Insulin resistance, endothelial function, angiogenic factors and clinical outcome in non-diabetic patients with chest pain without myocardial perfusion defects.

BACKGROUND: Patients with angina-like symptoms without myocardial perfusion scintigram (MPS)-verified abnormality may still be at risk for cardiovascular events. We hypothesized that insulin resistance could play a role in this population even without diagnosed diabetes. We further explored physiological and blood biomarkers, as well as global gene expression patterns that could be closely related to impaired glucose homeostasis to deepen our mechanistic understanding. METHODS: A total of 365 non-diabetic patients with suspected myocardial ischemia referred to MPS were enrolled and followed up regarding event-free survival with a median time of 5.1 years. All patients underwent endothelial function assessment by reactive hyperemic index (RHI) using EndoPAT and extensive biomarker analysis. Whole blood global gene expression pathway analysis was performed in a subset of patients. RESULTS: Homeostasis model assessment of insulin resistance (HOMA-IR) added independent prognostic value in patients without myocardial perfusion defects. In a multivariable analysis, HOMA-IR was inversely associated with low RHI. Furthermore, elevated HOMA-IR was associated with decreased levels of vascular endothelial growth factor D, stem cell factor and endocan as well as to increased level of interleukin-6. Global gene expression pathway analysis of whole blood cells showed that high HOMA-IR and impaired endothelial function were associated with upregulated pro-inflammatory pathways and down-regulated eukaryotic initiation factor-2 pathway. CONCLUSIONS: Insulin resistance measured by HOMA-IR is associated with endothelial dysfunction and confers independent prognostic information in non-diabetic patients with chest pain without myocardial perfusion defects. Increased systemic pro-inflammatory state and decreased levels of pro-angiogenic vascular growth factors may be important underlying molecular mechanisms.

A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling.

Evaluation and optimization of physicochemical and metabolic properties of compounds are a crucial component of the drug development process. Continuous access to this information during the design-make-test-analysis cycle enables identification of chemical entities with suitable properties for efficient project progression. In this study, we describe an integrated and automated assay panel (DMPK Wave 1) that informs weekly on lipophilicity, solubility, human plasma protein binding, and metabolic stability in rat hepatocytes and human liver microsomes. All assays are running in 96-well format with ultraperformance liquid chromatography-mass spectrometry (MS)/MS as read-out. A streamlined overall workflow has been developed by optimizing all parts of the process, including shipping of compounds between sites, use of fit-for-purpose equipment and information systems, and technology for compound requesting, data analysis, and reporting. As a result, lead times can be achieved that well match project demands across sites independently of where compounds are synthesized. This robust screening strategy is run on a weekly basis and enables optimization of structure-activity relationships in parallel with DMPK properties to allow efficient and informed decision making.

Intra-colonic administration of the TLR7 agonist R-848 induces an acute local and systemic inflammation in mice.

Imidazoquinoline compounds, such as resiquimod (R-848), are well known topically active immune modifiers that bind to toll-like receptor 7 (TLR7). The aim of this study was to characterize the R-848 induced inflammatory response in mice and to validate the response using methyl-prednisolone and anti-TNF antibody. Intra-colonic application of R-848 to BALB/c mice induced a systemic transient elevation of TNF, CXCL1, IL-6, and IL-12p40 and a colonic elevation of cytokines/chemokines and iNOS, without infiltration of immune cells or epithelial destruction. Treatment with methyl-prednisolone or anti-TNF antibody attenuated the systemic (TNF, IL-6, IL-12p40, and CXCL1) and local (colonic TNF and iNOS mRNA expression) response induced by R-848. In summary, intra-colonic administration of R-848 induces an acute systemic and local inflammatory response, which can be attenuated by steroids or anti-TNF antibody. We suggest that the R-848 inflammatory model can be useful in future validation of new drugs for gastrointestinal inflammatory conditions.

Acute colonic ischaemia in rats results in long-term structural changes without alterations of colonic sensitivity.

Colonic ischaemia and mast cells have been involved in the pathophysiology of the functional gastrointestinal disorder irritable bowel syndrome, although the cause-effect relationships remain unknown. We assessed long-term histopathological and functional changes associated to an acute ischaemic episode (1 h) of the colon, followed by 8-week recovery, in rats. Functional colonic alterations [sensitivity during colorectal distension (CRD), compliance and propulsive motility] were assessed regularly during the recovery. Colonic histopathology (presence of inflammation, morphometric alterations and variations in neuronal density in the enteric nervous system) 8-week postischaemia was assessed. Following ischaemia, none of the functional parameters tested (motility, sensitivity and compliance) were affected. At necropsy, the colon presented an overall normal appearance with an increase in weight of the ischaemic area (mg/cm: 99 +/- 6; P < 0.05 vs. control: 81 +/- 4 or sham ischaemia: 81 +/- 3). Histopathological evaluations revealed the presence of a local infiltrate of mast cells in the area of ischaemia (nb of mast cells: 142 +/- 50; P < 0.05 vs. control, 31 +/- 14 or sham ischaemia: 40 +/- 16), without other significant alterations. Animals subjected to colonic ischaemia and treated 8 weeks later with the mast cell degranulator, compound 48/80, showed no changes in CRD-related pain responses. These studies show that acute colonic ischaemia is associated with the presence of a long-term local infiltration of mast cells, located within the serosa and muscle layers, despite the absence of functional changes, including colonic sensitivity. Considering the important pathophysiological functions of mast cells, the observed mast cell infiltration may be involved in ischaemia-induced functional changes yet to be characterized.

Predicting and monitoring colitis development in mice by micro-computed tomography.

