Role of Endotoxin-Directed Innate Immunity in Acute Graft-Versus-Host Disease (aGVHD)

Ofer Levy M.D., Ph.D.

Children’s Hospital Boston

Funded in December, 2006: $690000 for 3 years


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Determining How a Bacterial Toxin Helps Initiate Graft Versus Host Disease in Transplant Patients

This study will explore the role of a bacterial toxin (endotoxin), which normally resides in the intestine but enters the bloodstream as a consequence of chemo- and radio-therapy in patients with leukemia or lymphoma undergoing bone marrow transplantation, in initiating Graft-versus-Host Disease (GVHD) in transplant recipients.

Bone marrow transplant is the preferred treatment for patients with bone marrow cancers, including leukemia and lymphoma, who are not cured by conventional chemotherapy and for patients with genetic defects effecting the bone marrow elements (white cells, red cells, and platelets). These transplants are often lifesaving, but they frequently result in severe and sometimes deadly GVHD, in which donor immune T cells (white blood cells) recognize host tissues as foreign and mount an inflammatory attack against the patients' tissues.

Prior to transplantation, radiation and chemotherapy is used to reduce or try to eliminate the patient's own immune cells, so that they do not attack the transplanted bone marrow. This gives the transplanted immune cells (which are produced in bone marrow) the opportunity to take hold and fight the cancer. Chemo- and radiation therapy weakens the patient's immune cells and damages the intestinal lining, allowing bacterial endotoxins that normally reside in the intestine to seep into the patient's bloodstream and initiate inflammatory responses that can trigger GVHD.

Consortium researchers will examine how bacterial endotoxin helps to trigger GVHD. They will use complementary areas of expertise, including oncology/hematology (Dana-Farber), infectious diseases (Children's Hospital Boston), and microbiology (University of Iowa), to examine how GVHD is initiated in bone marrow transplant recipients. Their study will be conducted as part of an ongoing pilot clinical trial, funded by XOMA L.L.C., of Berkeley California, of the effectiveness of an experimental agent known as recombinant (because it is manufactured using recombinant DNA technology) bactericidal/permeability-increasing protein (BPI) (rBPI21) in preventing GVHD. This agent is a natural host defense protein that can neutralize the harmful effects of endotoxin and is produced by human white blood cells. Of note, BPI is severely depleted during chemotherapy, as shown by the investigators in preliminary studies.

While XOMA will assess how effective (rBPI21) is in preventing GVHD, consortium researchers will examine how (rBPI21) may work, and whether it neutralizes the effects of the bacterial endotoxin in transplant patients. Consortium researchers hypothesize that the endotoxin molecule, located on the surface of intestinal bacteria, seeps into the patient's bloodstream and induces an immune “cytokine” (called tumor necrosis factor-α or TNF-α) to initiate inflammation. The inflammatory response, they hypothesize, then activates donor T cells to attack the recipient's inflamed tissues, producing GVHD. They further hypothesize that replenishing BPI, which is depleted by the chemotherapy, by intravenous administration of rBPI21 may neutralize this bacterial molecule, preventing this chain of events and reducing the occurrence of GVHD. They will analyze blood samples from patients undergoing transplantation and receiving (rBPI21) to see if their hypotheses are correct.

Significance: The research may reveal how bacterial endotoxin, which seeps from the intestine into the bloodstream in bone marrow transplant patients whose own immune system has been suppressed, helps to trigger GVHD, and how an experimental treatment may interrupt this process to prevent GVHD.


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Role of Endotoxin-Directed Innate Immunity in Acute Graft-Versus-Host Disease (aGVHD)

Myeloablative hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for both malignant and benign diseases of the bone marrow. However, HSCT is frequently complicated by acute graft-versus-host disease (aGVHD) wherein donor T lymphocytes target recipient tissue antigens. Animal models and human correlative data suggest that lipopolysaccharides (LPS, or “endotoxin”) derived from Gram-negative bacteria play a key role in HSCT-provoked aGVHD. Conditioning regimen-induced damage to the intestinal tract allows increased translocation of LPS from intestinal bacterial flora, triggering production of pro-inflammatory cytokines (e.g., TNF-α that activate allo-immune reactions culminating in aGVHD.

Our preliminary data indicate humoral and cellular changes consistent with LPS-induced systemic inflammation, including up-regulation of Toll-like receptor 4 (TLR4), a component of the LPS receptor. We also find that myeloablation-related neutropenia results in severely reduced plasma levels of bactericidal/permeability-increasing protein (BPI), a potent neutrophil-derived endotoxin-neutralizing host protein, and that individuals with lower baseline levels of plasma BPI have a significantly increased risk for aGVHD. Thus, both published and preliminary data suggest that replenishing BPI might shield patients from endotoxin and thereby reduce incidence of aGVHD.

