ITI Seed Grants 2008 - 2010

In November 2007 ITI established its first Interdisciplinary Research Program Awards in conjunction with the Beckman Center for Molecular and Genetic Medicine (CMGM) and the Mary Hewitt Loveless, M.D. Endowment.  The goals of this initiative were to support ground breaking studies in the areas of immunity, trasnplantation, and infection.  In addition the proposals had to show an emphasis on interdisciplinary medicine,  have a disease focus, and make use of the the Human Immune Monitoring Center (HIMC).  ITI funded eight projects which are outlined below.

On February 27, 2009 the eight seed grant recipients presented a one year progress report of their projects in the Clark Auditorium.  Below are summaries of the eight projects as they were submitted initially in March 2008 along with the one year progress updates presented this past February.

1) Immunophenotyping of Subjects with Near-fatal Food Allergy
Kari Nadeau (Pediatrics), Edgar Engleman (Pathology), Stephen Galli (Pathology, Microbiology and Immunology), and Mindy Tsai (Pathology)

March 30, 2009 (One year report)
Over the past year, we have made progress on all of the specific aims that were proposed in the ITI grant for Anaphylactic Food Allergies. Dr. Nadeau has met with Drs. Engleman and Galli throughout the past year to foster collaboration to enable a food allergy research group to continue at Stanford. All monies from the ITI grant have been used for supplies for basic science work in the lab and have not been used for any clinical trial purposes. The clinical trials mentioned in this report are funded separately; however, we are using blood samples from the subjects enrolled in those trials to assess whether food desensitization protocols decrease levels of pro-allergic cytokines and basophil activation markers and whether the diminution of these markers is correlated with conversion of the immune system from a state of sensitization for anaphylaxis to tolerance.

This past year, to address our overall objective, the Nadeau laboratory has collected 35 blood samples (goal=60) from children and adults with anaphylactic food allergies, 41 blood samples subjects with non-anaphylactic food allergies (goal=60), and 43 blood samples from age matched healthy controls (non allergic) (goal=60) under an IRB-approved Stanford protocol (#8629, Nadeau PI). These samples have been separated into PBMCs and plasma, deidentified, coded and stored in a database in the Nadeau laboratory using CryoTrack software. They are available for collaborations with any interested Stanford faculty. We continue to collect and store samples daily.
We appreciate the funding from the ITI Food Allergy Seed Grant and have made significant progress thus far. Our study addresses critical barriers in moving the field of diagnosing and treating food allergy forward.   We hope to be able to submit a manuscript this year and an R21 grant to be able to proceed with studies to enhance our understanding of mechanisms of tolerance vs. allergy induction in individuals with anaphylactic food allergies.

March 1, 2008 (Initial proposal)
Food allergy is emerging as a major public health problem; it affects 3-4% of adults and 6-8% of children in the United States and appears to be increasing in prevalence. For example, the prevalence of peanut allergy increased from 0.4% in 1997 to 0.8% in 2002. The fraction of new patients with the diagnosis of anaphylactic food allergy (i.e., a history of anaphylaxis to a food allergen with confirmatory diagnostic testing) referred to the Allergy, Asthma and Immunology LPCH/Stanford service has increased from 14% in 1998 to 67% in 2006. Approximately 50% of food allergic subjects will have an anaphylactic reaction. Allergies to peanuts, shellfish, and tree nuts, the most common causes of life-threatening allergic reactions, persist throughout life in the majority of subjects. Despite the risk of anaphylaxis, there is currently no cure for food allergy. Anaphylactic food allergies are increasing in incidence and prevalence, and there is a great need for more accurate and rapid diagnostics, as well as for more effective therapies. Accordingly, this scientific area is ideal for translational research using a multidisciplinary approach in the ITI. The long-term objective of this project is to establish a database available to the ITI and Stanford research community on subjects with near-fatal (anaphylactic) food allergies, with non-anaphylactic food allergies, and healthy controls. This will improve our efforts to understand the mechanisms underlying the development of anaphylactic food allergies and to improve treatment options for this disorder. In the future, the database could be further supplemented through clinical, translational, and basic science research at Stanford/ITI and at the individual laboratories of the multidisciplinary allergy research team within the ITI (Drs. Engleman, Galli, and Nadeau). The proposed project includes 2 specific aims. Our first aim in using the database is to define phenotypic subtypes of: 1) subjects with anaphylactic vs. non-anaphylactic food allergies, 2) subjects with transient vs. durable anaphylactic food allergies, and 3) subjects treated with anti-IgE vs. placebo for anaphylactic food allergies. Since immune tolerance to food allergens is mainly mediated by immune cell interactions with T cells, in our second aim, we propose to study immune cells from subjects with anaphylactic food allergy (vs. those from control groups) to determine if their immune cell-T cell interactions, differ from those in non-anaphylactic food allergy subjects and healthy controls.

2) Development of a Microbial Antigen Array for Multiplex, Longitudinal, Analysis of Exposure to Infectious Agents
Julie Parsonnet (Infectious Diseases), David Miklos (Blood and Marrow Transplantation)

March 2009 (One year report)
The Specific Aims of this project are:

  • To select microbial antigens (viral, bacterial, fungal and parasitic) for inclusion in a microbial antigen array.
  • To develop and test the performance characteristics of this array
  • To identify patient populations for array study.

