Microbial Spectrum of Intra-Abdominal Abscesses in Perforating Crohn's Disease: Results from a Prospective German Registry.

BACKGROUND: Intra-abdominal abscesses [IAAs] are common life-threatening complications in patients with Crohn's disease [CD]. In addition to interventional drainage and surgical therapy, empirical antibiotic therapy represents a cornerstone of treatment, but contemporary data on microbial spectra and antimicrobial resistance are scarce. METHODS: We recruited 105 patients with CD and IAAs from nine German centres for a prospective registry in order to characterize the microbiological spectrum, resistance profiles, antibiotic therapy and outcome. RESULTS: In 92 of 105 patients, microbial investigations of abscess material revealed pathogenic microorganisms. A total of 174 pathogens were isolated, with a median of 2 pathogens per culture [range: 1-6]. Most frequently isolated pathogens were E. coli [45 patients], Streptococcus spp. [28 patients], Enterococci [27 patients], Candida [13 patients] and anaerobes [12 patients]. Resistance to third-generation cephalosporins, penicillins with beta-lactamase inhibitors and quinolones were observed in 51, 36 and 35 patients, respectively. Seven patients had multiple-drug-resistant bacteria. Thirty patients received inadequate empirical treatment, and this was more frequent in patients receiving steroids or immunosuppression [37%] than in patients without immunosuppression [10%: p = 0.001] and was associated with a longer hospital stay [21 days vs 13 days, p = 0.003]. CONCLUSION: Based on antimicrobial resistance profiles, we herein report a high rate of inadequate empirical first-line therapy for IAAs in CD, especially in patients receiving immunosuppression, and this is associated with prolonged hospitalization.

Vedolizumab in the treatment of chronic, antibiotic-dependent or refractory pouchitis.

BACKGROUND: The most common complication after ileal pouch anal anastomosis in up to 50% of patients is an acute pouchitis. The majority of patients respond to antibiotic treatment. However, 10%-15% develops chronic antibiotic-dependent or refractory pouchitis which is usually hard to treat. AIM: To evaluate the effectiveness of vedolizumab in patients with chronic pouchitis. METHODS: Patients with chronic antibiotic-dependent or refractory pouchitis were treated with vedolizumab (300 mg at week 0, 2, 6 and 10) in 10 IBD centres and retrospectively registered. Data were recorded until week 14 of vedolizumab treatment. In total 20 patients (12 male, median age 43 years) were included for analysis. The effectiveness was measured using the Oresland Score (OS) at week 2, 6, 10 and 14 and the pouch disease activity index (PDAI) at week 0 and 14. RESULTS: The mean OS declined from 6.8 (range 2-12) to 3.4 (range 0-11). Concordantly, the mean PDAI after 14 weeks of treatment dropped from 10 (range 5-18) to 3 (range 0-10). Only three patients reported moderate side effects. No serious side effects were recorded. In addition, symptomatic co-medication such as loperamide and tincture of opium could be terminated in 8 out of 12 patients as well as antibiotic treatment could be stopped in 17 out of 19 patients. CONCLUSION: Our data indicate that vedolizumab could be an option in the treatment of patients with chronic, antibiotic-dependent or refractory pouchitis.

Comorbidity in acute pancreatitis relates to organ failure but not to local complications.

OBJECTIVE: Organ failure and local complications contribute to morbidity and mortality in acute pancreatitis. Comorbidity is known to be related to organ failure. The impact of comorbidity on local complications has not yet been delineated. Moreover, it is not clear if the outcome of first-attacks and acute-on-chronic episodes, respectively, differs from outcome in all episodes. METHODS: Consecutive cases of confirmed acute pancreatitis in a four-year period were reviewed. Charlson comorbidity index (CCI), complications (organ failure and local complications), disease severity (according to the revised Atlanta Classification), need for intensive care, and mortality were derived from the charts. RESULTS: A total of 391 episodes of acute pancreatitis were included. Patients with organ failure were significantly older (P< 0.001) und had a higher CCI (P< 0.001) than patients without organ failure. Patients with and without local complications did not significantly differ in age or CCI. The complication rate of the entire cohort (n = 391; 47.1 %) was comparable with the complication rate of first-attacks (n = 269; 46.8 %) and acute-on-chronic episodes (n = 68; 47.1 %). The majority of the twelve deceased patients had been old and/or chronically ill. Six of these patients had an advanced malignant disease. CONCLUSIONS: Comorbidity and age clearly are contributors to organ failure and mortality. Local complications occur independently of age and concomitant diseases. The overall complication rate is not significantly influenced by preceding inflammation of the pancreas. To further improve care in patients with acute pancreatitis special attention should be given to old and multi-morbid patients.

