Immobilized chemokine fields and soluble chemokine gradients cooperatively shape migration patterns of dendritic cells.

Chemokines orchestrate immune cell trafficking by eliciting either directed or random migration and by activating integrins in order to induce cell adhesion. Analyzing dendritic cell (DC) migration, we showed that these distinct cellular responses depended on the mode of chemokine presentation within tissues. The surface-immobilized form of the chemokine CCL21, the heparan sulfate-anchoring ligand of the CC-chemokine receptor 7 (CCR7), caused random movement of DCs that was confined to the chemokine-presenting surface because it triggered integrin-mediated adhesion. Upon direct contact with CCL21, DCs truncated the anchoring residues of CCL21, thereby releasing it from the solid phase. Soluble CCL21 functionally resembles the second CCR7 ligand, CCL19, which lacks anchoring residues and forms soluble gradients. Both soluble CCR7 ligands triggered chemotactic movement, but not surface adhesion. Adhesive random migration and directional steering cooperate to produce dynamic but spatially restricted locomotion patterns closely resembling the cellular dynamics observed in secondary lymphoid organs.

Distinct Disease Phenotype of Ulcerative Colitis in Patients With Coincident Primary Sclerosing Cholangitis: Evidence From a Large Retrospective Study With Matched Cohorts.

BACKGROUND: Primary sclerosing cholangitis is a classical extraintestinal manifestation in patients with ulcerative colitis. However, the impact of primary sclerosing cholangitis on the disease course is incompletely understood. OBJECTIVE: This study aimed to assess the impact of primary sclerosing cholangitis on disease phenotype and its course in patients with ulcerative colitis. DESIGN: This is a retrospective study with 3:1 matched cohorts. SETTINGS: Tertiary care center's electronic database was used for data analysis from 2000 and 2018. PATIENTS: Of 782 patients with ulcerative colitis, 77 patients who had coincident primary sclerosing cholangitis were included. MAIN OUTCOME MEASURES: The primary outcomes evaluated were disease characteristics including colonic disease activity, temporal change of disease course, colorectal neoplasia, and colectomy rates. RESULTS: Disease activity during acute flares, assessed by the complete Mayo score, was significantly lower in patients with primary sclerosing cholangitis (6.2 vs 7.3; p < 0.001). In addition, disease activity in patients with primary sclerosing cholangitis was decreased, especially within the first 10 years after disease onset, and biological therapy with anti-tumor necrosis factor and anti-integrin agents was commenced less frequently (22% vs 35%; p = 0.043) and later (10-year risk: 17.4% vs 27.8%; p = 0.034). Patients with primary sclerosing cholangitis were younger at colitis diagnosis (23.3 vs 29.3 years; p < 0.001) and had more extensive disease (75% vs 46%; p < 0.001). Colorectal cancer was more frequently detected in patients with coincident primary sclerosing cholangitis (6/77 vs 16/705; p = 0.016). Colectomy rates did not differ between both groups (14.3% vs 14.5%; p = 0.56). In contrast, patients with ulcerative colitis had to undergo surgery more frequently because of therapy-refractant inflammation, whereas surgery due to neoplasia development was increased in patients with coincident primary sclerosing cholangitis (p = 0.013). LIMITATIONS: The study was limited by its retrospective design. CONCLUSION: Patients who have ulcerative colitis with coincident primary sclerosing cholangitis develop a distinct disease course characterized by an earlier disease onset and lower disease activity, but more frequent extensive disease manifestation and higher risk for colorectal cancer. See Video Abstract at http://links.lww.com/DCR/B45. FENOTIPO DE ENFERMEDAD DISTINTIVO DE LA COLITIS ULCERATIVA EN PACIENTES CON COLANGITIS ESCLEROSANTE PRIMARIA CONCOMITANTE: EVIDENCIA DE UN ESTUDIO RETROSPECTIVO GRANDE CON COHORTES EMPAREJADAS: La colangitis esclerosante primaria es una manifestación extraintestinal clásica en pacientes con colitis ulcerativa. Sin embargo, el impacto de la colangitis esclerosante primaria en el curso de la enfermedad no es comprendido completamente.Evaluar el impacto de la colangitis esclerosante primaria en el fenotipo y curso de la enfermedad en pacientes con colitis ulcerativa.Este es un estudio retrospectivo con cohortes emparejadas 3:1.La base de datos electrónica de un centro de atención terciaria se utilizó para el análisis de datos de 2000 a 2018.782 pacientes con colitis ulcerativa, 77 padecían colangitis esclerosante primaria concomitante y fueron incluidos.Se evaluaron las características de la enfermedad, incluida la actividad de enfermedad colónica, el cambio temporal del curso de la enfermedad, la neoplasia colorrectal y las tasas de colectomía.La actividad de la enfermedad durante los brotes agudos, evaluada por la puntuación completa de Mayo, fue significativamente menor en pacientes con colangitis esclerosante primaria (6.2 vs 7.3; p < 0.001). Además, la actividad de la enfermedad en pacientes con colangitis esclerosante primaria se redujo especialmente en los primeros 10 años después del inicio de la enfermedad, y la terapia biológica con agentes anti-TNF y anti-integrina se inició con menos frecuencia (22% vs 35%; p = 0.043) y más tarde (riesgo a 10 años: 17.4% vs 27.8%; p = 0.034). Los pacientes con colangitis esclerosante primaria eran más jóvenes en el momento del diagnóstico de colitis (23.3 vs 29.3 años; p < 0.001) y tenían enfermedad más extensa (75% vs 46%; p < 0.001). El cáncer colorrectal se detectó con mayor frecuencia en pacientes con colangitis esclerosante primaria concomitante (6/77 vs 16/705; p = 0.016). Las tasas de colectomía no fueron diferentes entre ambos grupos (14.3% vs 14.5%; p = 0.56). En contraste, los pacientes con colitis ulcerativa tuvieron que someterse a cirugía con mayor frecuencia debido a inflamación refractaria a la terapia, mientras que el desarrollo de neoplasia se incrementó en pacientes con colangitis esclerosante primaria concomitante (p = 0.013).El estudio estuvo limitado por su diseño retrospectivo.Los pacientes con colitis ulcerativa con colangitis esclerosante primaria concomitante desarrollan un curso de enfermedad distintivo caracterizado por un inicio temprano de la enfermedad y una menor actividad de la enfermedad, pero con manifestación de enfermedad extensa más frecuente y un mayor riesgo de cáncer colorrectal. Vea el resumen en video en http://links.lww.com/DCR/B45.

