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This mosaic was created by General Surgeon Dr. Harold Berg (1918-2002). Close to 200 of Dr. Berg’s mosaics are on display in Louisville and around the world. A native of Brooklyn, NY, Dr. Berg graduated from the University of Louisville School of Medicine in 1941. He had an established general surgery practice and began teaching at the University of Louisville in 1961. Dr. Berg never accepted payment for any of his art works, which were always thoroughly researched and freely given out of respect to the recipients. Shown here is a mosaic of Dr. Abraham Flexner, who was born in Louisville (Ky) and is known for his 1910 publication titled the “Flexner Report.” An emphasis in the report was for increased faculties at university hospitals dedicated to teaching and research. Our dedication to this Louisville tradition is reflected in the research efforts of our PIs. Several of our faculty investigators have received awards from the National Institutes of Health. The research descriptions below are derived from the National Institutes of Health Research Portfolio Online Reporting Tool (RePORT), which is available at http://www.projectreporter.nih.gov/reporter.cfm.

PI Name: Jorge G. Gomez-Gutierrez, PhD
Title: Targeting Melanoma Hypoxia with Lactic Acid Bacterium L. Lactis

Grant Number: 1R21CA210202-01A1
Fiscal Year Search: : 2017

https://projectreporter.nih.gov/project_info_description.cfm?aid=9302876&icde=33489391

DESCRIPTION (provided by applicant):

Melanoma is the most aggressive form of skin cancer and, if disseminated through the dermis, has a poor prognosis with a high mortality rate. Hypoxia is a component of the tumor microenvironment, which reduces efficacy of both immuno- and chemo-therapies resulting in poor clinical outcome. Therefore, we will exploit the hypoxic microenvironment as a target for gene therapy, utilizing commensal facultative anaerobic bacteria. The overall goal of this proposal is to develop an effective and safe delivery system for cancer gene therapy by targeting the hypoxic tumor microenvironment with food-grade lactic acid bacteria (LAB) Lactococcus lactis (L. lactis). The use of LAB, as opposed to other bacteria, represents a much more desirable strategy to deliver therapeutic genes than attenuated pathogenic strains, as there is no risk of reversion or potential to instigate host reactions in immunocompromised patients. Because identification of tumor-specific accumulation and biodistribution of gene delivery vehicles in vivo is essential for translation of these agents to the clinic, we will utilize a newly emerging technology, multispectral optoacoustic tomography (MSOT). MSOT is a hybrid modality that detects sound waves generated by the absorption of electromagnetic energy, thus enabling the capability for 3D high-resolution at depth and in real time. Our preliminary data indicates that L. lactis expresses high levels of -galactosidase (LacZ), and in combination with 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside (X-gal), produces a strong blue color to facilitate detection of L. lactis using MSOT. Given these findings, we hypothesize that L. lactis will preferentially colonize the hypoxic areas of the tumor microenvironment to deliver therapeutic genes in a safe, tumor-specific, and effective manner. We propose two aims: (1) Evaluate the efficacy of L. lactis as a gene delivery vehicle in melanoma cells in vitro, and (2) determine the biodistribution and tumor-specific accumulation of L. lactis in metastatic melanoma-bearing mice. The successful completion of this proposal will substantially influence the field of gene therapy, specifically utilization of facultative bacteria as delivery agents for tumor-specific targeting of melanoma.

PI Name: Suzanne Ildstad
Title: Induction of donor tolerance in renal transplants

Grant Number: R42DK074331-04
Fiscal Year Search: 2013

http://projectreporter.nih.gov/project_info_details.cfm?aid=8333414&icde=16967176

DESCRIPTION (provided by applicant):

