Ammon B. Peck - US grants

Oxalobacter formigenes, a recently identified bacterium that colonizes the gastrointestinal (GI) tracts of all vertebrates, has gained considerable attention due to its important symbiotic relationship with its hosts in regulating oxalic acid absorption in the intestines as well as oxalic acid levels in plasma two factors associated with the risk to develop calcium-oxalate kidney stones. Oxalic acid is a by-product of metabolism and a common constituent of most diets; however, if permitted to accumulate, it can cause numerous pathological conditions, including hyperoxaluria, cardiac conductance disorders, calcium oxalate stones, renal failure, death and possibly inflammatory bowel disease and vulvovestibulitis. Despite the potential significance of O. formigenes in controlling enteric hyperoxaluria, and thus oxalate-related disorders, research has been limited due to inherent difficulties in culturing and identifying this bacterium. Nevertheless, preliminary studies already suggest a correlation between the number of colony forming units of Oxalobacter sp. per gram feces and the frequency of recurrent urolithiasis, inflammatory bowel disease and hyperoxaluria/nephrocalcinosis in cystic fibrosis. With the development of a rapid and sensitive DNA-based detection system highly specific for O. formigenes, we are now able to investigate the correlation between an increased risk for urolithiasis and the absence of O. formigenes from the GI tract. Using the laboratory rat as a model, we propose to: 1) study the colonization of non-colonized rats with various sub-strains of O. formigenes to determine if variations exist in the efficacy of different strains of O. formigenes to colonize and if primary colonization precludes secondary colonizations, 2) determine whether O. formigenes can prevent hyperoxaluria and subsequent formation of calcium-oxalate crystals in rats colonized with various sub-strains of the bacterium and challenged with high oxalate-containing diets, and 3) examine the relationship between diet and/or antibiotic treatment with subsequent decolonization of the GI tract by O. formigenes known to occur in human populations and whether decolonization of the GI tract is irreversible. Results from these studies should provide insight into the role O. formigenes plays in regulating oxalate homeostasis in vertebrates, including humans, and should offer new modalities in prevention therapy for oxalate-associated disorders, such as recurrent kidney stone formation.

DESCRIPTION (provided by applicant): Sjogren's syndrome, one of the connective tissue autoimmune diseases, presents clinically as a loss of exocrine secretory function due to autoaggression primarily against the salivary and lacrimal glands. The mechanism(s) underlying the tissue specific destruction of target organs in Sjogren's syndrome is not understood at the present time. Our studies have now established the NOD (non-obese diabetic) and NOD.B10.H2b mice as the best animal models for secondary and primary Sjogren's syndrome, respectively. To date, the NOD mouse remains the only animal identified to develop the corresponding pathophysiology of salivary gland secretion loss and lacrimal tear production in conjunction with the histological observation of lymphocytic infiltration of the exocrine tissues. Results of our studies have novel implications in how we define the autoimmune disease paradigm with the observation of exocrine gland cellular alterations which develop in the absence of a functional lymphocyte system (NOD-scid). Autoimmune diseases, such as IDDM, IBD, and Hashimoto's thyroiditis, similar to our studies in both the NOD-scid and NOD parental strain, indicate nonimmune factors, as a consequence of genetically programmed loss of glandular differentiated function or homeostasis, appear to contribute to initiating the disease process. Using a second congenic strain lacking B-cells (NOD.Igu null), the loss of secretory function was shown to be independent of T-cells but not autoantibodies. To elucidate further the mechanism(s) responsible for the loss of exocrine target tissue function of Sjogren' s syndrome-like disease in the NOD model, the following specific aims are proposed: 1. Define the role of regulatory effector cytokines modulating the humoral response in autoimmune exocrinopathy through the analysis of specific knockout mice; and 2. Define the components of the humoral immune response promoting exocrine gland destruction, using the NOD mouse models of Sjogren's syndrome. These studies will expand on our novel observations which established the NOD mouse as an appropriate model for the study of Sjogren's syndrome-like pathology by analyzing the role of the humoral immune system components and effector cytokines in the precipitation of exocrine tissue dysfunction. By using a global approach to the analyses of this autoimmune disease involving a coordinated evaluation of both the lacrimal and salivary glands, the results from this research will provide innovative insights into the mechanism(s) triggering autoimmune attack on specific tissues and further suggest novel strategies for the control of this tissue destruction.