Digital single-operator cholangioscopy: a useful tool for selective guidewire placements across complex biliary strictures.

BACKGROUND: Treatment of biliary strictures is challenging. Digital single-operator cholangioscopes (SOCs) equipped with an improved imaging quality, were recently introduced and may be useful for selective guidewire placement in difficult biliary strictures. METHODS: A total of 167 digital SOC procedures performed between 2015 and 2018 were retrospectively analyzed for successful guidewire placements across biliary strictures. Only cases with previous failed conventional guidewire placement approaches were included. RESULTS: In total, 30 examinations with a digital SOC-assisted guidewire placement across biliary strictures, performed in 23 patients, were identified. In 52% of all patients, the stricture was benign with post-liver-transplant strictures (75%) as the most frequent finding; in 48% of all patients the stricture was malignant with cholangiocellular carcinoma as the most frequent type (64%). Guidewire placement was successful in 21 of 30 procedures (70%). According to a subgroup analysis, digital SOC-assisted guidewire placements were significantly more successful in patients with benign strictures than those in patients with malignant strictures (88.2% vs. 46.2%; p = 0.02). Furthermore, the technical success rate tended to be increased in cases of initial examinations (78.3%) than in patients with repeated examinations (42.9%; p = 0.15). Adverse events, such as post-interventional pancreatitis or cholangitis as well as severe bleeding occurred in 16.7% of all examinations. CONCLUSIONS: Digital SOC-assisted guidewire placements have high technical success rates, especially in benign biliary strictures. This technique can help to avoid more invasive procedures such as percutaneous transhepatic or endoscopic ultrasound-guided biliary drainage.

Reprogramming of monocytes by GM-CSF contributes to regulatory immune functions during intestinal inflammation.