Renal transplantation is the preferred therapeutic approach for end organ failure. However, the chronic use of immunosuppressive agents is critical to prevent rejection. The drugs are costly ($15,000-25,000/year) and have significant toxicities including opportunistic infection, an increased rate of malignancy, nephrotoxicity, and other end organ damage. The induction of donor-specific tolerance would address these limitations. Bone marrow chimerism induces tolerance to transplanted organs. However, the toxicity and complications associated with conventional hematopoietic stem cell transplants (HSCT), primarily graft-versus-host disease (GVHD) and the need for a matched donor, has limited the therapeutic application of HSCT to tolerance induction. We have identified and patented a novel tolerogenic bone marrow cell population of CD8+/TCR- facilitating cells (FC) that enhances engraftment of stem cells in mismatched recipients without causing GVHD. The discovery of FC opens the door to employing HSCT as a viable cell-based approach for tolerance induction. The product, FCRx, addresses the major challenges preventing the widespread use of HSCT for tolerance induction. FCRx is a bioengineered bone marrow product that includes hematopoietic stem cells (HSC) and FC, but avoids GVHD in mismatched recipients. In phase I of this proposal, we achieved our proposed milestones, demonstrating that we could reliably produce and transport FCRx and safely infuse it into nonmyeloablatively conditioned renal transplant recipients. We demonstrated that the proprietary FCRx procedure can routinely produce a graft with defined 12-TCR+ T cell composition and enriched for HSC and engraftment-enhancing FC. The ultimate goal is the induction of immune tolerance and the elimination of or reduction in the need for expensive and harmful immunosuppressive drugs. We have developed a nonmyeloablative conditioning regimen with 200 cGy TBI/fludarabine/cyclophosphamide/MMF/prograf to avoid the toxicity of conditioning. Since completing Phase I, we have addressed a major concern expressed by the reviewers regarding our ability to enroll subjects and have now successfully transplanted 6 living donor kidney patients, all of whom demonstrated donor chimerism at one month post-transplant. The approaches for collection, shipping, and FCRx preparation have also been approved by the FDA. Three patients show laboratory evidence of donor-specific tolerance, and immunosuppression is being weaned. The remaining subjects are early in follow-up. The reproducibility and quality assurance of the FCRx process will lead to its successful commercial launch. In phase II we will increase the pace of transplants and demonstrate that FCRx consistently achieves engraftment of HLA mismatched HSC in nonmyeloablatively conditioned kidney transplant recipients, induces donor-specific tolerance without causing significant GVHD, and reduces or eliminates the need for long-term immunosuppression. A recent meeting with FDA classified our product as Phase II, another milestone that will enhance commercialization.

PI Name: Suzanne Ildstad
Title: Delayed tolerance induction in living related donor renal transplant recipients

Grant Number: R41AI098336-01
Fiscal Year Search: 2013

http://projectreporter.nih.gov/project_info_details.cfm?aid=8252790&icde=16967176

DESCRIPTION (provided by applicant):

We have developed a protocol that reproducibly induces transplantation tolerance in individuals undergoing a living related donor kidney transplant. Three subjects have been off all immunosuppression for 10 months, 2 months, and 1 month respectively and have remained chimeric with stable renal function. Two additional chimeric subjects are in various stages of withdrawal from immunosuppression. The goal of this proposal is to expand this protocol to individuals who have already been transplanted and have a living donor that could subsequently provide a bone marrow stem cell product for their recipient (delayed tolerance). This work is important because solid organ, vascularized composite tissue, and islet transplantation currently require nonspecific immunosuppressive agents indefinitely for graft maintenance. These agents have significant toxicities, including nephrotoxicity, opportunistic infections, increased rate of malignancy, diabetes, and hypertension. It has been known for over 50 years that bone marrow chimerism induces tolerance to transplanted organs, cells, and tissues. However, the requirement for close HLA matching and toxicity of ablative bone marrow transplantation has limited the widespread application of this approach. The induction of donor-specific tolerance through durable chimerism in mismatched recipients while avoiding graft-versus-host disease (GVHD) would impact organ and tissue recipients as well as for individuals with hemoglobinopathies, inherited metabolic disorders, and autoimmune disorders. We have strong preliminary data in 8 living donor kidney hematopoietic stem cell transplant (HSCT) recipients, 1 metachromatic leukodystrophy recipient, and 1 sickle cell disease transplant recipient that we can safely establish durable chimerism in highly mismatched donor/recipient pairs. We have successfully established high levels of donor chimerism without GVHD in up to 1/6 HLA matched unrelated donor/recipient pairs. Seven of the eight renal transplant recipients were transplanted with cryopreserved G-CSF mobilized HSCT processed to remove GVHD-producing cells and retain tolerogenic CD8+/TCR- graft facilitating cells (the FCRx). Unfortunately, this successful "simultaneous" stem cell/kidney approach does not address individuals who have already received a transplanted organ and who have a living donor willing to donate bone marrow. This proposal modifies this approach to accommodate induction of "delayed tolerance" in a phase I/II protocol. The findings from this study will directly translate to deceased donor organ transplantation. This is of high priority because most organ donors are deceased. Therefore, this novel approach to expand the FCRx technology to delayed tolerance will represent a major milestone in potential commercialization. PUBLIC HEALTH RELEVANCE: Dependence upon anti-rejection drugs in clinical transplantation is expensive and results in significant morbidity and limitation in graft survival. Donor specific transplant tolerance would eliminate the need for drug- based immunosuppression, but has been an elusive goal in the clinic for over a half century. We have developed a potentially transformative approach to achieving transplant tolerance in solid organ transplant recipients based upon a rationally bioengineered stem cell product, termed FCRx. This proposal will determine the safety and efficacy of FCRx for delayed tolerance induction in kidney transplant recipients and will also be directly applicable also to deceased donor organ allograft recipients which comprise the majority of organ transplants.

