The Oxysterol Synthesising Enzyme CH25H Contributes to the Development of Intestinal Fibrosis.

Intestinal fibrosis and stenosis are common complications of Crohn's disease [CD], frequently requiring surgery. Anti-inflammatory strategies can only partially prevent fibrosis; hence, anti-fibrotic therapies remain an unmet clinical need. Oxysterols are oxidised cholesterol derivatives with important roles in various biological processes. The enzyme cholesterol 25-hydroxylase [CH25H] converts cholesterol to 25-hydroxycholesterol [25-HC], which modulates immune responses and oxidative stress. In human intestinal samples from CD patients, we found a strong correlation of CH25H mRNA expression with the expression of fibrosis markers. We demonstrate reduced intestinal fibrosis in mice deficient for the CH25H enzyme, using the sodium dextran sulphate [DSS]-induced chronic colitis model. Additionally, using a heterotopic transplantation model of intestinal fibrosis, we demonstrate reduced collagen deposition and lower concentrations of hydroxyproline in CH25H knockouts. In the heterotopic transplant model, CH25H was expressed in fibroblasts. Taken together, our findings indicate an involvement of oxysterol synthesis in the pathogenesis of intestinal fibrosis.

Genetic polymorphisms associated with smoking behaviour predict the risk of surgery in patients with Crohn's disease.

BACKGROUND: Smoking is a strong environmental factor leading to adverse outcomes in Crohn's disease, but a more benign course in ulcerative colitis. Several single nucleotide polymorphisms (SNPs) are associated with smoking quantity and behaviour. AIM: To assess whether smoking-associated SNPs interact with smoking to influence the clinical course of inflammatory bowel diseases. METHODS: Genetic and prospectively obtained clinical data from 1434 Swiss inflammatory bowel disease cohort patients (821 Crohn's disease and 613 ulcerative colitis) were analysed. Six SNPs associated with smoking quantity and behaviour (rs588765, rs1051730, rs1329650, rs4105144, rs6474412 and rs3733829) were combined to form a risk score (range: 0-12) by adding the number of risk alleles. We calculated multivariate models for smoking, risk of surgery, fistula, Crohn's disease location and ulcerative colitis disease extent. RESULTS: In Crohn's disease patients who smoke, the number of surgeries was associated with the genetic risk score. This translates to a predicted 3.5-fold (95% confidence interval: 2.4- to 5.7-fold, P<.0001) higher number of surgical procedures in smokers with 12 risk alleles than individuals with the lowest risk. Patients with a risk score >7 had a significantly shorter time to first intestinal surgery. The genetic risk score did not predict surgery in ulcerative colitis or occurrence of fistulae in Crohn's disease. SNP rs6265 was associated with ileal disease in Crohn's disease (P<.05) and proctitis in ulcerative colitis (P<.05). CONCLUSIONS: SNPs associated with smoking quantity is associated with an increased risk for surgery in Crohn's disease patients who smoke. Our data provide an example of genetics interacting with the environment to influence the disease course of inflammatory bowel disease.

Disease Management Project Breast Cancer in Hesse - 5-Year Survival Data: Successful Model of Intersectoral Communication for Quality Assurance.

Introduction: The Disease Management Project Breast Cancer (DMP Breast Cancer) was first launched in Hesse in 2004. The project is supported by the health insurance companies in Hesse and the Professional Association of Gynaecologists in Hesse. The aim is to offer structured treatment programmes to all women diagnosed with breast cancer in Hesse by creating intersectoral cooperations between coordinating clinics, associated hospitals and gynaecologists in private practice who registered in the DMP programme. Method: Between 1 January 2005 and 30 June 2011, 13 973 women were enrolled in the DMP programme. Results: After data cleansing, survival rates were calculated for a total of 11 214 women. The 5-year overall survival (OS) rate was 86.3 %; survival rates according to tumour stage on presentation were 92.2 % (pT1) and 82.3 % (pT2), respectively. The impact of steroid hormone receptor status on survival (87.8 % for receptor-positive cancers vs. 78.9 % for receptor-negative cancers) and of age at first diagnosis on survival (≤ 35 years = 91 %) were calculated. Conclusion: The project showed that intersectoral cooperation led to significant improvements in the quality of treatment over time, as measured by quality indicators and outcomes after treatment.

