm for 0.5 hours followed by 20 ppm for 3.5 hours. This cycle was repeated for a total of 6 complete cycles over a 24-hour period. This procedure exposed the microorganisms to a total of 900 ppm-hours, which is less than the 960 ppm-hours of gNO exposure when delivered at a clinically safe, 40 ppm, continuous flow. 37 3.3.3.2 N O z exposure All of the studies with liquid media used low flow rates of 2 Lpm, which resulted in concomitant high levels of N O z formation. N 0 2 in high concentrations causes pulmonary edema (Brown et ai, 1983). It was noted that at 2 Lpm the N 0 2 concentration was between 11-16 ppm during the studies. Increasing gas flow to 10 Lpm reduced the level of N 0 2 to conform to with an acceptable level below 6 ppm (see Section 3.3.1.3). However, it was noted during preliminary pilot tests that there was excessive evaporation from the wells. To confirm that the cidal effect being observed was not due to the formation of N 0 2 , the experiment was repeated using the same protocol with S. aureus and P. aeruginosa, but instead of N O , N 0 2 at 20 ppm was titrated into the treatment chamber. 3.3.4 Measuring the Protective Effect of Mycothiol Against Nitric Oxide Damage in Mycobacteria 3.3.4.1 Strains and Microbiology Techniques Three unique phenotypes of M. smegmatis were used for the studies: a wild-type mc2155 which produces normal levels of MSH, a MshA" mutant lacking the gene for transcribing MSH, and a complemented MshA" mutant which has a gene spliced back in that should restore the MSH producing phenotype. Mutagenesis of M. smegmatis mc2155 for the MshA gene was constructed previously (Newton et.al, 2003) by a transposon mutation technique. The abbreviations used in this document for the three M. smegmatis strains are designated as wild-type mc2155, the mutant deficient in MSH is designated as MshA" and the complemented MshA" strain with the restored MSH producing ability is cMshA". Al l exposures to nitric oxide or control gas described below were performed with the validated methodology reported in the Section 3.2. Fresh M. smegmatis strains were grown in 7H10 medium with 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.05% Tween 20. Kanamycin (25 mg/ml) and hygromycin (50 mg/ml) were used when required to sustain growth of mutant strains. Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). A sample loop of bacterial colonies was removed aseptically from the prepared plates 38 and placed in a solution of 7H9 medium, 0.05% Tween 20 and OADC. The vials were placed in a shaker incubator, 250 revolutions/min, at 37°C until the cell density registered an absorbance at 600nm of 0.8-1.2 (for log phase assay) or 1.5 (for stationary phase). Suspensions were adjusted to a 1.0 reading with 0.1% proskaurer beck media and 0.01% Tween-20 (PBT) and placed back in the shaker incubator for 30 minutes to acclimatize. According to previous laboratory data, this yielded 108 cfu/ml per milliliter (cfu/ml). The suspension was then diluted 1:1000 with sterile saline to 20 ml in a sterile flask. Aliquots of 3 ml were pipetted into each of three wells of a six-well plate. One plate with three wells each was prepared for the treatment chamber and another one for the control exposure chamber. The plates were then placed into the exposure chambers and exposed for 12 hours to either 200 ppm gaseous nitric oxide (gNO) in air or to air alone as the control. With a micropipette and sterile tips, duplicate samples of 0.1ml, 0.01ml, and 0.001ml were drawn off from each of the three sample wells at time intervals of 0, 1, 2, 3, 4, 5, 6, 9, 10, 11 and 12 hours and then plated onto plates with 7H10 agar medium and placed in a conventional incubator at 37°C for 72 hours. The same individual for all studies visually counted cfus as an endpoint. Each study was repeated three times. Viable colony counts to detenriine the actual number of bacteria used in the final inoculum were performed using serial sevenfold dilutions of the final inoculum in 1.5 ml centrifuge plastic tubes by adding 100 | iL of the inoculum to 900 [iL of PBT. With a micropipette and sterile tips, 50 pJL of the 4, 5, 6 and sevenfold inoculate dilutions were plated onto 7H10 medium with 1% glucose standard sized agar petri dishes. These dilutions were then spread with a sterile loop, being careful not to touch the sides of the plate. Duplicate plates for each test were performed. The plates were incubated for 72 hours at 37° C in a conventional incubator. Cfus were then counted and the final cfu/ml calculated. 3.3.4.2 Mycothiol (MSH) Measurement Measurement technique for determination of MSH levels in M. smegmatis was performed using the procedure describe by Rawat et al, (2002), modified from a previously described technique (Newton et al., 1996; Anderberg et al, 1998). Labeling of cell extracts with monobromonimane (mBBr) to determine the MSH content was performed after the 39 intervention with either gNO or medical grade air. Exposure was terminated at 8 hours to ensure that complete bacterial eradication by gNO was not achieved. Viable counts were performed at the beginning of the study and at the end of the exposure period to verify that the mycobacteria were still alive and to ascertain the colony count in order to calculate the mycothiol (MSH) level per cfu. Determining viable colony counts prior to MSH assaying ensured confirmation that the numbers of living cells were similar to the control levels of cells. After exposure, 15 ml of solution from the six-well plates containing suspended cultures of approximately 108 cfu/ml in stationary phase in saline were collected in polypropylene tubes and then centrifuged for 15 minutes at 3500 RPM. Cell pellets were resuspended in 0.5ml of warm 50% acetonitrile-water containing 2 mM mBBr (Calbiochem, San Diego, CA) and 20 mM HEPES (pH 8). The suspensions were incubated for 15 minutes at 60°C in a water bath and then cooled on ice. A 2- to 5-u.l aliquot of 5 M H C L or 5 M trifluoroacetic acid was added to produce the final acidic pH, The cell debris in each sample was then pelleted by centrifugation (14,000 x g, 5 minutes) in a microcentrifuge. MSH assay was performed to determine the level of MSH in the cells using a dedicated high-pressure liquid chromatographic (HPLC) procedure. HPLC analysis of the MSH was carried out by injecting 25 LIL of a 1:4 dilution of the samples in 10 mM H C L onto a Beckman Ultrasphere Octyldecyl Silane column (5 mm; 250 by 4.6 mm). Elution was accomplished with 0.25% glacial acetic acid (pH 3.6; buffer A) and 95% methanol (buffer B) by using the following gradient: at 0 minutes, 10% buffer B; at 15 minutes, 18% buffer B; at 30 minutes, 27% buffer B; at 32 minutes, 100% buffer B; at 34 minutes, 10% buffer B; and at 60 minutes, 10% buffer B (reinjection). The flow rate was 1 ml/min, and fluorescence detection was as described previously (Anderberg, et al., 1998). MSH levels were determined by integration of the chromatograph peaks using a commercial software program (32 karat HPLC Software V 5.0, Beckman, USA). 3.3.5 Statistics The results from all of the experiments were analyzed using the unpaired student t-test for comparison between any two groups, and by nonparametric equivalents of A N O V A for multiple comparisons. P < 0.05 was considered to indicate statistical significance. Unless otherwise indicated, results are presented as mean ± standard deviation for at least three 40 independent data. A commercially available statistics/graphics package was used to analyze and graph the data (Graphpad-Prism V 3.0, GraphPad Softward Inc., USA). 41 4.0 R E S U L T S 4.1 V A L I D A T I N G T H E S T A B I L I T Y O F T H E I N V I T R O D E L I V E R Y S Y S T E M To validate the in vitro cell exposure device, various parameters were monitored to test the experimental variation in the delivery system as well as the internal variation between the treated and control exposure chambers (chamber 1 vs. 2). The gNO system was monitored during a 72-hour continuous exposure to a pre-set concentration of 20, 50, 80 and 200 ppm gNO. The N O concentration within the exposure chamber was recorded and had a maximum variation witiiin ± 5% of the pre-set concentration (Figure 10a). A comparison of 80 ppm gNO concentration between the two chambers for 72 hours of delivery did not reveal a significant difference in concentration (unpaired student t-test, p > 0.05). Similar results were obtained at 20, 50, and 200 ppm gNO concentrations (data not shown). The delivery system was capable of mamtaining a steady N O concentration for the duration of each experiment, averaging 78 ± 1.0 ppm. The buildup of N 0 2 was below 3.0 ppm for the duration of the study (Figure 10B). Overall N 0 2 levels remained consistently low for the duration of each exposure and averaged 2.0 ±0 .1 ppm, well below the standard for N O inhalation therapy set by the Occupational Safety and Health Administration (OSHA, 1998). The partial pressure of 0 2 within the system was maintained at a consistent level and did not fluctuate more than 0.5% for the duration of exposure (Figure 10C). The gNO delivery system maintained consistent temperature levels inside both exposure chambers at 36.9 + 0.4 °C (Figure 10D). These results confirm that the cell exposure device consistently maintained a set of environmental variables that are typical specifications for a conventional incubator. In addition, there were no differences in these ambient variables between the two chambers. The designed exposure device allows petri dishes (solid media), six-well plates (liquid media containing planktonic bacterial suspensions) and flasks containing cells (human fibroblasts in liquid media), to be tested with only one variable change, such as gNO dose, while keeping all other external environmental variables constant. The advantage of this system is that it creates conventional incubator conditions, and allows for gNO to be administered at a specific dosage to the treatment exposure chamber while air is administered to the control chamber. 42 CL ^ 80 o 2 75 -•—* c: . o 70 c o o 6 5 cn 60 f; > : t - S - : - t ^ 7 1 - B a m^-v 20 40 60 80 2.5 E §: 2.0 o ro CD o c 1.5 1.0 8 0 5 CM 9 0.0 Time (hr) * B 20 40 Time (hr) 60 4 3 80 Figure 10. Determination of the reproducibility of the gNO delivery system. Various parameters of the gNO delivery system were monitored during 72 hours of continuous exposure to a pre-set concentration of gNO (80 ppm). A: The N O concentration in the exposure chamber was monitored with an electrochemical analyzer (AeroNOx, PulmoNOx Medical Inc., Alberta). The maximum variation observed was within ± 5% of the pre-set concentration. B: The buildup of nitrogen dioxide was monitored during the 72-hour exposure. The N O z level remained below 3.0 ppm for the duration of this study. C: The partial pressure of oxygen in the exposure chamber was measured using the AeroNOx device. The partial pressure of O z in the exposure chamber remained constant for the duration of exposure. D: The gNO delivery system was able to maintain consistent temperature levels inside the exposure chambers at 36.9 ± 0.4 °C. Data are presented as single data points from chamber 1 (---•---) and chamber 2 ( m ). 44 4.1.1 Determining the Kinetics of Nitric Oxide Uptake Since studies on solid media in petri dishes could only indicate how many colonies were able to grow in the presence of gNO compared to air, the only conclusion that could be drawn from solid media studies was that gNO had a bacteriostatic effect. The only way to measure bacteriocidal effect is to use a known concentration of bacteria in suspension, expose the suspension to gNO, and enumerate the surviving bacteria. However, in order to do the study in liquid (saline), it was important to verify the gNO concentration that would result from gNO dissolving in saline. Figure 11 shows that gNO diffused proportionally in a linear fashion into a variety of liquid media. o o 0 8 16 24 32 40 48 Time (Hour) Figure 11. Nitric oxide gas delivery rate in various culture media. The uptake of gNO was estimated by monitoring nitrite and nitrate (NOx) concentrations in the solutions. NO x levels were measured using Griess reagent, following continuous exposure to 200 ppm gNO in saline (~~^  ), TSB ( ),Dulbecco's modified Eagle's medium (DMEM) f h and MB 7H9 ( ). Data are presented as means and bars represent standard deviations, from 3 independent studies. Where bars are not visible, they are smaller than the data point. The transfer or uptake rates of gNO into saline, TSB, D M E M , and MB 7H9 are shown in Table 2. The averaged transfer or uptake rate of N O in three selected culture media (TSB, D M E M , and MB 7H9) was calculated to be 2.78 (SD=0.71) uM/hour for 20 ppm and 78.2 45 (SD=7.0) [xM/hour in 200 ppm exposures. At a 4 hours exposure of 200 ppm gNO, 232 (SD=23.2) uM of N O had diffused into the saline. The uptake or transfer rate at 4 hours was estimated to be 192.5 (SD=56.6) uM. The gNO uptake or transfer rate was obtained from the slope of the best-fit linear regression lines for normal saline. The coefficients of determination (R2) for the curves from which this value were calculated, was well above 90%. Table 2. Calculated uptake or transfer rate of gNO into different liquid media. Liquid gNOUptake or Transfer Rate (uM/hour)1 20 ppm 200 ppm Saline 1.42 (0.97) 22.5 (0.98) TSJ3 2.34 (0.98) 75.4 (0.99) MB 7H9 2.72 (0.92) 86.6 (0.99) D M E M 3.32 (0.98) 88.4 (0.99) 1. Values were calculated from the slopes of the best-fit linear regression curves shown in Figure 11. Numbers in parentheses represent the coefficients of determination (R ). Bacterial growth is affected by the pH of the growth medium. In unbuffered aqueous media, dissolved N O creates an acidic environment that can kill some bacteria. To evaluate the effect of gNO on bacteria, the pH of the suspension medium was monitored during gNO administration. The results in Figure 12 indicate that saline exposed to 200 ppm gNO for up to 48 hours did not show a significant change in pH (unpaired student t-test, p > 0.05) compared to saline exposed to medical air alone (control). Similar results were observed for TSB, D M E M and MB 7H9 (data not shown). We concluded therefore that the effects described in the following sections were not a result of gNO causing acidification of the liquid media. 46 0 24 48 Time (hr) Figure 12. Measurement of the p H of saline following exposure to 200 ppm g N O . Grey bars represent medical air-exposed saline, and black bars represent gNO-exposed saline. Capped bars show the SD (n=3). 4.1.2 Cell Growth and Viability 4.1.2.1 Bacterial Growth in the in vitro cell exposure device The growth of E. coli, S. aureus, and P. aeruginosa was tested inside the exposure chamber, in the absence of N O gas, to evaluate cell viability and proliferation. For each organism, three separate experiments were performed with four test plates (TSA) per experiment. Bacteria inoculated on TSA were incubated for 24 hours in either the exposure chamber or a conventional laboratory incubator (Forma Scientific, Marietta, OH). Colonies were counted at the end of the incubation period. The results, summarized in Figure 13, indicated that E. coli, S. aureus, and P. aeruginosa grew as well in the exposure device as in a conventional incubator. The mean relative viabilities + SD of E. coli, S. aureus, and P. aeruginosa grown in the exposure chamber relative to the conventional incubator were 100.2 ± 8.7, 107.5 ± 7.5, and 134.9 ± 18.0, respectively. 47 E. coli ATCC 25922 1 2 S. aureus ATCC 25923 T T ••111 i P t l l l l l T - illiisi Illi(IIlSg§ •RSI Wmmmm 1 2 P. aeruginosa ATCC 27853 48 Figure 13. Bacterial cell viability in the gNO exposure chamber. Three separate growth experiments (designated 1, 2, and 3) were carried out for each organism, in quadruplicate. Treated groups were exposed to medical air inside the gNO exposure chamber for 24 hours at 37°C, and control groups were incubated in a conventional incubator for 24 hours at 37°C. Bacterial viability is expressed as the percent of colonies counted in the treated group relative to the control group. Grey bars represent the means and capped bars represent the SD. There was no significant difference in growth between the gNO exposure device and the conventional incubator. We concluded that the exposure device could be used to grow bacteria normally in the absence of gNO, and that any growth effects observed in subsequent experiments were not due to incubation in the exposure chamber. 4.1.2.2 Skin Cell Growth in the in vitro cell exposure device To assess the viability of mammalian cultured cells incubated in the exposure chamber in the absence of gNO a fibroblast proliferation assay was performed. Skin fibroblasts (2.5x104 cells per flask) were seeded into four 75 cm 2 vented flasks and incubated in the exposure chamber for 96 hours. Cells were exposed to 10.0 L P M of medical air containing 5% C O z at 37°C and 90% relative humidity inside the chamber. Control skin fibroblast cultures were incubated in a conventional humidified incubator with 5% C O z at 37°C. On day 4 post-exposure, all groups of cells were trypsinized and the total cell count obtained using a hemocytometer (Reichert, Buffalo, NY). Cell proliferation data are presented in Figure 14A. Fibroblasts incubated in the exposure chamber reached an average total cell count of 1.02xl05 cells per flask. The average total cell count for cells grown in the exposure chamber was very similar (p>0.05) to that of the control group, which was 1.00 xlO 5 cells per flask. The plating efficiency (expressed as a percentage) was calculated as the ratio of total cells per flask post-incubation to the number of cells originally plated. The 96-hour plating efficiencies for the skin fibroblasts grown in the exposure chamber and the conventional incubator were 413% and 415%, respectively. 49 To evaluate the fibroblasts' ability to re-attach to culture plates as an indication of healthy cell function, their cell attachment capacity in one-hour was evaluated immediately following growth in the exposure chamber or conventional incubator. As shown in Figure 14B, no significant difference was found in cell attachment capacity between gNO and control groups {p > 0.05). Approximately 70% of the trypsinized cells were able to re-attach in the exposure chamber group compared to 75% of cells able to re-attach in the control group. A further assessment of normal cell morphology was done using light microscopy after the 96-hour incubation in the exposure chamber or the conventional cell incubator (Figure 14C). In both the exposure chamber group and the control group, spindle-shaped fibroblasts appeared healthy and firmly attached to the flasks. The number of dead cells (white dots in Figure 14C) floating in the media was minimal in both groups. We therefore concluded that the exposure chamber could be used to grow skin fibroblasts in the absence of gNO, and that effects observed in subsequent experiments are not due to incubation in the exposure chamber. 50 51 Figure 14. Viability of primary human fibroblast culture incubated in the exposure chamber in the absence of gNO. A: Fibroblast cell proliferation was evaluated following 4-day incubation in the exposure chamber (grey bars) or in a conventional cell culture incubator (black bars). B: The fibroblasts' attachment capacity in one-hour was evaluated immediately following 24-hour incubation in the exposure chamber or the conventional incubator. In A and B, bars represent the mean of at least 3 measurements and capped lines represent the SD. C: Microscopic evaluation of fibroblast morphology for cells grown in the exposure chamber (gNO chamber) or in a conventional incubator (Control). 52 4.2 BACTERIOSTATIC ACTION OF GASEOUS NITRIC OXIDE ON S. A UREUS AND P. AER UGINOSA 4.2.1 Determining the effect of gNO on P. aeruginosa growth The effect of gNO on P. aeruginosa growth was evaluated using the gNO exposure device, which was designed to deliver gNO or medical air (control gas) in two separate chambers capable of housing petri dishes or microtitre dishes (Section 3.2). P. aeruginosa ATCC 27853 was spread on TSA, in quadruplicate, and the plates incubated in either the gNO exposure chamber or the medical air (control) chamber for 24 hours at 37°C. Three independent experiments were performed for each gNO concentration. Bacterial survival was calculated as cfus growing in the presence of gNO compared to cfu growing on plates in the control exposure chamber, and expressed as a percent of control. As shown in Figure 15, the mean P. aeruginosa survival rates for plates exposed to 50 and 80 ppm gNO were 80.7% (12.4) and 63.9% (15.0), respectively (SD appears in parentheses). For gNO deliveries of 50 and 80 ppm, there was no significant difference in survival between the gNO-treated bacteria and the control, medical air-treated bacteria (p>0.05). When the gNO concentration in the exposure chamber was raised to 120 and 160 ppm for 24 hours, the survival of P. aeruginosa dropped to 10.8% (3.4) and 14.4% (1.3), respectively. There were significant differences between all the bacterial survival means for 50, 80, 120 and 160 ppm gNO concentrations (p<0.05). There were no significant differences between the bacterial survival means for all plates incubated in the control chamber. There was no difference in the number and size of control colonies when they were incubated in a conventional incubator for an additional 24 hours. Control colonies coalesced and could not be counted because of their large size and the number of colonies. These results indicated that control colonies continued to grow whereas the colonies exposed to gNO did not increase in size. This experiment clearly demonstrates that P. aeruginosa growth is inhibited as the concentration of gNO is increased from 50 to 160 ppm. These results provide qualitative evidence that gNO had a dose-dependent effect on P. aeruginosa growth and may even have had a bacteriostatic effect. However, conclusions about the quantitative effect and bacteriocidal effect cannot be ascertained from these studies. 53 120 -i o tn 0 -I 1 , 1 1 0 40 80 120 160 gNO dose (ppm) F I G U R E 15. Pseudomonas aeruginosa survival on solid media when grown in the presence of g N O . Growth of P. aeruginosa was assessed at various delivered N O concentrations and compared to controls (P. aeruginosa grown i n the presence of delivered medical air). Bars represent the mean of at least 3 measurements and capped lines represent the SD. 4.2.2 Determining the effect of g N O on S. aureus growth The exposure device described in section 3.2 was used to expose S. aureus to various concentrations of gNO in the exposure chamber or medical air in the control chamber. S. aureus ATCC 25923 was spread on TSA, in quadruplicate, and the plates incubated in either the gNO exposure chamber or the medical air (control) chamber for 24 hours at 37°C. Three independent experiments were performed for each gNO concentration. S. aureus was exposed to 50, 80, 120 and 160 ppm gNO (Figure 16). Bacterial survival was calculated as cfus growing in the presence of gNO compared to cfus growing on plates in the control exposure chamber, and expressed as a percent of control (Figure 16). Mean survival rates for S. aureus were 97.1% (21.5), 86.5% (15.2), 55.3% (10.9) and 0% (0.0), respectively (SD appears in parentheses). There was a significant difference in survival between these gNO dose groups (p< 0.0001). A l l of the controls in each of the dosage studies grew normally as compared to the gNO treatment groups. Similar to the P. aeruginosa studies, there was no 54 difference in the numbers and size of the gNO-exposed colonies when they were incubated in a conventional incubator for an additional 24 hours. Control colonies coalesced and could not be counted because of their large size and the number of colonies. We concluded that control colonies continued to grow whereas the colonies exposed to gNO did not increase in size. This experiment clearly demonstrates that S. aureus growth is inhibited as the concentration of gNO is increased from 50 to 160 ppm for 24 hours. These results provide qualitative evidence that gNO had a dose-dependent effect on S. aureus growth and may even have had a bacteriostatic effect. However, conclusions about the quantitative effect and bacteriocidal effect cannot be ascertained from these studies. F I G U R E 16. S. aureus survival on solid media when grown in the presence of gNO. Growth of S. aureus was assessed at various delivered N O concentrations and compared to controls (5. aureus grown in the presence of delivered medical air). Bars represent the mean of at least 3 measurements and capped lines represent the SD. 55 4.2.3 Timed exposure study A timed exposure study was performed to determine the time it would take for the highest optimal dose of gNO (160 ppm) to have a similar bacteriostatic effect as observed in the previous study (4.2.2). S. aureus was grown as described in section 4.2.2. The greatest bacteriostatic effect occurred between 8 and 12 hours of exposure (Figure 17). The survival rate for 4, 8, 12, 16 and 20 hours was 66.1% (4.8), 52.0% (14.4), 10.0% (5.0) and 4.8% (4.6), respectively (SD appears in parentheses). The differences between the mean survival rates at each time were significant (p<0.001). The most dramatic decrease in survivability occurred between 8 and 12 hours. This led us to conclude that, over time, 160 ppm gNO had a bacteriostatic effect on S. aureus. These results, although qualitative in design, suggested that quantitative studies on the effect of gNO on bacteria were warranted. C o o CD > -i—< s_ ai > CO S. aureus (xlO4 cfu/ml) 10 15 Time (hours) 25 F I G U R E 17. Timed exposure of S. aureus to 160 ppm gNO. The growth of 5. aureus was assessed over 20 hours and compared to controls (S. aureus grown in the presence of delivered medical air). Symbols represent the mean of at least 3 measurements and capped lines represent the SD. 56 4.3 B A C T E R I O C I D A L A C T I O N O F GASEOUS NITRIC O X I D E O N A V A R I E T Y O F P A T H O G E N I C B A C T E R I A Bacteriocidal effect of 200 ppm gNO was tested exstensively on P. aeruginosa and S. aureus ATCC strains. Five and four complete studies in triplicate were performed respectively. Sampling rimes were performed at every 30 minute intervals. Testing protocol was utilized as described in section 3.3.3. To determine if gNO would be bacteriocidal for bacteria affecting patients, we tested the effects of gNO on pathogenic bacteria isolated from infected patient isolates (McMullin et al., in press). Ten patients were recruited from the 24-bed multidisciplinary ICU at Vancouver General Hospital. The average age of the study population was 47.9 (±18.8) years and ranged from 23 years to 68 years. Eight participants were male and two were female. The primary admitting diagnosis for six of the patients was trauma and the other four were admitted to the ICU following a surgical procedure. Radiographic evidence from each of the subjects' lower lung lobes supported a diagnosis of pneumonia. The radiographic changes observed were new or worsening infiltrates. Of the ten patients recruited, seven had left lower lobe infiltrates, one had right lower lobe infiltrates, and two patients had infiltrates in both lower lobes. The sputum cultures for three of the patients were classified as normal flora, one sputum culture did not grow microorganisms, and the remaining six patients' cultures produced a range of drug resistant bacteria: Klebsiella pneumoniae (3 patients), Serratia marcescens, Enterobacter aerogenes, Haemophilus influenzae, Stenotrophomonas (previously Xanthomonas) maltophilia and Acinetobacter baumanii. The H. influenzae isolate was tested for sensitivity to gNO as per the protocol described in section 4.2.1, however the post-treatment colonies were not H. influenzae. Although gNO showed 100% killing of the organism after four hours, this result was excluded from the analysis because we could not conclusively link the patient to this specific isolate. C. albicans, Group B Streptococcus, E. coli, MRSA, a multi-drug resistant P. aeruginosa from a cystic fibrosis patient, M. smegmatis, the bacteria tested in the pilot studies, and the other nine pathogens isolated from ICU patients were suspended in saline and exposed to 203 (±10.5) ppm gNO at a temperature of 37°C (±1.2) with an R H % of 57% (±7.7). There were no 57 significant differences between temperature and R H % (p >0.05) between the control chamber and the gNO exposure chamber. All bacteria, with the exception of the P. aeruginosa and S. aureus ATCC strains and M. smegmatis, were reported by the V G H laboratory to be resistant to at least one or more antibiotics. These bacteria represented several problematic clinical pathogens that are difficult to treat and often develop drug resistance. A dose of 200 ppm gNO was chosen to study the effect of gNO on bacteria viability because previous data demonstrated 95% bacteriostasis at 160 ppm and it was predicted that 200 ppm would attain a 100% bacteriocidal effect. The dose was limited to 200 ppm gNO based on animal toxicology data that indicated this would be a safe dose (FDA-FOI, 2000). Safe gNO dosage will be discussed in more detail in section 5 of the discussion. 