Jan Heidemann

Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury

Chronic hepatitis C virus (HCV) infection is characterized by inflammatory liver damage and is associated with a high risk of development of cirrhosis and hepatocellular carcinoma. Although histological examination of liver biopsies is currently the gold standard for the detection of early liver damage, there is a strong need for better noninvasive methods. We recently demonstrated that the proapoptotic activation of caspases is considerably enhanced in histological sections from HCV‐infected liver tissue, suggesting an important role of apoptosis in liver damage. Here, we investigated whether caspase activation is detectable also in sera from patients with chronic HCV infection. Using a novel enzyme‐linked immunosorbent assay that selectively recognizes a proteolytic neoepitope of the caspase substrate cytokeratin‐18, we demonstrate that caspase activity is markedly increased in the sera of HCV patients. Interestingly, while 27% of patients with chronic HCV infection showed normal aminotransferase levels despite inflammatory and fibrotic liver damage, more than 50% of those patients exhibited already elevated serum caspase activity. Moreover, 30% of patients with normal aminotransferase but elevated caspase activity revealed higher stages of fibrosis. In conclusion, compared with conventional surrogate markers such as aminotransferases, detection of caspase activity in serum might be a more sensitive method of detecting early liver injury. Thus, measurement of caspase activity might provide a novel diagnostic tool, especially for patients with normal aminotransferases but otherwise undiagnosed histologically active hepatitis and progressive fibrosis. (HEPATOLOGY 2004;40:1078–1087.)

Phagocyte‐specific S100 proteins are released from affected mucosa and promote immune responses during inflammatory bowel disease

Phagocyte‐derived S100 proteins are endogenous activators of innate immune responses. S100A12 binds to the receptor for advanced glycation end‐products, while complexes of S100A8/S100A9 (myeloid‐related proteins, MRP8/14; calprotectin) are ligands of toll‐like receptor 4. These S100 proteins can be detected in stool. In the present study we analyse the release of S100A12 and MRP8/14 from intestinal tissue. Specimens from patients with Crohns disease (CD; n = 30), ulcerative colitis (UC; n = 30), irritable bowel syndrome (IBS; n = 30) or without inflammation (n = 30) were obtained during endoscopy. After 24 h culture, S100A12 and MRP8/14 were analysed in supernatants. Endoscopic, histological, laboratory and clinical disease activity measures were documented. We found an increased spontaneous release of S100A12 from tissue in inflammatory bowel disease (IBD). The release of S100A12 into the supernatants was 28‐fold enhanced in inflamed tissue when compared to non‐inflamed tissue (mean 46.9 vs. 1.7 ng/ml, p < 0.0001). In active CD, release of S100A12 and MRP8/14 was strongly dependent on localization, with little release from sites of active ileal inflammation compared to colonic inflammation. This difference was more pronounced for S100A12 than for MRP8/14. S100A12 and MRP8/14 provoked up‐regulation of adhesion molecules and chemokines on human intestinal microvascular endothelial cells (HIMECs) isolated from normal colonic tissue. The direct release of phagocyte‐derived S100 proteins from inflamed tissues may reflect secretion from infiltrating neutrophils (S100A12) and also monocytes or epithelial cells (MRP8/14). Via activation of pattern recognition receptors, these proteins promote inflammation in intestinal tissue. The enhanced mucosal release can explain the correlation of fecal markers with disease activity in IBD. Copyright

Constrictive pericarditis, still a diagnostic challenge: comprehensive review of clinical management

The diagnosis of constrictive pericarditis (CP) continues to be a challenge in the modern era. Understanding the pathophysiology and integrating the results of invasive and non-invasive techniques are important in the differential diagnosis of CP and e.g. restrictive cardiomyopathy. New echocardiographic techniques such as tissue Doppler imaging (TDI) and 2D-speckle tracking, dual-source CT (computed tomographic imaging) and especially tagged cine-MRI (magnetic resonance imaging) with the analysis of phase contrast angiography sequences are promising novel approaches. Pericardiectomy in experienced centers with complete decortication (if technically feasible) is the treatment of choice for CP and it results in symptomatic relief in most patients. However, some patients may not benefit from pericardiectomy and this may be due to myocardial compliance abnormalities, myocardial atrophy after prolonged constriction, residual constriction or other myocardial processes. An important predictor of long-term outcome after pericardiectomy is the etiology of the pericardial disease. The overall mortality in the current literature is nearly 5-6%. Survival with post-surgical CP is worse than with idiopathic CP, but significantly better than with post-radiation CP.