Human and murine studies showed that GM-CSF exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF-activated monocytes (GMaMs) in vivo. We used microarray technology and functional assays to characterize GMaMs in vitro and used a mouse model of colitis to study GMaM functions in vivo. GM-CSF activates monocytes to increase adherence, migration, chemotaxis, and oxidative burst in vitro, and primes monocyte response to secondary microbial stimuli. In addition, GMaMs accelerate epithelial healing in vitro. Most important, in a mouse model of experimental T cell-induced colitis, GMaMs show therapeutic activity and protect mice from colitis. This is accompanied by increased production of IL-4, IL-10, and IL-13, and decreased production of IFN-γ in lamina propria mononuclear cells in vivo. Confirming this finding, GMaMs attract T cells and shape their differentiation toward Th2 by upregulating IL-4, IL-10, and IL-13 in T cells in vitro. Beneficial effects of GM-CSF in Crohn's disease may possibly be mediated through reprogramming of monocytes to simultaneously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limit excessive inflammation.

The risk of colorectal cancer in patients with ulcerative colitis.

BACKGROUND AND AIM: Ulcerative colitis increases the risk of developing dysplasia and colitis-associated cancer (CAC). The purpose of this study was to determine the risk factors as well as protective measures for disease burden, need for colectomy and the development of CAC in ulcerative colitis (UC) patients. METHODS: A cohort of n = 434 UC patients was evaluated. Data analysis was performed by univariate and multivariate logistic regression. Odds ratios (OR) and 95 % confidence intervals (CI) were calculated, and significance was assessed by the likelihood ratio test. RESULTS: Mean patient age at UC diagnosis was 45.7 ± 15.1 years which manifested mainly as pancolitis (47 %) or left-sided colitis (45.2 %). CAC was detected in ten patients (2.3 %). UC disease duration was strongly associated with the risk of CAC (P < 0.0014); disease duration between 9 and 15 years: OR of 2.5 (95 % CI 0.2-41.1), more than 15 years: OR of 21.4 (95 % CI 2.6-173.6). The risk of developing dysplasia (low-grade intraepithelial neoplasia, LGIEN and high-grade intraepithelial neoplasia, HGIEN) or the need to undergo colectomy was also significantly related to disease duration (P = 0.006, P = 0.002, respectively). Established anti-inflammatory medication (e.g., 5-ASA, anti-TNF-α) significantly reduced the risk of both dysplasia and CAC (P = 0.02). CONCLUSIONS: Despite the use of modern therapies for UC, CAC rates remain high. In our study, risk factors included disease duration while anti-inflammatory therapies reduced the risk. Effective control of the intestinal inflammation also reduced the disease burden as indicated by decreased risk of requiring colectomy, underscoring the need for sufficient surveillance and anti-inflammatory therapies.

Emergency critical care: closing the gap between onset of critical illness and intensive care unit admission.

Critical illness is an exquisitely time-sensitive condition and follows a disease continuum, which always starts before admission to the intensive care unit (ICU), in the majority of cases even before hospital admission. Reflecting the common practice in many healthcare systems that critical care is mainly provided in the confined areas of an ICU, any delay in ICU admission of critically ill patients is associated with increased morbidity and mortality. However, if appropriate critical care interventions are provided before ICU admission, this association is not observed. Emergency critical care refers to critical care provided outside of the ICU. It encompasses the delivery of critical care interventions to and monitoring of patients at the place and time closest to the onset of critical illness as well as during transfer to the ICU. Thus, emergency critical care covers the most time-sensitive phase of critical illness and constitutes one missing link in the chain of survival of the critically ill patient. Emergency critical care is delivered whenever and wherever critical illness occurs such as in the pre-hospital setting, before and during inter-hospital transfers of critically ill patients, in the emergency department, in the operating theatres, and on hospital wards. By closing the management gap between onset of critical illness and ICU admission, emergency critical care improves patient safety and can avoid early deaths, reverse mild-to-moderate critical illness, avoid ICU admission, attenuate the severity of organ dysfunction, shorten ICU length of stay, and reduce short- and long-term mortality of critically ill patients. Future research is needed to identify effective models to implement emergency critical care systems in different healthcare systems.

Converse regulation of CCR7-driven human dendritic cell migration by prostaglandin E2 and liver X receptor activation.