4.3.1 Exposing bacteria to 200 ppm gNO A series of experiments were designed to test the response of different clinical bacterial isolates to exposure to 200 ppm gNO. Bacteria suspended in saline at a concentration of 105 cfu/ml were exposed to 200 ppm gNO or medical air (control) and their survival monitored over time. P. aeruginosa and S. aureus studies were performed 4 and 5 times respectively with a minimum of triplicate measurements. Other studies were performed at least once, with at least two replicate measurements for each time point. In all experiments, the concentrations of the controls remained the same or increased when exposed to medical air only. Each gNO-exposed organism exhibited a 4- to 6-Log10 reduction in concentration, mdicating that 200 ppm gNO had a significant bacteriocidal effect. Figure 18 shows the survival curves for each isolate exposed to 200 ppm gNO and the medical air control. A. £ LL O re > "E 3 C/) 1.0x1007 1.0x1006 1.0x1005-i 1.0x1004 1.0x1003-1.0x1002-1.0x1001-1.0x1000-0.0 S. aureus 9 9-— i — 2.5 5.0 Time (Hr) 58 B . 1.0x1007 i.oxio°H 1.0x1005 B i.oxio 0 4-1.0x1003-1.0x1002-1.0X1001-1.0x10°° £ 3 > E 3 CO Time (hr) 1.0X1007 £ u. o > 3 09 1.0x1000 MRSA -1- - » - - ^ * \ - ~ — * ^ \ \ \ V X 0.0 2.5 5.0 Time (hrs) D . 1.0x10°7 j l.Oxio°H 3 1.0x10°54 it S i 1.0X10°4-| > 3 m 1.0x1003-1.0x1002-1.0x1001-1.0x10°° Time (Hrs) 59 £ CO > 3 1.0x1007 1.0x10°°-S. marcescens \ \ \ \ \ \ V \ \ \ \ \ \ _^ 0.0 2.5 5.0 Time (hr) 1.0x10° 7 1.0x10°H 1.0x10° 5 Si 1.0x1004-| 1.0x10° 3 1.0x10° 2-| 1.0x10° 1-| 1.0x10°° £ 3 2 *> >— 3 CO 2.5 5.0 Time (hr) D UL > 3 CO 1.0x10° 7 1.0x10°H 1.0x10°°-S.maltophilia \ \ \ \ \ 0.0 2.5 Times (hr) 5.0 6 0 H _1 E 5 L L o ro > 3 1.0x1007-1.0x1006-1.0x1005-1.0X1004-1.0x1003-1.0x1002-1.0x1001-1.0x1000-E. aerogenes — o.o 2.5 5.0 Time (hr) E 3 ro > > 3 C/5 1.0x1007 1.0x1006 1.0x1002-1.0x1000-A. baumanii *• * V \ \ V \ \ 0.0 2.5 5.0 Time (hr) E L L o ro > 3 to 1.0x1007 1.0x10° 6-1.0x10° 5-1.0x1004-1.0x1003-1.0x10° 2-1.0x10° 1-1.0x10°°-0.0 2.5 C. albicans — i — 5.0 Time (hr) 61 1.0x1007 1.0x1006 1.0x1005-1.0X1004-1.0x1003-1.0x1002-1.0x1001-1.0x10°° 0.0 Group B Streptococcus 2.5 5.0 Time (hr) 1.0x10°7 1.0x10°6-i 1.0X10°H 1.0x1004 1.0x10°3-1.0x10°2-1.0x1001-1.0x10°°-0.0 E. coli 2.5 5.0 Time (hr) 1.0x10°7 1.0x10°6-1.0x1005-1.0x1004-1.0x10°3-1.0x10°2 1.0x10°1 1.0x10°° — i — 2.5 M. smegmatis • H i 5.0 7.5 Time (hrs) 10.0 62 Figure 18. Survival curves for microorganisms exposed to 200 ppm gNO. Survival curves after continuous exposure to 200 ppm gaseous nitric oxide (red triangles) or medical air (blue squares) for a representative group of bacteria and a yeast. Panel A: S. aureus (ATCC 25923), B: S. aureus (clinical); C: S. aureus (MRSA); D: P. aeruginosa (ATCC 27853); E: S. marcescens; F; K pneumoniae; G: S. maltophilia; H : E. aerogenes; I: A. baumanii; J: C. albicans; K: Group B Streptococci; L/.E. coli; and M ; M. smegmatis. Symbols represent the mean of at least 3 measurements or the average of 2 measurements. Capped lines represent the SD. No SD is shown for symbols resulting from the average of 2 measurements. The graphs in Figure 18 were analyzed to determine the latency period (LP,), 100% lethal dose of gNO (LD1 0 0) and the slope between LP, and L D 1 0 0 . This analysis is depicted in Figure 19. The LP, was defined as the time taken for the bacterial concentration to decrease to the closest value to 1 log,0. The data point immediately prior to the first data point below the 1 log10-threshold was used for LP,. The L D 1 0 0 was defined as the time at which no further growth was observed. Effect of 200 ppm gNO on Serratia marcescens C F U , .JL 1 log io t 2" 1 . 0 x 1 0 ' E _» i_o*io0!M 1.0x10 1.0x10 ° 3 -* , v b i u i , u » i u — ®;" = = = = = h - A 3 C O 1 .0x10 0 2 -1 .0x10 0 1 - | 1 .0x10 °°-0.0 2.5 Time (hr) LP, 5.0 A t Point A Alog(survival) Point B LD l on Figure 19. Survival curve analysis for S. marcescens exposed to 200 ppm gNO. See text for explanation of abbreviations and calculations. 63 The slopes shown in Table 3 (between LP t and LD 1 ( I 0 ) represent the bacterial death rate or bacteriocidal rate. A higher absolute value (or a more negative number) indicated faster killing. The slope was calculated using the data point immediately prior to the first data point below the 1 log 1 0 threshold (Point A in Figure 19) and the L D 1 0 0 data point (Point B in Figure 19). The co-ordinates of Points A and B, and the formula used to calculate the slope were: Point A: [LP t , log(survivalA)] Point B: [ L D 1 0 0 , log(survivalB)] Slope = [ log(survivalA) - log(survivalB) ] / [ LP t - L D 1 0 0 ] = Alog(survival) / At The ppm hours (ppm-hrs) value was calculated by multiplying the gNO dose by the time of exposure as per the following calculation: Totalp p m_h r s (200 ppm) (duration of gNO exposure uni t /LD 1 0 0 in hours) Table 3 summarizes the results of PPM-hrs, LP e , L D 1 0 0 and the slope of LP t and L D 1 0 0 for each isolate tested. These results showed that the average latent period for all organisms was 3.1 hours (SD=1.6) with an average L D 1 0 0 of 5.1 (SD=1.8) hours. The average ppm hours of gNO required to achieve 100% killing was 1013 hours (SD=366). There was no significant difference (p>0.05) between bacteria with regard to the slope between LP t and L D 1 0 0 indicating that cell death was equally abrupt for all bacteria tested. There was no significant difference for PPM-hrs, LP t and L D 1 0 0 (p>0.05) between the isolates, except for the mycobacterial strain M. smegmatis which had a highly significant difference compared to the mean for PPM-hrs, LP t and LD100(p<0.001). These experiments confirmed that continuous exposure of these microorganisms to 200 ppm gNO resulted in complete killing. For most organisms, this killing effect had a 1-4 hour latent period, followed by rapid cell death. The only exception was M. smegmatis which had a prolonged latent period, approximately twice that of the other organisms. 64 Table 3. Survival curve analysis for microorganisms exposed to 200 ppm gNO Bacteria No. of Tests Latent Period* (hrs) LD100 (Hrs) PPM Hours LPt - LD100 Slope S. aureus (ATCC) 5 3 4 800 -4.6 P. aeruginosa (ATCC) 4 1 3 600 -4.2 MRSA 2 3 5 1000 -2.6 Serracia sp. 2 4 6 1200 -2.5 S. aureus (Clinical) 2 3 4 800 -4.7 Klebsiella sp. #7 2 3 6 1200 -1.7 Klebsiella sp.#2 2 2 5 1000 -1.5 Klebsiella sp. #3 2 3 6 1200 -1.4 S. maltophilia 2 2 4 800 -2.3 Enterobacter sp. 2 4 6 1200 -2.4 Acinetobacter sp. 2 4 6 1200 -2.5 Candida albicans 2 2 4 800 -1.7 Mycobacterium smegmatis 3 7 10" 2000 -1.8 E. coli 2 3 5 1000 -3.0 Group B Streptococci 5 1 2 400 -6.1 Average N/A 3.00 5.07 1013.33 -2.85 SD N/A 1.46 1.83 366.19 1.39 * > 1 SD; ** > 2 SD 65 This page is intentionally Blank. 4.3.2 Intermittent exposure of bacteria to 160 ppm gNO This study was designed to determine the time required to achieve an L D 1 0 0 for various bacteria, using intermittent, rather than continuous, exposure to gNO. A potentially useful clinical treatment cycle for gNO inhalation therapy would be 160 ppm gNO for 30 minutes followed by 20 ppm gNO for 3.5 hours. Exposing bacteria continuously to 200 ppm for 5 hours resulted in a completely bacteriocidal effect on most bacteria tested (section 4.3.1). The calculated dose- time was 200 ppm x 5 hours or 1000 ppm-hour. We calculated the total ppm-hours for the intermittent regimen as follows: Total ppm-hours = [160 ppm x 0.5 hours) + (20 ppm x 3.5 hours)] 4 (cycles until L D ] 0 0 ) = 1000 ppm-hours The resulting survival curves for each organism are shown in Figure 20^  The PPM-hrs, LP t , L D 1 0 0 and the slope of LP t and L D 1 0 0 for each organism are summarized in Table 4. Intermittent exposure of bacteria to gNO using this regimen resulted in complete bacterial killing for S. aureus, P. aeruginosa, E. coli, Group B Streptococci and an antibiotic-resistant P. aeruginosa strain from an anonymous cystic fibrosis patient, obtained through informed consent. These results showed that the average latent period for all organisms was 8.8 hours (SD=5.2). The average L D 1 0 0 was 16.0 (SD=6.3) hours (Table 4). These experiments determined that repeated cycles of 160 ppm gNO for 30 minutes followed by 20 ppm gNO for 3.5 hours resulted in complete cell death for the bacteria tested. 67 Bacteria No. of Assays Latent Period (hrs) LD100 (Hrs) PPM Hours L P t - LD100 Slope P. aeruginosa (ATCC) 3 8 16 600 -0.6 S. aureus (ATCC) 3 4 12 450 -0.6 P. aeruginosa (clinical) 3 12 20 750 -0.8 E.coli 3 16 24 900 -0.7 Group B Strep 3 4 8 300 -1.5 Average N/A 8.80 16.00 600.00 -0.85 SD N/A 5.22 6.32 237.17 0.36 Table 4. Survival curve analysis for bacteria exposed to 4 cycles of 160 ppm gNO for 30 minutes followed by 20 ppm gNO for 3.5 hours, during a 24-hour period. 68 This page is intentionally Blank. A B 3 LL O ro > 3 to £ LL o "ro > 3 to 1.0x1007 1.0*10°H 1.0X1005-( 1.0x1004-1.0x1003-1.0x1002-1 1.0X1001-1.0x10°° 1.0x1007 i.oxio°H 1.0x10° 5 1.0x10° 4-1.0x1003-1.0x1002-1 1.0x1001-1.0x10°° 5 P. aeruginosa t i •* »-160 20 ( O O o </> 0) ro T3 •o 3 10 h r 15 20 1.0x1007 1.0x1006 1.0x1005-O 1.0x1004-> 1.0x10°3-1.0x10°2-1.0x10°1-1.0x10°° E 3 3 w hr 70 D 1.0*1007 3 1.0x1006-E 3 1.0x1005 LL Si 1.0x1004-re > 3 to 1.0x1003-1.0x10°°-E. co// —*— I •1 'TT TT » rr \ T! 1 6 0 " —4 i i;— —i — —4 i— CQ z o D O CO QJ CQ CD 9^ 3 10 20 30 hr 1.0x10°7 1.0x10°6 1.0x10°H Si LOxlO0 4 j> 1.0x1003-1.0x10°2-1.0x10°1-1.0x10°° E 3 3 CO IT TT --* * \-i T -Group B Streptococcus TT TT f» -i T - * t 160 20 CQ z O D o (fl fi) J ! T3 "D 3 10 20 30 hr Figure 20. Bacterial survival curves after intermittent exposure to high and low doses of gNO. Bacteria were exposed to repeated cycles of 160 ppm gNO for 30 minutes followed by 20 ppm gNO for 3.5 hours (red triangles) or to medical air continuously (blue squares), for a total of 24 hours. The cycle of the intermittent gNO dose is also shown (green inverted triangle). Panel A: P. aeruginosa (ATCC 27853; B: S. aureus (ATCC 25923); C: P. aeruginosa (cystic fibrosis isolate); D: E. coli; E: Group B Streptococcus. 71 4.3.3 Results from N 0 2 Exposure N O reacts with oxygen to form nitrogen dioxide (NO,). N O , has been reported to cause deleterious effects on host cells at high levels (Stavert and Lehnert, 1990; Menzel, 1993; Horvath etal., 1978). During the previous studies, N O , levels were approximately 6 ppm but when total gas flow decreased to 2 Lpm the N O , values were noted to be approximately 16 ppm (Section 4.3). At no point during the studies using either 160 or 200 ppm gNO, did the N O z value approach zero. To ensure that the bacteriocidal effect observed in these studies was due to N O and not NO, , bacteria were exposed to 20 ppm N O , continuously for 8 hours and bacterial survival was monitored as previously described (Section 4.3.1). As shown in Figure 20, exposure to 20 ppm N O , did not kill S. aureus (ATCC 25923) or P. aeruginosa (ATCC 27853), while the gNO-exposed organisms were killed as before. This demonstrated that the bacteriocidal effect observed using 200 ppm gNO was not due to concomitant delivery of NO,. These results indicated that 20 ppm N O , had no bacteriocidal effect on S. aureus or P. aeruginosa. A E o CO > 3 Effect of 200 ppm gNO and 20 ppm N02 on P. aeruginosa (ATCC#27853) 1 . 0 x 1 0 0 7 LOx-IO06 1 . 0 X 1 0 0 5 1 . 0 X 1 0 0 4 1 . 0 X 1 0 0 3 1 . 0 * 1 0 ° N 1 . 0 x 1 0 0 1 1 . 0 x 1 0 ° 0 . 0 2.5 5.0 Time(hr) —•— Cont ro l (air) gNO-Tx (200 ppm) — - Cont ro l (air) — NO2-Tx(20ppm) 72 B E Li. o re > > 3 CO Effect of 200 ppm gNO and 20 ppm N 0 2 on S.aureus (ATCC#25923) 1 . 0 x 1 0 ° ° -5.0 •—-Control (air) gNO-Tx(200 ppm) ——Control (air) — NO2-Tx(20 ppm) Time (hrs) Figure 21. Survival curves for N O z exposure. Panel A: Survival curve for P. aeruginosa exposed to 20 ppm N 0 2 . Panel B: Survival curve for S. aureus exposed to 20 ppm N 0 2 . Results are superimposed over survival curve for organisms exposed to 200 ppm gNO (n=3). 4.4 MYCOTHIOL PROTECTS MYCOBACTERIA AGAINST NITRIC OXIDE DAMAGE Our previous results indicated that A l smegmatis, a strain of mycobacteria, was less susceptible to gNO compared to the other bacteria we studied (Figure 18-M). The reason for this is unknown. However, because some mycobacteria persist and survive within the harsh environment of the macrophage it has been proposed that mycobacteria have developed a number of detoxifying pathways to protect themselves against RNIs such as NO. A few of these putative detoxification mechanisms involves MSH. To provide evidence that MSH is important in the detoxification of N O we conducted studies using three different strains of M. smegmatis mc2155. One set of experiments were carried out with an M. smegmatis mutant containing a transposon insertion in mshA that results in lower MSH production. Figure 22 shows that the mshA mutant was more 73 susceptable to gNO compared to the wild-type strain of M. smegmatis. As determined by viable counts after exposure to 200 ppm gNO, the LP, for M. smegmatis mc2155 was 6 hours while the LP, for the mshA mutant was only 4 hours (Table 5). These differences demonstrated that the mshA mutant was more susceptible to gNO than the wild-type M. smegmatis mc2155, which implied that MSH played an important role in protecting mycobacteria from gNO toxicity. This study was performed only once with triplicate samples. Bacteria Latent Period (hrs) L D 1 0 0 (Hrs) PPM Hours L P , - L D 1 0 0 Slope mc2155 6 8 1600 -2.9 mshA 4 8 1600 -0.2 complemented mshA 6 9 1800 -0.6 Table 5. Summary of survival curves for M. smegmatis exposure to gNO. M. smegmatis strains were exposed to 200 ppm gNO for 11 hours. Wild-type, mc2155; mshA, MSH-deficient transposon mutant; complemented mshA, transposon mutant complemented with a wild-type copy of mshA on a plasmid. To establish that the mshA gene was responsible for the reduced MSH levels that caused increased sensitivity to gNO, a complemented isogenic mshA mutant in an M. smegmatis mc 2 l 55 background was evaluated for survival after gNO exposure. As shown in Figure 23, exposure of M. smegmatis mc2155 and the complemented mutant to 200 ppm gNO resulted in a steep bacterial killing curve after 7 hours while a steep killing curve for the mshA mutant occurred at 5 hours. A l l strains remained viable throughout the experiment when exposed only to medical air (controls). The complemented mshA mutant had similar sensitivity to 200 ppm gNO as M. smegmatis mc2155. The latent period for the complemented mshA mutant was also noted to return to similar baseline levels observed for the M. smegmatis mc2155. Together, these results suggest that the lack of MSH could be responsible for the increased sensitivity of M. smegmatis to gNO. This series of experiments demonstrates that MSH at least partially protects M. smegmatis from gNO toxicity. 74 1.0x10°°-^ 0.0 2.5 5.0 7.5 Time (hrs) - 9 10.0 12.5 Figure 22. Survival curve for M. smegmatis strains exposed to gNO or medical air. The wild-type (mc2155) and MSH-deficient M. smegmatis mutant were exposed to 200 ppm gNO at 37°C. Filled squares, mc2155 exposed to medical air; open squares mc2155 exposed to 200 ppm gNO; filled circles mshA mutant exposed to medical air; open circles mshA mutant exposed to 200 ppm g N O (N=3). 75 1 . 0 * 1 0 0 7 --1 c I . O x l O 0 6 ^ c L O x l O 0 5 -u. O L O x l O 0 4 -ival " I . O x l O 0 3 -> 3 L O x l O0 2 -1 . 0 * 1 0 0 1 -1 . 0 x 1 0 ° ° -0.0 12.5 Figure 23. Survival curves for M. smegmatis wild-type, mshA mutant, and complemented strains exposed to gNO. The sensitivities of the mshA mutant, the wild-type strain mc2155, and the complemented mshA mutant to 200 ppm gNO were compared. Filled blue squares, mc2155 exposed to medical air; open blue squares, mc2155 exposed to 200 ppm gNO; filled green circles, mshA transposon mutant exposed to medical air; open green circles, mshA transposon mutant exposed to 200 ppm gNO; filled red triangles, complemented mshA mutant exposed to medical air; open red triangles, complemented mshA mutant exposed to 200 ppm gNO. We tested to see if MSH could protect A l smegmatis from the bacteriocidal effect of 200 ppm gNO. M. smegmatis was exposed to either 200 ppm gNO or medical air (control) and the amount of MSH was determined to see if there was a detectable difference between the gNO-exposed bacteria and the control bacteria. We sought to determine that MSH was depleted on treatment of M. smegmatis with gNO just prior to reaching the rapid cell death phase. M. smegmatis was grown to O D 6 0 0 of 1.0 (approximately 2.25 x 107 cfu/ml) in MB 76 7H9, resuspended in sterile saline and exposed to 200 ppm gNO or medical air in the exposure chamber. Viable bacteria were enumerated before and after exposure by colony counts. The results shown in Table 6 demonstrate that the starting viable bacterial concentration was 2.6 x 107 cfu/ml (SD=0.1). After exposing the bacteria to 200 ppm gNO for 8 hours, the concentration of M. smegmatis was 2.5 xlO 7 cfu/ml(SD=0.0), suggesting the lethal effect of gNO was not yet detectable. An MSH assay was performed to determine the level of MSH in the M. smegmatis that were exposed to 200 ppm gNO or the medical air control. Figure 24 provides an illustration of the chromatograph, demonstrating that the MSH peak was significantly decreased after exposure to gNO, compared to the MSH level in M. smegmatis exposed to medical air. The MSH concentration in controls exposed only to medical air, were 10.3 nmoles/107 cells (SD=0.9). Bacteria exposed to 200 ppm gNO had an MSH concentration of 6.2 nmoles/107 cells (SD=2.5). This was a decrease of approximately 39% in cells exposed to gNO and is statistically significant (p<0.05). To confirm that exposure to gNO resulted in a time-dependent decrease in MSH a series of studies were performed (3.3.4.2) utilizing 400 ppm gNO to amplify the results as seen in the above MSH assay. Four separate studies were done with exposure to 400ppm gNO for 2, 3.5, 5 and 7 hours. The concentration of M. smegmatis exposed were 4.6 x 107 cfu/ml (SD=1.3) at 0 hours, 3.1 x 107 cfu/ml (SD=6.8) at 2 hours, 4.8 x 106 cfu/ml (SD=8.5) at 3.5 hours, 1.5 x 104 cfu/ml (SD=5.0) at 5 hours and no growth 7 hours. Figure 25 demonstrates that MSH was depleted over time and was nearly zero coninciding with complete cell death at 5 hours. 77 s D o d -D o aa -O < 200 ppm gNO / \ / / Mycothiol \ Control / \ J , /V/ Mycothiol J standard / \ Time (minutes) Figure 24. HPLC chromatograph of MSH measurement from M. smegmatis. MSH was measured after exposure of M. smegmatis mc2155 to 200 ppm gNO or medical air (control) for 8 hours. J2 > o CD r-_J o o »-~ o £ a. O V) o a> t l c 1 2 3 4 5 6 7 8 Time (hours) T x - 4 0 0 p p m g N O ~ - Control - Air Figure 25. MSH measurement from M. smegmatis. MSH was measured after exposure of M . smegmatis mc2155 to 400 ppm gNO or medical air (control) for 2, 3.5, 5 and 7 hours. 7 8 This page is intentionally Blank. S a m p l e S a m p l e O D 6 0 0 Sam pie V o l (mL) •Cel ls X1 0* A re a N E M N E T A re a C on v. F a c t o r pm oles Inj. Vo l . (uL) C o n e , of Inj. S a m p l e lu l l ) DM. F a c t o r S a m p l e C o n e , l u l l ) Sam pie Vo l . (mL) nm oles cfu 10 7 nm oles/cfu per 10 ' P1 Area M yc o th io1 /P1 R a tio Tx-a 1 9 2.25 1 9847 260 19587 50.43 388.38 50 7.77 4 31.07 0.5 15.54 1 .7 9.14 1 3527 1 .467 Tx-b 1 9 2.25 11001 260 10741 50.43 212.98 50 4.26 4 1 7.04 0.5 8.52 1 .7 5.01 12612 0.872 Tx-c 1 9 2.25 10134 260 9874 50.43 1 95.78 50 3.92 4 1 5.66 0.5 7.83 1 .7 4.61 14351 0.706 Control -a 1 9 2.25 29647 245 29402 50.43 582.99 50 1 1 .66 4 46.64 0.5 23.32 2.10 11.10 9506 3.119 Control -b 1 9 2.25 27992 245 27747 50.43 550.18 50 1 1 .00 4 44.01 0.5 22.01 2.10 10.48 1 0092 2.774 Control -c 1 9 2.25 24744 245 24499 50.43 485.77 50 9.72 4 38.86 0.5 19.43 2.10 9.25 9738 2.541 ' A s s u m p t i o n that O .D. 600nm/mL of 1.0 represents 2 .5X10 8 ce l l s M y c o t h i o l A r e a ( P 1 ) A v e S D T X 1 3496.7 869.9 C o n t r o l 9778.7 295.1 n m o l e s p e r 1 0 e 7 c e l l s A v e S D T X 6.3 2.5 C o n t r o l 10.3 0.9 M y c o t h lo l/P1 R a t i o A v e S D T X 1 .0 0.4 C o n t r o l 2.8 0.3 Table 6. M S H measurements and calculations. The process of calculating M S H concentration is shown. The Beckman Coulter 32 karat H P L C software program calculated the area of the M S H peak by integration. The conversion factor (Conv. Factor) is calculated using an 8.6 nmole M S H standard in an independent assay, as baseline comparison (see Figure 24, bottom line). The MSH:P1 ratio allows relative M S H levels to be compared to an internal standard. 80 5.0 DISCUSSION The experiments described in this thesis were designed to determine if gNO was bacteriocidal for drug-resistant isolates important in respiratory and wound infections. For these studies, a novel in vitro gNO delivery device was designed, tested and validated. Delivery of gNO to bacteria in culture using this device provided a methodology to advance research in this area. The lethality of gNO was tested on a representative number of bacterial isolates associated with drug resistant, nosocomial, respiratory tract and/or dermal wound infections. Most isolates showed high susceptibility. However, M. smegmatis showed less susceptibility. We demonstrated an important mechanism by which bacteria, specifically M. smegmatis, resist gNO-induced cell death. We showed that MSH produced by M. smegmatis became depleted on prolonged exposure to gNO. • We further demonstrated thztMycobacteria were less susceptible to gNO damage because of their MSH, which maintains the redox balance in the cell, thereby reducing nitrosative and oxidative stress. • Finally we demonstrated that an MSH-deficient M. smegmatis mutant was more susceptible to gNO damage than the wild-type mc2155 strain. 