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        "1266",
        "original Application",
        "The COUNCII. Fou -",
        "Densed",
        "\"10 KANT NTREET",
        "as too",
        "NRW YORK. N.Y. 10032",
        "and too much",
        "(212)",
        "of projects",
        "weil",
        "Application for Research Gront",
        "(Use exiro pages os needed)",
        "12-15-78",
        "1. Principal Investigator (give title and degrees):",
        "John E. Repine, MD",
        "John R. Hoidal,MD",
        "Assistant Director, Webb-Waring",
        "and",
        "Assistant Professor of",
        "Lung Institute",
        "Medicine",
        "Associate Professor of Medicine",
        "2. Institution & address:",
        "Univerisity of Colorado Medical Center",
        "4200 East Ninth Avenue",
        "Denver, Colorado, 80262",
        "3. Department(s) where reseorch will be done or colloboration provided.",
        "Webb-Waring Lung Institute",
        "and",
        "Department of Medicine",
        "4. Shorl title of study:",
        "The Role of Phagocytes in the Development of Centrilobular Emphysema",
        "5. Proposed starting date:",
        "July 1,1979.",
        "6. Estimated time to complete:",
        "one year",
        "7. Brief description of specific research aims:",
        "(1.) To determine whether there are differences in the abilities of lung phagocytes",
        "from human cigarette smokers and nonsmeking control subjects to make factors",
        "which attract monocytes and/or neutrophils.",
        "(2.) To determine whether there are differences in the oxygen(o,) uptake, super-",
        "oxide anion(0, ), hydrogen peroxide(H,0, hydroxyl radicaI(.OH), superoxide",
        "dismutase (sob) and catalase activities of the lung phagocytes from human",
        "cigarette smokers and nonsmokers.",
        "(3.) To determine whether alterations in the numbers and/or biochemical activities",
        "of lung phagocytes affect the development of centrilobular emphysema (CLE)",
        "a",
        "using chronic cadmium chloride(CdC1,) induced CLE in the rat as a model of",
        "suitable rn'",
        "I CLE seen in human cigarette smokers.",
        "&s' melity",
        "The leag tchm goal of the project is to determine the role of lung phagocytes",
        "in",
        "the develcoment of the CLE which is very frequently found in individuals who",
        "smoke cigarettes. If we can determine a relationship between cigarette smoking,",
        "the numbers and/or activities of lung phagocytes, and the development of CLE, we",
        "will be in a position to use this knowledge to test and find methods of altering",
        "the activities of lung phagocytes in order to medife or prevent the development",
        "of CLE.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
      ]
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        "2a.",
        "8.Brief statement of working hypothesis:",
        "The frequent occurence of CLE in cigarette smokers is well established but the",
        "mechanism responsible for this association is unknown(1-3) It seems reasonable",
        "to postulate that cigarette smoking causes an increase in the numbers of lung",
        "phagocytes by stimulating lung phagocytes to make factors which attract monocytes",
        "and/or neutrophils into the lung and that cigarette smoking stimulates lung phagocyte",
        "to make increased amounts of highly-reactive o, by-products which damage the lung",
        "phagocytes themselves and/or neighboring lung tissue. Injury to the phagocytes",
        "by o, by-products causes phagocytes to release enzymes which provide addj tional",
        "damage to the lung. These findings support our hypethesis.",
        "(1.)Individuals who smoke cigarettes have increased numbers of lung phagocytes,",
        "pigment-lader macrophages and to a lessor degree neutrophils, which are",
        "strategically located at the primary sites(peripheral airways) of the",
        "development of CLE(4-13).",
        "(2.)Stimulated lung macrophages have been shown under certain conditions to",
        "make factors which attract monocytes and neutrophils(14-16).",
        "(3. Stimulated phagocytes are fully capable of making and releasing extra-",
        "cellularly large quantities ef toxic O2 bv-products and granular enzymes",
        "(17-29).",
        "(4.)0, by-products from neutrophils can injure neutrophalls(30) and other",
        "tissues (31-35). Enzymes from phagocytes can damage lung tissue (36-37).",
        "(5.)The biochemical and enzymatic activities of macrophages from the lungs",
        "of cigarette smokers appear to be increased(38-40)",
        "Our second working hypothesis is that the rat model of CLE caused by chronic in-",
        "haltion of CdCl is an appropriate approach for studying CLE which is frequently",
        "2",
        "seen in individuals who smoke cigarettes. These findings support this observation.",
        "(1. Chronic exposure of rats to CdC1, causes CLE which closely resembles",
        "morphologically the CLE of humañ cigarette smokers(41)",
        "(2.) Chronic expesure of rats to CdC1, increases the numbers of phagocytes in the",
        "lung and the locations, numbers, and types of these phagocytes are similar",
        "to the findings in human cigarette smokers (41-44),",
        "(3.) Chronic exposure of rats to CdC1, appears to increase the biochemical and",
        "enzymatic activities of lung phagocytes so that they resemble the activated",
        "lung phagocytes of human cigarette smokers (45).",
        "(4.)Cigarette smok.e is the major source of .Cd exposure in humans (46).",
        "(5.)Cd is present in a dose-severity relationship in the lungs of cigarette",
        "smokers with (47).",
        "(6.)Workers chronically exposed to CdC1, develop CLE which is similar to CLE",
        "found in human cigarette smokers (48=50).",
        "(7.)Cd decreases the activities of SOD and catalase, two enzymes found in",
        "phagocytes, which serve to detoxify O, and H,0,, respectively (50-52).",
        "(8.)Animal models using exposure to cigarêtte smöke are expensive, technically",
        "difficult, require administration of cigarette smoke for long durations,",
        "and",
        "often don't develop the exact changes seen in the lung pathology or",
        "lung phagocytes seen in human cigarette smokers (53).",
        "When 1) the increased numbers of phagocytes (?5X or more) in the lungs of cigarette",
        "smokers, 2) the frequent infections of older cigarette smokers which stimulate the",
        "biochemical activities of lung phagocytes(?2X), and 3) the increased biochemical",
        "activities of phagocytes even in the healthy cigarette smokers (?2X) are considered,",