To date, we have identified a panel of 400 microbial agents for the array and have tested 30 of them with 60 pending in the next array.  We have found that persons with known prior exposure to specific infections or vaccines have higher IgG antibody responses to the corresponding antigens on the array than do young infants without known disease or vaccination.  We are currently revising controls so that we can titrate normal antibody responses. We have put in several proposals to look longitudinally at the cumulative role of infections in obesity and asthma. 

March 2008 (Initial proposal)
From infancy, humans are bombarded with microbial agents. With each infectious exposure, our immune system learns, and the host adjusts its response to the microbial world without and within. An accumulating body of evidence suggests that the infectious exposures we have from birth-and even before birth-may, in the end, determine our ability to mount an effective immune response to vaccines, to avoid autoimmune diseases, or even play an important part in chronic processes such as obesity, diabetes, and atherosclerosis. In the past, evaluation of infectious disease causation has been limited to individual pathogen-by-pathogen approaches. When faced with a clinical scenario-be it acute or chronic--investigators have "placed their bets" on a select group of microbial agents, hoping they hit the right ones. Such a focused hit or miss approach cannot identify complex sequences and interactions of infection that lead to alterations in immune response or to chronic diseases. Faced with this dilemma, the NIH has embarked on an endeavor to catalogue the microbes and the microbial genomes in and on the human body. Yet dissecting the microbiota and the microbiome individual-by-individual and anatomical-site-by-anatomical-site will yield only a cross-sectional view of our life in the microbial world. Left unexplored is the "archeology" of the human-microbial experience -knowing how individuals have traveled through their microbial lifetime to their current state. The quantity of infectious exposures, their sequence and their timing in human development-by altering immune response, host metabolic pathways, or ecologic niches -may ultimately play a critical role in determining health and disease status. Fortunately, our exposures leave footprints-i.e. antibodies--in our immunologic memory. Even better, these manifold antibody footprints can be simultaneously evaluated through new microarray technologies. With funding from Beckman and the ITI, we propose to develop a multiplex ELISA-type microarray of approximately 1000 microbial antigens to assess cumulative microbial infection throughout life. These arrays can then be applied longitudinally in population samples to assess questions related to infectious exposure, immune response and disease. Further, once the arrays are created and validated, they can be provided to investigators world-wide to inexpensively test critical regional and cultural differences that influence such things as vaccine efficacy, allergy, auto-immune diseases, and diseases of unknown etiology. Temporal and cross-sectional patterns of humoral immune response to infection, we believe, will ultimately provide important insights both into the etiology of disease and maintenance of health.

3) MiRNA Expression and Dysregulation in Autoimmunity
Chan-Zheng Chen (Microbiology and Immunology), William Robinson (Immunology and Rheumatology), David Hirschberg (HIMC), Mark Davis (Immunology), Mark Genovese (Immunology and Rheumatology), and PJ Utz (Immunology and Rheumatology)

March 2009 (One year report)
The Proposed research will develop and validate platforms for genome-side miRNAs expression analyses of normal and aberrant human lymphocytes. We hope to identify miRNA signatures that may be useful as diagnostic or prognostic marks for autoimmune patients.  We have made significant progresses in all the proposed aims. (1) We have systematically cloned miRNAs from various developmental mouse T cell populations, including double-negative 1 (DN1), DN2, DN3, DN4, CD4 single positive (SP), and CD8 SP thymocytes, CD4+ naïve T cells, TH1, and TH2 cells. A total of 297 unique miRNAs, including 214 known miRNA genes and 83 new miRNA genes were found in these T cell populations. The fact that 83 of the 214 miRNAs from these T cell populations are unknown supports our hypothesis that a large number of miRNAs expressed in purified T cell progenitors or mature T cells are yet to be identified. Numerous miRNAs are expressed in a stage-specific fashion in T and B cells, suggesting specific functional roles. This systematic “miRNAome” analysis in immune cells will allow us to develop quantitative PCR probes and miRNA chips comprising all “immune miRNAs,” thus setting a solid foundation for profiling miRNA expression in autoimmune patients and identifying aberrantly expressed miRNAs.  (2) B-cell receptor (BCR) induced apoptosis is crucial for the establishment and maintenance of the normal immune system by eliminating self-reactive lymphocytes. To study to what extend miRNAs are involved in BCR induced apoptosis we used the murine WEHI-231 B-cell lymphoma line as a model. We worked with the HIMC and tested miRNA microarray (Agilent), miRNA qPCR (Applied biosystems), and FlexmiR  (Luminex). Expression profiling showed that 33 miRNAs were differentially expressed upon BCR stimulation. Interestingly, rescue from apoptosis via CD40 co-stimulation almost completely prevented these changes in miRNA expression. We were able to show that overexpression of several of the deregulated miRNAs negatively affected cell growth. In conclusion, we have tested various expression platforms and confirmed that several candidate miRNAs play roles in BCR induced apoptosis. (3) Finally, we took both candidate and global approaches to characterize miRNA expression in autoimmune patients. To this end, we are investigating whether miRNAs are involved in regulating growth of the synovial lining to form pannus tissue and the production of TNF by macrophage in RA.  To identify miRNAs that might be involved in regulating these pathogenic processes, we utilized Agilent microarrays to profile miRNAs expressed by fibroblast-like synoviocytes (FLS) and macrophage derived from patients with RA. Analysis of the miRNA expression datasets revealed up-regulation or down-regulation of specific miRNAs in response to TNF stimulation. Our results support the published observation of miR155 up-regulation in the cellular response to TNF, and identify multiple additional candidate miRNAs that could play a role in aberrant fibroblast proliferation and TNF production in RA.

