Approaches to Integrating Biomarkers Into Clinical Trials and Care Pathways as Targets for the Treatment of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: There is no consensus on the best way to integrate biomarkers into inflammatory bowel disease (IBD) research and clinical practice. The International Organization for the Study of Inflammatory Bowel Disease aimed to outline biomarker definitions, categories, and operating properties required for their use in registration trials and clinical practice. Using fecal calprotectin as an example, we provide a framework for biomarker development and validation in patients with IBD. METHODS: We reviewed international society guidelines, regulatory agency guidance documents, and standardized reporting guidelines for biomarkers, in combination with publications on fecal calprotectin levels in patients with IBD. We assessed the validity of fecal calprotectin to serve as a surrogate biomarker of IBD activity and outlined a framework for further validation and development of biomarkers. RESULTS: No endpoints have been fully validated as surrogates of risk of disease complications; mucosal healing is the most valid endpoint used to determine risk of disease complications. Fecal level of calprotectin has not been validated as a biomarker for IBD activity because of lack of technical and clinical reliability, assessment of performance when used as a replacement for endoscopy, and assessment of responsiveness to changes in disease states. The level of fecal calprotectin can be used only as a prognostic factor for disease recurrence in patients in remission after medical or surgical treatment. CONCLUSIONS: We reviewed guidelines, regulatory documents, and publications to identify properties required for the development of biomarkers of IBD activity and areas in need of clarification from regulatory agencies and societies. We propose a path forward for research of biomarkers for IBD.

Outcome of patients with ischemic-like cholangiopathy with secondary sclerosing cholangitis after liver transplantation.

BACKGROUND AND AIMS: Sclerosing cholangitis in critically ill patients (SC-CIP) with sepsis and acute respiratory distress syndrome (ARDS) is a cholestatic liver disease with a rapid progression to liver cirrhosis and hepatic failure. Data on outcome of these patients after liver transplantation (LT) are sparse. PATIENTS AND METHODS: Eleven patients (46 ± 12 years; mean labMELD-score: 27 ± 7) with SC-CIP underwent LT. Six patients had severe polytrauma with multiple bone fractures, sepsis and ARDS. Five non-traumatic patients acquired SC-CIP during long-term intensive-care-unit stays due to sepsis and ARDS. Time to diagnosis, the microbiologic results and the survival rates after LT were evaluated. RESULTS: SC-CIP was diagnosed by endoscopic retrograde cholangiopancreatography (ERCP) within 3 ± 1 months after manifestation of cholestasis and histologically confirmed in explanted livers. The predominant microorganisms isolated in bile were: Enterococcus and Candida albicans. Mean follow-up after LT was 28 ± 20 months. One female patient (non-traumatic) died due to sepsis 26 days after LT. All other patients left the hospital alive, but two (non-traumatic) patients died from sepsis, and one (traumatic) patient died in a hemorrhagic shock, thereafter. Seven of 11 patients (5 with polytrauma) are still alive and have a good quality of life. The survival of the SC-CIP patients after LT was comparable with that of patients transplanted due to alcoholic liver cirrhosis. CONCLUSION: SC-CIP develops rapidly within several months. Enterococcus and C. albicans were the main isolated microorganisms in the bile. Sepsis was the main cause of death after LT. Overall, SC-CIP is a good indication for LT in selected patients.

Alterations in mechanical properties of mesenteric resistance arteries in experimental portal hypertension.