5.1 VALIDATION OF AN IN VITRO SYSTEM TO EXPOSE CELLS TO GASEOUS NITRIC OXIDE To systematically evaluate the effects of gNO on a variety of cell types, a new methodology was designed, validated and used. Specifically, a gNO delivery system, or in vitro cell exposure device, was designed to deliver constant levels of gNO, or other complex gas mixtures, from an exogenous source (800 ppm N O cylinder) to a heated, humidified chamber suitable for growing bacterial and/or mammalian cell cultures. To validate the properties of the exposure device, the distribution and reproducibility of several factors such as the concentration of N O , oxygen (Oj), nitrogen dioxide (NO^, nitrite and nitrate in solution, pH of exposed solutions, chamber temperature and humidity were monitored and evaluated. This exposure device was 81 found to be capable of mamtaining steady-state conditions over several days, for all the parameters noted above. The cell exposure device also allowed each of the above parameters to be independently controlled and adjusted. The cell exposure device provided easily reproducible exposure conditions for all experiments described. The cell exposure device used in these studies incorporated a number of innovative improvements over previous delivery systems. The exposure device provided two exposure chambers with identical conditions, wherein cells could be incubated in the presence of gNO in one chamber and in the absence of gNO in the other chamber. Each exposure chamber could hold up to three microtitre dishes or 9 petri dishes. Humidity, gas mixtures and flow rates, and temperature could all be easily adjusted. A vacuum pump was incorporated at the exhaust manifold to create a slight negative pressure inside the system. This ensured that a unidirectional flow of gas was maintained that minimized cross-contarnination due to back-flow inside the exposure chamber. In addition, each chamber was easily accessible and the device was simple to use. To ensure the accuracy of NO, N 0 2 , O z and C 0 2 concentrations in the gas mixtures, an adjustable gas dilution manifold with digital flowmeters was incorporated into the system. The digital flowmeter was able to detect a flow as low as 0.01 LPM, which allowed the delivery of highly accurate, consistent, and reproducible flow rates. These parameters were critical for reproducing experimental conditions, as flow rate is a potential contributing factor in time-dependent survival and growth of cells (Aufderheide et al., 2002). The dilution manifold also allowed up to 5 different gases to be mixed simultaneously, and had two independent ports for each exposure chamber. Gases were sampled from the exposure chambers, directly over the incubating culture dishes, ensuring an accurate representation of the local atmospheric conditions affecting the bacterial cultures. Another important modification was the incorporation of in-line gas humidifiers to mix and adjust relative humidity of gas mixtures prior to cell exposure. Bacteria cells will survive and replicate under low humidity, as described for Long's and Hoehn's delivery devices (Long et al., 1999; Hoehn et al., 1998). This is not the case for mammalian cell cultures, however, which are very sensitive to the effects of drying. Under normal physiologic conditions, both 82 mammalian and bacterial cells were exposed to approximately 60% R H at 37°C, which is considered an optimal level for cell cultures (Aufderheide et al, 2002). For cells cultured in aqueous media, the liquid layer can act as a physical barrier through which gases must diffuse. To characterize the uptake or transfer of gNO into different culture media, production of nitrite and nitrate (stable end-products of N O oxidation) were monitored in saline, TSB, D M E M , and MB 7H9 for 48 hours. Other studies have demonstated that the volume of the culture media (depth of the aqueous layer) affected gas uptake or transfer (Behnke et al., 1998; Ritter et al., 2001). For this reason, the volume of culture medium in flasks and 6-well microtitre plates was kept to a minimum (5 ml and 3 ml, respectively), to maintain cell viability. The key to characterizing the N O uptake or transfer rate in culture media is accounting for the reaction of N O with 0 2 , and subsequent reactions with water molecules. The oxidation of N O by 0 2 in aqueous solution is shown below (Wang and Deen, 2003): 2 NO + 02 > 2 N02 (1) NO + N02 < > N2 <93 (2) N203 + H20 > 2NO- + 2H+ (3) It is important to note that gNO was the only source of N O added to the system when compared to control conditions, and other intermediates (N0 2 and N 2 0 3 ) are present only in trace amounts (Lewis and Deen, 1994). Since nitrite (N02~) is the stable end product, its rate of formation is controlled by the reaction shown in equation (1). Under these conditions, as claimed by Wang and Deen (2003), reactions (1) to (3) can be simplified to: 4NO + 02+ H20 > 4N02 + 4H+ (4) The change in N0 2 " concentration will correlate directly with the amount of exogenous N O delivered to the aqueous system. Analysis and explanation of the lower uptake or transfer rates observed in saline solution compared to the more chemically complex culture media, is curious. We speculated that N O may be buffered by the minerals and substrates in the media. 83 We thought that N O might bind to these compounds until the measurement was made (section 2.2.1.6) when the bound N O may have been released. This release may have produced a spuriously high result. Recendy, Keynes et al, has reported that there are NO-consuming ingredients that remove N O in tissue culture medium (Keynes et al, 2003). They observed that superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments. Unfortunately this information was not available to us at the time of our experiments. In future studies addition of superoxide dismutase might be used to reverse this reaction. The observed linear increase in N O x concentration indicated consistent delivery and uptake or transfer of N O into the aqueous phase (Figure 11). This relationship made it possible to indirecdy calculate the concentration of N O in solution at any time point. One critical test to validate the use of the exposure device was a test of its capacity to provide conditions for prokaryotic and eukaryotic cell growth. We compared cellular growth, for both bacteria and human skin fibroblasts, in the exposure device versus a conventional incubator. The viability and growth pattern observed for cells grown inside the exposure device, closely resembled the viability and growth pattern observed for cells grown in the conventional incubator (control). In the case of all three bacterial strains tested, the average of three independent experiments showed growth at least equal to growth in the conventional incubator (Figure 13, Section 4.1.2.1.). This was not surprising, as conditions such as temperature, humidity, and airflow were optimized for bacterial growth in the new cell exposure device. To establish and test optimal growth conditions for mammalian cell cultures, we chose to work with human, punch biopsy-obtained, fibroblast cultures. Fibroblasts were used because of access to samples, flexibility of fibroblasts in culture, and the availability of a well-characterized, established fibroblast model (Ghahary et al., 1997). In a series of bacterial plating efficiency experiments, equivalent growth rates were observed in both the exposure device and a conventional incubator. The plating efficiency measured the number of colonies originating from single cells after growth in each of the incubators. This test sensitive and is often used to determine the nutritional requirement of cells, measuring the effects of growth factors, and the toxicity of applied agents (Mather and Roberts, 1998). Fibroblast health was assessed by determining the cellular attachment capability. Fibroblast 84 cellular attachment capability, following incubation in the exposure device, was not significantly different when cells were grown in a conventional incubator. Normal fibroblast reattachment was another indication that the exposure device provided a suitable environment for growth of mammalian cells. Microscopic evaluation of fibroblast morphology illustrated the characteristically elongated and narrow fibroblast shape for the cells grown in the exposure chamber (Lombello et al., 2000). Fibroblasts reached a high degree of confluency in 4 of days incubation, with no difference in the number of dead cells compared to the control. These successful outcomes of cell morphology, confluency, and survival, all confirmed the ability of the cell exposure device to function in a manner equivalent to a conventional incubator. Once it was determined that the cell exposure device could provide suitable conditions for the studies, it was important to ensure that the device would not produce toxic amounts of N 0 2 , which could adversely affect cell growth or introduce external errors. As Menzel previously hypothesized, N O exposed to O a in the air could contribute to formation of N 0 2 , which might be toxic to cells (Menzel, 1993). The formation of N O z is dependent on the N O and O z concentrations as well as the time of interaction (Hoehn et al., 1998). For this reason, the gNO flow rate in the pilot studies (3.3.3.2) was initially set to rriinimize the accumulation of N 0 2 . At a flow rate of 10.0 L P M and up to 200 ppm of continuous gNO delivery, the maximum level of N 0 2 measured in the system was below 6.0 ppm. This level of N 0 2 was within an acceptable clinical range for inhaled N O therapy (Roberts et al., 1997), and has been demonstrated to have no bacteriostatic effect (Mancinelli and McKay, 1983). However, at this flow rate, the media evaporated too quickly, so the flow rate was lowered to 2 L P M . Studies were done to confirm that levels as high as 20 ppm N O z had no bacteriocidal effect on the bacteria studied (Section 3.3.3.2). Decreasing the pH of growth media results in decreased bacterial growth (Mancinelli and McKay, 1983). We determined that there was no significant decrease in the pH of the growth media during the delivery of 20 ppm N 0 2 . The largest decrease in pH, following 48 hours of gNO exposure in the exposure device, was observed for MB 7H9 medium, with the pH of the medium dropping less than one pH unit (data not shown). Based on findings in previous studies (Iyengar et al., 1987) and those observed in this study, the pH change observed was unlikely to have a toxic effect on the bacteria or human fibroblast cells. It was surprising that 85 the pH did not decrease further; it is possible that the buffering capacity of the media prevented a significant decrease in pH due to dissolved gNO or N 0 2 . The results of these experiments characterized and validated the properties of the gNO exposure device. The data suggested that the test gas could be delivered in a controllable and reproducible manner for long-term exposure experiments. The results confirmed that cultured cells could be incubated in the device, exposed to medical air as a control, for at least 96 hours while preserving cell viability. The kinetics of gNO uptake or transfer were characterized in the system by measuring the concentration of nitrite and nitrate in solution, allowing gNO delivery to be accurately measured, indirecdy. The exposure device also allowed continuous monitoring of several parameters such as concentrations of NO, N O z , and O z , temperature, and humidity levels. Another advantage of this gNO exposure device was its mobility and flexibility, which allowed the cell cultures to be grown and maintained, independent of an external incubator or biosafety cabinet. In conclusion, the gNO cell exposure device was validated for bacterial and cell culture growth. This device increased our ability to carry out in vitro experiments analyzing the biological effects of gNO. We concluded that the effect of gNO on bacteria or eukaryotic cells would not be unduly influenced by any errors introduced through use of the exposure device. 5.2 DETERMINING THE BACTERIOSTATIC ACTION OF GASEOUS NITRIC OXIDE ON S. aureus AND P. aeruginosa The worldwide rapid increase of bacterial resistance to numerous antibiotics is stimulating the development of new antibacterial therapeutics (Tenover and Hughes, 1996; Marcola, 2000). In evaluating gNO as a potential antibacterial therapeutic, the first step was to design a simple study to determine what dose or concentration of delivered gNO, if any, would be bacteriocidal. Once an optimal dose was estimated, then a timed, bacterial killing study would be conducted. Data collected from these initial studies provided the framework for refining the methodology for further studies on bacterial killing (Section 3.3.3). For the initial pilot studies, two bacterial isolates were selected based on two hypothetical clinical applications of gNO. It was speculated that gNO could be inhaled into the lungs for respiratory infections or delivered directly to the surface of infected skin wounds. Bacteria 86 associated primarily with pulmonary disease or with surface wound infections were chosen for our study. Initially, these two bacteria would be used to evaluate the effectiveness of gNO as an antibacterial agent. A literature search was done to establish which bacteria would be most suitable to study, and the two bacteria selected were P. aeruginosa (respiratory tract infections) and S. aureus (surface wound infections). The rationale for using P. aeruginosa in this study is that it is a problematic opportunistic pathogen that is difficult to treat because of its resistance to antibiotics. It can be acquired in the hospital and can cause severe respiratory infections. P. aeruginosa is associated with a high mortality in patients with cystic fibrosis, severe burns, and in cancer and AIDS patients who are immunosuppressed (Speert, 2002). The clinical problems associated with this pathogen are many, as it is notorious for its resistance to antibiotics due to the permeability barrier afforded by its outer membrane lipopolysaccharide (LPS). The tendency of P. aeruginosa to colonize surfaces as a biofilm makes the cells even more impervious to therapeutic concentrations of antibiotics. The futility of treating P. aeruginosa infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with resistant strains. Alternate antibacterial therapies for this bacteria would have a major impact, as the Centers for Disease Control estimates that the overall incidence of P. aeruginosa infections in US hospitals averages about 0.4 percent (4 per 1000 discharges), and this pathogen accounts for 10.1 percent of all hospital-acquired infections. S. aureus was selected as the second test isolate for this study because staphylococci are important human pathogens causing severe infections mcluding endocarditis, pneumonia, sepsis and toxic shock. MetHcillin-resistant S. aureus (MRSA) is now one of the most common causes of nosocomial infections worldwide, causing up to 89.5% of all staphylococcal infections (Narezkina et al., 2002; Milind and Dekbhile, 2003). Community outbreaks of MRSA have also become increasingly frequent (Rosenberg, 1995). The usual treatment for these infections is the administration of glycopeptides (Vancomycin and Teicoplanin). Methicillin resistance in S. aureus has been reported for two decades, but the emergence of glycopeptide-resistance in S. aureus—namely glycopeptide intermediate S. aureus (GISA)—has been more recent (Hiramatsu, 1997). Glycopeptides are given only parenterally, and have many toxic side effects (Hamilton-Miller, 2002). The recent isolation of the first clinical Vancomycin-87 resistant S. aureus (VRSA) from a patient in the USA has heightened the importance and urgency of developing new antibacterial agents (Bardey, 2002). Most new drugs entering the market are modifications of existing classes of antibacterial agents (ie; Tetracyclines, Teicoplanin), however, novel therapeutic targets are preferred. Linezolide is an alternative to treat MRSA but in spite of its short therapeutic milieu, adverse effects, mainly hematologic, are akeady reported, (Champney and Miller, 2002; Gerson et al, 2002). For the pilot studies, 104 cfu P. aeruginosa and S. aureus were spread on agar plates. The plates were then exposed to various concentrations of gNO in the exposure device and the effect on colony growth was evaluated. These studies demonstrated that gNO concentrations above 160 ppm reduced the number of growing colonies by greater than 90%. Subsequent studies indicated that the time required to achieve this affect was between 8-12 hours. These preliminary studies provided the rationale and justification to systematically study the effect of gNO as a potential antibacterial agent for reducing pulmonary infections and/or infected wounds. The results from the pilot studies confirmed that gNO had an inhibitory effect on P. aeruginosa and S. aureus. The data provided preliminary evidence that with increasing gNO dose or increasing time of gNO exposure, bacteriocidal activity also increased. As the concentration of gNO increased, the number of colonies that grew on the plates decreased. Although the data indicated a trend towards only 5-10% bacterial survival, and although some plates had no colony growth, a 100% bacteriocidal effect could not be confirmed. Some bacteria may have survived because the agar media may have reacted with the gNO and mitigated its bacteriocidal effect. Of significance, was the observation that the bacterial colonies were unchanged in size and number after being transferred to a conventional incubator for 24 hours, whereas control colonies not exposed to gNO increased in number and size such that they could not be counted. This strongly suggested that gNO exposure prevented bacterial growth, and may have resulted in bacterial cell death during gNO exposure. Although this data suggested that gNO may be bacteriocidal, these initial studies could not confirm this quantitatively. Gaseous N O was observed to have at least a bacteriostatic effect on P. aeruginosa and S. aureus, and as a result, subsequent studies were designed to further quantify the bacteriocidal effects of gNO. 88 5.3 B A C T E R I O C I D A L A C T I O N O F GASEOUS NITRIC OXIDE O N A V A R I E T Y O F P A T H O G E N I C B A C T E R I A The purpose of this study was to determine the period of time required to produce a bacteriocidal effect by delivering 200 ppm gNO to a representative collection of antibiotic resistant Gram-positive and Gram-negative bacteria associated with clinical infection. For the following studies, a bacteriocidal effect was defined as a decrease in bacterial concentration of more than 3 log 1 0 cfu/ml. C. albicans, MRSA, a particularly resistant strain of P. aeruginosa from a cystic fibrosis patient, Group B Streptococcus, and M. smegmatis were included to determine the effectiveness of gNO against a representative variety of microorganisms. The results from these studies would be an important step in laying the foundation for potential use of gNO as an antibacterial agent, specifically for use against bacteria associated with clinical infections. A concentration of 200 ppm was selected for the testing of the isolates after reviewing both the published literature and the pilot studies conducted at a range of lower concentrations of 50, 80, 120, and 160 ppm. We had observed (Section 5.2) that at 80 ppm gNO exposure of P. aeruginosa, the bacteriostatic effect occurred at 14 hours. The goal of this study was to demonstrate that gNO had a 100% bacteriocidal effect, and at higher concentrations of gNO this would be more demonstrable. Long et al demonstrated a decrease of 2 Log 1 0 for M. tuberculosis using 90 ppm gNO but not a 100% bacteridical effect (Long et al, 1999). Their results suggested that a bacteriocidal effect would not have occurred if we had used a dose as low as 80 ppm. However, it cannot be concluded from the data reported here that using lower concentrations of gNO for extended periods would not have had a bacteriocidal effect. Further studies were required to determine if gNO given at lower doses for longer periods of time would be as effective. These studies showed that 200 ppm gNO had a significant bacteriocidal effect on all isolates tested since all organims experienced at least a 3 log1 0 reduction in cfu/ml. In further support of these results, every test resulted in complete Willing of the bacteria. These killing curves were characterized by a latent period, when bacterial concentration was unaffected. This latent period was followed by a rapid loss of viability, resulting in a steep killing curve. Al l bacteria 89 tested, mcluding antibiotic resistant strains, as well as yeast and mycobacteria were susceptible to 200 ppm gNO. Of importance, was the observation that the multi-drug resistant bacterial strains were equally susceptible to gNO. This is the first time that a specific concentration of gNO has been shown to direcdy exhibit a non-specific lethal effect on a variety of potentially pathogenic microorganisms. These results support observations previously described in the literature, that N O acts as an antibacterial agent (reviewed by DeGroote and Fang, 1999). This study also detected a significant increase in the latency period for mycobacteria compared to all other organisms. The longer latency period suggested that mycobacteria have a more potent mechanism that protects the cell from gNO cytotoxicity. Further research would be needed to elucidate the mechanism involved in protecting mycobacteria from gNO. The next set of studies indicated that a continuously high concentration of gNO (200 ppm) was not required to achieve the desired bacteriocidal effect (Section 4.3.2). An intermittent, cyclic regimen of 160 ppm gNO for 30 minutes, followed by 3.5 hours of 20 ppm gNO also resulted in 100% killing. The cyclic gNO regimen extended the latency time (L,) from 2-5 hours to a range of 16-22 hours. The reason for this was unclear, however, there may be a predictable dose-time value (time of exposure for a particular gNO dose) at which L D 1 0 0 would occur. This idea was confirmed when we calculated the number of ppm hours required to achieve L D 1 0 0 . In the case of all organisms tested, similar ppm-hours of gNO values value were obtained (Table 4). The results are very encouraging from a toxicity and safety standpoint, both because a time to L D 1 0 0 can be approximated and because the cyclic administration of high and low respiratory doses of gNO would be far less likely to expose a patient to significant amounts of toxic N O oxidation products. The data suggested that there was a dose- and time-dependent gNO threshold within the cell that, once reached, resulted in rapid cell death. One possibility was that this threshold occurred at the point when the organism's normal N O detoxification pathways were overwhelmed. Further exploration of this idea occurs in Section 4.4 by examining the key role of MSH in the detoxification gNO in M. smegmatis strains. W hypothesized that the non-specific, 100% bacteriocidal effect observed in these experiments was attributed to N O rather than N 0 2 . As N 0 2 is an oxidation product of NO, it was important to rule out N 0 2 as the mediator of the effects observed. Bacteria were exposed to 90 20 ppm N 0 2 (Section 4.3.3). Bacterial viability was not affected by exposure to N 0 2 . Some reports in the literature suggest that these levels of N 0 2 may cause cytotoxicity in both bacterial cells and eukaryotic cells (Mancinelli and McKay, 1983). Those studies, however, were performed at an acidic pH, perhaps resulting in conditions where N 0 2 was more toxic to the cells. Another possibility is that, even though N O z was direcdy measured in gaseous form within the exposure chambers, it did not diffuse into the saline solution in significant concentrations. In this series of studies, the pH was not measured because it was previously demonstrated that the pH of the saline remained above 6.5 after 10 hours of exposure to 200 ppm gNO in this experimental system. Although unlikely, a drop in the pH of the bacterial suspension as a result of N O exposure may have occurred, and this may have contributed to the bacteriocidal effect. In the future, separate pH studies will be undertaken to determine if this is a possibility. Results from these studies showed that mycobacteria were reduced in concentration from 105 cfu/ml to zero cfu/ml in 8 hours. Our results are significandy different from those reported in previous studies. Long et al., demonstrated only a 2-log10 reduction in colony counts (cfu) following exposure to 90 ppm gNO of M. tuberculosis over a 48 hour period (Long et al., 1999). An article stated that exposure of 90 ppm gNO would result in a 150 nM N O concentration in the aqueous phase (Nathan et al., 2004). At this level, a 100% bacteriocidal effect could not be achieved. In the studies reported here, the aqueous concentration of N O achieved during 8 hours of exposure to 200 ppm gNO, was 344 LIM . These conditions eradicated mycobacteria and suggested that a gNO concentration approximately 1000 times the chemotherapeutic levels for M. tuberculosis (reported by Long et al, 1999) was likely achieved during this study. With this high a potency, it was not a surprise that a 100% bacteriocidal effect was achieved for all organisms tested (reported by Long et al, 1999). The exposure device designed for these studies has enabled direct measurement of gNO delivery in ppm and we have verified the actual level of N O that diffused into the saline solution during the exposure period. Results from this study suggest that concentrations of gNO greater than physiologic concentrations have a non-specific bacteriocidal effect against a representative collection of drug-resistant, gram positive and gram negative bacteria. We acknowledge that the number of organisms tested was limited, but was representative of clinically important pathogens and 91 opportunistic pathogens. In contrast to these findings, early work by Tarnir showed that N O had a specific bacteriocidal effect on Salmonella typhimurium but was ineffective against fi. coli. (Tamir et al, 1993). Hoehn and coworkers studied the effect of 40 ppm, 80 ppm and 120 ppm gNO on five tracheal isolates of ventilated pre-term and term infants, including S. aureus, fi. coli, P. aeruginosa, S. epidermidis and group B Streptococcus (Hoehn et al, 1998). They found that gNO had no effect on S. aureus, fi. coli and P. aeruginosa, but did reduce colony counts of S. epidermidis and group B Streptococcus. Contrary to