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Studies on the Role of Interleukin-2 in the Management of HIV Infection
The Characterization of Pneumocystis carinii Surface Antigens
A Comparison of Two Strains of E. coli to Produce Septic Shock in Dogs
Investigations of New Therapies in Septic Shock
Effect of Nitric Oxide Synthase Inhibitors In Vivo Tumor Necrosis Factor-induced Myocardial Depression
Effect of Reconstituted High-density Lipoproteins in a Canine Model of Septic Shock
Characterization of Cytokine Responses During Pneumocystis carinii Pneumonia
Study of Control of Cytosolic Phospholipase A2 Gene Expression in Airway Epithelial Cells
Study of Salvage Regimens for Patients with HIV Infection Demonstrating Virologic Failure with Licensed Protease Inhibitors
A Controlled Trial of Tyrosine Kinase Inhibitors in a Canine Model of Septic Shock
Retrospective Assessment of Pulmonary Infection in Patients with Chronic Granulomatous Disease
Inflammatory Responses to Bronchial Endotoxin Instillation in Humans
Study of Control of Cytosolic Phospholipase A2 Activity
Phase I-II Trial of a Genetic Vaccine for HIV Infection
The Influence of Previous Infection on the Host Defense Effects of Granulocyte Colony-stimulating Factor in a Rat Model of Sepsis
Reactive Oxygen Species Signaling by Endothelial Nitric Oxide Synthase
Study to Assess the Utility of Oral Washes to Diagnose Pneumocystis
Physiologic Effects of Inhaled Nitric Oxide, Nitroglycerin, and Placebo in Study Subjects with Sickle Cell Anemia
Studies of Lymphocyte Kinetics in Healthy and HIV-infected Patients
Molecular Studies of Human Pneumocystis carinii
The Influence of Fluid Administration on the Effects of Tumor Necrosis Factor Soluble Receptor in Gram-negative Sepsis
Tyrphostin AG 556 Therapy Adjusted to Severity of Illness of New Therapies in Septic Shock
Effects of L-Arginine on Endotoxin-mediated Inflammatory Responses
Effects of Inhaled Nitric Oxide on Pulmonary Inflammatory Responses
Role of Nitric Oxide in Regulating Inflammation and Gene Expression
Magnetic Resonance Imagery Study of Avascular Necrosis of the Hip in Asymptomatic HIV-infected Patients
Study of the Control of p11 Protein Production
Endothelial Cell Response to Oxidative Stress
The Functional Genomics of Critical Illness
The Influence of Site, Severity, and Type of Infection on the Effects of an Endotoxin Analogue in Sepsis
The Effects of Tumor Necrosis Factor Monoclonal Antibodies in Wild Type and Tumor Necrosis Factor Knock Out Mice
The Influence of Site, Severity, and Type of Infection on the Effects of a Tyrosine Kinase Inhibitor in Sepsis
Gene Expression in Humans Challenged with Endotoxin
Functional Gene Expression in Humans Challenged with Endotoxin
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00036-13 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Studies on the Role of Interleukin-2 in the Management of HIV Infection
Principal Investigator:
J.A. Kovacs, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
H. Masur, M.D., CCMD
D. Chaitt, CCMD
Collaborating Unit:
LIR, NIAID (H.C. Lane, M.D.; M. Polis, M.D.; J. Falloon, M.D.; R. Davey, M.D.;
J. Tavel, M.D.; S. Vogel)
Staff-Years:
1.0
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Interleukin-2 (IL-2) is a cytokine with important regulatory properties for both T and B cells. The current studies were undertaken to evaluate IL-2 in the treatment of HIV infection. Our studies initially focused on patients with CD4 counts about 200 cells/mm3, administering IL-2 for 5 days approximately every 2 months at doses ranging from 6 to 18 million units/d.
The courses of IL-2 were well-tolerated, although most of the patients required dosage reductions due to IL-2 related adverse effects. Sustained improvement in CD4 number was seen primarily in patients with greater than 200 CD4 cells/mm3. There also was a transient increase in viral load as measured by the bDNA assay seen at day 6 to day 8 following initiation of IL-2 therapy. Responses in CD4 number were less common in patients with lower baseline CD4 counts.
Based on the preliminary results seen in our open trial, we undertook a randomized trial to evaluate IL-2 therapy in patients with CD4 counts above 200 cells/mm in combination with currently approved antiretroviral therapies. The study opened in April 1993 and was completed in February of 1995, with 60 patients enrolling. This study also showed in a controlled setting that intermittent therapy with IL-2 can lead to a substantial and sustained increase in CD4 cell counts without leading to an increase in plasma viral load. More recently, we have focused on improving the tolerance of IL-2, by decreasing the dose and duration of therapy, and by evaluating alternative methods of administering IL-2. We had enrolled patients in an extension phase of ongoing studies to determine whether administration of corticosteroids with IL-2 can lead to improved tolerance of IL-2 without interfering with the immunomodulatory effects. This phase has been discontinued due to the occurrence of avascular necrosis of the hip in some patients receiving prednisone. These studies are potentially important because they are the first ones to suggest that immunomodulating agents combined with antiretroviral agents may have a benefit in patients with HIV infection.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00037-13 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
The Characterization of Pneumocystis carinii Surface Antigens
Principal Investigator:
J.A. Kovacs, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
L. Bishop, CCMD
G. Kutty, CCMD
Collaborating Unit:
None
Staff-Years:
1.0
Human Subjects:
(a) Human subjects x (b) Human Tissue (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Pneumocystis carinii is a major opportunistic pathogen of immunocompromised patients. Because P. carinii cannot be reliably cultured, molecular approaches have been utilized to identify and characterize antigens of this organism. Recombinant antigens can then be used to examine host immune responses to P. carinii infections. We have an ongoing project to characterize the antigens of both rat and human P. carinii. We have previously purified the major surface glycoprotein (MSG) of both rat and human pneumocystis using HPLC. It is necessary to use P. carinii from both sources because antigenically they are different and specifically the major surface antigen in rat and human P. carinii are clearly different. Subsequently, we identified a number of clones from a cDNA library of rat P. carinii that contain genes encoding for the MSG. These clones are clearly related but not identical, demonstrating that multiple genes encode the MSG. We have continued studies to characterize potential antigens of P. carinii genes. We have cloned a number of human P. carinii MSG genes and have produced a recombinant fragment encoding a highly conserved region of human P. carinii MSGs that can be expressed at high levels. Preliminary studies have demonstrated that most humans have antibodies to the recombinant peptide, supporting the hypothesis that most humans have been previously infected with P. carinii. Over the past year we have expressed a full-length MSG in two fragments. We are currently attempting to purify these fragments, and will then use them in ELISA assays. We ultimately plan to use this panel of recombinant peptides to examine cellular and antibody responses to P. carinii in healthy and immunocompromised patients. In addition, we have begun studies to characterize other P. carinii antigens. We have identified two families of genes related to the MSG whose expression is regulated differently from MSG genes. We have also identified a family of genes in rat P. carinii, encoding proteases related to the yeast kexin, which functions as a pro-protein processing enzyme. This protease appears to be expressed on the surface of rat P. carinii. The goal of this study is to better understand the pathogenesis of P. carinii pneumonia with the hope that we can use this information to control or prevent this disease.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00045-12 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
A Comparison of Two Strains of E. coli to Produce Septic Shock in Dogs
Principal Investigator:
C. Natanson, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Banks, Ph.D., CCMD
S. Solomon, Ph.D., CCMD
Collaborating Units:
LCI, NIAID (T.A. Russo, M.D.)