Histopathological evidence of invasive gastric mucormycosis after transarterial chemoembolization and liver transplantation.

We describe a case of a 62-year-old diabetic woman with hepatocellular carcinoma due to chronic hepatitis B virus infection. Two weeks after orthotopic liver transplantation, endoscopy for massive upper gastrointestinal bleeding revealed a large necrotic area in the gastric fundus. The patient underwent emergency resection. Histopathologically, angioinvasive mold infection compatible with mucormycosis was diagnosed in a large area of necrosis, mimicking an atypically localized gastric ulcer. Foreign bodies originating from transarterial chemoembolization (TACE) performed 7 and 8 months earlier and 40 days before transplantation were identified in the submucosal tissue. The patient was treated with liposomal amphotericin B (LAB) for 5 weeks, followed by 7 weeks of posaconazole. Follow-up biopsies after 1 and 5 months confirmed successful treatment. Review of the radiological images of the TACE procedure showed that some of the TACE material had been diverted to the stomach via an accessory gastric branch originating from the left hepatic artery. TACE agents may be associated with chronic, refractory gastroduodenal ulcers. We hypothesize that the ischemic lesion was first colonized with presumed Mucorales mold and invasive growth was promoted by the posttransplantation immunosuppression. Careful exploration of extrahepatic collaterals during TACE may prevent this complication.

Toward more accurate measurements of anorectal motor and sensory function in routine clinical practice: validation of high-resolution anorectal manometry and Rapid Barostat Bag measurements of rectal function.

BACKGROUND: Measurements of anorectal function using high-resolution anorectal manometry (HR-ARM) and rectal barostat technology provide more reliable results than standard ARM with an elastic balloon; however, HR-ARM results have not been compared to ARM and standard barostat protocols are impractical in routine clinical practice. The aim of this study was to validate HR-ARM against standard ARM and standard barostat against a novel Rapid Barostat Bag (RBB) measurement and elastic balloon measurements of rectal function. METHODS: Twenty-six healthy volunteers (15 female, 11 male, 19-52 years) were studied. Measurements of anal function and simulated defecation were compared for 12-sensor HR-ARM and 6-sensor standard ARM using line plots from the same recording. Rectal capacity, compliance, and sensation (volume threshold) were measured by elastic balloon, standard barostat, and RBB methods using stepwise inflation of a 700-mL polyethylene bag to 40 mmHg distension by electronic barostat and handheld syringe monitored by sphygmo-manometer, respectively. Results are reported as mean ± SD. Bland-Altman plots and correlation coefficients (r) for measurements were calculated. KEY RESULTS: There was excellent agreement between HR- and standard ARM measurements (r > 0.86, <25 mmHg difference) and between standard barostat and RBB measurements of rectal capacity (r = 0.97, <25 mL difference). Correlation coefficients of threshold volumes for initial perception, urgency and discomfort were 0.37, 0.71, and 0.95, respectively. No significant correlation was present with elastic balloon measurements. Time to complete studies was shorter for HR-ARM than standard ARM and for RBB than standard barostat in historical controls. CONCLUSIONS & INFERENCES: HR-ARM with RBB measurements of anorectal function provides quick and reasonably accurate measurements of continence function suitable for use in routine clinical practice (ClinicalTrial.gov NCT01456442).

[MR imaging of lymph nodes using Gadofluorine M: feasibility in a swine model at 1.5 and 3T]. / MR-Bildgebung von Lymphknoten mit Gadofluorine M in einem Schweinemodell bei 1,5 and 3T.

