Gerald Weissmann

ARTHRITIS TRAINING GRANT

(2001)

Project Number :	2T32AR007176-22
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

ARTHRITIS TRAINING GRANT

(2001)

Project Number :	5T32AR007176-23
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

ARTHRITIS TRAINING GRANT

(2001)

Abstract :

The purpose of this program is to prepare individuals for research and training in the broad area of rheumatology. The major effort will be directed to the training of M.D.'s both in clinical rheumatology and a laboratory discipline so that they can assume responsible positions in rheumatology divisions in a medical school. Each fellow will be expected to become proficient in all areas of clinical rheumatology and, in addition, to acquire an in-depth training in immunology cell biology, electron microscopy, or biochemistry. Because of the nature of our staff, we also attract a number of Ph.D.'s for training in our laboratories. It is our hope that many of them, too, will continue their interest in the rheumatic diseases and will apply their skills in the future to studies in this area. In addition, we are responsible for the training of students, house staff, and postgraduate physicians in rheumatic diseases and basic sciences related to them.

Project Number :	5T32AR007176-24
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

ARTHRITIS TRAINING GRANT

(2001)

Abstract :

The purpose of this program is to prepare individuals for research and training in the broad area of rheumatology. The major effort will be directed to the training of M.D.'s both in clinical rheumatology and a laboratory discipline so that they can assume responsible positions in rheumatology divisions in a medical school. Each fellow will be expected to become proficient in all areas of clinical rheumatology and, in addition, to acquire an in-depth training in immunology cell biology, electron microscopy, or biochemistry. Because of the nature of our staff, we also attract a number of Ph.D.'s for training in our laboratories. It is our hope that many of them, too, will continue their interest in the rheumatic diseases and will apply their skills in the future to studies in this area. In addition, we are responsible for the training of students, house staff, and postgraduate physicians in rheumatic diseases and basic sciences related to them.

Project Number :	5T32AR007176-25
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

ARTHRITIS TRAINING GRANT

(2001)

Abstract :

The purpose of this program is to prepare individuals for research and training in the broad area of rheumatology. The major effort will be directed to the training of M.D.'s both in clinical rheumatology and a laboratory discipline so that they can assume responsible positions in rheumatology divisions in a medical school. Each fellow will be expected to become proficient in all areas of clinical rheumatology and, in addition, to acquire an in-depth training in immunology cell biology, electron microscopy, or biochemistry. Because of the nature of our staff, we also attract a number of Ph.D.'s for training in our laboratories. It is our hope that many of them, too, will continue their interest in the rheumatic diseases and will apply their skills in the future to studies in this area. In addition, we are responsible for the training of students, house staff, and postgraduate physicians in rheumatic diseases and basic sciences related to them.

Project Number :	5T32AR007176-26
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

LUNG INFLAMMATION

(2000)

Abstract :

We will determine how regulatory molecules released from resting and stimulated endothelial cells (+/-TNFa, Il- 1, endotoxin) modify neutrophil functions in Arthus and Shwartzman-like models of tissue injury. Using intact human neutrophils, granule-free cytoplasts, cell free systems, liposomes of varying physical structure, and human umbilical vein endothelial cells, we will test four novel hypotheses. Specific Aim I. To test the hypothesis that the Yin/Yang control of neutrophil function by cAMP/cGMP is exerted at the level of raf phosphorylation in the MAP-kinase cascade. Neutrophils, their cytoplasts and cell-free extracts exposed to the three classes of activators will be studied kinetically (5 sec to 5 min) for delta-psi, [Ca]i phospholipid turnover, lysosomal enzyme release, translocation of 47phox and 67phox components of the NADPH oxidase and ras-related proteins, carboxy methylation, cAMP, cGMP and phosphorylation/ dephosphorylation patterns studied of raf, CD11b/CD 18, MAP kinases +/- inhibitors. (calyculin, genestein, okadaiate, etc.). Specific Aim II. To test the hypothesis that endothelial cells release autocoids (adenosine, PGI2 and NO) that inhibit neutrophil-mediated endothelial activation (judged by translocation of NFkB, induction of COX II, NO synthases, expression of E-selectin, VCAM-1, ICAM-1). Neutrophils or cytoplasts will be incubated with endothelial cells that do or do not form the autocoid (+/- adenosine deaminase, +/- COX II, NO synthase inhibitors). Endothelial cell activation will be determined by electrophoretic mobility shift, Northern Blot and FACS. Specific Aim III. To test the hypothesis that lipid metabolites formed in the course of neutrophil/endothelial interactions, e.g. arachidonate (20:4) and phosphatidate (PA), regulate the association of ras-related proteins with their inhibitor/chaperones (GDI's) and with the plasmalemma. In intact cells, cytoplasts and cell-free, oxidase-competent systems we will study membrane/cytosol distribution, GDI association and phospholipid-dependent isoprenyl-directed carboxy methyltransferase (piCMT) activity with respect to O2- and/or aggregation, phosphorylation patterns in response to added 20:4 (+/- other eicosanoids, HODEs) or PA (+/- other phospholipids). Specific Aim IV. To test the hypothesis that the physical state of phospholipids (lamellar vs Hex(II)) as regulated by phospholipases A, C and D regulates a) the association of prenylated proteins with the plasmalemma and b) the effect of autocoids (PGE1, 14,15 di-HETEs, 13-HODE, 22:6, lipoxins) on the activity of piCMT and the oxidase system in natural and artificial bilayers. Lamellar (phosphatidyl choline, palmitoyl-oleyl ethanolamine) liposomes will be compared with Hex(II) forming liposomes (PA +/- Ca, dioleyl ethanolamine) for presentation to PGI2 receptors and reconstitution of the O2- system.

Project Number :	5R01HL019721-23
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	LBPA
Project Terms :	Arthus phenomenon, Shwartzman phenomenon, inflammation, lung injury, neutrophil, nitric oxide, vascular endothelium arachidonate, cyclic AMP, cyclic GMP, endotoxin, interleukin 1, leukocyte activation /transformation, mitogen activated protein kinase, phosphatidate, phospholipid, phosphorylation, prostaglandin, prostaglandin receptor, tumor necrosis factor alpha cell free system, flow cytometry, gel mobility shift assay, human subject, liposome, northern blotting

LUNG INFLAMMATION

(1999)

Abstract :

We will determine how regulatory molecules released from resting and stimulated endothelial cells (+/-TNFa, Il- 1, endotoxin) modify neutrophil functions in Arthus and Shwartzman-like models of tissue injury. Using intact human neutrophils, granule-free cytoplasts, cell free systems, liposomes of varying physical structure, and human umbilical vein endothelial cells, we will test four novel hypotheses. Specific Aim I. To test the hypothesis that the Yin/Yang control of neutrophil function by cAMP/cGMP is exerted at the level of raf phosphorylation in the MAP-kinase cascade. Neutrophils, their cytoplasts and cell-free extracts exposed to the three classes of activators will be studied kinetically (5 sec to 5 min) for delta-psi, [Ca]i phospholipid turnover, lysosomal enzyme release, translocation of 47phox and 67phox components of the NADPH oxidase and ras-related proteins, carboxy methylation, cAMP, cGMP and phosphorylation/ dephosphorylation patterns studied of raf, CD11b/CD 18, MAP kinases +/- inhibitors. (calyculin, genestein, okadaiate, etc.). Specific Aim II. To test the hypothesis that endothelial cells release autocoids (adenosine, PGI2 and NO) that inhibit neutrophil-mediated endothelial activation (judged by translocation of NFkB, induction of COX II, NO synthases, expression of E-selectin, VCAM-1, ICAM-1). Neutrophils or cytoplasts will be incubated with endothelial cells that do or do not form the autocoid (+/- adenosine deaminase, +/- COX II, NO synthase inhibitors). Endothelial cell activation will be determined by electrophoretic mobility shift, Northern Blot and FACS. Specific Aim III. To test the hypothesis that lipid metabolites formed in the course of neutrophil/endothelial interactions, e.g. arachidonate (20:4) and phosphatidate (PA), regulate the association of ras-related proteins with their inhibitor/chaperones (GDI's) and with the plasmalemma. In intact cells, cytoplasts and cell-free, oxidase-competent systems we will study membrane/cytosol distribution, GDI association and phospholipid-dependent isoprenyl-directed carboxy methyltransferase (piCMT) activity with respect to O2- and/or aggregation, phosphorylation patterns in response to added 20:4 (+/- other eicosanoids, HODEs) or PA (+/- other phospholipids). Specific Aim IV. To test the hypothesis that the physical state of phospholipids (lamellar vs Hex(II)) as regulated by phospholipases A, C and D regulates a) the association of prenylated proteins with the plasmalemma and b) the effect of autocoids (PGE1, 14,15 di-HETEs, 13-HODE, 22:6, lipoxins) on the activity of piCMT and the oxidase system in natural and artificial bilayers. Lamellar (phosphatidyl choline, palmitoyl-oleyl ethanolamine) liposomes will be compared with Hex(II) forming liposomes (PA +/- Ca, dioleyl ethanolamine) for presentation to PGI2 receptors and reconstitution of the O2- system.

Project Number :	5R01HL019721-22
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	LBPA
Project Terms :	Arthus phenomenon, Shwartzman phenomenon, inflammation, lung injury, neutrophil, nitric oxide, vascular endothelium arachidonate, cyclic AMP, cyclic GMP, endotoxin, interleukin 1, leukocyte activation /transformation, mitogen activated protein kinase, phosphatidate, phospholipid, phosphorylation, prostaglandin, prostaglandin receptor, tumor necrosis factor alpha cell free system, flow cytometry, gel mobility shift assay, human subject, liposome, northern blotting

LUNG INFLAMMATION

(1999)

Abstract :

We will determine how regulatory molecules released from resting and stimulated endothelial cells (+/-TNFa, Il- 1, endotoxin) modify neutrophil functions in Arthus and Shwartzman-like models of tissue injury. Using intact human neutrophils, granule-free cytoplasts, cell free systems, liposomes of varying physical structure, and human umbilical vein endothelial cells, we will test four novel hypotheses. Specific Aim I. To test the hypothesis that the Yin/Yang control of neutrophil function by cAMP/cGMP is exerted at the level of raf phosphorylation in the MAP-kinase cascade. Neutrophils, their cytoplasts and cell-free extracts exposed to the three classes of activators will be studied kinetically (5 sec to 5 min) for delta-psi, [Ca]i phospholipid turnover, lysosomal enzyme release, translocation of 47phox and 67phox components of the NADPH oxidase and ras-related proteins, carboxy methylation, cAMP, cGMP and phosphorylation/ dephosphorylation patterns studied of raf, CD11b/CD 18, MAP kinases +/- inhibitors. (calyculin, genestein, okadaiate, etc.). Specific Aim II. To test the hypothesis that endothelial cells release autocoids (adenosine, PGI2 and NO) that inhibit neutrophil-mediated endothelial activation (judged by translocation of NFkB, induction of COX II, NO synthases, expression of E-selectin, VCAM-1, ICAM-1). Neutrophils or cytoplasts will be incubated with endothelial cells that do or do not form the autocoid (+/- adenosine deaminase, +/- COX II, NO synthase inhibitors). Endothelial cell activation will be determined by electrophoretic mobility shift, Northern Blot and FACS. Specific Aim III. To test the hypothesis that lipid metabolites formed in the course of neutrophil/endothelial interactions, e.g. arachidonate (20:4) and phosphatidate (PA), regulate the association of ras-related proteins with their inhibitor/chaperones (GDI's) and with the plasmalemma. In intact cells, cytoplasts and cell-free, oxidase-competent systems we will study membrane/cytosol distribution, GDI association and phospholipid-dependent isoprenyl-directed carboxy methyltransferase (piCMT) activity with respect to O2- and/or aggregation, phosphorylation patterns in response to added 20:4 (+/- other eicosanoids, HODEs) or PA (+/- other phospholipids). Specific Aim IV. To test the hypothesis that the physical state of phospholipids (lamellar vs Hex(II)) as regulated by phospholipases A, C and D regulates a) the association of prenylated proteins with the plasmalemma and b) the effect of autocoids (PGE1, 14,15 di-HETEs, 13-HODE, 22:6, lipoxins) on the activity of piCMT and the oxidase system in natural and artificial bilayers. Lamellar (phosphatidyl choline, palmitoyl-oleyl ethanolamine) liposomes will be compared with Hex(II) forming liposomes (PA +/- Ca, dioleyl ethanolamine) for presentation to PGI2 receptors and reconstitution of the O2- system.

Project Number :	2R01HL019721-19
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	LBPA
Project Terms :	Arthus phenomenon, Shwartzman phenomenon, inflammation, lung injury, neutrophil, nitric oxide, vascular endothelium arachidonate, cyclic AMP, cyclic GMP, endotoxin, interleukin 1, leukocyte activation /transformation, phosphatidate, phospholipid, phosphorylation, prostaglandin, prostaglandin receptor, tumor necrosis factor alpha cell free system, flow cytometry, gel mobility shift assay, human subject, liposome, northern blotting

LUNG INFLAMMATION

(1999)

Project Number :	5R01HL019721-20
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	LBPA

LUNG INFLAMMATION

(1999)

Abstract :

We will determine how regulatory molecules released from resting and stimulated endothelial cells (+/-TNFa, Il- 1, endotoxin) modify neutrophil functions in Arthus and Shwartzman-like models of tissue injury. Using intact human neutrophils, granule-free cytoplasts, cell free systems, liposomes of varying physical structure, and human umbilical vein endothelial cells, we will test four novel hypotheses. Specific Aim I. To test the hypothesis that the Yin/Yang control of neutrophil function by cAMP/cGMP is exerted at the level of raf phosphorylation in the MAP-kinase cascade. Neutrophils, their cytoplasts and cell-free extracts exposed to the three classes of activators will be studied kinetically (5 sec to 5 min) for delta-psi, [Ca]i phospholipid turnover, lysosomal enzyme release, translocation of 47phox and 67phox components of the NADPH oxidase and ras-related proteins, carboxy methylation, cAMP, cGMP and phosphorylation/ dephosphorylation patterns studied of raf, CD11b/CD 18, MAP kinases +/- inhibitors. (calyculin, genestein, okadaiate, etc.). Specific Aim II. To test the hypothesis that endothelial cells release autocoids (adenosine, PGI2 and NO) that inhibit neutrophil-mediated endothelial activation (judged by translocation of NFkB, induction of COX II, NO synthases, expression of E-selectin, VCAM-1, ICAM-1). Neutrophils or cytoplasts will be incubated with endothelial cells that do or do not form the autocoid (+/- adenosine deaminase, +/- COX II, NO synthase inhibitors). Endothelial cell activation will be determined by electrophoretic mobility shift, Northern Blot and FACS. Specific Aim III. To test the hypothesis that lipid metabolites formed in the course of neutrophil/endothelial interactions, e.g. arachidonate (20:4) and phosphatidate (PA), regulate the association of ras-related proteins with their inhibitor/chaperones (GDI's) and with the plasmalemma. In intact cells, cytoplasts and cell-free, oxidase-competent systems we will study membrane/cytosol distribution, GDI association and phospholipid-dependent isoprenyl-directed carboxy methyltransferase (piCMT) activity with respect to O2- and/or aggregation, phosphorylation patterns in response to added 20:4 (+/- other eicosanoids, HODEs) or PA (+/- other phospholipids). Specific Aim IV. To test the hypothesis that the physical state of phospholipids (lamellar vs Hex(II)) as regulated by phospholipases A, C and D regulates a) the association of prenylated proteins with the plasmalemma and b) the effect of autocoids (PGE1, 14,15 di-HETEs, 13-HODE, 22:6, lipoxins) on the activity of piCMT and the oxidase system in natural and artificial bilayers. Lamellar (phosphatidyl choline, palmitoyl-oleyl ethanolamine) liposomes will be compared with Hex(II) forming liposomes (PA +/- Ca, dioleyl ethanolamine) for presentation to PGI2 receptors and reconstitution of the O2- system.