Splanchnic vasodilation is the pathophysiological hallmark in the development of the hyperdynamic circulatory syndrome in liver cirrhosis and portal hypertension. This has been attributed so far mainly to a marked vascular hyporeactivity to endogenous vasoconstrictors. However, myogenic tone and vessel stiffness have not been addressed in mesenteric arteries in liver cirrhosis. CCl(4)(-)-induced ascitic cirrhotic (LC) and age-matched control rats, portal vein-ligated (PVL) rats, and sham-operated rats were investigated. Third-order mesenteric resistance arteries were studied under no-flow conditions using a pressure myograph measuring media thickness and lumen diameter in response to incremental increases in intramural pressure, from which wall mechanics were calculated. Electron microscopy was used for investigation of wall ultrastructure, especially the fenestrae in internal elastic lamina (IEL). In PVL animals, no significant change in passive vessel strain, stress, media-to-lumen ratio, or cross-sectional area was noted. In contrast, in LC rats, vessel strain was markedly elevated compared with healthy control rats, indicating a marked reduction in vessel stiffness. In addition, the strain-stress curve was shifted to the right, and the elastic modulus in dependency on vessel stress decreased, demonstrating predominantly structure-dependent factors to be involved. The media-to-lumen quotient was not significantly altered, but cross-sectional area was highly increased in LC rats, indicating hypertrophic outward remodeling. These findings were paralleled by enlarged fenestrae in the IEL but no change in thickness of IEL or proportion of extracellular matrix or vascular smooth muscle in LC rats. We concluded that, in long-standing severe portal hypertension such as ascitic LC but not in short-term conditions such as PVL, mesenteric resistance arteries exhibit vascular remodeling and markedly less resistant mechanical properties, leading to decreased vessel stiffness accompanied by structural changes in the IEL. This may well contribute to the maintenance and severity of splanchnic arterial vasodilation in LC.

Transabdominal ultrasound with echoenhancement by contrast media in the diagnosis of hepatocellular carcinoma.

According to the European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Diseases (AASLD), ultrasound (US) is the recommended tool for surveillance of patients at risk of developing hepatocellular carcinoma (HCC). Larger HCCs can be diagnosed with a high accuracy by conventional US. However, the differentiation of smaller malignant lesions in cirrhotic livers can be improved by contrast-enhanced ultrasound (CEUS). Second-generation contrast agents consisting of microbubbles enable us to visualize specific tumor vascularization patterns. With CEUS, it is not only possible to detect and characterize HCC nodules, but to control the effects of ablation techniques of HCC as well, evaluating the former lesion with respect to complete necrosis or residual viable tumor. Limitations of CEUS are its inability to characterize lesions distant to the applicator. Moreover, so far the use of contrast agents in US did not result in increased sensitivity in the detection of small HCCs (<1 cm). Thus, there is currently no indication to use contrast agents to increase the detection rate of HCC in patients undergoing US surveillance.

Toll-like receptor ligands cause proinflammatory and prodiabetic activation of adipocytes via phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase but not interferon regulatory factor-3.

Here, we aim to investigate the mechanisms of Toll-like receptor (TLR)-induced prodiabetic and proinflammatory activation of adipocytes and to detect differences in the responsiveness of TLRs to their respective ligands between adipocytes isolated from inflamed vs. noninflamed adipose tissue. Experiments using specific ligands for all known TLRs were performed in murine 3T3-L1 adipocytes and in human adipocytes isolated from noninflamed and inflamed adipose tissue. IL-6 and monocyte chemoattractant protein-1 (MCP-1) release were measured by ELISA. The expression of the signal transduction proteins phospho-extracellular signal-regulated kinase (P-Erk), P-c-Jun N-terminal kinase (JNK), and P-interferon regulatory factor-3 was investigated by Western blot analysis. Additionally, functional inhibitors of MAPK kinase-1/-2 and JNK-1/-2 were used in the stimulation experiments. Activation of TRL4 by lipopolysaccharide (LPS) and TLR1/2 by Pam(3)Cys up-regulates IL-6 and MCP-1 release in adipocytes via specific activation of Erk. Stimulation of adipocytes by macrophage activating lipopeptide-2 (MALP-2) induces MCP-1 but has no effect on IL-6 release. This stimulatory effect on MCP-1 release is antagonized by inhibition of both mitogen-activated protein kinase-1/-2 and JNK-1/-2. Phosphorylation of Erk and JNK is up-regulated after stimulation by MALP-2. In human adipocytes isolated from noninflamed adipose tissue, LPS and Pam(3)Cys, but not MALP-2, are potent inducers of IL-6 and MCP-1. MALP-2 is able to induce IL-6 and MCP-1 release in adipocytes isolated from inflamed adipose tissue, whereas these adipocytes lost their ability to respond to LPS. The present results point to a role of the adipose tissue in innate immunity. TLR-ligand-induced proinflammatory and prodiabetic activation of adipocytes might couple visceral adipose tissue dysfunction with insulin resistance and type 2 diabetes mellitus.