PI Name: Suzanne T. Ildstad
Title: Training Program in Transplantation

Grant Number: T32HL076138
Fiscal Year Search: 2013

http://www.projectreporter.nih.gov/project_info_description.cfm?aid=7893659&icde=5639745

DESCRIPTION (provided by applicant):

This is a resubmission of a competing renewal for a highly successful training program in transplantation immunology and stem cell biology. Over the past 11 years, a dynamic group of internationally recognized investigators in transplantation and stem cell biology were strategically recruited to the University of Louisville which is increasingly recognized for its comprehensive translational research in composite tissue allotransplantation, tolerance, and stem cell-mediated regenerative medicine. This training program directly addresses the mission of the NIH training grant program to help ensure that a diverse and highly trained workforce is available to assume leadership roles related to the nation's biomedical research agenda. All positions have been filled with outstanding applicants to date including six women (one minority), thereby addressing the major concern of all three reviewers. Applicants must have a Ph.D., D.V.M., or M.D. They are required to submit a CV, publication list, three letters of recommendation, and a statement of the specific research proposed. An Executive Training Committee comprised of the program Director and senior trainers selects the trainees. Trainees assemble a program and undertake basic research with one trainer and one or more co-mentors. Evaluation of the Training Program occurs on several levels, including manuscript and abstract submissions; oral presentations and participation at research seminar series; and completion and submission of an NRSA postdoctoral individual training grant or equivalent (AHA, JDRF, ASH, or other foundation or society fellowships) within the second year of training. The overall progress of this Training Program is assessed by an External Advisory Board through semiannual meetings and feedback. The External Advisory Board also assists in minority recruitment. The faculty in this training program has the expertise and breadth of background to successfully train fellows in well-equipped, centrally-located laboratories, it is our goal that our trainees will be qualified to assume leadership positions related to transplantation and stem cell biology in industry, academia, and biotechnology. Our established track record demonstrates that we are well on our way to achieving this goal.

PI Name: Sufan Chien, MD
Title: A new technique for treating hemorrhagic shock

Grant Number: R43HL114235
Fiscal Year Search: 2013

http://projectreporter.nih.gov/project_info_description.cfm?aid=8461125&icde=16967230&ddparam=&ddvalue=&ddsub=&cr=1&csb=default&cs=ASC

DESCRIPTION (provided by applicant):

The specific aims of this proposal are to use a newly developed intracellular energy delivery technique to enhance resuscitation, and to study the relationship between tissue high-energy phosphate supplementation and tissue necrosis and apoptosis during hemorrhagic shock. In most western countries, trauma/hemorrhage is the leading cause of death up to the age of 40. The most common consequence of severe trauma and hemorrhage is shock, which is a hypovolemic condition in which oxygen delivery to the body is inadequate for the generation of the adenosine triphosphate (ATP) and adenosine diphosphate (ADP) necessary to maintain the function and structural integrity of tissues. Treatment strategies for shock have changed very little in the past half century and the incidence of mortality and morbidity remains very high. Depletion of high-energy phosphates during hemorrhagic shock was described decades ago, and direct administration of free Mg-ATP was reported to increase tissue ATP concentrations, tissue and mitochondrial Mg levels, and cellular functions by some authors. However, this approach is controversial, not only because the protective effects cannot always be duplicated by other scientists, but also because the half life of ATP is very short in the blood circulation-less than 40 seconds. Furthermore, it i well known that highly charged molecules like ATP do not normally cross the cell membrane. We have developed a technique for intracellular Mg-ATP delivery (named ATP- vesicles or VitaSolTM). Preliminary results have indicated a very promising effect in several models of tissue hypoxia and ischemia, including shock. Our hypothesis is that the replenishment of intracellular ATP levels alleviates or eliminates many of the detrimental effects caused by hemorrhagic shock, thereby increasing survival time. This Phase I proposal has two aims: 1) To establish the effect of VitaSolTM treatment on hemorrhagic shock; and 2) To investigate the relationship between tissue high- energy phosphate contents and necrosis and apoptosis during hemorrhagic shock, and the effect of VitaSolTM treatment on these changes. These studies have not been performed in the past. The success of this project will likely provide a totally new therapeutic approach for treatment of severe trauma and shock. Further study of this new energy delivery technique may also benefit various clinical conditions, such as coronary heart disease, stroke, spinal cord injury, cardiopulmonary bypass, organ transplant, chronic wounds, and many other conditions where ischemia is involved. The potential impact on medicine is high.