In this context, a pilot phase I/II study is planned to examine the safety, tolerability, and pharmacokinetics of rBPI21, a 21 kDa recombinant N-terminal fragment of human BPI with an excellent safety profile and in vivo LPS-neutralizing activity, administered to individuals undergoing myeloablative allogeneic HSCT. We propose to take advantage of this pilot study as a unique opportunity to probe the relationship between endotoxin-directed innate immunity and aGVHD in patients undergoing HSCT. As rBPI21 is a selective inhibitor of endotoxin, our approach will assess the role of endotoxemia in triggering innate immune responses in vivo during HSCT.

We propose to examine banked samples from a cohort of HSCT donor:patient pairs (N = 150) to determine if common BPI genotypes are associated with aGVHD, a screen that may, for the first time, identify BPI genotypes that modify risk of aGVHD. A potential mechanism for such association will be explored by correlating aGVHD-associated BPI genotypes with BPI expression.
Comparison of blood plasma derived from control (observational study, N = 30) and treated (rBPI21 pilot study, N = 30) cohorts with respect to total and bioactive endotoxin, as well as plasma-mediated modulation of exogenous endotoxin bioactivity will characterize endotoxin-directed humoral innate immunity during myeloablative HSCT. In addition, measurement of in vivo production (plasma samples) of LPS-modulating acute phase reactants (LPS-binding protein and soluble CD14) and cytokines (IL-6, MCP-1, and sTNFRs) with distinct temporal patterns of up-regulation after HSCT will reveal the impact of endotoxin neutralization on the post-HSCT inflammatory response in vivo.

Finally, we will determine the effects of rBPI21 infusion on functional expression of the tri-partite endotoxin receptor composed of membrane CD14/TLR4/MD-2. Surface expression of CD14, MD-2 and TLR4, as well as spontaneous and endotoxin-induced production of intracellular TNF-a ,will be characterized by flow cytometry, defining relationships between bioactivity of endotoxin and endotoxin receptor expression.

Overall these studies will provide fresh insights into the role of endotoxin-directed innate immunity in triggering aGVHD.


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Ofer Levy M.D., Ph.D.

Ofer Levy, M.D., Ph.D. is a Principal Investigator in the Department of Medicine, Division of Infectious Diseases, Children's Hospital Boston and an Assistant Professor at Harvard Medical School. He is a graduate of the Bronx High School of Science, Yale College (B.S. in Molecular Biophysics and Biochemistry), and the Medical Scientist (M.D./Ph.D.) Training Program at New York University. Dr. Levy completed both a Residency in Pediatrics and a Fellowship in Pediatric Infectious Diseases at Children's Hospital Boston.

His research laboratory studies molecular mechanisms of innate immunity in immuno-compromised patients, including newborns and those undergoing chemotherapy, with a goal of employing innate defense molecules to prevent and/or treat infections in these susceptible populations.

Eva C. Guinan, M.D., is an Attending Physician in the Department of Pediatrics, Dana-Farber Cancer Institute and the Department of Medicine, Division of Hematology/Oncology, Children's Hospital Boston and an Associate Professor of Pediatrics at Harvard Medical School. She is a graduate of Harvard College (B.A. in Biochemistry), and Harvard Medical School. Dr. Guinan completed a Residency in Pediatrics at Children's Hospital Boston and a combined Pediatric Hematology/Oncology Fellowship at Children's and the Dana-Farber Cancer Institute.

Her research laboratory has a primary focus on induction of alloantigen-specific tolerance via costimulatory blockade as a means of limiting transplant-related immunosuppression. She works in collaboration with Dr. Ofer Levy on studies of innate immunity in human transplantation, with a goal of employing innate defense molecules to prevent regimen-related toxicity in susceptible populations.

Jerrold Weiss, Ph.D., is Professor of Internal Medicine and Microbiology at the Roy J. and Lucille A. Carver College of Medicine, at the University of Iowa. Dr. Weiss received his Ph.D. in Microbiology at the New York University School of Medicine.

His lab has long been interested in the molecular and cellular mechanisms of innate host defenses against invading bacteria, including the molecular mechanisms by which specific host molecules recognize specific bacterial products to mediate recognition and response to infection. During his tenure at NYU, Dr. Weiss and his mentor and colleague Dr. Peter Elsbach discovered and characterized the bactericidal/permeability-increasing protein (BPI), a potent antibiotic-like protein that recognizes Gram-negative bacterial endotoxins and thereby targets bacteria for elimination and neutralizes the potent pro-infammatory endotoxin. These properties have led to pre-clinical and clinical development of a bio-active recombinant fragment of BPI (rBPI21) for use in clinical circumstances when normal mechanisms of control of infection and of endotoxin are insufficient. This may include the acute immuno-pathology (i.e., acute Graft vs. Host Disease) that can follow bone marrow transplantation. More recent studies at the University of Iowa have focused on the molecular bases of the potent pro-inflammatory actions of endotoxin.