the above findings, the results presented here show 100% bacteriocidal effect on all bacteria tested, mcluding S. aureus, fi. coli, group B Streptococcus and P. aeruginosa. The experiments described in this thesis likely resulted in higher sustained intracellular gNO concentrations than in previous studies reported in the literature. The use of the in vitro cell exposure device in these studies also allowed precise control of many variables that may not have been controlled in previously reported studies. Future studies should test bacteria such as S. typhimurium, which was previously reported to be resistant to N O damage and toxicity. We predicted that the general bacteriocidal effect of gNO reported here would also be true for S. typhimurium. The results reported in this thesis indicate that it would be worthwhile to explore the clinical application of gNO for the treatment of wound infections. Treating infected wounds with 160-200 ppm gNO may be the most prudent initial indication, until safety issues regarding methemoglobinemia and N O z are addressed for inhaled gNO therapy. For gNO, Long et al. demonstrated a time- and concentration-dependent bacteriocidal effect on M. tuberculosis in vitro (Long et al., 1999). However, the true effect of 90 ppm gNO for 48 hours may have been underestimated since the bacteria were plated on TSA. The TSA may have mitigated the bacteriocidal effect of gNO through buffering capacity or provision of nutrients to the bacteria. These results raise the question as to whether the results in our in vitro studies will translate to an in vivo model, because the bacteria were suspended in saline and not media. For the experiments described in this thesis, saline was selected as the bacterial suspension medium because it would not mask the direct effect of gNO as a bactericide. A rich growth medium might have introduced external variables ie. increased buffering capacity or chemical reaction with the gNO (Mancinelli and McKay, 1983). Furthermore, suspending the bacteria in 92 saline may more realistically represent a hostile, nutrient limited, host environment. In saline, the bacteria were static but remained viable. This methodology was intended to control for confounding variables related to replication and allowed for examination of the direct effect of gNO on bacteria. Growth media would also provide metabolites and replenish nutrients that may have allowed production of enzymes to protect the bacteria from oxidative and nitrosative damage, thereby masking the effect of gNO. Saline also provided a liquid environment for the study rather than solid media and hense gNO may transfer more readily. A liquid environment is more like Webert's and Jean's approach, using animal models (Webert et al., 2000; Jean et al., 2002 ), rather than Hoehn's and Long's studies using growth plates for testing (Hoehn et al., 1998; Long et al., 1999). There are some limitations of this in vitro test model. Specifically, lung and wound infections are more complex than planktonic bacteria suspended in saline. Further research should be conducted on bacterial cells growing as a biofilm phenotype and not restricted to planktonic cells suspended in aqueous solution (Costerton et al., 1999). The concentrations of M. smegmatis used in this study were twice that reported in Long's study of approximately 0.1 ml of 103 cfu/ml (Long et. al., 1999). The definition of infection used for the studies reported here, was when the bacterial concentration reached 105 cfu/ml (Bowler, 2003). For this reason, a target of 105 cfu/ml was selected for all studies in this thesis. In these studies, after a latency period, rapid cell death was observed during a 10-hour period (Section 4.4) instead of the cell death observed at 48 hours in Long's work (Long, et al, 1999). This confirmed that 200 ppm gNO was bacteriocidal to M. smegmatis mc 2l 55. It is possible that the M. tuberculosis used in Long's studies may be more resistant to gNO than M. smegmatis. Nevertheless, M. smegmatis has been typically used in preliminary studies in tuberculosis research, because it has similar biochemical characteristics as virulent strains of M. tuberculosis, but is safer to work with. An obvious question is whether host cells could survive the bacteriocidal gNO concentrations used in these studies. To answer this question, we have just completed studies using our cell exposure device described, to demonstrate that human fibroblasts are indeed able to survive an atmosphere of 200 ppm gNO for up to 96 hours (data not shown). The potential toxic effect on human skin cells was investigated by exposing human fibroblasts to 200 ppm gNO in the 93 exposure device for 24, 48 and 96 hours. Following gNO exposure, fibroblast proliferation, morphology, viability, migration, and expression of collagen and collagenase were evaluated (data not shown). These experiments determined that fibroblasts exposed to gNO were viable and functional because collagen and collagenase levels increased or were similar to the controls. In addition, preliminary results from separate studies demonstrated that human alveolar monocytes and macrophages also tolerated 200 ppm gNO for 24 hours. These studies are still underway, and the results will be published separately. The studies reported here indicate that supraphysiologic levels of N O are bacteriocidal for representative strains of drug-resistant bacteria. This bactericial effect appears to be rapid and non-specific. However, a number of questions arise: How does gNO kill bacteria so rapidly? What is the host detoxification mechanism that allows 200 ppm gNO to eliminate bacteria yet preserve the host cell's integrity? Why are mycobacteria more resistant to the bacteriocidal effect of gNO than other bacteria? This could be a key to understanding the bacteriocidal effect of gNO. To elucidate the potential antibacterial mechanism of gNO action, we studied its effect on M. smegmatis, which was observed to have decreased susceptibility to gNO compared to other bacteria (Section 4.4). 5.4 UNDERSTANDING D E F E N S E M E C H A N I S M S BY W H I C H E U K A R Y O T E S AND M A M M A L S P R O T E C T T H E M S E L V E S AGAINST NITRIC OXIDE D A M A G E In life, the epithelial cells of the human body are constandy challenged by exposure to a broad range of bacteria. The host response to these routine challenges typically keeps these potential pathogens in check. In the host, increasing N O production by phagocytes may act as an antibacterial defense. When host defense mechanisms are inadequate to prevent the proliferation of bacteria, an infection ensues. The experiments described in section 4.3 indicated that during gNO exposure,- there was a latent period followed by rapid bacterial killing by gNO for a representative range of bacteria, including drug-resistant strains. This antimicrobial activity appears to be broad, non-organism specific, and very effective. In tuberculosis infections, a few of the bacteria are able to survive and even reside within macrophages where N O is present in high concentrations. Even though the latent period was 94 more than twice as long as for other bacteria, even the mycobacteria tested in our studies failed to survive exogenous gNO exposure. During infection, host cells—particularly macrophages—are exposed to N O and survive, even at concentrations capable of killing mycobacteria. The difference in the response of host cells and bacteria to gNO is important to characterize, as it may provide information about how different organisms have evolved and adapted to survive oxidative or nitrosative assault. Normal host metabolic processes and host defense processes both expose mammalian cells to NO, and there are detoxification mechanisms that manage the potentially destructive action of N O on these cells. Higher order organisms, like humans, have very high levels of GSH, a low molecular weight thiol that serves as a key electron donor for reducing proteins and free radicals. In contrast, most prokaryotes have little, if any, GSH (Dolphin et al, 1989). When high concentrations of N O are produced by phagocytic cells during a host immune response, endogenous GSH protects the host cells by reacting with N O to produce S-Nittosoglutathione (GSNO). Bacteria may use a similar scavenging process until all GSH is consumed (Figure 3, Section 1.5), at which point, the bacterial cell would become vulnerable to gNO. The normal oxidative and nitrosative protection pathways in bacteria may not be sufficient to defend against continuous exposure to 200 ppm gNO. Eukaryotes can cope with exposure to 200 ppm gNO for up to five-fold longer than bacteria since no deleterious effects were observed in human skin fibroblasts exposed to gNO for 24 hours (data not shown). Bacteria, with little or no GSH, are unable to protect themselves against gNO-mediated damage, which rapidly destroys the cellular respiratory and replicating machinery. The results reported in this thesis support this theory. A latent period was observed when no bacteriocidal effect occurred, for all bacteria tested. This latent period may coincide with the availability of GSH to detoxify the exogenous gNO. At a gNO threshold of approximately 900-1000 ppm-hrs, rapid cell death was observed for all bacterial strains tested, with a reduction of at least 3 log l t l cfu/ml. This concentration threshold may indicate the point at which all bacterial gNO detoxification pathways are saturated. The steep killing curve is consistent with the lethal action of unrestricted levels of ROIs and RNIs in the cytosol (Fang, 1999). This explanation is consistent with the prolonged latent period associated with 95 increasingly sophisticated detoxification pathways seen in higher organisms, such as eukaryotes. Experiments were designed to attempt to provide supportive data for this concept. Mycobacteria produce a specialized thiol, MSH, which protects the cell against gNO damage in the same way GSH protects eukaryotic cells. The presence of MSH may account for the prolonged latency period observed for M. smegmatis, compared to other bacteria exposed to the same gNO concentration. In the studies presented in Section 4.4, after the latent period, M. smegmatis was rapidly killed by gNO. I propose that the length of time M. smegmatis would be protected from gNO-mediated killing would depend on the amount of cytosolic MSH available. Once MSH is depleted, M. smegmatis should be vulnerable to the bacteriocidal action of ROIs and RNIs and rapid cell death would result. The studies reported here demonstrated that M. smegmatis was more resistant to gNO-mediated killing than other bacteria; the scientific literature supports this observation by suggesting that mycobacteria have a more highly developed mechanism for N O detoxification (Newton et al., 2000b). The methodology described in section 3.3.1 allowed further experiments to be done, examining how mycobacteria were protected against gNO damage, and how gNO was so effective as an antibacterial agent. The methodology may also help to define detoxification pathways that protect mycobacteria from host defenses and mechanisms of antibiotic resistance. Once detoxification pathways have been elucidated, new drug targets may be identified that will neutralize these defense mechanisms, making bacteria more susceptible to host immune defenses and/or drug therapy. 5.5 D E T E R M I N I N G T H E B A C T E R I A L M E C H A N I S M F O R P R O T E C T I O N AGAINST GASEOUS NITRIC O X I D E - M E D I A T E D K I L L I N G Bacteria that do not have GSH have other low-molecular weight thiols to protect themselves against oxidative assault (Newton et al., 1996). M smegmatis produces MSH which may act analogously to GSH to protect the cell from N O and other electrophilic molecules (Newton et al., 1996). It has been proposed that mycobacteria have the highest levels of MSH of any bacteria (Newton et al., 2002). Experiments were designed to show that MSH initially protects 96 the cell from gNO, but when MSH is depleted mycobacteria are as susceptible to gNO toxicity. The concentration of MSH in M. smegmatis was found to be in the nmol/ml range and decreased by 40% and almost 80% following exposure to 200 and 400 ppm gNO respectively (Table 6). Further, it was demonstrated that this was a time-dependent depletion of MSH when exposed to gNO (Figure 25). The cells were still viable when MSH was measured. This suggested that MSH decreased due to gNO exposure. Since some MSFI was still present after gNO exposure, albeit at a much lower concentration, it appears that even low levels of MSH provided protection against gNO. Altogether, these results provide evidence that MSH functions in protection of the cell from gNO insult. Results herein demonstrated that MSH-deficient M. smegmatis was more susceptible to gNO than the wild type parent and that complementation with a cloned mshA gene restored wild-type gNO resistance (Section 4.4). Accordingly, the MSH biosynthesis pathway may be important to protect M. smegmatis against gNO. These studies also showed that M. smegmatis was killed rapidly following the latency period, when exposed to 200 ppm gNO continuously for 10 hours. These results further suggest that MSH protected the cell from N O and suggest strongly that when MSH decreases below a critical level, the mycobacteria are extremely vulnerable and rapidly killed. These results support the findings of Rawat et al., (2002), who demonstrated a correlation between MSH depletion and toxin sensitivity in bacteria. Rawat used the same M. smegmatis MSH deficient mutants, and found that they were hypersensitive to alkylating agents, free radicals, and several antibiotics (Rawat et al., 2002). The data presented here provides additional proof that the MSH biosynthesis pathway is essential for protecting mycobacteria from ROIs and RNIs, specifically gNO. From these results we speculate that the reduced form of MSH probably protects the cell against free radicals by reacting with the N O entering the cell. There are two putative cellular MSH detoxification pathways for N O (Figure 26). In the first, N O and MSH would form an MS-NO conjugate, similar to other toxins, as shown in previous studies (Newton et al., 2000b). A key enzyme, mycothiol S-conjugate amidase (Mca) could then cleave the MS-NO-conjugate to produce glucosarninyl inositol (a substrate in the mycothiol biosynthesis pathway) and mercapturic acid (an N-acetylcysteine S-conjugate), which would then be excreted 97 (Newton et al., 2000b). The recycling of glucosaminyl inositol back into MSH biosynthesis is dependent on a multiplicity of enzymes and substrates. Figure 26. T w o potential cellular M S H detoxification pathways for N O . L E F T P A T H W A Y : El iminat ion of N O as mercapturic acid and the intracellular regeneration of M S H from GlcN-Ins . R I G H T : El iminat ion as nitrates and water (Vogt et al., 2003) with M S S M reduced back into M S H . Abbreviations: M S H , mycothiol; M c a , mycothiol S-conjugate amidase; N O , nitric oxide, O N O O " , peroxynitrite; M S S M , mycothiol disulphide; M S N O , S-nitrosomycothiol; GlcN-Ins , l-D-myo-inosityl-2-deoxy-D-glucopyranoside. Question marks denote theoretical pathways. An alternate mechanism by which gNO can be detoxified in the cell, is through metabolic pathways (Figure 26). MSH may oxidize N O (in the form of S-nitrosothiol) in the presence of S-mtrosomycothiol reductase (Patel and Blanchard, 1999). The proposed products of this reaction would be nitrates, water and mycothiol disulfide (MSSM). Another reductase (MSSM-reductase) may reduce MSSM to reduced MSH for further interaction with NO. 98 The experiments described above support the hypotheses that MSH is needed to protect M. smegmatis from endogenous N O toxicity. The results showed that when MSH was reduced or absent, the bacteria were even more susceptible to gNO, and supports the theory that the extent to which gNO is cytotoxic is dependent on the capacity of the cell to detoxify NO. The studies with M. smegmatis showed that mycobacteria appear to have a greater capacity to detoxify N O compared to other bacteria tested. This ability may have evolved as a survival response to the hostile phagosomal environment that mycobacteria face. The capacity to absorb N O influx is critical in bacterial defense against NO, and indicates that high concentrations of exogenous gNO, such as 200 ppm, can overwhelm this system and kill the bacteria. MSH is found in actinomycetes and may explain the innate capability of mycobacteria to resist host defenses. If MSH plays a role analogous to GSH and protects mycobacteria and other actinomycetes against N O toxicity, it may be possible to develop drugs that block enzymes in this N O detoxification pathway or overwhelm MSH action but have no adverse side effects on host cells. Perhaps gNO itself could be delivered safely and effectively either topically or as an inhaled agent. Further investigations in these areas are warranted. 99 5.6 M O V I N G T O T H E F U T U R E - T H E P O T E N T I A L U S E O F G A S E O U S N I T R I C O X I D E A S A T H E R A P E U T I C A G E N T The treatment of bacterial infections with antibiotics has been dominated by delivery via the parenteral or intravenous routes. These approaches require absorption and/or vascular delivery to the infected site for therapeutic effectiveness. They also expose the entire body to these therapeutic agents. The potential advantage of gNO is that it diffuses quickly into aqueous solution and can be delivered direcdy to the site of infection in its therapeutic form. That gNO had a bacteriocidal effect on organisms identified as causing pneumonia in mechanically ventilated patients, shows promise as a possible alternative or complement to the use of antibiotics to control infection. The key limitation of these studies for inhalational use of gNO is that the pathophysiology of nosocomial pneumonia (NP) is more complex than the model used in these experiments. As previously noted, most clinicians would be uncomfortable admimstering a concentration of gNO as high as 200 ppm to their patients. From a safety perspective, the greatest obstacles to gNO therapy are N O toxicity, N O z toxicity and methemoglobinemia. If these hurdles can be overcome then the future bodes well for gNO therapy. Potential applications for inhaled gNO are as a prophylactic treatment to decontaminate the airways and breatliing circuit to prevent the occurrence of NP or as a therapeutic agent once NP is suspected. Administration of gNO for NP could reduce the bacterial load, allowing the immune system and/or antibiotics to eliminate the infection. Administration of 200 ppm gNO may be more acceptable for non-inhalational applications such as the topical treatment of wound or skin infections. Methemoglobin formation or N O absorption through the skin or wound surface should be small, so there is no anticipated limitation to using this concentration. This approach is currendy being tested in preliminary clinical studies and is the foundation for future work (Section 6.2). The toxicology and pharmacokinetic report (FDA-FOI, 2000) for the FDA-approved gNO demonstrated that this drug is relatively non-toxic. In one study from this report, rats were exposed to room air or 80, 200, 300, 400, or 500 ppm N O for 6 hours per day for up to 7 days. There were no deaths below 300 ppm due to methemoglobinemia and the histological 100 data for the gNO-treated animals for all concentrations were similar to that in the control animals: no damage to the epithelium of the upper respiratory tract was found in any of the animals. There was no evidence of loss of or damage to cilia or ciliated cells and no evidence of toxicity was found in the mast cells. Electron microscopy data scored the epithelial edema for rats treated with 200 ppm as "moderate" at day one and "slight/mild" at day 7 . Another study in the same report exposed rats to room air, 40, 80, 160, 200 and 250 ppm N O for 6 hours/day for 28 days and observed no deaths below 200 ppm and the histology data indicated there was no evidence of damage. These studies strongly suggest that inhalational gNO at a concentration of 200 ppm could be safely delivered for short periods of time. Based on results from our intermittent studies, we would suggest a cyclic regimen of 160 ppm for 30 minutes and followed by 20 ppm for 3.5 hours. We have shown, in vitro, that this regimen had a complete bacteriocidal effect after only 24 hours. We predict that if these results project to clinical applications, a paradigm shift may occur in antibacterial therapy for a number of pulmonary diseases involving multi-drug resistant bacteria, such as nosocomial pneumonia. Other studies examining the effects of high dose N O exposures in animals have shown similar findings: toxicity due to N O is minimal if the duration of exposure is short. Stavert and Lehnert evaluated the acute exposure of rats to 500-1500 ppm N O (limiting N 0 2 to < 30 ppm) for 5-30 minutes, as well as to 10-100 ppm N 0 2 for 5 to 30 minutes (Stavert and Lehnert, 1990). There were no differences in histopathological evaluation between the N O -exposed animals and the room air-exposed control animals. In contrast, exposure to 50 ppm of N 0 2 for 30 minutes produced significant injury. Their conclusion was that short durations of very high concentrations of N O do not cause lung injury. The challenge will be to deliver gNO without the concomitant delivery of N 0 2 at toxic concentrations which may be accomplished by using pulsed-dose inhalational technology. We studied the delivery of gNO with pulsed-doses and found that this delivered high concentration bursts of gNO (80 ppm) during the first portion of the inhaled breath. Previously, pulsed-dose gNO did not appear advantageous, but now it may be useful for detemiining methods for safe and effective gNO delivery for pulmonary bacteriocidal indications. 101 Another concern is the toxic effect of methemoglobin accumulation. The amount of methemoglobin formed is in direct relation to the concentration and duration of inhaled gNO. High levels of methemoglobin block oxygen transport, leading to hypoxemia and death. The acceptable safe clinical concentration of methemoglobin in the blood is 2.5-5%. The predicted half-life of methemoglobin detected in the rats in study RDR-0075-DD was 182 minutes, but human data from the same report determined that the half-life of methemoglobin in blood was 1 hour (FDA-FOI, 2000). Healthy human volunteers have safely breathed 512 ppm gNO for up to 55 minutes at which time their methemoglobin levels reached 5% (Young, 1994). Accordingly, as long as there is at least 2 hours between doses of high gNO, the problem caused by high levels of methemoglobin should be nominal. The data from these FDA studies suggests that if 160 ppm gNO was inhaled for a short period of time (30 minutes), then the concentration of methemoglobin after 4 hours (and just prior to the next high dose of 160 ppm gNO) would only be 0.1%. Further, data in the FDA report indicated that inhalation of 40 ppm gNO continuously resulted in no difference in methemoglobin concentration between the control and gNO-exposed animals. The use of 80 ppm gNO continuous exposure has been reported in several human clinical trials and 40 ppm gNO delivered continuously for prolonged periods (days) is considered well within the range of safe therapeutic levels (Hurford, 2002; Kinsella, 1995; Zapol and Hurford, 1993). Since the patient's methemoglobin load would be a function of the ppm hours of N O exposure, a high-low gNO dosing strategy is proposed. The strategy used to effectively kill the bacteria in our intermittent studies (Section 3.3.3.1) would expose the patient to 900 ppm-hours per day (160 ppm x 0.5 hour x 6 + 20 ppm x 3.5 hours x 6 = 480 + 420 = 900 ppm-hours). This regimen would produce a lower methemoglobin burden than that of a patient exposed to 40 ppm continuously (40 ppm x 24 hours = 960 ppm-hours). Six cycles of the intermittent high-low gNO dose delivery regimen (160 ppm gNO for 0.5 hour followed by 20 ppm gNO for 3.5 hours) for 24 hours would keep the methemoglobin concentration under 2% (within OSHA guidelines). The safety and toxicological obstacles to gNO use as an inhaled bacteriocidal agent are not insurmountable. The studies reported here have shown that intermittent gNO exposure of 160 ppm for 0.5 hour followed by 20 ppm for 3.5 hours resulted in 100% bacterial killing in 4 to 6 cycles, or less than 24 hours. Non-specific cytotoxicity was observed as gNO delivered using this regimen achieved 100% killing for S. aureus P. aeruginosa, E. coli, Group B Streptococci and a 102 highly antibiotic-resistant strain of P. aeruginosa isolated from a cystic fibrosis patient. These results are only preliminary but are exciting nonetheless. This thesis has provided, for the first time, a validated system for direct study of the effect of gNO in vitro. This methodology provides a powerful tool to enhance our ability to study the effect of gNO on a wide variety of viruses, bacteria, and animal cells. The studies reported in this thesis have confirmed that N O is 100% bacteriocidal for a number of difficult-to-treat, drug-resistant pathogens, including two multi-drug resistant bacterial strains. These studies have shown that supraphysiologic concentrations of gNO are non-specific in activity and broad-spectrum in their ability to rapidly kill microorganisms. This thesis has also provided insight into bacterial detoxification pathways for gNO that enable them to resist host defenses. These studies provide the in vitro foundation to consider the use of gNO as a therapeutic antibacterial agent. It is my hope that ultimately, the knowledge derived from this thesis may provide the rationale to develop effective gNO treatments for pulmonary and wound infections caused by drug-resistant pathogens. 