NIAID (D.W. Alling, M.D., Ph.D.)
Univ. of Buffalo
Staff-Years:
2.5
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: This project has been completed and a paper published in the Journal of Infectious Diseases.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00073-12 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Investigations of New Therapies in Septic Shock
Principal Investigator:
C. Natanson, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
A. Hilton, CCMD
S. Richmond, CCMD
M. Fernandez, CCMD
S. Solomon, Ph.D., CCMD
S. Banks Ph.D., CCMD
P. Eichacker, M.D., CCMD
Collaborating Unit:
None
Staff-Years:
1.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Septic shock and related sequelae of infection (e.g., multiple organ system failure) are the most common cause of death in intensive care units. Deaths due to sepsis can occur in previously healthy individuals, in all age groups, and in a variety of common clinical settings. Some common predisposing conditions are premature neonates, previously healthy children with acquired infections (e.g., meningitis, pneumonia, upper respiratory infections), teenagers or young adults with trauma or cancer, and elderly patients with pneumonia or gall bladder disease. Half of all children or adults who acquire this septic shock die from the syndrome. Thus septic shock, which affects young children and the elderly alike (even those without predisposing illness), is a common and important clinical problem with substantial mortality and produces an great financial burden on society. Surprisingly little is known about the pathophysiology of this disease infection (organism virulence factors and toxins) and factors related to the host response (endogenous molecules that affect and modulate the inflammatory response). Thus successful treatment of the septic shock syndrome which reduces morbidity and mortality will result from curing the infection and interrupting the effects of these organism and host mediators. Using purpose bred beagles, the canine model of septic shock has successfully provided information on the pathophysiology and treatment of human disease. This model of acute and chronic infection simulates the course and cardiovascular changes seen routinely in children and adults with septic shock. Prior experiments using the model have established the role of specific bacteria (gram positive and gram negative), bacterial toxins (endotoxin), and host mediators to produce septic shock. Thus, the canine model has been highly successful in simulating the human disease and guiding therapy for humans. There are several therapies under investigation that might be effective in human septic shock. The canine model, which simulates the cardiovascular changes seen in children and adult humans with septic shock is ideally suited for pre-clinical trials of these new therapies. The canine model allows properly controlled trials to evaluate therapeutic mechanisms and adverse effects of therapies; that is not always possible in human studies. We are evaluating or will evaluate the following therapies for septic shock in the canine model: superoxide dismutase, Tyrosine kinase inhibitors, endotoxin precursors, bradykinin antagonists, dantrolene, soluble tumor necrosis factor receptor, antibodies to CD-18 receptors on white blood cells; ibuprofen; antibody to human tumor necrosis factor receptor, granulocyte stimulating factor; antibodies to platelet activating factor; antibodies to protein C; continuous arteriovenous hemofiltration; high-density lipoproteins; platelet activating-factor inhibitor.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00133-07 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Effect of Nitric Oxide Synthase Inhibitors In Vivo Tumor Necrosis Factor-induced
Myocardial Depression
Principal Investigator:
C.N. Natanson, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
A. Hilton, CCMD
S. Richmond, CCMD
M. Fernandez, CCMD
S. Solomon, Ph.D., CCMD
S. Banks Ph.D., CCMD
R.L. Danner M.D., CCMD
P.Q. Eichacker, M.D., CCMD
Collaborating Unit:
None
Staff-Years:
1.5
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The present investigation has been undertaken to determine, in vivo, if nitric oxide is responsible for cytokine-induced myocardial depression. The negative ionotropic effects of cytokines on the heart are believed to be mediated by nitric oxide, based on in vitro data. In isolated hamster cardiac papillary muscle, this negative ionotropic effect of cytokines can be blocked by N-G monomethyl-L-arginine (NMA), a nitric oxide synthase inhibitor. Because the in vitro data demonstrates that nitric oxide synthase inhibitors prevented tumor necrosis factor (TNF)-induced myocardial depression of rapid onset and reversal, we studied a low dose of recombinant human TNF challenge in canines. This TNF challenge produces significant, early, and short-lived myocardial depression (resolved by 24 h). Surprisingly, we found that NMA did not prevent the early (up to 6h) deleterious effects of TNF on cardiac function. In fact, during this time period, TNF and NMA effects on all cardiac and hemodynamic parameters were additive (i.e., NMA did not block TNF effects). However, 24 h after TNF infusion, NMA did ameliorate the effects of TNF on some parameters such as acid-base derangements and decreases in mean arterial pressure and systemic vascular resistance. These data suggest that the early phase of TNF-induced cardiac and vascular abnormalities may not be related to nitric oxide production. However, some of the later effects of TNF may be related to the production of nitric oxide. Given the suggestive finding of a beneficial effect of NMA 24 h post TNF infusion, we evaluated the effects of nitric oxide inhibition in the setting of higher doses of TNF causing longer-lasting myocardial depression. Previous experiments using TNF challenges in canines suggest that this is a reasonable hypothesis, i.e., there may be two phases of cardiac injury. In canines, there is an early (less than 8 h), dose-independent mechanism of myocardial depression and a late (greater than 24 h), dose-dependent mechanism of myocardial depression. It was hypothesized that the inhibition of nitric oxide synthesis might not be advantageous early when myocardial depression is dose dependent. Therefore, we studied both prophylactic treatment (pretreatment) or therapeutic treatment with NMA (post-treatment after TNF challenge examining both early- and late-time points). Treatment with NMA lowered measures of nitric oxide production in both early and late-time points. At early-time points, NMA given either therapeutically or prophylactically did not prevent the adverse affects of TNF. However at 24 hour, after reversal of the NMA with l-arginine, the natural substrate for nitric oxide production, prophylactic NMA ameliorated the decline in cardiac function seen with TNF challenge. These data suggest a dual effect of TNF on cardiac function. The early effect appears to be nitric oxide independent, while the later effect appears to be nitric oxide dependent. In future studies, we plan to confirm these findings in actual bacterial infection-induced myocardial depression models. NMA is being used with cytokine therapy for cancer patients to inhibit their cardiovascular toxicity and studies are planned in AIDS patients to do the same. These studies will help determine the advisability of this approach.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00134-07 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Effect of Reconstituted High-density Lipoproteins in a Canine Model of Septic
Shock
Principal Investigator:
C. Natanson, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
R.L. Danner, M.D., CCMD
P.Q. Eichacker, M.D., CCMD
J. Sevransky, M.D., CCMD
Collaborating Units:
The Rogosin Institute
The New York Hospital-Cornell Medical Center
Staff-Years:
2.5
Human Subjects:
(a) Human subjects (b) Human Tissue x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: This project has been completed and a manuscript has been published in the Journal of Pharmacology and Experimental Therapeutics 1999;288(1):107-113.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00146-07 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Characterization of Cytokine Responses During Pneumocystis carinii Pneumonia
Principal Investigator:
J.A. Kovacs, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
V. Vestereng, M.D., CCMD
Collaborating Unit:
LIR, NIAID (I. Sereti, M.D.)