Innate immunity and adipocyte function: ligand-specific activation of multiple Toll-like receptors modulates cytokine, adipokine, and chemokine secretion in adipocytes.

The aim of this study was to analyze Toll-like receptor (TLR) expression in preadipocytes and mature adipocytes and to investigate whether TLR ligands influence the release of cytokines, chemokines, and adipokines. Murine 3T3-L1 preadipocytes and mature adipocytes were used for stimulation experiments. The effects of lipopolysaccharide (LPS), flagellin, Poly (U), Poly (I:C), macrophage-activating lipopeptide-2 (MALP2), Pam3Cys, and CpG on the release of interleukin-6 (IL-6), resistin, and monocyte chemoattractant protein-1 (MCP-1) were determined by enzyme-linked immunosorbent assay (ELISA). Nuclear translocation and promoter binding of NFkappaB were analyzed by electrophoretic mobility shift assays. TLR expression was investigated by reverse-transcriptase (RT-PCR). All TLRs except TLR5 and TRL7 are expressed in the stromal vascular cell (SVC) fraction and in mature adipocytes of different fat stores. Whereas basal and LPS-induced IL-6 release is higher in preadipocytes, basal and LPS-induced MCP-1 release is higher in mature adipocytes. Mature adipocytes respond to corticosterone regarding MCP-1 and resistin release. The ligands for TLRs influence IL-6, MCP-1, and resistin release differentially. Some of these ligands induce nuclear translocation and promoter binding of NFkappaB. Besides TLR5, that is not expressed in mature adipocytes, all TLR family members are involved. There exists a functional TRL pathway in adipocytes that connects innate immunity with adipocyte function. As a consequence, the role of the adipose tissue in both immunity and metabolism has to be investigated in future studies. The results of this approach will help to explain the metabolic changes such as insulin resistance observed during infection and the immunological phenomena such as macrophage infiltration of adipose tissue seen in obesity.

Regulation and function of collagenous repeat containing sequence of 26-kDa protein gene product "cartonectin".

OBJECTIVE: Collagenous repeat containing sequence of 26-kDa protein (CORS-26) was identified as a new gene transcript expressed in cartilage with unknown function. It was the aim of this study to investigate expression, regulation, and function of CORS-26 in adipocytes. RESEARCH METHODS AND PROCEDURES: CORS-26 mRNA and protein expression was studied by reverse transcriptase-polymerase chain reaction, Western blot analysis, and quantitative real-time polymerase chain reaction. Transcriptional regulation was studied by electrophoretic mobility shift assay and luciferase reporter gene assay. The adipocytic secretion of adiponectin and resistin was measured by enzyme-linked immunosorbent assay. RESULTS: CORS-26 mRNA is absent in 3T3-L1 preadipocytes and adipocytes after 48 hours of differentiation. CORS-26 mRNA was induced from Day 4 to Day 9 of adipocyte differentiation. CORS-26 protein was induced in mature adipocytes. Peroxisome proliferator-activated receptor (PPAR) gamma (but not PPARalpha) in nuclear extracts prepared from adipocytes was shown to bind specifically to a putative peroxisome proliferator response element-one-half-site located at -641/-596 bp. Increasing doses of the ligands troglitazone (1, 10, 20 microM) and fenofibrate (50, 100, 200 microM) but not 15-deoxy-prostaglandin (J(2)) (0.5, 1.0, 2.5 microM) resulted in a significant reduction of both promoter activity and the amount of mRNA expression. Recombinant CORS-26 significantly stimulated the adipocytic secretion of adiponectin and resistin in a dose-dependent manner. DISCUSSION: The mRNA and protein expression profile puts CORS-26 in the adipocytokine family. Cartonectin is negatively regulated by exogenous, but not endogenous, PPARgamma ligands. Because CORS-26 up-regulates adipokine secretion, it might be involved in metabolic and immunologic pathways.