Migration and homing of DCs to lymphoid organs is pivotal for inducing adaptive immunity and tolerance. DC homing depends on the chemokine receptor CCR7. However, expression of CCR7 alone is not sufficient for effective DC migration. A second signal, mediated by prostaglandin E(2) (PGE(2)), is critical for the development of a migratory DC phenotype. PGE(2) is important for inducing efficient immune responses, but, if deregulated, contributes to chronic inflammation, autoimmune diseases through Th17-cell development and tumorigenesis. In contrast, activation of liver X receptor (LXR)α has recently been shown to interfere with CCR7 expression and migration of DCs resulting in a reduced immune response. Here, we demonstrate that PGE(2) downregulates LXRα expression in human monocyte derived as well as ex vivo DCs. Moreover, PGE(2) stimulation dampens LXR activation, auto-regulation and LXR-mediated gene transcription. Consequently, we show that PGE(2) enhances CCR7 expression and migration of LXR-activated DCs. Furthermore, we provide evidence that PGE(2) signaling and LXR activation specifically elicit converse effects on CCR7 expression and DC migration. In contrast, production of MMP9, CCL4, COX-2, and IL-23 is solely regulated by PGE(2) , but not by LXR activation, offering new perspectives for therapeutic interventions.

Distinct modulation of chemokine expression patterns in human monocyte-derived dendritic cells by prostaglandin E(2).

Dendritic cells (DCs) are key in regulating immune responses. DCs reside in tissues facing the environment and sample their surrounding for pathogens. Upon pathogen encounter, DCs mature and migrate into secondary lymphoid organs. Distinct maturation signals dictate the ability of DCs to produce distinct patterns of chemokines that orchestrate immunity. Prostaglandin E(2) (PGE(2)) is produced during inflammation and modulates DC functions. We demonstrate that PGE(2) modulates distinct chemokine expression patterns of human monocyte-derived (Mo) DCs upon maturation with various stimuli. PGE(2) dampened early production of the inflammatory chemokines CCL2, CCL4, CCL5 and attenuated the expression of the T cell attractant CXCL10. In contrast, PGE(2) enhanced CXCL8 production early during maturation, whereas CXCL16 levels were continuously elevated, contributing to innate immune cell recruitment. Moreover, PGE(2) induces transcription of the homeostatic chemokines CCL17 and CCL22. Finally, mature MoDCs produced the homing chemokine CCL19 and its expression was down-regulated by PGE(2).

ABO incompatibility and mild factor VIII deficiency in the neonate: Not too much of a problem but a challenge on ECMO.

Coagulopathy and bleeding on extracorporeal membrane oxygenation (ECMO) contribute to a worse outcome, and hyperbilirubinemia is an additional threat for newborn babies. We report a case of a newborn boy with congenital diaphragmatic hernia (CDH) associated with ABO incompatibility and an inherited mild hemophilia A. Due to respiratory failure he needed ECMO on his first day of life. During ECMO an exchange transfusion was performed after an extensive hyperbilirubinemia had evolved. Thereafter severe bleeding occurred, and a very low factor VIII level was found causative for that. After factor VIII substitution bleeding was under control and the baby eventually could be weaned from ECMO, underwent corrective surgery, and recovered.

Prostaglandin E(2) enhances T-cell proliferation by inducing the costimulatory molecules OX40L, CD70, and 4-1BBL on dendritic cells.

Dendritic cell (DC)-based immunotherapy of malignant diseases relies on 2 critical parameters: antigen transport from the periphery to draining lymph nodes and efficient priming of primary and stimulation of secondary immune responses. Prostaglandin E(2) (PGE(2)) signaling has been shown to be pivotal for DC migration toward lymph node-derived chemokines in vitro and in vivo. Here, we demonstrate that PGE(2) induced the expression of the costimulatory molecules OX40L, CD70, and 4-1BBL on human DCs. Short triggering by PGE(2) early during DC maturation was sufficient to induce the costimulatory molecules. The expression of the costimulatory molecules was independent of the maturation stimulus but strictly dependent on PGE(2) on both monocyte-derived (Mo) DCs and peripheral blood myeloid (PB) DCs. PGE(2)-matured MoDCs showed enhanced costimulatory capacities resulting in augmented antigen-specific CD4(+) and CD8(+) T-cell proliferation in primary and recall T-cell responses. Blocking OX40/OX40L signaling impaired the enhanced T-cell proliferation induced by PGE(2)-matured MoDCs. Moreover, MoDCs matured in the presence of PGE(2) induced the expression of OX40, OX40L, and CD70 on T cells facilitating T-cell/T-cell interaction that warrant long-lasting costimulation. This newly identified parameter will help to further optimize DC-based immunotherapy.

