Relationship to Other NIH Traininq Proqrams at MGH

The MGH has 18 NIH-sponsored training grants. These programs include the following fields or areas:

1. radiological sciences
2. nuclear magnetic resonance
3. endocrinology and diabetes
4. nephrology
5. immunology and tumor biology
6. cell and molecular training for cardiovascular biology
7. connective tissue structure, function and disease
8. basic science research training for anesthetists
9. digestive diseases
10. AIDS
11. transplantation biology
12. cancer biology
13. lung cell and molecular biology
14. integrative pathophysiology of solid tumor
15. molecular imaging research
16. cardiac MR and CT
17. reproductive and developmental biology
18. burns and trauma

The research in the training program continues to be primarily focused on the MGH Burn Research Center activities with selected research opportunities in the Center for Engineering in Medicine. Although the fields of expertise of the training faculty cover a broad spectrum of problems related to burns and trauma, the varied research interests of the faculty might be broadly categorized into two areas:

• Alterations in metabolism produced by burn injury (Tompkins, Burke, Hales, Martyn, Avruch, Fischman, Toner, Yarmush, and Kelleher)
• Bioengineering applications in the treatment of injury including tissue engineering and artificial organ development (Tompkins, Burke, Ausubel, Stephanopoulos, Toner, and Yarmush)

The MGH Burn Research Center's unifying hypothesis underlying the plan of research is that severe burn injury initiates a unique series of changes in both the homeostasis of nitrogen metabolism and that of the major energy yielding substrates, glucose and lipids. Severe injury also profoundly alters the integration of interorgan cooperation in overall nitrogen and energy economy of the host. The net effects of these changes are an overall nitrogen catabolic state that seriously compromises wound healing and recovery and which is refractory to treat with current therapies. These changes lead to a functional redistribution of nitrogen (amino acids and proteins) and substrate metabolism among the wounded tissues and major body organs. This redistribution results in a quantitative reordering of the usual pathways of carbon and nitrogen flow within and among regions of the body and results in depletion of required substrates and cofactors in key organs. The liver is a particularly key component in this metabolic response because it compensates for the changes occurring extrahepatically and it is an organ that also orchestrates the whole body response. A major focus of our research concerns the impact of nutritional, hormonal, and pathophysiological factors on in vivo aspects of substrate metabolism in human subjects.

The main metabolic focus of the Center has been strengthened with the addition of several investigators over the past several years. Since 1997 Dr. Avruch, an internationally recognized expert in signal transduction pathways for insulin, has been included in the training faculty staff from his work with the Center. Because insulin is a very important anabolic signal in human physiology and potentially in the pathophysiology of burn injury, his contributions may be particularly timely and beneficial because of the recently identified intermediate cellular cascades (p38 and SAPK) and their potentially important physiological roles in post- injury metabolism. Dr. Jeeva Martyn has joined the Center as an investigator and has brought with him a wealth of expertise in trauma-related cell injury, particularly cell death by apoptosis and molecular mechanisms to inhibit apoptotic cell death. The addition of these two investigators has allowed the Center to address the metabolic changes at the organ and tissue levels in addition to the more traditional whole-body level. The recent recruitment of Dr. Joanne Kelleher, a well respected PhD investigator in isotope kinetics and intermediary metabolism, complements our well-established team of investigators in tracer studies and mass spectroscopy.

Project 1. Tissue-Specific Metabolic Response to Injury (Tompkins, Martyn, Fischman)

The major focus of this project is to explore simultaneously or in closely-related studies, the energy (oxygen utilization), glucose, and fatty acid metabolism, as well as muscle protein synthesis in major organs that are heterogenously affected by severe burn injury. The purpose is to understand how these organs and tissues (cardiac and skeletal muscle, liver, and kidney) interact at a metabolic level in the response of the whole body to the stress induced by a severe burn. A major and exciting new application for this project is the use of PET technology, which has not been applied previously in the study of burn trauma metabolism.

Project 2. Molecular Mechanisms of Burn Induced Insulin Resistance (Tompkins, Avruch, Toner, Fischman)

The metabolic alterations quantified and described at the in vivo level under Project 1 are hypothesized to be due, in part, to insulin function and cellular reaction to this normally anabolic signal. Project 2 explores in depth the underlying mechanisms for "burn-induced insulin resistance" and its metabolic sequelae. The aim is achieved by studies of insulin binding and receptor status and intracellular signaling processes in model system and, to a more limited extent in burn patients, using PET and euglycemic insulin clamp techniques. The project also studies the beneficial effects of insulin administration at molecular and in vivo levels to formulate new therapies aimed at preventing the burn-induced wasting syndrome. Specifically, the project focuses on the possibility that insulin and insulinomimetic drugs, such as vanadium compounds, may prevent the wasting syndrome by virtue of an inhibitory action on one or more of the catabolic system present in muscle.