Improvement in the routine diagnostic assessment of the liver by high-resolution sonography: an analysis of 999 cases.

OBJECTIVE: High-frequency ultrasound transducers have been helpful in certain settings of transabdominal ultrasound examination, and their role in the evaluation of the liver surface in patients with cirrhosis is well documented. However, their value in the routine assessment of the liver has not yet been analysed systematically. The aim of this pilot study was to clarify whether the additional use of high-frequency ultrasound as compared to the standard 3.5 MHz-transducer is of any benefit. MATERIAL AND METHODS: A total of 999 patients from a tertiary care medical centre were examined with a wideband 3.5 MHz- and a high-frequency transducer (band width 4.5 to 10 MHz) with tissue harmonic imaging using one of two high-end ultrasound machines (Siemens Sonoline Elegra or Hitachi EUB-8500). Findings on hepatic pathologies were collected on a standardized documentation sheet and were evaluated using descriptive statistics. RESULTS: In all, 948 patients showed a plain liver surface when the 3.5 MHz transducer was used, whereas this was only true for 862 patients examined with the high-frequency probe. Using the 7.5 MHz probe, the structure of the liver parenchyma appeared to be homogeneous (n=800; 80.1%) less often than when the 3.5 MHz probe (n=822; 82.3%) was used. More cases of liver cirrhosis were suspected with the high-frequency probe (n=66; 6.6% as compared with n=49; 4.9%). In 85 patients (8.5%) new hepatic pathologies were described which had not been detected with the 3.5 MHz probe. The examiners judged the high-frequency examination to be helpful in 284 cases. The time needed for the additional examination ranged between 0.5 and 10 min (mean: 2.2 min). CONCLUSIONS: This study demonstrates that the additional use of a high-frequency transducer during routine abdominal examinations reveals new hepatic pathologies in a significant proportion of examined patients, without substantial prolongation of the overall examination period.

Enhanced Y1-receptor-mediated vasoconstrictive action of neuropeptide Y (NPY) in superior mesenteric arteries in portal hypertension.

BACKGROUND/AIMS: Vascular hyporeactivity to catecholamines contributes to arterial vasodilation and hemodynamic dysregulation in portal hypertension. Neuropeptide Y (NPY) is a sympathetic neurotransmitter facilitating adrenergic vasoconstriction via Y1-receptors on the vascular smooth muscle. Therefore, we investigated its role for vascular reactivity in the superior mesenteric artery (SMA) of portal vein ligated (PVL) and sham operated rats. METHODS: In vitro perfused SMA vascular beds of rats were tested for the cumulative dose-response to NPY dependent on the presence and level of alpha1-adrenergic vascular tone (methoxamine MT: 0.3-10 microM). Moreover, the effect of NPY (50 nM) on vascular responsiveness to alpha1-adrenergic stimulation (MT: 0.3-300 microM) was evaluated. Y1-receptor function was tested by Y1-selective inhibition using BIBP-3226 (1 microM). RESULTS: NPY dose-dependently and endothelium-independently enhanced MT-pre-constriction in SMA. This potentiation was increasingly effective with increasing adrenergic pre-stimulation and being more pronounced in PVL rats as compared to sham rats at high MT concentrations. NPY enhanced vascular contractility only in PVL rats correcting the adrenergic vascular hyporeactivity. Y1-receptor inhibition completely abolished NPY-evoked vasoconstrictive effects. CONCLUSIONS: NPY endothelium-independently potentiates adrenergic vasoconstriction via Y1-receptors being more pronounced in portal hypertension improving mesenteric vascular contractility and thereby correcting the splanchnic vascular hyporeactivity. This makes NPY a superior vasoconstrictor counterbalancing arterial vasodilation in portal hypertension.