DESCRIPTION (provided by applicant): Sjogren's syndrome, an autoimmune disease that is one of the leading causes of salivary gland inflammation and dysfunction, leads to severe dryness of the oral cavity. Dry mouth is thought to result from a genetic predisposition that, in association with environmental stimuli, results in a chronic immune attack against specific (auto)-antigens expressed in salivary gland tissue. Although HLA inheritance has been recognized as an important risk factor for most autoimmune diseases, as yet no HLA genotype has been identified as being associated with an increased risk for developing Sjogren's syndrome. Non-HLA genes also appear to contribute to the genetic predisposition in humans, but identification of non-HLA genes only complicates5ability to understand the genetic basis of Sjogren's syndrome. Animal models of autoimmune disease provide an excellent resource for identifying genetic pathways responsible for underlying pathogenesis. We have popularized the NOD mouse as a model for Sjogren's syndrome as this mouse develops progressive lymphocytic infiltration, cytokine and autoantibody production in the exocrine glands concomitant with decreased exocrine gland secretions. Using the multiple congenic strains of NOD now available, two NOD-derived loci, designated Aec1 and Aec2 (autoimmune exocrinopathy genetic regions 1 and 2) containing insulin dependent diabetic loci Idd3 on chromosome 3 and Idd5 on chromosome 1, respectively, have been identified. These two intervals appear to act in an additive and hierarchical manner to control the epithelial cell pathology, subsequent accumulation of lymphocytic infiltrates, and the eventual loss of secretory function of the salivary (and lachrymal) glands in the NOD mouse. We have successfully developed the C57BL/6.NOD- Aecl Aec2 mouse which recapitulates the complete disease phenotype observed in the parental NOD mouse. To further map the chromosomal intervals, we propose to generate recombinant inbred (RI) strains of the C57BL/6.NOD- Aecl Aec2 mouse. For Specific Aim t, aset of RI strains of C57BL/6.NOD- Aecl Aec2 mice will be generated to fine-map the Aecf and Aec2 genetic regions to identify specific intervals associated with the development of autoimmune exocrinopathy in the NOD mouse. For Specific Aim 2, cDNA microarray technology will be used to identify candidate genes within these intervals responsible for immune and non-immune components resulting in autoimmune exocrinopathy by comparing expression levels of transcripts from disease-susceptible versus non-susceptible RI mice. Results from these studies will provide insight into the genetic mechanism(s) underlying the pathogenesis of Sjogren's syndrome important to the long-term goal of developing targeted preventive or ready intervention strategies.

DESCRIPTION: Sjogren's syndrome is a human autoimmune disease characterized by exocrine dysfunction resulting from the action of1 a chronic autoimmune attack against the lacrimal and salivary glands. It is one of the leading causes of salivary gland inflammation and dysfunction, leading to severe dryness of the ocular and oral surfaces. Diagnosis of Sjogren's syndrome includes detection of leukocytic infiltrates in the minor salivary glands, the presence of rheumatoid factor, hypergammaglobulinemia, specific anti-nuclear autoantibodies, loss of stimulated fluid secretion, and complaints of dry eyes and/or dry mouth. Over the past several years, the NOD mouse has been shown to exhibit numerous disease manifestations that parallel Sjogren's syndrome, including loss of stimulated fluid secretion concomitant with the appearance of leukocyte infiltrates in the lacrimal and salivary glands. Studies of autoimmune exocrinopathy in NOD mice have revealed that the disease can be separated into two phases: Phase 1 is characterized by pathophysiological and biochemical changes in the exude glands whose occurrence are independent of the autoimmune attack. Phase 2 is characterized by the appearance of leukocytic infiltrates in the exocrine glands, proinflammatory responses, autoantibodies to multiple acinar cell components, loss of acinar cell mass, and decline in exocrine function. The use of specific gene knockout mice congenic with the parental NOD mouse strain indicated that loss of exocrine function was dependent, first, on the presence of B cells and, second, expression of the intedeukin-4 (IL-4) cytokine. Subsequent studies suggested that IL-4 exerted its activity by regulating antibody isotypic class switching in B ceils. Nevertheless, IL-4 can activate two distinct signaling pathways in B ceils: the STAT-6 and the IRS pathways. The STAT-6 signaling pathway is involved in antibody class switching, while the IRS pathway is involved in B cell maturation and clonal deletion. Thus, the purpose of the current R21 grant application is to initiate studies examining the mechanism(s) by which IL-4 elicits its effector function in the development of exocrine gland dysfunction and decreased fluid secretion. Specifically, we plan to (t) document the autoimmune phenotype of a newly constructed NOD.B10.H-2b.B/c-STAT6 knockout mouse line and compare this phenotype to NOD.B10.H-2b, NOD.B10.H-2b.lgp KO and NOD.B10.H-2b.IL4 KO mice, and (2) identify if the IL-4 dependent development of exocrine gland dysfunction is due to activation of the STAT-6, IRS or both signal transduction pathway(s). Results from these studies will provide insight into the role of the cytokine, IL-4, and its effector mechanism in regulating development of the pathogenesis of Sj[unreadable]gren's syndrome. Identification of this effector mechanism could be important to the long-term goal of developing targeted preventive or early intervention strategies.