Human Intestinal Microvascular Endothelial Cells Express Toll-Like Receptor 5: A Binding Partner for Bacterial Flagellin

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.

Mechanisms of Endotoxin Tolerance in Human Intestinal Microvascular Endothelial Cells

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6–60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-κB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24–48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-κB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.

The intestinal microvasculature as a therapeutic target in inflammatory bowel disease.

Abstract:  Chronic inflammation is a complex biologic process which involves immune as well as non‐immune cells including the microvasculature and its endothelial lining. Growing evidence suggests that the microvasculature plays an integral role in the pathophysiology of inflammatory bowel disease (IBD; Crohns disease and ulcerative colitis). The microvasculature contributes to chronic inflammation through altered leukocyte recruitment, impaired perfusion, and angiogenesis leading to tissue remodeling. These diverse areas of IBD microvascular biology represent therapeutic targets that are currently undergoing investigation.

Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling

The immunosuppressive agent cyclosporin A (CsA), a calcineurin inhibitor which blocks T cell activation has provided the pharmacologic foundation for organ transplantation. CsA exerts additional effects on non-immune cell populations and may adversely effect microvascular endothelial cells, contributing to chronic rejection, a long-term clinical complication and significant cause of mortality in solid-organ transplants, including patients with small bowel allografts. Growth of new blood vessels, or angiogenesis, is a critical homeostatic mechanism in organs and tissues, and regulates vascular populations in response to physiologic requirements. We hypothesized that CsA would inhibit the angiogenic capacity of human gut microvessels. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were used to evaluate CsAs effect on four in vitro measures of angiogenesis, including endothelial stress fiber assembly, migration, proliferation and tube formation, in response to the endothelial growth factor VEGF. We characterized the effect of CsA on intracellular signaling mechanisms following VEGF stimulation. CsA affected all VEGF induced angiogenic events assessed in HIMEC. CsA differentially inhibited signaling pathways which mediated distinct steps of the angiogenic process. CsA blocked VEGF induced nuclear translocation of the transcription factor NFAT, activation of p44/42 MAPK, and partially inhibited JNK and p38 MAPK. CsA differentially affected signaling cascades in a dose dependent fashion and completely blocked expression of COX-2, which was integrally linked to HIMEC angiogenesis. These data suggest that CsA inhibits the ability of microvascular endothelial cells to undergo angiogenesis, impairing vascular homeostatic mechanisms and contributing to the vasculopathy associated with chronic rejection.

Cyclosporine A Enhances Leukocyte Binding by Human Intestinal Microvascular Endothelial Cells through Inhibition of p38 MAPK and iNOS PARADOXICAL PROINFLAMMATORY EFFECT ON THE MICROVASCULAR ENDOTHELIUM

The calcineurin inhibitor cyclosporine A (CsA) modulates leukocyte cytokine production but may also effect nonimmune cells, including microvascular endothelial cells, which regulate the inflammatory process through leukocyte recruitment. We hypothesized that CsA would promote a proinflammatory phenotype in human intestinal microvascular endothelial cells (HIMEC), by inhibiting inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an important mechanism in limiting endothelial activation and leukocyte adhesion. Primary cultures of HIMEC were used to assess CsA effects on endothelial activation, leukocyte interaction, and the expression of iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the expression of iNOS in tumor necrosis factor-α/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK phosphorylation in activated HIMEC, a key pathway in iNOS expression, but failed to inhibit NFκB activation. These studies demonstrate that CsA exerts a proinflammatory effect on HIMEC by blocking iNOS expression. CsA exerts a proinflammatory effect on the microvascular endothelium, and this drug-induced endothelial dysfunction may help explain its lack of efficacy in the long-term treatment of chronically active inflammatory bowel disease.

Detection and differentiation of inflammatory versus fibromatous Crohn's disease strictures: prospective comparison of 18F-FDG-PET/CT, MR-enteroclysis, and transabdominal ultrasound versus endoscopic/histologic evaluation.