Project Number :	5R01HL019721-21
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	LBPA
Project Terms :	Arthus phenomenon, Shwartzman phenomenon, inflammation, lung injury, neutrophil, nitric oxide, vascular endothelium arachidonate, cyclic AMP, cyclic GMP, endotoxin, interleukin 1, leukocyte activation /transformation, mitogen activated protein kinase, phosphatidate, phospholipid, phosphorylation, prostaglandin, prostaglandin receptor, tumor necrosis factor alpha cell free system, flow cytometry, gel mobility shift assay, human subject, liposome, northern blotting

MECHANISMS OF ARTHRITIS

(1998)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	5R37AR011949-30
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	NSS
Project Terms :	joint disorder, osteoarthritis, rheumatoid arthritis acylation, alternatives to animals in research, antibody receptor, antiinflammatory agent, aspirin, autoimmune disorder, bacterial antigen, blood lipoprotein, cartilage, cytochalasin, diacylglycerol, enzyme mechanism, immune complex disease, immunosuppressive antileukocyte serum, ionomycin, leukocyte, leukotriene, lipoxygenase, macrophage, membrane protein, microtubule, neutrophil, peptidase, phosphatidate phosphatase, phospholipase D, phospholipid, protein kinase C Porifera, centrifugation, fluorescence microscopy, human tissue, ion exchange chromatography, laboratory rabbit, monoclonal antibody, nuclear magnetic resonance spectroscopy, radioimmunoassay, scanning electron microscopy

MECHANISMS OF ARTHRITIS

(1997)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	4R37AR011949-26
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	NSS
Project Terms :	joint disorder, osteoarthritis, rheumatoid arthritis acylation, alternatives to animals in research, antibody receptor, antiinflammatory agent, aspirin, autoimmune disorder, bacterial antigen, blood lipoprotein, cartilage, cytochalasin, diacylglycerol, enzyme mechanism, immune complex disease, immunosuppressive antileukocyte serum, ionomycin, leukocyte, leukotriene, lipoxygenase, macrophage, membrane protein, microtubule, neutrophil, peptide hydrolase, phosphatidate phosphatase, phospholipase D, phospholipid, protein kinase C Porifera, centrifugation, fluorescence microscopy, human tissue, ion exchange chromatography, laboratory rabbit, monoclonal antibody, nuclear magnetic resonance spectroscopy, radioimmunoassay, scanning electron microscopy

MECHANISMS OF ARTHRITIS

(1997)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	5R37AR011949-27
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	NSS
Project Terms :	joint disorder, osteoarthritis, rheumatoid arthritis acylation, alternatives to animals in research, antibody receptor, antiinflammatory agent, aspirin, autoimmune disorder, bacterial antigen, blood lipoprotein, cartilage, cytochalasin, diacylglycerol, enzyme mechanism, immune complex disease, immunosuppressive antileukocyte serum, ionomycin, leukocyte, leukotriene, lipoxygenase, macrophage, membrane protein, microtubule, neutrophil, peptide hydrolase, phosphatidate phosphatase, phospholipase D, phospholipid, protein kinase C Porifera, centrifugation, fluorescence microscopy, human tissue, ion exchange chromatography, laboratory rabbit, monoclonal antibody, nuclear magnetic resonance spectroscopy, radioimmunoassay, scanning electron microscopy

MECHANISMS OF ARTHRITIS

(1997)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	5R37AR011949-28
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	NSS
Project Terms :	joint disorder, osteoarthritis, rheumatoid arthritis acylation, alternatives to animals in research, antibody receptor, antiinflammatory agent, aspirin, autoimmune disorder, bacterial antigen, blood lipoprotein, cartilage, cytochalasin, diacylglycerol, enzyme mechanism, immune complex disease, immunosuppressive antileukocyte serum, ionomycin, leukocyte, leukotriene, lipoxygenase, macrophage, membrane protein, microtubule, neutrophil, peptide hydrolase, phosphatidate phosphatase, phospholipase D, phospholipid, protein kinase C Porifera, centrifugation, fluorescence microscopy, human tissue, ion exchange chromatography, laboratory rabbit, monoclonal antibody, nuclear magnetic resonance spectroscopy, radioimmunoassay, scanning electron microscopy

MECHANISMS OF ARTHRITIS

(1997)

Project Number :	5R37AR011949-29
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	NSS

ARTHRITIS

(1996)

Project Number :	5T32AR007176-19
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

ARTHRITIS

(1996)

Project Number :	5T32AR007176-20
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

ARTHRITIS

(1996)

Project Number :	2T32AR007176-17
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

ARTHRITIS

(1996)

Project Number :	5T32AR007176-18
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

ARTHRITIS

(1996)

Project Number :	5T32AR007176-21
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	AMS

LUNG INFLAMMATION

(1994)

Abstract :

Neutrophils mediate tissue injury in two classical models of experimental pathology: the Arthus lesion(1903) and the Shwartzman phenomenon (1927). The aim of this proposal is to explain in the language of 1989 how vascular injury induced by immune complexes and complement (the Arthus lesion) differs from the induced by endotoxin, cytokines and complement (the Shwartzman phenomenon). We will study neutrophil/endothelial cell interactions in vitro to test novel hypotheses of "triggering" and activation" of neutrophils. We will determine the functional effects on human neutrophils and cytoplasts of "defensive" molecules release from activated vs. resting endothelial cells. A variety of endothelial cells (eg HUVEC) will be exposed to endotoxin or cytokines and the release measured of a) prostacyclin b) adenosine c) endothelium-derived relaxation factor (EDRF, or NO) and d) sphingosine (an endogenous inhibitor of O2 generation). The effects of these molecules will be studied on neutrophils or cytoplasts activated by chemoattractants or immune complexes with respect to a) heterotypic adherence (to HUVEC), b) cell-cell aggregation, c) disaggregation d) chemotaxis in several systems, and e) release of O-2 H2 O2 and lysosomal enzymes. We will also expose neutrophils activated by chemoattractants and immune complexes to PGI2 adenosine, NO and sphingosine. Stimulus/response coupling will be examined with attention to receptors for iC3b and immune complexes (FcRII,FcRIII), cytosolic pH and free calcium (pH, and Cai) 45Ca fluxes, membrane viscosity, membrane potential (delta psi), crosslinking of cytoskeletal proteins, microtubule assembly, phospholipid remodelling, and protein phosphorylation. We will compare the susceptibility of resting HUVEC with cells activated by endotoxin or cytokines (IL-1, TNF and IFN) to killing by neutrophils exposed to chemoattractants (C5a, LTB, FMLP). We will study whether immune complexes present a) in the bulk phase b) on the HUVEC monolayer, or c) in the subphase, provoke neutrophil-mediated cell injury despite release of "defensive" molecules from HUVEC. Finally, using marine sponge cells and human neutrophils, we will study whether 20.4 and other cis unsaturated fatty acids activate a novel GTP-binding protein the function of which is mimicked by phospatidic acid. These studies will employ novel liposomal methods for phospholipid presentation. Thus we will be able to define the roles played by cytokine activated endothelial cells (the Shwartzman model) and immune complexes (the Arthus model) in neutrophil-mediated blood vessel injury.

Project Number :	5R01HL019721-18
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	Arthus phenomenon, Shwartzman phenomenon, inflammation, lung disorder, neutrophil, vascular endothelium adenosine, alternatives to animals in research, cell cell interaction, complement, complement receptor, cytokine, endotoxin, guanine nucleotide binding protein, immune complex, immunopharmacology, leukocyte activation /transformation, nitric oxide, respiratory pharmacology, vasodilator Porifera, cell free system, histochemistry /cytochemistry, human tissue, radiotracer, salt water environment, tissue /cell culture

LUNG INFLAMMATION

(1994)

Abstract :

Neutrophils mediate tissue injury in two classical models of experimental pathology: the Arthus lesion(1903) and the Shwartzman phenomenon (1927). The aim of this proposal is to explain in the language of 1989 how vascular injury induced by immune complexes and complement (the Arthus lesion) differs from the induced by endotoxin, cytokines and complement (the Shwartzman phenomenon). We will study neutrophil/endothelial cell interactions in vitro to test novel hypotheses of "triggering" and activation" of neutrophils. We will determine the functional effects on human neutrophils and cytoplasts of "defensive" molecules release from activated vs. resting endothelial cells. A variety of endothelial cells (eg HUVEC) will be exposed to endotoxin or cytokines and the release measured of a) prostacyclin b) adenosine c) endothelium-derived relaxation factor (EDRF, or NO) and d) sphingosine (an endogenous inhibitor of O2 generation). The effects of these molecules will be studied on neutrophils or cytoplasts activated by chemoattractants or immune complexes with respect to a) heterotypic adherence (to HUVEC), b) cell-cell aggregation, c) disaggregation d) chemotaxis in several systems, and e) release of O-2 H2 O2 and lysosomal enzymes. We will also expose neutrophils activated by chemoattractants and immune complexes to PGI2 adenosine, NO and sphingosine. Stimulus/response coupling will be examined with attention to receptors for iC3b and immune complexes (FcRII,FcRIII), cytosolic pH and free calcium (pH, and Cai) 45Ca fluxes, membrane viscosity, membrane potential (delta psi), crosslinking of cytoskeletal proteins, microtubule assembly, phospholipid remodelling, and protein phosphorylation. We will compare the susceptibility of resting HUVEC with cells activated by endotoxin or cytokines (IL-1, TNF and IFN) to killing by neutrophils exposed to chemoattractants (C5a, LTB, FMLP). We will study whether immune complexes present a) in the bulk phase b) on the HUVEC monolayer, or c) in the subphase, provoke neutrophil-mediated cell injury despite release of "defensive" molecules from HUVEC. Finally, using marine sponge cells and human neutrophils, we will study whether 20.4 and other cis unsaturated fatty acids activate a novel GTP-binding protein the function of which is mimicked by phospatidic acid. These studies will employ novel liposomal methods for phospholipid presentation. Thus we will be able to define the roles played by cytokine activated endothelial cells (the Shwartzman model) and immune complexes (the Arthus model) in neutrophil-mediated blood vessel injury.

Project Number :	2R01HL019721-14
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, HYPERSENSITIVITY, ANAPHYLAXIS, ARTHUS PHENOMENON, HYPERSENSITIVITY, ANAPHYLAXIS, SHWARTZMAN PHENOMENON, RESPIRATORY DISORDERS, LUNG DISORDERS CELL-CELL INTERACTION, IMMUNITY, CELLULAR, LEUKOCYTE ACTIVATION, TRANSFORMATION AND PROLIFERATION, IMMUNITY, CYTOKINES, IMMUNOLOGY, ANTIGEN-ANTIBODY REACTIONS, IMMUNE COMPLEXES, IMMUNOLOGY, ANTIGENS BACTERIAL, ENDOTOXINS, IMMUNOLOGY, COMPLEMENT, PROTEINS, BINDING PROTEINS, GUANINE NUCLEOTIDE BINDING PROTEINS, PURINE NUCLEOSIDES, ADENINE NUCLEOSIDES, ADENOSINE, RECEPTORS, COMPLEMENT RECEPTORS, RESPIRATORY SYSTEM PHARMACOLOGY, immunopharmacology ANIMALS, INVERTEBRATES, SPONGES, CELL-FREE SYSTEMS, HISTOCHEMISTRY AND CYTOCHEMISTRY, HUMAN, HUMAN TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PR OJECT, RADIOTRACERS, TISSUE (CELL) CULTURE, WATER ENVIRONMENT, AQUATIC ORGANISMS, MARINE

LUNG INFLAMMATION

(1994)

Abstract :

Neutrophils mediate tissue injury in two classical models of experimental pathology: the Arthus lesion(1903) and the Shwartzman phenomenon (1927). The aim of this proposal is to explain in the language of 1989 how vascular injury induced by immune complexes and complement (the Arthus lesion) differs from the induced by endotoxin, cytokines and complement (the Shwartzman phenomenon). We will study neutrophil/endothelial cell interactions in vitro to test novel hypotheses of "triggering" and activation" of neutrophils. We will determine the functional effects on human neutrophils and cytoplasts of "defensive" molecules release from activated vs. resting endothelial cells. A variety of endothelial cells (eg HUVEC) will be exposed to endotoxin or cytokines and the release measured of a) prostacyclin b) adenosine c) endothelium-derived relaxation factor (EDRF, or NO) and d) sphingosine (an endogenous inhibitor of O2 generation). The effects of these molecules will be studied on neutrophils or cytoplasts activated by chemoattractants or immune complexes with respect to a) heterotypic adherence (to HUVEC), b) cell-cell aggregation, c) disaggregation d) chemotaxis in several systems, and e) release of O-2 H2 O2 and lysosomal enzymes. We will also expose neutrophils activated by chemoattractants and immune complexes to PGI2 adenosine, NO and sphingosine. Stimulus/response coupling will be examined with attention to receptors for iC3b and immune complexes (FcRII,FcRIII), cytosolic pH and free calcium (pH, and Cai) 45Ca fluxes, membrane viscosity, membrane potential (delta psi), crosslinking of cytoskeletal proteins, microtubule assembly, phospholipid remodelling, and protein phosphorylation. We will compare the susceptibility of resting HUVEC with cells activated by endotoxin or cytokines (IL-1, TNF and IFN) to killing by neutrophils exposed to chemoattractants (C5a, LTB, FMLP). We will study whether immune complexes present a) in the bulk phase b) on the HUVEC monolayer, or c) in the subphase, provoke neutrophil-mediated cell injury despite release of "defensive" molecules from HUVEC. Finally, using marine sponge cells and human neutrophils, we will study whether 20.4 and other cis unsaturated fatty acids activate a novel GTP-binding protein the function of which is mimicked by phospatidic acid. These studies will employ novel liposomal methods for phospholipid presentation. Thus we will be able to define the roles played by cytokine activated endothelial cells (the Shwartzman model) and immune complexes (the Arthus model) in neutrophil-mediated blood vessel injury.

Project Number :	5R01HL019721-17
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	Arthus phenomenon, Shwartzman phenomenon, inflammation, lung disorder, neutrophil, vascular endothelium adenosine, cell cell interaction, complement, complement receptor, cytokine, endotoxin, guanine nucleotide binding protein, immune complex, immunopharmacology, leukocyte activation /transformation, nitric oxide, respiratory pharmacology, vasodilator Porifera, cell free system, histochemistry /cytochemistry, human tissue, radiotracer, salt water environment, tissue /cell culture

LUNG INFLAMMATION

(1994)

Abstract :

Neutrophils mediate tissue injury in two classical models of experimental pathology: the Arthus lesion(1903) and the Shwartzman phenomenon (1927). The aim of this proposal is to explain in the language of 1989 how vascular injury induced by immune complexes and complement (the Arthus lesion) differs from the induced by endotoxin, cytokines and complement (the Shwartzman phenomenon). We will study neutrophil/endothelial cell interactions in vitro to test novel hypotheses of "triggering" and activation" of neutrophils. We will determine the functional effects on human neutrophils and cytoplasts of "defensive" molecules release from activated vs. resting endothelial cells. A variety of endothelial cells (eg HUVEC) will be exposed to endotoxin or cytokines and the release measured of a) prostacyclin b) adenosine c) endothelium-derived relaxation factor (EDRF, or NO) and d) sphingosine (an endogenous inhibitor of O2 generation). The effects of these molecules will be studied on neutrophils or cytoplasts activated by chemoattractants or immune complexes with respect to a) heterotypic adherence (to HUVEC), b) cell-cell aggregation, c) disaggregation d) chemotaxis in several systems, and e) release of O-2 H2 O2 and lysosomal enzymes. We will also expose neutrophils activated by chemoattractants and immune complexes to PGI2 adenosine, NO and sphingosine. Stimulus/response coupling will be examined with attention to receptors for iC3b and immune complexes (FcRII,FcRIII), cytosolic pH and free calcium (pH, and Cai) 45Ca fluxes, membrane viscosity, membrane potential (delta psi), crosslinking of cytoskeletal proteins, microtubule assembly, phospholipid remodelling, and protein phosphorylation. We will compare the susceptibility of resting HUVEC with cells activated by endotoxin or cytokines (IL-1, TNF and IFN) to killing by neutrophils exposed to chemoattractants (C5a, LTB, FMLP). We will study whether immune complexes present a) in the bulk phase b) on the HUVEC monolayer, or c) in the subphase, provoke neutrophil-mediated cell injury despite release of "defensive" molecules from HUVEC. Finally, using marine sponge cells and human neutrophils, we will study whether 20.4 and other cis unsaturated fatty acids activate a novel GTP-binding protein the function of which is mimicked by phospatidic acid. These studies will employ novel liposomal methods for phospholipid presentation. Thus we will be able to define the roles played by cytokine activated endothelial cells (the Shwartzman model) and immune complexes (the Arthus model) in neutrophil-mediated blood vessel injury.