Staff-Years:
0.5
Human Subjects:
(a) Human subjects (b) Human Tissue x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Pneumocystis carinii is a major pathogen of patients with HIV infection. The immune responses to P. carinii are poorly understood, but cytokines may play a role in both clearing P. carinii infection and in the hypoxia associated with P. carinii pneumonia that may be exacerbated following initiation of therapy. We are using the scid mouse model, as well as other immunodeficient mice, to further evaluate the role of individual cytokines and other immunoregulatory molecules in modulating P. carinii infection. We are in the process of developing techniques that will allow assessment of which cytokines are produced in response to P. carinii antigens. It is hoped that these studies will provide insights into the role of cytokines in P. carinii pneumonia, and may provide mechanisms for increasing clearance of P. carinii or decreasing the inflammation that may be causing hypoxia.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00149-07 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Study of Control of Cytosolic Phospholipase A2 Gene Expression in Airway Epithelial
Cells
Principal Investigator:
J.H. Shelhamer, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
M. Cowan, M.D., CCMD
M. Lawrence, M.S., CCMD
R. Pawliczak, M.D., Ph.D., CCMD
Collaborating Unit:
None
Staff-Years:
2.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The 5' promoter region of the cytosolic phospholipase A2 (cPLA2) gene has been cloned and sequenced. The promoter for the cPLA2 gene does not have a TATA box but is inducible. Reporter genes with inserts extending from the 5' portion of the promoter region to the first intron have been made and reporter genes with mutations in a putative initiator region have been utilized to characterize the control mechanisms important in expression of this gene. Sequences important in control of transcription have been identified. A minimal promoter sequence has been identified. Nucleotides within the initiator region which are critical to basal transcription are under study. An initiator element at the transcription start site is critical for initiation of transcription. Further, a sequence of nucleotides 30-36 bases 5' to the transcription start site is critical to the initiation function. Two series of nucleotide repeats have also been identified. These repeats appear to act to down- regulate basal transcription rates.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00155-05 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Study of Salvage Regimens for Patients with HIV Infection Demonstrating Virologic
Failure with Licensed Protease Inhibitors
Principal Investigator:
H. Masur, M.D., Chief (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Unit:
LIR, NIAID (J. Falloon, M.D.; H.C. Lane, M.D.; S. Vogel, R.N.)
Staff-Years:
1.0
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Highly active antiretroviral therapy has revolutionized the therapy of HIV disease, yet a substantial fraction of patients either never respond, or lose virologic control during the first 3 years of therapy. In this study, patients who have failed HAART were treated with three agents. The study had two major goals: (1) to determine the response rate to these agents, and (2) to determine what clinical and laboratory parameters, especially genotypic and phenotypic analysis, are useful for predicting response. Accrual of 98 patients has been completed; all have reached 48 weeks of followup. Results show that phenotypic assays are useful for predicting response to salvage regimens, and that toxicity is common, especially for patients with high viral loads. Genotypic assays were also useful for predicting response, but their interpretation is much more complex than for phenotypic assays. A final manuscript is being prepared.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00164-05 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
A Controlled Trial of Tyrosine Kinase Inhibitors in a Canine Model of Septic
Shock
Principal Investigator:
C. Natanson, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
R.L. Danner, M.D., CCMD
P.Q. Eichacker, M.D., CCMD
M. Solomon, M.D., CCMD
S. Solomon, Ph.D., CCMD
Collaborating Unit:
None
Staff-Years:
1.0
Human Subjects:
(a) Human Subject
(b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Septic shock appears to result from excessive release of cytokines [e.g., tumor necrosis factor-a (TNF-a), IL-2, etc.] and other pro-inflammatory substances [e.g., nitric oxide (NO)] from cells of the monocyte/macrophage lineage in response to infection or lipopolysaccharide (LPS) administration. The production of these cytokines, as well as their action, is mediated by signal transduction events which induce protein tyrosine phosphorylation. Theoretically, inhibition of protein tyrosine phosphorylation may be beneficial in sepsis. These compounds would block the potentially high cytokine production which is dependent on tyrosine phosphorylation. These protein kinase inhibitors would block both activation or production of cytokinase by bacterial products and the effects of cytokines on target cells. Tyrphostins AG 126 and AG 556 are both protein kinase inhibitors and have been shown to improve outcome in small animal models during both LPS and live bacterial challenge. Further, both AG 126 and AG 556 have been shown to inhibit LPS-induced TNF production from dog peripheral blood mononuclear cells, in vitro. Studies in large animal model are needed before we can begin human clinical trials to establish efficacy and safety of these compounds. In collaboration with Dr. Novogrodsky and his colleagues, we evaluated AG 126 and AG 556 in our canine peritonitis model. In a controlled clinical trial in 100 animals over 6 months, AG 556 but not AG 126 significantly improved survival and prevented multiorgan failure during canine septic shock. This therapeutic agent (AG556) is proceeding to human clinical trials.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00165-06 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Retrospective Assessment of Pulmonary Infection in Patients with Chronic Granulomatous
Disease
Principal Investigator:
F.P. Ognibene, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
J. Gallin, M.D., OD
M. Gladwin, M.D., CCMD
A. Slonim, M.D., CCMD
Collaborating Unit:
NIAID (S. Holland, M.D.; E. DeCarlo, R.N.)
Staff-Years:
0.2
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Patients with chronic granulomatous disease (CGD) frequently develop pulmonary infections. In many patients with CGD, an accurate diagnosis is difficult to establish due to the scarcity of organisms in pathologic specimens. This study assesses, in a retrospective manner, all procedural as well as diagnostic data in patients with CGD and pulmonary disease. The study assesses the utility of sputum, bronchoscopy (lavage and transbronchial biopsy), CTT-guided transthoracic needle aspiration, and open-lung biopsy in establishing either a definitive or presumptive pulmonary diagnosis.
Data collection for the research project has been completed. The data are now being entered into a database in order to facilitate manipulation and analysis. There are a total of 50 patients for whom the clinical characteristics, radiologic, microbiologic, and cytopathologic results of pulmonary infections are available. To date, the microbiologic and radiologic data have been catalogued. Over 600 microbiologic procedures and 1,800 radiologic procedures have been entered. At the completion of the project, a diagnostic, evidenced-based algorithm will be available to aid in the diagnosis of pulmonary infections in patients with CGD.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00171-05 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Inflammatory Responses to Bronchial Endotoxin Instillation in Humans
Principal Investigator:
A.F. Suffredini, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
R.L. Danner, M.D., CCMD
J.H. Shelhamer, M.D., CCMD
M. Tropea, CCMD
H. Preas, M.D., ANES
N. O'Grady, M.D., CCMD
Collaborating Units:
DCS, NCI (A. Filie, M.D.)