March 2008 (Initial proposal)
Recent discoveries have revealed widespread post-transcriptional genetic programs controlled by microRNAs (miRNAs), an abundant class of ~ 22 nucleotides small regulatory RNAs, and their crucial roles in animal development. miRNA expression levels are tightly regulated so that the miRNAs can act in concert to regulate immune cell formation and function. Recently, we have demonstrated that T cell sensitivity to antigens is intrinsically regulated by a miRNA during T cell development and maturation, demonstrating that miRNAs have critical roles in the development and function of immune cells. Moreover, misregulation of miRNA expression may contribute to the pathogenesis of autoimmune diseases and various hematopoietic disorders, such as leukemias and lymphomas. Thus, systematic analysis of miRNA gene expression profiles using comprehensive miRNA microarrays may be used to establish a miRNA taxonomy of human immune systems under normal or autoimmune conditions. The proposed research will develop and validate platforms for genome-wide miRNAs expression analyses of normal and aberrant human lymphocytes. We hope to identify miRNA signatures that may be useful as diagnostic or prognostic marks for autoimmune patients.

4) Comprehensive Immunophenotyping of Patients with Primary Immunodeficiency
David Lewis (Pediatrics), PJ Utz (Immunology and Rheumatology), Kari Nadeau (Pediatrics)

March 2009 (One year report)
This project is casting a wide cellular and molecular net to understand the cause of immune and autoimmune dysfunction in adult and pediatric patients with primary immunodeficiencies. The central aim is to comprehensively immunophenotype common variable immunodeficiency (CVID) patients with and without autoimmune disease, comparing these samples with those of a) healthy controls without CVID or other primary immunodeficiency or autoimmunity; and b) controls without PID who have apparently isolated autoantibody disease. With the goal of testing potentially defective tolerance mechanisms in CVID, we have performed studies to examine CD4 T-cell output from the thymus to determine if reduced output is associated with CVID and, in particular, associated autoimmune disease. We have analyzed a group of CVID patients with and without autoimmune disease as well as healthy age-matched controls and have found a very striking deficit in the circulating number of naïve CD4 T cells in the patients. This deficit is associated with both decreases in the expression of surface protein tyrosine kinase 7 (PTK7), a marker for recent thymic emigrants of the CD4 T-cell lineage (1). In parallel with these studies, conventional and phospho-flow cytometric analysis were used to analyze CVID patients with and without autoimmune disease to determine if impaired regulatory T-cell (Treg) function was associated with the risk of autoimmunity. Patients with autoimmune disease had significant impairment of Treg activity compared to CVID patients without autoimmune disease or to healthy controls. Tregs from the autoimmune CVID group also had reduced expression of several proteins that play a role in regulatory function (FoxP3, granzyme A) or helping maintain cytokine responsiveness of Tregs (e.g., phospho-tyrosine-STAT5) compared to the other two groups. Together, these results show a striking association of decreased Treg function and expression of functional proteins and the risk of autoimmune disease in CVID (2). We have also developed a specialized autoantibody array that includes a diverse group of antigens that are commonly observed in more severe autoimmune diseases, such as systemic lupus erythematosus (SLE), e.g., ribonucleoproteins, as well as several cytokines. Our initial experiments have validated the array with patients that were previously identified as clinically having acquired auto-antibodies against IFN-g, which presented as severe non-tuberculous Mycobacterial infections. With validation of these assays complete, plasma from the full cohort of CVID patients (autoimmune versus non-autoimmune) and age-matched healthy controls will be analyzed for autoantibodies. We anticipate that there will be a direct association between the degree of impairment of RTE output, Treg activity, and number of positive autoantibodies per CVID patient. A high priority for further studies will be to extend the phospho-flow analysis to the evaluation of T-cell, B-cell, and monocyte responsiveness to cytokines using the Human Immune Monitoring Core.

March 2008 (Initial proposal)
This project will cast a wide cellular and molecular net to understand the cause of dysfunction of the immune system in patients with primary immunodeficiencies. Primary immunodeficiencies are inherited disorders in which patients have an inability to mount immune responses to either microbes or to vaccines. One of the most common of primary immunodeficiencies is common variable immunodeficiency (CVID), which afflicts about 1:10,000-20,000 adults as well as a substantial number of children. Despite many years of work >85% of cases of CVID have no definable genetic cause, and the reason these patients are unable to make specific antibodies to pathogens and vaccines is unclear. Moreover, CVID is also challenging to clinicians in that patients not only have more frequent and severe infections than normal, but also have a substantially increased risk of autoimmune disease of a type in which the body makes antibodies against its own tissues (autoantibodies) and cancer. The reasons for the greater risk of these non-infectious complications of CVID are also unknown. To understand both the cause and nature of the immune dysfunction and it complications in CVID, we will comprehensively determine the frequency of the known types of white blood cells, the proteins and RNAs that they express, and the ability of these cells to respond internally to activating signals they receive from outside. We will also comprehensively evaluate the types of antibodies in the blood of these patients, particularly focusing on autoantibodies. All of these analyses will compare CVID patients without autoimmune complications with 1) CVID patients with autoimmune complications; 2) healthy age-matched controls; and 3) patients without CVID that have autoimmune disease. The ultimate goal is to define new cellular and molecular “signatures” of the many parameters that will be analyzed that will predict which patients with CVID will develop autoimmune disease and which individuals in families with a history of CVID are at risk of developing the disease. This project brings together the individual expertise that the investigators have in performing flow cytometry and RNA analysis (Lewis), autoantibody analysis (Utz), and analysis of signaling within the cell by flow cytometry (Nadeau). All three of the investigators also see patients with this disorder. The project also involves physicians who are involved in the care of non-CVID patients with autoimmune disease, and the studies will be greatly facilitated by the use of the Human Immune Monitoring Core directed by Dr. David Hirschberg.