Project 3. Molecular Basis of Hepatic Hypermetabolism in Burns (Yarmush, Stephanopoulos, Tompkins, Toner, Kelleher)

The overall aim of this project is to identify the cellular and molecular events that account for the catabolic state of nitrogen (amino acid) metabolism in the liver of the burned host. A liver perfusion technique is integrated into kinetic and substrate balance studies of amino acid metabolism and further supplemented by an assessment of the regulation and level of activity of key enzymes involved in the nitrogen economy of the liver cell. Studies are also underway to correlate the expression of key metabolic enzymes with the activation of AP-1, ATF-1, and NF-κB in muscle and the gluconeogenic organs after burn injury, and investigate the role of the stress-activated transcription factors in the metabolic response to burn and trauma. These studies enhance an interpretation of the findings emerging from studies in humans (healthy volunteers and burn patients) and potentially provide a better basis for study design of future studies in vivo of burn metabolism.

Project 4. Smoke Inhalation and the Mechanisms of Smoke Injury (Hales, Fischman, Tompkins)

Smoke injury is a leading cause of death in fire victims even in the absence of thermal injury. Some of the major toxins in smoke have been identified and the pathways to pulmonary injury initiated by these toxins are multiple and complex. However, one pathway, the arachidonic acid cascade, is certain to play a prominent role in the inflammation and pulmonary edema leading to pulmonary injury. It is our hypothesis that manipulation of the arachidonic acid cascade could lead to new therapeutic measures to alleviate the mortality and morbidity associated with smoke inhalation. Furthermore, there are few pharmacological mediators that can alter this pathway with specificity and unwanted side effects. Thus, we propose to test the use of a novel class of agents known as gene-based therapeutics, which have the potential to manipulate the arachidonic acid cascade with great specificity. Delivery of the gene-based therapeutics is mediated by cationic liposomes that have been previously demonstrated to be effective at delivering genes to the lung. Selected gene delivery to the lung should allow us to answer important basic questions regarding the role of key enzymes in the arachidonic acid pathway and their contribution to the injurious as well as protective responses after smoke injury. Furthermore, gene delivery to the lung facilitates the development of gene-based therapeutics designed to selectively inhibit the injurious response or enhance the protective response of the arachidonic acid pathway in smoke injury.

Bioengineering Applications in the Treatment of Injury, Including Tissue Engineering and Artificial Organ Development

Bioengineering is a dynamic and expanding field that strives to make improvements in patient care and quality of life through the application of principles and tools of the physical and biological sciences. MGH and SHC have been at the forefront of the biomedical engineering research as it applies to burns and trauma since 1970’s, starting with the collaboration between Professors John Burke at MGH/SHC and Ionnas Yannas at MIT to develop the first tissue-engineered skin for the treatment of burn injury. This tradition has continued and significantly expanded into other areas, including genetically modified skin, hepatic tissue engineering and the development of bioartificial liver assist systems, cryopreservation and desiccation of living tissue engineered systems, bioinformatics and gene expression studies, microfluidic and micropatterning approaches for cellular studies, among others.

Currently, five of the eleven Center faculty members have doctoral degrees in engineering and/or physical sciences. Furthermore, the Center for Engineering in Medicine (CEM) recently established the first hospital-based Microscale Engineering Core Facilty (μECF). This novel research and training facility empowers the MGH Burn Research Center and CEM faculty with a collection of engineering tools primarily to create living cell-based microdevices with a broad range of applications varying from diagnostic, tissue-engineered products, cell-based high-throughput screening tools, and basic biology tools. The potential for the BioMEMS facility lies in the fact that miniaturized components can be batch-fabricated using the tools developed in microelectronics industry (e.g., photolithography, etching, surface deposition, etc.) and then merged together with living systems to probe, analyze, and perturb cellular behavior at the micron or submicron scales. Examples of the bioengineering research opportunities in burns and trauma include:

1. Development of a bioartificial liver
2. Development of skin replacement materials
3. Living cell arrays for functional genomics
4. Bioinformatics approaches to determine the set of phenotypes observed in the immuno-inflammatory host response to injury

The liver is an indispensable, complex organ vital to the functions of metabolism, excretion, detoxification, storage, and phagocytosis. At present, the only treatments offered for liver failure are supportive care and liver transplants. Although a number of liver substitutes have been exploited including hemodialysis, hemoperfusion over various sorbents, exchange, transfusions, and plasma cross perfusions, none of these treatments have met with any consistent success. The proposed work provides a systematic analysis of the fundamental principles involved in the design of a hybrid artificial liver, including determination of effects of cell-cell, cell-extracellular matrix, and cell-fluid interaction on long-term viability and function. The effects of culture substrate, culture geometry, and culture medium on each cell type will be investigated. Potentially useful surface coatings for the devices including the various types of collagen, tissue fibronectin, laminin, and glucosaminoglycan in different ratios and (micro) patterns are used. In addition, we have developed theoretical models that relate important experimental parameters to liver function. Models consider various types of transport and reaction mechanisms including convection, diffusion, receptor-mediated endocytosis, intracellular processing and sorting, enzyme kinetics, protein secretion, and small molecule excretion. These studies provide the basic information for rational design of an effective liver support device for the management of post-burn liver failure.

