10 th International Conference Joseph D. Brain Molecular and - - PDF document

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Joseph D. Brain 10th International Conference Molecular and Integrative Physiological Sciences Program Neonatal and Childhood Department of Environmental Health Pulmonary Vascular Disease Harvard University Conference T.H. Chan School of Public Health Saturday, March 11, 2017 665 Huntington Avenue Boston, MA 02115 RELATIONSHIPS BETWEEN INHALED AEROSOL DEPOSITION IN THE LUNGS AND HUMAN LUNG PHYSIOLOGY IN CHILDREN AND ADULTS I. Introduction

• A. Comparison of the inner and outer environment
• B. Major surfaces of the body: skin, respiratory tract, G.I. tract

II. Skin

• A. Histology and thickness of the skin
• B. Basic properties of cell membranes
• C. Kinds of materials that penetrate skin
• D. Microbial flora

III. Gastrointestinal Tract

• A. Anatomy and physiology of the G.I. tract
• 1. Secretion and motility
• 2. Digestion and absorption
• B. Kinds of materials that are absorbed from the small intestine
• C. Microbial flora

IV. The Respiratory Tract

• A. Introduction
• B. Basic anatomy and histology of the respiratory tract
• C. Respiratory physiology
• D. Microbial flora

V. Deposition of Therapeutic Bioaerosols

• A. Characterization and collection of aerosols
• 1. Mass
• 2. Size distribution
• 3. Physical and chemical analyses
• B. Forces acting to deposit particles in the lungs:
• 1. Inertial
• 2. Gravitational
• 3. Diffusional
• 4. Interception, thermal, electrical and magnetic forces
• C. Factors determining the effectiveness of these forces:
• 1. Aerosol characteristics
• 2. Breathing pattern
• 3. Anatomy of the respiratory system

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VI. Clearance of Aerosols from the Lungs

• A. Clearance from the ciliated regions of the lungs: mucus transport
• 1. Frequency and quality of the ciliary beat
• 2. Quantity and rheological properties of the mucus
• B. Clearance of particles from the non-ciliated regions of the lungs
• 1. Role of alveolar macrophages
• 2. Lymphatic drainage
• C. Cough

VII. Fate of Aerosols that Gain Access to the Blood

• A. The Circulation
• 1. Anatomy and physiology
• 2. Overall patterns
• B. Mononuclear phagocyte system
• C. I.V. drugs targeted to the lungs, e.g., α-1 antitrypsin

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Inhaled Therapeutic Aerosols: Relevant Principles Human lungs, because of their primary function of gas exchange, are brought into intimate contact with inhaled particles. In every environment, particles enter the lungs. We do not live in a sterile

• environment. Only in recent centuries have we emphasized air pollution particles and exposures in

the work environment. Infectious aerosols have always been presentThe mechanisms which are pertinent to particle deposition and clearance will now be described and the relationship of these mechanisms to aerosol retention will be presented. Deposition Deposition is the process that determines the fraction of the inspired particulates that will be caught in the respiratory tract and thus fail to exit with the expired air. Several distinct processes operate to move particles suspended in the inspired air toward the surface of the respiratory tract: inertial forces, sedimentation, Brownian diffusion and interception. It is likely that all particles deposit after touching a surface; thus the site of initial deposition is the site of contact. Inertia refers to the tendency of moving particles to resist changes in direction and speed. Repeated branching in the airways cause sudden changes in the direction of air-flow; however, because of inertia, particles tend to continue in their original direction, crossing air-flow streamlines and eventually impacting on the airway walls. Gravity accelerates falling bodies downward, and terminal settling velocity is reached when viscous resistive forces are equal and opposite in direction to gravitational forces. Respirable particles reach this constant terminal or sedimentation velocity in less than 0.1 msec. Thus, particles are removed as their terminal velocity causes them to strike the airway walls or alveolar surfaces. Aerosols also undergo Brownian diffusion, a motion caused by collisions of gas molecules with particles suspended in the air; this motion causes the particles to cross streamlines and thus increases the probability they will deposit. The effectiveness of these deposition mechanisms depends on: (1) the anatomy of the respiratory tract, (2) the effective aerodynamic diameters of the particles, and (3) the pattern of breathing. These factors determine the fraction of the inhaled particles that are deposited as well as the site of deposition. The anatomy of the respiratory tract is important since it is necessary to know the diameters of the airways, the frequency and angles of branching, and the average distances to the alveolar walls. Furthermore, along with the inspiratory flow rate, airway anatomy specifies the local linear velocity of the air stream and the character of the flow. A significant change in the effective anatomy of the respiratory tract occurs when there is a switch between nose and mouth breathing. There are inter- and intra-species differences in lung morphometry; even within the same individual, the dimensions of the respiratory tract vary with changing lung volume, with aging, and with pathological processes. The effective aerodynamic diameters of the particles affect the magnitude of forces acting on them. For example, while inertial and gravitational effects increase with increasing particle size, diffusion produces larger displacements as particle size decreases. Effective aerodynamic diameter is a function of particle size, shape, and density. In order to predict deposition patterns, it is essential to