5) A Systems Biology Approach for Discovery of Biomarkers for Inflammatory Pain
Martin Angst (Anesthesiology), Atul Butte (Pediatrics), David Schneider (Microbiology and Immunology), David Clark (Anesthesiology), Eliza Chakravarti (Immunology and Rheumatology), and Brendan Carvalho (Anesthesiology)

March 2009 (One year report)
The specific aims of this seed grant effort are:
1. Test the hypothesis that distinct gene expression patterns can be identified with aid of the GEO database that separate diseases associated with pain and inflammation from diseases not associated with pain and inflammation.

2. Use diverse model systems to test the hypothesis that certain gene expression patterns obtained from the bioinformatics analysis of the human GEO datasets are causally linked to the genesis of pain and inflammation. In addition, generate new gene expression and cytokine data to provide a cross-species comparison for inflammatory pain biomarkers.

The investigators who received this seed grant are members of the working group “Inflammation and Pain” of ITI. Members of the working group report have made significant progress towards the discovery of pain biomarkers.
Bioinformatic efforts (Atul Butte): Gene expression data for 175 ICD9-coded human diseases were retrieved from the GEO data base and correlated with the “painfulness” of these diseases. So far, “painfulness” was inferred via a particular search strategy in PubMed and by accessing data on disease-specific analgesic consumption (health care databases). A number of genes were identified that are significantly associated with the “painfulness” of a disease (hypothesis 1), providing first evidence for the utility of used bioinformatics approach.
Model in fruit flies (David Schneider): Efforts were directed towards developing an assay measuring the effects of an innate immune response on pain behavior. A quantitative feeding assay was adapted by spiking food with wasabi (nociceptive substance) but failed due to a more complex feeding behavior than suggested by the literature. Other assays are currently considered.
Model in rodents (David Clark): Efforts were directed towards developing models of incisional pain, a surrogate for pain associated with surgery. Cytokines, chemokines and trophic factors were measured in 16 strains and their association with pain was tested. So far, IL-1b was studied further. Injection of IL-1b into mouse hind paws caused a pain response, haplotypic genetic mapping identified a significant association with the gene coding for caspase, and inhibition of caspase-1 reduced IL-1b production as well as the pain response. Results indicate the suitability of this approach for testing the functional relationship between wound mediators and pain (hypothesis 2).
Models in humans (Martin Angst & Brendan Carvalho): Significant progress has been made in measuring locally-released inflammatory and pain mediators in humans under in-vivo conditions. Micro-catheter techniques allowed sampling from experimental sunburns in human volunteers and from wounds in patients undergoing surgery. The release characteristics of 33 mediators and alterations of such release in response to pharmacological interventions have successfully been described. Results highlight the utility of these models for future hypotheses testing (hypothesis 2).

Outlook Year 2
Data mining will be refined and expanded to other microarray and clinical data bases. Bioinformatic efforts represent a full-time effort, and a full time posthoc has jointly been hired.  A second assay in fruit flies will be developed. Model development in rodents by the Clark laboratory will continue and include gene chip studies. Finally, a series of hypotheses generated with outlined bioinformatics approach will be tested in suitable animal /human models.

March 2008 (Initial proposal)
Pain is universal in human experience, and attempts to alleviate pain using multiple modalities (drugs, surgery, complementary medicine, etc.) account for a large percentage of health care costs. While much is known about the molecular biology and physiology of pain, adequate treatment of pain remains problematic. For example, at least one third of all surgical patients suffer from moderate to severe postoperative pain. Inadequately treated pain is associated with increased postoperative morbidity and mortality and a higher incidence of chronic pain as a result of surgery. Pain is a highly individual and subjective experience, making it difficult to quantify and generalize from one person to another or even within the same person under different conditions. Our long-term goal is to discover biomarkers that provide a more quantitative and mechanistic basis for the diagnosis and evaluation of pain, leading ultimately to new, more targeted and effective treatments. In this project, we propose to analyze existing national public datasets using novel bioinformatics tools to look for patterns of gene expression that differ between human diseases associated with pain and inflammation and those diseases not associated with pain and inflammation. We will then use different laboratory model systems (fruit fly, mouse) to test whether these human gene expression patterns can be causally linked to the genesis of pain and inflammation. In addition, we will generate new data on gene expression and levels of cytokines (inflammatory molecules) to provide a cross-species comparison for pain biomarkers. The investigators on this project comprise a true interdisciplinary team whose expertise and research interests include bioinformatics, model system genetics/molecular biology/physiology, human experimental pain models, as well as clinical investigations and medical practice related to painful and inflammatory diseases and conditions. In parallel with the studies in model organisms supported by this ITI grant, we will leverage other internal seed funding to examine human gene products of interest under experimental conditions (e.g. experimental sunburn) in the human pain laboratory or under clinical conditions in patients suffering from acute or chronic pain and inflammatory diseases. The release of gene products will be correlated to measures such as disease activity (e.g. pain, serum levels of different inflammatory molecules) or responses to medical interventions (e.g. drug therapy). Thus this project represents a truly translational enterprise whereby data flows from human studies to non-human model systems and back to clinical practice, with the hope of ultimately leading to more targeted and personalized medical care.