6.0 F U T U R E W O R K The work in this thesis showed that gNO can act as an antibacterial agent to lower the bacterial load associated with respiratory tract and/or dermal wound infections. The in vitro work presented in this thesis will need to be augmented with safety studies to provide evidence that the technique to delivery 200 ppm gNO is indeed possible. The groundwork done in this thesis has already provided strong rationale for commencement of animal efficacy and safety studies for application of gNO for non-healing infected wounds. Additional research is needed to explore the potential of gNO for pulmonary infections. This section will discuss the future work that will be undertaken in this area, not only in the continued exploration of the potential use of gNO as an antibacterial therapy, but for respiratory therapy in general. Each of the following sections will briefly describe the work that either will or can be undertaken to continue, both specific to this project, and in the field in general. 103 6.1 M E T H O D O L O G Y F O R G N O A N A L Y S I S A N D S T U D I E S O N A N T I B A C T E R I A L A C T I V I T Y O F G N O A seminal methodology was created and validated in this thesis for testing the effect of gNO on bacteria and other cell lines. This methodology has many potential applications for future work. Until now, studies on the effect of gNO on cells, has been hampered by a multiplicity of mdirect techniques that only extrapolate putative effect of gNO on cells. This thesis provides supporting data on the direct effect of gNO on cell survival and provides a validated methodology to continue work in this area on any number of cell lines in the future. The device is currently being utilized at two other institutions in North America to continue this work, by further quantifying the impact of gNO on various bacterial strains. It is also being used to test the effect of gNO on other cell types including human cell lines like fibroblasts, monocytes, endothelial, keratinocytes and epithelial cells. This work may even be expanded to evaluate the effects of various gases, such as helium, carbon dioxide and carbon monoxide, on cell lines. Research is being planned to use this methodology to evaluate drugs in combination with gNO. We anticipate that this methodology will focus on examuiing cells infected with viruses and intracellular pathogens as well as tumor cells. In addition, we anticipate continuing studies on the mechanism of gNO cytotoxicity in bacteria, especially mycobacteria, and to study the effect of gNO on the detoxification pathway in particular. More work will be performed on the gNO susceptibility in macrophages, specifically, to examine if we can kill the mycobacteria while keeping the macrophage alive. The methodology validated in this thesis is providing a tool to continue research into how mycobacteria protect themselves from high levels of N O produced by the macrophage. Future studies could examine the effect of first inhibiting proteasome function, then exposing the mycobacteria to gNO. Studies are currently in progress to evaluate 12 strains of 104 mycobacteria that are deficient in the production of the various key enzymes in the putative detoxification pathways. Inhibiting or using mutant species that reduce key enzymes such as mycothiol S-conjugate amidase and MSH thiol transferase, which function in the mycothiol detoxification pathway, could be studied with this methodology. Measuring the levels of mercapturic acid after exposing M. smegmatis to gNO and mass spectrophotometric analysis or nuclear magnetic resonance of the products would provide valuable insight into the proposed detoxification pathway. We are continuing work to ascertain if a treatment regimen can be developed for inhalational use. Our work has shown preliminary findings that ppm-hours of around 900 is effective as an bacteriocidal agent. Much more work is required to find a dosing regimen that will allow gNO to be inhaled in such a way that the high concentrations needed for bacteriocidal activity will not cause concomitant hypoxemia due to methemoglobinemia and N O and N 0 2 toxicity. Work is in progress to use the methodology to run a series of dosing studies to determine the optimal regimen. Additional work is in progress to specifically evaluate the effect of gNO on bacterial pathogens related to drug resistant strains of bacteria that have already caused the deaths of their hosts. Research has commenced to now evaluate the effect of gNO on biofilms, specifically on phenotypes of mucous and biofilm producing Burkerbolderia cepacia from cystic fibrosis patients. In is our intent to identify the optimal dosage of gNO to kill mycobacteria. We will then evaluate the effect of gNO on THP1 human alveolar macrophages, and determine the effect of gNO on macrophages that have been infected with mycobacteria. This research will allow us to determine if there is a possibility that gNO could potentially be used for tuberculosis treatment at an early stage of infection. Our future work using this device will be to make slight modifications so that rodents could be housed in the exposure cylinders. Ariimal studies are currendy being designed to evaluate the safety of gNO on lung tissue using various treatment regimens. Safety studies will measure methemoglobin levels, myeloperoxidases, serum N O x levels and other markers. 105 These results will be required prior to any evaluation of gNO potential in the human clinical model. This cell exposure device also has many important uses for others research as well. For instance very little work has been done to evaluate the effect of gNO on various materials used in conjunction with inhaled nitric oxide therapy. The device has already been used in material compatibility testing for PulmoNOx Medical Inc. and Viasys Healthcare. Other proprietary work has been done with endotracheal tabes to test the reaction with various concentrations and conditions associated with gNO. Because of the versatile design, this device can be used to test the impact of any kinds of gas or gas combinations. For instance the F i 0 2 can be adjusted between 0 and 1.0. Anerobic conditions were not used during the studies herein, however, anaerobic gas mixtures can easily be mixed and provided as test parameters dictate. For studies requiring high oxygen concentrations to study superoxide ions, this device may prove very useful. Macrophages require 5% carbon monoxide to remain viable during studies and this has been easily accomplished already with the device. 6.2 IN VIVO STUDIES In order to work towards validating the use of gNO for in vivo studies, we are currently working on the toxicology and safety issues related to the delivery of gNO to a wound. Animal studies have already been completed looking at the effect on bacterial load and showed a highly significant reduction in bacterial load in comparison to controls. Further histological evaluation showed that gNO treated tissue had less inflammatory bodies correlating with the decreased bacterial load. Animal studies showed that 200 ppm gNO delivered over five days to an open wound did not result in significant change in methemoglobin nor serum N O x levels. Results from these studies strengthened our hypothesis that gNO was an antibacterial agent for reducing bacterial load associated with wound infections (data not shown). 106 Systemic N O uptake from skin exposure is negligible as the surface area is miniscule when compared to the surface area of the lungs (which are equal to the surface area of a tennis court). Although 200 ppm gNO can be safely used for topical applications, it may not be acceptable for inhalational applications. The highest continuous level of inhaled N O generally reported has been 100 ppm (Francoe et al., 1998). There are reports that high levels (1000 ppm) of inhaled N O may be tolerated for short periods of time (Stavert and Lehnert, 1990). Additionally, cigarette, cigar and pipe smoke produce between 150 to 1200 ppm N O (Haagen-Amit, 1959). Stavert and Lehnert carefully controlled for contamination of the inhaled N O by N O z and reported that rats exposed to 500 ppm N O for 30 minutes showed no increases in extravascular lung water and had no evidence of lung injury (Stavert and Lehnert, 1990). Further strength to support our hypothesis was completed recentiy by successfully using gNO at 200ppm to treat a two-year old non-healing wound that had not responded to any therapy including antibiotics and silver ion dressings. This case study has been accepted for publication in the Journal of Cutaneous Medicine and Surgery. A copy of the manuscript is included in Appendix 2. This result was so promising that we designed a clinical trial entided "The effects of topical application of ViaNOx-H (gaseous nitric oxide) on the biofilm and wound healing in chronic non-healing wounds of the lower extremities" and submitted it for Institutional Ethics Approval (IRT3). We are pleased to report at the final hour of this thesis that the study has been approved and that industry is supporting our efforts to continue to further validate the hypothesis of this thesis. More work is needed to validate the hypothesis with regard to the use of gNO as an antibacterial agent for the respiratory tract. Long et ai, in an abstract at the American Thoracic Society meeting presented data that showed that 80 ppm gNO delivered to patients with infectious tuberculosis was unsuccessful (Long et ai, 2001). This suggests that higher levels of gNO may be needed to reduce the bacterial load in the human lung. Further work is required to identify a safe delivery regimen to deliver intermittent doses of gNO for identifying safe inhaled levels. Our preliminary results of delivery 160 ppm for 30 minutes and then 20 ppm for 3.5 hours showed promise. However, to deliver even this regimen is a formidable challenge that will keep us busy for some time to come. We are exploring high-107 low dosing regimens within the exposure device and industry has committed to support animal in vivo work if we can solve this challenge. Pulsed-dose delivery that delivers only gNO during the first 25% of an inspiratory breath may provide the answer for delivery of high doses of gNO safely to the lung. With the promising results in the wound healing area this supports the rationale for continued research in respiratory therapy use of gNO. This thesis has opened the door for studying the multifaceted aspects of gNO using a systematic validated methodology. 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This matrix used articles ranging from 1996 to 1998 that were referenced in the Bacteria section of Table II in Chapter 12 of Nitric Oxide and Infection (Fang, 1999). From 1999 to mid-2004, a M E D L I N E search was performed with the key words, "nitric and oxide and [bacterium species]". The following are the species that were searched for, with the number of cited articles in parentheses when the search was perfomed (June 2004): Bacillus (89), Brucella (16), Burkholderia (7), Chlamydia (24), Clostridium (23), Ehrlichia (7), Enterococcus (2) , Escherichia (474), Francisella (4), Helicobacter (113), Klebsiella (13), Listeria (53), Micrococcus (1), Mycobacterium (234), Mycoplasma (16), Pseudomonas (106), Rickettsia (4), Salmonella (154), Shigella (7), Staphylococcus (71), and Yersinia (9). It was observed that the frequency of published research related to antibacterial and Nitric Oxide research has grown significantly, perhaps exponentially. Articles that did not direcdy report on the antibacterial properties of Nitric Oxide were filtered out and not reported in the matrix. In the first round of filtering, each title was considered for relevance. Following this, each abstract was considered and full articles were searched for further details. 128 Bacillus Author. Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Weikert LF. Lopez JP. Abdolrasulnia R. Chroneos ZC. Shepherd VL. Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway. American Journal of Physiology -Lung Cellular & Molecular Physiology 279(2):L216-23. 2000 Bacillus Calmette-Guerin (BCG) none Rat macrophages were incubated with BCG in the presence and absence of SP-A. The release of TNF-alpha and NO were measured. NO was inhibited by L-NMMA. n/a Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. 129 Brucella Author . Tit le. Source Year Microbes Ki l led / Inhibited Microbes Not Ki l led / Inhibited Substance Used / Methodology Summary Concentrat ion Addi t ional Notes / Resu l ts Gross A, Bertholet S, Mauel J, Dornand J. Impairment of Brucella growth in human macrophagic cells that produce nitric oxide. Microb Pathog 36(2):75-82. 2004 Brucella suis none Using DFGiNOS U937 macrophagic cells engineered to produce NO and U937 cells activated by ligation of IgE receptors, it was shown that the intracellular development of Brucella was impaired in human macrophages, which produced NO. n/a n/a Wang M, Qureshi N, Soeurt N, Splitter G. High levels of nitric oxide production decrease early but increase late survival of Brucella abortus in macrophages. Microb Pathog 31(5):221-30. 2001 Brucella abortus none E. coli LPS and IFN-gamma were used to increase iNOS expression. 10 ng/ml E. coli LPS and 25 U/ml IFN-gamma NO accelerates B. abortus killing, but not to completion Gross A, Spiesser S, Terraza A, Rouot B, Caron E, Dornand J. Expression and bactericidal activity of nitric oxide synthase in Brucella su/s-infected murine macrophages. Infect Immun 66(4): 1309-16. 1998 Brucella suis none The expression and activity of iNOS in both gamma interferon (I FN) treated and untreated murine macrophages infected with the gram-negative bacterium Brucella suis was examined. iNOS was inhibited by L-NAME. variable B. su/s-infected murine macrophages, the posttranscriptional regulation of iNOS necessitates an additive signal triggered by macrophage Fc receptors. They also support the possibility that in mice, NO favors the elimination of Brucella, providing that IFN-and antibrucella antibodies are present. 130 Burkholderia Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Utaisincharoen P, Anuntagool N, Limposuwan K, Chaisuriya P, Sirisinha S. Involvement of beta interferon in enhancing inducible nitric oxide synthase production and antimicrobial activity of Burkholderia pseudomallei-infected macrophages. Infect lmmun71(6):3053-7. 2003 Burkholderia pseudomallei none Exogenous IFN-gamma added to upregulate IRF-1 and iNOS production. NO inhibitor L-NAME was also used in this study. 100 U/ml IFN-gamma Without exogenous IFN-gamma B. pseudomallei was not significantly killed, due to a low iNOS expression. Smith AW, Green J, Eden CE , Watson ML. Nitric oxide-induced potentiation of the killing of Burkholderia cepacia by reactive oxygen species: implications for cystic fibrosis. J Med Microbiol 48(5):419-23. 1999 Burkholderia cepacia none The combined effect of NO, superoxide and H202 was examined against B. cepacia. n/a NO and hydrogen peroxide appear to be the bactericidal mediators. Miyagi K, Kawakami K, Saito A. Role of reactive nitrogen and oxygen intermediates in gamma interferon-stimulated murine macrophage bactericidal activity against Burkholderia pseudomallei. Infect Immun 65(10):4108-13. 1997 Burkholderia pseudomallei none Examined RNI and ROI contributions to bactericide. IFN-gamma-activated macrophages inhibited bacteria growth. Nitrite and bactericidal activity inhibited by L-NMMA, SOD and catalase. Bacteria killed by chemically generated NO (Sodium Nitrite) and superoxide anion in a macrophage-free system. n/a Nitrite solution (latter part of study) was tested at pH of 4, 5, 6, 7. 131 Chlamydia Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed/ Inhibited Substance Used / Methodology Summary > NO Concentration Additional Notes / Results Chesebro BB, Blessing E, Kuo CC , Rosenfeld ME, Puolakkainen M, Campbell LA. Nitric oxide synthase plays a role in Chlamydia pneumoniae-induced atherosclerosis. Cardiovasc Res 60(1):170-4. 2003 Chlamydia pneumoniae none 3 groups of C. pneumoniae infected mice and 3 uninfected, atherogenic-diet-fed groups were tested: eNOS-deficient, iNOS-deficient, and control. n/a Production of NO by eNOS protects against development of fatty streak lesions in uninfected hyperlipidemic mice, but does not offer additional protection in infected hyperlipidemic mice, while iNOS may play a protective role, thus limiting chlamydial exacerbation of fatty streak lesions. Liuba P, Pesonen E, Paakkari I, Batra S, Andersen L, Forslid A, Yla-Herttuala S, Persson K, Wadstrom T, Wang X, Laurini R. Co-infection with Chlamydia pneumoniae and Helicobacter pylori results in vascular endothelial dysfunction and enhanced VCAM-1 expression in apoE-knockout mice. J Vase Res 40(2):115-22. 2003 Chlamydia pneumoniae and Helicobacter pylori none apoE-KO Mice were infected with either C. pneumoniae or H. pylori, or with both C. pneumoniae and H. pylori. L-NAME was used to inhibite NO. Nitrite/nitrate levels were measured. n/a Co-infection of apoE-KO mice with C. pneumoniae and H. pylori seems to be associated with impaired bioactivity of endothelial NO. Van Nerom A, Ducatelle R, Charlier G, Haesebrouck F. Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide. Dev Comp Immunol 24(4):417-32. 2000 Mycoplasma hyorhinis and M. gallisepticum Chlamydia psittaci Purified turkey monocytes were inoculated (in vitro) with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. L-NAME inhibited NO. n/a n/a 132 Chlamydia con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Igietseme J U , Uriri IM, Chow M, Abe E, Rank RG. Inhibition of intracellular multiplication of human strains of Chlamydia trachomatis by nitric oxide. Biochem Biophys Res Commun 232(3):595-601. 1997 Chlamydia trachomatis none Study evaluates the susceptibility of human isolates of C trachomatis to NO delivered by G S N O (NO donor) or the induction of the epithelial iNOS system by a cytokine-secreting T cell clone. GSNO: 5-50 uM Treatment of infected epithelial cells with 50 uM G S N O resulted in significant inhibition (approx. 70%) of chlamydial multiplication, while the NO scavenger, myoglobin plus ascorbate, could reverse the effect demonstrating that NO could directly inhibit human strains of Chlamydia. Chen B, Stout R, Campbell WF. Nitric oxide production: a mechanism of Chlamydia trachomatis inhibition in interferon-gamma-treated RAW264.7 cells. F E M S Immunol Med Microbiol 14(2-3):109-20. 1996 Chlamydia trachomatis none IFN-gamma and LPS induced nitrite production; L-NMMA inhibited nitrite production (and restored C trachomatis replication) IFN: 100 U/ml; LPS: 100 ng/ml; L-NMMA: 0.1-10 mM Cells treated with both IFN and L P S induced highest nitrite production (99.96 nmol/ml) and lowest CT recovery (2.4 log reduction). Igietseme J U . The molecular mechanism of T-cell control of Chlamydia in mice: role of nitric oxide. Immunology 87(1):1-8. 1996 Chlamydia trachomatis none IFN-gamma, anti-IFN-gamma antibodies, TNF-alpha, and NG-monomethyl-L-arginine monoacetate (MLA, NOS inhibitor). n/a n/a Igietseme J U , Uriri IM, Hawkins R, Rank RG. Integrin-mediated epithelial-T cell interaction enhances nitric oxide production and increased intracellular inhibition of Chlamydia. J Leukoc Biol 59(5):656-62. 1996 Chlamydia trachomatis none IFN-gamma producing T-cell clones cultured with epithelial cells to enhance NO production n/a n/a 133 Ehrlichia Author. Title. Source Year.; Microbes Killed/ Inhibited Microbes Not Killed/ Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Ganta RR, Wilkerson MJ, Cheng C, Rokey AM, Chapes SK. Persistent Ehrlichia chaffeensis infection occurs in the absence of functional major histocompatibility complex class II genes. Infect Immun 70(1):380-8. 2002 Ehrlichia chaffeensis none Toll-like receptor 4 (tlr4)- or major histocompatibility complex class II (MHC-ll)-deficient mice were used in these studies. n/a Macrophage activation and cell-mediated immunity, orchestrated by CD4(+) T cells, are critical for conferring resistance to E. chaffeensis. Banerjee R, Anguita J , Fikrig E. Granulocytic ehrlichiosis in mice deficient in phagocyte oxidase or inducible nitric oxide synthase. Infect Immun 68(7):4361-2. 2000 Granulocytic Ehrlichiosis none NOS2-deficient mice had delayed clearance of the agent of human granulocytic ehrlichiosis (HGE) in comparison to control or phox-deficient mice. n/a Reactive nitrogen intermediates may play a role in the early control of HGE. Gokce HI, Woldehiwet Z. Lymphocyte responses to mitogens and rickettsial antigens in sheep experimentally infected with Ehrlichia (Cytoecetes) phagocytophila. Veterinary Parasitology 83(1 ):55-64. 1999 none Ehrlichia phagocytophila The addition of the prostaglandin inhibitor, indomethacin, or the nitric oxide inhibitor, N(G)-monomethyl-L-arginine, had no significant effect on the suppressive effects of £. phagocytophila on lymphocyte reactivity to the mitogens. 300 uM (L-NMMA) n/a 134 Enterococcus Author. Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Simsek 1, Mas MR, Yasar M, Ozyurt M, Saglamkaya U, Deveci S, Comert B, Basustaoglu A, Kocabalkan F, Refik M. Inhibition of inducible nitric oxide synthase reduces bacterial translocation in a rat model of acute pancreatitis. Pancreas 23(3):296-301. 2001 Enterococcus sp. and Escherichia coli and Staphylococcus sp. and Proteus none S-methylisothiourea, an iNOS inhibitor, was used to improve the course of acute pancreatitis in rats. n/a Bacteria levels were lower; however, mortality was not affected. 135 Escherichia Author. Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Rudkowski J C , Barreiro E, Harfouche R, Goldberg P, Kishta O, D'Orleans-Juste P, Labonte J , LesurO, Hussain SN. Roles of iNOS and nNOS in sepsis-induced pulmonary apoptosis. Am J Physiol Lung Cell Mol Physiol 286(4):L793-800. 2004 Escherichia coli none Pulmonary apoptosis was measured in a rat model of E. coli lipopolysaccharide (LPS)-induced sepsis in the absence and presence of the selective iNOS inhibitor 1400W. n/a NO derived from iNOS plays an important protective role against sepsis-induced pulmonary apoptosis. Klink M, Cedzynski M, St Swierzko A, Tchorzewski H, Sulowska Z. Involvement of nitric oxide donor compounds in the bactericidal activity of human neutrophils in vitro. J Med Microbiol 52(Pt 4):303-8. 2003 Escherichia coli and Proteus vulgaris and Salmonella Anatum none Role of NO donors, SNP and SIN-1, in bacteria killing by neutrophils was tested. 10-1000 uM (donors) NO donors, S N P and SIN-1, enhanced the ability of neutrophils to kill bacteria. However, strains differed in their susceptibility to this process. Jana B, Andronowska A, Kucharski J . Involvement of nitric oxide in inflammation of ovaries in gilts. Reprod Biol 2(1):73-85. 2002 Escherichia coli and Staphylococcus aureus and Corynebacterium pyogenes none NADPH-diaphorase and an iNOS were demonstrated in porcine ovaries after unilateral infusion of bacteria into the hilus of an ovary. n/a n/a Tafalla C, Novoa B, Figueras A. Production of nitric oxide by mussel (Mytilus galloprovincialis) hemocytes and effect of exogenous nitric oxide on phagocytic functions. Comp Biochem Physiol B Biochem Mol Biol 132(2):423-31. 2002 none Escherichia coli The ability of mussel hemocytes to produce NO in response to phorbol myristate acetate was determined using the Griess reaction. NO inhibitor L-NMMA and NO donors, SNAP and GTN, were used. n/a NO exogenously produced by S N A P significantly inhibited the chemiluminescent response of mussel hemocytes, whereas it did not have a significant effect on the capability of these cells to phagocytose bacteria. 136 Escherichia con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Zhang B, Cao GL, Cross A, Domachowske JB, Rosen G M . Differential antibacterial activity of nitric oxide from the immunological isozyme of nitric oxide synthase transduced into endothelial cells. Nitric Oxide 7(1):42-9. 2002 Staphylococcus aureus and Escherichia coli none Primary cultures of endothelial cells, grown on the three-dimensional matrix Gelfoam where they take on the morphology of these cells in vivo, were used to test phagocytosis of S. aureus and two strains of E. coli. n/a S. aureus and E. coli induced separate microbicidal mechanisms of endothelial cells Carlsson S, Wiklund NP, Engstrand L, Weitzberg E, Lundberg JO. Effects of pH, nitrite, and ascorbic acid on nonenzymatic nitric oxide generation and bacterial growth in urine. Nitric Oxide 5(6):580-6. 2001 Escherichia coli and Pseudomonas aeruginosa and Staphylococcus saprophytics none NO formation and bacterial growth in mildly acidified human urine containing nitrite and the reducing agent vitamin C was studied. pH and nitrite levels were varied. n/a Large amounts of NO were produced and bacteria was markedly reduced with nitrite in mildly acidified urine. Inhibition was enhanced by ascorbic acid. Hardwick JB , Tucker AT, Wilks M, Johnston A, Benjamin N. A novel method for the delivery of nitric oxide therapy to the skin of human subjects using a semi-permeable membrane. Clin Sci (Lond) 100(4):395-400. 2001 Staphylococcus aureus and Escherichia coli none A chemical system using sodium nitrite and ascorbic acid to produce NO on the skin surface was administered with a selectively permeable, hydrophilic polyester co-polymer membrane system. The study measured vasodilation and anti-microbial effects of the system. 1 - 1000 mM sodium nitrite and ascorbic acid Potent anti-microbial properties of NO were seen at concentrations of nitrite above 50 mM 137 Escherichia con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary i > - NO Concentration Additional Notes / Results Simsek 1, Mas MR, Yasar M, Ozyurt M, Saglamkaya U, Deveci S, Comert B, Basustaoglu A, Kocabalkan F, Refik M. Inhibition of inducible nitric oxide synthase reduces bacterial translocation in a rat model of acute pancreatitis. Pancreas 23(3):296-301. 