[unreadable] DESCRIPTION (provided by applicant): Oxalobacter formigenes is a gram-negative, anaerobic bacterium inhabiting the gastrointestinal tracts of most, it not all, vertebrates, including man. This bacterium plays an important symbiotic relationship with its hosts by regulating the absorptive and secretory pathways for oxalic acid in the intestines, thereby maintaining homeostatic levels in plasma. The absence of this bacterium from the gut has been shown to correlate with hyperoxaluria and an increased frequency of first-time and recurrent calcium oxalate kidney stones; consequently, the absence of O. formigenes appears to be a risk factor for urolithiasis. Epidemiological studies of O. formigenes colonization in humans have pointed to several important observations: humans normally become colonized as young children; virtually all children can become colonized naturally; colonization rates in adult populations ranges only between 60-80% worldwide; and, colonization is especially sensitive to changes in the GI tract and the use of antibiotics. In 2003, clinical trials using O.formigenes in the treatment of hyperoxaluria in children with primary hyperoxaluria provided encouraging results, thus raising the specter that this bacterium may be effective in treating calcium oxalate kidney stone disease with enteric hyperoxaluria as an underlying cause. One such population is the subgroup of irritable bowel disease (IBD) patients that develop kidney stones. The studies being proposed in this exploratory R21 grant application are in response to NIDDK Program Announcement PA-03-065, one goal of which is to develop new animal models that may help clarify etiologic factors underlying oxalate stone disease in patients with bowel disease and the identification of improved prevention and therapy options. To this end, we are proposing to determine if (a) the IL-10 gene KO mouse, known to share many features with human Crohn's disease, represents an adequate animal model for the study of hyperoxaluria and calcium oxalate kidney stone disease associated with IBD, and (b) O. formigenes is an effective intervention treatment for reducing hyperoxaluria and/or calcium oxalate crystaluria under conditions that may exist in human patient populations with IBD. By defining the inter-relationships between O. formigenes and the host when the host has IBD, we will be able to define limitations and/or the possible risks of using this bacterium in "probiotic-like" therapies to treat hyperoxaluria in patients with IBD. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Over the past several years, the TH1/TH2 paradigm forming the basis of T cell immunology has expanded rapidly from the discovery of TH17 cells, a subset of CD4+ T memory cells characterized by their unique ability to secrete IL-17 family cytokines. Most importantly, TH17 cells appear to be intimately involved in innate immunity and autoimmunity. Sjvgren's syndrome (SjS) is an autoimmune disease affecting primarily of the salivary and lacrimal glands characterized by exocrine gland dysfunction. In recent years, NOD and NOD-derived mice have been shown to exhibit a disease that closely parallels SjS, including the loss of fluid secretions concomitant with the appearance of leukocyte infiltrates within the salivary and lacrimal glands. This autoimmune exocrinopathy has been separated into two major phases: first, numerous pathophysiologic changes occur within the exocrine glands independent of any autoimmune attack, and second, a progressive chronic autoimmune response resulting in loss of acinar cell mass and decline in exocrine function. Immunohistochemical staining of submandibular glands from C57BL/6.NOD-Aec1Aec2 mice (as well as salivary gland biopsies from SjS patients) has now shown strong positive staining for both IL-17 and IL-23 within the lymphocytic foci, plus a diffuse, sparsely staining of epithelial tissues. Temporal expressions of IL-17 and IL-23 in submandibular glands of C57BL/6.NOD-Aec1Aec2 mice correlated with expression of ROR3t, the TH17 cell master control gene. At the same time, more recent work has shown that IL-27, the cytokine that down-regulates TH17 activity, is expressed at very low levels in the exocrine glands. These results suggest that the TH17/IL-17/ IL-23 system is not only up-regulated in the exocrine glands of C57BL/6.NOD-Aec1Aec2 mice (and SjS patients) at time of disease, but may contribute to the clinical manifestations of the disease. In this grant application, we propose to study the importance