6) Definition of the Ligand for CD83 and its Signaling Pathway
C. Garrison Fathman (Immunology and Rheumatology), and Garry Nolan (Microbiology and Immunology)

March 2009 (One year report)
Specific Aims
1.  To identify the ligand on PBCs that binds sCD83
2.  To assess the mode of action (MoA) of sCD83 in vitro by genomic and proteomic assays       using T cell activation models.

The initial experiments to identify the ligand(s) of CD83 used a variety of soluble human and murine CD83 analogs that were screened on 3T3 fibroblast cells transduced with a dendritic–B lymphocyte cDNA expression library. The few positive clones corresponded to Fc receptor interactions inherent in the screen design. An alternative approach that bypassed this problem identified Ly6e. However, this gene did not bind CD83 in a different assay performed subsequently. A more targeted approach was based on the hypothesis that CD83 was a member of the CD28 family, and might bind a B7 analog. To test this hypothesis, Multiple B7 family members were cloned, expressed in the 3T3 cells, and verified they bound their known ligands, and assayed for their ability to bind sCD83. No positive hits were observed with this approach. Soluble forms of B7 family members were also made with fluorophore tags in an attempt to stain CD83 bearing cells, but this also failed. TAP system (tandem affinity purification) was also introduced. The full length or extracellular domain of CD83 was transduced into Jurkat or HPB-ALL cells which were reported to express the putative CD83 ligand and we then tried to immunoprecipitate the putative CD83 ligand and identify it by MS but this also failed.
During the screening process, a number of forms of sCD83, including material provided to us by Argos, were screening in vitro for their ability to inhibit murine mixed lymphocyte and in vivo in collagen induced arthritis in animal models. No inhibition was observed with any protein preparations in any of the in vitro or in vivo models, including several preparations of the identical constructs reported to be active in the literature.  Furthermore, none of the screened phospho-epitopes were activated by sCD83 in murine PBC in vitro or following sCD83 administration in vivo.
A fundamental conundrum of the screening process was that because there is no known function for CD83, there was no method to determine whether the various recombinant proteins used in the assays were biologically active or correctly folded. In addition, soluble forms of CD83 produced by bacteria, which lack glycosylation, were used in most of the screening assays. The choice to use these reagents was driven by a report by Steinkasserer that bacterially produced soluble CD83 reduced the symptoms of EAE in mice. Bacterially produced protein also was the basis for the solution of the crystal structures of CD28, CTLA-4 and PD-1.
Related work in our lab has established that E3 ligase GRAIL can interact with and ubiquitinate full length, membrane bound CD83. In an effort to establish an in vitro assay for 08functional sCD83, the extracellular domains of GRAIL and sCD83 have been expressed and purified from CHO cells. Unfortunately, in collaboration with Chris Garcia’s lab, no association between the purified proteins was observed using Plasmon Resonance. The sCD83 we prepared contained a cysteine to serine mutation at a residue proximal to the membrane-spanning region in an effort to limit aggregation of the molecule; aggregation had been seen in earlier work. When analyzed using gel filtration, this cys to ser substituted bacterial produced molecule was a monomer, and it is possible that a multimeric form of sCD83 is required for biological activity.
Our current hypothesis is that CD83 binds its ligand with sufficiently low affinity that only a multimeric interaction will be apparent in the absence of a biological membrane. To test this hypothesis, CD83 with a carboxyl leucine zipper is being expressed with the expectation that a biologically active species will be a dimer or a higher aggregate.  Once bioactivity can be established, ligand screening and immunotherapy trials in preclinical models will be reinstituted.

March 2008 (Initial proposal)
Studies carried out under the IRP award will be performed by a clinician scientist, Dr. C. Garrison Fathman MD Professor of medicine and chief of the Division of Immunology and Rheumatology, and by a basic scientist, Dr. Garry Nolan PhD, Associate professor of Microbiology and Immunology. These studies combine animal models of disease prevention with studies on mechanism of action of a novel immunotherapeutic, a soluble form of a cell surface molecule, CD83. An immunoregulatory activity for the soluble form of the protein, sCD83, recently has been demonstrated in a number of studies in animal models. The most relevant for disease prevention was the suppression of the induction of autoimmunity in a mouse model of multiple sclerosis, EAE. Mice immunized with an encephalogenic peptide and treated with three intraperitoneal (ip) injections of sCD83 around the day of immunization with MOG remained essentially disease free, whereas immunized control mice developed expected clinical signs of EAE. Moreover, when these sCD83-treated mice were immunized a second time with MOG in adjuvant 4 weeks later to induce a relapse clinical phase, the previously treated sCD83 treated group was still totally protected. These data suggested a long-lasting immunoregulatory effect of sCD83 treatment in vivo. Perhaps more importantly, sCD83 treatment was able to inhibit clinical symptoms and progression of EAE in treatment of mice that had already presented with clinical symptoms. Mice treated with sCD83 at the height of their clinical disease score recovered from paralysis at an accelerated rate and demonstrated only a very mild disease after re-administration of the immunizing peptide. These data demonstrate that ip administration of sCD83 can both prevent and treat EAE. Studies in the Stanford IRP award have two specific aims: 1. To identify the ligand on DCs that binds sCD83 where the overall strategy will involve both random screening methods and shortcuts exploring known ligands of the CD28 family to identify the protein product (ligand) that binds to sCD83; and, 2. To assess the mode of action (MoA) of sCD83 in vitro by genomic and proteomic assays using T cell activation models. Briefly, the cells that express the CD83 ligand, presumably dendrititc cells, will be co-cultured with sCD83 and placed in culture with activated T cells that will be subjected to phospho-flow analysis in collaboration with the Nolan lab to look for changes in cellular signaling. Finally, cDNA microarray analysis will be performed on the sCD83 treated and resting DCs as well as T cells cultured with the sCD83 and treated and untreated DCs. These studies intend to identify the cell surface product (ligand) that is bound by sCD83 to result in immunomodulation and to study the mechanism of action of this effect.

