Dysregulated NOD2 predisposes SAMP1/YitFc mice to chronic intestinal inflammation.

Nucleotide-binding oligomerization domain-containing 2 (NOD2) is an intracellular receptor that plays an essential role in innate immunity as a sensor of a component of the bacterial cell wall, muramyl dipeptide (MDP). Crohn's disease (CD)-associated NOD2 variants lead to defective innate immune responses, including decreased NF-κB activation and cytokine production. We report herein that SAMP1/YitFc (SAMP) mice, which develop spontaneous CD-like ileitis in the absence of NOD2 genetic mutations, fail to respond to MDP administration by displaying decreased innate cytokine production and dysregulated NOD2 signaling compared with parental AKR control mice. We show that, unlike in other mouse strains, in vivo administration of MDP does not prevent dextran sodium sulfate-induced colitis in SAMP mice and that the abnormal NOD2 response is specific to the hematopoietic cellular component. Moreover, we demonstrate that MDP fails to enhance intracellular bacterial killing in SAMP mice. These findings shed important light on the initiating molecular events underlying CD-like ileitis.

Enhanced targeting of ultrasound contrast agents using acoustic radiation force.

Contrast-enhanced ultrasound has shown significant promise as a molecular imaging modality. However, one potential drawback is the difficulty that ultrasound contrast agents (UCA) may have in achieving adhesion to target molecules on the vascular endothelium. Microbubble UCA exhibit a lateral migration toward the vessel axis in laminar flow, preventing UCA contact with the endothelium. In the current study, we have investigated low-amplitude acoustic radiation as a mechanism to move circulating UCA toward targeted endothelium. Intravital microscopy was used to assess the retention of microbubble UCA targeted to P-selectin in the mouse cremaster microcirculation and femoral vessels. Acoustic treatment enhanced UCA retention to P-selectin four-fold in cremaster venules and in the femoral vein and 20-fold in the femoral artery. These results suggest acoustic treatment as a mechanism for enabling ultrasound-based molecular imaging in blood vessels with hemodynamic and anatomical conditions otherwise adversarial for UCA retention.

Binding of function-blocking mAbs to mouse and human P-selectin glycoprotein ligand-1 peptides with and without tyrosine sulfation.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin-expressing endothelial cells under shear flow. Function-blocking monoclonal antibodies (mAbs) against mouse and human PSGL-1 recognize an anionic segment at the N-terminus of PSGL-1. High affinity interaction of PSGL-1 with P-selectin requires sulfation of tyrosines 46, 48, and 51 (human) or 54 and 56 (mouse). We tested binding of two anti-human (KPL1 and PL1) and two anti-mouse (4RA10 and 2PH1) PSGL-1 mAbs to synthetic peptides of N-terminus of human and mouse PSGL-1 and found binding to be independent of tyrosine sulfation. In peptide-blocking experiments, sulfated and nonsulfated human and mouse peptides competed with antibody binding to PSGL-1 expressed on myeloid cells. Arylsulfatase treatment significantly reduced P-selectin binding but had no effect on antibody binding. Our data show, in three independent assay systems, that function-blocking antibodies to mouse or human PSGL-1 do not require sulfation of N-terminal tyrosines for binding.

Role of platelets in the development of atherosclerosis.

Platelets are blood cell fragments that originate from the cytoplasm of megakaryocytes in the bone marrow and circulate in blood to play a major role in the hemostatic process and in thrombus formation after an endothelial injury. Recent studies have provided insight into platelet functions in inflammation and atherosclerosis. A range of molecules, present on the platelet surface and/or stored in platelet granules, contributes to the cross-talk of platelets with other inflammatory cells during the vascular inflammation involved in the development and progression of atherosclerosis. This review discusses the nature of these molecules and the mechanisms involved in the participation of platelets in atherosclerosis, with emphasis on P-selectin, platelet-monocyte interactions, chemokines, and inflammatory cytokines.

Cerebral vasospasm after subarachnoid hemorrhage: putative role of inflammation.