        "the potential for cigarette smoke induced phagocyte mediated lung injury may be of",
        "amplified significance.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
      ]
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        "2b.",
        "H",
        "16135",
        "9.Details of experimental design and procedures:",
        "A. Research Plan: This section will be divided into sections on experimental",
        "design, detailed technical aspects, and anticipated problems.",
        "1. Experimental Design: We will perform experiments to provide answers to",
        "the following questions.",
        "a. Are there differences in the ability of lung phagocytes from human",
        "cigarette smokers or nonsmokers to make factors which attract",
        "monocytes and/or neutrophils?",
        "(1.)We will search(using Boyden chambers) for factors which attract",
        "monocytes and neutrophils in the lavages from the lungs of",
        "asymptomatic, voung age matched and nonsmoking control",
        "subjects.",
        "(2.)We will remove phagocytes from the lungs of human smokers and",
        "nonsmokers, incubate them alone or with non chemotactic stimulants",
        "such as latex particles,collect the supernatants from these",
        "mixtures, and test them for chemotactic activity.",
        "b. Are there differences in the biochemical activities of lung phagocytes",
        "from human cigarette smokers or nonsmokers?",
        "(1.)We will measure the O,uptake, ,H2021 *Oh, SOD, and catalase",
        "activities of phagocytes from the lüngs of sFokers and nonsmokers.",
        "(2.)We will measure malondialdehyde, SOD, and catalase activities in",
        "the lavages from the lungs of human smokers and nonsmokers.",
        "(3.)We will measure the Cd levels of triply-washed phagocytes from",
        "the lungs of smokers and nonsmokers and correlate these levels",
        "with the biochemical activities of the lung phagocytes.",
        "c. Do alterations in the numbers and biochemical activities of lung",
        "phagocytes alter the development of CLE?",
        "(1.)We will determine the numbers and biochemical activities of washed",
        "phagecytes from the lungs of rats exposed to various concentrations",
        "of CdC1. and correlate these findings with the degree of CLE",
        "observed morphologically.",
        "(2.)We will also measure malondialdehyde,SoD, and catalase activities",
        "of lavage fluids from the lungs of CdC1, treated experimental",
        "and control rats.",
        "(3.)We will measure the Cd levels of the lavage fluids and the washed",
        "phagocytes and correlate these findings with the biochemical",
        "measurements and the morphologic determinations of CLE.",
        "(4.)We will perform manipulations, alone or in combination, to alter",
        "the numbers and activities of phagocytes in the lungs of CdC1,",
        "treated rats and correlate these findings with changes in the",
        "development or severity of CLE.",
        "(a.) IImmunization with BCG or Freunds adjuvant (increases the",
        "numbers and biochemical activities of phagocytes, 54)",
        "(b.)exposure to 100%0, (increases numbers and activities,54).",
        "c.)nitrogen mustard or cytoxan (decreeses the numbers,! 55).",
        "it.)diethyldithiocarbamate (DDC) treatment (inhibits SOD,56).",
        "(e.)aminotriazole (AMT)treatment (inhibits catalase, 57).",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
      ]
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        "2c.",
        "K once",
        "9. continued.",
        "2. Detailed Technical Aspects:",
        ".Selection of human subjects and recovery of lung phagocytes: Lung",
        "phagocytes will be obtained by standard sterile saline lavage using a modified flexible",
        "Tiberoptic bronchoscope which can be wedged and sealed in the appropriate pulmonary",
        "subsegment thereby elminating contamination by upper airway celle or substances (58).",
        "Dr. Hoidal has performed more than 2000 of these procedures without complication.",
        "The",
        "procedure has been approved by the Committee for Research on Human Volunteers for the",
        "University of Colorado. We have chosen to compare young asymptomatic, age matched",
        "smoking and nonsmoking individuals because we are interested in the earliest changes",
        "occuring in the phapocytes of cigarette smokers and want to avoid confounding variables",
        "such as infection, lung dysfunction, or malignancy, which are commonly found in the",
        "older, cigarette smoking individuals. A majority of the lavage fluid is retrievable",
        "by gentle aspiration and contains the lung phagocytes.",
        "b.Care and exposure of rats to CdC1,: Cesarean-derived pathogen",
        "controlled male Sprague Dawley(275-325gm)rats will be obtained from a commercial",
        "supplier and kept in clean plastic cages fitted with filters and housed in temp-",
        "erature controlled animal quarters. Rats will be fed antibiotic free rat chow and",
        "given water ad libitum. Groups of rats will be exposed to polydispersed CdC1, (41)",
        "aerosol (1x10 M) in saline using a deVilbiss 35A ultrasonic nebulizer. Rats are exposed",
        "for 1 hr/day for 0,40r9 days in a specially designed exhaust hood. Control rats are",
        "exposed to saline in similar fashion. Two hours, 2,5, or 10 days after the last",
        "exposure to CdC1, or saline, the experimental and control rats are weighed, anesthet-",
        "ized by intraperitoneal injection of sodium pentobarbital, and exsanguinated by aortic",
        "transection. In our pilot studies these condicions produced changes in the lung",
        "pathology and inflammatory cells which closely resembled conditions present in",
        "human cigarette smokers.",
        ".Recovery of phagocytes and preparation of rat lungs for evaluation",
        "of CLE: After sacrifice, experimental and control rats will be divided into two groups.",
        "In one half of the rats the lungs will be removed, weighed, and lavaged with sterile",
        "saline (measured amounts). For some experiments, homogenates will be made of lavaged",
        "phagocytes, lungs previously lavaged for phagocytes, and total un-lavaged lungs. These",
        "studies will be done to give an indication of the relative contribution of lung",
        "phagocyte and non-lung phagocyte components to the products in the lavage fluid.",
        "Lungs from the other half of the experimental and control rats will be fixed by constan",
        "25cm. pressure inflation with glutaraldehyde for 2 days(59). Following fixation the",
        "lungs are cut sagitally, processed for histologic staining at 5u, and stained with",