Temple Univ. (R. DeLaCadena, M.D.)
Univ. Hospital Geneva (J. Pugin, M.D.)
Staff-Years:
1.75
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Administration of endotoxin to humans allows a unique way to evaluate the early inflammatory reactions that occur during infection. Characterizing these responses and the mechanisms that control them is important because these inflammatory responses contribute to the development of septic shock and organ failure.
Under protocol 92-CC-0141, the effects of direct instillation of endotoxin into lung subsegments will be evaluated. Sequential bronchoalveolar lavage will be performed at 2, 6, 24, 48, or 72 hours after endotoxin instillation. Analyses will include the following: bronchoalveolar lavage for acute phase cytokines; flow cytometry of neutrophils and lymphocyte subpopulations; and systemic and inflammatory effects including acute phase cytokine release, recruitment of cells from the marrow and the initiation of acute phase protein release. An in vitro bilayer model of the alveolar blood interface has been designed to facilitate discovery of mechanisms that recruit inflammatory cells to the lung. These observations will be useful in defining important events in the initiation and resolution of acute lung inflammation to bacterial endotoxin.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00182-05 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Study of Control of Cytosolic Phospholipase A2 Activity
Principal Investigator:
J.H. Shelhamer, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Logun, M.S., CCMD
X. Huang, M.D., CCMD
Collaborating Unit:
None
Staff-Years:
1.0
Human Subject:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The activity of cytosolic phospholipase A2 (cPLA2) may be altered by calcium or by phosphorylation of serines in the cPLA2 molecule. A dual hybridization system in yeast was used to identify protein-protein interactions which might also be involved in the modulation of cPLA2 activity. Using this system, a member of the S-100 family of protein was identified as interacting with cPLA2. Ongoing studies include production of recombinant protein and modulation of enzyme function by oxidant molecules.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00183-05 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Phase I-II Trial of a Genetic Vaccine for HIV Infection
Principal Investigator:
J.A. Kovacs, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
H. Masur, M.D., CCMD
G. Kelly, R.N., CCMD
Collaborating Units:
LIR, NIAID (H.C. Lane, M.D.; M. Polis, M.D.; J. Falloon, M.D.; R. Davey, M.D.;
J. Tavel, M.D.)
Dept. of Pathology, Univ. of Pennsylvania (D. Weiner, Ph.D.)
Wyeth-Lederle Vaccines (R. Ginsberg, M.D.)
Staff-Years:
0.5
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Development of a vaccine able to prevent HIV infection would play a major role in managing the spread of HIV. A recently developed class of vaccines that use DNA as the immunizing agent offers a promising approach because genetic vaccination in animal models results in a broader immune response, including the induction of cytotoxic T-cell responses, than protein vaccines can achieve. Apollon, Inc. (now part of Wyeth Ayerst Vaccines) has developed a genetic vaccine encoding the envelope protein of HIV-1 that has been shown to be immunogenic in animal studies and has undergone early evaluation as an immunomodulating agent in HIV-infected patients. In collaboration with Apollon, Inc., we have undertaken a study in up to 41 individuals not infected with HIV. The study will involve administration of four doses of the genetic vaccine or a similarly constituted vehicle control over a 6-month period, with a followup of 6 additional months. Four dose levels of the vaccine will be used. To date, 27 volunteers have enrolled in the study and immunizations have been well tolerated. The study will focus on evaluating the safety and immunogenicity of this candidate vaccine, including its ability to induce cytotoxic T-cell responses.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00184-04 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
The Influence of Previous Infection on the Host Defense Effects of Granulocyte
Colony-stimulating Factor in a Rat Model of Sepsis
Principal Investigator:
P.Q. Eichacker, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Parent, D.V.M., CCMD
R. Danner, M.D., CCMD
Collaborating Unit:
Amgen
Staff-Years:
2.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Studies suggest that the production of endogenous anti-inflammatory agents during sepsis reduces host defense and predisposes to subsequent infection. Whether such a state of immunosuppression during sepsis actually exists or is reversible with the administration of proinflammatory agents is unclear. However, use of recombinant granulocyte colony-stimulating factor (G-CSF) to augment host defense following the onset of sepsis in critically ill patients has been proposed. Using a rat model, we plan to determine whether an initial episode of infection and sepsis augments the host defense effects of G-CSF during a subsequent episode.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00188-03 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Reactive Oxygen Species Signaling by Endothelial Nitric Oxide Synthase
Principal Investigator:
R.L. Danner, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
W. Wang, Ph.D., CCMD
S. Wang, M.D., CCMD
E. Nishanian, M.D., CCMD
A.D.P. Cintron, M.T (ASCP), CCMD
Collaborating Unit:
None
Staff-Years:
4.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Nitric oxide synthases (NOS), the enzymes responsible for nitric oxide (NO) production from the substrate L-arginine, are also NADPH oxidases. In cell-free systems, some of these enzymes have been shown to produce reactive oxygen species such as superoxide. In this investigation, we transfected monoblastoid U937 cells with human endothelial NOS (eNOS) and found that TNFa production was increased, but that this effect was not related to NO production. Further work found that eNOS upregulates TNFa by producing a reactive oxygen species (ROS), (J Biol Chem, 2000). Recent experiments have demonstrated that eNOS upregulation of TNFa occurs through superoxide-dependent activation of p44/42 mitogen activated protein kinase (Abstract, FASEB J, 1999). Future work will focus on the switching mechanism(s) that regulate eNOS to produce either NO or ROS. A closely related effort will examine eNOS modulation of inflammatory responses in endothelial cells and the relative roles played by NO and ROS.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00189-03 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Study to Assess the Utility of Oral Washes to Diagnose Pneumocystis
Principal Investigator:
H. Masur, M.D., Chief (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
J. Kovacs, M.D., CCMD
S. Huang, M.D., CCMD
S. Fischer, M.D., CP
V. Gill, Ph.D., CP
J. Parker, CP
Collaborating Units:
LIR, NIAID (H.C. Lane, M.D.)
Johns Hopkins Univ. (J. Maenza, M.D.; R. Brower, M.D.)
Washington Hospital Center (D. Lucey, M.D.)
Staff-Years:
1.0
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
x (a1) Minors (a2) Interviews
Summary of Work: This study is part of a 15-year project to develop less invasive methods to diagnose pneumocystis pneumonia and to predict responses to therapy. Oral washes, induced sputum, and bronchalveolar lavage are being collected from patients with immunosuppressive diseases and respiratory syndromes. Samples for control patients are being collected as well.
First, a polymerase chain reaction (PCR) technique using a unique major surface glycoprotein primer is being used in conjunction with a published primer to develop a method adaptable to clinical laboratories that is highly specific and sensitive. It is hoped that oral wash can replace sputum as the sample of choice. Second, mutations associated with drug resistance are being assessed in all organisms identified to determine the epidemiology and clinical importance of such mutations. Third, markers of strain variation are being assessed to elucidate pathogen epidemiology.
