List of Training Laboratories Immunobiology of Trauma

The following laboratories are available for training by fellows participating in the 2 year Immunobiology of Trauma training grant (T32 GM 086308). Potential new fellows should contact the laboratories to discuss potential projects within each laboratory.

Pulmonary Injury group
Avrum Spira, M.D., and Jerome Brody, M.D. The lab strives to apply and develop computational tools that can be used to mine data from high-throughput translational research studies, by combining expertise in designing and running genome-wide studies of gene and protein expression on clinical specimens with high- throughput data storage and analysis capabilities. The scientists affiliated with our lab combine talents of molecular biologists with mathematicians, statisticians, epidemiologists and computer scientists. We currently train Bioinformatics, Pathology and Genetic/Genomic graduate students and clinician- scientists in the application of these high-throughout tools to clinical studies.

The primary research focus of the lab is to determine how cigarette smoking affects intra-thoracic (lobar bronchi) and extra-thoracic (buccal mucosa and nasal mucosa) airway epithelial cell gene expression and to use this information to develop a non-invasive genomic biomarker for lung cancer that can identify that subset of smokers who have, or are at risk for developing, lung cancer. Our lab has also begun to explore how this molecular “field of injury” in airway epithelium reflects information about the perturbation of specific oncogenic pathways within an individual, potentially allowing personalized genomic approaches to chemoprophylaxis and therapy. This airway “field of injury” concept is also being extended to identify biomarkers for and pathways perturbed in Chronic Obstructive Lung Disease (COPD), as well as identify non-invasive measures of the biological response to tobacco exposure that can be applied to large-scale population studies as part of the NIH/NIEHS Genes and Environment Initiative. Additionally, we are interested in understanding the underlying mechanisms of smoking-related disease risk and are seeking to identify microRNA changes and DNA polymorphisms that are predictive of the gene-expression changes that characterize the airway field of injury. These sophisticated approaches could be used to identify molecular signatures in the trauma patient which predict poor outcome.

Wellington V. Cardoso, M.D., Ph.D., Research in the laboratory focuses on the mechanisms that regulate embryonic lung development. The overall long-term goal is to understand how lung progenitor cells form and ultimately give rise to the airways and alveoli. We have been particularly interested in the mechanisms by which molecules, such as retinoids, fibroblast growth factors and transforming growth factor beta factors influence this process. We are also investigating the role of heparan sulfate proteoglycans in modulating Fgf responses in lung developmental programs. To achieve these goals we combine classical cell and organ culture techniques with gene discovery tools, including DNA microarrays, computational analyses, large scale in situ hybridization and mouse transgenic/knockout technologies. This research provides insights into basic mechanisms of lung development and stem cell biology that are critical to understand lung disease states and repair. Acute lung injury is a frequent complication of the traumatized patient and the methods in the laboratory may offer insights into the basic mechanisms of disease.

William Cruikshank, Ph.D. and David Center, M.D. The lab has spent the last 20 years investigating cytokine mediators that modulate asthmatic inflammation. Our recent work has focused on the recruitment and activation of T regulatory cells in the lung. We have identified two factors, interleukin-16 (IL-16) and agonists of the histamine 4 receptor (H4R), that when administered in the asthma model suppress asthmatic inflammation and airway hyper-reactivity. An H4R agonist reduces acute inflammation while IL-16 promotes tolerance to antigenic stimulation in a chronic model of asthma. We have identified that both factors induce Treg cell recruitment. Agonists of the H4R selectively recruit Treg cells into the lung while IL-16 induces Treg cell recruitment to the draining lymph nodes for the lung. A fellow supported from this training grant would have the opportunity to further develop murine models of acute lung injury and induced tolerance with respect to the role of IL-16 and histamine in regulating Treg cell recruitment and resolution of acute lung injury, a feared complication of traumatic injury. They would gain the expertise needed to run and evaluate the animal models in addition to developing a solid immunological understanding of lung inflammation and mediators of resolution.These techniques and methods would be directly applicable to the acute lung injury observed in the traumatized patient.