MicroRNA-320a Monitors Intestinal Disease Activity in Patients With Inflammatory Bowel Disease.

OBJECTIVES: In patients with inflammatory bowel disease (IBD), a treat-to-target treatment strategy requires tight monitoring of disease activity. Noninvasive biomarkers may help to monitor the intestinal disease activity. We demonstrated recently that peripheral microRNA (miR)-320a expression in mice follows the course of experimental colitis. The aim of this study was to evaluate the potential of miR-320a to monitor the disease activity in patients with IBD, to predict the course of disease, and to distinguish IBD from infectious colitis. METHODS: The miR-320a levels were prospectively assessed by quantitative real-time polymerase chain reaction analysis of peripheral blood samples from 40 patients with Crohn's disease (CD) and 37 patients with ulcerative colitis (UC) as well as from 19 healthy control individuals and 7 patients with infectious colitis. Disease activity was quantified by appropriate clinical disease indices and endoscopic scoring systems. RESULTS: When compared with healthy controls, miR-320a blood levels were significantly increased in patients with active CD and UC (16.1 ± 2.6 vs 2,573 ± 941; vs 434 ± 96; both P < 0.001) and patients with IBD in remission (316 ± 251 [CD] and 91 ± 29 [UC]; both P < 0.001). In patients with CD, miR-320a levels showed a strong correlation with the endoscopic disease activity (r = 0.76; P < 0.001). Similarly, in patients with UC, we detected a significantly enhanced miR-320a expression, which was highest in patients with severe endoscopic disease activity (eMayo = 0-1: 66 ± 16 vs eMayo = 2: 352 ± 102; vs eMayo = 3: 577 ± 206; both P < 0.001). Finally, miR-320a blood expression in patients with active CD and UC significantly increased compared with patients with infectious colitis (63 ± 13, P < 0.001). DISCUSSION: MiR-320a expression in peripheral blood from patients with IBD follows the clinical and endoscopic disease activities and may help to distinguish IBD from infectious colitis.

Inhibition of transcription and DNA replication by the papillomavirus E8-E2C protein is mediated by interaction with corepressor molecules.

Papillomavirus genomes replicate as nuclear plasmids at a low copy number in undifferentiated keratinocytes. Papillomaviruses encode the E1 and E2 proteins that bind to the origin of replication and are required for the activation of replication. In addition to E2, several papillomaviruses express an E8-E2C protein, which is generated by alternative splicing and functions as a transcriptional repressor and inhibitor of the E1/E2-dependent replication of the viral origin. Previous analyses suggested that the E8 domain functions as a transferable repression domain. In this report we present evidence that the E8 domain is responsible for the interaction with cellular corepressor molecules such as histone deacetylases, the histone methyltransferase SETDB1, and the TRIM28/KAP-1/TIF1beta/KRIP-1 protein. Whereas the interaction with histone deacetylases is involved only in transcriptional repression, the interaction with TRIM28/KAP-1/TIF1beta/KRIP-1 contributes to the inhibition of E1/E2-dependent replication. The corepressor TRIM28/KAP-1/TIF1beta/KRIP-1 has been described to be part of multicomponent complexes involved in transcriptional regulation and functions as a scaffold protein. Since neither histone deacetylases nor the histone methyltransferase SETDB1 appears to be involved in the inhibition of E1/E2-dependent replication, most likely the modification of non-histone proteins contributes to the replication repression activity of E8-E2C.

Target-Specific Fluorescence-Mediated Tomography for Non-Invasive and Dynamic Assessment of Early Neutrophil Infiltration in Murine Experimental Colitis.