2001 Enterococcus sp. and Escherichia coli and Staphylococcus sp. and Proteus none S-methylisothiourea, an iNOS inhibitor, was used to improve the course of acute pancreatitis in rats. n/a Bacteria levels were lower; however, mortality was not affected. St John G, B ro tN, Ruan J , Erdjument-Bromage H, Tempst P, Weissbach H, Nathan C. Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates. Proc Natl Acad Sci USA 98(17):9901-6. 2001 Escherichia coli and Mycobacterium tuberculosis none The role of nitrite- and GSNO-produced NO was examined using MsrA-deficient E. coli and M. tuberculosis. n/a Nitrite and G S N O kill E. coli by intracellular conversion to peroxynitrite, intracellular Met residues in proteins constitute a critical target for peroxynitrite, and MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage. Suzuki S, Togari H, Yamaguchi N, Haas KM. Nitric oxide inhalation and nitric oxide synthase inhibitor supplement for endotoxin-induced hypotension. Pediatrlnt 43(4):343-9. 2001 Escherichia coli none Piglets were administered with E. coli lipopolysaccharide, then after 60 mins., inhaled NO, The non-control group also had L-NNA administered. Inhaled NO: 20 ppm for 60 or 120 mins. The combined therapy was effective against pulmonary hypertension 138 Escherichia con't Author. Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Kim SO, Orii Y, Lloyd D, Hughes MN, Poole RK. Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide. F E B S Lett 445(2-3):389-94. 1999 none Escherichia coli NO donors used: GSNO, SNP, and SNAP. Also, 20%-NO / 80%-N2 gas bubbled in solution. Hmp reacts with oxygen to turn NO into N 2 0 under anoxic growth conditions. 0.2 or 0.3 m M NO (final concentration) n/a Nagata K, Yu H, Nishikawa M, Kashiba M, Nakamura A, Sato EF, Tamura T, Inoue M. Helicobacter pylori Generates Superoxide Radicals and Modulates Nitric Oxide Metabolism. J Biol Chem 273(23):14071-14073. 1998 Helicobacter pylori and Escherichia coli none NO solution: prepared by bubbling Argon and then NO gas through 50 mM H E P E S - N a O H buffer, pH 7.4, for 15 minutes at 100 ml/min. The saturated NO solution (1.9 mM) was then kept on ice and used for experiments within 3h. 1.9 mM solution NO directly inhibited E. coli respiration, wheras it rapidly reacted with endogenously generated superoxide radicals in H. pylori. The resulting peroxynitrite inactivated the respiration of H. pylori. Marcinkiewicz J . Nitric oxide and antimicrobial activity of reactive oxygen intermediates. Immunopharmacology 37(1 ):35-41. 1997 Escherichia coli none Treated E. coli with G S N O (NO donor) and H202 or HOCI. pH and concentrations varied GSNO: variable E. coli was not inhibited under certain conditions, i.e., 10-30 M H O C I + 1000M GSNO; 1000M GSNO; small, sublethal amounts of H202 + GSNO) Dykhuizen RS, Frazer R, Duncan C, Smith CC , Golden M, Benjamin N, Leifert C. Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense. Antimicrob Agents Chemother 40(6):1422-5. 1996 Escherichia coli and Salmonella typhimurium and S. enteritidis and Shigella sonnei and Yersinia enterocolitica none The study examines the antimicrobial effect of acidified nitrite in vitro. pH was varied: 2.1, 3.0, 3.7, 4.2, 4.8, 5.4 variable (order of .1, 1 and 10 umol/mL) Susceptibility to the acidified nitrate solutions ranked as follows: Y. enterocolitica > S. enteritidis > S. typhimurium = Shigella sonnei > E. coli 139 Escherichia con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Hausladen A, Privalle CT, Keng T, DeAngelo J , Stamler JS. Nitrosative stress: activation of the transcription factor OxyR. Cell 86(5):719-29. 1996 Escherichia coli none SNO-Cy s and RSNO (NO donor with short half-life) + GSH = G S N O (NO donor) 0.01-2 mM S N O -Cys n/a 140 Francis elk Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Bosio CM , Elkins KL. Susceptibility to secondary Francisella tularensis live vaccine strain infection in B-cell-deficient mice is associated with neutrophilia but not with defects in specific T-cell-mediated immunity. Infect Immun 69(1): 194-203. 2001 Francisella tularensis none B-cell-deficient (BKO) mice vs. wild-type mice. Nitrite levels were monitered. n/a The control of F. tularensis live vaccine strain growth appeared to depend primarily on gamma interferon and nitric oxide and was similar in wild-type and B K O mice Cowley SC, Myltseva SV, Nano FE. Phase variation in Francisella tularensis affecting intracellular growth, lipopolysaccharide antigenicity and nitric oxide production. Mol Microbiol 20(4):867-74. 1996 Francisella tularensis Francisella tularensis Phase variation of F. tularensis postphones antimicrobial action of macrophage-produced NO. n/a n/a 141 Helicobacter Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Liuba P, Pesonen E, Paakkari 1, Batra S, Andersen L, Forslid A, Yla-Herttuala S, Persson K, Wadstrom T, Wang X, Laurini R. Co-infection with Chlamydia pneumoniae and Helicobacter pylori results in vascular endothelial dysfunction and enhanced VCAM-1 expression in apoE-knockout mice. J Vase Res 40(2): 115-22. 2003 Chlamydia pneumoniae and Helicobacter pylori none apoE-KO Mice were infected with either C. pneumoniae or H. pylori, or with both C. pneumoniae and H. pylori. L-NAME was used to inhibite NO. Nitrite/nitrate levels were measured. n/a Co-infection of apoE-KO mice with C. pneumoniae and H. pylori seems to be associated with impaired bioactivity of endothelial NO. Obonyo M, Guiney DG, Fierer J , Cole SP. Interactions between inducible nitric oxide and other inflammatory mediators during Helicobacter pyloriinfection. Helicobacter 8(5):495-502. 2003 none Helicobacter pylori Wild-type and iNOS gene deficient mice were infected with H. pylori strain SS1. n/a iNOS does not influence expression of inflammatory mediators in the early stages of H. pylori infection in mice. von Bothmer C. Edebo A. Lonroth H. Olbe L. Pettersson A. Fandriks L. Helicobacter pylori infection inhibits antral mucosal nitric oxide production in humans. Scand J Gastroenterol 37(4):404-8. 2002 Helicobacter pylori none Study investigates actual NO production in the human antrum in situ, on H. py/orApositive and -negative volunteers. n/a NO synthase expression is increased in H. pyforf-infected antral mucosa. However, NO synthesis is restricted owing to the presence of pathogen-induced competitive NOS inhibitors such as methylated arginines. 142 Helicobacter con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Gobert AP, McGee DJ, Akhtar M, Mendz GL, Newton J C , Cheng Y, Mobley HL, Wilson KT. Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: A strategy for bacterial survival. Proc Natl Acad Sci USA 98(24): 13844-9. 2001 none Helicobacter pylori Wild-type strains of H. pylori were compared with and H. pylori with an inactivated rocF gene. H. pylori inhibits NO production by activated macrophages at physiologic concentrations of L-arginine. n/a Macrophages cocultured with rocF-deficient H. pylori generated substantially higher levels of NO and resulted in efficient killing of the bacteria, whereas wild-type H. pylori exhibited no loss of survival under these conditions. Potter CL, Hanson P J . Exogenous nitric oxide inhibits apoptosis in guinea pig gastric mucous cells. Gut 46(2):156-62. 2000 Helicobacter pylori Helicobacter pylori Primary cultures of guinea pig gastric mucosal cells were exposed to the NO donor SNAP for 24 hours. 1 mM S N A P Exogenous NO inhibited apoptosis in guinea pig gastric mucous cells. NO, if from NOS during infection with H pylori, may therefore counter the proapoptotic effects of this pathogen. Dykhuizen RS, Fraser A, McKenzie H, Golden M, Leifert C, Benjamin N. Helicobacter pylori is killed by nitrite under acidic conditions. Gut 42(3):334-7. 1998 Helicobacter pylori none Addition of nitrite to acidic solutions (pH 2) resulted in complete kill of H. pylori within 30 minutes exposure time. 1 mM nitrite. Effective at concentrations > 500 uM Acid alone allowed the organism to survive. Nagata K, Yu H, Nishikawa M, Kashiba M, Nakamura A, Sato EF, Tamura T, Inoue M. Helicobacter pylori Generates Superoxide Radicals and Modulates Nitric Oxide Metabolism. J Biol Chem 273(23):14071-14073. 1998 Helicobacter pylori and Escherichia coli none NO solution: prepared by bubbling Argon and then NO gas through 50 mM H E P E S - N a O H buffer, pH 7.4, for 15 minutes at 100 ml/min. The saturated NO solution (1.9 mM) was then kept on ice and used for experiments within 3h. 1.9 mM solution NO directly inhibited E. coli respiration, wheras it rapidly reacted with endogenously generated superoxide radicals in H. pylori. The resulting peroxynitrite inactivated the respiration of H. pylori. 143 Klebsiella Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Brock TG, McNish RW, Mancuso P, Coffey MJ, Peters-Golden M. Prolonged lipopolysaccharide inhibits leukotriene synthesis in peritoneal macrophages: mediation by nitric oxide and prostaglandins. Prostaglandins Other Lipid Mediat71(3-4):131-45. 2003 none Klebsiella pneumoniae NOS was induced by lipopolysaccharide (LPS). Macrophages were treated with NO donor SNAP. NOS was inhibited by L-NMMA and L-NIL. n/a Macrophages exposed to prolonged LPS demonstrated impaired killing of K. pneumoniae and the combination of NOS and C O X inhibitors restored killing to the control level. Hickman-Davis JM , O'Reilly P, Davis IC, Peti-Peterdi J , Davis G, Young KR, Devlin RB, Matalon S. Killing of Klebsiella pneumoniae by human alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 282(5):L944-56. 2002 Klebsiella pneumoniae none Putative mechanisms by which human surfactant protein A effects killing of K. pneumoniae by human alveolar macrophages (AMs) isolated from bronchoalveolar lavagates of patients with transplanted lungs was investigated. NO inhibitor, L-NMMA, and NO donors, P A P A N O N O and SIN-1, were also used. 100 u.M PAPANONOate, 1 mM or 500 \iM SIN-1 SP-A mediates pathogen killing by AMs from transplant lungs by stimulating phagocytosis and production of ROI and RNI. 144 Legionella Author, Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary Concentration Additional Notes / Results Neumeister B, Bach V, Faigle M, Northoff H. Induction of iNOS in human monocytes infected with different Legionella species. F EMS Microbiol Lett 7;202(1):31-8. 2001 none Legionella pneumophila and L. micdadei and L. longbeachae and L. gormanii and L. steigerwaltii Stimulating NO production with 1,25-dihydroxyvitamin D3 and inhibiting NO production by L-NMMA were not able to modify the intracellular multiplication of legionellae within (human) Mono Mac 6 cells. n/a n/a Rajagopalan-Levasseur P, Lecointe D, Bertrand G, Fay M, Gougerot-Pocidalo MA. Differential nitric oxide (NO) production by macrophages from mice and guinea pigs infected with virulent and avirulent Legionella pneumophila serogroup 1. Clin Exp Immunol 104(1):48-53. 1996 Legionella pneumophila Legionella pneumophila Ex vivo production of NO by peritoneal macrophages of mice and guinea pigs. Virulent and avirulent strains of Leg. pneumophila tested. n/a mixed anti-microbial effects of NO 145 Listeria Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO '"' Concentration Additional Notes / Results Remer KA, Pfister H, Fatzer R, Leib SL, Jungi TW. [The role of nitric oxide in Listeria encephalitis of ruminants and in rats intracisternally infected with Listeria monocytogenes]. [German]. Berliner und Munchener Tierarztliche Wochenschrift 115(7-8):259-66. 2002 Listeria encephalitis none Measured iNOS levels in sheep, goats, and cattle brains, which had succumed to L. encephalitis. Then rats were studied. iNOS inhibitor L-NIL and the radical scavenger PBN resulted in rapid death of the treated animals. n/a Results suggest that reactive oxidants other than NO are also involved in controlling brain infection of Listeria. Ouadrhiri Y, Sibille Y, Tulkens PM. Modulation of intracellular growth of Listeria monocytogenes in human enterocyte Caco-2 cells by interferon-gamma and interleukin-6: role of nitric oxide and cooperation with antibiotics. J Infect Dis 180(4):1195-204. 1999 Listeria monocytogenes none IFN-gamma and interleukin IL-6 considerably reduced the bacterial intracellular growth, an effect largely abolished by NO inhibitor L-MMA. Both cytokines caused overexpression of iNOS. n/a n/a Ogawa R, Pacelli R, Espey MG, Miranda KM, Friedman N, Kim SM, Cox G, Mitchell JB, Wink DA, Russo A. Comparison of control of Listeria by nitric oxide redox chemistry from murine macrophages and NO donors: insights into listeriocidal activity of oxidative and nitrosative stress. Free Radic Biol Med 30(3):268-76. 2001 Listeria monocytogenes none The effects of NO exposure were examined, either delivered by NO donors (PAPA/NO and DEA/NO) or generated in situ within ANA-1 murine macrophages, on L. monocytogenes growth. n/a Nitrosative chemistry was not dependent upon nor mediated by interaction with reactive oxygen species, but resulted solely from NO and intermediates related to nitrosative stress. 146 Listeria con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Remer KA, Jungi TW, Fatzer R, Tauber MG, Leib SL. Nitric oxide is protective in listeric meningoencephalitis of rats. Infect Immun 69(6):4086-93. 2001 Listeria monocytogenes none Infant rats were infected intracisternally to generate experimental listeric meningoencephalitis and treated with the selective iNOS inhibitor, L-NIL. n/a Both reactive oxygen and NO. contribute to Listeria growth control. Akaki T, Sato K, Shimizu T, Sano C, Kajitani H, Dekio S, Tomioka H. Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids. J Leukoc Biol 62(6):795-804. 1997 Listeria monocytogenes (Lm) and Mycobacterium avium complex none Microbicidal activities of RNI, ROI, and free fatty acids against Mycobacterium avium complex (MAC) and the mode of macrophage (mphi) production of these effectors was studied. RNI specifics: scavengers for ROI or RNI, NOS inhibitors and acidified NaN02. n/a RNI with FFA temporarily participate in mphi-mediated killing of M A C in the relatively early phase after M A C stimulation. With Lm as a target organism, RNI + FFA and RNI + H202-halogenation gave a synergistic effect, whereas FFA + H202 -halogenation showed antagonism in exerting bactericidal activity. 147 Mycobacterium Author, Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Coban AY. Bayramoglu G. Ekinci B. Durupinar B. [Antibacterial effect of nitric oxide]. [Turkish] Mikrobiyoloji Bulteni. 37(2-3):151-5. 2003 Mycobacterium tuberculosis none Antibacterial effects of NO were tested using NO donor DETA-NO against 4 strains of multi-drug resistant M. tuberculosis and multiple isolates of Klebsiella, E. coli, Staphylococcus, Enterobacter, Pseudomonas and Proteus. n/a NO was more effective on multi-drug resistant M. tuberculosis strains than the other bacterial species. Cooper AM, Adams LB, Dalton DK, Appelberg R, Ehlers S. IFN-gamma and NO in mycobacterial disease: new jobs for old hands. Trends Microbiol 10(5):221-6. 2002 Mycobacterium avium M. leprae and M. tuberculosis In several murine models of mycobacterial infection, the role of IFN-gamma induced NO was measured. n/a n/a Da Si lva TR, De Freitas JR, Silva QC, Figueira CP, Roxo E, Leao SC, De Freitas LA, Veras PS. Virulent Mycobacterium fortuitum restricts NO production by a gamma interferon-activated J774 cell line and phagosome-lysosome fusion. Infect Immun 70(10):5628-34. 2002 none Mycobacterium fortuitum An in vitro assay was used to compare macrophage responses to opaque and transparent Mycobacterium fortuitum variants. NO donor SNAP and IFN-gamma were used to produced NO. SNAP: 100 uM n/a St John G, Brot N, Ruan J , Erdjument-Bromage H, Tempst P, Weissbach H, Nathan C. Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates. Proc Natl Acad Sci USA 98(17):9901-6. 2001 Escherichia coli and Mycobacterium tuberculosis none The role of nitrite- and GSNO-produced NO was examined using MsrA-deficient E. coli and M. tuberculosis. n/a Nitrite and G S N O kill E. coli by intracellular conversion to peroxynitrite, intracellular Met residues in proteins constitute a critical target for peroxynitrite, and MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage. 148 Mycobacterium con't Author. Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Wang CH, Kuo HP. Nitric oxide modulates interleukin-1beta and tumour necrosis factor-alpha synthesis, and disease regression by alveolar macrophages in pulmonary tuberculosis. Respirology 6(1):79-84. 2001 Mycobacterium tuberculosis none Purified alveolar macrophages were retrieved by bronchoalveolar lavage from TB patients and normal subjects, and cultured in the presence or absence of a NO inhibitor, L-NMMA. n/a The enhanced NO generation by macrophages of TB patients may play an autoregulatory role in amplifying the synthesis of pro-inflammatory cytokines, probably through the activation of NF-kappaB. Adams LB, Job CK, Krahenbuhl JL. Role of inducible nitric oxide synthase in resistance to Mycobacterium leprae in mice. Infect Immun 68(9):5462-5. 2000 Mycobacterium leprae none iNOS gene deficient mice were used in vivoXo test the role of RNI during infection by M. leprae. n/a RNI appear to have an important role in macrophage-mediated resistance to M. leprae, but infection may also be controlled in its absence. Garcia I. Guler R. Vesin D. Olleros ML. Vassalli P. Chvatchko Y. Jacobs M. Ryffel B. Lethal Mycobacterium bovis Bacillus Calmette Guerin infection in nitric oxide synthase 2-deficient mice: cell-mediated immunity requires nitric oxide synthase 2. 2000 Mycobacterium bovis BCG none NOS2-deficient mice were used in vivo to test the role of NO. n/a Infected wild-type mice survived and NOS2-deficient mice died. Jagannath C, Sepulveda E, Actor JK, Luxem F, Emanuele MR, Hunter RL. Effect of poloxamer CRL-1072 on drug uptake and nitric-oxide-mediated killing of Mycobacterium avium by macrophages. Immunopharmacology 48(2): 185-97. 2000 Mycobacterium avium none Ploxamer CRL-1072 was tested to induce production of mRNA for iNOS and NO by U937 cells. iNOS inhibitor, NMMA, was used reverse NO-induced macrophage killing of bacteria. n/a CRL-1072 enhances ability of NO-induced macrophages to kill ingested organisms. 149 Mycobacterium con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Majumdar S, Gupta R, Dogra N. Interferon-gamma- and lipopolysaccharide-induced tumor necrosis factor-alpha is required for nitric oxide production: tumor necrosis factor-alpha and nitric oxide are independently involved in the killing of Mycobacterium microti in interferon-gamma- and lipopolysaccharide-treated J774A.1 cells. Folia Microbiol (Praha) 45(5):457-63. 2000 Mycobacterium microti none Cells containing antisense TNF-alpha plasmid were used to determine the role of endogenous TNF-alpha on nitric oxide production as well as on the growth of Mycobacterium microti in IFN-gamma and lipopolysaccharide treated cells. L-NMA was used to inhibit NO production. n/a TNF-alpha and NO are independently involved in the killing of intracellular M. microti with IFN-gamma and L P S Gomes MS, Florido M, Pais TF, Appelberg R. Improved clearance of Mycobacterium avium upon disruption of the inducible nitric oxide synthase gene. J Immunol 162(11 ):6734-9. 1999 Mycobacterium avium Mycobacterium avium iNOS gene deficient mice were used in vivo and in vitro to test the role of NO during infection by M. avium. n/a NO had no effect in vitro. In vivo, the infection progressed at similar rates for both groups of mice for 2 months. After 2 months, however, iNOS gene deficient mice were more efficient in bacteria clearing. Long R, Light B, Talbot JA. Mycobacteriocidal action of exogenous nitric oxide. Antimicrob Agents Chemother 43(2):403-5. 1999 Mycobacterium tuberculosis none Exogenous Nitric Oxide, in vitro; < 24 hours of exposure < 100 ppm n/a 150 Mycobacterium con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Chen L, Xie QW, Nathan C. Alkyl hydroperoxidase reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates. Mol Cell 1:795-805. 1998 Salmonella typhimurium and Mycobacterium tuberculosis none Test if AhpC could protect cells from reactive nitrogen intermediates (RNI). RNI is delivered exogenously or produced endogenously by transfected NOS at pH 5 and 7 various concentrations of nitrite, nitrate, G S N O Resistance to RNI appears to be a physiologic function of ahpC. AhpC is the most widely distributed gene known that protects cells directly from RNI, and provides an enzymatic defense against an element of antitubercular immunity. Akaki T, Sato K, Shimizu T, Sano C, Kajitani H, Dekio S, Tomioka H. Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids. J Leukoc Biol 62(6):795-804. 1997 Listeria monocytogenes (Lm) and Mycobacterium avium complex none Microbicidal activities of RNI, ROI, and free fatty acids against Mycobacterium avium complex (MAC) and the mode of macrophage (mphi) production of these effectors was studied. RNI specifics: scavengers for ROI or RNI, NOS inhibitors and acidified NaN02. n/a RNI with FFA temporarily participate in mphi-mediated killing of MAC in the relatively early phase after MAC stimulation. With Lm as a target organism, RNI + FFA and RNI + H202-halogenation gave a synergistic effect, whereas FFA + H202-halogenation showed antagonism in exerting bactericidal activity. Zhao B, Collins MT, Czuprynski C J . Effects of gamma interferon and nitric oxide on the interaction of Mycobacterium avium subsp. paratuberculosis with bovine monocytes. Infect Immun 65(5):1761-6. 1997 Mycobacterium avium subsp. Paratuberculosis and Mycobacterium avium (similar results) none, except under certain condition (see additional notes) L-NMMA (inhibitor), rIFN-gamma, and SNAP (donor) N02 < 5 pM/well M. avium subsp. Paratuberculosis was killed by SNAP-generated NO, but killing of greater than 1 log of M. avium subsp. Paratuberculosis cells required approx. 200 pM nitrite. 151 Mycobacterium con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary "'A>NO Concentration Additional Notes / Results Arias M, Rojas M, Zabaleta J , Rodriguez J l , Paris SC, Barrera LF, Garcia LF. Inhibition of virulent Mycobacterium tuberculosis by Bcg(r) and Bcg(s) macrophages correlates with nitric oxide production. J Infect Dis 176(6):1552-8. 1997 Mycobacterium tuberculosis none The in vitro ability of B10R and BIOS murine macrophages to inhibit M. tuberculosis and to produce NO in response to infection and IFN-gamma was compared. NMMA and L-arginine also used in study. N02 < 90 uM none MacMicking JD, North RJ, LaCourse R, Mudgett JS , Shah SK, Nathan CF. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci USA 94(10):5243-8. 1997 Mycobacterium tuberculosis none Tests were performed with NOS2-deficient mice, NO inhibitor NIL (pH 2.7), and NO donor RSNO. Mycobacterium tuberculosis replication was measured in the lungs. 1.5-200 u.M RSNO NOS2 identified as a critical host gene for tuberculostasis Rhoades ER, Orme IM. Susceptibility of a panel of virulent strains of Mycobacterium tuberculosis to reactive nitrogen intermediates. Infect Immun 65(4):1189-95. 1997 Mycobacterium tuberculosis none All of the strains tested were killed by levels of RNI generated by the acidification of NaN02 to pH 6.5 or 5.5, and the strains exhibited a range of tolerance to lower concentrations of RNI. 10 mM NaN02 Under stringent conditions, RNI can kill M. tuberculosis, but under less harsh, more physiological conditions, the effects of RNI range from partial to negligible inhibition. Chan J , Tian Y, Tanaka KE, Tsang MS, Yu K, Salgame P, Carroll D, Kress Y, Teitelbaum R, Bloom BR. Effects of protein calorie malnutrition on tuberculosis in mice. Proc Natl Acad Sci USA 93(25):14857-61. 1996 Mycobacterium tuberculosis none Mice were malnourished with a low-protein diet. IFN-gamma, TNF-alpha, iNOS levels were monitored. n/a Decrease in NO corresponded with increase in infection 152 Mycoplasma Author, Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Hickman-Davis JM . Gibbs-Erwin J . Lindsey JR. Matalon S. Role of surfactant protein-A in nitric oxide production and mycoplasma killing in congenic C57BL/6 mice. Am J Respir Cell Mol Biol 30(3):319-25. 2004 Mycoplasma pulmonis none Role of surfactant protein A studied in regulating NO production using SP-A deficient mice. Nitrite/nitrate levels monitored. n/a SP-A suppressed NO in the absence of bacteria and increased NO in the presence of bacteria. Van Nerom A, Ducatelle R, Charlier G, Haesebrouck F. Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp:. the importance of nitric oxide. Dev Comp Immunol 24(4):417-32. 2000 Mycoplasma hyorhinis and M. gallisepticum Chlamydia psittaci Purified turkey monocytes were inoculated (In vitro) with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. L-NAME inhibited NO. n/a n/a Ribeiro-Dias F, Russo M, Marzagao Barbuto JA, Fernandes do Nascimento FR, Timenetsky J , Jancar S. Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages toward YAC-1 tumor cells through production of NO. J Leukoc Biol 65(6):808-14. 1999 Mycoplasma arginini none NO produced by NO donor SNP and inhibited by L-NAME or aminoguanidine Variable concentrations of S N P ; 1 0 m M L -NAME; 1 mM aminoguanidine M. arginini activates thioglycollate-elicited murine macrophages for NO and T N F release increasing their cytotoxic activity toward Y A C -1 cells. This activity is dependent on NO but not TNF release 153 Mycoplasma con't Author. Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Hickman-Davis J , Gibbs-Erwin J , Lindsey JR, Matalon S. Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages by production of peroxynitrite. Proc Natl Acad Sci USA 96(9):4953-8. 1999 Mycoplasma pulmonis none SP-A deficient and iNOS deficient mice were used in in vivo study. NO generated by SIN-1 (1 mM or 200 u.M) and PAPA in in vitro study. 