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describe the distribution of aerodynamic diameters of aerosols as well as the mean of the aerodynamic diameter. The remaining factor affecting deposition is the breathing pattern. Minute volume defines the average flow velocity of the aerosol-containing air in the lung and the total number of particulates to which the lung will be exposed. Respiratory frequency will affect the residence time of aerosols in the lungs and hence the probability of deposition by gravitational and diffusional forces. Changing lung volume will alter the dimensions of the airways and parenchyma. The ICRP lung model of 1966 provides some predictions for the deposition fraction (collection efficiency) of particles for an adult human breathing a 1,450 ml tidal volume, 15 times a minute. Deposition in the nasopharynx ranges from 50.2% of the inspired particles with 2.0 um MMAD to 95.6% of 20 um particles. Deposition in the tracheobronchial compartment decreases from 3.61 to 1.03% as the MMAD increases from 2.0 um to 20 um and finally, deposition in the pulmonary compartment decreases from 21 to 2.6% as MMAD increases from 2.0 um to 20.0 um. Clearance Clearance refers to the dynamic processes that physically expel particulates from the respiratory tract; it is the output of particulates previously deposited. Highly soluble particles dissolve rapidly and are absorbed into the blood from the respiratory tract. Their metabolism and excretion resemble that of an intravenously injected dose of the same material. Although some of this discussion applies to bioaerosols, we also emphasize unique aspects of respiratory defense mechanisms which are critical to keeping our lungs clean and sterile. Some secretions of the upper respiratory tract such as airway mucus may have bactericidal or antiviral

• properties. Especially, the ability of phagocytic cells looms large. Their ability to migrate, bind,

and ingest deposited bioaerosols is critical. After phagosomes containing pathogens are created, preformed lysosomes fuse to create a phagolysosome. Within that “intracellular stomach” pathogens are killed by reactive oxygen species. After pathogens are killed, they are then digested by lytic enzymes. Ultimately, their constituents are recycled or metabolized. Clearance can refer to the disappearance of materials which are measured because of their physical or chemical

• properties. It also describes in situ mechanisms where pathogens are killed, digested, and gradually

disappear. Ciliated Regions Less soluble particles that are deposited on the mucus blanket covering pulmonary airways are moved toward the pharynx by the cilia. Also present in this moving carpet of mucus are cells and particles which have been transported from the non-ciliated alveoli to the ciliated airways. Similarly, particles deposited on the ciliated mucus membranes of the nose are propelled toward the pharynx. There, mucus, cells, and debris coming from the nasal cavities and the lungs meet, mix with salivary secretions, and enter the gastrointestinal tract after being swallowed. A number of factors can affect the speed of mucus flow. They may be divided into two categories: those affecting the cilia themselves and those affecting the properties of the mucus. The following aspects of ciliary action may be affected: the number of strokes per minute, the amplitude of each

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stroke, the time course and form of each stroke, the length of the cilia, the ratio of ciliated to non-ciliated areas, and the susceptibility of the cilia to intrinsic and extrinsic agents that modify their rate and quality of motion. The characteristics of the mucus may become critically important. The thickness of the mucus layer and its rheological properties may undergo wide variations; for example chronic exposure to cigarette smoke or to irritant gases in the work place can induce significant changes in airway mucus. Non-Ciliated Regions Particles deposited in the non-ciliated portion of the lungs are either moved toward the ciliated region, primarily within alveolar macrophages, or they enter the alveolar wall and accumulate in connective tissue, especially lymph nodes. Particles remaining on the surface are cleared with a biological half-time estimated to be twenty-four hours in humans, while particles that have penetrated into "fixed" tissues are cleared with half-times ranging from a few days to thousands of