Project Number :	5R01HL019721-16
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	Arthus phenomenon, Shwartzman phenomenon, inflammation, lung disorder, neutrophil, vascular endothelium adenosine, cell cell interaction, complement, complement receptor, cytokine, endotoxin, guanine nucleotide binding protein, immune complex, immunopharmacology, leukocyte activation /transformation, nitric oxide, respiratory pharmacology, vasodilator Porifera, cell free system, histochemistry /cytochemistry, human tissue from nonrelated source, radiotracer, salt water environment, tissue /cell culture

LUNG INFLAMMATION

(1994)

Abstract :

Neutrophils mediate tissue injury in two classical models of experimental pathology: the Arthus lesion(1903) and the Shwartzman phenomenon (1927). The aim of this proposal is to explain in the language of 1989 how vascular injury induced by immune complexes and complement (the Arthus lesion) differs from the induced by endotoxin, cytokines and complement (the Shwartzman phenomenon). We will study neutrophil/endothelial cell interactions in vitro to test novel hypotheses of "triggering" and activation" of neutrophils. We will determine the functional effects on human neutrophils and cytoplasts of "defensive" molecules release from activated vs. resting endothelial cells. A variety of endothelial cells (eg HUVEC) will be exposed to endotoxin or cytokines and the release measured of a) prostacyclin b) adenosine c) endothelium-derived relaxation factor (EDRF, or NO) and d) sphingosine (an endogenous inhibitor of O2 generation). The effects of these molecules will be studied on neutrophils or cytoplasts activated by chemoattractants or immune complexes with respect to a) heterotypic adherence (to HUVEC), b) cell-cell aggregation, c) disaggregation d) chemotaxis in several systems, and e) release of O-2 H2 O2 and lysosomal enzymes. We will also expose neutrophils activated by chemoattractants and immune complexes to PGI2 adenosine, NO and sphingosine. Stimulus/response coupling will be examined with attention to receptors for iC3b and immune complexes (FcRII,FcRIII), cytosolic pH and free calcium (pH, and Cai) 45Ca fluxes, membrane viscosity, membrane potential (delta psi), crosslinking of cytoskeletal proteins, microtubule assembly, phospholipid remodelling, and protein phosphorylation. We will compare the susceptibility of resting HUVEC with cells activated by endotoxin or cytokines (IL-1, TNF and IFN) to killing by neutrophils exposed to chemoattractants (C5a, LTB, FMLP). We will study whether immune complexes present a) in the bulk phase b) on the HUVEC monolayer, or c) in the subphase, provoke neutrophil-mediated cell injury despite release of "defensive" molecules from HUVEC. Finally, using marine sponge cells and human neutrophils, we will study whether 20.4 and other cis unsaturated fatty acids activate a novel GTP-binding protein the function of which is mimicked by phospatidic acid. These studies will employ novel liposomal methods for phospholipid presentation. Thus we will be able to define the roles played by cytokine activated endothelial cells (the Shwartzman model) and immune complexes (the Arthus model) in neutrophil-mediated blood vessel injury.

Project Number :	5R01HL019721-15
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, HYPERSENSITIVITY, ANAPHYLAXIS, ARTHUS PHENOMENON, HYPERSENSITIVITY, ANAPHYLAXIS, SHWARTZMAN PHENOMENON, RESPIRATORY DISORDERS, LUNG DISORDERS CELL-CELL INTERACTION, IMMUNITY, CELLULAR, LEUKOCYTE ACTIVATION, TRANSFORMATION AND PROLIFERATION, IMMUNITY, CYTOKINES, IMMUNOLOGY, ANTIGEN-ANTIBODY REACTIONS, IMMUNE COMPLEXES, IMMUNOLOGY, ANTIGENS BACTERIAL, ENDOTOXINS, IMMUNOLOGY, COMPLEMENT, PROTEINS, BINDING PROTEINS, GUANINE NUCLEOTIDE BINDING PROTEINS, PURINE NUCLEOSIDES, ADENINE NUCLEOSIDES, ADENOSINE, RECEPTORS, COMPLEMENT RECEPTORS, RESPIRATORY SYSTEM PHARMACOLOGY, immunopharmacology ANIMALS, INVERTEBRATES, SPONGES, CELL-FREE SYSTEMS, HISTOCHEMISTRY AND CYTOCHEMISTRY, HUMAN, HUMAN TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PR OJECT, RADIOTRACERS, TISSUE (CELL) CULTURE, WATER ENVIRONMENT, AQUATIC ORGANISMS, MARINE

MECHANISMS OF ARTHRITIS

(1992)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	2R37AR011949-21
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	BIO
Project Terms :	SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID, SKELETAL DISORDERS, JOINT DISORDERS ACYLS, ACYLATION-DEACYLATION, ANTIBIOTICS, IONOMYCIN, BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CELL COMPONENTS, CYTOSKELETON, MICROTUBULES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME MECHANISMS, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), LIPIDS, GLYCERIDES, DIGLYCERIDES, DIACYLGLYCEROL, LIPOPROTEINS BLOOD, OXYGENASES, LIPOXYGENASE, PHENYLCARBOXYLATES, SALICYLATES, ACETYL-, PHOSPHOLIPASE, PHOSPHATIDYLCHOLINE PHOSPHATIDOHYDROLASE, PHOSPHOLIPIDS, PHOSPHOMONOESTERASES, PHOSPHATIDATE PHOSPHOHYDROLASE, PHOSPHOTRANSFERASES-ATP, PROTEIN KINASES, PROTEIN KINASE C, PROTEASES AND PEPTIDASES, PROTEINS, MEMBRANE PROTEINS, RECEPTORS, ANTIBODY RECEPTORS, SKELETAL SYSTEM, CARTILAGE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN ANIMALS, CHORDATES, MAMMALS, LAGOMORPHS, ANIMALS, INVERTEBRATES, SPONGES, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, NMR, HUMAN, HUMAN TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PR OJECT, IMMUNOLOGICAL PREPARATIONS, MONOCLONAL ANTIBODIES, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, OPTICS, MICROSCOPY, ELECTRON SCANNING, OPTICS, MICROSCOPY, FLUORESCENCE, PHYSICAL SEPARATION, CENTRIFUGATION, PHYSICAL SEPARATION, CHROMATOGRAPHY, DEAE CELLULOSE

MECHANISMS OF ARTHRITIS

(1992)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	5R37AR011949-25
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	BIO
Project Terms :	joint disorder, osteoarthritis, rheumatoid arthritis acylation, antibody receptor, antiinflammatory agent, aspirin, autoimmune disease, bacterial antigen, blood lipoprotein, cartilage, cytochalasin, diacylglycerol, enzyme mechanism, immune complex disease, immunosuppressive antileukocyte serum, ionomycin, leukocyte, leukotriene, lipoxygenase, macrophage, membrane protein, microtubule, neutrophil, peptide hydrolase, phosphatidate phosphatase, phospholipase D, phospholipid, protein kinase C Porifera, centrifugation, fluorescence microscopy, human tissue, ion exchange chromatography, laboratory rabbit, monoclonal antibody, nuclear magnetic resonance spectroscopy, radioimmunoassay, scanning electron microscopy

MECHANISMS OF ARTHRITIS

(1992)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	5R37AR011949-24
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	BIO
Project Terms :	joint disorder, osteoarthritis, rheumatoid arthritis acylation, antibody receptor, antiinflammatory agent, aspirin, autoimmune disease, bacterial antigen, blood lipoprotein, cartilage, cytochalasin, diacylglycerol, enzyme mechanism, immune complex disease, immunosuppressive antileukocyte serum, ionomycin, leukocyte, leukotriene, lipoxygenase, macrophage, membrane protein, microtubule, neutrophil, peptide hydrolase, phosphatidate phosphatase, phospholipase D, phospholipid, protein kinase C Porifera, centrifugation, diethylaminoethyl cellulose chromatography, fluorescence microscopy, human tissue from nonrelated source, laboratory rabbit, monoclonal antibody, nuclear magnetic resonance spectroscopy, radioimmunoassay, scanning electron microscopy

MECHANISMS OF ARTHRITIS

(1992)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	5R37AR011949-23
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	BIO
Project Terms :	SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID, SKELETAL DISORDERS, JOINT DISORDERS ACYLS, ACYLATION-DEACYLATION, ANTIBIOTICS, IONOMYCIN, BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CELL COMPONENTS, CYTOSKELETON, MICROTUBULES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME MECHANISMS, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), LIPIDS, GLYCERIDES, DIGLYCERIDES, DIACYLGLYCEROL, LIPOPROTEINS BLOOD, OXYGENASES, LIPOXYGENASE, PHENYLCARBOXYLATES, SALICYLATES, ACETYL-, PHOSPHOLIPASE, PHOSPHATIDYLCHOLINE PHOSPHATIDOHYDROLASE, PHOSPHOLIPIDS, PHOSPHOMONOESTERASES, PHOSPHATIDATE PHOSPHOHYDROLASE, PHOSPHOTRANSFERASES-ATP, PROTEIN KINASES, PROTEIN KINASE C, PROTEASES AND PEPTIDASES, PROTEINS, MEMBRANE PROTEINS, RECEPTORS, ANTIBODY RECEPTORS, SKELETAL SYSTEM, CARTILAGE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN ANIMALS, CHORDATES, MAMMALS, LAGOMORPHS, RABBITS (LABORATORY), ANIMALS, INVERTEBRATES, SPONGES, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, NMR, HUMAN, HUMAN TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PR OJECT, IMMUNOLOGICAL PREPARATIONS, MONOCLONAL ANTIBODIES, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, OPTICS, MICROSCOPY, ELECTRON SCANNING, OPTICS, MICROSCOPY, FLUORESCENCE, PHYSICAL SEPARATION, CENTRIFUGATION, PHYSICAL SEPARATION, CHROMATOGRAPHY, DEAE CELLULOSE

MECHANISMS OF ARTHRITIS

(1992)

Abstract :

We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20: SPECIFIC AIM 1: In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down. SPECIFIC AIM 2: To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and "site-directed" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids. SPECIFIC AIM 3: To resolve the "leukotriene B4 paradox", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes. SPECIFIC AIM 4: We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two. SPECIFIC AIM 5: We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as "phylogenetic controls." Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).

Project Number :	5R37AR011949-22
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	BIO
Project Terms :	SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID, SKELETAL DISORDERS, JOINT DISORDERS ACYLS, ACYLATION-DEACYLATION, ANTIBIOTICS, IONOMYCIN, BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CELL COMPONENTS, CYTOSKELETON, MICROTUBULES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME MECHANISMS, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), LIPIDS, GLYCERIDES, DIGLYCERIDES, DIACYLGLYCEROL, LIPOPROTEINS BLOOD, OXYGENASES, LIPOXYGENASE, PHENYLCARBOXYLATES, SALICYLATES, ACETYL-, PHOSPHOLIPASE, PHOSPHATIDYLCHOLINE PHOSPHATIDOHYDROLASE, PHOSPHOLIPIDS, PHOSPHOMONOESTERASES, PHOSPHATIDATE PHOSPHOHYDROLASE, PHOSPHOTRANSFERASES-ATP, PROTEIN KINASES, PROTEIN KINASE C, PROTEASES AND PEPTIDASES, PROTEINS, MEMBRANE PROTEINS, RECEPTORS, ANTIBODY RECEPTORS, SKELETAL SYSTEM, CARTILAGE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN ANIMALS, CHORDATES, MAMMALS, LAGOMORPHS, ANIMALS, INVERTEBRATES, SPONGES, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, NMR, HUMAN, HUMAN TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PR OJECT, IMMUNOLOGICAL PREPARATIONS, MONOCLONAL ANTIBODIES, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, OPTICS, MICROSCOPY, ELECTRON SCANNING, OPTICS, MICROSCOPY, FLUORESCENCE, PHYSICAL SEPARATION, CENTRIFUGATION, PHYSICAL SEPARATION, CHROMATOGRAPHY, DEAE CELLULOSE

ARTHRITIS

(1991)

Project Number :	5T32AR007176-16
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	ADDK

ARTHRITIS

(1991)

Project Number :	2T32AR007176-12
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	ADDK

ARTHRITIS

(1991)

Project Number :	5T32AR007176-13
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	ADDK

ARTHRITIS

(1991)

Project Number :	5T32AR007176-14
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	ADDK

ARTHRITIS

(1991)

Project Number :	5T32AR007176-15
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	ADDK

CELL/PROTEIN INTERACTIONS IN CONNECTIVE TISSUE DISEASES

(1989)

Abstract :

The program consists of four major areas: 1) Structural and functional studies of connective tissue, serum and membrane proteins including cartilage constituents and fibronectin. 2) Humoral immunity including the structure, function, biosynthesis and genetic control of immunoglobulins and factors involved in the regulation of antibody synthesis. In parallel and complementing this, there is an active program devoted to the structure of certain complement components and their interaction with cellular receptors. Clinically, these studies converge on patients with immune complex diseases and patients with lymphoproliferative or plasma cell disorders. 3) Cellular mechanisms in diseases. In particular there is a multidisciplinary collaborative effort to define subsets of monocytes, to study the role of surface enzymes in normal and disordered monocyte function, and to correlate the structure and function of various subsets of lymphocytes. These studies focus on the amyloid diseases and various lymphoid neoplasms. 4) Genetics, development and membrane structure. The main focus of this work is to evaluate the role of genetic factors in resistance to leukemia and to study various surface components of lymphoid cells coded by the major histocompatibility locus that behave as differentiation markers and may play a role in lymphocyte function and cell-cell interaction.

Project Number :	5P01AR001431-33
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	SRC
Project Terms :	CONNECTIVE TISSUE DISORDERS, RHEUMATIC DISORDERS, IMMUNOLOGY, ANTIGENS, SURFACE ANTIGENS, immunogenetics, immunopathology

CELL/PROTEIN INTERACTIONS IN CONNECTIVE TISSUE DISEASES

(1989)

Abstract :

The program consists of four major areas: 1) Structural and functional studies of connective tissue, serum and membrane proteins including cartilage constituents and fibronectin. 2) Humoral immunity including the structure, function, biosynthesis and genetic control of immunoglobulins and factors involved in the regulation of antibody synthesis. In parallel and complementing this, there is an active program devoted to the structure of certain complement components and their interaction with cellular receptors. Clinically, these studies converge on patients with immune complex diseases and patients with lymphoproliferative or plasma cell disorders. 3) Cellular mechanisms in diseases. In particular there is a multidisciplinary collaborative effort to define subsets of monocytes, to study the role of surface enzymes in normal and disordered monocyte function, and to correlate the structure and function of various subsets of lymphocytes. These studies focus on the amyloid diseases and various lymphoid neoplasms. 4) Genetics, development and membrane structure. The main focus of this work is to evaluate the role of genetic factors in resistance to leukemia and to study various surface components of lymphoid cells coded by the major histocompatibility locus that behave as differentiation markers and may play a role in lymphocyte function and cell-cell interaction.

Project Number :	5P01AR001431-32
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	SRC
Project Terms :	CONNECTIVE TISSUE DISORDERS, RHEUMATIC DISORDERS, IMMUNOLOGY, ANTIGENS, SURFACE ANTIGENS, immunogenetics, immunopathology

CELL/PROTEIN INTERACTIONS IN CONNECTIVE TISSUE DISEASES

(1989)

Abstract :

The program consists of four major areas: 1) Structural and functional studies of connective tissue, serum and membrane proteins including cartilage constituents and fibronectin. 2) Humoral immunity including the structure, function, biosynthesis and genetic control of immunoglobulins and factors involved in the regulation of antibody synthesis. In parallel and complementing this, there is an active program devoted to the structure of certain complement components and their interaction with cellular receptors. Clinically, these studies converge on patients with immune complex diseases and patients with lymphoproliferative or plasma cell disorders. 3) Cellular mechanisms in diseases. In particular there is a multidisciplinary collaborative effort to define subsets of monocytes, to study the role of surface enzymes in normal and disordered monocyte function, and to correlate the structure and function of various subsets of lymphocytes. These studies focus on the amyloid diseases and various lymphoid neoplasms. 4) Genetics, development and membrane structure. The main focus of this work is to evaluate the role of genetic factors in resistance to leukemia and to study various surface components of lymphoid cells coded by the major histocompatibility locus that behave as differentiation markers and may play a role in lymphocyte function and cell-cell interaction.