Alan Fine, M.D., The ongoing focus of the lab remains the development and application of techniques to identify endogenous lung stem cells, and to identify non-lung sources of stem cells that have reparative properties. We have extended our program into 2 new areas: how the lung modulates hematopoeitic cell phenotypes, and an examination into the contribution of hematopoeitic cells to pulmonary artery structure. The objective of this work is to further understand the basic molecular signals that regulate how the lung responds to insults, and how inflammation is locally controlled. These investigations have profound implications for understanding acute lung injury and for the development of cell-based treatments for related syndromes. Together, these projects provide the basis for a variety of projects that directly relate to the focus of this T32 proposal. Resolution of acute lung injury to restore full pulmonary function would be a significant advance in the treatment of the traumatized patient.

Darrell Kotton, M.D., The laboratory is focused on stem cell biology and gene transfer techniques applicable to the study and therapy of lung disease. The lab employs embryonic stem cells as an ex vivo model of lung epithelial development, and utilizes in vivo transplantation techniques, including single cell transplantation, to test the potential of various stem cell populations to reconstitute lung tissues after injury in mouse models. A fellow or post-doctoral trainee rotating through the laboratory will be immersed in the field of stem cell biology and exposed to hands-on training with a variety of stem cell populations, including bone marrow-derived stem cells as well as embryonic stem cells. Because many projects in the lab involve rigorous methods required for optimal stem cell purification, the trainee will also learn to utilize and interpret experiments featuring these techniques. In addition, the Kotton laboratory specializes in the genetic manipulation of stem cells using lentiviral transduction methods that do not alter stem cell capacity. Molecular cloning, viral packaging, and in vitro and in vivo tests of these lentiviral vectors is experienced by trainees in the Kotton laboratory.

Wound Healing

Louis Gerstenfeld Ph.D. The studies focus on bone repair after trauma or surgical treatment. All of our ongoing research uses translational research techniques with human surgical techniques adapted to animal models to address our research goals. We use a combination of state of the art assessments tools to examine fracture and bone repair in vivo including micro-computer assisted tomography (CT) biomechanical testing for strength and material property and novel tools of three dimensional tissue reconstruction of sequential histological specimens. We use cell based models of primary cells isolated from the bone marrow and fracture callus and several skeletal cells lines to compliment our animal studies. We use all forms of state of the art molecular analysis both in vivo and in vitro including transgenic animal models, retroviral knock down strategies and various approaches of mRNA expression analysis including from RT-PCR, large scale transcriptional profiling to approach our research questions. There are multiple research areas of study in our laboratory.

  1. We have numerous ongoing projects covering a diverse set of pre-clinical trials of biological compounds, small molecule pharmaceuticals and cell based therapies that are directed at enhancing fracture healing. Our pre-clinical trials of compounds and drugs has involved our laboratory in the basic development of both Bone Morphogenetic Protein (BMPs) and PTH as therapeutics for improvements in bone healing as well as in studies that have shown that non-steroidal anti-inflammatory agents and cyclo-oxygenase inhibitors exert inhibitory effects on bone repair.
  2. Our basic research studies in fracture healing focus on three project areas. One set of projects examines the role of the innate immune system as a primary initiator in bone regeneration after injury and have primarily focused on the role of tumor necrosis factor (TNF) and Fas mediated cell apoptosis. A second project area is focused on the VEGF signaling function during fracture healing. Our final set of basic science projects focuses on the role of inherited genetics in bone formation and fracture healing. This project makes use of inbred strains of mice that have definable variations in bone quality as assessed by variations in geometric characteristics and material property intrinsic to mineral and matrix properties. This project specifically examines how genetic variations effect fracture healing. The identification of specific types of structural and biomechanical traits that both relate to rates of fracture healing and specific biological processes that occur during fracture healing have the potential to be developed into clinically usable assays. These assays may be prognostic of slow or fast fracture healing identify those at risk for poor bone quality to delayed healing.

