Thesis (Ph. D.)--Drexel University, 2006. / Includes abstract and vita. Includes bibliographical references (leaves 131-148).

Economic inluence in Czech republic

Methotrexate (MTX) is an antagonist of folic acid metabolism that was initially designed to treat malignancies, including childhood leukemia. After its anti-inflammatory properties were discovered, use of MTX became widespread in the treatment algorithms for several autoimmune illnesses, including the inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC). The specific cause(s) of IBD in general, and CD in specific, have not yet been fully elucidated. However, most investigators agree that the pathogenesis is likely due to a combination of genetic vulnerability and precipitating environmental exposures. Some of these identifiable modifiers include adherence to a low fiber diet, vitamin D deficiency, smoking, and an alteration in the diversity of the gut microbiome in response to viral or bacterial illness or antibiotic medications. Disease activity in patients with CD, particularly during clinical trials, is assessed using composite indices, including the Crohn’s Disease Activity Index (CDAI), Crohn’s Disease Endoscopic Index of Severity (CDEIS), and the Short Inflammatory Bowel Disease Questionnaire (SIBDQ). A wide range of medications is used to induce and maintain long-term remission in patients with active CD. These include corticosteroids, immunosuppressive agents (thiopurines and MTX) and several classes of biologics such as TNF-inhibitors, anti-IL-12/23 and anti-adhesion molecules. TNF-inhibitors are often used as first-line biologic therapies in patients with IBD because they have been in widespread use for over two decades and therefore afford the clinician a more data-driven consideration of risk to benefit ratio when discussing treatment options with their patients. However, there is a relatively high rate of primary non-response and acquired secondary loss of response to TNF-inhibitors. A secondary loss of response often results from the production of neutralizing antibodies, referred to as Antibodies to Infliximab (ATI). In response, concomitant low-dose oral MTX therapy has been employed by clinicians to reduce the immunogenicity of biologic therapy. In addition, previous studies have also demonstrated the efficacy of MTX monotherapy in maintaining clinical remission in patients with CD when delivered at relatively higher doses. However, there is no consensus on the proper dosing or route of administration (oral or parenteral) of MTX. This knowledge gap has resulted in inconsistent clinical practice across physicians and institutions. Pharmacologic studies have identified the metabolic pathways underpinning the mechanism of action of MTX. It is generally understood that MTX in its native form is free to move across cell membranes in a bidirectional manner. It is only after MTX has been glutamated (MTX-PG) that it is “caged” within the cell and can exert its effects. MTX can be glutamated on up to five discrete sites, each contributing to its stereospecificity and membrane permeability. A better understanding of this process may inform the development of rational dosing and pharmacokinetic-based treatment algorithms that provide patients with a sufficient MTX (and subsequently MTX-PG) level required to achieve anticipated clinical efficacy but not so high as to contribute undo morbidity to treated patients. Therefore, optimizing MTX/MTX-PG dosing can significantly advance the utility of this immunomodulatory pathway to treat IBD and other autoimmune disorders. MTX can be administered orally or parenterally, the latter being delivered by either subcutaneous or intramuscular injection. Previous studies have demonstrated increased bioavailability of the drug at higher doses when delivered via the parenteral route. The native form of MTX has a short half-life and is eliminated from the body within 24 hours. Instead, it is the active metabolites of MTX which are retained for more extended periods of time and are ultimately responsible for the anti-inflammatory effects in the body. A MTX molecule can have anywhere from 1 and 5 glutamyl groups attached to it, denoted as MTX-PG1, MTX-PG 2, MTX-PG3, MTX-PG 4, and MTX-PG 5. The mechanism(s) by which MTX-PG moieties contribute anti-inflammatory activity is not fully understood but remains an area of active research. Several studies have explored the association between disease activity and erythrocyte MTX-PG levels. While initial results were mixed, more recent prospective cohort studies in patients with Rheumatoid Arthritis (RA) found an inverse relationship between intracellular levels of longer-chain MTX-PG (MTX-PG3, MTX-PG4, and MTX-PG5) and disease activity. This finding has raised the possibility that monitoring MTX-PG levels could be used as a clinical tool to optimize MTX therapy for patients. However, several key issues persist, including high interpatient variability in MTX-PG levels. Similar studies in patients with CD have thus far been scarce. More prospective studies are needed to explore the utility of MTX-PG pharmacokinetics as a useful biomarker and clinical tool to develop an individualized approach to managing patients with Crohn’s disease.