Project Number :	5P01AR001431-31
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	SRC
Project Terms :	CONNECTIVE TISSUE DISORDERS, RHEUMATIC DISORDERS, IMMUNOGENETICS (GENERAL), IMMUNOLOGY, ANTIGENS, SURFACE ANTIGENS (GENERAL), IMMUNOPATHOLOGY (GENERAL)

CELL/PROTEIN INTERACTIONS IN CONNECTIVE TISSUE DISEASES

(1989)

Abstract :

The program consists of four major areas: 1) Structural and functional studies of connective tissue, serum and membrane proteins including cartilage constituents and fibronectin. 2) Humoral immunity including the structure, function, biosynthesis and genetic control of immunoglobulins and factors involved in the regulation of antibody synthesis. In parallel and complementing this, there is an active program devoted to the structure of certain complement components and their interaction with cellular receptors. Clinically, these studies converge on patients with immune complex diseases and patients with lymphoproliferative or plasma cell disorders. 3) Cellular mechanisms in diseases. In particular there is a multidisciplinary collaborative effort to define subsets of monocytes, to study the role of surface enzymes in normal and disordered monocyte function, and to correlate the structure and function of various subsets of lymphocytes. These studies focus on the amyloid diseases and various lymphoid neoplasms. 4) Genetics, development and membrane structure. The main focus of this work is to evaluate the role of genetic factors in resistance to leukemia and to study various surface components of lymphoid cells coded by the major histocompatibility locus that behave as differentiation markers and may play a role in lymphocyte function and cell-cell interaction.

Project Number :	5P01AR001431-30
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	SRC
Project Terms :	CONNECTIVE TISSUE DISORDERS, RHEUMATIC DISORDERS, IMMUNOGENETICS (GENERAL), IMMUNOLOGY, ANTIGENS, SURFACE ANTIGENS (GENERAL), IMMUNOPATHOLOGY (GENERAL)

CELL/PROTEIN INTERACTIONS IN CONNECTIVE TISSUE DISEASES

(1989)

Abstract :

The program consists of four major areas: 1) Structural and functional studies of connective tissue, serum and membrane proteins including cartilage constituents and fibronectin. 2) Humoral immunity including the structure, function, biosynthesis and genetic control of immunoglobulins and factors involved in the regulation of antibody synthesis. In parallel and complementing this, there is an active program devoted to the structure of certain complement components and their interaction with cellular receptors. Clinically, these studies converge on patients with immune complex diseases and patients with lymphoproliferative or plasma cell disorders. 3) Cellular mechanisms in diseases. In particular there is a multidisciplinary collaborative effort to define subsets of monocytes, to study the role of surface enzymes in normal and disordered monocyte function, and to correlate the structure and function of various subsets of lymphocytes. These studies focus on the amyloid diseases and various lymphoid neoplasms. 4) Genetics, development and membrane structure. The main focus of this work is to evaluate the role of genetic factors in resistance to leukemia and to study various surface components of lymphoid cells coded by the major histocompatibility locus that behave as differentiation markers and may play a role in lymphocyte function and cell-cell interaction.

Project Number :	2P01AM001431-29
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	SRC
Project Terms :	CONNECTIVE TISSUE DISORDERS, RHEUMATIC DISORDERS, IMMUNOGENETICS (GENERAL), IMMUNOLOGY, ANTIGENS, SURFACE ANTIGENS (GENERAL), IMMUNOPATHOLOGY (GENERAL)

LUNG INFLAMMATION

(1989)

Abstract :

We address the role of phagocytes in acute and chronic lung injury. In acute injury, neutrophils, activated by humoral factors (e.g. C5a), aggregate, release lysosomal enzymes, generate O2- and form eicosanoids mainly via lipoxygenase pathways. It is not clear, however, which of these mediators is critical to tissue injury marked by leukoaggregation of the Adult Respiratory Distress Syndrome. We will compare intact neutrophils and their granule-free cytoplasts (neutroplasts) for their capacity to injure endothelial substrates in vitro. Neutroplasts cannot release lysosomal proteases or myeloperoxidase, but can aggregate and generate O2-. We can thus determine whether lysosomal proteases and the myeloperoxidase/H2O2 system are necessary for endothelial injury, or whether aggregation and O2- generation suffice. Differences with respect to lipoxygenase products formed by intact neutrophils and neutroplasts will permit dissection of the role of neutrophil eicosanoids in cell activation and endothelial injury. Since platelets augment neutrophil aggregation and cytotoxicity, we will determine how platelet-neutrophil interactions influence formation of leukotriene B4 (LTB4) and its antagonist, 5S, 12SdiHETE, and whether these products interact with several protein kinases and phosphoprotein phosphatases. we will also study in vitro correlates of chronic lung disease by exposing neutrophils and mononuclear phagocytes to crystals of silica and asbestos. We will determine relations between the membranolytic potential of crystals and their capacity to release LTB4 from phagocytes-as urate crystals can from neutrophils. We will determine whether colchicine, recently found to be a specific inhibitor of LTB4 synthesis in urate-stimulated neutrophils, inhibits LTB4 formation after silica or asbestos. Finally, we will test a novel hypothesis as to the mode of action of nonsteroidal antiinflammatory drugs (NSAID). Cyclooxygenase products (e.g. PGE2, PGI2) inhibit phagocyte functions whereas some lipoxygenase products (esp. LTB4) are potent stimulants. But other lipoxygenase products (5S, 12S, diHETE or 14, 15 diHETE) antagonize LTB4. We propose that exogenous NSAID divert arachidonate to yield antiinflammatory eicosanoids: "the body's own NSAID." These products will be sought in phagocytes treated with aspirin-like drugs. We will test this hypothesis by means of marine sponge cells which do not respond to stable prostaglandins, but whose LTB4-mediated aggregation is, surprisingly, inhibited by exogenous NSAID.

Project Number :	5R01HL019721-13
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELLS, PHAGOCYTES, DISEASES, CELLULAR LEVEL STUDIES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS, RESPIRATORY DISORDERS, LUNG DISORDERS ALKALOIDS, COLCHICINE, BENZOPYRROLE CARBOXYLIC ACIDS, INDOMETHACIN, BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, BLOOD CELLS, MONOCYTES, BLOOD PLATELETS, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, CELL COMPONENTS, LYSOSOMES, CELL-CELL INTERACTION, CELL-CELL INTERACTION, CELL AGGREGATION, DISEASES, CHRONIC DISEASES, ENZYME INHIBITORS, ELASTASE INHIBITORS, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, FATTY ACIDS, EICOSANOIDS, PROSTAGLANDINS, FATTY ACIDS, PROPIONIC ACID, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, OXYGENASES, LIPOXYGENASE, PHENYLCARBOXYLATES, IBUPROFEN, PHENYLCARBOXYLATES, SALICYLATES, PHOSPHOTRANSFERASES-ATP, ATP:PROTEIN PHOSPHOTRANSFERASE, PHOSPHOTRANSFERASES-ATP, PROTEIN KINASES, PROTEASES AND PEPTIDASES, PURINE NUCLEOSIDES, ADENINE NUCLEOSIDES, ADENOSINE, RESPIRATORY DISORDERS, ENVIRONMENTAL POLLUTANTS ASSOCIATED, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, immunopharmacology ANIMALS, INVERTEBRATES, SPONGES, HISTOCHEMISTRY AND CYTOCHEMISTRY, HUMAN, HUMAN TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PR OJECT, OPTICS, MICROSCOPY, ELECTRON, PHYSICAL SEPARATION, CHROMATOGRAPHY, THIN LAYER, RADIOTRACERS, SILICATES, SILICATES, ASBESTOS, TISSUE (CELL) CULTURE, WATER ENVIRONMENT, AQUATIC ORGANISMS, MARINE

LUNG INFLAMMATION

(1989)

Abstract :

We address the role of phagocytes in acute and chronic lung injury. In acute injury, neutrophils, activated by humoral factors (e.g. C5a), aggregate, release lysosomal enzymes, generate O2- and form eicosanoids mainly via lipoxygenase pathways. It is not clear, however, which of these mediators is critical to tissue injury marked by leukoaggregation of the Adult Respiratory Distress Syndrome. We will compare intact neutrophils and their granule-free cytoplasts (neutroplasts) for their capacity to injure endothelial substrates in vitro. Neutroplasts cannot release lysosomal proteases or myeloperoxidase, but can aggregate and generate O2-. We can thus determine whether lysosomal proteases and the myeloperoxidase/H2O2 system are necessary for endothelial injury, or whether aggregation and O2- generation suffice. Differences with respect to lipoxygenase products formed by intact neutrophils and neutroplasts will permit dissection of the role of neutrophil eicosanoids in cell activation and endothelial injury. Since platelets augment neutrophil aggregation and cytotoxicity, we will determine how platelet-neutrophil interactions influence formation of leukotriene B4 (LTB4) and its antagonist, 5S, 12SdiHETE, and whether these products interact with several protein kinases and phosphoprotein phosphatases. we will also study in vitro correlates of chronic lung disease by exposing neutrophils and mononuclear phagocytes to crystals of silica and asbestos. We will determine relations between the membranolytic potential of crystals and their capacity to release LTB4 from phagocytes-as urate crystals can from neutrophils. We will determine whether colchicine, recently found to be a specific inhibitor of LTB4 synthesis in urate-stimulated neutrophils, inhibits LTB4 formation after silica or asbestos. Finally, we will test a novel hypothesis as to the mode of action of nonsteroidal antiinflammatory drugs (NSAID). Cyclooxygenase products (e.g. PGE2, PGI2) inhibit phagocyte functions whereas some lipoxygenase products (esp. LTB4) are potent stimulants. But other lipoxygenase products (5S, 12S, diHETE or 14, 15 diHETE) antagonize LTB4. We propose that exogenous NSAID divert arachidonate to yield antiinflammatory eicosanoids: "the body's own NSAID." These products will be sought in phagocytes treated with aspirin-like drugs. We will test this hypothesis by means of marine sponge cells which do not respond to stable prostaglandins, but whose LTB4-mediated aggregation is, surprisingly, inhibited by exogenous NSAID.

Project Number :	5R01HL019721-12
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELLS, PHAGOCYTES, DISEASES, CELLULAR LEVEL STUDIES (GENERAL), DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), PATHOLOGY A STUDY SECTION, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) ALKALOIDS, COLCHICINE, BENZOPYRROLE CARBOXYLIC ACIDS, INDOMETHACIN, BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, BLOOD CELLS, MONOCYTES, BLOOD PLATELETS, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, CELL COMPONENTS, LYSOSOMES, CELL-CELL INTERACTION, CELL-CELL INTERACTION, CELL AGGREGATION, DISEASES, CHRONIC (GENERAL), ENZYME INHIBITORS, ELASTASE INHIBITORS, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, FATTY ACIDS, EICOSANOIDS, PROSTAGLANDINS, FATTY ACIDS, PROPIONIC ACID, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, OXYGENASES, LIPOXYGENASE, PHENYLCARBOXYLATES, IBUPROFEN, PHENYLCARBOXYLATES, SALICYLATES, PHOSPHOTRANSFERASES, ATP:PROTEIN PHOSPHOTRANSFERASE, PHOSPHOTRANSFERASES, PROTEIN KINASES (GENERAL), PROTEASES AND PEPTIDASES, PURINE NUCLEOSIDES, ADENINE NUCLEOSIDES, ADENOSINE, RESPIRATORY DISORDERS, ENVIRONMENTAL POLLUTANTS ASSOCIATED, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, immunopharmacology HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, OPTICS, MICROSCOPY, ELECTRON, PHYSICAL SEPARATION, CHROMATOGRAPHY, THIN LAYER, RADIOTRACERS, SILICATES, SILICATES, ASBESTOS, SPONGES, TISSUE (CELL) CULTURE, WATER ENVIRONMENT, AQUATIC ORGANISMS, MARINE

LUNG INFLAMMATION

(1989)

Abstract :

We address the role of phagocytes in acute and chronic lung injury. In acute injury, neutrophils, activated by humoral factors (e.g. C5a), aggregate, release lysosomal enzymes, generate O2- and form eicosanoids mainly via lipoxygenase pathways. It is not clear, however, which of these mediators is critical to tissue injury marked by leukoaggregation of the Adult Respiratory Distress Syndrome. We will compare intact neutrophils and their granule-free cytoplasts (neutroplasts) for their capacity to injure endothelial substrates in vitro. Neutroplasts cannot release lysosomal proteases or myeloperoxidase, but can aggregate and generate O2-. We can thus determine whether lysosomal proteases and the myeloperoxidase/H2O2 system are necessary for endothelial injury, or whether aggregation and O2- generation suffice. Differences with respect to lipoxygenase products formed by intact neutrophils and neutroplasts will permit dissection of the role of neutrophil eicosanoids in cell activation and endothelial injury. Since platelets augment neutrophil aggregation and cytotoxicity, we will determine how platelet-neutrophil interactions influence formation of leukotriene B4 (LTB4) and its antagonist, 5S, 12SdiHETE, and whether these products interact with several protein kinases and phosphoprotein phosphatases. we will also study in vitro correlates of chronic lung disease by exposing neutrophils and mononuclear phagocytes to crystals of silica and asbestos. We will determine relations between the membranolytic potential of crystals and their capacity to release LTB4 from phagocytes-as urate crystals can from neutrophils. We will determine whether colchicine, recently found to be a specific inhibitor of LTB4 synthesis in urate-stimulated neutrophils, inhibits LTB4 formation after silica or asbestos. Finally, we will test a novel hypothesis as to the mode of action of nonsteroidal antiinflammatory drugs (NSAID). Cyclooxygenase products (e.g. PGE2, PGI2) inhibit phagocyte functions whereas some lipoxygenase products (esp. LTB4) are potent stimulants. But other lipoxygenase products (5S, 12S, diHETE or 14, 15 diHETE) antagonize LTB4. We propose that exogenous NSAID divert arachidonate to yield antiinflammatory eicosanoids: "the body's own NSAID." These products will be sought in phagocytes treated with aspirin-like drugs. We will test this hypothesis by means of marine sponge cells which do not respond to stable prostaglandins, but whose LTB4-mediated aggregation is, surprisingly, inhibited by exogenous NSAID.

Project Number :	5R01HL019721-11
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELLS, PHAGOCYTES, DISEASES, CELLULAR LEVEL STUDIES (GENERAL), DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), PATHOLOGY A STUDY SECTION, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) ALKALOIDS, COLCHICINE, BENZOPYRROLE CARBOXYLIC ACIDS, INDOMETHACIN, BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, BLOOD CELLS, MONOCYTES, BLOOD PLATELETS, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, CELL COMPONENTS, LYSOSOMES, CELL-CELL INTERACTION, CELL-CELL INTERACTION, CELL AGGREGATION, DISEASES, CHRONIC (GENERAL), ENZYME INHIBITORS, ELASTASE INHIBITORS, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, FATTY ACIDS, EICOSANOIDS, PROSTAGLANDINS, FATTY ACIDS, PROPIONIC ACID, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, OXYGENASES, LIPOXYGENASE, PHENYLCARBOXYLATES, IBUPROFEN, PHENYLCARBOXYLATES, SALICYLATES, PHOSPHOTRANSFERASES, ATP:PROTEIN PHOSPHOTRANSFERASE, PHOSPHOTRANSFERASES, PROTEIN KINASES (GENERAL), PROTEASES AND PEPTIDASES, PURINE NUCLEOSIDES, ADENINE NUCLEOSIDES, ADENOSINE, RESPIRATORY DISORDERS, ENVIRONMENTAL POLLUTANTS ASSOCIATED, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, immunopharmacology HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, OPTICS, MICROSCOPY, ELECTRON, PHYSICAL SEPARATION, CHROMATOGRAPHY, THIN LAYER, RADIOTRACERS, SILICATES, SILICATES, ASBESTOS, SPONGES, TISSUE (CELL) CULTURE, WATER ENVIRONMENT, AQUATIC ORGANISMS, MARINE

LUNG INFLAMMATION

(1989)

Abstract :

We address the role of phagocytes in acute and chronic lung injury. In acute injury, neutrophils, activated by humoral factors (e.g. C5a), aggregate, release lysosomal enzymes, generate O2- and form eicosanoids mainly via lipoxygenase pathways. It is not clear, however, which of these mediators is critical to tissue injury marked by leukoaggregation of the Adult Respiratory Distress Syndrome. We will compare intact neutrophils and their granule-free cytoplasts (neutroplasts) for their capacity to injure endothelial substrates in vitro. Neutroplasts cannot release lysosomal proteases or myeloperoxidase, but can aggregate and generate O2-. We can thus determine whether lysosomal proteases and the myeloperoxidase/H2O2 system are necessary for endothelial injury, or whether aggregation and O2- generation suffice. Differences with respect to lipoxygenase products formed by intact neutrophils and neutroplasts will permit dissection of the role of neutrophil eicosanoids in cell activation and endothelial injury. Since platelets augment neutrophil aggregation and cytotoxicity, we will determine how platelet-neutrophil interactions influence formation of leukotriene B4 (LTB4) and its antagonist, 5S, 12SdiHETE, and whether these products interact with several protein kinases and phosphoprotein phosphatases. we will also study in vitro correlates of chronic lung disease by exposing neutrophils and mononuclear phagocytes to crystals of silica and asbestos. We will determine relations between the membranolytic potential of crystals and their capacity to release LTB4 from phagocytes-as urate crystals can from neutrophils. We will determine whether colchicine, recently found to be a specific inhibitor of LTB4 synthesis in urate-stimulated neutrophils, inhibits LTB4 formation after silica or asbestos. Finally, we will test a novel hypothesis as to the mode of action of nonsteroidal antiinflammatory drugs (NSAID). Cyclooxygenase products (e.g. PGE2, PGI2) inhibit phagocyte functions whereas some lipoxygenase products (esp. LTB4) are potent stimulants. But other lipoxygenase products (5S, 12S, diHETE or 14, 15 diHETE) antagonize LTB4. We propose that exogenous NSAID divert arachidonate to yield antiinflammatory eicosanoids: "the body's own NSAID." These products will be sought in phagocytes treated with aspirin-like drugs. We will test this hypothesis by means of marine sponge cells which do not respond to stable prostaglandins, but whose LTB4-mediated aggregation is, surprisingly, inhibited by exogenous NSAID.