1 mM SIN-1; 100 pM PAPA NONOate Peroxynitrite generation by AMs is necessary for the killing of a pathogen in vitro and in vivo. Hickman-Davis JM , Lindsey JR, Zhu S, Matalon S. Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages. Am J Physiol 274(2, Pt 1):L270-7. 1998 Mycoplasma pulmonis none Alveolar macrophages from mice, activated with IFN-gamma and incubated with surfactant protein A, produced NO and decreased mycoplasma CFUs by 8 3 % in 6hrs. When nitrite/nitrate was inhibited by L-NMMA, no bacteria were killed. n/a n/a 154 Pseudomonas Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Coban AY. Bayramoglu G. Ekinci B. Durupinar B. [Antibacterial effect of nitric oxide]. [Turkish] Mikrobiyoloji Bulteni. 37(2-3):151-5. 2003 Mycobacterium tuberculosis none Antibacterial effects of NO were tested using NO donor DETA-NO against 4 strains of multi-drug resistant M. tuberculosis and multiple isolates of Klebsiella, E. coli, Staphylococcus, Enterobacter, Pseudomonas and Proteus. n/a NO was more effective on multi-drug resistant M. tuberculosis strains than the other bacterial species. Darling KE, Evans TJ. Effects of nitric oxide on Pseudomonas aeruginosa infection of epithelial cells from a human respiratory cell line derived from a patient with cystic fibrosis. Infect Immun 71(5):2341-9. 2003 Pseudomonas aeruginosa none Using cells transfected with human iNOS cDNA, the effect of NO on P. aeruginosa replication, adherence,, and internalization was studied. n/a n/a Nablo BJ , Schoenfisch MH. Antibacterial properties of nitric oxide-releasing sol-gels. J Biomed Mater Res 67A(4):1276-83. 2003 Pseudomonas aeruginosa none Various Sol-gel surfaces were tested: DET3, AHAP3, AEMP3, AEAP2. flow rate = 200 mL/min Sol-gel surfaces capable of NO release decrease bacterial adhesion by 3 0 % to 9 5 % relative to controls. Dukelow AM, Weicker S, Karachi TA, Razavi HM, McCormack DG, Joseph MG, Mehta S. Effects of nebulized diethylenetetraamine-NONOate in a mouse model of acute Pseudomonas aeruginosa pneumonia. Chest 122(6):2127-36. 2002 Pseudomonas aeruginosa none Mice received no treatment, nebulized DETA-NO (NO donor) at 4 h and 12 h, or continuous inhaled NO for 24 h until they were killed at 24 h. Inhaled NO: 10 or 40 ppm; DETA-NO: 12.5 or 125 pmol The antibacterial effect of DETA-NO in vivo and in vitro is due, in large part, to the DETA nucleophile moiety and is independent of NO, suggesting a limited therapeutic role for exogenous NO in pneumonia. 155 Pseudomonas con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary Concentration Additional Notes / Results Jean D, Maitre B, Tankovic J , Meignan M, Adnot S, Brun-Buisson C, Harf A, Delclaux C. Beneficial effects of nitric oxide inhalation on pulmonary bacterial clearance. Crit Care Med 30(2):442-7. 2002 Pseudomonas aeruginosa none Rats were exposed to 0 2 or 02+NO inhalation during 24 hrs. Alveolar neutrophil levels, bacterial clearance and protein concentration were monitored. In vitro experiments were also performed using NO donor, SIN-1 or DPTA. Inhaled NO: 10 ppm NO inhalation resulted in a direct bactericidal effect and an influx of alveolar neutrophils in clearing bacteria. Carlsson S, Wiklund NP, Engstrand L, Weitzberg E, Lundberg JO. Effects of pH, nitrite, and ascorbic acid on nonenzymatic nitric oxide generation and bacterial growth in urine. Nitric Oxide 5(6):580-6. 2001 Escherichia coli and Pseudomonas aeruginosa and Staphylococcus saprophytics none NO formation and bacterial growth in mildly acidified human urine containing nitrite and the reducing agent vitamin C was studied. pH and nitrite levels were varied. n/a Large amounts of NO were produced and bacteria was markedly reduced with nitrite in mildly acidified urine. Inhibition was enhanced by ascorbic acid. Webert KE, Vanderzwan J , Duggan M, Scott JA, McCormack DG, Lewis JF , Mehta S. Effects of inhaled nitric oxide in a rat model of Pseudomonas aeruginosa pneumonia. Crit Care Med 28(7):2397-405. 2000 Pseudomonas aeruginosa none The effects of inhaled NO on pulmonary infections, leukocyte infiltration, and NOS activity was studied. Rats were exposed to inhaled NO or air for 24 hrs before being killed. In vitro effect of ambient NO on P. aeruginosa was also measured. Inhaled NO: 40 ppm; ambient NO: 40 ppm In vitro study confirmed a delayed antibacterial effect of NO. 156 Rickettsia Author. Title. Source Year Microbes Killed/ Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Feng HM, Walker DH. Mechanisms of intracellular killing of Rickettsia conorii\r\ infected human endothelial cells, hepatocytes, and macrophages. Infect Immun 68(12):6729-36. 2000 Rickettsia conorii none R. conor//-i nfected endothelial cells (HUVECs) stimulated with cytokines (TNF-alpha, IFN-gamma, and IL-1beta) and a chemokine (RANTES) were tested. NO was inhibited NMMLA. variable Human cells are capable of controlling rickettsial infections intracellularly by one or a combination of three mechanisms involving NO synthesis, hydrogen peroxide production, and tryptophan degradation. Turco J , Liu H, Gottlieb SF, Winkler HH. Nitric oxide-mediated inhibition of the ability of Rickettsia prowazekii to infect mouse fibroblasts and mouse macrophagelike cells. Infect Immun 66(2):558-66. 1998 Rickettsia prowazekii none Fibroblastic L929 cells treated with crude lymphokine preparations or with IFN-gamma and TNF-alpha nitrite and 0.5 mM NO in solution NO released from appropriately stimulated potential host cells kills extracellular rickettsiae and thus prevents the rickettsiae from infecting the cells Walker DH, Popov VL, Crocquet-Valdes PA, Welsh C J , Feng HM. Cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells. Lab Invest 76(1 ):129-38. 1997 Rickettsia conorii none NO production and rickettsial survival and growth were examined under four different experimental conditions: (a) no cytokine treatment, (b) treatment with IFN-[gamma] and TNF-[alpha], (c) treatment with cytokines and NMMLA, and (d) treatment with sodium nitroprusside, a source of NO. 100 pM NMMLA; 100 pM sodium nitroprusside n/a 157 Salmonella Author. Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Abu-Shakra A. The mutagenic activity of the S-nitrosoglutathione/glutathione system in Salmonella typhimurium TA1535. Mutat Res 539(1-2):203-6. 2003 Salmonella typhimurium none S-nitrosoglutathine (GSNO) and reduced glutathione (GSH) were tested for mutagenicity against strain Salmonella typhimuriumtMSSb 10 - 50 uM per plate Neither G S N O nor G S H were mutagenic when tested alone. In combination, the GSNO/GSH system induced a positive mutagenic response Coban AY, Durupinar B. The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro. Mem Inst Oswaldo Cruz 98(3):419-23. 2003 Salmonella typhimurium none The effect of NO alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin was investigated in vitro. NO donor DETA-NO was dissolved in 0.1 M NaOH with antibiotics. n/a Combinations of DETA-NO with these agents were less effective than DETA-NO alone, against some isolates. Eriksson S, Chambers BJ, Rhen M. Nitric oxide produced by murine dendritic cells is cytotoxic for intracellular Salmonella enterica sv. Typhimurium. Scand J Immunol 58(5):493-502. 2003 Salmonella typhimurium none The role of dendritic cells in producing iNOS and NO for killing bacteria was investigated. iNOS was inhibited by N-monomethyl-l-arginine. Cells from iNOS gene-deficient mice were used. n/a DC-derived NO was exerted a bactericidal effect, whereas the effect of NO in macrophage-like cells was bacteriostatic. Klink M, Cedzynski M, St Swierzko A, Tchorzewski H, Sulowska 2. Involvement of nitric oxide donor compounds in the bactericidal activity of human neutrophils in vitro. J Med Microbiol 52(Pt 4):303-8. 2003 Escherichia coli and Proteus vulgaris and Salmonella Anatum none Role of NO donors, SNP and SIN-1, in bacteria killing by neutrophils was tested. 10-1000 uM (donors) NO donors, S N P and SIN-1, enhanced the ability of neutrophils to kill bacteria. However, strains differed in their susceptibility to this process. 158 Salmonella con't Author. Title. Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary Concentration Additional Notes / Results Alam MS, Akaike T, Okamoto S, Kubota T, Yoshitake J , Sawa T, Miyamoto Y, Tamura F, Maeda H. Role of nitric oxide in host defense in murine salmonellosis as a function of its antibacterial and antiapoptotic activities. Infect Immun 70(6):3130-42. 2002 Salmonella enterica none iNOS-deficient mice were infected with an avirulent or virulent Salmonella enterica serovar Typhimurium strain n/a NO has a direct antimicrobial effect as well as cytoprotective actions for infected host cells, possibly through its antiapoptotic effect. Vazquez-Torres A, Jones-Carson J , Mastroeni P, Ischiropoulos H, Fang FC. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro. J Exp Med 192(2):227-36. 2000 Salmonella typhimurium none Congenic iNOS-/-, gp91phox-/-, and doubly immunodeficient iNOS -/-qp91phox-/- mice (gene-deficient mice) n/a IFN-gamma appears to augment antibacterial activity predominantly by enhancing NO production, although a small iNOS-independent effect was also observed. Crawford MJ, Goldberg DE. Role for the Salmonella flavohemoglobin in protection from nitric oxide. J Biol Chem 273(20):12543-7. 1998 Salmonella typhimurium none Acidified nitrite and S-nitrosothiols: GSNO, SNAC nitrite: 3mM, 30mM; plate cultures: 10mM GSNO; liquid cultures: 500uM G S N O S. typhimurium strain displays an increased sensitivity to acidified nitrite and S-nitrosothiols 159 Salmonella con't Author. Title. Source Year. Microbes Killed / Inhibited Microbes Not Killed/ Inhibited Substance Used / Methodology Summary NO -Concentration Additional Notes / Results Chen L, Xie QW, Nathan C. Alkyl hydroperoxidase reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates. Mol Cell 1:795-805. 1998 Salmonella typhimurium and Mycobacterium tuberculosis none Test if AhpC could protect cells from reactive nitrogen intermediates (RNI). RNI is delivered exogenously or produced endogenously by transfected NOS at pH 5 and 7 various concentrations of nitrite, nitrate, G S N O Resistance to RNI appears to be a physiologic function of ahpC. AhpC is the most widely distributed gene known that protects cells directly from RNI, and provides an enzymatic defense against an element of antitubercular immunity. De Groote MA, Ochsner UA, Shiloh MU, Nathan C, McCord JM , Dinauer MC, Libby S J , Vazquez-Torres A, Xu Y, Fang FC. Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase. Proc Natl Acad Sci USA 94(25): 13997-4001. 1997 Salmonella typhimurium none Salmonella deficient in Cu,Zn-SOD tested. G S N O (NO donor) and SIN-1, and Xanthine oxidase (XO) and SPER/NO (NO donor) were used in tests. Killing blocked by L-NMMA and aminoguanidine. 500 mM GSNO, 500 mM SIN-1, 0.1 unit/ml XO and 1 mM SPER/NO Effect on sodC mutant Salmonella: SPER/NO - no significant effect; XO - approx. 9 7 % decrease in sodC after 2 hours; XO and SPER/NO -approx. 99.97% decrease after 2 hours Umezawa K, Akaike T, Fujii S, Suga M, Setoguchi K, Ozawa A, Maeda H. Induction of nitric oxide synthesis and xanthine oxidase and their roles in the antimicrobial mechanism against Salmonella typhimurium infection in mice. Infect Immun 65(7):2932-40. 1997 Salmonella typhimurium none xanthine oxidase (XO) and DETC (NO donor) tested seperately and together DETC: 400 mg/kg Both XO and NO play an important role in the antimicrobial mechanism 160 Salmonella con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed/ Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Dykhuizen RS, Frazer R, Duncan C, Smith CC , Golden M, Benjamin N, Leifert C. Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense. Antimicrob Agents Chemother 40(6):1422-5. 1996 Escherichia coli and Salmonella typhimurium and Salmonella enteritidis and Shigella sonnei and Yersinia enterocolitica none The study examines the antimicrobial effect of acidified nitrite in vitro. pH was varied: 2.1, 3.0, 3.7, 4.2, 4.8, 5.4 v riable (order of .1,1 and 10 umol/mL) Susceptibility to the acidified nitrate solutions ranked as follows: Y. enterocolitica > S. enteritidis > S. typhimurium = Shigella sonnei > E. coli De Groote MA, Testerman T, Xu Y, Stauffer G, Fang FC. Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium. Science 272(5260):414-7. 1996 Salmonella typhimurium none Role of homocysteine investigated. NO's antimicrobial activity was demonstrated using GSNO, SNAC, SIN-1 and DETA-NO; aminoguanidine inhibited NO synthesis and increased S. typhimurium survival. 1 mM G S N O n/a Meli R, Raso GM, Bentivoglio C, Nuzzo I, Galdiero M, Di Carlo R. Recombinant human prolactin induces protection against Salmonella typhimurium infection in the mouse: role of nitric oxide. Immunopharmacology 34(1 ):1-7. 1996 Salmonella typhimurium none Role of recombinant human prolactin (rhPRL) studied. NO production was involved in the protective effect of rhPRL since pre-treatment of the animals with the NO inhibitor L-NAME completely reverted the protective activity, whereas D-NAME, the inactive isomer, was without effect. n/a n/a 161 Shigella Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed/ Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Biswas T. Role of porin of Shigella dysenteriae type 1 in modulation of lipopolysaccharide mediated nitric oxide and interleukin-1 release by murine peritoneal macrophages. F E M S Immunol Med Microbiol 29(2):129-36. 2000 Shigella dysenteriae, type 1 none Porin was added to macrophages in tissue culture medium with or without NMMA to assess the specificity of NO release. Cells were incubated with or without porin or porin-NMMA alone or in combination with any one of the modulators I FN, LPS, LPA, PS. 250 M NMMA; 100 U/ml I FN; 10g/ml LPS; 200 ng/ml LPA; 200 ng/ml PS. n/a Balter-Seri J , Yuhas Y, Weizman A, Nofech-Mozes Y, Kaminsky E, Ashkenazi S. Role of nitric oxide in the enhancement of pentylenetetrazole-induced seizures caused by Shigella dysenteriae. Infect Immun 67(12):6364-8. 1999 none Shigella dysenteriae 60R sonicate NO inhibitors: SMT, NNA SMT: 2.0 mg/kg of body weight; NNA: 2.5 mg/kg of body weight Findings indicate that NO, induced by S. dysenteriae 60R sonicate, is involved in enhancing the susceptibility to seizures caused by S. dysenteriae Dykhuizen RS, Frazer R, Duncan C, Smith CC, Golden M, Benjamin N, Leifert C. Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense. Antimicrob Agents Chemother 40(6):1422-5. 1996 Escherichia coli and Salmonella typhimurium and Salmonella enteritidis and Shigella sonnei and Yersinia enterocolitica none The study examines the antimicrobial effect of acidified nitrite in vitro. pH was varied: 2.1, 3.0, 3.7, 4.2, 4.8, 5.4 variable (order of .1, 1 and 10 umol/mL) Susceptibility to the acidified nitrate solutions ranked as follows: Y. enterocolitica > S. enteritidis > S. typhimurium = Shigella sonnei > E. coli 162 Staphylococcus Author. Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Roth MD, Whittaker K, Salehi K, Tashkin DP, Baldwin GC. Mechanisms for impaired effector function in alveolar macrophages from marijuana and cocaine smokers. J Neuroimmunol 147(1-2):82-6. 2004 Staphylococcus aureus none Alveolar macrophages were recovered from nonsmokers, regular tobacco smokers, marijuana smokers, or crack cocaine smokers, and evaluated for their production of NO and the role of NO as an antimicrobial effector molecule. L-NMMA inhibited NO. n/a The host defense mechanism is suppressed by habitual exposure to inhaled marijuana or crack cocaine in vivo. Coban AY. Bayramoglu G. Ekinci B. Durupinar B. [Antibacterial effect of nitric oxide]. [Turkish] Mikrobiyoloji Bulteni. 37(2-3):151-5. 2003 Mycobacterium tuberculosis none Antibacterial effects of NO were tested using NO donor DETA-NO against 4 strains of multi-drug resistant M. tuberculosis and multiple isolates of Klebsiella, E. coli, Staphylococcus, Enterobacter, Pseudomonas and Proteus. n/a NO was more effective on multi-drug resistant M. tuberculosis strains than the other bacterial species. Jana B, Andronowska A, Kucharski J . Involvement of nitric oxide in inflammation of ovaries in gilts. Reprod Biol 2(1):73-85. 2002 Escherichia coli and Staphylococcus aureus and Corynebacterium pyogenes none NADPH-diaphorase and an iNOS were demonstrated in porcine ovaries after unilateral infusion of bacteria into the hilus of an ovary. n/a n/a Rachlis A, Watson JL, Lu J , McKay DM. Nitric oxide reduces bacterial superantigen-immune cell activation and consequent epithelial abnormalities. J Leukoc Biol 72(2):339-46. 2002 Staphylococcus aureus enterotoxin B none The ability of NO to alleviate SEB-induced epithelial dysfunction and immune cell activation was examined. Human peripheral blood mononuclear cells were activated by SEB for 24 h with or without the NO donors, SNAP and spermine-NONOate. These findings suggest a beneficial role for NO in inflammation by reducing immune cell activation and thus ameliorating consequent physiological abnormalities, in this instance, perturbed epithelial permeability and active ion transport. 163 Staphylococcus con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Zhang B, Cao GL, Cross A, Domachowske JB, Rosen GM. Differential antibacterial activity of nitric oxide from the immunological isozyme of nitric oxide synthase transduced into endothelial cells. Nitric Oxide 7(1):42-9. 2002 Staphylococcus aureus and Escherichia coli none Primary cultures of endothelial cells, grown on the three-dimensional matrix Gelfoam where they take on the morphology of these cells in vivo, were used to test phagocytosis of S. aureus and two strains of E. coli. n/a S. aureus and E. coli induced separate microbicidal mechanisms of endothelial cells Carlsson S, Wiklund NP, Engstrand L, Weitzberg E, Lundberg JO. Effects of pH, nitrite, and ascorbic acid on nonenzymatic nitric oxide generation and bacterial growth in urine. Nitric Oxide 5(6):580-6. 2001 Escherichia coli and Pseudomonas aeruginosa and Staphylococcus saprophytics none NO formation and bacterial growth in mildly acidified human urine containing nitrite and the reducing agent vitamin C was studied. pH and nitrite levels were varied. n/a Large amounts of NO were produced and bacteria was markedly reduced with nitrite in mildly acidified urine. Inhibition was enhanced by ascorbic acid. Hardwick JB, Tucker AT, Wilks M, Johnston A, Benjamin N. A novel method for the delivery of nitric oxide therapy to the skin of human subjects using a semi-permeable membrane. Clin Sci (Lond) 100(4):395-400. 2001 Staphylococcus aureus and Escherichia coli none A chemical system using sodium nitrite and ascorbic acid to produce NO on the skin surface was administered with a selectively permeable, hydrophilic polyester co-polymer membrane system. The study measured vasodilation and anti-microbial effects of the system. 1 - 1000 mM sodium nitrite and ascorbic acid Potent anti-microbial properties of NO were seen at concentrations of nitrite above 50 mM 164 Staphylococcus con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary Concentration Additional Notes / Results Simsek 1, Mas MR, Yasar M, Ozyurt M, Saglamkaya U, Deveci S, Comert B, Basustaoglu A, Kocabalkan F, Refik M. Inhibition of inducible nitric oxide synthase reduces bacterial translocation in a rat model of acute pancreatitis. Pancreas 23(3):296-301. 2001 Enterococcus sp. and Escherichia coli and Staphylococcus sp. and Proteus none S-methylisothiourea, an iNOS inhibitor, was used to improve the course of acute pancreatitis in rats. n/a Bacteria levels were lower; however, mortality was not affected. Weller R, Price RJ, Ormerod AD, Benjamin N, Leifert C. Antimicrobial effect of acidified nitrite on dermatophyte fungi, Candida and bacterial skin pathogens. J Appl Microbiol 90(4):648-52. 2001 Staphylococcus aureus and Propionibacterium acnes Streptococcus pyogenes Organisms were cultured in nitrite before being transferred to recovery medium. Addition of nitrite increased the antimicrobial activity of acid solutions against all organisms tested, except for Strep, pyogenes. pH, concentration and culture time were varied Nitrite was also found to increase antimicrobial activity in acid solutions against three fungis: Trichophyton mentagrophytes, T. rubrum and Candida albicans. McKay DM, Lu J , Jedrzkiewicz S, H o W , Sharkey KA. Nitric oxide participates in the recovery of normal jejunal epithelial ion transport following exposure to the superantigen, Staphylococcus aureus enterotoxin B. J Immunol 163(8):4519-26. 1999 Staphylococcus aureus none Mice were treated with S. aureus enterotoxin B alone or in combination with an inhibitor of the inducible form of NO synthase (iNOS), L-NIL. n/a The beneficial effect of NO in this model system is probably via regulation of TNF-alpha and IFN-gamma production. 165 Staphylococcus con't Author, Title, Source Year Microbes Killed / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary Concentration Additional Notes / Results Sakiniene E, Bremell T, Tarkowski A. Inhibition of nitric oxide synthase (NOS) aggravates Staphylococcus aureus septicaemia and septic arthritis. Clin Exp Immunol 110(3):370-7. 1997 Staphylococcus aureus none Mice treated with NOS inhibitors: N(G)-monomethyl-L-arginine or N(omega)-nitro-L-arginine methyl ester n/a NOS inhibitor-treated mice had 10 times less bacteria killed than in controls. Kaplan SS, Lancaster J R Jr, Basford RE, Simmons RL. Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide. Infect Immun 64(1):69-76. 1996 Staphylococcus aureus none SNAP (NO donor), xanthine oxidase, hypoxanthine 0.01 -1 mM SNAP Prolonged exposure to NO causes a delayed loss of viability 166 Yersinia Author, Title. Source Year,, Microbes KLlled / Inhibited Microbes Not Killed / Inhibited Substance Used / Methodology Summary NO Concentration Additional Notes / Results Fite A, Dykhuizen R, Litterick A, Golden M, Leifert C. Effects of ascorbic acid, glutathione, thiocyanate, and iodide on antimicrobial activity of acidified nitrite. Antimicrob Agents Chemother 48(2):655-8. 2004 Yersinia enterocolitica Yersinia enterocolitica The ability of various reducing agents and hydroxyacids to augment the antimicrobial activity of (dietary) acidified nitrite against Y. enterocolitica was tested. pH was varied. Nitrite: 0 - 1000 uM. Chemicals: 0 - 1000 pM. Ascorbic acid and glutathione reduced the activity of acidified nitrite against Y. enterocolitica, iodide and thiocyanate increased the antimicrobial activity, and hydroxyacids (citrate, lactate, and tartarate) had no measurable effects. Zhao YX, Lajoie G, Zhang H, Chiu B, Payne U, Inman RD. Tumor necrosis factor receptor p55-deficient mice respond to acute Yersinia enterocolitica infection with less apoptosis and more effective host resistance. Infect Immun 68(3):1243-51. 2000 Yersinia enterocolitica none TNF receptor p55 deficient mice were used to evaluated the antimicrobial role of TNF. n/a Yersinia can induce TNFRp55-mediated apoptosis of splenocytes in the acute phase of the infection and alteration of T-cell-generated cytokines can dramatically alter the early events in host defense against this pathogen. Dykhuizen RS, Frazer R, Duncan C, Smith C C , Golden M, Benjamin N, Leifert C. Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense. Antimicrob Agents Chemother 40(6):1422-5. 1996 Escherichia coli and Salmonella typhimurium and Salmonella enteritidis and Shigella sonnei and Yersinia enterocolitica none The study examines the antimicrobial effect of acidified nitrite in vitro. pH was varied: 2.1, 3.0, 3.7, 4.2, 4.8, 5.4 variable (order of .1, 1 and 10 pmol/mL) Susceptibility to the acidified nitrate solutions ranked as follows: Y. enterocolitica > S. enteritidis > S. typhimurium = Shigella sonnei > E. coli 167 Appendix 2 THE TREATMENT OF CHRONIC NON-HEALING LEG ULCERATION WITH GASEOUS NITRIC OXIDE - A CASE STUDY Chris C. Miller *; Minna K. Miller \ Abdi Ghaffari *; Brian Kunimoto § * Ph.D.