of this TH17/IL-17/ IL-23 - IL-27 interactive system in the development and onset of SjS. To achieve this goal two aims are advanced: (i) define and characterize the IL-23 secreting and CD4+ TH17 memory T cell populations infiltrating the salivary glands during development of SjS- like disease, and (ii) determine a possible regulatory potential of IL-27 on the CD4+ TH17 memory T cell populations for preventing development of SjS using a gene therapy approach in the C57BL/6.NOD-Aec1Aec2 mouse model of SjS. Results from this study are expected to provide insight into the inter-relationship(s) between the LF-associated TH17 / IL-23 system and its regulatory IL-27 system in the development and sustainability of the autoimmune response leading to salivary gland dysfunction. Identification of molecular and cellular mechanisms involved in the development and onset of SjS, focusing specifically on biological changes associated with secretory dysfunction at a molecular level, should point to a variety of new and novel pathways permitting development of immunotherapy. A gene therapy approach directed towards regulating the TH17/IL-17/ IL-23 system through IL-27 would be a highly feasible method if results of the current study provide evidence that TH17 cells play an active role in the development and/or onset of SjS PUBLIC HEALTH RELEVANCE: TH1/TH2 paradigm, forming the basis of T cell immunology for decades, has been challenged by the discovery of TH17 cells, a subset of CD4+ T memory cells characterized by their unique ability to secrete IL-17 family cytokines and apparently involved in autoimmunity. In this grant application, we propose to utilize a gene therapy approach to study the possible role of the TH17/IL-17/ IL-23 - IL-27 interactive system in the development and onset of Sjvgren's syndrome (SjS), an autoimmune disease leading to dry mouth and dry eye syndromes. Identification of molecular and cellular mechanisms involved in the development and onset of SjS, focusing specifically on biological changes regulated by TH17/IL-17/ IL-23 / IL-27 and associated with secretory dysfunction, should point to a variety of new and radical targets permitting development of immunotherapy to treat SjS.

ABSTRACT Hyperoxaluria, defined as the presence of excess oxalic acid and/or oxalate salts in urinary excretions, is a physiological condition associated with an increasing number of common and debilitating chronic diseases whose prevalences are increasing in both the United States and the rest of the world. Oxalic acid is a natural and abundant by-product of metabolism, but also a highly oxidized organic compound with powerful chelating activity that, in high concentrations, can cause death in both animals and humans due to its corrosive effects. More commonly, however, hyperoxaluria is now associated (or correlated) with a variety of pathological disorders, including cardiomyopathy, cardiac conductance disorders, urolithiasis, fungal infections, cystic fibrosis, colitis, primary hyperoxaluria type-I, pyridoxine deficiency, and steatorrhea. Thus, regulating oxalate levels in the body is receiving greater recognition as an important factor in controlling the effects of hyperoxaluria in multiple pathologies. Unfortunately, current testing for hyperoxaluria is expensive, time- consuming and mainly conducted by trained technicians in well-equipped laboratories. This SBIR proposal addresses a serious issue in the field of hyperoxaluria-associated pathologies: the lack of a simple, rapid point- of-care device to monitor oxalate levels in biological fluids. To address this deficiency, we propose to complete the development of a dipstick test that rapidly and accurately measures oxalate levels in urine. To obtain proof- of-concept, the team proposes the following Phase I Specific Aims: 1) Develop a dipstick test from which results can be obtained by visual reading, and 2) Perform in-vitro testing of the dipsticks to determine the sensitivity, specificity, accuracy, and reproducibility. Development of a dipstick for quantifying changes in oxalate levels, similar to the glucometer for measuring glucose levels, would represent a unique advancement for patients suffering with diseases ranging from calcium-oxalate kidney stone disease to cystic fibrosis and primary hyperoxaluria type 1. Our preliminary data suggest the stated aims will be readily achieved. Once proof-of-concept is confirmed, the team will move into Phase II, which will focus on clinical functionality and performance of the dipstick.