Paratuberculosis represents a major problem in farmed ruminants and at the present is considered a potential zoonosis. The disease is caused by Mycobacterium avium subsp. paratuberculosis, and susceptibility to infection is suspected to have a genetic component. Caspase recruitment domain 15 (CARD15) gene encodes for a cytosolic protein implicated in bacterial recognition during innate immunity. Crohn's disease (CD) is an idiopathic inflammatory bowel disease in humans comparable in many features to bovine paratuberculosis involving an abnormal mucosal immune response. The association between mutations in the CARD15 gene and increased risk of Crohn's disease has been described. The objective of this candidate gene case-control study was to characterize the distribution of three polymorphisms in the bovine CARD15 gene and test their association with paratuberculosis infection in cattle. Three previously reported single nucleotide polymorphisms (E2[-32] intron 1; 2197/C733R and 3020/Q1007L) were screened for the study population (431 adult cows). The statistical analysis resulted in significant differences in allelic frequencies between cases and controls for SNP2197/C733R (P < 0.001), indicating a significant association between infection and variant allele. In the analysis of genotypes, a significant association was also found between SNP2197/C733R and infection status (P < 0.0001); cows with the heterozygous genotype were 3.35 times more likely to be infected than cows with the reference genotype (P = 0.01). Results suggest a role for CARD15 gene in the susceptibility of cattle to paratuberculosis infection. These data contribute to the understanding of paratuberculosis, suggest new similarities with Crohn's disease and provide new information for the control of bovine paratuberculosis.

INTRODUCTION: Inflammatory bowel disease (IBD) is a growing public health concern with a pressing need for new diagnostic and disease activity biomarkers. Recently, studies have linked IBD disease factors to an imbalance in the gut’s reductive-oxidative (redox) defensive mechanisms and the resulting oxidative stress due to reactive oxygen species. With this new paradigm, direct measurement of redox status in the body could be utilized as a novel biomarker of disease activity for patients with IBD. OBJECTIVES: The goal of this study is to determine how gastrointestinal oxidative state relates to IBD disease activity. Additionally, this study aims to establish a reproducible and accurate measurement protocol for measuring redox status in the human body. METHODS: Patients with and without IBD admitted to Boston Children’s Hospital (Boston, MA) were enrolled in the study from October 2020 to February 2021. Stool and urine samples were collected from these patients and the oxidative status in these biosamples was measured via three redox measuring systems. RESULTS: Data suggests that relative stool oxidative state is more positive in patients with active disease states compared to controls. Also, a finalized protocol for the measurement of relative redox status in stool and urine was established in this study. CONCLUSION: With a reliable and accurate method of measurement established, the potential for relative redox status in the human body to serve as a predictive biomarker for IBD state is promising. Moving forward this study will focus on expanding the study’s size and types of samples to make more significant conclusions in the future. The usefulness of oxidative state in the body as a disease biomarker is just beginning to be realized.