PURPOSE: To investigate the potential of Gadofluorine M for targeted lymph node imaging in a human size animal model and on a clinical MR scanner at 1.5 and 3 T. MATERIALS AND METHODS: Pelvic and cervical lymph nodes in a swine model were investigated prior to and 24 hours after intravenous administration of 50 micromol/kg body weight Gadofluorine M, an experimental contrast agent. MR imaging was carried out on clinical 1.5 T and 3 T whole-body MR systems using clinically available coils and T 1-weighted sequences. The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) with respect to the surrounding tissue were assessed and compared using the Student's t-test. The Gd concentration in the lymph nodes (n = 43) was measured post mortem by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). RESULTS: Gadofluorine M allowed for high signal and high contrast visualization of lymph nodes in all stations on post-contrast images with a significantly increased SNR and CNR (SNR pelvic lymph nodes post vs. pre: 46 +/- 7 vs.14 +/- 3, SNR cervical lymph nodes post vs. pre: 105 +/- 64 vs. 32 +/- 21; CNR pelvic lymph node vs. muscle post vs. pre 28 +/- 5 vs. 0.2 +/- 0.5, CNR cervical lymph node vs. muscle post vs. pre 76 +/- 53 vs. 11 +/- 15, p < 0.05 for all comparisons). The SNR and CNR in the pelvis were further improved using 3 T compared to 1.5 T scanners (SNR lymph nodes 3 T vs. 1.5 T 84 +/- 6 vs. 46 +/- 7, CNR lymph node vs. muscle 3 T vs. 1.5 T 53 +/- 9 vs. 28 +/- 5 respectively, p < 0.05). A high concentration of Gd in the lymph nodes was found (149 +/- 25 mmol Gd/L). CONCLUSION: Gadofluorine M accumulates in the lymph nodes and allows for selective targeted high contrast MR imaging of lymph node tissue in a large animal model using clinically available MR imaging techniques. 3 T further improves SNR and CNR compared to 1.5 T.

[Billing based on a case-based lump sum for stroke. Did this lead to discharge of patients in a worse clinical condition?]. / Abrechnung mittels Fallpauschalen beim Hirninfarkt. Führte dies zu Entlassungen in klinisch schlechterem Zustand?

BACKGROUND: It has been supposed that the introduction of a new inpatient reimbursement system starting in 2004 in Germany using the German diagnosis-related groups (G-DRG) may lead to false incentives with encouragement of premature hospital discharge of patients. Exploring a large database on stroke patients, we addressed the question whether length of stay (LOS) and discharge in more severe condition were associated with the introduction of the G-DRG. We further examined other factors with probable effect on LOS such as variations of patient characteristics and treatment during the observation period. PATIENTS AND METHODS: All stroke patients treated in 2003-2006 in the German state of Hesse (6,100,000 inhabitants) were assessed with respect to stroke severity, symptoms on admission and discharge, LOS and stroke-related deficits on discharge. We compared LOS and outcome in 2003 (before introduction of the G-DRG) with 2004 when the G-DRG had recently been introduced and with 2006 when the G-DRG was already well established in the clinical routine. The effects of LOS and treatment year on outcome were assessed using a logistic regression model. RESULTS: During the observation period, we evaluated 37,396 stroke patients. The length of stay was reduced significantly from 12.2 to 10.4 days (p<0.001). Both severity of stroke on admission and outcome on discharge decreased during the observation period. A multivariate analysis revealed a minor but significant association [odds ratio (OR): 1.020 per day of hospital treatment; 95% confidence interval (CI): 1.016-1.024] of LOS on outcome. Treatment in 2006 compared to 2003 led to good outcome with an OR of 1.378 (95% CI: 1.279-1.485). Subgroup analysis limited to patients with severe stroke revealed that LOS was significantly lower in 2006 compared to 2003 also in this patient subgroup; moreover, the proportion of patients discharged with severe outcome was lower in 2006 compared to 2003. CONCLUSIONS: This study reveals a significant reduction of LOS during the years after introduction of the G-DRG. However, reduction of LOS was not associated with more severe outcome on discharge, possibly due to changes in stroke treatment implemented during the observational period. Our results do not support the conjecture that changes in the reimbursement system were associated with compromised patient care.

[High-resolution T1-weighted MR-lymphography of inguinal lymph nodes after interstitial application of Gadomer-17 in animal experiments]. / Hochauflösende T1-gewichtete MR-Lymphographie inguinaler Lymphknoten nach interstitieller Applikation von Gadomer-17 im Tierexperiment.