Thomas A. Einhorn, M.D. The current focus of the laboratory is to investigate the molecular mechanisms which underlie the repair and regeneration of skeletal tissues. These studies are particularly relevant in the aging population as the prevalence of osteoporosis increases. We approach this by establishing standardized models of fracture healing and distraction osteogenesis in the mouse and the rat, and examine tissue responses using a variety of techniques. Where possible, we use genetically manipulated animal models to delete, inhibit, or overexpress a particular gene and study the healing response. Current topics in our laboratory include the role of bone morphogenetic protein signaling in fracture healing and bone regeneration, the role of angiogenesis in bone regeneration via distraction osteogenesis, the relationship between tissue differentiation and the mechanical strain environment during fracture healing. Our goal is to elucidate the basic mechanisms which control the repair and regeneration of skeletal tissues in order to establish a foundation of knowledge to support studies on pharmacological treatments and tissue engineering technologies to enhance these processes.

Innate and Adaptive Immunity
Daniel G. Remick, M.D. The laboratory studies several aspects of the inflammatory response in different disease states including asthma and sepsis. Sepsis is a feared consequence of the hospitalized trauma patient and fellows working in my laboratory would participate in ongoing studies attempting to determine the injurious mechanisms of sepsis. We try to answer several questions including 1) What is the natural evolution of the inflammatory response in sepsis induced by cecal ligation and puncture? 2) What is the appropriate response that allows clearance of the infectious challenge without damaging the host? 3) Which aspects of the inflammatory response cause cell, tissue and organ injury? 4) What are the appropriate biomarkers that predict poor outcome in sepsis? 5) How can the septic response be modified to provide the best outcome? Fellows will be trained in a variety of specialized techniques including multiplex protein detection, measurements of small animal physiology such as temperature and motor activity and analysis of data. While the lab works in teams, each fellow will be given her/his own project addressing a specific issue. Training in the specialized methods will be done by either myself (small animal surgery), lead technician (multiplex protein detection), or graduate students (physiology measurements). In the past the lab has aggressively collected data to publish in the peer reviewed literature, a tradition we would continue.

James Becker, M.D., and Arthur Stucchi, Ph.D. The George Sears Surgical Research Laboratory was established in 1994 by the incoming and present Chair and Chief of Surgery, Dr. James Becker, to not only promote his mission of fostering academic research in the department, but also to provide research opportunities to young faculty and surgical residents wishing to explore or pursue careers in academic surgery. To achieve this goal, the lab was designed and equipped with state-of-the-art molecular biology and cell culture equipment with funding from the Ripple Foundation. The laboratory is now staffed with two full-time PhD level basic scientists, Dr. Arthur Stucchi, a biochemist and Dr. Karen Reed, a molecular biologist, one part-time PhD, one MS level scientist, and a number of technical support staff. The laboratory also participates in the training of MD/PhD and PhD students from the Department of Pharmacology, thus the fellows have the unique opportunity for mutual interactions with other basic scientists in training. Since 1996, 10 PGY2 surgical residents have undertaken 2 years of intensive, full-time research in the laboratory. Incoming fellows benefit from the collegial research environment that the surgical research lab cultivates. The research conducted by surgical fellows has resulted in over 34 published abstracts and presentations at national and international scientific meetings and more than 25 peer-reviewed papers in esteemed journals such as the Proceedings of the National Academy of Sciences, the American Journal of Physiology and the Annals of Surgery. These studies have also led to awards for fellows including “Best Poster Presentation” at the international meeting of the Peritoneal Access Society held in Belgium (September 2006), and “Best Poster Presentation” at the Annual Meeting of the Colon and Rectal Surgeons, Philadelphia, PA (May, 2005). Incoming fellows have the opportunity to work on a number of surgically related projects from a basic science perspective. One area involves adhesion formation following abdominal surgery. The laboratory is interested in understanding the molecular and cellular events that underlie adhesion formation in an effort to develop safe and effective means of postoperative adhesion prevention. We have demonstrated that statins and a substance P receptor antagonist reduce post-surgical adhesion formation in a rat model and that these responses involve tissue plasminogen activator (tPA), matrix metalloproteinases (MMPs) and oxidative stress. Studies are underway to determine how tPA, MMPs and oxidative stress intersect to regulate postoperative adhesion formation. The second area of research focuses on the etiology of inflammatory bowel disease (IBD). Specifically, the laboratory is interested in transcriptional mechanisms regulating TNF- production in stimulated colonic lamina propria macrophages and whether vitamin D can block this proinflammatory response. A broader understanding of the molecular mechanisms leading to the increased TNF- synthesis observed in IBD is fundamental to the development of more effective IBD therapeutic strategies. This laboratory offers the opportunity for surgical fellows to conduct meaningful and productive research projects. This fertile training ground and the scope of the research would be ideal for a fellow to investigate the immunobiology of the trauma patient.