Project Number :	5R01HL019721-10
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELLS, PHAGOCYTES, DISEASES, CELLULAR LEVEL STUDIES (GENERAL), DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), PATHOLOGY A STUDY SECTION, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) ALKALOIDS, COLCHICINE, BENZOPYRROLE CARBOXYLIC ACIDS, INDOMETHACIN, BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, BLOOD CELLS, MONOCYTES, BLOOD PLATELETS, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, CELL COMPONENTS, LYSOSOMES, CELL-CELL INTERACTION, CELL-CELL INTERACTION, CELL AGGREGATION, DISEASES, CHRONIC (GENERAL), ENZYME INHIBITORS, ELASTASE INHIBITORS, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, FATTY ACIDS, EICOSANOIDS, PROSTAGLANDINS, FATTY ACIDS, PROPIONIC ACID, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, OXYGENASES, LIPOXYGENASE, PHENYLCARBOXYLATES, IBUPROFEN, PHENYLCARBOXYLATES, SALICYLATES, PHOSPHOTRANSFERASES, ATP:PROTEIN PHOSPHOTRANSFERASE, PHOSPHOTRANSFERASES, PROTEIN KINASES (GENERAL), PROTEASES AND PEPTIDASES, PURINE NUCLEOSIDES, ADENINE NUCLEOSIDES, ADENOSINE, RESPIRATORY DISORDERS, ENVIRONMENTAL POLLUTANTS ASSOCIATED, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, immunopharmacology HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, OPTICS, MICROSCOPY, ELECTRON, PHYSICAL SEPARATION, CHROMATOGRAPHY, THIN LAYER, RADIOTRACERS, SILICATES, SILICATES, ASBESTOS, SPONGES, TISSUE (CELL) CULTURE, WATER ENVIRONMENT, AQUATIC ORGANISMS, MARINE

LUNG INFLAMMATION

(1989)

Abstract :

We address the role of phagocytes in acute and chronic lung injury. In acute injury, neutrophils, activated by humoral factors (e.g. C5a), aggregate, release lysosomal enzymes, generate O2- and form eicosanoids mainly via lipoxygenase pathways. It is not clear, however, which of these mediators is critical to tissue injury marked by leukoaggregation of the Adult Respiratory Distress Syndrome. We will compare intact neutrophils and their granule-free cytoplasts (neutroplasts) for their capacity to injure endothelial substrates in vitro. Neutroplasts cannot release lysosomal proteases or myeloperoxidase, but can aggregate and generate O2-. We can thus determine whether lysosomal proteases and the myeloperoxidase/H2O2 system are necessary for endothelial injury, or whether aggregation and O2- generation suffice. Differences with respect to lipoxygenase products formed by intact neutrophils and neutroplasts will permit dissection of the role of neutrophil eicosanoids in cell activation and endothelial injury. Since platelets augment neutrophil aggregation and cytotoxicity, we will determine how platelet-neutrophil interactions influence formation of leukotriene B4 (LTB4) and its antagonist, 5S, 12SdiHETE, and whether these products interact with several protein kinases and phosphoprotein phosphatases. we will also study in vitro correlates of chronic lung disease by exposing neutrophils and mononuclear phagocytes to crystals of silica and asbestos. We will determine relations between the membranolytic potential of crystals and their capacity to release LTB4 from phagocytes-as urate crystals can from neutrophils. We will determine whether colchicine, recently found to be a specific inhibitor of LTB4 synthesis in urate-stimulated neutrophils, inhibits LTB4 formation after silica or asbestos. Finally, we will test a novel hypothesis as to the mode of action of nonsteroidal antiinflammatory drugs (NSAID). Cyclooxygenase products (e.g. PGE2, PGI2) inhibit phagocyte functions whereas some lipoxygenase products (esp. LTB4) are potent stimulants. But other lipoxygenase products (5S, 12S, diHETE or 14, 15 diHETE) antagonize LTB4. We propose that exogenous NSAID divert arachidonate to yield antiinflammatory eicosanoids: "the body's own NSAID." These products will be sought in phagocytes treated with aspirin-like drugs. We will test this hypothesis by means of marine sponge cells which do not respond to stable prostaglandins, but whose LTB4-mediated aggregation is, surprisingly, inhibited by exogenous NSAID.

Project Number :	2R01HL019721-09
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELLS, PHAGOCYTES, DISEASES, CELLULAR LEVEL STUDIES (GENERAL), DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), PATHOLOGY A STUDY SECTION, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) ALKALOIDS, COLCHICINE, BENZOPYRROLE CARBOXYLIC ACIDS, INDOMETHACIN, BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, BLOOD CELLS, MONOCYTES, BLOOD PLATELETS, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, CELL COMPONENTS, LYSOSOMES, CELL-CELL INTERACTION, CELL-CELL INTERACTION, CELL AGGREGATION, DISEASES, CHRONIC (GENERAL), ENZYME INHIBITORS, ELASTASE INHIBITORS, FATTY ACIDS, PROPIONIC ACID, FATTY ACIDS, UNSATURATED, LEUKOTRIENES, FATTY ACIDS, UNSATURATED, PROSTAGLANDINS, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, OXYGENASES, LIPOXYGENASE, PHENYLCARBOXYLATES, SALICYLATES, PHOSPHOTRANSFERASES, ATP:PROTEIN PHOSPHOTRANSFERASE, PHOSPHOTRANSFERASES, PROTEIN KINASES (GENERAL), PROTEASES AND PEPTIDASES, PURINE NUCLEOSIDES, ADENINE NUCLEOSIDES, ADENOSINE, RESPIRATORY DISORDERS, ENVIRONMENTAL POLLUTANTS ASSOCIATED, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, immunopharmacology HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, OPTICS, MICROSCOPY, ELECTRON, PHYSICAL SEPARATION, CHROMATOGRAPHY, THIN LAYER, RADIOTRACERS, SILICATES, SILICATES, ASBESTOS, SPONGES, TISSUE (CELL) CULTURE, WATER ENVIRONMENT, AQUATIC ORGANISMS, MARINE

MECHANISMS OF ARTHRITIS

(1987)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	5R01AR011949-20
ICD :	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIBODIES, ANTIBODY RECEPTORS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

MECHANISMS OF ARTHRITIS

(1987)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	5R01AM011949-19
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIBODIES, ANTIBODY RECEPTORS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

MECHANISMS OF ARTHRITIS

(1987)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	2R01AM011949-16
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIBODIES, ANTIBODY RECEPTORS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

MECHANISMS OF ARTHRITIS

(1987)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	5R01AM011949-17
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIBODIES, ANTIBODY RECEPTORS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

MECHANISMS OF ARTHRITIS

(1987)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	5R01AM011949-18
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIBODIES, ANTIBODY RECEPTORS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

ARTHRITIS

(1986)

Project Number :	5T32AM007176-08
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	SRC

ARTHRITIS

(1986)

Project Number :	5T32AM007176-09
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	SRC

ARTHRITIS

(1986)

Project Number :	5T32AM007176-10
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	SRC

ARTHRITIS

(1986)

Project Number :	5T32AM007176-11
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	SRC

LUNG INFLAMMATION: LYSOSOMAL PROTEASES AND LIPOSOMES

(1984)

Abstract :

The aim of these experiments is to provide a quantitative model of lung inflammation, mediated by the endogenous proteases of lung phagocytes, which can be abrogated by means of aerosolized liposomes which will act as vectors for anti-proteases. Elaborating upon a new model for acute and chronic alveolitis in the rabbit, we will study tracheal aspirates from animals exposed to intratracheal zymosan particles, by biochemical and ultrastructural, cytochemical techniques. Lungs, tracheal aspirates and purified cells will be analyzed for collagenase, elastase, cathepsin G, cathepsin D, and histonase after their encounter with zymosan particles and/or activated complement components. The pattern will be studied of inhibition of these proteases by defined protease inhibitor (alpha 1-antitrypsin, pepstatin, leupeptin, antipain, etc.). We will determine both the complement and the granulocyte-dependence of this model lung inflammation, and how the balance between proteases and antiproteases determines such inflammatory stimuli as chemotaxis. After specific protease inhibitors, encapsulated in immunoglobulin coated liposomes, have been introduced to rabbit PMN and macrophages in vitro, we will study which are most effective at inhibiting intracellular and regurgitant proteases. These inhibitors will be entrapped in multilamellar and large unilamellar liposomes, and such liposomes, coated with aggregated rabbit IgG, will be instilled by Nebulizer into the experimentally inflamed lungs. Direct introduction via the airways should lead to the encounter of these Ig-coated vectors with the Fc receptor-bearing, inflammatory cells of the lung. Thus the phagocytes may be tricked into ingesting the means of their own disarmament. The ultimate goal is to provide a method for the treatment of genetic and acquired lung inflammation in man by means of inhaled liposome dispersions containing protease inhibitors.

Project Number :	2R01HL019721-04
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, CELL COMPONENTS, LYSOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), IMMUNOPATHOLOGY, DIAGNOSIS, PATHOLOGY STUDY SECTION, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, MONOCYTES, CELLS, PHAGOCYTES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DRUGS VEHICLES, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNOLOGY, ANTIGENS MICROBIAL, ZYMOSAN, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, IMMUNOPHARMACOLOGY, MODELS, BIOLOGICAL, PHYSICAL PROPERTIES, AEROSOLS, RESPIRATORY CIRCULATORY DISORDERS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, enzyme replacement therapy HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN SUBJECTS, VOLUNTEERS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON, RADIOISOTOPES, IODINE, RADIOTRACERS, TISSUE (CELL) CULTURE

LUNG INFLAMMATION: LYSOSOMAL PROTEASES AND LIPOSOMES

(1984)

Abstract :

Non-cytotoxic release of lysosomal enzymes from polymorphonuclear leukocytes (PMNs) may mediate lung inflammation in hypersensitivity vasculitis, shock lung, and emphysema. We will test the possibility that protease inhibitors, appropriately presented to PMN by encapsulating the inhibitors in immunoglobulin-coated liposomes, will modify damage to lung vasculature in vitro by lysosomal proteases. Some soluble or particulate immune reactants (C5a, aggregated IgG, Sepharose-IgG, Sepharose-3b, endotoxin) provoke the exocytosis of the contents of both azurophile and specific granules of PMNs; others (phorbol myristate acetate, concanavalin A) the release only of specific granule contents. After determining which stimuli provoke the release of known human PMN proteases (cathepsin D, cathepsin G, collagenase, elastase) we will assess, by means of inhibitors (e.g. delta-I antitrypsin, peptsatin, phosphoramidon, elastatinal), which of these enzymes are most likely responsible for vessel injury. Inhibitors will be tested for their capacity to protect isolated vessel strips, endothelial cell cultures, and lung fragments from injury (assessed biochemically and ultrastructurally) induced by PMN secretions and purified enzymes. These inhibitors will be presented in two ways: either dissolved in the fluid medium around PMNs and/or the purified protease or as encapsulated in IgG-coated liposomes fed to PMNs before these cells are permitted to secrete proteases upon immune stimulation. Finally, we will determine whether IgG-coated liposomes bearing protease inhibitors can be taken up by PMNs in vitro and in vivo, subsequently to inhibit PMN-dependent lung injury. These studies should permit a rational approach to PMN-dependent lung inflammation and lead to therapeutic intervention in genetic deficiencies of protease inhibitors.

Project Number :	5R01HL019721-05
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, CELL COMPONENTS, LYSOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), IMMUNOPATHOLOGY, DIAGNOSIS, PATHOLOGY A STUDY SECTION, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, MONOCYTES, CELLS, PHAGOCYTES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DRUGS VEHICLES, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNOLOGY, ANTIGENS MICROBIAL, ZYMOSAN, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, IMMUNOPHARMACOLOGY, MODELS, BIOLOGICAL, PHYSICAL PROPERTIES, AEROSOLS, RESPIRATORY CIRCULATORY DISORDERS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, enzyme replacement therapy HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN SUBJECTS, VOLUNTEERS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON, RADIOISOTOPES, IODINE, RADIOTRACERS, TISSUE (CELL) CULTURE

LUNG INFLAMMATION: LYSOSOMAL PROTEASES AND LIPOSOMES

(1984)

Abstract :

Non-cytotoxic release of lysosomal enzymes from polymorphonuclear leukocytes (PMNs) may mediate lung inflammation in hypersensitivity vasculitis, shock lung, and emphysema. We will test the possibility that protease inhibitors, appropriately presented to PMN by encapsulating the inhibitors in immunoglobulin-coated liposomes, will modify damage to lung vasculature in vitro by lysosomal proteases. Some soluble or particulate immune reactants (C5a, aggregated IgG, Sepharose-IgG, Sepharose-3b, endotoxin) provoke the exocytosis of the contents of both azurophile and specific granules of PMNs; others (phorbol myristate acetate, concanavalin A) the release only of specific granule contents. After determining which stimuli provoke the release of known human PMN proteases (cathepsin D, cathepsin G, collagenase, elastase) we will assess, by means of inhibitors (e.g. delta-I antitrypsin, pepstatin, phosphoramidon, elastatinal), which of these enzymes are most likely responsible for vessel injury. Inhibitors will be tested for their capacity to protect isolated vessel strips, endothelial cell cultures, and lung fragments from injury (assessed biochemically and ultrastructurally) induced by PMN secretions and purified enzymes. These inhibitors will be presented in two ways: either dissolved in the fluid medium around PMNs and/or the purified protease or as encapsulated in IgG-coated liposomes fed to PMNs before these cells are permitted to secrete proteases upon immune stimulation. Finally, we will determine whether IgG-coated liposomes bearing protease inhibitors can be taken up by PMNs in vitro and in vivo, subsequently to inhibit PMN-dependent lung injury. These studies should permit a rational approach to PMN-dependent lung inflammation and lead to therapeutic intervention in genetic deficiencies of protease inhibitors.

Project Number :	5R01HL019721-06
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, CELL COMPONENTS, LYSOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), IMMUNOPATHOLOGY, DIAGNOSIS, PATHOLOGY A STUDY SECTION, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, MONOCYTES, CELLS, PHAGOCYTES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DRUGS VEHICLES, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNOLOGY, ANTIGENS MICROBIAL, ZYMOSAN, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, IMMUNOPHARMACOLOGY, MODELS, BIOLOGICAL, PHYSICAL PROPERTIES, AEROSOLS, RESPIRATORY CIRCULATORY DISORDERS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, enzyme replacement therapy HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN SUBJECTS, VOLUNTEERS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON, RADIOISOTOPES, IODINE, RADIOTRACERS, TISSUE (CELL) CULTURE

LUNG INFLAMMATION: LYSOSOMAL PROTEASES AND LIPOSOMES

(1984)

Abstract :

Non-cytotoxic release of lysosomal enzymes from polymorphonuclear leukocytes (PMNs) may mediate lung inflammation in hypersensitivity vasculitis, shock lung, and emphysema. We will test the possibility that protease inhibitors, appropriately presented to PMN by encapsulating the inhibitors in immunoglobulin-coated liposomes, will modify damage to lung vasculature in vitro by lysosomal proteases. Some soluble or particulate immune reactants (C5a, aggregated IgG, Sepharose-IgG, Sepharose-3b, endotoxin) provoke the exocytosis of the contents of both azurophile and specific granules of PMNs; others (phorbol myristate acetate, concanavalin A) the release only of specific granule contents. After determining which stimuli provoke the release of known human PMN proteases (cathepsin D, cathepsin G, collagenase, elastase) we will assess, by means of inhibitors (e.g. delta-I antitrypsin, pepstatin, phosphoramidon, elastatinal), which of these enzymes are most likely responsible for vessel injury. Inhibitors will be tested for their capacity to protect isolated vessel strips, endothelial cell cultures, and lung fragments from injury (assessed biochemically and ultrastructurally) induced by PMN secretions and purified enzymes. These inhibitors will be presented in two ways: either dissolved in the fluid medium around PMNs and/or the purified protease or as encapsulated in IgG-coated liposomes fed to PMNs before these cells are permitted to secrete proteases upon immune stimulation. Finally, we will determine whether IgG-coated liposomes bearing protease inhibitors can be taken up by PMNs in vitro and in vivo, subsequently to inhibit PMN-dependent lung injury. These studies should permit a rational approach to PMN-dependent lung inflammation and lead to therapeutic intervention in genetic deficiencies of protease inhibitors.