PURPOSE: To evaluate the microstructural anatomy of inguinal lymph nodes in pigs after interstitial MR-lymphography with the dendritic contrast agent Gadomer-17. MATERIAL AND METHODS: High-resolution T1-weighted MR-lymphography was performed in inguinal lymph nodes of 10 domestic pigs (39 - 46 kg) after subcutaneous injection of 10 mumol/kg body weight Gadomer-17 in the hind legs of the animals. A 1.5T MR scanner and a ring-shaped surface coil were used. Two different high-resolution gradient-echo sequences with additionally reconstructed maximum-intensity projections were evaluated in a total of 20 lymph nodes. The high-resolution MR-findings were correlated with the histologic sections of the excised inguinal lymph nodes. RESULTS: Coronal T1-weighted 3D gradient-echo images (TR = 20 msec, TE = 6.1 - 8.3 msec, FA = 20 degrees ) with a slice thickness of 1 mm, a field-of-view of 120 mm and a matrix size of 256 x 256 (reconstructed to 1024 x 1024 voxels) yielding a reconstructed in-plane resolution of 117 x 117 microm (2) were best suited for the high-resolution MR lymphography of inguinal lymph nodes and enabled the differentiation of the hyperintense lymph node sinuses and hypointense lymphoid parenchyma of each lymph node (100 %). Even dilated lymphatic vessels evident in the histologic specimen were best demonstrated on the MIP images. CONCLUSION: High-resolution interstitial MR lymphography with Gadomer-17 allows the visualization of different tissue compartments of inguinal lymph nodes. This new technique is feasible on a routine 1.5T scanner and may offer potential for the detection of micrometastases in lymph nodes of cancer patients.

T1-weighted MR-lymphography after intramammary administration of Gadomer-17 in pigs.

PURPOSE: To evaluate the enhancement of regional lymph nodes and lymphatic vessels after intramammary injection of Gadomer-17. MATERIALS AND METHODS: T1-weighted MR-lymphography was performed in 8 domestic pigs after intramammary injection of 5 - 10 micromol/kg bodyweight (bw) Gadomer-17 on a 1.5 T-MR scanner. T1-weighted 3D gradient-echo images were acquired 10 to 120 minutes after contrast material injection in coronal and sagittal planes. The contrast enhancement of the draining lymphatic system was qualitatively assessed by two radiologists applying a three grade scale. RESULTS: T1-Weighted MR-lymphography after intramammary injection of Gadomer-17 was feasible in each pig. A dose of 5 micromol/kg body weight Gadomer-17 was best suited for MR-lymphography of the mammary line and the draining lymphatic system. Enhancement of regional lymph nodes and lymphatic vessels was classified as good in 5, and excellent in three cases. CONCLUSIONS: Intramammary injection of Gadomer-17 allows for good quality T1-weighted MR-lymphography of mammary gland draining regional lymph nodes and lymphatic vessels. This new technique might offer potential for the evaluation of the sentinel lymph node in patients with breast cancer.

Posttranslational protein translocation across the membrane of the endoplasmic reticulum.

Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.

Gadofluorine 8: initial experience with a new contrast medium for interstitial MR lymphography.

RATIONALE AND OBJECTIVES: Differential diagnosis of malignant and beign lymph nodes is still a problem in lymphographic imaging modalities. Plain magnetic resonance imaging (MRI) and computed tomography (CT) are inadequate for detecting metastases in normal-sized lymph nodes and for differentiating enlarged nodes. Therefore it is important to have a contrast agent that accumulates in healthy lymphatic tissue but does not accumulate in metastatic deposits. METHODS: The lymphographic contrast agent Gadofluorine 8 (Schering AG, Berlin, Germany) is a lipophilic but water-soluble gadolinium complex. Lymphographic effects were investigated in guinea pigs, dogs, and tumor-bearing rabbits after interstitial (subcutaneous or intracutaneous) injection. MR imaging was performed using T1-weighted gradient-echo sequences until 120 min after administration. RESULTS: After interstitial injection Gadofluorine 8 accumulates in regional lymph nodes, resulting in a pronounced increase in signal intensity in the lymph nodes. Differentiation between normal and metastatic lymph nodes was achieved. CONCLUSIONS: Gadofluorine 8 is an innovative contrast agent that can distinguish between normal and tumorous lymph nodes in interstitial MR lymphography.