Specimens are being obtained from NIH and the Washington Hospital Center. Results from 30 patients with morphologic evidence of PCP, and 250 smear-negative immunosuppressed patients show that the oral wash has a 70 percent positive-predictive value and a 99 percent negative-predictive value. The oral wash, tested by PCR, is a useful screening test for PCP. A quantitative assay is being developed to increase the positive-predictive value of this test.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00190-03 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Physiologic Effects of Inhaled Nitric Oxide, Nitroglycerin, and Placebo in Study
Subjects with Sickle Cell Anemia
Principal Investigator:
M. Gladwin, M.D. (Senior Staff Fellow)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
F.P. Ognibene, M.D. (Medically Responsible Investigator), CCMD
J.H. Shelhamer, M.D., CCMD
Collaborating Units:
NIDDK (A.N. Schechter, M.D.; G.P. Rodgers, M.D.; C.T. Noguchi, Ph.D.)
NHLBI (A.H. Aletras, Ph.D.; R.S. Balaban, Ph.D.; R. Cannon, M.D.; A.E. Arai,
M.D.; C.A. Combs, Ph.D.; B. Hrinczenko, M.D.)
Staff-Years:
0.5
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Sickle cell anemia is an autosomal recessive disorder and the most common genetic disease affecting African Americans. Approximately 0.15 percent of African Americans are homozygous for sickle cell disease, and 8 percent have sickle cell trait. Acute pain crisis and acute chest syndrome (ACS) are common complications of sickle cell anemia. Inhaled nitric oxide (NO) has been proposed as a possible therapy for ACS. Anecdotally, NO has been described to rapidly improve the hypoxemia and the clinical course of ACS. Furthermore, a number of recent studies have suggested that NO may have a favorable impact on sickle hemoglobin at the molecular level and could improve the abnormal microvascular perfusion that is characteristic of sickle cell anemia.
This clinical trial was designed to evaluate the physiologic and molecular effects of inhaled NO and a currently available, safe, FDA-approved medication, nitroglycerin, that is a nitric oxide donor (i.e., a source of NO after metabolism in the body), in study subjects with and without sickle cell anemia. Whole blood was analyzed to characterize the metabolism of NO and NO donors, the molecular interactions between hemoglobin and NO, the duration of effect of these therapies on hemoglobin oxygen affinity and other properties of the erythrocyte and intracellular hemoglobin (including the solubility of deoxy sickle hemoglobin).
We found that during NO inhalation at 80 ppm, NO binds to the heme of hemoglobin and is delivered to the peripheral circulation. The amount delivered is sufficient to restore regional blood flow to the forearm during NO synthase inhibition (measured by strain-gauge plethysmography). This may prove an effective therapy to increase regional blood flow during sickle cell pain crisis and after vascular procedures such as angioplasty.
We also characterized the effect of NO delivery on microvascular perfusion in study subjects with and without sickle cell anemia by magnetic resonance imaging (MRI) of lower extremity skeletal muscle enhancement during first passage of intravenously injected gadolinium contrast. Perfusion measurements were paired with 31-phosphorus magnetic resonance spectroscopy (31-P-MRS) study of the concentration of muscle high-energy phosphate compounds. We were unable to appreciate changes in blood flow in our pilot study using this imaging modality.
This ongoing project will allow three major assessments: (1) the characterization of the microvascular perfusion at rest and during exercise in study subjects with sickle cell anemia, (2) the effects of NO on red cell and hemoglobin function and skeletal muscle perfusion in normal study subjects (without sickle cell anemia), and finally, (3) the effects of NO on red cell and hemoglobin function and skeletal muscle perfusion in study subjects with sickle cell anemia. Our hypothesis is that one or more of these effects could be of potential therapeutic benefit to sickle cell anemia patients.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00191-03 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Studies of Lymphocyte Kinetics in Healthy and HIV-infected Patients
Principal Investigator:
J.A. Kovacs, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
H. Masur, M.D., CCMD
G. Kelly, CCMD
Collaborating Units:
LIR, NIAID (H.C. Lane, M.D.; R. Davey, M.D.; J. Falloon, M.D.; M. Polis, M.D.;
J. Tavel, M.D.; B. Herpin)
SAIC (R. Lempicki, Ph.D.; M. Baseler, Ph.D.; J. Aldesberger)
Staff-Years:
0.5
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Understanding the rate of lymphocyte replication and destruction in HIV-infected patients, as well as the effects of therapy on lymphocyte replication should lead to a better understanding of the mechanisms behind the immunodeficiency induced by HIV. Little is known about the replication rate in healthy and HIV-infected patients. Two approaches are being used to address this issue. (1) Healthy and HIV-infected patients will receive up to 5 days of continuous infusions with [6,6-2H2]-glucose, a nonradioactive, stable isotope of glucose that is safe to administer. The deuterium is incorporated into DNA via metabolism of glucose to ribose and incorporation into nucleotides. The rate of incorporation can be measured in subpopulations of cells to determine the rate of replication of those cells, and the rate of loss of the incorporated deuterium can be used to examine the turnover rate of the replicated cells. (2) Administering bromodeoxyuridine (BrDU; 200 mg/m2), an analogue of thymidine, to HIV-infected patients. BrDU is incorporated into DNA and incorporation can be measured using an anti-BrDU monoclonal antibody. By FACS analysis, both surface markers and BrDU can be measured. Thus, FACS analysis can be used to directly measure subpopulations of cells that have replicated. To date, 25 patients have been enrolled in these studies. Techniques for measuring incorporation have been developed and validated for both methods. Studies are ongoing to evaluate lymphocyte replication in various populations. These two approaches complement each other and should provide information about lymphocyte kinetics that will have relevance to HIV infection and other disease states.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00192-03 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Molecular Studies of Human Pneumocystis Carinii
Principal Investigator:
J.A. Kovacs, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
M. Liang, M.D., CCMD
G. Kutty, CCMD
Collaborating Unit:
CP, CC (V. Gill, Ph.D.; M. Larsen, M.D.)
Staff-Years:
1.5
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Pneumocystis carinii infections remain common in HIV-infected patients despite the broad use of highly active antiretroviral therapies and prophylactic regimens. Studies of human P. carinii are focusing on two areas: diagnosis and evaluation for potential resistance to therapy. To try to develop highly sensitive, non-invasive diagnostic methods, we have been evaluating polymerase chain reaction (PCR) using primers based on the major surface glycoprotein (MSG) genes of human P. carinii. This is a family of genes that are closely related and that encode an important surface protein of P. carinii. PCR using primers based on this gene is potentially a highly sensitive method since this is a multicopy gene (estimated at greater than 100 copies/genome). We have been evaluating the diagnostic potential using a conserved region of the gene family. Our studies have shown that the sensitivity of MSG-based primers is greater than that of previously utilized primers. We are currently evaluating these primers prospectively in collaboration with the Microbiology Department. Because human P. carinii cannot be cultured, we cannot directly determine if resistance to commonly used therapeutic agents is developing. However, molecular techniques can be used to identify mutations that may confer resistance in genes that are targets of therapeutic agents. The most commonly used agent to treat P. carinii pneumonia is the combination of trimethoprim, which targets dihydrofolate reductase (DHFR), and sulfamethoxazole, which targets dihydropteroate synthase (DHPS). We have cloned the human P. carinii DHFR gene, and have examined (by PCR and sequencing) the P. carinii DHFR and DHPS genes of a variety of human isolates from patients with P. carinii pneumonia. DHPS mutations were found in about one-third of patients, while no mutations have been found to date in the DHFR gene. We have also expressed recombinant human P. carinii DHFR and characterized the kinetics of this enzyme. These studies should provide improved diagnostic methods for PCP and insights into the reasons for therapy or prophylaxis failures.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00195-03 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
The Influence of Fluid Administration on the Effects of Tumor Necrosis Factor
Soluble Receptor in Gram-negative Sepsis
Principal Investigator:
P.Q. Eichacker, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
Q. Xizhong, M.D., CCMD
C. Parent, D.V.M., CCMD
R. Danner, M.D., CCMD
C. Natanson, M.D., CCMD
Collaborating Unit:
Immunex, Inc.
