LABORATORY SERVICES RESEARCH PROGRAM
Willy A. Flegel, MD
Immunogenetics and molecular immunohematology are the translational research topics of the Laboratory Services Section (LSS) in the Department of Transfusion Medicine (DTM). Molecular methods were introduced first in HLA laboratories, and red cell genotyping followed approximately by the year 2000. Clinical applications have been expanded and refined ever since. We promote moving transfusion medicine laboratories rapidly into the molecular arena, because molecular tools have been shown to improve patient care to a level that cannot be achieved by any available serology alone.
Milestones that we accomplished include: the elucidation of the physical structure of the RH gene locus, which led to the understanding of the molecular basis and mechanism of the RHD deletion (see Figure 1); the determination of RHCE as the ancestral RH gene, while the RHD is the duplicated gene; the molecular bases of the weak D phenotypes and the prevalent DEL phenotype; the definition of ERMAP as the gene encoding the Scianna blood group antigens; and the application of red cell genotyping in hematopoietic progenitor cell transplantation.
Figure 1. Molecular basis of Rh negative. (A) The physical structure of RHD and RHCE genes on the short arm of chromosome 1. (B) An unequal crossing-over event between the 2 Rhesus boxes (triangles) was triggered by their nucleotide sequence homology. (C) Resolving the crossed-over chromosome yields the RH gene structure of the extant RHD negative haplotype that is prevalent in humans. Adapted from Blood 95(2000)3667 [disclaimer].
Our current research interests include: red cell genotyping; relationship of genotype, phenotype and function of blood group proteins; stem cell surface antigens in vivo and during ex vivo expansion; and immunogenetics in hematopoietic stem cell transplantation focusing on HLA and KIR genes. We develop molecular immunohematology and immunogenetics methods for blood transfusion, hematopoietic progenitor cell transplantation and immune monitoring, and apply these methods to clinical services for patient care. The projects represent translational research in support of NIH Clinical Center patients, often in collaboration with other NIH investigators.
Our translational research has led to several methods with clinical applications. Their implementation, for example by commercially available kits and platforms, has increased blood component safety. The availability varies among health-care systems, notably between North America and Europe. The advent of such Conformité Européenne (CE)-labeled test kits has rendered it technically and legally possible, within the specifications of the CE-certification process for in vitro diagnostic devices in the European Union, to replace several blood group serology tasks by red cell genotyping; this is not the case in the US at this time.
Strengthening the clinical approach by using molecular technologies that were previously restricted to applied research fits well into DTM’s goals of providing cutting edge clinical services. DTM laboratory services are offered nationwide as a strong reference laboratory resource for selected immunohematology and HLA work. We will demonstrate the applicability of the available technologies in the population, improve available technologies, facilitate implementation in national blood group laboratories and establish a dataset to gauge the cost-benefit. We envision molecular typing as the future for resolving several remaining issues in blood component compatibility. Molecular typing benefits many patient groups, such as pregnant women and patients with sickle cell disease, who are vulnerable to immunization or subsequently become difficult to supply with compatible blood.
LSS is a research environment combining access to unique patient and blood donor specimens with an established expertise in molecular technology and a primary interest in translational research.
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BLOOD SERVICES RESEARCH PROGRAM
Susan F. Leitman, M.D.
The Blood Services Section is responsible for maintaining a volunteer donor registry of whole blood and apheresis donors sufficient to meet 100% of the transfusion needs of Clinical Center patients; for maintaining a research donor registry sufficient to meet 100% of the needs of NIH investigators for blood components for in vitro research use; for providing therapeutic apheresis services and blood stem cell collections to support clinical transplant and other patient-care protocols; and for maintenance of one of the largest hospital-based National Marrow Donor Program registries in the country. In line with these functions, the section conducts investigator-initiated and collaborative research focused in four areas:
- Establishing programs to study and treat iron deficiency and other eligibility determinants in volunteer blood donors, with the goal of maximizing blood donor eligibility status.