Project Number :	5R01HL019721-07
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, CELL COMPONENTS, LYSOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), IMMUNOPATHOLOGY, DIAGNOSIS, PATHOLOGY A STUDY SECTION, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, MONOCYTES, CELLS, PHAGOCYTES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DRUGS VEHICLES, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNOLOGY, ANTIGENS MICROBIAL, ZYMOSAN, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, MODELS, BIOLOGICAL, PHYSICAL PROPERTIES, AEROSOLS, RESPIRATORY CIRCULATORY DISORDERS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, enzyme replacement therapy, immunopharmacology HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN SUBJECTS, VOLUNTEERS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON, RADIOISOTOPES, IODINE, RADIOTRACERS, TISSUE (CELL) CULTURE

LUNG INFLAMMATION: LYSOSOMAL PROTEASES AND LIPOSOMES

(1984)

Abstract :

Non-cytotoxic release of lysosomal enzymes from polymorphonuclear leukocytes (PMNs) may mediate lung inflammation in hypersensitivity vasculitis, shock lung, and emphysema. We will test the possibility that protease inhibitors, appropriately presented to PMN by encapsulating the inhibitors in immunoglobulin-coated liposomes, will modify damage to lung vasculature in vitro by lysosomal proteases. Some soluble or particulate immune reactants (C5a, aggregated IgG, Sepharose-IgG, Sepharose-3b, endotoxin) provoke the exocytosis of the contents of both azurophile and specific granules of PMNs; others (phorbol myristate acetate, concanavalin A) the release only of specific granule contents. After determining which stimuli provoke the release of known human PMN proteases (cathepsin D, cathepsin G, collagenase, elastase) we will assess, by means of inhibitors (e.g. delta-I antitrypsin, pepstatin, phosphoramidon, elastatinal), which of these enzymes are most likely responsible for vessel injury. Inhibitors will be tested for their capacity to protect isolated vessel strips, endothelial cell cultures, and lung fragments from injury (assessed biochemically and ultrastructurally) induced by PMN secretions and purified enzymes. These inhibitors will be presented in two ways: either dissolved in the fluid medium around PMNs and/or the purified protease or as encapsulated in IgG-coated liposomes fed to PMNs before these cells are permitted to secrete proteases upon immune stimulation. Finally, we will determine whether IgG-coated liposomes bearing protease inhibitors can be taken up by PMNs in vitro and in vivo, subsequently to inhibit PMN-dependent lung injury. These studies should permit a rational approach to PMN-dependent lung inflammation and lead to therapeutic intervention in genetic deficiencies of protease inhibitors.

Project Number :	5R01HL019721-08
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, CELL COMPONENTS, LYSOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), IMMUNOPATHOLOGY, DIAGNOSIS, PATHOLOGY A STUDY SECTION, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, MONOCYTES, CELLS, PHAGOCYTES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, DRUGS VEHICLES, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNOLOGY, ANTIGENS MICROBIAL, ZYMOSAN, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, COMPLEMENT, ANAPHYLATOXINS, PHYSICAL PROPERTIES, AEROSOLS, RESPIRATORY CIRCULATORY DISORDERS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, RESPIRATORY SYSTEM PHARMACOLOGY, RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, enzyme replacement therapy, immunopharmacology HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN SUBJECTS, VOLUNTEERS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON, RADIOISOTOPES, IODINE, RADIOTRACERS, TISSUE (CELL) CULTURE

REGULATION OF PMN'S IN IMMUNE COMPLEX DISEASE

(1983)

Project Number :	5P50AI017365-030003
ICD :	NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Project Terms :	BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS) CARDIOVASCULAR DISORDERS, VASCULITIS, CARDIOVASCULAR DISORDERS, VASCULITIS, ARTERITIS, CELL BIOLOGY STUDY SECTION, CELL INGESTION, PHAGOCYTOSIS, CONNECTIVE TISSUE DISORDERS, LUPUS ERYTHEMATOSUS SYSTEMIC, ENVIRONMENT, ORIENTATION, CHEMOTAXIS, IMMUNOLOGY, ANTIGEN-ANTIBODY REACTIONS, IMMUNE COMPLEXES, POPULATION STUDIES HUMAN, EPIDEMIOLOGY, POPULATION STUDIES HUMAN, LONGITUDINAL STUDY, SKIN DISORDERS, ERYTHEMA MULTIFORME, cell adhesion HUMAN, CLINICAL

MECHANISMS OF ARTHRITIS

(1982)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	2R01AM011949-11
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN SUBJECTS, VOLUNTEERS, HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION*

MECHANISMS OF ARTHRITIS

(1982)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	5R01AM011949-12
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN SUBJECTS, VOLUNTEERS, HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

MECHANISMS OF ARTHRITIS

(1982)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	5R01AM011949-13
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN SUBJECTS, VOLUNTEERS, HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

MECHANISMS OF ARTHRITIS

(1982)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	5R01AM011949-14
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIBODIES, ANTIBODY RECEPTORS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

MECHANISMS OF ARTHRITIS

(1982)

Abstract :

Rheumatoid arthritis is, at least in part, associated with immune-complex induced synovitis. Immune complexes and complement components (e.g. C5a, C3a, and C3b) cause the non-cytotoxic release of lysosomal proteases from both polymorphonuclear leukocytes and macrophages. To modify immune tissue injury of joint structures, we will introduce into the offending inflammatory cells protease inhibitors specific for the proteases of phagocytes, by means of liposomes. These vectors will be introjected into PMN's and macrophages in two ways: a) by coating with aggregated immunoglobulins so as to promote uptake via Fc receptors into the lysosomal apparatus, and b) by preincorporating the fusogen, lysolecithin into the liposomal bilayer so as to engender fusion of multilamellar liposomes directly with the plasma membrane of macrophages, which contain non-lysosomal proteases. After establishing that phagocytes after in vitro exposure to immune reactants, release proteases (collagenase, elastase, cathepsins G and D) capable of degrading cartilage slices, isolated proteoglycans, and of affecting normal synovial cells in culture, we will determine which specific protease inhibitors prevent phagocyte-induced connective tissue injury. The appropriate inhibitors (alpha 1-antitrypsin, pepstatin, phosphoramidon, elastatinol) will then be incorporated into liposomes. Liposome-encapsulated inhibitors will also be instilled into the knee joints of rabbits in which immune complex arthritis has been induced by means of BSA/anti-BSA and peroxidase complexes, to determine whether immune injury of joints can also be aborted in vivo. By means of ultrastructural cytochemistry, we will determine the fate of both antigen and antibody in the models of arthritis, as well as the cellular localization of inhibitor-laden liposomes used to modify immune joint injury. These studies should not only clarify the role of lysosomal and non-lysosomal proteases in immunologically induced arthritis, but define the discrete contributions of PMN and macrophage proteases to connective tissue degradation induced by immune complexes and complement components.

Project Number :	5R01AM011949-15
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD AND RE SYSTEM, IMMUNOHEMATOLOGY (GENERAL), BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), ENZYME MECHANISMS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNITY, IMMUNOSUPPRESSION, ALLOANTISERA, ANTILEUKOCYTIC, IMMUNOLOGY, ANTIBODIES, ANTIBODY RECEPTORS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS), MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PROTEASES AND PEPTIDASES, PROTEASES AND PEPTIDASES, CATHEPSIN B, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, PROTEOGLYCANS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE, TISSUE (CELL) CULTURE, TOXICOLOGY, MYCOTOXINS, CYTOCHALASIN HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CENTRIFUGATION

REGULATION OF PMN'S IN IMMUNE COMPLEX DISEASE

(1981)

Project Number :	5P50AI017365-020003
ICD :	NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Project Terms :	BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS) CARDIOVASCULAR DISORDERS, ARTERITIS, CELL BIOLOGY STUDY SECTION, CELL INGESTION, PHAGOCYTOSIS, CONNECTIVE TISSUE DISORDERS, LUPUS ERYTHEMATOSUS SYSTEMIC, ENVIRONMENT, ORIENTATION, CHEMOTAXIS, IMMUNOLOGY, ANTIGEN-ANTIBODY REACTIONS, IMMUNE COMPLEXES, POPULATION STUDIES HUMAN, EPIDEMIOLOGY, POPULATION STUDIES HUMAN, LONGITUDINAL STUDY, SKIN DISORDERS, ERYTHEMA MULTIFORME, cell adhesion HUMAN, CLINICAL

REGULATION OF PMN'S IN IMMUNE COMPLEX DISEASE

(1980)

Project Number :	1P50AI017365-010003
ICD :	NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Project Terms :	BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, IMMUNOPATHOLOGY, IMMUNE COMPLEX DISEASES (SEE ALSO SPECIFICS) CARDIOVASCULAR DISORDERS, ARTERITIS, CELL BIOLOGY STUDY SECTION, CELL INGESTION, PHAGOCYTOSIS, CONNECTIVE TISSUE DISORDERS, LUPUS ERYTHEMATOSUS SYSTEMIC, ENVIRONMENT, ORIENTATION, CHEMOTAXIS, IMMUNOLOGY, ANTIGEN-ANTIBODY REACTIONS, IMMUNE COMPLEXES, POPULATION STUDIES HUMAN, EPIDEMIOLOGY, POPULATION STUDIES HUMAN, LONGITUDINAL STUDY, SKIN DISORDERS, ERYTHEMA MULTIFORME, cell adhesion HUMAN, CLINICAL

EXPERIMENTAL THERAPY OF GENETIC STORAGE DISEASES

(1979)

Abstract :

The objectives of the proposal are to test the following hypotheses: a) Enzyme replacement therapy of lysosomal storage disease (e.g. Tay-sachs, Gauchers disease) is best achieved if the missing enzyme is presented to cells in a kind of biological "spansule" (liposome) which has surface ligands to engender uptake by cells possessing specific receptors. b) That uptake of Ig-coated liposomes by phagocytes from patients with Tay-Sachs disease or peroxidase deficiency into their lysosomal system provides the means to bring enzymes into every organ and tissue, since circulating leukocytes percolate through these after emigrating from post-capillary venules. That the experimental model we have already exploited, peroxidase deficiency of Mustelus canis, can be used to test this enzyme delivery system. c) That means can be found selectively to release the newly acquired enzymes from the lysosomal system of circulating phagocytes without generalized cytotoxicity, immunogenicity of the foreign protein, and with some tissue specificity. d) That Ig-coated liposomes exceed in their capacity to correct enzyme deficits in man and experimental animals the capacity of free enzymes or of enzymes entrapped in uncoated liposomes.

Project Number :	1R01GM023211-01
ICD :	NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG :	CTY
Project Terms :	METABOLIC DISORDERS INBORN, LYSOSOMAL DISORDERS, METABOLIC DISORDERS INBORN, SPHINGOLIPIDOSES, GANGLIOSIDE GM 2 TYPE I, MOLECULAR CYTOLOGY STUDY SECTION, enzyme replacement therapy BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LIPOSOMES, CELL INGESTION, PHAGOCYTOSIS, DRUGS RECEPTORS, DRUGS, PHARMACOLOGY, BIOAVAILABILITY, METABOLIC DISORDERS INBORN, SPHINGOLIPIDOSES, GLUCOSYLCEREBROSIDE, MODELS, BIOLOGICAL, PEROXIDASES, MYELOPEROXIDASE EYE CIRCULATION, BLOOD-AQUEOUS BARRIER, FISH, ELASMOBRANCHS, HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, HYDROGEN, TRITIUM, MAMMALS, RODENTS, MYOMORPHA, MICE (LABORATORY), OPTICS, FLUOROMETRY, RADIOISOTOPES, IODINE, RADIOISOTOPES, TECHNETIUM, RADIOTRACER, TISSUE (CELL) CULTURE, enzyme substrate

EXPERIMENTAL THERAPY OF GENETIC STORAGE DISEASES

(1979)

Abstract :

The objectives of the proposal are to test the following hypotheses: a) Enzyme replacement therapy of lysosomal storage disease (e.g. Tay-Sachs, Gauchers disease) is best achieved if the missing enzyme is presented to cells in a kind of biological "spansule" (liposome) which has surface ligands to engender uptake by cells possessing specific receptors. b) That uptake of Ig-coated liposomes by phagocytes from patients with Tay-Sachs disease or peroxidase deficiency into their lysosomal system provides the means to bring enzymes into every organ and tissue, since circulating leukocytes percolate through these after emigrating from post-capillary venules. That the experimental model we have already exploited, peroxidase deficiency of Mustelus canis, can be used to test this enzyme delivery system. c) That means can be found selectively to release the newly acquired enzymes from the lysosomal system of circulating phagocytes without generalized cytotoxicity, immunogenicity of the foreign protein, and with some tissue specificity. d) That Ig-coated liposomes exceed in their capacity to correct enzyme deficits in man and experimental animals the capacity of free enzymes or of enzymes entrapped in uncoated liposomes.

Project Number :	5R01GM023211-02
ICD :	NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG :	CTY
Project Terms :	METABOLIC DISORDERS INBORN, LYSOSOMAL DISORDERS, METABOLIC DISORDERS INBORN, SPHINGOLIPIDOSES, GANGLIOSIDE GM 2 TYPE I, MOLECULAR CYTOLOGY STUDY SECTION, enzyme replacement therapy BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LIPOSOMES, CELL INGESTION, PHAGOCYTOSIS, DRUGS, PHARMACOLOGY, BIOAVAILABILITY, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, METABOLIC DISORDERS INBORN, SPHINGOLIPIDOSES, GLUCOSYLCEREBROSIDE, MODELS, BIOLOGICAL EYE CIRCULATION, BLOOD-AQUEOUS BARRIER, FISH, ELASMOBRANCHS, HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, HYDROGEN, TRITIUM, MAMMALS, RODENTS, MYOMORPHA, MICE (LABORATORY), OPTICS, FLUOROMETRY, RADIOISOTOPES, IODINE, RADIOISOTOPES, TECHNETIUM, RADIOTRACER, TISSUE (CELL) CULTURE, enzyme substrate

EXPERIMENTAL THERAPY OF GENETIC STORAGE DISEASES

(1979)

Abstract :

The objectives of the proposal are to test the following hypotheses: a) Enzyme replacement therapy of lysosomal storage disease (e.g. Tay-Sachs, Gauchers disease) is best achieved if the missing enzyme is presented to cells in a kind of biological "spansule" (liposome) which has surface ligands to engender uptake by cells possessing specific receptors. b) That uptake of Ig-coated liposomes by phagocytes from patients with Tay-Sachs disease or peroxidase deficiency into their lysosomal system provides the means to bring enzymes into every organ and tissue, since circulating leukocytes percolate through these after emigrating from post-capillary venules. That the experimental model we have already exploited, peroxidase deficiency of Mustelus canis, can be used to test this enzyme delivery system. c) That means can be found selectively to release the newly acquired enzymes from the lysosomal system of circulating phagocytes without generalized cytotoxicity, immunogenicity of the foreign protein, and with some tissue specificity. d) That Ig-coated liposomes exceed in their capacity to correct enzyme deficits in man and experimental animals the capacity of free enzymes or of enzymes entrapped in uncoated liposomes.