Background: Differentiation between inflammatory and fibromatous strictures in Crohns disease (CD) is difficult but crucial for therapeutic decisions. The aim of this study was to assess the best noninvasive imaging method for the detection and differentiation of inflammatory and fibromatous stenoses in CD in comparison to endoscopic and histologic evaluation. Methods: Patients with suspected CD strictures were included. According to a formalized endoscopic and histologic protocol, strictures were classified as inflammatory, mixed, and fibrostenotic. Strictures were further analyzed using fluorine 18‐labeled fluoro‐2‐deoxy‐D‐glucose (18FDG) / positron emission tomography (PET) low‐dose computed tomography (CT), magnetic resonance (MR) enteroclysis and transabdominal ultrasound using standardized scoring systems. Results: Thirty patients with 37 strictures were evaluated (inflamed n = 22; mixed n = 12, fibromatous n = 3). 18FDG‐PET/CT detected 81%, MR‐enteroclysis 81%, and ultrasound 68% of the strictures. Correct differentiation rates of strictures were 57% for MRE, 53% for 18FDG‐PET/CT, and 40% for ultrasound. Differences of detection rates and differentiation rates were not statistically significant. When combining transabdominal ultrasound with 18FDG‐PET/CT or MR‐enteroclysis all strictures that required invasive treatment were detected. Conclusions: Detection rates of the strictures were not significantly different between 18FDG‐PET/CT, MR‐enteroclysis, and ultrasound. Despite good stricture detection rates relating to our gold standard, 18FDG‐PET/CT nor MR‐enteroclysis nor ultrasound can accurately differentiate inflamed from fibrotic strictures. A combination of MR‐enteroclysis and ultrasound as well as a combination of 18FDG‐PET/CT and ultrasound resulted in a 100% detection rate of strictures requiring surgery or endoscopic dilation therapy, suggesting the combination of these methods as an alternative to endoscopy at least in the group of patients not able to perform an adequate bowel preparation. (Inflamm Bowel Dis 2012;)

Vascular cell adhesion molecule-1 expression in human intestinal microvascular endothelial cells is regulated by PI 3-kinase/Akt/MAPK/NF-κB: inhibitory role of curcumin

Endothelial activation and surface expression of cell adhesion molecules (CAMs) is critical for binding and recruitment of circulating leukocytes in tissues during the inflammatory response. Endothelial CAM expression plays a critical role in the intestinal microvasculature in inflammatory bowel disease (IBD), as blockade of leukocyte alpha4-integrin binding by gut endothelial CAM ligands has therapeutic benefit in IBD. Mechanisms underlying expression of vascular cell adhesion molecule (VCAM)-1, a ligand for alpha4-integrin in primary cultures of human intestinal microvascular endothelial cells (HIMEC) has not been defined. We investigated the effect of curcumin, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), and mitogen-activated protein kinase (MAPK) inhibitors on VCAM-1 expression and function in HIMEC. CAM expression was assessed and HIMEC-leukocyte adhesion was visualized under static and flow conditions. Western blotting and in vitro kinase assays were used to assess Akt and MAPK activation. Nuclear factor-kappaB (NF-kappaB) activation and nuclear translocation of its p65 subunit were determined. Tumor necrosis factor (TNF)-alpha/lipopolysaccharide (LPS)-induced VCAM-1 expression in HIMEC was suppressed by Akt small-interfering RNA, curcumin, and inhibitors of NF-kappaB (SN-50), p38 MAPK (SB-203580) and PI 3-kinase/Akt (LY-294002). VCAM-1 induction was partially suppressed by p44/42 MAPK (PD-098059) but unaffected by c-Jun NH2-terminal kinase (SP-600125) inhibition. Curcumin inhibited Akt/MAPK/NF-kappaB activity and prevented nuclear translocation of the p65 NF-kappaB subunit following TNF-alpha/LPS. At physiological shear stress, curcumin attenuated leukocyte adhesion to TNF-alpha/LPS-activated HIMEC monolayers. In conclusion, curcumin inhibited the expression of VCAM-1 in HIMECs through blockade of Akt, p38 MAPK, and NF-kappaB. Curcumin may represent a novel therapeutic agent targeting endothelial activation in IBD.