PI Name: Jason Wayne Smith
Title: Adjunctive Peritoneal Resuscitation in Hemorrhagic Shock

Grant Number: 5K23GM095859-02
Fiscal Year Search: 2013

http://projectreporter.nih.gov/project_info_description

DESCRIPTION (provided by applicant):

This K23 proposal will enable Jason W. Smith, MD, to achieve his goal of becoming an independent investigator focusing on clinical and translational research in hemorrhagic shock and inflammation. Dr. Smith will conduct a prospective clinical study evaluating the effects of adjunctive direct peritoneal resuscitation (DPR) on patients undergoing damage control surgery and hemorrhagic shock. Highlights of Dr. Smith's career development plan include: (1) obtaining a PhD in Physiology and Biophysics focusing on microcirculation and hemorrhagic shock, (2) experience running a prospective clinical trial at a large academic hospital, and (3) technical laboratory training in experimental methods that can be applied across a wide range of future experiment subjects. These activities will occur in the setting of the University of Louisville and the University Of Louisville Hospital Trauma Institute and will be monitored quarterly by an advisory committee. An estimated 46 million Americans suffer a traumatic accident yearly. A number of those patients will suffer hemorrhagic shock and its sequelae as a result of their injury. Our preliminary data document (i) improvement in patient outcomes using adjunctive peritoneal resuscitation, and (ii) improvement in liver blood flow after administration of DPR. Our hypothesis is that DPR therapy will result in improvement in mechanisms postulated to play a role in the development and progression of systemic inflammatory response due to hemorrhagic shock. To test this hypothesis, we will perform a prospective randomized controlled, mechanistic study of DPR administration in 100 patients undergoing damage control surgery for hemorrhagic shock over a 30 month period. The expected primary endpoints are an (i) increase in hepatic blood flow during resuscitation in patients treated with DPR , (ii) reduction in cellular edema and hepatocyte necrosis similar to our laboratory findings, and (iii) a reduction inflammatory response and chemotactic mediators compared to the conventional resuscitation group. Additionally, expected secondary clinical endpoints are (i) improved time to abdominal closure, (ii) decreased fluid resuscitation requirements with decreased vascular permeability, and (iii) a reduction in post-operative complications following damage control surgery. These studies utilize state of-the art well validated techniques in a translational approach to develop an enhanced understanding of a potential innovative therapy for hemorrhagic shock. PUBLIC HEALTH RELEVANCE: This K23 proposal will enable Jason W. Smith, MD, to achieve his goal of becoming an independent investigator focusing on clinical and translational research in hemorrhagic shock and inflammation. We will perform a randomized controlled study of adjunctive peritoneal resuscitation in patient suffering from hemorrhagic shock secondary to traumatic injury during the 30 month study period.

PI Name: H. Sam Zhou
Title: Adenovirus E1B55K Functions Related to Oncolytic Replication

Grant Number: R01CA129975
Fiscal Year Search: 2013

http://www.projectreporter.nih.gov/project_info_description.cfm?aid=7777755&icde=5639745

DESCRIPTION (provided by applicant):