The role of neutrophils in the pathogenesis of inflammatory bowel disease (IBD) is still only incompletely understood. Here, we evaluated target-specific fluorescence-mediated tomography (FMT) for visualization of neutrophil infiltration in murine experimental DSS-induced colitis. Colitis was assessed using clinical, endoscopic, and histopathological parameters. Intestinal neutrophil infiltration was determined at day 0, 4, and 10 by targeted FMT after injection of a neutrophil-specific fluorescence-labelled monoclonal antibody (Gr-1). Complementary, immunofluorescence tissue sections with Gr-1 and ELISA-based assessment of tissue myeloperoxidase (MPO) served as the gold standard for the quantification of neutrophil infiltration. Colitic animals showed decreasing body weight, presence of fecal occult blood, and endoscopic signs of inflammation. FMT revealed a significantly increased level of fluorescence only four days after colitis induction as compared to pre-experimental conditions (pmol tracer 73.2 ± 18.1 versus 738.6 ± 80.7; p < 0.05), while neither body weight nor endoscopic assessment showed significant changes at this early time. Confirmatory, post-mortem immunofluorescence studies and measurements of tissue MPO confirmed the presence of increased neutrophil infiltration in colitic mice compared to controls. Concluding, Gr-1 targeted FMT can detect early colonic infiltration of neutrophils in experimental colitis even before clinical symptoms or endoscopic alterations occur. Therefore, FMT might be an important tool for repetitive and non-invasive monitoring of inflammatory cell infiltrate in intestinal inflammation.

Percutaneous-transhepatic-endoscopic rendezvous procedures are effective and safe in patients with refractory bile duct obstruction.

Background: Percutaneous-transhepatic-endoscopic rendezvous procedures (PTE-RVs) are rescue approaches used to facilitate biliary drainage. Objective: The objective of this article is to evaluate the safety and the technical success of PTE-RVs in comparison with those of percutaneous transhepatic cholangiographies (PTCs). Methods: Percutaneous procedures performed over a 10-year period were retrospectively analyzed in a single-center cohort. Examinations were performed because of a previous or expected failure of standard endoscopic methods including endoscopic retrograde cholangiography (ERC) or balloon-assisted ERC to achieve biliary access. Results: In total, 553 percutaneous procedures including 163 PTE-RVs and 390 PTCs were performed. Overall, 71.3% of the patients suffered from malignant disease with pancreas-carcinoma (32.8%) and cholangio-carcinoma (19.0%) as the most frequent, while 28.7% of the patients suffered from benign disease. Many patients had a postoperative change in bowel anatomy (50.8%).PTC had a higher technical success rate (89.7%); however, the technical success rate of PTE-RVs was still high (80.4%; p < 0.003). Overall complications occurred in 23.5% of all procedures. Significantly fewer complications occurred after performing PTE-RVs than after PTCs (16.6% vs 26.4%; p = 0.037). Conclusion: Beside a high technical efficacy of PTE-RVs, significantly fewer complications occur following PTE-RVs than following PTCs; thus, PTE-RV should be preferred over PTC alone in selected patients.

Syndecan-4 Modulates Epithelial Gut Barrier Function and Epithelial Regeneration in Experimental Colitis.

Background: The transmembrane heparan sulfate proteoglycan Syndecan-4 (Sdc4) plays an important role in the regulation of various inflammatory disorders. However, the involvement of Sdc4 in intestinal inflammation remains unknown. Therefore, we assessed the impact of Sdc4 deficiency on experimental colitis and epithelial wound healing in vitro and in vivo. Methods: Dextran sulfate sodium (DSS)-induced colitis was monitored in wild type and Sdc4-deficient (Sdc4-/-) mice by assessment of body weight, histology, inflammatory cellular infiltration, and colon length. Syndecan-4 expression was measured by immunohistochemistry, Western blot, and quantitative real-time PCR. Epithelial permeability was evaluated by Evans blue measurements, Western blot, and immunohistological analysis of tight junction protein expression. Impact of Sdc4 on epithelial wound healing was determined by scratch assay in vitro and by colonoscopy following mechanical wounding in vivo. Results: In Sdc4-/- mice, colitis-like symptoms including severe weight loss, shortened colon length, histological damage, and invasion of macrophages and granulocytes were markedly aggravated compared with wild type (WT) animals. Moreover, colonic epithelial permeability in Sdc4-/- mice was enhanced, while tight junction protein expression decreased. Furthermore, Sdc4-/- colonic epithelial cells had lower cell proliferation and migration rates which presented in vivo as a prolonged intestinal wound healing phenotype. Strikingly, in WT animals, Sdc4 expression was reduced during colitis and was elevated during recovery. Conclusions: The loss of Sdc4 aggravates the course of experimental colitis, potentially through impaired epithelial cell integrity and regeneration. In view of the development of current treatment approaches involving Sdc4 inhibition for inflammatory disorders like arthritis, particular caution should be taken in case of adverse gastrointestinal side-effects.