        "Hematoxalin-Phloxin-Safranin.",
        "d. Evaluation of inflammation and CLE morphologically: Following fix-",
        "ation, sections from the rat lungs will be evaluated in double blind fashion and",
        "scored individually for the presence of mural inflammatory cells, peribronchiolar",
        "fibrosis, and eptihelial metaplasia. Multiple sections from each lung and each group",
        "of rats will be evaluated. The extent of CLE will be measured in standard fashion",
        "by determining the mean linear intercepts in 20 randomly selected fields from 4 section",
        "from each lung (60).",
        "e Isolation and characterization of phagocytes from lung lavages:",
        "Phagocytes are isolated from lung lavages by centrifugation at 400xg, resuspended in",
        "Hanks' Balanced Salt Solution(HBSS), and differentially counted by hemocytometer",
        "Macrophages are also quantitated and characterized using trypan blue exclusion (viabilit",
        "esterase, Wright's Giemsa, and acid phosphatase staining. When indicated and necessay,",
        "neutrophils and macrophages can be separated by Ficoll-Hypaque centrifucation(61) and",
        "macrophage sf rom lymphocytes with a cell density separator (62)",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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        "2d.",
        "H",
        "9. continued",
        "E.Measurement of chemotactic activities: We will collect and test",
        "supernatants from the lung lavages for their ability to attract monocytes and/or",
        "neutrophils in vitro using Boyden chamber technique(53-64). Monocytes and neutrophils",
        "will be separated from heparinized dextran sedimented human blood using Ficoll-Hypaque",
        "centrifugation as previously described (63-64). After placing the cells in the top",
        "compartment of the chamber, we will count the numbers of cells which move through",
        "separating Millipore filters (8u for monocytes, 5u for neutrophils) in response to",
        "various concentrations of lavage supernatants, HBSS(random locomotion), or known",
        "stimuli, such as C5a, from zymosan activated serum, or synthetic chemotactic peptides",
        "(65) in the bottom compartment (stimulated locomotion, chemotaxis) The chambers are",
        "then incubated for 3 hours at 37°c. After incubation, the filters are removed, rinsed",
        "and stained in a holding rack. The filters are then placed upside down on a microscop",
        "slide and permanently mounted for counting. The number of phagocytes that traverse the",
        "filter in 5HPF are counted. The chemotactic factors from lung macrophages will be",
        "characterized with respect to heat stability, molecular weight, and ether extractabilit",
        "as described(14-15).",
        "g .Preparation of serum, heat-killed bacteria, and PMA: The subsequent",
        "studies of lung phagocyte biochemistry will be done with unstimulated or bacteria or",
        "PMA stimulated phagocytes. Serum(for preopsonization, 66) is obtained from 5 normal",
        "human subjects or 5 rats, allowed to clot, rimmed, recovered by centrifugation, pooled",
        "and then frozen at -70°C for not more than one month. Fresh homologous serum will be",
        "used in some experiments. Heat-killed bacteria (HKB) are prepared by heating at 70°C",
        "for 30 minutes twice washed Staphylococcy : aureus, 502A which has been grown for 17 hrs",
        "at 37°c in Penassay broth (67) Suspensions of HKB are sonicated and diluted with HBSS",
        "to yield bacteria to macrophage ratios of 100:1 and bacteria to neutrophil ratios of",
        "50:1 ( optimal ratios). MA(12-0-tetradeconyl phorbol-13-acetate, MW616) is dissolved it",
        "dimethyl sulfoxide(De(so) at a stock concentration of 2mg/ml and stored in the dark at",
        ".°c for two weeks cr until use. Jus: before usé, PMA is diluted with HBSS and used at",
        "a concentration of Jug/ml(67-69).",
        "h.Measurement of oxygen consumption:0, uptake is assayed on a Yellow",
        "Springs biologic 0, electrode attached to a Beckman recorder (67-69). Mixing vials are",
        "maintained at 37°C by a circulating water bath and stirred with a magnetically driven",
        "stirring bar. Rates of o, uptake are calculated from the maximum slope over a 10 min.",
        "6",
        "period from a 3ml vial containing 10x10 phagocytgs alone , with preopsonized HKB,",
        "or",
        "PMA. Data are expressed as 010,/consumed/hr/5x10 phagocytes.",
        "1.Measurement of superoxide anion release:0, release is determined b",
        "measuring",
        "SOD inhibitable horse heart ferricytochrome c réduction spectrophoto-",
        "netrically at 550nn(68-70). Reaction mixtures (1m1) contain 5x10 phagocytes in HBSS.",
        "In some samples, HKB or PMA are added. One of a pair of mixtures is incubated for 20",
        "min. at 37 C;the other is kept on ice and used as a blank. Specificity of cytochrome",
        "C reduction is determined by using identical mixtures which also contain 50ug/ml SOD.",
        "After incubation, the mixtures are rapidly cooled to 4°C and centrifuged at 800xg for",
        "20 min. Supernatants are assayed spectrophotometrically and the results are copverted",
        "-I",
        "to nanomoles of SOD inhibitable cytochrome C reduced/20min. (E -2.1 x 10 m em \").",
        "550nm",
        "j.Measurement of hydrogen peroxide production:H,0 is measured using",
        "the scopoletin assay(71).Briefly the 460nm emission from reduced scopoletin excited by",
        "light is extinquished when scopoletin is oxidized by in the presence of horse-",
        "radish peroxidase(MPO). The loss of flourescence is proportionel to the concentration",
        "cf H.o. The reaction mixtures contain 5x10 phagocytes in HBSS, HKD or PMA solutions",
        "202",
        "at 37°C and 10mN scopoletin to which 22mM HPO is added. The system is standardized",
        "with known amounts of #2°2°",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
      ]
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        "K 00316135",
        "2e.",
        "9. continued.",
        "k.Measurement of ethylene generation: OH is a powerful O2 by-product",
        "generated under appropriate conditions by the interaction of 0, and H2O, It is",
        "believed that ethylene produced by phagocytes may represent .OR(19,20) or singlet",
        "o, (21). We will use the standard gas chromatographic analysis of ethylene produced",
        "by",
        "phagocytes with and without inhibitors (19-21). We will again study phagocytes",
        "alone, following stimulation with HKB or PMA, with and without inhibitors such as",
        "SOD, catalase, ethancl, tryptophan, benzoate, thiourea, DABCO, azide. and histidine.",