• days. Therefore, the probability of particle penetration is critical in determining the clearance of

particles from the non-ciliated regions of the lungs. Particles removed by alveolar macrophages show a variety of patterns and half-lives which are dependent upon particle number, size, shape and surface reactivity. However, generally alveolar macrophages act to decrease the probability of particle penetration, thereby aiding clearance. These free cells, ultimately derived from the hematopoietic system, play the primary role in removal of dust particles and potentially pathogenic micro-organisms from the alveoli. Most free cells containing the deposited particles eventually reach the ciliated region of the lungs and are eliminated into the pharynx and swallowed. Pathophysiology of Pulmonary Macrophages In addition to a variety of protective postures that help the host, macrophages may also participate in the pathogenesis of lung disease. Inhaled cytotoxic, radioactive, or carcinogenic particles become concentrated in pulmonary macrophages because macrophages are actively phagocytic. What begins as a diffuse and uniform exposure becomes highly localized and nonuniform. Macrophages may also metabolize chemicals and change them to more toxic forms. Another way in which macrophages may be involved is through diminution or failure of their defensive role. Macrophage function can be compromised by environmental insults and pathological changes. Such diverse agents as silica, immunosuppressives, ethanol intoxication, cigarette smoke, nitrogen dioxide, ozone, and hyperoxia can depress the ability of pulmonary macrophages to protect their host. Pulmonary macrophages not only fail but may contribute directly to the pathogenesis of pulmonary

• diseases. Two important examples involve pulmonary connective tissue. Connective tissue proteins

have an essential role in lung structure and function. Collagen and elastin help maintain alveolar, airway, and vascular stability, limit lung expansion, and contribute to lung recoil at all lung

• volumes. Two groups of lung disease are associated with aberrations of normal collagen and elastin

balance: emphysematous and fibrotic disorders.

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Release of lysosomal enzymes, particularly proteases, from activated macrophages and leukocytes promotes the development of emphysema. Release is a consequence of cell death, cell injury, exocytosis, or regurgitation while feeding. Increased deposition of particles acts to recruit additional macrophages and thus may reinforce the effect. Oxidant gases which inactivate endogenous anti-proteases may further disturb the normal balance of proteolytic and antiproteolytic forces. Fibrogenesis also involves macrophage damage. Dead or dying macrophages may release substances that attract fibroblasts and elicit fibrogenic responses. These mediators may be chemotactic for fibroblasts; they also stimulate their proliferation, and their rate of collagen

• formation. Dust particles which are cytotoxic and durable are retained in the lungs and

stimulate persistent production of excess collagen in the alveolar wall. In such diseases as asbestosis and silicosis, progressive fibrogenesis may continue long after inhalation of dust particles has stopped. The response of the lungs to inhaled antigens and allergens is also emerging as an important area that involves macrophages. Considerable evidence is available showing that inhaled

• rganic chemicals, dusts, molds, and animal proteins can cause a variety of lung responses

such as allergic asthma, extrinsic allergic alveolitis, immune complex disease, and other

• phenomena. Yet, little is known about the degradation of proteins deposited on the

respiratory tract surfaces. Probably, in most instances macrophages and mucociliary transport defend the body against excessive antigenic stimulation. However, there may be other circumstances when clearance pathways cooperate with the immune system and preserve and present immunogenic molecules to the immune system. Thus the issue of how and when pulmonary macrophages suppress or enhance the immunogenicity of antigens must be confronted. In conclusion, macrophages serve as a key element in the defense of the respiratory tract. They are also capable of injuring the host while exercising their defensive role. Investigations should continue into the ultrastructural and biochemical features of normal pulmonary macrophages as well as their alterations after exposure to physical, chemical, and infectious agents. Retention The actual amount of a substance in the respiratory tract at any time is called the retention. When the exposure is continuous, the equilibrium concentration (achieved when the clearance rate matches the deposition rate) is also the retention. Thus, the relative rate constants of deposition and clearance determine the equilibrium levels; it is the equilibrium level, or retention integrated over time, and the properties of the particle that are presumably related to the probability of a pathological or therapeutic response. For nonliving particles, clearance results in decreasing amounts of material. Retention as a function of increasing time is a decreasing fraction of the amount initially deposited. In contrast, many pathogens are capable of replicating within the respiratory tract. Thus, sometimes following deposition

• f bioaerosols, the number of pathogens which can be cultured from the lung may actually

exceed the number initially deposited.