7) Cryptic Translation of MHC I-presented Peptides by Specialized Ribosomes
Peter Sarnow (Microbiology and Immunology), and Joseph Puglisi (Structural Biology)

March 2009 (One Year report)
It has been observed that cryptic translation products can be generated by an unconventional translation mechanism that decodes reading frames from CUG instead of AUG start codons. These findings have consequences on the observed rapid presentation of MHC class I peptides during microbial infections or cellular stress. Our hypothesis is that specialized ribosome populations are used to start translation at CUG codons. We have established a translation system in which AUG- and CUG-initiating ribosomes can be purified and have evidence that these two ribosome populations have different sensitivities to compounds that interfere with translational initiation and are composed of few different polypeptides. The identification of these peptides by mass spectrometry and their functional roles is being investigated.

March 2008 (Initial proposal)
This research project employs cell-based and biochemical assays to quantify this effect and to compare the ribosomes that selectively translate mRNAs from AUG and CUG codons. Single-molecule assays are used to provide an understanding of the dynamics of this unusual initiation mechanism. Our goal is to identify the mechanism by which a specialized set of ribosomes generate from non-conventional CUG start codons cryptic translation products that are presented by MHC class I molecules on the cell surface to immuno-surveillant CD8 T cells.

8) Prediction and Mechanisms of Transplantation Tolerance
Minnie Sarwal (Pediatrics), Edgar Engleman (Pathology), and Samuel Strober (Immunology and Rheumatology)

March 2009 (One year report)
Progress in Gene Array Monitoring for Induced Transplant Tolerance (Sarwal Lab)
The Agilent 44K human genome microarray platform was used for these studies. We have developed a test that is able to discriminate kidney transplant recipients with normal graft function, off immunosuppressive drugs, with clinical graft tolerance, from transplant patients with chronic rejection and normal healthy controls. In the studies in this seed grant, to date, we have performed arrays on all 8 patients and full analysis of results is available.
Samples have been banked at pre and post-immunosuppression weaning, giving us a unique opportunity to study patients with stable graft tolerance, off immunosuppression. A minimal gene set of 21 unique genes was identified by PAM in a 3 phenotypes classification of operational tolerance, chronic rejection and normal healthy controls and each sample is given a prediction probability score based on the prediction. In the first year we have accomplished the following: a). Patients with stable induced tolerance are more similar to patients with operational tolerance, rather than patients with chronic rejection by the hierarchical clustering based on the top 21 unique genes. Similar signaling pathways seem to play a role in operational and induced tolerance patients. It is thus possible that expansion of a subset or subsets of regulatory cells in the circulation, driven by the same cytokine milieu in tolerant patients, supports the maintenance of tolerance, be it naturally acquired or induced by the HCT protocol. b).The distinct gene-set of 21 unique genes, can segregate stable induced tolerance from patients who did not acquire stable tolerance in the HCT protocol. Two of the 3 patients analyzed so far who failed to achieve stable tolerance more closely resemble patients with chronic rejection, whereas 5 of the 5 patients analyzed so far with operational tolerance have prediction probability scores of 80-90% that match tolerance. c).Serial, longitudinal gene expression analysis across the discriminant gene-set for tolerance, can evaluate the evolution of tolerance and for the first time, see a “change” in prediction scores for tolerance. This is the first attempt at longitudinal testing for the acquisition of the “tolerant" pattern, since previous studies were performed on groups of patients who were off drugs and found to maintain good graft function retrospectively. Thus, the serial monitoring of the gene arrays before, during, and after immunosuppressive drug withdrawal may be of help in determining the presence and kinetics of the tolerant state in the patients enrolled in our prospective tolerance induction protocol.

March 2008 (Initial proposal)
The ultimate goal of our research program is to develop and optimize tolerance induction protocols for transplant patients that are efficacious, safe, and clinically applicable. The gold standard definition of clinical allograft tolerance is normal graft function, without any immunosuppression with demonstration of donor antigen specific unresponsiveness of the immune system of the recipient. Our strategy in this proposal is to foster collaborative research that promotes the integration of Stanford clinical trials of tolerance induction in recipients of kidney, liver, and bone marrow transplants with new immune monitoring procedures developed at Stanford including gene arrays and phosho flow cytometry that can predict the tolerant state so that immunosuppressive drugs can be safely withdrawn. We have developed a test by gene arrays that is able to discriminate kidney transplant recipients with normal graft function, off immunosuppressive and with clinical graft tolerance from conventional transplant patients on immunosuppressive drugs with normal graft function or with chronic rejection. A key goal in transplantation is to attempt to use this assay to predict this state by longitudinal testing on transplant recipients who evolve from a non-tolerant state to a tolerant state. This has not been done before. We propose to thus take a correlative assay and turn it into a longitudinal predictive assay. We plan to do similar testing of changes in recipient intracellular signaling pathways measured by phospho flow cytometry in response to stimulation by donor alloantigen. We propose to do this by initially taking patients enrolled in a prospective tolerance induction protocols and test their gene and phospho flow patterns that change from a non-tolerant to a tolerant signature, concomitant with the development of clinical allograft tolerance .These assays will be used to provide improved future guidelines for the safe withdrawal of all immunosuppressive drugs from transplant recipients.