        "In addition we have been working the past year and one half on a new assay for .OH",
        "radical which we feel will be more specific than conventional assays. The work on this",
        "approach will be completed in all likelihood before the start of this grant.",
        "1.Measurement of superoxide dismutase:SOD is determined by measuring",
        "the rate of inhibition of the spontaneous oxidation of pyrogallo] at 41 5nm in Tris",
        "buffer,pH8.3(72) Briefly, samples from homogenates of phagocytes or concentrated",
        "lavages are added to a stock solution of pyrogallol in dilute HCI. The resultant",
        "mixture is then added to a 3cc. of the Tris buffer and the rate of oxidation of pyro-",
        "gallol is recorded on a Beckman recorder- attached to a spectrcphotometer. The degree",
        "of inhibition of the rate of oxidation is compared to a purified standard.",
        "m.Measurement of catalase:Catalase is measured using a biologic O2",
        "probe attached to a recorder to determine the 0, produced by the catalase sample",
        "upon addition of Sul of H.O. (73). The linear increase in 0, concentration is recorded",
        "for 2 min. A standard curve is constructed using purified commercial bovine liver",
        "catalase. The results are expressed as units/mg protein/min.",
        "n.Measurement of malondialdehyde:MI is a by-product of the reaction",
        "of free radicals with lipids and is measured by a standard method(74). Reaction mix-",
        "tures are incubated and then terminated by the addition of 0.5%(w/v) trichloroacetic",
        "acid per ml. of incubation mixture. Then, 1 ml of 0.5% 2-thiobarbituric acid is added",
        "per ml. of the incubation mixture and the tube is heated at 60 for 90 min. After",
        "cooling, 1 ml of 70. trichloracetic acid is added to each tube. Then, 3 ml chloroform",
        "is added and the tubes are shaken to remove dispersed lipid. The samples are then",
        "centrifuged a.d read spectrophotometrically at 532nm.",
        "Measurement of cadmium:Cd is measured using the established",
        "technique(75). Tissues and samples for measurement of Cd must be handled and prepared",
        "for Cd analysis with care to avoid contamination. Samples are dried at 100°C for 24hr",
        "and ashed with the temperature carefully controlled at 450°C for 48 hr. The ash is",
        "then dissolved in 5ml of 6N nitric acid and assayed in duplicate for cadmium by",
        "atomic absorbtion spectroscopy, using the Perkin Elmer 403 with an attached deuterium",
        "arc for background collection. A number of other ions such as zinc, sodium, potassium,",
        "and calcium can also be measured by this technique aad used for control purposes.",
        "p.Analyzation of data and statistics:Granted that the initial studies",
        "will involve extensive data collection, we have already performed enough experiments",
        "to focus our investigation and allow us to persue times and concentrations of CdC1,",
        "which produce changes typical of CLE in cigarette We will perform the4",
        "studies with large groups of rats and undertake enough experiments so that statistical",
        "analysis can be applied appropriately to determine differences between experimental",
        "and control situations (76).",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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        "KBo 116139",
        "2f.",
        "9. continued.",
        "3.Anticipated Problems. An attempt has been made to discover potential",
        "problems in order to find methods by which they",
        "might be minimized or accounted for in interpret-",
        "ation.",
        "3. Spedific phagocyte causing CLE: Macrophages and/or neutrophils",
        "could be responsible for the CLE found in cigarette smokers. In the present studies",
        "we have elected to initially not try to distinquish between the roles played by each",
        "cell individually until we ':ave clearly documented that phagocytes play a role in CLE.",
        "Techniques are available for separating macrophages from neutrophils so that we can",
        "undertake this clarification of our findings at the appropriate time. The evidence",
        "available suggests that macrophages and/or neutrophils could be involved in the de-",
        "velopment of CLE. The normal lung contains macrophages and few if any neutrophils.",
        "Lavages of normal nonsmoking individuals yield on the average 20 million macrophages",
        "with less than 1%: neutrophils. Lavages of young, asymptomatic smokers yield 100",
        "million macrophages with 5-8% neutrophils. In our pilot studies we have found that",
        "rats exposed to large concentrations of CdC12 acutely develop lung injury but more of",
        "a panlobular type. In contrast rats exposed to smaller concentrations of CdC1, in a",
        "chronic fashion develop CIE and lavages contain markedly increased numbers of macro-",
        "phages(5-9x) and approxinately 5% neutrophils. While the increased numbers of macro-",
        "phages relative to neutrophils clearly favors this cell type as a prime factor, we",
        "know that the biochemical activities of stimulated neutrophils is 3-6X the metabolic",
        "activities of macrophages. We will keep the various numbers and biochemical activities",
        "of these two lung phagocytes in mind and not attribute lung injury to one or the other",
        "of them until we have firm evidence to support their involvement.",
        "b. Infection:The presence of lung infection in humans or animals pro-",
        "foundly affects the nature of airway inflammation, the function of lung phagocytes, and",
        "the development of CLF. To minimize this problem we will use rats. Our previous",
        "work has shown that there are similar patterns in the metabolic activities of macro-",
        "phages from the lungs of humans and rats and that there is a low incidence of infection",
        "in rats. We will take every precaution to keep animals free of infection. However,",
        "we will still continue to culture lavage materials from the lungs of experimental and",
        "control subject to monitor this problem.",
        "c. Non-representative nature of the lavage samples: Another concern is",
        "that the phagocyte content of the lavage returns do not accurately reflect the phago-",
        "cytes present in the total lung. This problem is amplified because the cells in the",
        "lavages come from all areas of the lung and not just areas of inflammation or injury.",
        "Fortunately, CLE is a diffuse process. Attempts to determine the validity of this",
        "concern by obtaining cells from minced lungs and comparing the numbers and types and",