IL-10 downregulates CXCR3 expression on Th1 cells and interferes with their migration to intestinal inflammatory sites.

Inflammatory bowel disease (IBD) is characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the etiology is poorly understood, it is widely accepted that loss of tolerance is involved in the development of IBD. Therefore, re-establishing tolerance or gut homeostasis is one of the key features in the development of new therapeutic strategies. Here we show that antigen targeting to DEC-205 on dendritic cells leads to an interleukin (IL)-10-dependent downregulation of C-X-C chemokine receptor 3 (CXCR3) expression on differentiated antigen-specific T helper type 1 (Th1) cells in vivo. This downregulation interferes with the migration of Th1 cells into the gut and protects mice against severe acute and relapsing intestinal inflammation. Moreover, CD4(+)CXCR3(+) T cells are highly enriched in the inflamed mucosa of IBD patients. Interference with this pathway may therefore be a promising approach for the treatment of IBD. In conclusion, we propose a hitherto undescribed mechanism by which IL-10 can act on effector T cells and orchestrate intestinal immune responses.

Long-term Outcomes in Steroid-refractory Ulcerative Colitis Treated with Tacrolimus Alone or in Combination with Purine Analogues.

BACKGROUND AND AIMS: Tacrolimus is recommended for the treatment of steroid-refractory ulcerative colitis (UC). Concomitantly started purine analogues (PAs) are used for the maintenance of remission, though their therapeutic relevance remains uncertain. Here we studied the role of PAs in the long-term outcome of steroid-refractory UC after tacrolimus treatment. METHODS: In five centres, charts of tacrolimus-treated UC patients with a steroid-refractory moderate to severe course were reviewed. Long-term efficacy was determined by colectomy rates and clinical remission in cases of colectomy-free survival for 3 months. RESULTS: We identified 156 patients (median age 34 years) with a median Lichtiger score of 12 (4-17) and pancolitis (E3) in 65% (101). The Kaplan-Meier curve for colectomy-free survival after month 3 showed a benefit in the PA group (p = 0.02). In patients treated with PA clinical remission was achieved in 82% (65/79) vs 67% (39/58) in those not treated with PA (p = 0.02). Time to colectomy was 2 years (median, 0.7-5.8) in the PA group and 0.8 years (0.3-4.7) in the group not treated with PAs (p = 0.02). Time to relapse was 1.2 years (median, 0.3-6.2) in patients with PA treatment and 0.5 years (0.3-3.9) in those without PA treatment (p = 0.05). Overall, clinical remission was achieved in 67% (104/156) of patients. Colectomy was performed in 29% (45/156) 0.5 years (median, 0.04-5.79) after initiation of tacrolimus. Ten (6%) patients had to stop tacrolimus due to adverse events and two (without PA treatment) died. CONCLUSIONS: Our study supports the efficacy of tacrolimus in steroid-refractory UC. Purine analogues appear to be beneficial for the long-term outcome of these patients.

Inflammatory bowel diseases influence major histocompatibility complex class I (MHC I) and II compartments in intestinal epithelial cells.

Antigen presentation by intestinal epithelial cells (IEC) is crucial for intestinal homeostasis. Disturbances of major histocompatibility complex class I (MHC I)- and II-related presentation pathways in IEC appear to be involved in an altered activation of CD4(+) and CD8(+) T cells in inflammatory bowel disease. However, a comprehensive analysis of MHC I- and II-enriched compartments in IEC of the small and large bowel in the healthy state as opposed to inflammatory bowel diseases is lacking. The aim of this study was to characterize the subcellular expression of MHC I and II in the endocytic pathway of IEC throughout all parts of the intestinal tract, and to identify differences between the healthy state and inflammatory bowel diseases. Biopsies were taken by endoscopy from the duodenum, jejunum, ileum and colon in healthy individuals (n = 20). In Crohn's disease (CD), biopsies were obtained from the ileum and colon and within the colon from ulcerative colitis (UC) patients (n = 15). Analysis of IEC was performed by immunoelectron microscopy. MHC I and II were identified in early endosomes and multi-vesicular, multi-lamellar, electrondense and vacuolar late endosomes. Both molecules were enriched in multi-vesicular bodies. No differences were found between the distinct parts of the gut axis. In CD and UC the expression of MHC I and II showed a shift from multi-vesicular bodies towards the basolateral membranes. Within the multi-vesicular bodies, MHC I and II moved from internal vesicles to the limiting membranes upon inflammation in CD and UC. MHC I- and II-enriched compartments in IEC were identical in all parts of the small and large bowel. CD and UC appear to modulate the MHC I- and II-related presentation pathways of exogenous antigens in IEC.