Two major "hallmarks" of all cancer cells include dysregulated cell cycle and inhibited apoptosis, both of which are also involved in adenovirus (Ad) infection. These two processes are primarily conducted by viral oncoproteins E1A and E1B. Since the function of viral E1 protein is similar to that of cancer cellular factors that promote proliferation and inhibit apoptosis, viruses with mutations in oncoprotein E1A and E1B can selectively replicate in cancer cells. One such mutant with deletion of E1B55K, known as dl1520, has been used in clinical trials documenting oncolytic effects. However, the mechanism of viral oncolytic replication has not been well characterized, and the therapeutic efficacy needs improvement. It is generally believed that the major role of E1B55K is to bind to and inhibit p53 activation, but many studies have documented that E1B55K-mediated p53 inactivation is not required for virus replication. Our laboratory has shown: (1) that the limited spread of mutant viruses in large tumors is a key factor in decreased therapeutic efficacy; (2) increased Ad E1A expression enhances virus-oncolytic replication; and (3) apoptosis caused by E1B deletion can partially decrease virus replication but does not change the final outcome of virus-mediated cancer killing. Our recent studies have revealed that E1B55K has a novel function in the induction of cyclin E and other cell cycle-related genes. Most importantly, we also observed that increased cyclin E expression is correlated with virus replication efficiency. E1B55K-induced cyclin E expression is required for virus replication in normal cells, but is not necessary in cancer cells. We hypothesize that E1B55K may target cellular factor(s) to increase cyclin E expression, and this factor(s) may already be activated in cancer cells. Thus, cyclin E dysregulation in cancer cells may be the molecular basis for oncolytic replication of E1B55K-deleted viruses. Our research team will (1) identify cellular factors targeted by E1B55K for cyclin E induction, (2) define the mechanism by which E1B55K activates cyclin E expression, and (3) determine the relationship between cyclin E overexpression and oncolytic replication of E1B-deleted viriuses. If we confirm that oncolytic replication relies on cyclin E expression and cell proliferation, patients with aggressively growing tumors and dysregulated cyclin E should greatly benefit from this adenoviral therapy. The long-term goal of this work is to increase the efficacy of oncolytic cancer gene therapy. PUBLIC HEALTH RELEVANCE: Adenoviruses lacking an important regulative protein-E1B55K-still can amplify in some cancer cells. Therefore, the E1B55K mutant dl1520 has been used in clinical trials. It is important to understand the E1B55K function and the selective replication of E1B55K-deleted dl1520 in cancer cells. Our recently published studies have revealed that E1B55K has a novel function in the induction of cyclin E expression, which is crucial for DNA replication. Cancer cells generally express high levels of cyclin E or have a dysregulation of the gene. We reason that viral E1B55K may activate some cellular factors that increase cyclin E expression for viral DNA replication. Cancer cells may already have the factors activated; therefore cancer cells do not require the E1B55K function. The study of this possibility is very important. If we confirm that mutated virus replication relies on cyclin E expression, patients with aggressively growing tumors and dysregulated cyclin E should greatly benefit from this adenoviral therapy. The long-term goal of this work is to increase the efficacy of oncolytic cancer gene therapy.

PI Name: Sufan Chien, MD
Title: Intracellular Energy Delivery and Diabetic Wounds

Grant Number: R01DK074566
Fiscal Year Search: 2010

http://www.projectreporter.nih.gov/project_info_description.cfm?aid=7803637&icde=5639745

DESCRIPTION (provided by applicant):

The long-term goal of our program is to develop a safe and effective technique to combat various tissue ischemic damages. The specific aim of this proposal is to use our newly developed proprietary intracellular energy delivery technique to promote healing of diabetic wounds. Of the 17 million Americans with diabetes, approximately 2.5 to 4.5 million will develop a chronic wound in their lifetime. The overall cost of diabetic foot problems, including loss of productivity, could be as high as $20 billion per year. Despite thousands of dressing products developed to treat wounds, none have shown consistent effect. We propose a new approach for chronic wounds. Our central hypoth- esis is that wound tissue hypoxia results in depletion of adenosine triphosphate (ATP), which is the fundamental cause of non-healing chronic wounds, and a direct intracellular ATP delivery will improve microenvironment of wound tissue and facilitate healing process. Direct energy supply for wound treatment has never been attempted before, and the relationship between increased energy supply and wound healing process is entirely unknown. During the tenure of the Pi's NIH grant entitled "Enhanced glycolysis for hypothermic heart preservation", a new technique for direct intracellular delivery of ATP has been developed in which a special carrier is used to encapsulate ATP. The composition of this carrier is similar to the cell membrane. When the carrier meets with the cell membrane, it fuses with it and delivers the contents into the cytosol. Preliminary results indicate that this new energy delivery technique can provide significant protection to ischemic cells and tissues. The technique has shown very promising effects on normal and ischemic wounds. Three US patents and more than 12 international patents have been filed and the innovation has also been reported to the NIH. Our preliminary results also indicated that high-energy phos- phate contents were severely depleted in human chronic wounds, and treatment with ATP-vesicles in animal wounds increased tissue high-energy contents. Five hypotheses will be tested: (1) high-energy phosphate contents are decreased in chronic diabetic wounds; 2) an ischemic wound model created using a minimally invasive surgical technique can be tolerable to diabetic animals; 3) intracellular ATP delivery will increase wound tissue energy levels to facilitate healing; (4) by providing energy to wound tissue, improved healing is achieved through coordinated upregulation of growth factors and other healing mechanisms; and (5) direct intracellular energy delivery will enhance wound healing by improved tissue perfusion. These issues have not been explored in the past, but our preliminary results have established the basis for the success of this project. The expansion of usage of the direct intracellular energy delivery is likely to have a major impact on medicine. It will not only improve chronic wound care, but also help our treatment to various ischemic conditions, such as severe trauma, shock, stroke, heart attack, spinal cord injury, cardiopulmonary bypass, organ transplant, and many other acute and chronic ischemic diseases.