Institutes of Medicine Seed Grant Fund in Translational Research

The Institutes of Medicine Seed Grant Fund in Translational Research  has been established at the School of Medicine to support important translational research in each of the five Institutes of Medicine.  Each year for five years, a $25,000 seed grant will support one research project in one of the five Institutes, with the award rotating each year to a new Institute until all five Institutes have received a year of seed funding.  The Dean of the School of Medicine determined that this year (2008-2009) the seed grant will be given to the Institute for Immunity, Transplantation and Infection (ITI).  ITI received 34 proposals which were reviewed by the Director of ITI, Mark Davis, and the ITI Steering Committee.   They have decided to award the seed grant to Shirit Einav, Medical Fellow in Jeffrey Glenn’s lab. Her project is entitled: “Novel Mechanisms for HCV Induced Cellular Transformation.”

There were many good proposals that were submitted and two runners-up were selected, the funding of which will come from ITI and other sources.  They are:

  • Petr Broz, Post Doctoral Scholar in Denise Monack’s lab. His project is entitled: “Characterization of Innate Immune Pathways Leading to Inflammasome Activation.”
  • David Pride, Medical Fellow in David Relman’s lab.  His project is “Oral Bacteriophage Communities.”

Below are abstracts of the three projects starting with the Seed Grant winner:

Novel mechanisms for HCV induced cellular transformation
Shirit Einav, MD
Chronic infection with the hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC). HCC is the sixth most common cancer, but the third leading cause of cancer death in the world. The incidence of HCC and the mortality rate associated with it are increasing dramatically. While chronic inflammation, fibrosis and liver cell proliferation are considered the major factors contributing to the development of HCC, there is accumulating data supporting direct viral effects. Despite advances in the understanding of HCV's biology, mechanisms whereby the virus promotes cellular transformation remain poorly understood. Current therapies are inadequate for the majority of HCV patients with or without HCC. HCC is the most common indication for liver transplantation. Our long-term goals are to better understand the HCV life cycle and the viral factors that mediate development of HCC and to translate this knowledge into novel antiviral strategies that would potentially inhibit not only viral replication but also viral-mediated cellular transformation. In this proposal we seek to study the role of viral factors in mediating development of HCV induced HCC – the leading indication for liver transplantation, by studying their effect on host cellular factors (cytokines, other host cell proteins and microRNA). Thus, this proposal integrates the three disciplines of the ITI:

Infection,Transplantation and Immunology.
While previously characterized by a complete lack of known function, the HCV non-structural (NS) protein NS4B has emerged as a key player in viral replication and in cellular transformation. NS4B was previously shown by others to transform NIH3T3 cells when co-expressed with the Ha-ras gene. While the mechanism(s) for NS4B’s transformation activity remains unknown, disrupting this function may help inhibiting development of HCC. We have previously identified a nucleotide binding motif (NBM) within NS4B. This motif consists of a set of conserved amino acids found in both the GTP-binding members of the G-protein family, as well as in the superfamily of viral proteins with nucleotide binding domains. The most highly-conserved element within these nucleotide-binding domains of GTP-binding proteins is the A motif. Other conserved elements known from crystal structures to participate in nucleotide binding include the G, PM2 and B motifs. We have shown that NS4B’s NBM mediates both binding and hydrolysis of GTP and HCV RNA replication. Furthermore, we have recently shown that NS4B can mediate cellular transformation and tumor formation in mice independently of the Ha-ras gene. Similar to the NBM of human oncogenes, such as ras, known to mediate malignant transformation, the NBM was found to mediate NS4B’s role in cellular transformation.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting messenger RNAs through translational repression or RNA degradation. Data is accumulating in regard to the role miRNA play in the settings of infection and cancer. miRNAs interact with classic oncogenes and tumor suppressor networks and contribute to the initiation and progression of many human malignancies. Recently, Randall, et al. showed that HCV replication modulates the expression of a limited set of host miRNAs in replicon cells. Nevertheless, the temporal effects of HCV replication on miRNAs profile haven’t been studied yet. Furthermore, the involvement of various miRNA in HCV induced oncogenesis hasn’t been studied either. We, in collaboration with Dr. Andrew Fire’s laboratory, have recently profiled the miRNA expression following HCV infection. A significant increase in the frequency of two miRNAs was detected several days post infection. One of these miRNAs is known to be involved in development of tumors. We have shown that NS4B but not the other non-structural HCV proteins known to transform cells (NS3 and NS5A), is capable of inducing this miRNA levels. Furthermore, the levels of this miRNA were found to be elevated in NS4B transformed cell lines in correlation with NS4B’s levels.

Our overall hypothesis is that nucleotide binding and hydrolysis leading to induction of miRNA are essential for mediating NS4B’s role in cellular transformation and oncogenesis. Thus to the extent viral factors contribute to the increased risk of HCC, approaches designed to disrupt these functions may be potentially used to inhibit HCV associated HCC. Pharmacologic inhibition of the NBM may also inhibit HCV replication.

These results shed light on the mechanism by which NS4B mediates cellular transformation and have exciting implications. The NBM is conserved across natural HCV isolates suggesting that it is essential for infection in vivo. The involvement of miRNAs in HCV induced transformation represents a novel mechanism of viral-mediated carcinogenesis. Finally, these results may also lead to development of novel strategies for prevention and treatment of HCC in HCV patients.