Minor cartilage collagens type IX and XI are expressed during embryonic stem cell-derived in vitro chondrogenesis.

Cartilage development is a complex process that can be analyzed using numerous model systems. We have previously shown that in vitro differentiation of murine embryonic stem (ES) cells via embryoid bodies (EBs) recapitulates the cellular differentiation steps of chondrogenesis. However, differentiated chondrocytes lose their characteristic phenotype when they are kept in monolayer culture. This dedifferentiation process is one of the main obstacles of cartilage tissue engineering and could not be analyzed using the EB model system. The aim of this study was to further characterize the chondrogenic nodules derived by in vitro-differentiation of murine ES cells for the distribution of collagen types II, IX and XI in comparison to in vitro dedifferentiating primary chondrocytes from murine embryonic ribs. Expression of cartilage collagens and other extracellular matrix proteins was analyzed using immunostaining, cytochemical stainings and quantitative RT-PCR. We show that ES cell-derived chondrocyte differentiation starts with mesenchymal condensations synthesizing high amounts of fibronectin. Later, the matrix of the mature cartilage nodules consists of type II collagen, proteoglycans and the minor collagens type IX and XI. The nodules show a three-dimensional structure with multiple layers of collagen type II-positive cells. At late differentiation stages these chondrocytes were located at lateral regions of the nodules. Similar to the distribution pattern of collagen type II positive cells, the cells staining positive for collagen type IX and XI were present in the surface regions, but not in the central areas of the chondrogenic nodules. During cultivation of the primary murine rib chondrocytes expression of chondrogenic marker genes such as collagen type II and aggrecan declined and many chondrocytes lost characteristic cartilage matrix proteins and converted to an elongated, fibroblastoid shape with prominent actin stress fibers. Chondrogenic differentiation of murine ES cells combined with monolayer culture of embryonic rib chondrocytes is a valuable tool to study changes in the expression pattern during differentiation and dedifferentiation of chondrocytes.

Short-term efficacy of tacrolimus in steroid-refractory ulcerative colitis - experience in 130 patients.

BACKGROUND: Steroid-refractory ulcerative colitis (UC) remains a challenging condition warranting surgery upon failure of pharmacological treatment. Calcineurin inhibitors or infliximab are alternatives in this situation. Data on the efficacy and safety of tacrolimus in this setting are limited. AIM: To study the short-term efficacy and safety of tacrolimus in moderate-to-severe steroid-refractory UC. The role of thiopurines in this situation and predictors of colectomy were evaluated. METHODS: In three centers, all charts from tacrolimus-treated patients with steroid-refractory UC were reviewed. Efficacy was assessed by colectomy-free survival and clinical remission at 3 months. RESULTS: We identified 130 patients with pancolitis in 75 (59%), left-sided disease in 35 (27%) and proctitis in 18 patients (14%) (disease localisation not obtainable in two patients). The median age was 40 (range: 18-81). Clinical activity according to the median Lichtiger score decreased from 13 (range: 4-17) at baseline to 3 (0-14) at week 12. Eighteen patients underwent colectomy within the first 3 months of treatment with tacrolimus (14%). Clinical remission was achieved in 94 patients (72%) in this period. Thiopurines given in parallel to tacrolimus tended to limit colectomy and significantly increased remission (P = 0.002) in the short-term. No other predictors of colectomy or remission were identified. Side effects were noticed in 53% of patients and no severe events occurred. CONCLUSION: This large survey confirms the efficacy and safety of tacrolimus in patients with steroid-refractory ulcerative colitis.

[CEUS as a tool to uncover an unusual cause of obstructive jaundice]. / KM-verstärkter Ultraschall zur Erkennung der seltenen Ursache eines obstruktiven Ikterus.