Staff-Years:
2.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Despite encouraging results in preclinical studies, clinical experience with anti-inflammatory in patients with sepsis have been disappointing to date. Tumor necrosis factor soluble receptor (TNFsr) is one agent. In early preclinical work utilizing endotoxin or gram-negative bacteria infusion challenges, TNFsr was protective and improved survival. This protection appeared to be related to improvements in cardiovascular function with TNFsr. However, in clinical sepsis trials this agent was not beneficial and in some cases appeared to have harmful effects. These finding suggest that factors not identified in preclinical studies may have altered the effects of TNFsr in clinical studies. One such factor may be the presence or absence of other supportive therapies. In clinical studies, subjects also have cardiovascular support with fluid administration. The presence of such support in clinical studies may have negated the beneficial effects of TNFsr on cardiovascular function and possibly accentuated adverse effects it may have had on host defense. In the present study, using a multifactorial design, we are testing the effects of fluid administration on TNFsr with both intravascular and extravascular infection in a rat model of sepsis.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00198-03 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Tyrphostin AG 556 Therapy Adjusted to Severity of Illness of New Therapies in
Septic Shock
Principal Investigator:
C. Natanson, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
A. Hilton, CCMD
S. Richmond, CCMD
M. Fernandez, CCMD
J. Sevransky, M.D., CCMD
S. Solomon, Ph.D., CCMD
S. Banks, Ph.D., CCMD
P. Eichacker, M.D., CCMD
Collaborating Unit:
None
Staff-Years:
3.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Septic shock appears to result from excessive release of cytokines [e.g., tumor necrosis factor-a (TNF-a), IL-2, etc.] and other pro-inflammatory substances [e.g., nitric oxide (NO)] from cells of the monocyte/macrophage lineage in response to infection or lipopolysaccharide (LPS) administration. The production of these cytokines, and their action, is mediated by signal transduction events that induce protein tyrosine phosphorylation. Theoretically, inhibition of protein tyrosine phosphorylation may be beneficial in sepsis. These compounds would block the potentially high cytokine production that is dependent on tyrosine phosphorylation. These protein kinase inhibitors would block both activation and production of cytokines by bacterial products and the effects of cytokines on target cells. Tyrphostins AG 126 and AG 556 are both protein kinase inhibitors and have been shown to improve outcome in small animal models during both LPS and live bacterial challenge. Further, both AG 126 and AG 556 have been shown to inhibit LPS-induced TNF production from dog peripheral blood mononuclear cells, in vitro. In collaboration with Dr. Novogrodsky and his colleagues, we evaluated AG 126 and AG 556 in our canine peritonitis model. In a controlled clinical trial in 100 animals over 6 months, AG 556 but not AG 126 significantly improved survival and prevented multiorgan failure during canine septic shock.
Recent analysis of animal experimental data suggests that the effect of anti-inflammatory agents is dependent in part on the underlying infectious burden of the animal. It appears that studies in which controls exhibited high mortality showed improved survival in response to anti-inflammatory therapy. Conversely, studies in which controls exhibited lower mortality suggested that anti-inflammatory agents had no benefit, and possibly some harm. Therefore, it is possible that the reason that human clinical trials in sepsis have shown no benefit is that the anti-inflammatory agents have been given to individuals with varying degrees of illness, and that a subgroup of patients with higher burden of illness might be helped by anti-inflammatory therapy.
This study is designed to examine the effect of titrating AG 556 to the severity of illness in canines infected with high- and low-infectious burdens. In our canine model of peritonitis, cohorts of animals with either high or low burdens of E. coli peritonitis clots will be studied. We will compare the efficacy with standard dose 2.5 mg/kg AG 556 to placebo, to titrated dosing 1mg/kg and then 1 or 4 mg/kg depending upon the blood pressure of animals at the 6 h time point. This study is the first study in an animal model to examine whether the utility of anti-inflammatory therapy is dependent upon the burden of infectious agent, and has potential implication for human clinical trials of anti-inflammatory agents in sepsis.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01156-02 CCM
October 1, 1999
to September 30, 2000
Title of Project:
Effects of L-Arginine on Endotoxin-mediated Inflammatory Responses
Principal Investigator:
A.F. Suffredini, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Fiuza, M.D., CCMD
R.L. Danner, M.D., CCMD
M. Tropea, CCMD
H. Preas, M.D., ANES
Collaborating Unit:
None
Staff-Years:
1.75
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: This project has been discontinued due to ambiguity of animal toxicity with high doses of L-arginine.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01157-02 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Effects of Inhaled Nitric Oxide on Pulmonary Inflammatory Responses
Principal Investigator:
A.F. Suffredini, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Fiuza, M.D., CCMD
M. Tropea, CCMD
Collaborating Unit:
None
Staff-Years:
1.75
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Inhaled nitric oxide (NO) diminishes inflammatory responses in vitro and in some animal models of lung inflammation. We are studying the mechanisms involved in NO modulation of local pulmonary inflammation in humans.
Evidence suggest that NO can modulate the inflammatory response in experimental lung inflammation. Nitric oxide donors inhibit inflammatory cytokine production by human alveolar macrophages in vitro, prevents IL-1 induced neutrophil accumulation and edema in isolated rat lungs, and blocked increases in pulmonary lavage neutrophils, protein, and lung myeloperoxidase content in septic swine. Only limited data are available in humans treated with inhaled nitric oxide for acute lung injury. After 4 days of inhaled NO, patients had a reduction of BAL neutrophil spontaneous H2O2 production, CD11b/CD18 expression, and less IL-6 and IL-8 in BAL fluid compared to patients who did not receive inhaled NO. Nitric oxide remains under investigation for adjunctive therapy for acute lung injury.