Cerebral vasospasm is a common, formidable, and potentially devastating complication in patients who have sustained subarachnoid hemorrhage (SAH). Despite intensive research efforts, cerebral vasospasm remains incompletely understood from both the pathogenic and therapeutic perspectives. At present, no consistently efficacious and ubiquitously applied preventive and therapeutic measures are available in clinical practice. Recently, convincing data have implicated a role of inflammation in the development and maintenance of cerebral vasospasm. A burgeoning (although incomplete) body of evidence suggests that various constituents of the inflammatory response, including adhesion molecules, cytokines, leukocytes, immunoglobulins, and complement, may be critical in the pathogenesis of cerebral vasospasm. Recent studies attempting to dissect the cellular and molecular basis of the inflammatory response accompanying SAH and cerebral vasospasm have provided a promising groundwork for future studies. It is plausible that the inflammatory response may indeed represent a critical common pathway in the pathogenesis of cerebral vasospasm pursuant to SAH. Investigations into the nature of the inflammatory response accompanying SAH are needed to elucidate the precise role(s) of inflammatory events in SAH-induced pathologies.

Dynamics of in silico leukocyte rolling, activation, and adhesion.

BACKGROUND: We present a multilevel, agent based, in silico model that represents the dynamics of rolling, activation, and adhesion of individual leukocytes in vitro. Object-oriented software components were designed, verified, plugged together, and then operated in ways that represent the molecular and cellular mechanisms believed responsible for leukocyte rolling and adhesion. The result is an in silico analogue of an experimental in vitro system. The experimentally measured, phenotypic attributes of the analogue were compared and contrasted to those of leukocytes in vitro from three different experimental conditions. RESULTS: The individual in silico dynamics of "rolling" on simulated P-selectin, and separately on simulated VCAM-1, were an acceptable match to individual in vitro distance-time and velocity-time measurements. The analogues are also able to represent the transition from rolling to adhesion on P-selectin and VCAM-1 in the presence of GRO-alpha chemokine. The individual in silico and in vitro behavioral similarities translated successfully to population level measures. These behavioral similarities were enabled in part by subdividing the functionality of the analogue's surface into 600 independent, "cell"-controlled, equally capable modules of comparable functionality. CONCLUSION: The overlap in phenotypic attributes of our analogue with those of leukocytes in vitro confirm the considerable potential of our model for studying the key events that determine the behavioral outcome of individual leukocytes during rolling, activation, and adhesion. Our results provide an important foundation and framework for future in silico research into plausible causal links between well-documented, subcellular molecular level events and the variety of systemic phenotypic attributes that distinguish normal leukocyte adhesion from abnormal disease-associated adhesion.

Targeting mucosal addressin cellular adhesion molecule (MAdCAM)-1 to noninvasively image experimental Crohn's disease.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is the second most common chronic inflammatory disorder worldwide; however, a noninvasive means of accurately assessing the severity and extent of intestinal inflammation is currently not available. The aim of the present study was to develop a noninvasive imaging modality to detect and evaluate ileitis in SAMP1/YitFc (SAMP) mice. METHODS: An image-enhancing ultrasound (US) contrast agent, consisting of encapsulated gaseous microbubbles (MB), was developed specifically to bind mucosal addressin cellular adhesion molecule-1 (MAdCAM-1), a mucosal-restricted addressin up-regulated during gut inflammation. MAdCAM-1-targeted MB (MB(M)) were tested for binding specificity on MAdCAM-1 protein and tumor necrosis factor (TNF)-stimulated SVEC4-10 endothelial cells using an in vitro flow chamber assay and for their ability to detect and quantify ileitis by intravital microscopy and transabdominal US. RESULTS: Under in vitro flow conditions, a 100-fold increase in MB(M) binding was observed on MAdCAM-1 protein compared with nonspecific MB (P < .001). TNF-stimulated endothelial cells bound significantly more MB(M) vs nonspecific MB (P < .001), which was abrogated after preincubation with anti-MAdCAM-1 antibodies (P < .001). In vivo, MB(M) specifically accumulated in focal areas of ileal inflammation and produced stronger acoustic echoes, measured by average video intensity, in SAMP vs uninflamed AKR mice (P < .001) or SAMP given nonspecific MB (P < .001). MB(M)-specific video intensity showed a strong positive correlation with total ileal inflammatory scores (R2 = 0.92). CONCLUSIONS: We have developed a novel intravascular US contrast agent targeting MAdCAM-1 that specifically detects and quantifies intestinal inflammation in experimental ileitis, providing the potential for a reliable, noninvasive means to diagnose and monitor disease in patients with IBD.