- Developing a comprehensive program for management of patients with hereditary hemochromatosis in the Blood Center, involving clinical and molecular studies of response to phlebotomy therapy.
- Optimizing the safety and efficacy of apheresis donations.
- Collaborating with NIH investigators to perform prospective trials of therapeutic apheresis and evaluate novel hematopoietic transplantation strategies.
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CELL PROCESSING RESEARCH PROGRAM
David Stroncek, MD
New Assays for Assessing Cellular Therapies
The production of cellular therapies is complex. Most clinical cell therapy products require cell mobilization, collection, subset isolation, in vitro or in vivo stimulation, and culture over a period of several days. The production of some cellular therapies involves serial isolation steps and multiple stimulation and/or culturing steps. Cellular therapy product manufacture is further complicated by donor or patient genetic and physiological heterogeneity. The final product is often markedly different from the starting material.
Despite these complexities cellular therapies must be of high quality. Cellular therapies must provide the desired clinical affect without resulting in adverse effects. An adequate dose of cells must be provided to each patient, each product must meet release specifications, and lot-to-lot variation should be minimized.
In order to produce consistently high quality products, quality assurance has become a critical part of cellular therapy laboratories. Cell therapies must be safe, pure, sterile, stable, and potent. To ensure that cell therapies have these properties, they are tested in many ways both during and at the end of their production. The analysis of cell therapies has become an important part of the delivery of effective clinical cellular therapies.
The goal of these studies is to develop novel methods to assess the quality of cellular therapies. Traditional analytic assays including ELISA, flow cytometry, and ELISPOT have many limitations. Better assays are needed to assess the complex and multiple functions of cellular therapy products, some of which are not well understood. Gene expression profiling using microarray technology has been widely and effectively used to assess changes of cells in response to stimuli and to classify cancers. Preliminary studies have shown that the expression of noncoding micro RNA which play an important role in cellular development, differentiation, metabolism and signal transduction can distinguish different types of stem cells and leukocytes. Both gene and micro RNA expression profiling have the potential to be important tools for assessing cellular therapies. In addition, these assays could be used to identify biomarkers that correlate with the safety, purity, and potency of cellular therapies; validate that therapies prepared using different methods are equivalent; and assess changes in the cellular therapy manufacturing process. The development of these assays will lead to safer and more effective therapies for all patients being treated with cellular therapies.
- Develop novel molecular methods to access cellular therapies
- Identify and validate biomarkers for cell potency, purity, and viability
- Validate biomarker expression by the cellular therapies with their clinical effectiveness
Development of New Cellular Therapies
The cell therapy laboratory invests a considerable amount of resources in the development of new cellular therapies. While the development of most of these cellular therapies are initiated in the laboratory of clinical investigators, the Cell Processing Laboratory must scale up and validate these procedures. For many protocols this process involves considerable scientific, medical, regulatory and laboratory input from the Cell Processing Section.
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CLINICAL STUDIES SUBSECTION (CSS)
Harvey J. Alter, MD
Chief, Clinical Studies Subsection
Associate Director for Research
CSS conducts a large-scale, investigator-initiated, and collaborative research program. The long-term goals of this research program are to: 1) study and prevent transfusion-associated infections; in the past, these studies focused on transfusion-associated hepatitis, but have now expanded to investigate all potential transfusion-transmitted agents; 2) explore new technologies for the detection and study of blood-transmitted agents including highly sensitive antibody assays and gene array technology; 3) characterize novel and established blood-transmitted agents, their infectivity, and their susceptibility to inactivation/neutralization; 4) prospectively study donors infected with hepatitis C virus to determine the natural history and long-term outcome of this infection; 5) study the immunologic, virologic and gene-associated differences between HCV chronic carriers and those who spontaneously recover; 6) study neutralizing antibodies to HCV and develop a hepatitis C hyper-immune globulin; 7) develop an HCV vaccine model; 8) develop a cell line susceptible to HCV infection using siRNAs to block critical steps in the innate immune system; 8) study the mechanisms underlying accelerated fibrosis progression in persons transplanted for HCV-related end-stage liver disease.