Background: Crohn's Disease (CD), a chronic inflammatory bowel disease (IBD), is thought to be multifactorial, involving an interaction between genetic susceptibility, undefined environmental triggers, and immune-mediated tissue injury. Biochemical and other molecular approaches identified isolates from intestinal tissues of patients with CD as Mycobacterium avium subsp paratuberculosis (MAP), the causative agent of Johne's disease, a granulomatous bowel disease in ruminants similar to CD. MAP has been identified directly in resected tissues of increasing numbers of CD patients at a frequency significantly higher than those of controls. Treatment of CD patients, which depends on the location and severity of disease, complication, and response to previous treatment is most often to control the disease. There is no cure. Diagnosis of this disease requires a series of tests including upper gastrointestinal endoscopy and colonoscopy. These tests are expensive, inconvenient and require hospitalization. Objective: A blood serologic test is sought for diagnosis of CD patients infected with MAP. Methods: The recombinant E. coli clone pBl 1 containing a 1,302 bp MAP DNA insert and expressing a 20 kD protein has been grown, induced by arabinose and then harvested by centrifugation. Protein extracts were prepared, quantitated and then subjected to Isoelectricfocussing (IEF) in ampholyte buffer pH 3-10. Twenty fractions were collected, quantitated and then analyzed on SDS-P AGE by silver staining and Imrnuboblotting. The immunoblots were screened with anti-express IgG monoclonal antibodies. Fractions containing the semipurified 20 KD protein were analyzed by immnoblot against 85 sera specimens with 1:30 dilution (43 CD patients and 42 controls). Both IgG and IgA response in each patient was determined. Results: Of 20 fractions collected, fractions 5 and 6 with a PI ranging from 4.18 to 5.01 reacted with the anti-express IgG antibodies. p20 with a 20 kD molecular weight was confirmed. These fractions contained fewer proteins bands with p20 being dominant. Of 43 CD sera specimens, 74% contained IgG response and only 50% contained IgA response to p20. On the other hand, of 42 controls, only 17% contained IgG and. 50 % contained IgA response.against p20 antigen. Conclusion: p20 reacted with CD IgG sera with frequency much higher than control sera (74% versus 17%) indicating a great potential for using p20 as a reagent in a quantitative ELISA assay for specific diagnosis of CD patients. Additionally, the data add strong support to MAP role in CD pathogenesis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been suggested as a causative agent of Crohn's disease (CD). Despite a long-term debate to prove this possibility, the role of this bacterium in the pathogenesis of CD is still a subject of controversy. The objective of the study was to develop a serodiagnostic assay for the diagnosis of CD in humans. METHODS: In the present study, five different ELISA assays were accessed: 1) IDEXX, a commercially available kit for the diagnosis of Johne's disease in ruminants; 2) an in-house developed assay based on total MAP cytoplasmic proteins, and three other assays based on recombinant MAP recombinant antigens a) a 23 kDa antigen, pB11/B7, b) a 35 kDa antigen, P35 and c) a 36 kDa antigen, P36. The last three proteins were identified from an expression genomic library of MAP that was constructed in our laboratory. A total of 43 sera samples were analyzed in this study, which included 14 CD patients, 14 Ulcerative Colitis (UC) patients, and 13 non-inflammatory bowl disease (IBD) patients. lmmunoblot and silver stain analyses were performed to confirm protein identity and purity. ELISA was developed and used to analyze the level of anti-MAP lgG antibodies in sera from patients. RES UL TS: The rate of positive ELISA results is based on previously published interpretation criteria. ELISA results using the IDEXX kit showed 12/14 (85.7%) positive for CD as compared to 7/13 (53.8%) for non-lBD and 6/14 (42.9%) for uc.· 8/14 (57.1%) of the CD sera were positive with the ELISA results based on MAP cytoplasmic proteins compared with 6/13 (46.2%) of non-lBD and 10/14 (71.4%) of UC. Further analyzing the recombinant proteins, when two out of three assays were used 12/14 (85.7%) CD (P<0.05), 0/13 (0.0%) non-lBD, and 1/14 (7.7 %) UC were positive. Moreover, when all three recombinant proteins are utilized for analysis, the specificity of the test greatly increased, giving 13/14 (92.9%) positive for CD, 3/14 (21.4%) for UC and 2/14 (14.3%) for non-lBD. CONCLUSION: MAP recombinant proteins, pB11/B7, p35, and p36 showed a strong reactivity with diagnosed CD patients while excluding healthy individuals and other IBD patients. In addition, they served as a great tool to distinguish between CD and UC patients. A larger sample size needs to be tested, none the less this data strengthens the role of MAP in CD etiology and suggests a great potential for using the recombinant-based assays for diagnosis and treatment monitoring of patients with inflammatory bowel disease.