Specific expression and regulation of the new melanoma inhibitory activity-related gene MIA2 in hepatocytes.

The novel human gene MIA2 encoding a melanoma inhibitory activity (MIA) homologous protein was identified by a GenBank(TM) search. MIA2, together with MIA, OTOR, and TANGO, belongs to the novel MIA gene family sharing important structural features, significant homology at both the nucleotide and protein levels, and similar genomic organization. In situ hybridization, reverse transcriptase-PCR, and Northern blots presented a highly tissue-specific MIA2 expression pattern in the liver. Promoter studies analyzing transcriptional regulation of MIA2 revealed an HNF-1-binding site at position -236 controlling hepatocyte-specific expression. Mutation of the site led to a complete loss of promoter activity in HepG2 cell. Further sites detected in the MIA2 promoter were consensus binding sites for SMAD and STAT3, Consistently, stimulation of MIA2 mRNA expression occurred by treatment with interleukin-6, transforming growth factor-beta, and conditioned medium from activated hepatic stellate cells. In accordance with these results, MIA2 mRNA was found to be increased in liver tissue of patients with chronic hepatitis C infection compared with controls. MIA2 mRNA levels were significantly higher in patients with severe fibrosis or inflammation than in patients with less severe fibrosis or inflammation. In summary our data indicate that MIA2 represents a potential novel acute phase protein and MIA2 expression responds to liver damage. The increased transcription in more severe chronic liver disease suggests that MIA2 may serve as a marker of hepatic disease activity and severity.

Autocrine fibronectin-induced migration of human colonic fibroblasts.

OBJECTIVES: A central event during wound repair is the migration of activated fibroblasts to the wound area. Thus far, the mechanisms inducing migration of colonic lamina propria fibroblasts (CLPF) have not been studied in detail. Previously, we have shown that CLPF secrete factors that are essential to their ability to migrate in response to different growth factors. METHODS: Primary human CLPF were obtained from endoscopic biopsies or surgical specimens taken from normal mucosa areas of patients undergoing surveillance colonoscopy or surgery for colorectal carcinoma. Migration assays of CLPF were performed in the modified 48-well Boyden chamber. RESULTS: Conditioned medium of CLPF collected after 24-h stimulated migration of CLPF (22 +/- 2 cells/ hpf). Filtration of conditioned medium through a 300-kDa filter reduced the migration-inducing potential in subsequent migration assays to 2 +/- 1 cells/hpf, filtration through a 100-kDa filter abolished migration of CLPF completely, indicating that large molecules such as extracellular matrix components could be responsible for the induction of CLPF migration. Enzyme-linked immunosorbent assays revealed the presence of fibronectin in conditioned medium (17.3 microg/ml). Immunoprecipitation of fibronectin in conditioned medium of CLPF reduced the migration-inducing potential by 63%. Addition of fibronectin to fibronectin-depleted conditioned medium reconstituted the migration. Dose-response assays with fibronectin (1-100 microg/ml) diluted in nonconditioned medium induced migration of CLPF in a dose-dependent manner. Maximum migration was induced with 25 microg/ml fibronectin (37 +/- 5 cells/hpf). CONCLUSION: Fibronectin is an autocrine and paracrine factor essential for intestinal fibroblast migration. Fibronectin induces migration of intestinal fibroblasts and is essential for their ability to migrate in response to different growth factors. A detailed understanding of the regulation of the migration of intestinal fibroblasts is necessary to gain further insights in the pathophysiology of stricture and fistula formation.