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Detection and characterization of murine colitis and carcinogenesis by molecularly targeted contrast-enhanced ultrasound.

AIM: To study mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) and vascular endothelial growth factor (VEGF)-targeted contrast enhanced ultrasound (CEUS) for the assessment of murine colitis and carcinogenesis. METHODS: C57BL/6 mice were challenged with 3% dextran sodium-sulfate (DSS) for three, six or nine days to study the development of acute colitis. Ultrasound was performed with and without the addition of unspecific contrast agents. MAdCAM-1-targeted contrast agent was used to detect and quantify MAdCAM-1 expression. Inflammatory driven colorectal azoxymethane (AOM)/DSS-induced carcinogenesis was examined on day 42 and 84 using VEGF-targeted contrast agent. Highly specific tissue echogenicity was quantified using specialized software. Sonographic findings were correlated to tissue staining, western blot analysis and immunohistochemistry to quantify the degree of inflammation and stage of carcinogenesis. RESULTS: Native ultrasound detected increased general bowel wall thickening that correlated with more progressed and more severe DSS-colitis (healthy mice: 0.3 mm ± 0.03 vs six days DSS: 0.5 mm ± 0.2 vs nine days DSS: 0.6 mm ± 0.2, P < 0.05). Moreover, these sonographic findings correlated well with clinical parameters such as weight loss (r2 = 0.74) and histological damage (r2 = 0.86) (P < 0.01). In acute DSS-induced murine colitis, CEUS targeted against MAdCAM-1 detected and differentiated stages of mild, moderate and severe colitis via calculation of mean pixel contrast intensity in decibel (9.6 dB ± 1.6 vs 12.9 dB ± 1.4 vs 18 dB ± 3.33, P < 0.05). Employing the AOM/DSS-induced carcinogenesis model, tumor development was monitored by CEUS targeted against VEGF and detected a significantly increased echogenicity in tumors as compared to adjacent healthy mucosa (healthy mucosa, 1.6 dB ± 1.4 vs 42 d, 18.2 dB ± 3.3 vs 84 d, 18.6 dB ± 4.9, P < 0.01). Tissue echogenicity strongly correlated with histological analysis and immunohistochemistry findings (VEGF-positive cells in 10 high power fields of healthy mucosa: 1 ± 1.2 vs 42 d after DSS start: 2.4 ± 1.6 vs 84 d after DSS start: 3.5 ± 1.3, P < 0.01). CONCLUSION: Molecularly targeted CEUS is a highly specific and non-invasive imaging modality, which characterizes murine intestinal inflammation and carcinogenesis in vivo.

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing.

The incidence of inflammatory bowel disease, i.e., Crohn's disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Diagnostic imaging advances in murine models of colitis.

Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are chronic-remittent inflammatory disorders of the gastrointestinal tract still evoking challenging clinical diagnostic and therapeutic situations. Murine models of experimental colitis are a vital component of research into human IBD concerning questions of its complex pathogenesis or the evaluation of potential new drugs. To monitor the course of colitis, to the present day, classical parameters like histological tissue alterations or analysis of mucosal cytokine/chemokine expression often require euthanasia of animals. Recent advances mean revolutionary non-invasive imaging techniques for in vivo murine colitis diagnostics are increasingly available. These novel and emerging imaging techniques not only allow direct visualization of intestinal inflammation, but also enable molecular imaging and targeting of specific alterations of the inflamed murine mucosa. For the first time, in vivo imaging techniques allow for longitudinal examinations and evaluation of intra-individual therapeutic response. This review discusses the latest developments in the different fields of ultrasound, molecularly targeted contrast agent ultrasound, fluorescence endoscopy, confocal laser endomicroscopy as well as tomographic imaging with magnetic resonance imaging, computed tomography and fluorescence-mediated tomography, discussing their individual limitations and potential future diagnostic applications in the management of human patients with IBD.