Crohn's Disease (CD) is a debilitating condition characterised by chronic intermittent intestinal inflammation. More than 90 genetic polymorphisms are associated with CD susceptibility, including several in genes of the innate immune system. Here I present a series of experiments designed to enhance our knowledge of the roles of CD-associated polymorphisms in pathogenesis. Many therapeutic regimens are employed in CD treatment, but patients' responses to treatment and disease progression vary widely. There is great interest in studying whether analysis of patients' genotype at CD-associated polymorphisms can be used to predict their disease course, and guide clinical decision-making. To answer these questions, it is essential to be able routinely and cost- effectively to genotype patients at the full range of known CD-associated polymorphisms. The first project presented here describes the design and initial successful testing of a CD-specific genotyping microarray for use in genotype-phenotype studies. The polymorphism most strongly associated with CD susceptibility is in the pattern recognition receptor NOD2; the remaining experiments presented here study the function of NOD2 in primary human monocyte-derived Dendritic Cells (DCs). First, a microarray study is presented which characterises global transcriptional responses to NOD2 stimulation in DCs. NOD2 stimulation is shown to enhance transcriptional changes induced by Toll-Like Receptor 2 stimulation, and NOD2-mediated transcriptional regulation is shown to be lost in DCs expressing CD-associated NOD2 variants. Second, experiments are presented which describe development of a new protocol for proteomic analysis of post-translational protein modifications, and which identify a number of novel candidate targets of NOD2 signalling in DCs. Finally, a project is presented which demonstrates for the first time that NOD2 stimulation induces autophagy in DCs, in an NF-kB and RIPK2-dependent pathway. CD-associated polymorphisms in NOD2 and ATG 16Ll abolish NOD2-mediated autophagy induction, resulting in impaired bacterial handling and antigen presentation.

NOD2 is an intracellular innate immune receptor expressed in dendritic cells and gastrointestinal epithelial cells. Polymorphisms in the NOD2 gene convey a strong predisposition to Crohn’s disease (CD), a form of inflammatory bowel disease. Understanding the function of NOD2, and in what way it is aberrant in the presence of NOD2 polymorphisms, would confer a valuable paradigm for understanding Crohn’s pathogenesis. CD is thought to arise both from defects in the gut mucosal barrier and from a dysregulated Th17 immune response to commensal gut flora. Aberrant expression of IL-23 is present in both human CD and in murine models of colitis. Wild-type NOD2 contributes to NFκB activation and pro-inflammatory cytokine production on recognition of its cognate ligand, a function that is lost in CD-associated mutations. How the predominantly loss-of-function CD-NOD2 contributes to the pro-inflammatory response present in Crohn’s is not yet understood. In this thesis a set of experiments is described that aim to shed light on the function of NOD2, firstly through identification of negative regulators of immune activation that are dependent on NOD2 for their expression. This work identifies the microRNAs that are expressed following NOD2 triggering in human dendritic cells. Specifically, up-regulation of the miR-29 family was found to be dependent on wild-type NOD2 function. A number of novel miR-29 targets and their functional consequences are presented, including the cytokine subunits IL-12p40 and IL-23p19, directly linking NOD2 polymorphisms and aberrant IL-23 expression. Secondly, a project aiming to identify components of the NOD2 signalling complex (or signalosome) is described. To this end I employed a model system that involved tagging NOD2, and stable expression in a human cell line. These clones were validated for expression and function before an immunoprecipitation protocol was optimised. Mass spectrometry analysis of these samples identified the known NOD2-interacting protein Erbin.

A system of ordinary differential equations is developed to model the effect of fatty acids on chronic intestinal inflammation that is typical of a Crohn’s patient. Several murine studies have shown an anti-inflammatory response when specific polyunsaturated fatty acids are included regularly in the diet. It is believed that the fatty acids serve as a specific ligand that activates the Peroxisome Proliferator Activated Receptor (PPAR) which is located on multiple cell types that are active in the inflammatory response. The binding of the PPAR results in a suppression of the inflammatory pathway. Results of the model indicate a muted inflammatory response when fatty acids are added regularly to the diet in mild to moderate cases of Crohn’s. Results of mathematical analysis show a stable fixed point with decreased inflammatory markers and pathogen levels when fatty acids are added regularly to the diet.