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BIBLIOGRAPHY I. Aerosol Generation, Deposition, and Clearance in the Respiratory Tract 1. Brain JD and Blanchard J. Deposition and Fate of Inhaled Pharmacological Aerosols. In: Spector, S. ed. New York: Marcel Dekker, Inc., pp. 157-213, 1995. 2. Brain JD and Blanchard JD. Mechanisms of particle deposition and clearance. In: Moren F, Dolovich MB, Newhouse MT, and Newman S, eds., Aerosols in Medicine. Principles, Diagnostics, and Therapy, 2nd ed. Amsterdam: Elsevier Science, pp. 117-156, 1993. 3. Brain JD, Curran MA, Donaghey T, Molina RM. Biologic responses to nanomaterials depend on exposure, clearance, and material characteristics. Nanotoxicology. 3:174-80, 2009. 4. Brain JD and Valberg PA. Aerosol deposition in the respiratory tract. Am. Rev. Resp. Dis. 120:1325-1373, 1979. 5. Brain JD, and Valberg PA. Models of lung retention based on the report of the ICRP Task

• Group. Arch. Environ. Health 28:1-11, 1974.

6. Brain JD. Inhalation, deposition and fate of insulin and other therapeutic proteins. Diabetes Technology & Therapeutics. 2007; 9 Suppl 1: S4-S15. 7. Brody AR, and Roe MW. Deposition pattern of inorganic particles at the alveolar level in the lungs of rats and mice. Am Rev Respir Dis. 128:724-9, 1983. 8. Darquenne C. Aerosol deposition in health and disease. J Aerosol Med Pulmn Drug Deliv. 25(3):140-7, 2012. 9. Choi HS, Ashitate Y, Lee JH, Kim SH, Matsui A, Insin N, Bawendi MG, Semmler-Behnke M, Frangioni JV, Tsuda A. Rapid translocation of nanoparticles from the lung airspaces to the body. Nature Biotechnology. 28(12):1300-1303, 2010.

• 10. Fabian P, Brain J, Houseman EA, Gern J, Milton DK. Origin of exhaled breath particles

from healthy and human rhinovirus-infected subjects. J Aerosol Med Pulm Drug Deliv. 2011 Jun;24(3):137-47.

• 11. Finlay WH, Martin AR. Recent advances in predictive understanding of respiratory tract
• deposition. J Aerosol Med Pulm Drug Deliv. 2008 Jun;21(2):189-206. Review. PubMed

PMID: 18518795.

• 12. Friedander SK. Smoke, dust and haze: Fundamentals of aerosol behavior. John Wiley &

Sons Pub. New York. 1977.

• 13. Heyder J, Blanchard JD, Feldman HA and Brain JD. Convective mixing in human

respiratory tract: estimates with aerosol boli. J. Appl. Physiol. 64(3): 1273-8, 1988.

• 14. Heyder J, Gebhart J, Rudolf G, Schiller CF, and Stahlhofen W. Deposition of particles in

the human respiratory tract in the size range 0.005-15 um. J. Aerosol Sci. 17:811-825, 1986.

• 15. Hinds WC. Aerosol technology: properties, behavior, and measurement of airborne
• particles. New York: John Wiley & Sons, Inc., 1999.
• 16. ICRP Publication 66: Human Respiratory Tract Model for Radiological Protection,

Pergamon, Vol. 24, 1994.

• 17. Johnson GR, Morawska L. The mechanism of breath aerosol formation. J Aerosol Med

Pulm Drug Deliv., 22(3):229-237, 2009.

• 18. Kreyling WG, André S, Collier CG, Ferron GA, Métivier H, Schumann G. Interspecies

comparison of lung clearance after inhalation of monodisperse; solid cobalt oxide aerosol

• particles. J. Aerosol Sci., 22:509-35, 1991.

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• 19. Leith DE, Butler JP, Sneddon SL, Brain JD. Cough. In Handbook of Physiology, The

Respiratory System (ed. A. P. Fishman), Sect. 3, vol III, Chapt. 20, pp. 315-336. Bethesda, MD, Am. Physiol. Soc. 1986.

• 20. Malashenko A, Tsuda A, Haber S. Propagation and breakup of liquid menisci and aerosol

generation in small airways. J. Aerosol Med. Poulmn. Drug Deliv., 22(4):341-353, 2009.