        "functions with lavage results have not revealed any remarkable differences. However,",
        "the approach is complicated by the contamination of lung minces with mar.; interstitial",
        "cells. Another reassurance that the lavage may accurately reflect lung conditions is",
        "our previous work which shows that relatively only a few phagocytes remain in the lung",
        "following lavage and the increases numbers of phagocytes are primarily in the",
        "bronchioles where we know that they are effectively recovered by lavage.",
        "d. Lack of specificity of some of the reagents: Some of the manipul-",
        "ations suggested to alter the numbers and/or biochemical activities of phagocytes in",
        "the lung are not specific for this feature alone we will run controls with the",
        "agents alone and consider this in our interpretation of the results. Likewise some",
        "of the reagents used to scavenge activated o, by-products are not entirely specific",
        "and interpretation of their specificity must be undertaken with considerable caution.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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        "ir",
        "2g",
        "continue",
        "9. continued.",
        "B. Importance of the Proposed Work:",
        "Centrilobular emphysema (CLE) resulting from cigarette smoking is an important",
        "and common clinical problem. The incidence, morbidity, and mortality of amokers.",
        "is very impressive. In the United States alone, approximately 50% of the population",
        "smokes cigarettes and mere than 25,000 people die of emphysema each year at an estimated",
        "cost of greater than 1.5 billion dollars (77). Since campaigns to encourage individuals",
        "to quit smoking cigarettes have beea unsuccessful(1) better understanding of the",
        "mechanisms linking cigarette smoking to emphysema need to be determined so that more",
        "effective methods of interrupting lung injury can be found. Additional information oi",
        "use may also come from the proposed studies. First, we may be able to determine if",
        "chere is a mechanism underlying the ability of certain individuals to smoke cigarettes",
        "and yet avoid any emphysema. If we can discover this information we will be in a",
        "position to test individuals and determine if they are unusually susceptible to the",
        "toxic effects of rigarette smoke. Second, we may be able to apply the information",
        "to improve understanding of the toxic effects of cigarette smoke on a number of other",
        "organs other than the lung such as the heart and the vessels. Third, since phagocytes",
        "are involved in the pathogenesis of a number of lung diseases which are not related to",
        "cigarette smoking, the proposed studies may provide information which may be basic",
        "to elucidating the role of phagocytes in a variety of lung injuries.",
        "c. Previous Work on this or Related Projects by the Applicants:",
        "We hove performed many investigations during the past seven years to determine",
        "the",
        "activities of lung phagocytes and these are summarized in the biographies of the",
        "applicants. We have also been working on this project for the last year. The most",
        "relevant work will be summarized below.",
        "(1.) Published studies: We have documented",
        "-",
        "(a.) that Human alveolar macrophages consume 0,, make 0, , and OU when",
        "stimulated by bacteria or phorbol myristate acetate: AM increase thesé activities.",
        "(b.)that the metabolic. activities of lung macrophages from rats, rabbits,",
        "and human subjects are similar.",
        "(c. ) that lung macrophages from cigarette smokers have decreased adherence",
        "and altered surface morphology.",
        "(2.) Unpublished preliminary studies: We have found",
        "(a.) that factors which attract neutrophils in vitro can be isolated from",
        "the lung lavages of animals exposed to 100% o, and that lung macrophages in culture in",
        "response to 100: 0, make factors which attract neutrophils in vitro.",
        "(b.) that malondialdehyde can be isolated from the lavages of the Jungs",
        "of",
        "animals exposed to 100% o, and in concentrations which exceed those found in control",
        "animals which have been exposed to room air.",
        "(c.) that there are increased numbers of macrophages and neutrophils in",
        "the",
        "lavages of the lungs of young, asymptematic cigarette smokers compared to non-",
        "smoking age~matched control subjects.",
        "(d.) that the 0,- production of macrophages from the young cigarette",
        "smokers is increased compared to macrophages from nonsmoking controls. This is in",
        "spite of the fact that the o, uptake of the smoker and nonsmoker macrophages are equal.",
        "The basis of this finding could be related to the decreased SOD activities we have",
        "found in the macrophaçes from the cigarette smokers. The differences in the biochemical",
        "activities of smoker macrophages can not be explained on the basis of cell size and/or",
        "protein differences in the smoker and nonsmoker macrophages. These findings support",
        "our assumption that Cd frem cigarette smoke may have altered the SOD content oi the",
        "lung macrophages and that this is reflected in increased production of C2-.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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        "2h",
        "(80316441",
        "9. continued.",
        "(e.) that rats exposed to chronic CdC1, develop CLE and changes in the",
        "numbers and biochemical activities of their lung phacocytes which closely resemble",
        "changes seen in human cigarette smokers. Specifically rats administered CdC12 have",
        "increased numbers of macrophages and the 0,- production of these macrophages is",
        "increased over levels produced by macrophages from saline exposed rats.",
        "D. Results of this or Similar Project Obtained by Others:",
        "The primary supporting evidence for this work has been provided in section",
        "8",
        "of the grant application. To our knowledge we are the only individuals pursuing",
        "the role of O2 by-products in the production of CLE using a comprehensive approach o",
        "comparing humän Sand rats exposed to CdC1,- There is however wide and increasing",
        "interest regarding the role of phagocytes in lung injury and the nature and mechanisr",
        "responsible for phagoryte locomotion, biochemical and enzymatic function and tissue",