Cell Biology
Katya Ravid, Ph.D. Adenosine has been described to control inflammation, but it has not been certain which of its receptors mediate this effect. We generated an A2b adenosine receptor (A2bAR) knockout/reporter gene knock-in mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared to age- sex- and strain-matched control mice. Augmented proinflammatory cytokines, such as TNF, and a consequent downregulation of IkB are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2bAR knockout mice. Exposure to endotoxin results in augmented proinflammatory cytokine levels in A2bAR null mice compared to control mice. Bone marrow transplantation indicated that bone marrow (and to a lesser extent vascular) A2bARs regulate these processes. Hence, the A2bAR is identified as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target. To further examine the functional significance of this receptor expression, we applied a femoral artery injury model to A2bAR knockout (KO) mice and showed that the A2bAR prevents vascular lesion formation in an injury model that resembles human restenosis post angioplasty. While considering related mechanisms, we noted in the injured KO mice higher levels of TNF-alpha, an up-regulator of CXCR4 and of VSMC proliferation. In accordance, CXCR4, which is known to attract progenitor cells during tissue regeneration, is upregulated in lesions of the KO mice. In addition, aortic VSMC derived from A2bAR KO mice display a greater proliferation rate, in comparison to controls. Bone marrow transplantation experiments indicated that the majority of the signal for lesion formation in the null mice originates from bone marrow cells. Thus, this study is the first to highlight the significance of the A2bAR in regulating CXCR4 expression in vivo, and in protecting against vascular lesion formation. Our investigations continue to explore the role of this and other adenosine receptor in the process of healing following various injury models. The role of adenosine receptors in neutrophil function in the traumatized patient is under active investigation by several scientists active in the field of trauma research.

Peter Burke, M.D., The research undertaken in the surgical Trauma Research Laboratory directed by Dr. Peter A. Burke focuses on developing a more in depth understanding of molecular events governing the regulation of the Acute Injury Response. Specifically the lab has been developing a better understanding of the transcriptional regulatory mechanisms that are responsible for orchestrating of the liver’s modulation from a steady (non-injury) state to that of the Acute Phase Response. The extensive changes in liver protein production brought about by the Acute Phase Response represent a change in liver phenotype and are regulated at the level of gene transcription. Multiple transcription factors involved in regulating acute phase genes are being studied. Specific focus on the liver specific transcript factors HNF-1 (Hepatocyte Nuclear Factor-One) HNF-3, HNF-4 and C/EBP (CAAT-Enhancer Binding Proteins) which are central to liver development and phenotype. The effects of injury on transcriptional factor binding, transactivation, post translational injury induced changes to transcriptional factor activities and the effect on the overall Acute Phase Response are being examined utilizing both in vitro and in vivo injury modules.