(c), Faculty of Experimental Medicine, University of British Columbia t BA, Faculty of Nursing, University of British Columbia * B .SC, Faculty of Sciences, University of Alberta § M.D., Dermatology, University of British Columbia ABSTRACT Despite recent advances in wound care, chronic non-healing ulcers of the lower extremities still present a huge challenge to modern society. Approximately four million people in United States alone suffer from chronic lower leg ulcers. Despite best clinical practice, a variety of factors such as infection or presence of bacterial biofilm, poor tissue perfusion, necrotic tissue, advanced age, and diabetes impair the healing process in these patients. Endogenously released nitric oxide (NO) has been shown to play a significant role in vasodilation, regulation of immune response and wound healing, and also acts as an antimicrobial agent in body's non-specific immune response. As a result, it was hypothesized that administration of exogenous gaseous nitric oxide (gNO) directly to a non-healing wound, may facilitate the body's response to heal wounds that express significant bacterial burden. In this case study, we report the application of gNO therapy to a two-year old non-healing chronic venous ulcer in a 55-years-old man with thirty years history of severe venous disease, both deep and superficial. gNO was applied at 200 ppm to the lower extremity using a gas diluting delivery system connected to a "single patient use" plastic boot. The patient received nocturnal treatment for 14 consecutive nights, with an average of 8.1 hours per treatment. The patient was monitored daily by a qualified nurse and the wound was assessed and photographed on day 0, 3, and 14, as well as 10 days , 6 and 26 weeks following cessation of treatment. At day 0, the wound was malodorous and covered by a bacterial biofilm with little healthy granulation tissue present. Following 3 days of gNO treatment, healthy granulation tissue was noted in the ulcer base with absence of malodorous odor. At day 14, the ulcer was clearly reduced in size and almost completely re-epithelialized. Assessment of the wound on day 10 and week 6 and 26 post-treatment did not reveal any deterioration in healing. This single case study demonstrated that 200 ppm gNO as a topical agent was well tolerated by the patient without any report of discomfort or side effect. The result of this wound healing approach is very promising and warrants future randomized controlled trials to explore the feasibility of gNO therapy. K E Y W O R D S : Nitric oxide gas, chronic wound healing, venous ulcer, lower leg ulcer, non-healing wound, bacterial biofilm, antimicrobial agent 168 This page is intentionally blank. INTRODUCTION Chron ic ulcers of the lower extremities are a significant public health problem. Bes ides the large f inancial burden p laced on the health care system for their treatment, they exert a heavy toll in human suffering. A s our population ages and with the current obesity crisis in North Amer i ca , venous, diabetic, and pressure ulcers are likely to become ever more common . Approximately 4 million (1% of population) people in the United States develop chronic lower leg ulcers, the majority c lassi f ied as diabetic or venous leg ulcers, and this number can cl imb to 4 - 5 % in older (>80 years of age) patients (1). Despi te recent advances in chronic wound care, many ulcers of the lower extremity will not heal . A variety of factors can potentially inf luence wound heal ing. T h e s e include presence of infection, necrotic t issue, poor t issue handl ing, and impaired t issue perfusion, and are inf luenced by other cl inical condit ions such as advanced age, d iabetes, and steroid administration (2). By definition, such ulcers show no ev idence of heal ing after 6 to 12 weeks of best cl inical practice (3). O n e of the major factors effecting non-heal ing chronic wounds is the presence of significant bacterial burden interfering with the normal p rocess of heal ing. Recent ly, it has been recognized that the wound bacterial burden may be composed of a bacterial "biofilm", which is a complex organized network of bacteria and tenacious film that is nearly impossible to eradicate with convent ional antibiotics (4, 5, 6). The pers istence of these biofilms disrupts the normal wound heal ing process (7). Strategies to deal with these biofilms usually consist of physical removal by curettage and the use of topical antimicrobial agents. T h e s e expens ive methods and products are often ineffective with frequent and relatively rapid reoccurrence, and clinical research is needed to formulate new approaches. O n e such potential approach may be the use of exogenous gaseous nitric oxide (gNO), identical to the endogenous ly produced molecule, which plays a critical role in various bodily functions, including the vasodi lat ion , neurotransmission, regulation of wound heal ing and regulation of immune response to infection as well as cytotoxicity toward various organisms including bacter ia (8,9). Its specif ic role in wound heal ing has been demonstrated to be important and related to vasodi lat ion, ang iogenes is , anti-inflammation and antimicrobial act ions (10, 11, 12, 17, 18). Prel iminary dose ranging investigations have shown the antibacterial effects of direct application nitric oxide to microorganisms and that the optimal exposure dose of 200 parts per million (0.02%) gaseous nitric oxide (gNO) had the predicted effect on common bacterial strains contributing to wound infections in both in vitro and in vivo animal models (19, 20), without any indication of systemic adverse effects (21). Paral lel in vitro studies on human dermal f ibroblasts exposed to 48 hours of cont inuous 200 ppm g N O showed no sign of reduced viability or loss of cel lular function (22). T h e s e encouraging results supported further exploration for the use of g N O in a cl inical appl icat ion. To our knowledge, direct application of gaseous nitric oxide to a non-heal ing wound has not been reported in the literature. The purpose of this pilot c a s e study was to evaluate the feasibility and potential role of short term gaseous nitric oxide in reducing the wound biofilm and thus improving wound heal ing status in a two-year old, non-heal ing chronic ulcer that had not previously responded to convent ional therapies. 170 Materials & Methods Nitric oxide gas (V iaNOx-H , V I A S Y S Heal thcare, U S A ) was appl ied to the lower extremity with use of a gas diluting delivery system (C idaNOx Delivery System) designed specif ical ly for the study (Pu lmoNOx Medica l Inc., Canada) . Th is C i d a N O x delivery system contains an internal air pump for dilution of the g N O and a flow control circuit to dilute the 800 parts per million (ppm) in the N O source cyl inder down to the therapeutic level of 200 ppm. The total f low from the sys tem was 1.0 liter per minute and included one-quarter of a liter per minute (250 mL/min) flow of g N O . Severa l internal pressure sensors assure the dilution flow is operat ional and monitor the sys tem. The flow of nitric oxide was limited to 250 mL/min by a mechanical ly set pressure regulator and a mechan ica l f lowmeter that have no external controls that could be changed by the patient. The concentrat ion of nitric oxide del ivered was assured by measurement of the C i d a N O x output with a calibrated nitric oxide analyzer (AeroNOx, Pu lmonox Medica l Inc, Canada ) that is approved for monitoring inhaled N O in human patients. The 200 ppm g N O from the C i d a N O x Delivery Sys tem f lowed out to a "single patient use" plastic boot that covered the patient's lower extremity. The boot had an inflatable cuff near the top that provided a low pressure sea l . A secondary air outlet from the C i d a N O x unit managed the inflation of the cuff. The patient connected the pump outlet to the cuff connector until it was inflated and then the connector was sea led , c losed with the provided c lamp. The g N O flow was then connected to the inlet connector near the toe of the boot and the return line to the connector near the top of the boot. The return line passed through the C i d a N O x unit and then out through a scavenger consist ing of charcoal and potassium permanganate that absorbs the nitrogen oxides. The C i d a N O x Delivery Sys tem had two toggle posit ions, one for delivery of g N O and the other for delivery of air only. At the end of the treatment period, the patient switched the delivery flow to air only so as to c lear the boot of remaining g N O before taking the boot off. C A S E S T U D Y This c a s e study involved a 55-year-old man with a thirty year history of severe venous d isease , both deep and superf icial, related to deep vein thrombophlebit is. Initially, while in his twenties, the patient deve loped bilateral non-heal ing venous leg ulcers that were surgical ly treated. The surgical si tes healed but the ulcers cont inued to recur. Approximately 26 months ago, the patient presented with a smal l ulcer located just below the medial mal leolus of the left ankle. Al though not increasing in s ize , this ulcer did not completely heal with two years of standard of care therapy. Most of the time, the wound base was covered with a biofilm, a tenacious yel low-colored, gel-l ike material. E d e m a control was maintained by using graduated compress ion stockings. Antimicrobial dress ings were tried including M a n u k a Honey, a starch iodine preparation ( lodosorb, Smith & Nephew, U S A ) and colloidal si lver (Aquacel A G , Conva tec U S A ) . His wound was frequently debr ided in order to physical ly remove the biofilm. Th is was general ly ineffective as the biofilm was frequently noted to be present again at the next visit. Twenty percent benzyol peroxide lotion was appl ied every few days in order to trigger the development of granulation t issue however this was ineffective as well. At t imes, there 171 would be improvement as the ulcer would appear to become covered with new skin only to break down weeks later. This poor progress to complete closure was noted despite wound care that addressed proper moisture balance, wound bed preparation, and treatment of the underlying disease. This failure of his wound to close had a significant impact on quality of life for this patient. He made clinic office visits at least once a month for the entire two years. The cost of the treatment, including the surgeon's time and treatment materials (several thousand dollars) put pressure on healthcare system as well as the patient, with him having to travel several hours each visit for treatment. As previous treatments proved ineffective, the patient was invited to participate in this investigational study. Following a discussion of the investigational therapy and potential risks, an informed consent was obtained. The patient was seen at the clinic where the wound was assessed and photographed (Figure 2). The treatment regimen was explained and the use of the CidaNOx Delivery System and boot was demonstrated. Arrangements were made to meet at the patient's home the following day to set up the equipment and for him to have a repeat training on the use of the treatment system. Training included use of the system as well as safety information on using the gas equipment. The patient was instructed to continue wearing supportive stockings and to use a hydrofiber dressing (Aquacel, Convatec, USA) on the wound when not receiving gNO treatment. During the gNO treatment, he removed the supportive stocking and replaced the Aquacel dressing with a porous, low adherence dressing (ETE, Molnlycke Health Care, Sweden), which had previously been shown to allow the diffusion of gNO through it (data not shown). The in vitro experience with various microorganisms suggested that it would take at least 8 hours to attain the cidal action of the gNO. However, since the limited animal experience suggested that 72 hour did not completely sterilize the wound site and we wanted to explore the potential for wound bed preparation and accelerated wound healing from prolonged use, we chose to extend the treatment beyond three days. It was decided to stop at 14 days to evaluate the short term effects and explore the possibility that the short term effects would improve the longer term outcome. The patient was encouraged to wear the gNO boot as often as possible during each 24-hour- period. As the patient worked during the day, it was decided that it would be most practical to wear the boot and receive the gNO treatments only while in bed at night. The patient recorded the date, time and duration of each treatment period on a data sheet, and any significant observations related to the wound, treatment or equipment. The wound size (cm2) was measured using digital photography and densitometry technique (Scion Image 3-4.02, Scion Corp., Maryland, USA). R E S U L T S O F T H E C A S E S T U D Y The patient self-administered the treatment for fourteen consecutive nights. The nocturnal treatment duration varied from 6.5 to 9.75 hours per treatment. The cumulative wound exposure to 200 ppm of gNO during the fourteen treatment periods was 105.25 hours. The wound was assessed and photographed on day 0 (Fig. 2A, pretreatment), day 3 (Fig. 2B, 172 following accumulat ive 24 hrs of g N O exposure) , and day 14 (Fig. 2C) . The wound was a lso a s s e s s e d and photographed ten days following the complet ion of the 14-day treatment (Fig. 2D) and in the sixth and twenty sixth week following the complet ion of the treatment (Fig. 2 E & F, respectively). During the active treatment period, the subject was a s s e s s e d with respect to the use of the C i d a N O x sys tem. The subject found the sys tem easy to use in a f ixed location, found the application of the bag comfortable and never reported any pain assoc ia ted with its use. He noted that the upper part of the bag somet imes sl ipped off and suggested that it could be better sea led . He suffered no bleeding ep isodes . Figure 2A shows the initial presentation of the ulcer prior to use of the g N O . The wound base was covered by a biofilm and there was little healthy granulation t issue present as well as there was no ev idence of new skin growth from the edges . The wound was malodorous. After 24 hours of N O exposure (3 days at 8 hours per day), for the first t ime, there was healthy granulation t issue noted in the ulcer base . There was also early ev idence of new skin growth from the edges observed. The malodorous odor was also absent. Concomitant ly, there was less biofilm present (Figure 2B) . At 14 days of therapy (Figure 2C) the ulcer clearly had diminished in s ize . By then, it had almost completely epithelial ized. Signif icant wound s ize reduction was observe as early as day 3 of g N O treatment (p = 0.014), with approximately 7 5 % reduction in wound area by end of g N O therapy at day 14 (Figure 3). The wound was further a s s e s s e d 10 days following cessat ion of g N O treatment (Fig. 2D). There did not appear to be any deterioration of the wound during this time although the ulcer was judged to be incompletely healed. No significant deterioration in wound s ize was observed in compar ison to last day of g N O treatment (Fig 3). S ix weeks later, the wound was judged to be about 9 0 % healed with no deterioration in wound s ize or epithelialization (Fig 2 E & Fig 3). At 26 weeks post N O discontinuation, the ulcer was noted to be completely healed and re-epithelial ized (Fig 2F) . Over the entire post-treatment time, there were no changes to the dressing regimen and no other antimicrobials or antibiotics were used. D I S C U S S I O N Success fu l wound heal ing involves numerous physiological responses, including the inflammatory response, ang iogenes is , the development of f ibrous t issue, and re-epithelial ization. N O is both directly and indirectly involved in each of these physiological p rocesses . Many wound-resident cel ls such as macrophages, neutrophils, endothelial cel ls , vascu lar smooth musc le cel ls , keratinocytes, lymphocytes, and fibroblasts have the ability to synthesize or affect the synthesis of N O (10). Severa l studies have suggested a role for N O in wound heal ing by showing that, in some situations, impaired wound heal ing results from the absence of N O (23-26). Speci f ical ly, some studies attempted to reverse impaired wound heal ing by administering dietary arginine or adenoviral-mediated express ion of human i N O S ( iNOS is a designat ion for " inducible" N O Synthetase enzyme. Here, the enzyme activity may be increased by the p resence of inflammatory subs tances or bacterial l ipopolysaccharides) in N O S - K O mice, with both showing some degree of s u c c e s s (24-26). N O acts by way of multiple mechan isms. S o m e are due to its chemical reaction with oxygen which forms free radicals, whereas others are due to its affinity with enzymes containing heme or Fe (iron). A l so N O may regulate express ion of var ious genes , whose 173 products are important in the heal ing process. The multiple sites of action of N O partially explain its potential ability to enhance wound heal ing. W o u n d macrophages have been shown to be a major component of i N O S express ion in the early inflammatory phase of repair. Fibroblasts express both e N O S and i N O S , and the N O that they produce participates in the regulation of col lagen synthesis by these cel ls. ( e N O S is the designat ion for "endothelial" N O S . This enzyme differs from i N O S in that it is not "inducible"; rather, its activity depends on intracellular calc ium ion levels.) Kerat inocytes at wound margins strongly express i N O S (27). The effect of N O on keratinocyte function appears to be dose-dependent . At low levels, increased proliferation is observed , whereas at high levels, cel lular proliferation is inhibited and differentiation occurs (28,13) A role for N O on keratinocyte function is suggested as i N O S inhibition results in significant impairment of re-epithelialization, whereas N O production is assoc ia ted with the acquisit ion of a locomotory phenotye (migration of cel ls toward center of the wound) (29). N O has also been shown to participate in the formation of new blood vesse ls , as pharmacologic inhibition of N O S prevents capil lary organizat ion in vitro (30). The effects of N O on ang iogenes is may be mediated v ia pro-angiogenic cytokines, such as vascu lar epithelial growth factor ( V E G F ) , whose activity is in part N O -dependent (31). The average time for ulcers that result from venous stasis d i sease to heal under optimal care, ranges from 12 to 16 weeks . Th is patient, who had a non-responsive ulcer for more than two years, exhibited a posit ive response to a brief exposure to gaseous nitric oxide. His wound dec reased in s ize , a granular base was establ ished, and the malodorous smel l was eradicated during this two-week period. Whether a longer exposure or a different concentration once the biofilm was el iminated would have made a difference in the c losure of the lesions, only further studies and randomized control led trials will be able to answer. C O N C L U S I O N In the single c a s e study we have descr ibed here, it was shown that 200 ppm g N O as a topical agent was well tolerated by the patient and augmented wound heal ing. G a s e o u s N O could be safely del ivered nocturnally at home, with good compl iance. There were no adverse side effects assoc ia ted with the treatment or its delivery. The result ach ieved in this single patient is very promising but warrants additional investigation. Currently, there is no avai lable or effective treatment for many types of chronic wounds. If results such as these continue to be favorable, g N O may become a valuable adjunct to the wound heal ing process . A C K N O W L E D G M E N T S W e would like to thank the team at Pu lmonox for their support of this study, in particular Mr. Robert Lee and Bruce Murray for their expert technical ass is tance in construction of the device. W e would a lso like to thank A lex Stenzler of V I A S Y S Healthcare for his invaluable input and encouragement . 174 REFERENCES 1. Krasner D. Chronic wound care: a clinical source book for healthcare professionals. Health Management Publications, USA, 1990. p12-18. 2. McCance KL and Heuther SE. Pathophysiology: The biologic basis for disease in adults and children. 3rd ed. Mosby, St. Louis, 1998:232-234. 3. Kunimoto B, Cooling M, Gulliver W, Houghton P, Orsted H, Sibbald RG. Best practices for the prevention and treatment of venous leg ulcers. Ostomy Wound Management 2001 ;47(2):34-46,48-50. 4. Hannson C, Hoborn J , Moller A, Swanbeck G. The microbial flora in venous leg ulcers without clinical signs of infection. Acta Dermato-Venerologica 1995;75:24-30. 5. Consterton JW, Stewart PS. Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318-1322 6. Consterton JW, Stewart PS. Battling Biofilms. Scientific American 2001 ;July:75-81 7. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001;358:135-138 8. Moncada S, palmer RMJ, Higgis EA. Nitric oxide. Physiology, Pathophysiology and Pharmacology, Pharmacol Rev 1991;43:109-134. 9. De Groote MA, Fang FC. NO inhibitions:antimicrobial properties of nitric oxide. Clinical Infectious Diseases 1995;21 (suppl 2):S162-165. 10. Witte MB, Barubul A. Role of nitric oxide in wound repair. The American Journal of Surgery 2002;183:406-412 11. Fang FC. Mechanisms of nitric oxide - related antimicrobial activity. American Society of Clinical Investigations 1997;99(12):2818-2825. 12. VazQuez-Torres A, Fang FC. Therapeutic applications of nitric oxide in infection in Nitric Oxide and Infection 1999 Plenum Publishers, New York pp 475-488. 13.0rmerod AD, Copeland P, Hay I, Husain A, Ewen WB. The inflammatory and cytotoxic effects of a nitric oxide releasing cream on normal skin. J Invest Dermatol 113:392-397,1999. 14. Bauer JA, Weisan R, Smith DJ. Evaluation of linear polyethyleneimine / NO adduct on wound repair: therapy versus toxicity. Wound Rep Reg 1998; 6:569-577. 15.Shabini M, PulferSK, Bulgrin JP, Smith DJ. Enhancement of wound repair with a topically applied nitric oxide-releasing polymer. Wound Rep Reg 1996;4:353-362. 16. Kavdia M, Nagarajan S, Lewis RS. Novel devices for the predictable delivery of nitric oxide to aqueous solutions. Chem Res Toxicol 1998 11:1346-1351. 17. Weller R, Ormerod AD, Hobson RP, Benjamin N. A randomised trial of acidified nitrite cream in the treatment of Tinea pedis. J . Am. Acad. Dermatol 1998;38:559-563. 18. Hardwick JBJ, Tucker AT, Wilks M, Johnston A, Benjamin N. A novel method for the delivery of nitric oxide therapy to the skin of human subjects using a semi-permeable membrane. Chemical Science 2001 ;(100):395-400. 19. Ghaffari A, Ardakani A, Neil DH, Miller CC. In vitro antibacterial effect of gaseous nitric oxide on common bacterial strains contributing to wound infections: Pseudomonas aeruginosa and Staphylococcus aureus. 2nd International Conference of Nitric Oxide, Prague, Czech Republic, 2002. 20. Ghahary A, Ghaffari A, Miller C. In Vivo effects of exogenous gaseous nitric oxide on Staphylococcus aureus infection in a healing rabbit wound model (abstract). 175 Wound Caring Symposium, Canadian Association of Wound Care, Vancouver, Canada; November, 2002: 23-24. 21. Ghaffari A, Miller C, Ardakani A, Kilani R, Karami A, Ghahary A. Exogenous nitric oxide gas in chronic wound infection. Wound Repair & Regeneration 2003; 11(2): A11. 22. Ghaffari A, Ardakani A, Miller C C, Ghahary A. Viability of Human Fibroblast Cells Exposed to Gaseous Nitric Oxide. 8th Annual Conference of the Canadian Association of Wound Healing, Vancouver, 2002. 23. Stallmeyer B, Kampfer H, Kolb N, Pheilshifter J , Frank S . The function of NO in wound repair: inhibition of iNOS severly impairs wound reepithelialization. J Invest Dermatol 1999; 113:1090-1098. 24. Shi HP, Most D, Efron DT, Witte MB, Barbul A. Supplemental L-arginine enhances wound healing in diabetic rats. Wound Rep Reg 2003; 11:198-203. 25. Barbul A, Fishel RS, Shimazu S, Wasserkrug HL, Yoshimura NN, Tao RC, Efron G. Intravenous hyperalimentation with high arginine levels improves wound healing and immune function. J Surg Res 1985; 38:328-334. 26. Yamasaki k, Edington HDJ, McClosky C, Tzeng E, Lizonova A, Kovesdi I, Steed DL, Billiar TR. Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest 1998; 101:967-971. 27. Wang R, Ghahary A, Shen YJ, Scott PG, Tredget EE. Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible NOS isoforms. J invest dermatol 1996; 106:419-427. 28. Krischel V, Bruch-Gerharz D, Suschek C, Kroncke KD, Ruzicka T, Kolb-Bachoften V . Biphasic effect of exogenous NO on proliferation and differentiation in skin-derived keratinocytes but not fibroblasts. J Invest Dermatol 1998; 111:286-291. 29. Noiri E, Peresleni T, Srivastava N, Weber P, Bahou WF, Peunova N, Goligorsky MS. NO is necessary for a switch from stationary to locomoting phenotype in epithelial cells. Am J Physiol 1996; 270:c794-802. 30. Lee PC, Salyapongse AN, Bragdon GA, Shears LL, Watkins SC, Edington HDJ, Billiar TR. Impaired wound healing and angiogenesis in eNOS-deficient mice. Am. J . Physiol. 1999;277(Heart Circ. Physiol 46):H1600-H1608. 31 .Frank S, Stallmeyer B, Kampfer H, Kolb N, Pfeilschifter J . Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair. FASEB 1999; 13:2002-2014. 176 F I G U R E L E G E N D S Figure 1. A: The gaseous nitric oxide delivery device consisting of a source cylinder of 800 parts per million (ppm) nitric oxide (ViaNOx-H, VIASYS Healthcare, USA). B: a gas diluting system (CidaNOx, PulmoNOx Medical Inc., Canada) providing 200 ppm nitric oxide gas balance air at 1 Lpm to a sealed plastic "boot." Figure 2. A: Pre-Treatment image (day 0) shows the initial presentation of the ulcer prior to use of the nitric oxide gas. The wound base is covered by a biofilm and there is little healthy granulation tissue present and no evidence of new skin growth from the edges. The wound was malodorous. B: Following 24 hours of nitric oxide gas at 200 ppm for 8 hours a night over 3 days there is healthy granulation tissue noted in the ulcer base. There is also early evidence of new skin growth from the edges. Concomitantly, there is less biofilm present. C: Following 14 days of treatment, the ulcer clearly is diminished in size. The ulcer is almost completely epithelialized. Nitric oxide treatment was discontinued at this time. D: 10 days following 14 days of treatment there does not appear to be any deterioration of the wound. E: 6 weeks following 14 days of treatment the wound is judged to be about 90% healed. No deterioration is noted and the ulcer is much healthier than on day 0. F: 26 weeks following 14 days treatment the wound is completely closed with healthy granulation tissue. Figure 3. wound size reduction following nitric oxide gas treatment. Significant decrease in area was observed following 3 and 14 days of gNO application to the wound. Wound status did not deteriorate after removal of treatment (arrow). Wound was completely healed following 26 weeks (186 days). Values are means and standard deviation from 3 independent assessments. * p = 0.019 vs day 0 ** p = 0.014 vs day 3 *** p < 0.01 vs day 3 F I G U R E S 177 *** 0 3 14 24 42 186 Day Figure 3. wound size reduction following nitric oxide gas treatment. Significant decrease in area was observed following 3 and 14 days of gNO application to the wound. Wound status did not deteriorate after removal of treatment (arrow). Wound was completely healed following 26 weeks (186 days). Values are means and standard deviation from 3 independent assessments. * p = 0.019MS dayO ** p = 0.014 vs day 3 *** p < 0.01 vs day 3 ) 179 

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