Characterization of innate immune pathways leading to inflammasome activation
Petr Broz, PhD
Bacterial infections still represent a major cause of death worldwide, especially in developing countries. In the developed world the emergence of antibiotic-resistant bacteria and the abandonment of vaccination has led to the re-appearance of infectious diseases. Indeed, the treatment of diseases caused by intracellular bacterial pathogens remains a major challenge for the biomedical community. For example, millions of human cases of Salmonellosis are reported worldwide every year, resulting in huge economic costs to society and thousands of deaths. Furthermore, there are 1.7 million deaths annually due to infections caused by Mycobacterium tuberculosis, the causative agent of Tuberculosis (WHO). Mammalian hosts have evolved mechanisms, collectively called the innate immune system, to detect and fight off many microbial infections quickly. In response, disease-causing bacteria have evolved specific mechanisms to survive in the host despite the presence of the mammalian immune system. I am interested in studying how the innate immune system recognizes bacterial pathogens, and how pathogens manipulate the host. To detect the presence of microbes, the host innate immune system relies on surveillance proteins, or receptors that recognize “foreign” microbial molecules. Some of these receptors are on the surface of host cells and detect extracellular microbes and their products, whereas other receptors are inside host cells and detect intracellular microbes and their products. Interestingly, the same receptors that are involved in the recognition of intracellular pathogens also recognize so-called “danger signals” resulting from cell injuries caused for example by the inhalation of airborne pollutants like asbestos and silica or from uric-acid crystals associated with gout. In addition mutations in these receptors have been shown to result in human disease. For example, mutations in Nod2 and NALP3, both intracellular receptors, are associated with Crohn’s disease, and the receptor NALP1 is associated with Vitiligo and other autoinflammatory diseases. Once these intracellular receptors recognize a microbial product or a danger signal, they trigger the assembly of an intracellular multiprotein complex termed the “inflammasome.” The inflammasome promotes the activation of caspase-1, an enzyme that is required for the processing and release of pro-inflammatory chemical messengers, called cytokines, such as interleukin (IL)-1b and IL-18, which alert other immune cells. Inflammasome activation can also lead to death of the infected cell, which is crucial in restricting intracellular replication of invasive bacterial pathogens such as Salmonella typhi, which causes typhoid fever, and Francisella tularensis, which causes tularemia, a highly infectious disease also known as “rabbit fever.” 

Despite the central role of the inflammasome complex in innate immunity, many aspects remain uncharacterized. Therefore, the aim of my project is to identify new inflammasome receptors and to characterize pathways that lead to inflammasome activation using in vitro and in vivo models of bacterial infections. In particular, I am interested in the molecular mechanisms that are used by macrophages (a crucial cell of the innate immune system) to recognize intracellular pathogens, such as Francisella tularensis and Salmonella typhi, and the subsequent activation of the inflammasome. The better understanding of the central inflammasome complex and the identification of receptors leading to its activation are of clinical relevance because the same receptors involved in detecting intracellular pathogens also recognize danger signals from cell injury and are involved in autoinflammatory diseases. These findings could lead to a better understanding of the pathogenesis of infectious diseases autoinflammatory diseases. Finally, this project could promote the development of novel immune modulators and/or anti-infectives.

Oral Bacteriophage Communities
David Pride, MD, PhD
Periodontitis is a state of the human oral cavity where there is inflammation of the supporting structures of the teeth. Unlike many other diseases, no specific bacterial cause has been attributed to periodontitis, and the prevailing belief is that the disease occurs as a result of changes in the oral bacterial community as a whole.  Periodontal disease often is associated with the presence of dental caries, which represents a disease condition affecting the structure of the teeth. Several different bacteria, including certain species of Streptococcus have been associated with dental caries, and promote the formation of dental caries through demineralization of the teeth through acid production. Bacteriophages are viruses that specifically infect bacteria and are not known to infect human cells. They represent the most abundant entities on the planet and have been found wherever bacteria exist. There are vast unexplored communities of bacteriophages that inhabit human bacterial communities, but because they do not directly infect human cells, they have not traditionally been thought to be involved in either promoting human health or disease. There have been numerous examples of changes in bacterial communities leading to human disease, such as antibiotic-associated diarrhea, where antibiotics eradicate beneficial gut bacteria, leading to an overgrowth of certain harmful bacteria. Bacteriophages, through their ability to enter human bacterial communities and eliminate certain bacteria, have the capacity to substantially alter bacterial communities, and thus, may play a role in human health and disease.

There have been no previous comprehensive studies of bacteriophage communities that inhabit humans.  We propose a multidisciplinary approach, which is the first of its kind, towards understanding whether bacteriophages play a role in human oral health and disease. We have recruited human subjects with both oral health and disease, and collected both saliva and dental plaque from each subject.  Our specific goals are:

  1. To develop techniques to isolate and describe bacteriophages from the human oral cavity,
  2. To identify bacteriophages that alter oral bacterial communities and play a role in oral health and disease, and
  3. To understand the interplay between oral bacteria and bacteriophages through analysis of certain mechanisms of competition between the two communities. Through sampling of the human oral cavity in states of health and disease and isolation of bacterial and bacteriophage communities, we believe we can gain a more comprehensive view of the role of bacteriophage communities in the human oral cavity, and gain novel insight into the impact bacteriophages have on oral bacteria and oral health.