PI Name: Brian G. Harbrecht, MD
Title: Hepatocyte Nitric Oxide Synthase Regulation by Glucagon and Insulin

Grant Number: R01DK055664
Fiscal Year Search: 2010

http://www.projectreporter.nih.gov/project_info_description.cfm?aid=7743768&icde=5639745

DESCRIPTION (provided by applicant):

The immune response to sepsis involves a series of complex, highly integrated homeostatic responses that, if prolonged and excessive, can lead to organ dysfunction and death. Nitric oxide (NO) synthesis is upregulated by sepsis in many tissues and is an essential component of the host immune response. Nitric oxide synthesis can be beneficial and improve immune and organ function, but if synthesis is excessive and prolonged, NO can promote organ injury, tissue inflammation, and death. NO is produced in hepatocytes by the inducible nitric oxide synthase (iNOS) that is stimulated by cytokines and proinflammatory stimuli. Excessive NO from iNOS produces cellular dysfunction and hepatic injury. Glucagon and cyclic adenosine monophosphate (cAMP) regulate hepatic iNOS expression in vitro and in vivo, and by doing so, decrease NO-mediated hepatic injury. Our preliminary data demonstrate that insulin also down-regulates cytokine-induced iNOS expression. Both glucagon and insulin alter specific intracellular signaling pathways in hepatocytes, but the mechanisms involved in the regulation of hepatocyte function in sepsis by glucagon and insulin, and specifically the regulation of hepatocyte iNOS expression, have not been identified. In this proposal, we will determine the mechanisms responsible for the regulation of hepatocyte iNOS expression by glucagon and insulin. In Aim I, we will continue our work in determining the mechanism for the glucagon and cAMP-induced inhibition of hepatocyte iNOS expression. We will focus on protein kinase A (PKA)-independent pathways induced by cAMP and evaluate the role of the guanine nucleotide exchange factor Epac and the role of calcium. In Aim II, we will determine the mechanisms responsible for the inhibition of iNOS by insulin. By defining how these hormones regulate hepatocyte iNOS expression, we will provide a framework for understanding the basic pathophysiologic cellular events in shock and sepsis that may lead to novel cellular-based therapies for critically ill patients. Project Narrative: Nitric oxide is synthesized in critically ill patients during septic shock, and when overproduced, can increase cellular dysfunction, tissue injury, and death. Glucagon and insulin primarily regulate blood glucose, which has become an important facet of the care of critically ill patients, but we have found that they also regulate hepatic nitric oxide production. We will determine the mechanisms responsible for the regulation of hepatocyte inducible nitric oxide synthase (iNOS) expression by glucagon and insulin. By defining these mechanisms, we will provide a framework for understanding the basic cellular events in shock and sepsis, which may lead to novel cellular-based therapies for critically ill patients.