A further goal of the laboratory is to develop a better understanding of the effects of injury severity on the kinetics and magnitude of the liver’s acute phase response and how injury severity affects transcription events regulating the acute phase response. In collaboration with the orthopedic trauma research laboratory directed by Dr. Tom Einhorn and Dr. Lou Gerstenfeld we are investigating the effects of injury and its severity on the bone marrow compartment looking specifically at the signals involved in progenitor cell proliferation and release into the circulation as well as investigating a role for progenitor cell in the regulation of the liver’s acute phase response.

Kenneth Walsh, Ph.D. Research in the Walsh laboratory has been focused in three areas. The major project investigates the signaling- and transcriptional-regulatory mechanisms that control both normal and pathological tissue growth in the cardiovascular system. Many of these studies involve analyses of the PI3- kinase/Akt/GSK/Forkhead signaling axis. This pathway is of critical importance in the regulation of organ growth and body size. Signaling through this pathway controls cellular enlargement (hypertrophy), cell death (apoptosis), and blood vessel recruitment and growth (angiogenesis). We have also shown that some of these signaling steps are important for cardiac hypertrophy during normal postnatal development, and that they regulate myocyte survival in models of heart disease.

The second project investigates the role of the immune system in vascular disease. The formation of atherosclerotic lesions involves inflammatory cell interactions within the endothelium and subsequent extravasation into the vessel wall. Accelerated atherosclerosis is a critical factor contributing to the stroke and coronary heart disease that is a major cause of death among young women with systemic lupus erythematosus (SLE). Currently, there is considerable controversy regarding the causes of accelerated atherosclerosis in patients with SLE, although chronic inflammation is likely to be a contributing factor. To better understand these pathophysiological processes we created a new mouse model of atherosclerosis where lesion formation appears to be more dependent upon immune function than the standard apoE-/-model. Our findings suggest the existence of positive feedback interactions between atherosclerosis and the immune system that can develop when peripheral tolerance is breached. Further application of this model could be valuable in delineating the roles of the immune system in modulating the development of atherosclerotic lesions. These findings may also help control the response to traumatic injury, particularly in the recovery phase.

The third project analyzes the actions of adiponectin on cardiovascular tissues. It is now recognized that adipose tissue functions as an endocrine organ and that obesity contributes to cardiovascular and metabolic disorders through an imbalance of cytokines. Adiponectin is an adipocyte-derived cytokine that is down-regulated in obese individuals. Hypo-adiponectinemia is an independent risk factor for the development of diabetes, hypertension and coronary artery disease. We have found that adiponectin has beneficial actions on the cardiovascular system by directly acting on the heart and blood vessels. These data indicate that short- term administration of adiponectin, a natural cardioprotectant, could have utility for the treatment of acute cardiovascular disease.
Recently, we have developed mouse models to study the interrelationships between adipose and skeletal muscle tissues. We have isolated a number of novel proteins that are secreted during muscle growth. Analysis of these factors could provide insights about the pathogenesis of metabolic and cardiovascular diseases, including those that occur in the traumatized patient.

Adam Lerner, M.D., The laboratory focuses on lymphoid signal transduction through the cAMP signaling pathway. A variety of cyclic nucleotide phosphodiesterase inhibitors are either already FDA approved (PDE3, PDE5) or in clinical development (PDE4, PDE7, PDE8). The laboratory has examined the potential clinical utility of PDE3, 4 and 7 inhibitors in management of B and T cell disorders. PDE4 plays a central role in the responses of monocytes, neutrophils, B cells and T cells to Gs-coupled GPCRs. PDE4 inhibitors are currently in late stage clinical trials for asthma and chronic obstructive disease as they are potent anti-inflammatory agents. Fellows in Dr. Lerner’s lab would examine the potential utility of targeting cyclic nucleotide phosphodiesterases in improving therapy for trauma patients.