Aneurysms within the visceral arteries are rare. Among these, aneurysms of the splenic artery occur most frequently followed by aneurysms of the hepatic arteries. An early diagnosis is easily missed and almost all patients become symptomatic with an acute rupture associated with high mortality. Here we demonstrate the case of a 76-year-old patient who presented with acute upper abdominal pain accompanied by a single episode of vomiting and pyrexia of 39 °C. Laboratory results presented the picture of an obstructive jaundice without evidence for accompanying pancreatitis. Inflammatory markers were within normal limits at onset, but increased dramatically within the next few days. An acute calculous cholecystitis was diagnosed on abdominal ultrasound whereas gastroscopy revealed no relevant changes. Computed tomography was suspicious for pancreatitis of the head with obstruction of the bile duct. Choledocholithiasis was ruled out by ERCP, but symptoms persisted despite papillotomy. Due to raising inflammatory markers and an ongoing impairment of the patients condition, an abdominal CT scan was repeated which revealed the suspicion of a ruptured aneurysm of the common hepatic artery. At the time of transferral we were able to confirm the diagnosis by contrast-enhanced ultrasound and angiography. The patient was immediately forwarded to surgery due to lack of satisfactory endovascular procedures. In summary, the patient suffered from a ruptured spurial aneurysm of the right gastric artery thereby obstructing the common bile duct. Beside CT scans and angiography, this case documents a pivotal role for contrast-enhanced ultrasound in the work-up of visceral artery aneurysms.

[Intensive care and inflammatory bowel diseases (IBD). The severe and fulminant flare]. / Intensivmedizin und chronisch entzündliche Darmerkrankungen (CED). Schwerer und fulminanter Schub.

Inflammatory bowel disease (IBD) is a non-curable disease. Although we seem to have a definitive surgical solution for ulcerative colitis, patients are at risk of suffering from acute and chronic pouchitis as well as peri- and postoperative complications. In Crohn's disease medical treatment is able to prolong surgery-free survival, but no definitive healing strategy is as yet at hand. Moreover, effective medication exposes patients to potentially life-threatening complications. The severely compromised patient in particular needs careful surveillance and requires a balanced treatment strategy endorsed by gastroenterologists and surgeons. Treatment is still aimed at suppressing immune mechanisms and is far from being causal. Nevertheless, this approach has resulted in a reduction in surgical procedures and hospitalizations. The following overview covers special issues related to strategies for severe and fulminant flares of inflammatory bowel disease.

Infliximab as rescue medication for patients with severe ulcerative/indeterminate colitis refractory to tacrolimus.

BACKGROUND: The calcineurin inhibitor tacrolimus and the anti-TNF-antibody infliximab are established options in steroid-refractory ulcerative colitis (UC). AIM: To evaluate the efficacy of infliximab-salvage therapy in patients with refractory UC failing to respond to tacrolimus. METHODS: Twenty-four patients were enrolled in this evaluation. Reasons for tacrolimus therapy were steroid-refractory disease in 19 patients and steroid-dependency in five patients. All patients receiving infliximab had tacrolimus-refractory active disease (Lichtiger score >10) and were treated with 5 mg/kg at weeks 0, 2 and 6 and every 8 weeks thereafter, if tolerated. RESULTS: Six of 24 patients (25%) achieved remission following infliximab infusion and four of 24 (17%) had an initial response only, but underwent proctocolectomy later because of loss of response (3) or development of a delayed hypersensitivity reaction (1). Fourteen patients (58%) completely failed to respond with 10 undergoing colectomy. Eight patients experienced side effects under infliximab, including two infectious complications (herpes zoster and herpes pneumonia). CONCLUSIONS: Infliximab offers a therapeutic option as rescue therapy in about a quarter of patients with active UC after failing to respond to tacrolimus. This benefit has to be weighed against the risks of infectious complications.

Cannabinoid type 1 receptor blockade induces transdifferentiation towards a brown fat phenotype in white adipocytes.