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INFECTIOUS DISEASE AND IMMUNOGENETICS SECTION (IDIS), DTM, CC, NIH
Ena Wang, MD
Acting Chief and Technical Supervisor
In the 2004 report entitled: “The Yin and Yang of intra-tumor inflammation”, we proposed the pursuit of clinical and experimental observations aimed at the identification of the mechanisms switching a chronic inflammatory process conducive to cancer growth into acute inflammation causing cancer rejection. We postulated that this “switch” could only be caught by observing the dynamic phase of tumor/host interaction during immunotherapy at the site where they matter: the tumor microenvironment (Figure 1) (1).
Figure 1. Postulated interactions between immune and cancer cells at various stages of carcinogenesis and progression; Mantovani A et al, Lancet 2008 (2). The IGL focuses on the “immunotherapy” stage.
Following these tracks, we collected during the last four years, snap shots of events occurring during tumor rejection in humans receiving immunotherapy or in animal models of immune rejection that lead to the identification of recurrent themes; based on these observations, we hypothesized that the mechanisms leading to immune-mediated tumor rejection a) are similar independent of treatment and b) parallel those causing acute allograft rejection, clearance of pathogen or flares of autoimmunity. Using the human cancer model, we observed the chain of events that precede, accompany and follow tumor rejection during systemic administration of interleukin (IL)-2, active specific anti-cancer vaccination for the treatment of metastatic melanoma or the local application of the Toll-like receptor (TLR)-7 agonist imiquimod for the treatment of basal cell carcinoma (BCC). Comparison of immunological signatures occurring in such conditions with signatures associated with immune-mediated, tissue-specific destruction (TSD) reported by others in the context of viral infection, allo-transplantation and autoimmunity, lead to the formulation of the “immunologic constant of rejection” hypothesis (3): immune-mediated tissue-specific destruction follows a common pathway independent of the originating cause and the disease context.
- Research Goals – The case for “Comparative Immunology”
We aim to expand these studies following two broad research interests: 1) confirm, validate and refine the immunologic constant of rejection hypothesis and, 2) identify therapeutic strategies aimed at the modulation of the common effector mechanisms leading to TSD that could be utilized for the treatment of a broad spectrum of diseases. These goals can be most effectively achieved through a combination of hypothesis-generating and hypothesis-testing steps.
We have consistently proposed that the study of complex human diseases can only be done effectively adopting a global, discovery-driven approach that takes into account the various components of human biology (genetic code, transcriptional patterns and protein/protein interactions) and the uncontrollable complexity of human disease affected by the distinct genetic background of the host and the rapidly evolving phenotypes of cancer or virally infected cells (19, 20). Thus, our major research goals can be separated into three categories: 1) Development of cost/effective strategies for the study of genomic/transcriptional/translational interactions; 2) analysis of the genetic background of the host and its influence on the natural history of disease; 3) analysis of evolving disease heterogeneity and its influence on its natural history.
As the scientific thinking evolved, we realized that our goals can be most effectively achieved through a “comparative immunology approach”. The term comparative immunology refers to parallel analyses of immunological phenomena across species; here, we suggest that a similar strategy could be adopted to identify similarities among immunologically-mediated phenomena independent of their causative mechanisms and evaluate what common minimal denominator sits as a sine qua non requirement for their determinism. Thus, we call for a paradigm shift in the way research is done in humans and for the determination of novel strategies discussed in the following section. A good example of the recognition this approach has received is a new NIH intra-mural initiative called the “Center for Human Immunology (CHI)” designed to tackle immunologic questions independent of clinical disciplines through an inter-institutional approach; Dr. Wang and I were asked to guide the development of the genomics program of CHI.
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