        "injury. We are also aware that the measurement of 0, by-products is a sophisticated",
        "undertaking. We will be aware cf any developments that occur in these areas and",
        "modify our approaches appropriately during the course of the investigations.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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        "21",
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        "2m",
        "73. Beers RF Jr, and Sizer IW: A spectrophotometric method for measuring the",
        "breakdown of hydrogen peroxide by catalase. J Biol Chem 195:133,1952.",
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        "Inquiry 13:15,1976.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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        "J.",
        "ir Spuc\" and available (when elsewhore tion item 2 indicates, state location;-",
        "For this project we will have access to the facilities of the Webb-Waring Lung",
        "Institute. These include a space assigned to us of approximately 3000 sq. ft.",
        "The Webb-Waring is equipped with sophisticated animal quarters and ecuipment for",
        "transmission electron microscopy There are also facilities for culture. The",
        "equipment available includes scintillation counters, centrifuges, microscopes,",
        "aerosol equipment, spectrophotometer; freezers, oxygen monitor, and sterilization",
        "equipmert. The University of Colorado is an excellent training center for pulmonary",
        "physicians so many patients are available for study by bronchoscopy. In addition",
        "there are a large number of investigators interested in macrophage function and",
        "structure, lung pathology, and cellular immunology in the Webb-Waring Lung Institute",
        "and the surrounding University of Colorado Medical Center and National Jewish",
        "Hospitals.",
        "11. Additional facilities required:",
        "None",
        "12. Biographical sketches of investigator(s) and other professional personnel (append):",
        "13. Publications: (five most recent and pertinent of investigator(s); append list. and provido reprints if available).",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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      "text": [
        "14. buage*",
        "A. Salunes (give nomes or \":o te recrunci\",",
        "Protessional (give % time a investigator(s)",
        "even if no salary requested)",
        "John E. Repine, M.D., Principal Investigator",
        "Technical",
        "Researcher I - to be recruited",
        "Fringe benefits",
        "Primitivo Romero, Research Animal Attendant 11",
        "Fringe benefits",
        "Sub-Total for A",
        "B. Consumoble supplies (by major categories)",
        "Animals",
        "Chemicals and Gases",
        "Glassware",
        "Syringes, Millipore filters",
        "Sub-Tetal for 6",
        "c. Grier expenses (itemize)",
        "Animal board",
        "Publication costs",
        "Equipment maintenance",
        "Running Total of A + B - c",
        "D. Permanent equipment (itemize)",
        "Sub-Total for D",
        ": ingirect costs (15% of A+B-C)",
        "Estimated future requirements:",
        "Salaries",
        "Consumoble Suppl.",
        "Other Eaponses",
        "Permanent Equip.",
        "Inance Costs",
        "T.",
        "Y",
        "12,730",
        "10,010",
        "7,150",
        "4,484",
        "34,374",
        "Y.ar 3",
        "14,130",
        "11,011",
        "7,865",
        "37,957",
        "4,951",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215",
        "% time",
        "Amcont",
        "H KB0316148",
        "-0-",
        "6,300",
        "50",
        "850",
        "3,732",
        "487",
        "11,369",
        "1,500",
        "2,000",
        "5,000",
        "600",
        "9,100",
        "4,500",
        "500",
        "1,500",
        "6,500",
        "Sub-Toral for c",
        "26,969",
        "4.045",
        "E",
        "31,014",
        "Total request"
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      "text": [
        "is understood that the investigator and institutional",
        "icers in applying ior o grant have read and accept",
        ": Council's \"Statement of Policy Contoining Conditions",
        "d Terms Under Which Project Grants Are Made.\"",
        "payable to",
        "University of Colorado Medical Center",
        "uling address for checks",
        "Howard T. Leavenworgh,Director_ OGC",
        "4200 Eant Ninth Avenue",
        "Denver, Colorado 80262",
        "16. Other sources of financial support.",
        "List financial support from all sources, including own institution, for this and related research projects",
        "Inclusive",
        "All of our present",
        "support for this and",
        "related projects is",
        "local (Minnesota)money",
        "which will not be",
        "available when we move",
        "to the Webb-Waring Lung",
        "Institute in Denver in",
        "July, 1979.",
        "PENDING OR PLANNED",
        "Source",
        "Title of Project",
        "(give grant numbers)",
        "Phagocyte Mediated Luns",
        "American Heart Assoc.",
        "Injury in 0,-Toxicity",
        "Mechanisms of 0,",
        "National Institutes of",
        "Induced Lung Injury",
        "Health",
        "Typed Name",
        "Signature Date",
        "Tolephone",
        "3.",
        "il E0316149",
        "CURRENTLY ACTIVE",
        "Source",
        "Title of Project",
        "Dates",
        "(give grant numbers)",
        "Amount",
        "Inclusive",
        "Amount",
        "Dates",
        "July 79-81.",
        "(20,000/yr",
        "July 79-81.",
        "50,000/yr",
        "I",
        "Principol investigators",
        "John R. Hoidal",
        "John E. Repine,",
        "Areo Code",
        "Nember",
        "Responsible officer of institution",
        "Typed Nome John W. Cowee",
        "Title Signate/ Chancellor Kath Data 12/19/78",
        "7682",
        "Tolephone",
        "Areo Code",
        "Number",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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      "text": [
        "5.",
        "80316149",
        "16. Other sources of financia: suppon",
        "List financiali support from all sources, including own institution, for this and related research projects.",
        "CURRENTLY ACTIVE",
        "Source",
        "inclusive",
        "Tutle of Project",
        "(give grant numbers)",
        "Amount",
        "Dates",
        "All of our present",
        "support for this and",
        "related projects is",
        "local (Minnesota) money",
        "which will not be",
        "available when we move",
        "to the Webb-Waring Lung",