• 21. Muhlfeld C, Gehr P, Rothen-Rutishauser B. Translocation and cellular entering mechanisms
• f nanoparticles in the respiratory tract. Swiss Med Wkly. 138(27-28):387-391, 2008.
• 22. Rostami AA. Computational modeling of aerosol deposition in respiratory tract: a review.

Inhal Toxicol. 2009 Feb;21(4):262-90. Review. PubMed PMID: 19235608.

• 23. Tsuda A, Rogers RA, Hydon PE and Butler JP. Chaotic mixing deep in the lung. Proc. Natl.
• Acad. Sci. USA. 99:10173-8, 2002.
• 24. Valberg PA. Determination of retained lung dose. In: Toxicology of Inhaled Materials, HP

Witschi and JD Brain, eds. New York: Springer-Verlag, pp. 57-91, 1985.

• 25. Zeltner TB, Sweeney TD, Skornik WA, Feldman HA, and Brain JD. Retention and

clearance of 0.9-mm particles inhaled by hamsters during rest or exercise. J Appl Physiol 70: 1137-45, 1991.

• II. Mucociliary Transport, Pulmonary Macrophages, and Lung Responses

1. Braiman A, Priel Z. Efficient mucociliary transport relies on efficient regulation of ciliary

• beating. Respir Physiol Neurobiol. 2008 Nov 30;163(1-3):202-7.

2. Brain JD. Environmental lung disease: exposures and mechanisms. Chest, 109: 74S-78S, 1996. 3. Brain JD. Macrophages in the respiratory tract. Chp. 14 in: Handbook of Physiology - Section 3: The Respiratory System. Vol. 1. Circulation and Nonrespiratory Functions. A.P. Fishman and A.B. Fisher, eds. Bethesda, MD; American Physiological Society, pp. 447-71, 1985. 4. Brain JD. Toxicological aspects of alterations of pulmonary macrophage function. Ann Rev Pharmacol Toxicol. 26:547-65, 1986. 5. Gardner DE, ed. Inhalation Toxicology, Vol. 12, Num. 1-2, Jan.-Feb., 2000. 6. Gehr P and Heyder J, eds. Particle-Lung Interactions. New York: Marcel Dekker, Inc., 2009. 7. Godleski J and Clarke R. Pathophysiological mechanisms of cardiopulmonary effects. In Particle-lung interactions. Gehr P and Heyder J, eds. New York: Marcel Dekker, Inc., pp. 577-601, 2000. 8. Smith DJ, Gaffney EA, Blake JR. Modelling mucociliary clearance. Respir Physiol

• Neurobiol. 2008 Nov 30;163(1-3):178-88.

9. Tilley AE, Walters MS, Shaykhiev R, Crystal RG. Cilia dysfunction in lung disease. Annu Rev Physiol. 2015;77:379-406.

• III. Health Outcomes and General Sources

1. Brain JD, Beck BD, Warren AJ and Shaikh RA, eds. Variations in susceptibility to inhaled pollutants: Identification, mechanisms and policy implications. The Johns Hopkins University Press, Baltimore, 502 pages, 1988.

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2. Brain JD, Kreyling WG, and Godleski JJ. Inhalation Toxicology. In: Hayes’ Principles and Methods of Toxicology. Sixth Edition. Edited By: A. Wallace Hayes and Claire L.

• Kruger. CRC Press: Boca Raton. 2014. Pages 1385-1444.

3. Busse WW and Mathur SK. The biology of asthma. Chp. 45 in: Fishman’s Pulmonary Diseases and Disorders, Volume 1. Fishman AP, Elias JA, Fishman JA, Grippi MA, Kaiser LR, Senior RM, eds. New York: McGraw-Hill, pp. 773-86, 2008. 1. Dockery DW, Cunningham J, Damokosh AI, .Neas LM, Spengler JD, Koutrakis P, Ware JH, Raizanne M and Speizer FE. Health effects of acid aerosols on North American children: respiratory symptoms. Environ. Health Perspect. 104:500-5, 1996. 2. Dockery DW. Health effects of particulate air pollution. Ann Epidemiol. 2009 Apr;19(4):257-63. Review. PubMed PMID: 19344865. 3. Heinrich J, Hoelscher B, Wjst M, Ritz B, Cyrys J, and Wichmann H. Respiratory diseases and allergies in two polluted areas in East Germany. Environ. Health Persp. 107(1):53- 62, 1999. 4. Lehman-McKeeman LD. Absorption, distribution, and excretion of toxicants. Chp. 5 in: Casarett & Doull’s Toxicology: The Basic Science of Poisons, 7th Edition, Curtis D. Klaassen, ed. New York: McGraw-Hill, pp. 131-60, 2007. 5. Mohr U, Dungworth D, Brain JD, Driscol KE, Grafstrom RC, Harris CC, eds. Relationships between respiratory disease and exposure to air pollution. Washington D.C.: ILSI Press, 1998. 6. Mohr U, Dungworth D, Kimmerle G, Lewkowski J, McClellan R, Stober W, eds. Inhalation