Interaction of BiP with the J-domain of the Sec63p component of the endoplasmic reticulum protein translocation complex.

Proteins of the Hsp70 family of ATPases interact with a conserved domain of their J-protein partners, the J-domain, to function in numerous cellular processes. We have studied the interaction of BiP, an Hsp70 family member in the lumen of the endoplasmic reticulum, with the J-domain of Sec63p, a component of the Sec complex involved in post-translational protein translocation across the endoplasmic reticulum membrane. In a real-time solid phase binding assay, BiP binds to the immobilized Sec complex or to a fusion protein of the J-domain and glutathione S-transferase in a reaction that requires ATP hydrolysis. In the final complex, BiP is bound in the ADP form with its peptide binding pocket occupied. An intact peptide binding pocket is required for this interaction. Our experiments suggest that the activation of BiP by the J-domain involves a transient contact between these components, and that in the absence of physiological substrates, J-activated BiP binds even to the J-proteins themselves.

BiP acts as a molecular ratchet during posttranslational transport of prepro-alpha factor across the ER membrane.

We have addressed the mechanism by which proteins are posttranslationally transported across the membrane of the yeast endoplasmic reticulum (ER). We demonstrate that BiP (Kar2p), a member of the Hsp70 family resident in the ER lumen, acts as a molecular ratchet during translocation of the secretory protein prepro-alpha factor through the channel formed by the Sec complex. Multiple BiP molecules associate with each translocation substrate following interaction with the J domain of the Sec63p component of the Sec complex. Bound BiP minimizes passive backward movements of the substrate through the channel, and BiP's subsequent dissociation results in a free polypeptide in the ER lumen. Antibodies against the substrate can replace BiP, indicating that a Brownian ratchet is sufficient to achieve translocation.

J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences.

Proteins of the Hsp70 family of ATPases, such as BiP, function together with J proteins to bind polypeptides in numerous cellular processes. Using a solid phase binding assay, we demonstrate that a conserved segment of the J proteins, the J domain, catalytically activates BiP molecules to bind peptides in its immediate vicinity. The J domain interacts with the ATP form of BiP and stimulates hydrolysis resulting in the rapid trapping of peptides, which are then only slowly released upon nucleotide exchange. Activation by the J domain allows BiP to trap peptides or proteins that it would not bind on its own. These results explain why BiP and probably all other Hsp70s can interact with a wide range of substrates and suggest that the J partner primarily determines the substrate specificity of Hsp70s.

Protein transport by purified yeast Sec complex and Kar2p without membranes.

Posttranslational protein translocation across the endoplasmic reticulum membrane of yeast requires a seven-component transmembrane complex (the Sec complex) in collaboration with the lumenal Kar2 protein (Kar2p). A translocation substrate was initially bound to the cytosolic face of the purified Sec complex in a signal-sequence-dependent but Kar2p- and nucleotide-independent manner. In a subsequent reaction, in which Kar2p interacted with the lumenal face of the Sec complex and hydrolyzed adenosine triphosphate, the substrate moved through a channel formed by the Sec complex and was released at the lumenal end. Movement through the channel occurred in detergent solution in the absence of a lipid bilayer.

Interstitial MR lymphography using Gd-carrying liposomes.

The detection of metastasis in lymph nodes is greatly enhanced by the use of contrast media. Interstitial injection of lymphography contrast agents requires only injection of a low dose of the agent, and leads to high accumulation in regional lymph nodes with only minor side effects. We were able to show the suitability of liposome-encapsulated gadobutrol as an interstitially injectable lymphography contrast agent in an experimental animal model. For screening of possible lymphotrophic compounds a guinea pig model was used. Accumulation of the contrast agent in 3 successive lymph node groups was determined 4 h after subcutaneous injection of the contrast agent (about 0.1 ml of a 30 mmol Gd/l solution resulting in a total dose of 10 mumol/kg) into the interdigital skin fold of a hind limb. The Gd concentration in the first lymph node group (popliteal) was 540 mumol/l (16% dose/g tissue), in the second group (inguinal): 260 mumol/l (8% dose/g tissue), and in the third group (iliac): 910 mumol/l (27% dose/g tissue). Moreover, this compound was completely eliminated and well tolerated.