Project 2. Development of Skin Replacement Materials (Burke, Toner, Tompkins)

A clinically successful artificial dermis has been developed in the laboratories of Drs. Burke and Yannas over the previous twenty-plus years. This material consists of a collagen and glycosaminoglycan coprecipitate bound to a medical-grade silicone membrane. Called Integra, this material received premarketing approval from the Federal Drug Administration in 1996 and has entered into the daily practice of burns care. Further development of this approach is being explored using genetically modified fibroblasts and keratinocytes develop a living skin material in vitro. Genetic modification strategies are also being pursued to impart tolerance to desiccation in living skin substitutes. Many species down-regulate their metabolism and enter into a state of stasis by desiccation (or anhydrobiosis). Studies with anhydrobiotic organisms have revealed a complex series of adaptive changes including the accumulation of large amounts of internal sugars (e.g., trehalose). Keratinocytes are genetically modified to produce pre-determined amounts of intracellular trehalose to induce desiccation tolerance for long-term storage of living skin substitutes at ambient temperatures. Another project within the scope of tissue engineering of the skin involves the development of an analogue of the basement membrane of skin with complex topographical features using microfabrication technologies. The overall goal of this project is to engineer skin with natural topographical features to increase the longevity of the skin substitutes and to provide a cosmetically more appealing skin substitute for burn victims.

Project 3. Living Cell Arrays for Functional Genomics Studies of Hypermetabolism (Yarmush, Stephanopoulos, Toner)

The overall goals of this project are to understand metabolic changes in cells and tissues on the molecular level and to investigate the role of temporal gene expression in determining the observed metabolic phenotype. Metabolic studies on the liver after injury have provided insights on the state of the organ; however, this approach offers limited information of the gene expression events that lead or sustain a particular metabolic state. The relationship between alterations in gene expression and the metabolic phenotype on the basis of their interactions and the feedback mechanisms involves need to be determined. By dynamically monitoring the behavior of multiple genes in a massively parallel and non-destructive manner in living liver cells, we expect to generate information that can be used for the development of integrated models of biological processes and disease states. To this end, micromachining and microfluidics techniques are utilized to develop a living cell array systems where the genes whose expression is altered by molecular mediators of the stress response are tagged with green fluorescence protein (GFP) to monitor the dynamic response of stress inducible genes in hepatocytes exposed to complex inputs. A bioinformatics component includes novel methodologies to analyze temporal data from massively parallel cell arrays.

Project 4. Bioinformatics Studies to Probe Immuno-Inflammatory Host Response to Injury (Tompkins, Yarmush, Stephanopoulos, Toner)

The goal of this project is to define the immuno-inflammatory response in circulating peripheral white blood cells in terms of their cellular and humoral phenotype. Microfluidic and imaging tools are utilized to sort white blood cells into homogeneous subpopulations without altering their phenotype. Protein analyses are performed on separated cells using both low and high throughput approaches to define proinflammatory and anti-inflammatory cytokines, leukocyte phenotypes, and cell signaling intermediates. High throughput microarray analysis is performed to determine gene expression patterns of homogeneous subpopulations of blood cells as a result of severe trauma. Bioinformatics and computational tools are then used to rigorously and mathematically analyze the gene expression data. These studies are conducted in both humans and murine injury models.

Trainee Participation in the Research

In the projects related to “Alterations in Metabolism Produced by Burn Injury,” the trainee is involved in human metabolic studies. The trainee learns study design and performs the clinical studies with the assistance of the investigators and collaborators. He or she performs mass spectrometric and nuclear magnetic resonance analyses to learn the principles and procedures as well as the interpretation of the primary analytic data. He or she also summarizes, evaluates, and prepares the data for presentation at informal seminars, scientific meetings, and for peer-reviewed publication. Through this exposure, the research fellow develops the skills and leadership to conduct careful, well-designed studies as an independent investigator.

In the projects related to “Bioengineering Applications in the Treatment of Injury,” instruction in basic science, engineering, and physical chemistry as well as in analytic techniques is provided. The trainee is integrated into the overall training program with full participation in all clinical and basic science aspects of our program. The trainee is provided access to all laboratories and tutors for special short interaction periods to learn specialized techniques not available in their major tutor’s laboratory. Dedicated teaching in scientific responsibility, microfabrication tools, literature evaluation and scientific writing is provided. For the research fellow to fully understand the fundamental physical principles, he or she should participate in formal course work. Following the training period, the trainee should be equipped with the necessary knowledge to perform individual research projects as an independent investigator.