• Toxicology. Springer-Verlag, New York. 307 pages, 1988.

4. Norris GS, YoungPong N, Koenig JQ, Larson TV, Sheppard L, and Stout JW. An association between fine particles and asthma emergency department visits for children in Seattle. Environ. Health Persp. 107(6):489-93, 1999. 5. Peters JM, Avol E, Gaudeman WJ, Linn WS, Navidi W, London SJ, Margolis H, Rappaport E, Vora H, Gong H, Jr. and D.C. Thomas. A study of twelve Southern California communities with differing levels and types of air pollution: II Effects on pulmonary

• function. Am. J. Respir. Crit. Med., 159:768-75, 1999.

6. Peters JM, Avol E, Navidi W, London SJ, Gaudeman WJ, Lurmann F, Linn WS, Margolis H, Rappaport E, Gong H, Jr. and Thomas DC. A study of twelve Southern California communities with differing levels and types of air pollution: I Prevalence of respiratory

• morbidity. Am. J. Respir. Crit. Med., 159:760-7, 1999.

7. Pope CA 3rd, Ezzati M, Dockery DW. Fine-particulate air pollution and life expectancy in the United States. N Engl J Med. 2009 Jan 22;360(4):376-86. PubMed PMID: 19164188. 8. Pope CA III. Epidemiology of fine particulate air pollution and human health: biologic mechanisms and who's at risk? Environ Health Perspect 2000; 108 Suppl 4:713-23. 7. Salem H and Katz SA, eds. Inhalation Toxicology. Taylor & Francis. New York. 2006. 8. Samet MS and Utell MJ. Indoor and outdoor air pollution. Chp. 60 in: Fishman’s pulmonary Diseases and Disorders, Volume 1. Fishman AP, Elias JA, Fishman JA, Grippi MA, Kaiser LR, Senior RM, eds. New York: McGraw-Hill, pp. 1009-36, 2008. 9. Spengler JD, Koutrakis P, Dockery DW, Raizanne M, and Speizer FE. Health effects of acid aerosols on North American children: air pollution exposures. Environ. Health

• Perspect. 104:492-9, 1996.

9. Weibel ER and Ochs M. Functional design of the human lung for gas exchange. Chp. 2 in: Fishman’s Pulmonary Diseases and Disorders, Volume 1. Fishman AP, Elias JA,

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Fishman JA, Grippi MA, Kaiser LR, Senior RM, eds. New York: McGraw-Hill, pp.23- 70, 2008.

• 10. Weiss ST and Apter J. Asthma: epidemiology. Chp. 46 in: Fishman’s Pulmonary Diseases

and Disorders, Volume 1. Fishman AP, Elias JA, Fishman JA, Grippi MA, Kaiser LR, Senior RM, eds. New York: McGraw-Hill, pp. 787-98, 2008.

• 10. Wichmann HE, Peters A. Epidemiological evidence of the effects of ultrafine particle
• exposure. Philos. Trans. R. Soc. Lond. A, 358: 2751-69, 2000.
• 11. Witschi HP, and Brain JD, eds. Toxicology of Inhaled Materials. Vol. 75 of Handbook of

Experimental Pharmacology. Springer-Verlag, Berlin. 553 pages, 1985.

• 12. Witschi HR, Pinkerton KE, Van Winkle LS, Last JA. Toxic responses of the respiratory
• system. Chp. 15 in Casarett & Doull’s Toxicology: The Basic Science of Poisons, 7th

Edition, Curtis D. Klaassen, ed. New York: McGraw-Hill, pp. 609-30, 2007.