        "Institute in Denver in",
        "July, 1979.",
        "PENDING OR PLANNED",
        "Source",
        "Inclusive",
        "Title of Project",
        "(give grant numbers)",
        "Amount",
        "Dates",
        "Phagocyte Mediated Luns",
        "20,000/yr",
        "July 79-81.",
        "American Heart Assoc.",
        "Injury in 0,-Toxicity",
        "Mechanisms of 02",
        "National Institutes of",
        "Induced Lung Injury",
        "50,000/yr",
        "July 79-81.",
        "Health",
        "is undersicod that the investigator and institutional",
        "Principal investigator s",
        "icers in opplying ior o grant have read and accept",
        "John E. Repine,",
        "John R. Hoidal",
        ": Council's \"Statement of Policy Contoining Conditions",
        "Typed Name",
        "id Terms Under Which Project Grants Are Mode.\"",
        "Signature paurteyne John Date",
        "R Handal",
        "Telephone",
        "Arec Code",
        "Number",
        "Gatemion",
        "asts payable IO",
        "Responsible officer of institution",
        "University of Colorado Medical Center",
        "Typod Name John W. Cowee",
        "aling address for checks",
        "Chancellor",
        "Howard T. Leavepworth, Director OGC",
        "4200 East Ninth Avenue",
        "Signatule Title our Kathe 7682 Date 12/19/28",
        "prepartion",
        "Tolephone",
        "Denver, Colorado 80262",
        "Areo Cede",
        "Number",
        "Enterioun",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
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      "text": [
        "6.",
        "H",
        "12. Biographical Sketches.",
        "Curriculum Vitae",
        "Repine, John, E., M.D.",
        "Assistant Director, Webb-Waring Lung Institute",
        "Associate Professor of Medicine",
        "University of Colorado Medical Center",
        "U.S. Citizen, born Rock Island, Illinois,December 26,1944 (age 33)",
        "Education and Professional Experience",
        "Degree",
        "Year",
        "University of Wisconsin, Madison, Wisconsin",
        "B.S.",
        "1966",
        "University of Minnesota, Minneapolis, Minnesota",
        "M.D.",
        "1971",
        "Internship, Internal Medicine Residency, and",
        "Pulmonary Medicine Fellowship-University of",
        "Minnesota Health Sciences Center",
        "1971-75",
        "Instructor, Dept. of Medicine, Univ. of Minnesota",
        "1974-75",
        "Asst. Prof. ,Dept. of Medicine, Univ. of Minnesota",
        "1975-78",
        "Assoc. Prof., Dept. of Medicine, Univ. of Minnesota",
        "1978-79",
        "Assec. Prof., Dept. of Medicine, Univ. of Colorado",
        "Asst. Dir. Webb-Waring Lung Institute",
        "1979-",
        "Honors",
        "Evans Scholar",
        "SAMA Scientific Exhibit Award",
        "Young Pulmonary Investigator Award(NIH)",
        "Mead-Johnson Grand award for Research by a Resident-SAMA-UTMB-1971",
        "Minnesota Heart Fellowship",
        "Basil O'Connor Starter Research Award - March of Dimes",
        "Graduate Research Award(NIH)",
        "Established Investigator Award (American Heart Associacion,1976-1981).",
        "Selected Bibliography-Published Manuscripts and manuscripts accepted for",
        "publication",
        "Repine JE, White JG, Clawson CC and Holmes BM: The influence of phorbol myristate",
        "acetate on oxygen consumption by polymorphonuclear leukocytes.J Lab Clin Med83:911,1974",
        "Repine JE, White JG, Clawson CC and Holmes BM: Effects of phorbol myristate acetate on",
        "the metabolism and ultrastructure of neutrophils in chronic granulomatous disease.",
        "J Clin Invest 54:83,1974.",
        "Repine JE, White JG, Clawson CC and Holmes BM: The influence of phorbol myristate",
        "acetate on the metabolism of neutrophils from carriers of sex-linked chronic",
        "granulomatous disease. J Lab Clin Med 85:82,1975.",
        "Repine JE, Clawson cc, White JG and Holmes BM: Spectrum of function of neutrophils from",
        "carriers of sex-linked chronic granulomatous disease. J Pediatrics 87:813,1975.",
        "Repine JE, Clawson cc, and Brunning RD: Primary leukocyte alkaline phosphatase",
        "deficiency in an adult with repeated infections. Brit J Hemat 34:87,1976.",
        "Repine JE, Clawson cc, Burchell HB, and White JG: Reversible neutrophil bactericidal",
        "defect in bacterial endocarditis. J Lab Clin Med 88:780,1976.",
        "Repine JE, Clawson cc. and Brunning RD: Abnormal pat tern of bacterial killing by",
        "neutrophils deficient in granules, alkaline phosphatase, and myeloperoxidase. J Lab",
        "Clin Med 88:788,1976.",
        "Repine JE, Clawson cc, and Friend PS: Influence of a deficiency of the second component",
        "of complement on the bactericidal activity of neutrophils in vitro. J Clin Invest 59:",
        "802,1977.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
      ]
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        "Repine JE and Clawson CC: Quantitative measurement of the bactericidal capability of",
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        "Repine JE, Clawson cc, Rasp FL, Sarosi, GA, and Hoidal JR: Defective neut rophil chemo-",
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        "neutrophil unstimulated and stimulated locomotion in vitro. JRES",
        "Rasp FL, Clawson cc, Hoidal JR, and Repine JE: Adherence of human alveolar macrophages",
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        "electron microscope comparison. JRES",
        "Rasp FL, Clawson CC, and Repine JE: Comparison of the adherence and morphology of",
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        "Academic Press, Inc, New York, NY.",
        "Abstracts not subsequentally published in full (Selected list)",
        "Rasp F1, Repine JE, Hoidal JR, and Sarosi GA: Decreased adherence in vitro of poly-",
        "morphonuclear leukocytes (PMN) from patients with invasive fungal infections (IFI)",
        "ARRD 115:199,1977.",
        "Beall GD, Repine JE, Hoidal JR, and Rasp FL: Generation of hydroxyl radical( OH) by",
        "humen alveolar macrophages(AM). ARRD 117:269,1978",
        "Hof DG, Repine JE, Petersen PK, Hoidal JR, and Quie PG: Opsonic requirements and kinetics",
        "of phagocytosis by alveolar macrophages (AM) ARRD117:275,1978.",
        "Fox RB, Eaton JW, Repine JE. and Brown DB: Ontogeny of cytoplasmic superoxide dis-",
        "mutase (SOD) activity in fetal and neonatal lung tissues. Amer Rev Resp Dis, April 1979.",
        "Fox RB, Hoidal JR, Takiff HE, and Repine JE: Pretreatment with Freund's adjuvant",
        "increases the numbers of alveolar macrophages and protects against pulmonary oxygen",
        "toxicity. Amer Rev Resp Dis, April , 1979.",