Project Number :	5R01GM023211-03
ICD :	NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG :	CTY
Project Terms :	METABOLIC DISORDERS INBORN, LYSOSOMAL DISORDERS, METABOLIC DISORDERS INBORN, SPHINGOLIPIDOSES, GANGLIOSIDE GM 2 TYPE I, MOLECULAR CYTOLOGY STUDY SECTION, enzyme replacement therapy BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, BLOOD CELLS, LEUKOCYTES, CELL INGESTION, PHAGOCYTOSIS, DRUGS, PHARMACOLOGY, BIOAVAILABILITY, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, METABOLIC DISORDERS INBORN, SPHINGOLIPIDOSES, GLUCOSYLCEREBROSIDE, MODELS, BIOLOGICAL EYE CIRCULATION, BLOOD-AQUEOUS BARRIER, FISH, ELASMOBRANCHS, HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, HYDROGEN, TRITIUM, MAMMALS, RODENTS, MYOMORPHA, MICE (LABORATORY), OPTICS, FLUOROMETRY, RADIOISOTOPES, IODINE, RADIOISOTOPES, TECHNETIUM, RADIOTRACERS, TISSUE (CELL) CULTURE, enzyme substrate

MODIFICATION OF TISSUE INJURY IN MYOCARDIAL INFARCTION

(1979)

Abstract :

In studies of the experimental pathology of tissue injury in myocardial infarction in dogs, we have shown an early loss of integrity of cell components. The membranes of myocardial cells fail as a barrier to ions and macromolecules with consequent swelling and functional disruption of cells and organelles, and lysosomes release into the cytosol, and surrounding tissue hydrolytic enzymes which may augment regional injury. The two processes appear interrelated and self-perpetuating since partially damaged cells may show injury to one or another compartment whereas cells in the area of necrosis always show injury to all compartments. Furthermore, the area of necrosis continues to expand for up to 18 hours after initiation of ischemia indicating that a mechanism exists for continued injury to tissues marginally perfused at the outset. Since lysosomal hydrolases are known to play an important role in tissue injury, we propose to study the role of lysosomes and lysosomal hydrolases in the augmentation of tissue injury in experimental myocardial infarction, and to study procedures which may protect the ischemic myocardial. This proposal is designed to test the following hypotheses: 1) that loss of integrity of myocardial cells and organelles is critical in the amplification of ischemic injury; 2) that hydrolytic enzymes released from lysosomes of myocardial cells damaged early in ischemia play a role in this amplification of tissue injury; and 3) that pharmacologic agents which affect tissue integrity can influence the development of necrosis. The techniques to be employed have been in use in our laboratories for a minimum of three years and include stable animal models for both short term and long term infarctions, as well as appropriate morphological, cytochemical and biochemical evaluations of the extent of injury.

Project Number :	1R01HL019072-01
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELL COMPONENTS, LYSOSOMES, HEART DISORDERS, CORONARY, MYOCARDIAL INFARCT, HEART DISORDERS, CORONARY, MYOCARDIAL ISCHEMIA AND HYPOXIA, HYDROLASES, PATHOLOGY STUDY SECTION ADRENAL CORTEX HORMONES ANALOGS, METHYLPREDNISOLONE, ADRENAL CORTEX HORMONES, GLUCOCORTICOIDS, CARBOHYDRASES, BETA-GLUCURONIDASE, CARDIOVASCULAR DISORDERS CHEMOTHERAPY, CARDIOVASCULAR SYSTEM, BLOOD SUPPLY, CELL COMPONENTS, INTRACELLULAR MEMBRANES, DISEASES, ACUTE (GENERAL), DISEASES, MOLECULAR LEVEL STUDIES (GENERAL), ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), HEART DISORDERS, ARRHYTHMIA, HEART, ENDOCARDIUM, HEART, MYOCARDIUM, MODELS, BIOLOGICAL, PHOSPHOMONOESTERASES, ACID PHOSPHATASE, PHOSPHOTRANSFERASES, ATP:CREATINE PHOSPHOTRANSFERASE, PROTEASES AND PEPTIDASES, VITAMIN A, heart revascularization DIAGNOSTIC TESTS, BIOPSY, HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), MAMMALS, CARNIVORES, DOGS, OPTICS, MICROSCOPY, ELECTRON

MODIFICATION OF TISSUE INJURY IN MYOCARDIAL INFARCTION

(1979)

Abstract :

In studies of the experimental pathology of tissue injury in myocardial infarction in dogs, we have shown an early loss of integrity of cell components. The membranes of myocardial cells fail as a barrier to ions and macromolecules with consequent swelling and functional disruption of cells and organelles, and lysosomes release into the cytosol, and surrounding tissue hydrolytic enzymes which may augment regional injury. The two processes appear interrelated and self-perpetuating since partially damaged cells may show injury to one or another compartment whereas cells in the area of necrosis always show injury to all compartments. Furthermore, the area of necrosis continues to expand for up to 18 hours after initiation of ischemia indicating that a mechanism exists for continued injury to tissues marginally perfused at the outset. Since lysosomal hydrolases are known to play an important role in tissue injury, we propose to study the role of lysosomes and lysosomal hydrolases in the augmentation of tissue injury in experimental myocardial infarction, and to study procedures which may protect the ischemic myocardial. This proposal is designed to test the following hypotheses: 1) that loss of integrity of myocardial cells and organelles is critical in the amplification of ischemic injury; 2) that hydrolytic enzymes released from lysosomes of myocardial cells damaged early in ischemia play a role in this amplification of tissue injury; and 3) that pharmacologic agents which affect tissue integrity can influence the development of necrosis. The techniques to be employed have been in use in our laboratories for a minimum of three years and include stable animal models for both short term and long term infarctions, as well as appropriate morphological, cytochemical and biochemical evaluations of the extent of injury.

Project Number :	5R01HL019072-03
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELL COMPONENTS, LYSOSOMES, HEART DISORDERS, CORONARY, MYOCARDIAL INFARCT, HEART DISORDERS, CORONARY, MYOCARDIAL ISCHEMIA AND HYPOXIA, HYDROLASES, PATHOLOGY STUDY SECTION ADRENAL CORTEX HORMONES ANALOGS, METHYLPREDNISOLONE, ADRENAL CORTEX HORMONES, GLUCOCORTICOIDS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CARBOHYDRASES, BETA-GLUCURONIDASE, CARDIOVASCULAR SYSTEM, BLOOD SUPPLY, CELL COMPONENTS, CELL COMPONENTS, INTRACELLULAR MEMBRANES, DISEASES, MOLECULAR LEVEL STUDIES (GENERAL), ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), HEART DISORDERS CHEMOTHERAPY, HEART DISORDERS, ARRHYTHMIA, HEART, ENDOCARDIUM, HEART, MYOCARDIUM, MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, MODELS, BIOLOGICAL, PHOSPHOMONOESTERASES, ACID PHOSPHATASE, PHOSPHOTRANSFERASES, ATP:CREATINE PHOSPHOTRANSFERASE, PROTEASES AND PEPTIDASES, VITAMIN A, calcium, heart revascularization DIAGNOSTIC TESTS, BIOPSY, HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HISTOPATHOLOGY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, MAMMALS, CARNIVORES, DOGS, OPTICS, MICROSCOPY, ELECTRON, PHYSICAL SEPARATION, CHROMATOGRAPHY, DEAE CELLULOSE, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL, TISSUE (CELL) CULTURE

LUNG INFLAMMATION: LYSOSOMAL PROTEASES AND LIPOSOMES

(1979)

Abstract :

Non-cytotoxic release of lysosomal enzymes from polymorphonuclear leukocytes (PMNs) may mediate lung inflammation in hypersensitivity vasculitis, shock lung, and emphysema. We will test the possibility that protease inhibitors, appropriately presented to PMN by encapsulating the inhibitors in immunoglobulin-coated liposomes, will modify damage to lung vasculature in vitro by lysosomal proteases. Some soluble or particulate immune reactants (C5a, aggregated IgG, Sepharose-IgG, Sepharose-3b, endotoxin) provoke the exocytosis of the contents of both azurophile and specific granules of PMNs; others (phorbol myristate acetate, concanavalin A) the release only of specific granule contents. After determining which stimuli provoke the release of known human PMN proteases (cathepsin D, cathepsin G, collagenase, elastase) we will assess, by means of inhibitors (e.g., delta-I antitrypsin, pepstatin, phosphoramidon, elastatinal), which of these enzymes are most likely responsible for vessel injury. Inhibitors will be tested for their capacity to protect isolated vessel strips, endothelial cell cultures, and lung gragments from injury (assessed biochemically and ultrastructurally) induced by PMN secretions and purified enzymes. These inhibitors will be presented in two ways: either dissolved in the fluid medium around PMNs and/or the purified proteases or as encapsulated in IgG-coated liposomes fed to PMNs before these cells are permitted to secrete proteases upon immune stimulation. Finally, we will determine whether IgG-coated liposomes bearing protease inhibitors can be taken up by PMNs in vitro and in vivo, subsequently to inhibit PMN-dependent lung injury. These studies should permit a rational approach to PMN-dependent lung inflammation and lead to therapeutic intervention in genetic deficiencies of protease inhibitors.

Project Number :	1R01HL019721-01
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	SSS
Project Terms :	CELL COMPONENTS, LYSOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, IMMUNOPATHOLOGY, DIAGNOSIS, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) BIOLOGICAL TRANSPORT, ACTIVE TRANSPORT, PINOCYTOSIS, BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, CELL COMPONENTS, LIPOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, PROPERDIN, IMMUNOPHARMACOLOGY, PROTEASES AND PEPTIDASES, CATHEPSINS, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, RESPIRATORY CIRCULATORY DISORDERS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY, SHOCK LUNG, enzyme replacement therapy HUMAN SUBJECTS, VOLUNTEERS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, OPTICS, MICROSCOPY, ELECTRON

MODIFICATION OF TISSUE INJURY IN MYOCARDIAL INFARCTION

(1979)

Abstract :

In studies of the experimental pathology of tissue injury in myocardial infarction in dogs, we have shown an early loss of integrity of cell components. The membranes of myocardial cells fail as a barrier to ions and macromolecules with consequent swelling and functional disruption of cells and organelles, and lysosomes release into the cytosol, and surrounding tissue hydrolytic enzymes which may augment regional injury. The two processes appear interrelated and self-perpetuating since partially damaged cells may show injury to one or another compartment whereas cells in the area of necrosis always show injury to all compartments. Furthermore, the area of necrosis continues to expand for up to 18 hours after initiation of ischemia indicating that a mechanism exists for continued injury to tissues marginally perfused at the outset. Since lysosomal hydrolases are known to play an important role in tissue injury, we propose to study the role of lysosomes and lysosomal hydrolases in the augmentation of tissue injury in experimental myocardial infarction, and to study procedures which may protect the ischemic myocardial. This proposal is designed to test the following hypotheses: 1) that loss of integrity of myocardial cells and organelles is critical in the amplification of ischemic injury; 2) that hydrolytic enzymes released from lysosomes of myocardial cells damaged early in ischemia play a role in this amplification of tissue injury; and 3) that pharmacologic agents which affect tissue integrity can influence the development of necrosis. The techniques to be employed have been in use in our laboratories for a minimum of three years and include stable animal models for both short term and long term infarctions, as well as appropriate morphological, cytochemical and biochemical evaluations of the extent of injury. BIBLIOGRAPHIC REFERENCES: Roos, D., Goldstein, I.M., Kaplan, H.B., Weissmann, G., Dissociation of phagocytosis, metabolic stimulation and lysosomal enzyme release in human leukocytes. Agents and Actions 6:256-262, 1976. Weissmann, G., The pharmacological control of immunologically-induced inflammation In: Infection and Immunology in the Rheumatic Diseases (D.C. Dumonde, ed.) Bleckwell Scientific Publications, Oxford, pp. 503-510, 1976.

Project Number :	5R01HL019072-02
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELL COMPONENTS, LYSOSOMES, HEART DISORDERS, CORONARY, MYOCARDIAL INFARCT, HEART DISORDERS, CORONARY, MYOCARDIAL ISCHEMIA AND HYPOXIA, HYDROLASES, PATHOLOGY STUDY SECTION ADRENAL CORTEX HORMONES ANALOGS, METHYLPREDNISOLONE, ADRENAL CORTEX HORMONES, GLUCOCORTICOIDS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CARBOHYDRASES, BETA-GLUCURONIDASE, CARDIOVASCULAR DISORDERS CHEMOTHERAPY, CARDIOVASCULAR SYSTEM, BLOOD SUPPLY, CELL COMPONENTS, INTRACELLULAR MEMBRANES, DISEASES, MOLECULAR LEVEL STUDIES (GENERAL), ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), HEART DISORDERS, ARRHYTHMIA, HEART, ENDOCARDIUM, HEART, MYOCARDIUM, MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, MODELS, BIOLOGICAL, PHOSPHOMONOESTERASES, ACID PHOSPHATASE, PHOSPHOTRANSFERASES, ATP:CREATINE PHOSPHOTRANSFERASE, PROTEASES AND PEPTIDASES, VITAMIN A, calcium, heart revascularization DIAGNOSTIC TESTS, BIOPSY, HISTOCHEMISTRY AND CYTOCHEMISTRY (GENERAL), HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, MAMMALS, CARNIVORES, DOGS, OPTICS, MICROSCOPY, ELECTRON, PHYSICAL SEPARATION, CHROMATOGRAPHY, DEAE CELLULOSE, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL, TISSUE (CELL) CULTURE

LUNG INFLAMMATION: LYSOSOMAL PROTEASES AND LIPOSOMES

(1979)

Abstract :

Non-cytotoxic release of lysosomal enzymes from polymorphonuclear leukocytes (PMNs) may mediate lung inflammation in hypersensitivity vasculitis, shock lung, and emphysema. We will test the possibility that protease inhibitors, appropriately presented to PMN by encapsulating the inhibitors in immunoglobulin-coated liposomes, will modify damage to lung vasculature in vitro by lysosomal proteases. Some soluble or particulate immune reactants (C5a, aggregated IgG, Sepharose-IgG, Sepharose-3b, endotoxin) provoke the exocytosis of the contents of both azurophile and specific granules of PMNs; others (phorbol myristate acetate, concanavalin A) the release only of specific granule contents. After determining which stimuli provoke the release of known human PMN proteases (cathepsin D, cathepsin G, collagenase, elastase) we will assess, by means of inhibitors (e.g., delta-I antitrypsin, pepstatin, phosphoramidon, elastatinal), which of these enzymes are most likely responsible for vessel injury. Inhibitors will be tested for their capacity to protect isolated vessel strips, endothelial cell cultures, and lung fragments from injury (assessed biochemically and ultrastructurally) induced by PMN secretions and purified enzymes. These inhibitors will be presented in two ways: either dissolved in the fluid medium around PMNs and/or the purified proteases or as encapsulated in IgG-coated liposomes fed to PMNs before these cells are permitted to secrete proteases upon immune stimulation. Finally, we will determine whether IgG-coated liposomes bearing protease inhibitors can be taken up by PMNs in vitro and in vivo, subsequently to inhibit PMN-dependent lung injury. These studies should permit a rational approach to PMN-dependent lung inflammation and lead to therapeutic intervention in genetic deficiencies of protease inhibitors.

Project Number :	5R01HL019721-02
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	SSS
Project Terms :	CELL COMPONENTS, LIPOSOMES, CELL COMPONENTS, LYSOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), IMMUNOPATHOLOGY, DIAGNOSIS, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) ADRENAL CORTEX HORMONES, HYDROCORTISONE, BIOLOGICAL TRANSPORT, ACTIVE TRANSPORT, PINOCYTOSIS, BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CARBOHYDRASES, HEXOSAMINIDASES, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, CELL COMPONENTS, MICROTUBULES (GENERAL), DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, PROPERDIN, IMMUNOPHARMACOLOGY, METAL COMPLEXES, LIGANDS, METALLOPROTEINS, CYTOCUPREINS, PROTEASES AND PEPTIDASES, CATHEPSINS, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, RESPIRATORY CIRCULATORY DISORDERS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY, SHOCK LUNG, SENSORY DEPRESSION, ANESTHESIA CONDUCTION, LOCAL, enzyme replacement therapy HEART DISORDERS, CORONARY, MYOCARDIAL INFARCT, HUMAN SUBJECTS, VOLUNTEERS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, METABOLIC DISORDERS INBORN, SPHINGOLIPIDOSES, GANGLIOSIDE GM 2 TYPE I, OPTICS, MICROSCOPY, ELECTRON

LUNG INFLAMMATION: LYSOSOMAL PROTEASES AND LIPOSOMES

(1979)

Abstract :

Non-cytotoxic release of lysosomal enzymes from polymorphonuclear leukocytes (PMNs) may mediate lung inflammation in hypersensitivity vasculitis, shock lung, and emphysema. We will test the possibility that protease inhibitors, appropriately presented to PMN by encapsulating the inhibitors in immunoglobulin-coated liposomes, will modify damage to lung vasculature in vitro by lysosomal proteases. Some soluble or particulate immune reactants (C5a, aggregated IgG, Sepharose-IgG, Sepharose-3b, endotoxin) provoke the exocytosis of the contents of both azurophile and specific granules of PMNs; others (phorbol myristate acetate, concanavalin A) the release only of specific granule contents. After determining which stimuli provoke the release of known human PMN proteases (cathepsin D, cathepsin G, collagenase, elastase) we will assess, by means of inhibitors (e.g., delta-I antitrypsin, pepstatin, phosphoramidon, elastatinal), which of these enzymes are most likely responsible for vessel injury. Inhibitors will be tested for their capacity to protect isolated vessel strips, endothelial cell cultures, and lung fragments from injury (assessed biochemically and ultrastructurally) induced by PMN secretions and purified enzymes. These inhibitors will be presented in two ways: either dissolved in the fluid medium around PMNs and/or the purified proteases or as encapsulated in IgG-coated liposomes fed to PMNs before these cells are permitted to secrete proteases upon immune stimulation. Finally, we will determine whether IgG-coated liposomes bearing protease inhibitors can be taken up by PMNs in vitro and in vivo, subsequently to inhibit PMN-dependent lung injury. These studies should permit a rational approach to PMN-dependent lung inflammation and lead to therapeutic intervention in genetic deficiencies of protease inhibitors.