We are evaluating the ability of NO to alter the inflammatory response associated with segmental endotoxin instillation. Twenty-four volunteers will be studied in a randomized fashion. An initial pilot study will be performed in eight subjects challenged with bronchial endotoxin instillation. Following the endotoxin instillation, four subjects will breathe NO (40 ppm), delivered by an anesthesia non-rebreathing face mask with a reservoir bag and four subjects will breathe room air through a similar mask. The subjects will breathe through the circuit for 6 hours. The lavage cells will be studied using cell culture, functional studies, surface markers and intracellular cytokines with flow cytometry, and mRNA expression. The lavage supernatant will be evaluated for various inflammatory mediators and markers of inflammatory cell activation. Sequential blood samples will be obtained for total leukocyte counts, as well as plasma levels of inflammatory mediators.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01158-02 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Role of Nitric Oxide in Regulating Inflammation and Gene Expression
Principal Investigator:
R.L. Danner, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Wang, M.D., CCMD
W. Wang, M.D., CCMD
E. Nishanian, M.D., CCMD
J. Zhang, Ph.D., CCMD
Collaborating Unit:
LHD, NIAID (H. Malech, M.D.)
Staff-Years:
4.0
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Nitric oxide (NO) is an important intercellular and intracellular messenger implicated in the pathogenesis of septic shock. Inhibition of NO synthase is under investigation as a treatment for hypotension in septic shock. In addition to the vasodilating effect of NO, this messenger also has effects on platelets and immune cells. In this investigation, we are examining the role of the NO pathway as a modulator of immune cell function and gene expression. We have been unable to create conditions under which human phagocytes, in particular neutrophils, endogenously produce NO (J Immunol: 1825, 1994). Therefore, the ability of NO produced by other cells, such as endothelium and epithelium, to alter the function of human phagocytes is being explored.
We have confirmed that NO regulates cytokine production using a U937 monocytic cell line transfected to express murine inducible NO synthase (Blood: 1160, 1997). Further investigation of this effect has resulted in the description of a cGMP-independent signaling pathway for NO (J Biol Chem: 5959, 1997). We have found that in addition to upregulating TNFa production (J Immunol: 4102, 1994) NO modulates IL-8 message transcription and release in human neutrophil preparations. However, contrary to other reports, NO does not directly alter neutrophil chemotaxis (J Infect Dis: 116, 1998). More recent work has identified a NO-response element in the TNFa promoter (J Biol Chem, 1999).
Recent experiments have generalized the role of this putative NO-response element to several unrelated promoters. Further, the importance of sequences flanking this NO-response element to its function are being investigated. Work with the IL-8 promoter suggest that this chemokine is regulated by NO through a mechanism that is also cGMP-independent, but distinct from the pathway that regulates TNFa. In a new phase of this project, expression microarrays will be used to define larger sets of genes regulated by NO and to dissect out the underlying mechanisms by which the regulation occurs.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01159-02 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Magnetic Resonance Imagery Study of Avascular Necrosis of the Hip in Asymptomatic
HIV-infected Patients
Principal Investigator:
H. Masur, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
J.A. Kovacs, M.D., CCMD
G. Kelly, R.N., CCMD
G. Joe, M.D., RM
L. Gerber, M.D., RM
M. Rick, M.D., CPD
E. Jones, DRD
Collaborating Unit:
LIR, NIAID (H.C. Lane, M.D.; M. Polis, M.D.; J. Falloon, M.D.; R. Davey, M.D.;
R. Walker, M.D.; J. Mican, M.D.)
Staff-Years:
0.5
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Avascular necrosis (AVN) of the hip has occasionally been reported in the setting of HIV infection. After we diagnosed AVN in two patients in May 1999, we became concerned that HIV-infected patients may be at higher risk for developing AVN than previously recognized. To examine this, we undertook a magnetic resonance imaging (MRI)-based study of asymptomatic HIV-infected patients to determine the prevalence of hip AVN in our clinic population. 15 of 339 (4.4 percent) patients had evidence of AVN by MRI: six had bilateral and nine had unilateral involvement. None of 118 HIV-negative volunteers had MRI evidence of AVN. Prospectively performed physical examinations did not distinguish HIV-infected patients with AVN from those without. Patients with osteonecrosis were more likely than patients without osteonecrosis to have used corticosteroids, lipid-lowering agents, and testosterone, and were more likely to routinely exercise by bodybuilding. Thus, HIV-1 infection should be included among medical conditions that predispose to the development of osteonecrosis. Long-term followup of this cohort will help determine the natural history of AVN in this population.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01160-02 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Study of the Control of p11 Protein Production
Principal Investigator:
J.H. Shelhamer, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
M. Gladwin, M.D., CCMD
M. Cowan, M.D., CCMD
X. Huang, M.D., CCMD
Collaborating Unit:
None
Staff-Years:
2.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: p11 is a protein which can bind to and inhibit cytosolic Phospholipase A2. Modulation of p11 levels might provide a way to control a variety of cellular functions. Control of p11 has been studied at the protein and mRNA level. The p11 5' promoter has been cloned, sequenced, and characterized. p11 protein production has been studied in response to dexamethasone and to retonoic acid. The effect of cytokine and growth factor stimulation of epithelial cells on p11 production is also being studied. Two manuscripts have been published and two are in preparation.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01163-01 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Endothelial Cell Response to Oxidative Stress
Principal Investigator:
J.H. Shelhamer, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
U. Nanavaty, M.D., CCMF
Collaborating Unit:
None
Staff-Years:
1.0
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The response of human lung epithelial cells and endothelial cells to oxidative stress is being studied at the level of cellular function and gene expression. Signal transduction pathways activated in response to oxidative stress and linked to these events are also under active investigation.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01164-01 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
The Functional Genomics of Critical Illness
Principal Investigator:
R.L. Danner, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
J. Zhang, Ph.D., CCMD
J.H. Shelhamer, M.D., CCMD
A.F. Suffredini, M.D., CCMD
C. Natanson, M.D., CCMD
P.Q. Eichacker, M.D., CCMD
A.D.P. Cintron, M.T., CCMD
M. Tomaska, M.Sc., CCMD
Collaborating Units:
NIAID (S. Holland, M.D.)
Boston Univ. (D. Golenbach, M.D.)
Washington Univ. (J.P. Cobb, M.D.)
Staff-Years:
10.0
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Critical illness syndromes, such as acute respiratory distress syndrome (ARDS), septic shock, myocardial depression, and multiple organ failure all share the hypothesis that the host response plays a central pathogenic role. In this project, CCMD has established the infrastructure necessary to define these pathogenic host responses at the level of gene expression across thousands of mRNA transcripts, simultaneously. This technology will be used to create a large critical illness functional genomics database using in vitro models, small animal (rat and mouse) models, endotoxin-challenged volunteers, and ultimately critically ill patients.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01165-01 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
The Influence of Site, Severity, and Type of Infection on the Effects of an
Endotoxin Analogue in Sepsis
Principal Investigator:
P.Q. Eichacker, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Solomon, Ph.D., CCMD
C. Natanson, M.D., CCMD
Collaborating Unit:
Essai Corp.