PI Name: James B. Hoying
Title: Fabricated Microvascular Networks

Grant Number: R01EB007556-05
Fiscal Year Search: 2010

http://www.projectreporter.nih.gov/project_info_description.cfm?aid=7822693&icde=5639745

DESCRIPTION (provided by applicant):

Fabricated Microvascular Networks. The importance of an effective vascular supply for tissue health is universally accepted. In developing strategies to build vasculatures for tissue engineering and other therapeutic applications, it is important to recognize that, foremost, the new vasculature must quickly provide sufficient blood flow to the target tissue to preserve cell viability. We have found that new microvessels formed in vitro can begin to carry blood within the first days following implantation. However, flow patterns are atypical and likely ineffective at establishing normoxia until many days later. The delay is primarily due to a lack of organization within the network at the time of implantation and the time needed to develop new mature inflow and outflow pathways. We hypothesize that pre-determining an appropriate network organization prior to implantation would reduce the amount of time needed for the new microvasculature to effectively perfuse a tissue. We have established generic technologies utilizing a direct-write tissue printing tool for patterning and organizing tissue components for tissue engineering applications. We propose to implement this technology to design and fabricate pre-patterned, 3-dimensional microvascular networks with pre-existing inflow and outflow pathways. Also, we will use an in vitro, intravascular-perfusion bioreactor system to establish flow through the networks to further organize and mature the microvascular networks prior to implantation. Computational modeling and physiological analyses serve to direct design strategies and characterize the architectures and functionality of the fabricated vasculatures both in vitro and in vivo. In addition to providing an enabling technology platform for assembling pre-determined microvascular networks, this work will provide a foundation from which to explore the importance of network architectures in vascular function.

PI Name: Suzanne T. Ildstad
Title: Tolerance induction to islet transplants

Grant Number: R01DK069766
Fiscal Year Search: 2009

http://www.projectreporter.nih.gov/project_info_description.cfm?aid=7674545&icde=5639745

DESCRIPTION (provided by applicant):

The focus of this proposal is to develop a novel "conditioning" approach that will replace myelotoxic agents to establish chimerism in NOD mice. We will induce immune deviation to promote host-versus-graft hyporesponsiveness, thereby giving the hematopoietic stem cell (HSC) an opportunity to engraft and establish subsequent self-perpetuating deletional tolerance to islet allografts. Our recent studies in a mouse model suggest that the primary role for conditioning for HSC transplantation is to suppress host-versus-graft alloreactivity, rather than to prepare vacant niches in the recipient's bone marrow compartment. This observation suggests that one could replace myelotoxic agents with antigen-specific approaches to induce host-versus-graft hyporeactivity or anergy at the time of HSC transplantation. As the mechanisms underlying T cell activation are defined, highly specific approaches to suppress this alloreactivity have emerged. In AIM I. we will ESTABLISH CHIMERISM THROUGH IMMUNE DEVIATION OF THE RECIPIENT. We will immunomodulate the recipient: (a) targeting alloreactive cells in the host microenvironment; (b) inducing anergy and/or antigen-specific apoptosis of alloreactive host cells; and (c) through generation of regulatory T cells (Treg), and develop a novel nonmyeloablative conditioning regimen to induce antigen-specific hyporesponsiveness to the HSC and islet allografts. Cell-based therapies have great potential for inducing transplantation tolerance. Of greatest interest are the new subpopulations of bone marrow-derived dendritic cells (DC) that have recently been shown to be potently tolerogenic in vitro under certain circumstances. We are the first to demonstrate an in vivo engraftment-enhancing effect for precursor plasmacytoid DC (p-preDC). The exploitation of this discovery in vivo and its potential to reduce the need for myelotoxic conditioning has not yet been tested. Hematopoietic growth factors have also been used to drive the immune response to a tolerogenic T helper 2 (Th2) phenotype through production of p-preDC or other tolerance-promoting cells (graft facilitating cells {FC}) that in turn generate Treg. In AIM II, we will USE PRE-TRANSPLANT IMMUNOMODULATION OF THE DONOR WITH HEMATOPOIETIC GROWTH FACTORS TO GENERATE TOLEROGENIC CELLS IN THE HSC ALLOGRAFT. We will use these factors and the cells they generate to modulate the tolerogenicity of the donor marrow inoculum in vivo to tip the immune milieu in favor of graft acceptance, enhancing bone marrow chimerism without myelotoxic conditioning. We will examine the mechanism by which this occurs and identify which cell types in the graft are critical to tolerance induction. P-preDC exposed to apoptotic donor antigens are potently tolerizing in vitro through generation of Treg. The therapeutic application of this approach has not been tested in vivo. In AIM III, we will USE EX VIVO IMMUNOMODULATION OF THE MARROW to expand p-preDC and FC and induce a tolerogenic inoculum for HSC transplantation.