BACKGROUND: Computed tomography (CT) has been developed as a tool for monitoring human inflammatory bowel disease (IBD). The aim of this study was to evaluate colon wall thickness as a noninvasive marker in the dextran sodium sulfate (DSS) mouse model of colitis using micro-CT. METHODS: Mice were examined by micro-CT 1, 2, or 4 times between day 0 (d0) and d26 after induction of colitis to document the kinetics of changes in colon wall thickness and its relation to colitis development. RESULTS: DSS-treated mice displayed a significantly thicker colon wall at all timepoints (days 5, 8, 12, 19, and 26) investigated compared to healthy controls. Colon wall thickness showed a good correlation to the macroscopic grading of colitis (r = 0.81). The increase in colon wall thickness occurred mainly during the acute phase of colitis (between days 5 and 12) and did not progress much further in the chronic phase of colitis (d26). Colon wall thickness at d26 was thereby predicted by measurements at d12. All mice did not respond equally to DSS and this difference was manifest during the first 2 weeks of colitis, providing an important tool in stratifying responders from nonresponders. CONCLUSIONS: While the potential impact of handling and anesthesia should be considered on repeated micro-CT, irradiation exposure during repeated micro-CT did not affect the development of colitis. Thus, the results suggest that micro-CT can be used for monitoring and prediction of the inflammatory response in mouse colitis in future therapeutic studies.

Validation of murine dextran sulfate sodium-induced colitis using four therapeutic agents for human inflammatory bowel disease.

Dextran sulfate sodium (DSS)-induced colitis is one of the most frequently used rodent models for inflammatory bowel disease (IBD). The aim of this study was to validate the murine DSS-induced colitis model using four therapeutic agents for IBD. C57BL/6 mice were exposed to 3% DSS for 5days followed by 7-9 days of water (acute inflammation) or 20-31 days of water (chronic phase). Clinical symptoms, plasma and colonic inflammatory markers and histology were assessed for the efficacy of cyclosporine A (CsA), methotrexate or anti-IL-12p40 in acute colitis and of anti-IL-12p40 or an agonistic anti-CD3 antibody in chronic colitis. Cyclosporine A and anti-IL-12p40 (in the acute phase) and anti-CD3 (in the chronic phase) treatment attenuated local cytokine levels, improved clinical symptoms (CsA and anti-IL-12p40) and histology (CsA and anti-CD3). Further, anti-IL-12p40 treatment was partly efficacious in the chronic phase, whereas methotrexate showed no efficacy in the acute colitis. Thus, three of the current tested agents showed efficacy in the disease model, arguing that DSS-induced colitis can be used as a relevant model for the translation of mice data to human disease.

Magnetic resonance imaging of experimental mouse colitis and association with inflammatory activity.

BACKGROUND: Ulcerative colitis and Crohn's disease are the major chronic inflammatory bowel diseases affecting the gastrointestinal tract in humans. Imaging techniques such as endoscopy and computed tomography are used to monitor disease activity. Magnetic resonance imaging (MRI) is emerging as a diagnostic modality, and studies have shown that MRI can be used in the diagnostic procedure of patients with inflammatory bowel disease. The aim of the present study was to investigate the role of MRI in quantitatively reflecting inflammation in an experimental mouse colitis model. METHODS: Colonic inflammation was induced by exposing mice to dextran sulfate sodium. MRI was used to assess colon wall thickness, T2-weighted (T2w) signal, and contrast-enhanced T1-weighted (T1w) signal in inflamed and healthy animals in vivo. Haptoglobin and interleukin-1beta served as systemic and local inflammatory markers, and macroscopic ex vivo scoring of the colon was performed to assess colonic inflammation. RESULTS: Dextran sulfate sodium-exposed animals displayed increased levels of inflammatory markers and higher inflammatory score compared with healthy animals. Colon wall thickness and contrast-enhanced T1w signal were significantly increased in dextran sulfate sodium-exposed compared with healthy animals. In addition, the T2w signal was positively correlated with haptoglobin levels and colon wall thickness in the inflamed animals. CONCLUSIONS: Our results show that MRI can be used to depict healthy and inflamed mouse colon and that the T2w signal, contrast-enhanced T1w signal, and colon wall thickness may be used to characterize inflammation in experimental colitis. These potential biomarkers may be useful in the evaluation of putative drugs in longitudinal studies in both mice and humans.

Local production of chemokines and prostaglandin E2 in the acute, chronic and recovery phase of murine experimental colitis.

Increased levels of chemokines and prostaglandins have been reported in patients with inflammatory bowel disease, although their changes during disease development are less understood. The aim of this study was to investigate the local production of nine selected chemokines and prostaglandin E(2) (PGE(2)) to elucidate their role in colitis progression in BALB/c and C57BL/6 mice exposed to dextran sulphate sodium. The acute inflammation in both strains was accompanied by a significant up-regulation of CXCL1, CXCL2/3, CXCL10, CCL2, CCL4 and CCL22 and a downregulation of PGE(2). In the recovery phase in BALB/c, one-week post-DSS, PGE(2) levels were significantly increased with a concomitant downregulation of CXCL1, CXCL2/3, CXCL10, CCL2, and CCL4. In contrast, in C57BL/6 mice CXCL1, CXCL2/3, CXCL10, CCL2, CCL3 and CCL4 production remained high during the chronic phase, without any up-regulation of PGE(2). In addition, CCL5 was significantly increased at d26 and 33 compared to d5. Interestingly, the number of macrophages was significantly increased during the acute phase, whereas T cells were significantly increased in both the acute and chronic phase in C57BL/6 mice. Thus, our results show that chemokines are produced in a dynamic manner during colitis progression.