Staff-Years:
2.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Clinical experience with anti-inflammatory agents in patients with sepsis has been disappointing to date. We have found that several factors such as the site, type, and severity of infection have important influences on many of these agents. Developing new agents which are impacted minimally by such factors, will increase the usefulness of this therapeutic approach. The role of endotoxin in the injury of sepsis is unclear. It is likely that under many circumstances it produces harmful effects. Targeting endotoxin rather than host mediators may be a more generally useful goal in sepsis. Antibodies against endotoxin have had little effect to date in sepsis because the toxic lipid portion of the molecule may be difficult to target. An alternative approach to neutralizing endotoxin uses analogue molecules which competitively inhibit cell signaling by endotoxin. We are currently performing a series of studies investigating the influence of site, severity, and type of infection on a competitive inhibitor of endotoxin.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01166-01 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
The Effects of Tumor Necrosis Factor Monoclonal Antibodies in Wild Type and
Tumor Necrosis Factor Knock Out Mice
Principal Investigator:
P. Eichacker, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Solomon, Ph. D., CCMD
X. Cui, M.D., Ph.D., CCMD
C. Parent, D.V.M, CCMD
Collaborating Unit:
None
Staff Years:
2.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of work: The primary purpose of this study is to compare the effects of chronic tumor necrosis factor (TNF) inhibition via gene deletion versus its acute inhibition with monoclonal antibodies (MAb), in a murine model of sepsis. Research has strongly suggested that activation of the host inflammatory response and release of host mediators, while important for host defense, if excessive could be important contributors to the pathogenesis of sepsis and septic shock. Based on preclinical sepsis models in which MAbs designed to inhibit specific host mediators like tumor necrosis factor (TNF) improved survival, similar MAbs have now been used in clinical sepsis trials. While the clinical experience with these agents has not been as successful as preclinical models suggested, studies we have done indicate that this approach is an effective one, if critical factors, importantly the severity of infection, are taken into account. Thus, use of MAbs in wild type animals appears to have been an effective approach for identifying critical host components in the pathogenesis of sepsis and septic shock. There is increasing interest in the use of murine knock-out models to also study the relevance of specific host protein products in the pathogenesis of acute diseases like sepsis. Whether the absence of a gene product since birth in an animal results in a similar response as its acute inhibition during a disease like sepsis is unclear. Lifetime absence related to gene deletion of a protein product protective under some circumstances may stimulate the growth of compensatory mechanisms during development. This compensation may confound studies assessing the influence the absence of the gene product has in question. The study will be executed in three parts: (1) Develop an antibiotic treated murine model of sepsis in wild type mice. (2) Test the susceptibility of TNF knock out mice to the model of sepsis developed for wild type mice. (3) Study the effects of TNF inhibition with anti-TNF MAbs during sepsis using doses of bacteria producing low, medium or high lethality rates in wild type versus TNF gene knock-out mice.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01167-01 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
The Influence of Site, Severity and Type of Infection on the Effects of a Tyrosine
Kinase Inhibitor in Sepsis
Principal Investigator:
P.Q. Eichacker, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Solomon, Ph.D., CCMD
C. Natanson, M.D., CCMD
Collaborating Unit:
Essai Corp.
Staff-Years:
2.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Clinical experience with anti-inflammatory agents in patients with sepsis has been disappointing to date. We have found that severity of infection has an important influence on many of these agents. Developing new agents which are impacted minimally by this factor, will increase the usefulness of this therapeutic approach. We are presently performing a series of studies investigating the influence of severity of infection on a recently developed tyrosine kinase inhibitor. In early studies with this agent, severity of inflammation related to endotoxin challenge did little to influence the beneficial effects of this agent.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01168-01 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Gene Expression in Humans Challenged with Endotoxin
Principal Investigator:
A.F. Suffredini, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
R.L. Danner, M.D., CCMD
J.H. Shelhamer, M.D., CCMD
C. Fiuza, M.D., Ph.D, CCMD
M. Tropea, MS, CCMD
Collaborating Unit:
NCI (L. Staudt, M.D., Ph.D.)
Staff-Years:
1.75
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Reliable biomarkers are needed to identify patients with sepsis who will benefit from anti-inflammatory therapies. In addition, recent observations suggest that novel mediators (i.e., calcitonin precursors, high mobility group-1 protein) play an important role in the pathogenesis of sepsis. In order to better characterize and discover new mediators and mechanisms involved in sepsis, we are using a model of inflammation based upon the administration of endotoxin, a bacterial wall component, to normal volunteers. By administering endotoxin either intravenously or via intrabronchial instillation, we are able to study early inflammatory events that occur in the blood and in the local environment of the lung. Intravenous endotoxin results in a systemic inflammatory response that is associated with the release of acute phase cytokines and activation of inflammatory cells and endothelium. Bronchial endotoxin instillation results in a localized neutrophil influx, increased permeability to protein, and acute inflammatory mediator release in the lung. The resolution of the inflammation in the lung is associated with apoptosis of neutrophils and a mononuclear cell influx over the following 48 hours.
Under protocol 92-CC-0141, the effects of endotoxin on gene expression will be studied using peripheral blood mononuclear cells and in separate studies, cells obtained with bronchoalveolar lavage from the lung. The temporal pattern of gene expression will be studied using cDNA microarrays. Spotted and oligonucleotide arrays will be performed on total mRNA extracted from these cells. These tools will be useful to study fundamental aspects of gene expression during exposure to bacterial products. It will provide one means of characterizing new mediators and mechanisms that are part of the acute phase response to bacterial products.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-01169-01 CCM
October 1, 1999 to
September 30, 2000
Title of Project:
Functional Gene Expression in Humans Challenged with Endotoxin
Principal Investigator:
A.F. Suffredini, M.D. (Senior Investigator)
CCMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Fiuza, M.D., Ph.D, CCMD
J.H. Shelhamer, M.D., CCMD M. Tropea, MS, CCMD
Collaborating Unit:
NCI (M. Bustin, Ph.D.)
Staff-Years:
1.75
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: High mobility group protein (HMG-1) is a non-histone DNA binding protein that facilitates transcription. Recently, investigators have shown that HMG-1 has other roles that may be critical in the development of sepsis and septic shock. HMG-1 is released as a late mediator of sepsis (i.e., after 8 to 15 hours) from mononuclear cells stimulated with TNF, IL-1, or endotoxin. It is detected in the blood of septic mice and in septic patients and it worsens outcome when given to septic mice. It plays a role in migrating axons of neurons in the developing brain and it activates plasminogen. Some of the actions are through the RAGE receptor (receptor for advanced glycation products) which plays a role in chronic inflammation in diabetes. This novel axis of inflammation remains to be characterized in human sepsis.
In order to study the target cells and contribution of HMG-1 to acute human inflammation, we are producing recombinant human HMG-1 in a bacterial expression system and are using the protein to study inflammatory responses in endothelium and mononuclear cells including alveolar macrophages. In addition, we are developing biologically active peptide fragments of the intact molecule in order to study structure function relationships. Cell lines and migrating human cells from humans challenged with endotoxin will be studied for the expression of RAGE and their responses to HMG-1. HMG-1 will also be studied in blood and inflammatory lavage obtained from volunteers challenged with endotoxin (protocol 92-CC-0141). Oligonucleotide gene arrays will be used to study the inflammatory axis initiated by HMG-1 on target cells. These data should provide important new information regarding the role of HMG-1 in acute human inflammation to bacterial products.
Index: Annual Report of Clinical Research Activities FY 2000
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