PI Name: Robert C.G. Martin
Title: Prevention of Reflux-Induced Esophageal Adenocarcinoma By Dietary Berries

Grant Number: R03CA137801
Fiscal Year Search: 2009

http://www.projectreporter.nih.gov/project_info_description.cfm?aid=7689145&icde=5640031

DESCRIPTION (provided by applicant):

Esophageal adenocarcinoma (EAC) has the fastest rate of increase among all cancers in America, yet there is very little research on the mechanisms and prevention of its development. EAC primarily affects White males and has one of the poorest prognoses among all cancers. Barrett's esophagus (BE) is a pre-cancerous lesion associated with the development of EAC. BE is caused by chronic heartburn, which affects more than 19 million Americans every year. Pharmaceutical treatments to cure heartburn do not appear to largely affect EAC incidence. New intervention strategies are urgently needed to address this issue. Commonly available berries, such as blueberries (BB) and black raspberries (BRB), are natural chemopreventative agents that can be easily incorporated into the American diet and used for clinical intervention. Moreover, dietary BRBs are highly effective in preventing squamous cell carcinoma (SCC), another type of esophageal cancer, the incidence of which is declining. We have developed an animal model that mimics the clinical development of EAC. In this model, chronic esophageal reflux resembling heartburn is induced in rats using a surgical procedure. More than one-third of the rats develop EAC at the end of 6 months. We can effectively use this model to study the preventive efficacy of dietary berries. Two types of berries have been chosen, since they differ widely in their phytochemical profiles. Our objective is to study the efficacy of dietary berries to prevent the development of reflux-induced EAC and to understand the mechanisms by which they do so. To achieve this, we plan to provide BB and BRB via the diet to male rats at a dose of 2.5%, which is equivalent of consuming one-half cup of dried berries every day. We will surgically-induce chronic reflux. At the end of 6 months, the rats that were provided with berry diet will be compared with those that did not receive intervention to evaluate the incidence of tumors. To further understand the mechanisms by which berries may prevent esophageal tumorigenesis, we plan to use intermediary monthly time points during the development of EAC to study the biomarkers that are altered. The first set of biomarkers deal with oxidative stress and will determine whether berries can induce cellular antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, which ultimately protect the cells from oxidative damage. Lipid peroxidation and oxidative DNA damage will be used as markers of oxidative stress. The second set of biomarkers deal with inflammation associated with BE. Nuclear factor kappa B (NF?B), a transcription factor that controls the expression of several cancer-inducing genes, is found to be activated by inflammation in BE. We will evaluate whether berries can prevent this activation. Thus, by following a two-part approach, we will be able to determine whether berries prevent EAC progression and the mechanisms involved in this prevention.

PI Name: Stuart Williams
Title: A Prevascularized Islet Immunoisolation Devcie

Grant Number: R01DK078175
Fiscal Year Search: 2010

http://www.projectreporter.nih.gov/project_info_description.cfm?aid=7786955&icde=5639745

DESCRIPTION (provided by applicant):

Beta-cell replacement therapy via islet transplantation remains a promising technology for the reversal of type 1 diabetes. A significant barrier to the clinical utilization of beta cell transplants has been the lack of a host-derived blood supply to maintain the viability and thus the function of transplanted cells. We have developed a new cell-based therapy for the generation of pre-vascularized tissue engineered constructs. We have also developed a new generation of biomaterials that support extensive neovascularization. The combined cell and material construct to be evaluated is termed a Prevascularized Immuno-Isolation Device or PVID. We propose to use these materials in the development of a new beta-cell immuno-isolation device to prolong beta cell viability and function. These constructs represent a pre-formed microcirculation that can be constructed from a patient's own fat-derived microvascular endothelial cells, avoiding the use of immuno- suppressive drugs. Specific aim 1 will evaluate the maturation of the microcirculation within a prevascularized construct following implantation in an animal model. Specific aim 2 will evaluate novel porous biomaterials and material surface modification to support the neovascularization of the porous material to assure perfusion of encapsulated islets. The biomaterial developed is a two component hybrid system that also provides immunoisolation for the encapsulated islets. Specific aim 3 will evaluate the viability and function of islets encapsulated in the prevascularized immunoisolation devices in an animal model of diabetes.