Linkage to peroxisome proliferator-activated receptor-gamma in SAMP1/YitFc mice and in human Crohn's disease.

BACKGROUND AND AIMS: Genetic predisposition is implicated strongly in Crohn's disease. Disease-associated mutations in NOD2/CARD15 , the best-studied susceptibility gene in this disorder, explain only a small fraction of the heritability. The SAMP1/YitFc (SAMP1/Fc) mouse strain expresses many features of Crohn's disease in humans. We bred SAMP1/Fc to disease-resistant AKR mice to identify additional susceptibility genes that may play a role in human disease. METHODS: Linkage disequilibrium mapping was performed in an (AKR x SAMP1/Fc) backcross to SAMP1/Fc, followed by sequencing, expression analysis using reverse transcription polymerase chain reaction (PCR) and immunohistochemistry, and functional testing in vivo of the regional candidate gene encoding the peroxisome proliferator-activated receptor gamma ( Pparg ). A cohort-based association study was performed in humans. RESULTS: We show that ileitis is blocked in SAMP1/Fc mice by inheritance of AKR alleles on chromosome 6 in the region of Pparg . Major differences in Ppargamma expression in the parental mouse strains are found specifically in the crypts of the small intestine, and treatment of ileitis-prone mice with a Ppargamma agonist decreased disease severity in susceptible mice expressing low levels of the protein. Rare alleles of PPARG are associated significantly with Crohn's disease in humans. CONCLUSIONS: We have identified Pparg as a susceptibility gene in both the SAMP/Yit mouse and in human Crohn's disease. Similarities between Crohn's disease and the SAMP1/Fc model suggest that the effect of this gene in humans may be mediated through regulation of PPARgamma activity in the crypts of the small intestine.

Alpha4beta1 integrin (VLA-4) blockade attenuates both early and late leukocyte recruitment and neointimal growth following carotid injury in apolipoprotein E (-/-) mice.

BACKGROUND: The alpha(4)beta(1) integrin (VLA-4) supports rolling and firm adhesion of leukocytes to inflamed tissues via ligation of VCAM-1 or fibronectin expressed on the activated endothelium. We tested the hypothesis that VLA-4 mediates leukocyte recruitment and neointimal growth after arterial injury in the atherosclerosis-prone apolipoprotein E (ApoE)-deficient mouse. METHODS: ApoE (-/-) mice fed a Western diet underwent air desiccation injury, and the expression patterns of VLA-4 and VCAM-1 were determined by immunohistochemistry (IHC). To determine the effect of targeted VLA-4 blockade on leukocyte recruitment and neointimal growth, ApoE (-/-) mice received an intraperitoneal injection of a VLA-4 neutralizing monoclonal antibody (PS/2) at the time of injury alone or over a prolonged administration course. Additional mice received an isotype control antibody. RESULTS: IHC demonstrated a marked increase in VLA-4 expression 7 days following injury. Prolonged administration of PS/2 resulted in a 72% reduction (p < 0.02) in neointimal growth 28 days following injury. IHC revealed a marked 95% reduction in neutrophil recruitment at 7 days and a 48% reduction in macrophage recruitment 28 days following injury with prolonged PS/2 administration. CONCLUSIONS: Prolonged VLA-4 blockade reduces leukocyte recruitment and neointimal growth following air desiccation injury in ApoE (-/-) mice. These findings demonstrate an important role for VLA-4 in the response to arterial injury.