        "Repine JE and Friend PS: Function of polymorphonuclear leukocytes(PMN) from a patient",
        "with an inherited absence of the fourth component of complement (c4). ARRD April, 1979.",
        "Rasp FL and Repine JE: Bacteria, fungi, and chemicals increase adherence of poly-",
        "morphonuclear leukocytes(PMN causing chemiluminescence(CL) and release of lysozyme(LY)",
        "Amer Rev Resp Dis April, 1979.",
        "Hoidal JR and Repine JE: A congenital defect in human alveolar macrophage(AM function:",
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        "Resp Dis April, 1979.",
        "Hoidal JR, Fox RB, Takiff HE, and Repine JE: Alveolar macrophages(AM) from young",
        "asymptomatic cigaretta smokers use equal amounts of oxygen(02) and glucose (1-14c)",
        "but smoker AM make more superoxide anion(0,- Amer. Rev Resp Dis April, 1979.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
      ]
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      "text": [
        "8.",
        "Curriculum Vitae",
        "Hoidal, John, R. M.D.",
        "Assistant Professor of Medicine",
        "U. s. Citizen, born akland,California, January 22, 1944(age34).",
        "Education and Professional Experience",
        "Degree",
        "Year",
        "Jamestown College, Jamestown, North Dakota",
        "B.S.",
        "1965",
        "University of Minnesota, Minneapolis, Minnesota",
        "M.D.",
        "1969",
        "Internship, Univeristy of Colorado Medical Center",
        "1969-70",
        "Resident, Internal Medicine, University of Minnesota",
        "1970-72",
        "Chief Resident, Internal Medicine, University of",
        "Minnesota",
        "1972",
        "Pulmonary Fellow, University of Minnesota",
        "1973-75",
        "Instructor, University of Minnesota",
        "1974-75",
        "Asst. Prof., University of Minnesota",
        "1975",
        "Honors",
        "Summa cum laude at Jamestown College",
        "Alpha Omega Alpha at University of Minnesota",
        "Board Certified Internal Medicine and Pulmonary",
        "Selected Bibliography- Published Manuscripts and manuscripts accepted for",
        "Publication",
        "Hoidal JR, Repine JE, Beall GD, Rasp FL, and White JG: The effect of phorbol myristate",
        "acetate on the metabolism and ultrastructure of human :liveolar macrophages. Amer.",
        "J.",
        "Path 91:469,1978",
        "Hoidal JR, Beall GD, Rasp FL, Holmes B, White JG, and Repine JE: Comparison of the",
        "metabolism of alveolar macrophages from humans, rats, and rabbits: Response to heat-",
        "killed bacteria or phorbol myristate acetate. J Lab Clin Med 92:787,1978.",
        "Ackerman SK, Friend PS, Hoidal JR, and Douglas SD: Production of C2 by human alveolar",
        "macrophages. Immunol 35:369,1978.",
        "Repine JE, Rasp FL, Clawson cc. Sarosi GA, and Hoidal JR: Defective neutrophil chemo-",
        "taxis is patients with bastomycosis: Evidence for a serum inhibitor. Amer Rev",
        "Resp Dis 118:325,1978.",
        "Beall GD, Repine JE, Hoidal JR, and Rasp FL: Chemiluminescence of human alveolar",
        "macrophages: Stimulation with heat-killed bacteria or phorbol myristate acetate",
        "Infect and Immunity 17:117,1977.",
        "Hoidal JR, White JG and Repine JE: Impairment of human alveolar macrophage oxygen",
        "consumption and superoxide anion production by local anesthetics used during",
        "bronchoscopy. Chest",
        "Repine JE, Davies SD, and Hoidal JR: Effect of C2 on the bactericidal activity and",
        "chemiluminescence responses of human neutrophils in vitro. Chest",
        "Repine JE, Hoidal JR, and White JG: Effect of temperature on the chemiluminescence of",
        "human neutrophils in vitro, In The Biology of Oxygen Radicals, W.S.Caughy,ed",
        "Academic Press, Inc, New York, NY.",
        "Rasp FL, Clawson cc, Hoidal, JR and Repine JE: Adherence of human alveolar macrophages",
        "and polymorphonuclear leukocytes from smokers and non-smokers in vitro. JRES",
        "Hoidal JR, White JG, and Repine JE: Influence of cationic local anesthetics on the",
        "metabolism and ultratructure of human alveolar macrophages. J Lab Clin Med",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
      ]
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      "score": [
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      "text": [
        "9.",
        "Abstracts not subsequently published in full (Selected List)",
        "Hoidal JR, Repine JE, Beall GD, and Rasp FL: Oxygen, free radical, and glucose meta-",
        "bolism of human alveolar macrophages stimulated by particulate or membrane active",
        "agents. Amer Rev Resp Dis 117:275,1978.",
        "Beall GD, Repine JE, Hoidal JR, Rasp FL, and White JG: Generation of hydroxyl radical",
        "by human alveolar macrophages. Amer Rev Resp Dis 117:269,1977.",
        "LeMarbe P, Hoidal JR, Besella R, and Rinehart J: Human pulmonary macrophage tumor",
        "cell cytotoxicity. Clin Res 26:669,1978.",
        "Rasp FL, Repine JE, Hoidal JR, and Sarosi GA: Decreased adherence in vitro of poly-",
        "morphonuclear leukocytes from patients with invasive fungal infections. Amer Rev",
        "Resp Dis 115:258,1977.",
        "Smith CM, Tukey DP, Mundshenk D, Repine JE, and Hoidal JR: Alveolar macre phages (AM)",
        "deformability: Measurement by positive pressure nucleopore filtration: Alteration by",
        "pharmacologic agents and application to AM from human cigarette smokers. Amer",
        "Rev Resp Dis April 1979.",
        "Davies SD, Hoidal JR, and Repine JE: Chemiluminescence(CL by human polymorphonuclear",
        "leukocytes (PMN) and monocytes(MN interacting with Cryptococcus neoformans: Primary",
        "dependence on direct (antibody independent) ictivation of the alternate pathway of",
        "complement (C). Amer Rev Resp Dis April, 1979.",
        "Takiff HE, Hoidal JR, Repine JE, and Fox RB: Search for different subpopulations of",
        "alveolar macrophages(AM) Study of AM kir using tritiated thymidune. Amer",
        "Rev Resp Dis April 1979.",
        "Hoidal JR and Repine JE: A congenital defect in human alveolar macrophage(AM) function",
        "Absence of increased (0,) uptake, superoxide anion(0,-) production, and glucose",
        "oxidetion by AM from a patient with chronic granulomatous disease(CGD) Amer Rev",
        "Resp Dis April, 1979.",
        "Hoidal JR, Fox RB, Takiff HE, and Repine JE: Alveolar macrophages(AM) from young",
        "asymptomatic cigarette smokers use equal amounts of oxygen(0,1 and glucose (1-14c),",
        "but smoker AM make more superoxide anion(0,-). Amer Rev Resp Dis, April, 1979.",
        "Source: https://www.industrydocuments.ucsf.edu/docs/gmnv0215"
      ]
    }
  ]
}