Project Number :	5R01HL019721-03
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	SSS
Project Terms :	BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, CELL COMPONENTS, LYSOSOMES, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), IMMUNOPATHOLOGY, DIAGNOSIS, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, LUNG DISORDERS (GENERAL) ADRENAL CORTEX HORMONES, HYDROCORTISONE, BIOLOGICAL TRANSPORT, ACTIVE TRANSPORT, PINOCYTOSIS, BIOLOGICAL TRANSPORT, SECRETORY MECHANISMS, BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, CARBOHYDRASES, HEXOSAMINIDASES, CARDIOVASCULAR SYSTEM, ENDOTHELIUM, CELL COMPONENTS, MICROTUBULES (GENERAL), DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN G, IMMUNOLOGY, COMPLEMENT, IMMUNOLOGY, PROPERDIN, IMMUNOPHARMACOLOGY, METAL COMPLEXES, LIGANDS, METALLOPROTEINS, CYTOCUPREINS, PROTEASES AND PEPTIDASES, CATHEPSINS, PROTEASES AND PEPTIDASES, COLLAGENASE, PROTEASES AND PEPTIDASES, ELASTASE, RESPIRATORY CIRCULATORY DISORDERS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, SHOCK LUNG, SENSORY DEPRESSION, ANESTHESIA CONDUCTION, LOCAL, enzyme replacement therapy HEART DISORDERS, CORONARY, MYOCARDIAL INFARCT, HUMAN SUBJECTS, VOLUNTEERS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, METABOLIC DISORDERS INBORN, SPHINGOLIPIDOSES, GANGLIOSIDE GM 2 TYPE I, OPTICS, MICROSCOPY, ELECTRON

MECHANISMS OF ARTHRITIS

(1977)

Abstract :

Studies of the structure and function of lysosomes will continue in four areas: 1) studies of the uptake, storage, and membrane-action of agents which stabilize or labilize the membranes of lysosomes; 2) studies of the regulation of endocytosis, intracellular distribution and degradation of antigenic macromolecules by macrophages; especially of the effects of cyclic AMP on the vacuolar system; 3) the formation of a lysosome model (liposome) composed of defined phospholipids, cholesterol, and membrane proteins which will enclose lysozyme, and 4) the effects of lysosomal proteases upon the proteins in intact nuclei or chromatin which retard the access of RNA polymerase to DNA. Each of these studies is a natural extension of previous work and is designed to answer questions raised by these earlier observations. Together they should elucidate means whereby the vacuolar system (a term which describes the shuttle and flow of lysosomal subgroups) is regulated. Since controlled hydrolysis of intracellular and extracellular macromolecules is the means whereby the cell handles foreign materials, processes antigens, remodels itself in cell division, and may indeed renew itself, these regulatory mechanisms deserve study. We will focus on those agents that regulate the flow of intracellular macromolecules from one compartment to the other, as well as those which act on membrane elements common to all components of the vacuolar system. Each regulatory factor will also be studied in a model, artificial system, recently devised, which bears many resemblances to natural biomembranes. BIBLIOGRAPHIC REFERENCES: Goldstein, I.M., Hoffstein, S.T. and Weissmann, G., Mechanisms of lysosomal enzyme release from human polymorphonuclear leukocytes: Effects of phorbol myristate acetate. J. Cell Biol. 66:647-652, 1975. Goldstein, I.M., Hoffstein, S.T. and Weissmann, G., Influence of divalent cations upon complement-mediated enzyme release from human polymorphonuclear leukocytes. J. Immunol. 115:665-670, 1975.

Project Number :	5R01AM011949-10
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME MECHANISMS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, MAMMALS, LAGOMORPHS, PHYSICAL SEPARATION, CENTRIFUGATION*

MECHANISMS OF ARTHRITIS

(1977)

Project Number :	2R01AM011949-06
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ADRENAL CORTEX HORMONES, CORTISONE, CELL COMPONENTS, LYSOSOMES, ENZYME MECHANISMS, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, PROTEOGLYCANS, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID, SKELETAL DISORDERS, JOINT DISORDERS ALLERGY AND IMMUNOLOGY STUDY SECTION, BLOOD CELLS, LEUKOCYTES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, IMMUNOLOGY, ANTIGENS BACTERIAL, MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, RADIOISOTOPES, ACTINIDE SERIES, THORIUM OXIDES, SKELETAL SYSTEM, CARTILAGE HUMAN, NONCLINICAL, MAMMALS, LAGOMORPHS, PHYSICAL SEPARATION, CENTRIFUGATION*

MECHANISMS OF ARTHRITIS

(1977)

Abstract :

This study is designed to investigate mechanisms underlying joint destruction in rheumatic diseases. Lysosomal enzymes will be purified and characterized as to their capacity to (1) break down protein polysaccharides; (2) induce fibroplasia; (3) act upon nuclear structures. In addition, we will attempt to study mechanisms whereby small, naive lymphocytes incapable of tissue injury are rendered rich in lysosomes which can subsequently both degrade connective tissue and induce fibroplasia. Finally we will study the effects of cortisone and chloroquine upon these processes, since these drugs stabilize lysosomes.

Project Number :	5R01AM011949-07
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME MECHANISMS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE HUMAN, NONCLINICAL, MAMMALS, LAGOMORPHS, PHYSICAL SEPARATION, CENTRIFUGATION*

MECHANISMS OF ARTHRITIS

(1977)

Abstract :

Studies of the structure and function of lysosomes will continue in four areas: 1) studies of the uptake, storage, and membrane-action of agents which stabilize or labilize the membranes of lysosomes; 2) studies of the regulation of endocytosis, intracellular distribution and degradation of antigenic macromolecules by macrophages; especially of the effects of cyclic AMP on the vacuolar system; 3) the formation of a lysosome model (liposome) composed of defined phospholipids, cholesterol, and membrane proteins which will enclose lysozyme, and 4) the effects of lysosomal proteases upon the proteins in intact nuclei or chromatin which retard the access of RNA polymerase to DNA. Each of these studies is a natural extension of previous work and is designed to answer questions raised by these earlier observations. Together they should elucidate means whereby the vacuolar system (a term which describes the shuttle and flow of lysosomal subgroups) is regulated. Since controlled hydrolysis of intracellular and extracellular macromolecules is the means whereby the cell handles foreign materials, processes antigens, remodels itself in cell division, and may indeed renew itself, these regulatory mechanisms deserve study. We will focus on those agents that regulate the flow of intracellular macromolecules from one compartment to the other, as well as those which act on membrane elements common to all components of the vacuolar system. Each regulatory factor will also be studied in a model, artificial system, recently devised, which bears many resemblances to natural biomembranes.

Project Number :	5R01AM011949-08
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY
Project Terms :	ALLERGY AND IMMUNOLOGY STUDY SECTION, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID ADRENAL CORTEX HORMONES, CORTISONE, BLOOD CELLS, LEUKOCYTES, CELL COMPONENTS, LYSOSOMES, CHOLESTANE SERIES, CHOLESTEROL, DISEASES, PATHOLOGIC PROCESSES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, ENZYME MECHANISMS, IMMUNOLOGY, ANTIGENS BACTERIAL, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, JOINT DISORDERS, SKELETAL SYSTEM, CARTILAGE HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, MAMMALS, LAGOMORPHS, PHYSICAL SEPARATION, CENTRIFUGATION*

MECHANISMS OF ARTHRITIS

(1977)

Project Number :	5R01AM011949-09
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	ALY

LYSOSOMAL PROTEASES AND CHRONIC PULMONARY DISEASE

(1976)

Abstract :

Recently, reasonable correlations have been drawn between the clinical presence of chronic obstructive pulmonary disease and the homozygous absence of alpha 1-antitrypsin in serum. Moreover, papain and ill-defined white cell extracts have produced chronic obstructive pulmonary disease in animals. We would like to demonstrate that one, well-characterized, enzyme in leukocyte lysosomes can produce experimental chronic obstructive pulmonary disease. This study will explore whether a neutral protease of leukocyte lysosomes can provoke the lesions of chronic obstructive pulmonary disease in animals, and to study the activity of this enzyme or its inhibitor in the peripheral blood leukocytes of patients with chronic obstructive pulmonary disease. Normal human subjects will be screened for the presence of neutral protease in lysosomes of PMN's of peripheral blood. We will also establish the presence and concentration of its specific, cytoplasmic inhibitor. The strategy behind these experiments is that it is entirely possible that there exists a population of patients with chronic obstructive pulmonary disease in whom disease is not due to insufficient inhibition of leukoprotease by serum alpha l-antitrypsin but due to the lack of a cytoplasmic inhibitor of the lysosomal enzyme. Should we find that there are clinical conditions associated with absence of this intracellular inhibitor we shall do genetic studies using subjects with the deficit as probands. Another part of the protocol describes studies in rats which will be treated with leukocyte lysosomal poteases prepared from rat, human and rabbit peripheral blood leukocytes. Groups will be treated with each of these enzyme preparations before and after admixture of cytoplasmic inhibitor sufficient to neutralize the protease activity. Should it prove possible to produce histologic changes resembling chronic obstructive pulmonary disease by both papain and lysosomal proteases, it will be possible to determine whether the inhibitor administered either intertracheally or systemically has the capacity to avert these changes. The strategy behind these experiments is that it should be possible to produce changes resembling chronic obstructive pulmonary diseases by means of a purified, isolated, lysosomal protease prepared from the leukocytes of various species. Should this prove possible, then all our previous work on mechanisms of lysosomal en (Text Truncated - Exceeds Capacity)

Project Number :	5R01HL015140-03
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELL COMPONENTS, LYSOSOMES, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), METABOLIC DISORDERS INBORN, LYSOSOMAL DISORDERS, PATHOLOGY STUDY SECTION, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, BRONCHITIS CHRONIC AND EMPHYSEMA BLOOD CELLS, LEUKOCYTES, ENZYME INHIBITORS, TRYPSIN INHIBITORS, ANTITRYPSIN, ENZYME MECHANISMS, METABOLIC DISORDERS CHEMOTHERAPY, METABOLIC DISORDERS INBORN DIAGNOSIS, MODELS, BIOLOGICAL, NUCLEOTIDES, NUCLEOSIDE MONOPHOSPHATES CYCLIC, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY (GENERAL), RESPIRATORY SYSTEM, ALVEOLAR MACROPHAGES, SLOW REACTING SUBSTANCES, PROSTAGLANDINS, enzyme replacement therapy BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, FISH, ELASMOBRANCHS, HUMAN, CLINICAL, MAMMALS, CARNIVORES, DOGS*

LYSOSOMAL PROTEASES AND CHRONIC PULMONARY DISEASE

(1976)

Abstract :

Recently, reasonable correlations have been drawn between the clinical presence of chronic obstructive pulmonary disease and the homozygous absence of alpha 1-antitrypsin in serum. Moreover, papain and ill-defined white cell extracts have produced chronic obstructive pulmonary disease in animals. We would like to demonstrate that one, well-characterized, enzyme in leukocyte lysosomes can produce experimental chronic obstructive pulmonary disease. This study will explore whether a neutral protease of leukocyte lysosomes can provoke the lesions of chronic obstructive pulmonary disease in animals, and to study the activity of this enzyme or its inhibitor in the peripheral blood leukocytes of patients with chronic obstructive pulmonary disease. Normal human subjects will be screened for the presence of neutral protease in lysosomes of PMN's of peripheral blood. We will also establish the presence and concentration of its specific, cytoplasmic inhibitor. The strategy behing these experiments is that it is entirely possible that there exists a population of patients with chronic obstructive pulmonary disease in whom disease is not due to insufficient inhibition of leukoprotease by serum alpha l-antitrypsin but due to the lack of a cytoplasmic inhibitor of the lysosomal enzyme. Should we find that there are clinical conditions associated with absence of this intracellular inhibitor we shall do genetic studies using subjects with the deficit as probands. Another part of the protocol describes studies in rats which will be treated with leukocyte lysosomal poteases prepared from rat, human and rabbit peripheral blood leukocytes. Groups will be treated with each of these enzyme preparations beforeand after admixtureof cytoplasmic inhibitor sufficient to neutralize the protease activity. Should it prove possible to produce histologic changes resembling chronic obstructive pulmonary disease by both paPain and lysosomal proteases, it will be possible to determine whether the inhibitor administered either intertracheally or systemically has the capacity to avert these changes. The strategy behind these experiments is that it should be possible to produce changes resembling chronic obstructive pulmonary diseases by means of a purified, isolated, lysosomal protease prepared from the leukocytes of various species. Should this prove possible, then all our previous work on mechanisms of lysosomal en (Text Truncated - Exceeds Capacity)

Project Number :	5R01HL015140-02
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA
Project Terms :	CELL COMPONENTS, LYSOSOMES, ENZYME INHIBITORS, PROTEASE INHIBITORS (GENERAL), METABOLIC DISORDERS INBORN, LYSOSOMAL DISORDERS, PATHOLOGY STUDY SECTION, PROTEASES AND PEPTIDASES, RESPIRATORY DISORDERS, BRONCHITIS CHRONIC AND EMPHYSEMA BLOOD CELLS, LEUKOCYTES, GENETICS, POPULATION GENETICS HUMAN, METABOLIC DISORDERS INBORN DIAGNOSIS, MODELS, BIOLOGICAL, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY (GENERAL) BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, HUMAN, CLINICAL, MAMMALS, LAGOMORPHS, MAMMALS, RATS*

LYSOSOMAL PROTEASES AND CHRONIC PULMONARY DISEASE

(1976)

Project Number :	1R01HL015140-01A1
ICD :	NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG :	PTHA

MECHANISMS OF ARTHRITIS

(1972)

Project Number :	5R01AM011949-05
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	GMA
Project Terms :	ADRENAL CORTEX HORMONES, CORTISONE, CELL COMPONENTS, LYSOSOMES, ENZYME MECHANISMS, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, MUCOPROTEINS, QUINOLINES, CHLOROQUINE, SKELETAL DISORDERS, ARTHRITIS, OSTEOARTHRITIS, SKELETAL DISORDERS, ARTHRITIS, RHEUMATOID, SKELETAL DISORDERS, JOINT DISORDERS BLOOD CELLS, LEUKOCYTES, CHOLESTANE SERIES, CHOLESTEROL, GENERAL MEDICINE STUDY SECTION, IMMUNOLOGY, ANTIGENS BACTERIAL, INJURIES, INFLAMMATION, ANTIINFLAMMATORY AGENTS, MODELS, BIOLOGICAL, NUCLEOTIDYLTRANSFERASES, RNA NUCLEOTIDYLTRANSFERASES, PHOSPHOLIPIDS, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, ADENOSINE MONOPHOSPHATE CYCLIC, SKELETAL SYSTEM, CARTILAGE, THOROTRAST HUMAN, NONCLINICAL, MAMMALS, LAGOMORPHS, PHYSICAL SEPARATION, CENTRIFUGATION*

MECHANISMS OF ARTHRITIS

(1972)

Project Number :	3R01AM011949-05S1
ICD :	NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & KIDNEY DISEASES
IRG :	GMA

NEW YORK UNIVERSITY

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