AIM: The endocannabinoid (EC) system is a major component in the control of energy homeostasis. It mediates a positive energy balance via central and peripheral pathways. Blockade of the cannabinoid type 1 receptor induces weight reduction and improves cardiovascular risk factors in overweight patients. Cannabinoid receptor type 1 (CB1R)-deficient mice are resistant to diet-induced obesity. The mechanisms responsible for these effects remain only partially elucidated. We hypothesized peripheral effects via direct modulation of adipocyte function to be an integral part of EC action on energy metabolism and insulin sensitivity. METHODS: SV40 immortalized murine white and brown adipocytes were used for all experiments. We investigated the effect of CB1R blockade by stimulating the cells acutely and chronically with rimonabant, a selective antagonist for the CB1R, or by knocking down the receptor with small interfering RNA (siRNA). Changes in thermogenic mRNA and protein expression as well as mitochondrial biogenesis and function were assessed by real-time RT-PCR, immunoblotting, fluorescent staining techniques, electron microscopy and by measuring oxygen consumption. RESULTS: Acute and chronic blockade of the CB1R with the selective antagonist rimonabant or by siRNA in murine white adipocytes strongly induced the thermogenic uncoupling protein-1 (UCP-1). UCP-1 expression was increased in a time- and dose-dependent manner both at the RNA and protein level. Furthermore, this effect was paralleled by enhanced peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) expression. In accordance with these findings, AMP-activated protein kinase (AMPK) phosphorylation was also increased after rimonabant treatment. Mitochondria-specific fluorescent staining demonstrated an augmentation in the number of mitochondria. This was confirmed by electron microscopy images. Moreover, rimonabant treatment enhanced the cytochrome c oxidase activity and increased cellular oxygen consumption. CONCLUSIONS: Taken together, our data demonstrate that inhibition of peripheral CB1R action in adipocytes directly promotes transdifferentiation of white adipocytes into a mitochondria-rich, thermogenic brown fat phenotype. Enhanced thermogenesis and insulin sensitivity may represent a peripheral mechanism contributing to weight loss and improved glucose homeostasis in rimonabant-treated patients.

Clinical trial: cyclophosphamide pulse therapy - a promising therapeutic alternative in refractory Crohn's disease.

BACKGROUND: In severe steroid-refractory Crohn's disease (CD), established therapies fail in a relevant proportion of patients. Recent pilot studies indicated the efficacy of cyclophosphamide pulse therapy in these patients. AIM: To provide further and substantial evidence for the rationale to apply cyclophosphamide pulse therapy as therapeutic option in severe courses of CD. METHODS: Fifteen patients with steroid-refractory (n = 13) or steroid-dependent (n = 2) CD received 2-6 (median 3) monthly pulses of 750 mg cyclophosphamide in an open-label fashion. Eleven patients were on concomitant immunosuppression (azathioprine/mercaptopurine n = 9; methotrexate n = 2). RESULTS: Thirteen of 15 patients (87%) had a clinical response (CDAI decrease >100). Ten patients (67%) went into remission (CDAI <150) after 8 weeks. Steroid-free remission was achieved in eight patients (54%). Two patients (13%) failed to respond. Median CDAI decreased from 420 (245-550) to 100 (26-538) at week 8. Remission lasted 16 months (median, range 4-40). In three patients, arthritis, erythema nodosum and episcleritis completely resolved. Cyclophosphamide pulse therapy administration was well tolerated in all subjects. CONCLUSIONS: Cyclophosphamide pulse therapy is safe and highly effective for induction and maintenance of remission in steroid-refractory/-dependent CD. There is a strong need for additional experience to improve the setting of the encouraging cyclophosphamide treatment in CD.

F-actin is a component of the karyosome in neuropteran oocyte nuclei.

The principles underlying the assembly of intranuclear compartments are only beginning to be understood. The karyosome is an organelle typical of oocyte nuclei. It represents the tightly packed oocyte chromosomes, arrested at the diplotene of meiotic prophase. It has been known from several insect orders that a prominent capsule of unknown materials is built around the karyosome in the course of previtellogenesis and vitellogenesis. Here we show that F-actin, detected by dye-coupled phalloidin, is a major molecular component of the karyosome capsule in Neuroptera. We investigated capsule formation in six species belonging to the family Chrysopidae. Though F-actin was present in the capsules of all six species there were striking interspecific differences in the morphological array of actin filaments and the developmental dynamics of actin deposition in the capsule. The potential biological significance of the karyosome capsule is discussed with respect to the presence of extrachromosomal rDNA in neuropteran oocytes and the molecular functions known from F-actin. Our results corroborate the still controversial hypothesis of a role for actin as a nuclear protein.

Modulation of antigen trafficking to MHC class II-positive late endosomes of enterocytes.

BACKGROUND & AIMS: Oral tolerance is recognized as a central immunoregulatory phenomenon. The mechanisms of its induction remain unclear, and the role of the intestinal epithelial cells that are able to present antigens to T lymphocytes is poorly understood. In this report, we analyze under in vivo conditions the intracellular targeting of mucosally administered ovalbumin (OVA) to major histocompatibility complex (MHC) class II antigen containing compartments of enterocytes and compare these pathways between BALB/c and SCID mice, the latter being unable to generate a transferable tolerogenic moiety after a feed of OVA. METHODS: OVA, lysosome-associated membrane proteins (LAMP-1), and MHC class II antigens were localized in jejunal biopsy specimens of BALB/c and SCID mice at 0, 5, 10, 20, 40, 60, and 120 minutes after a single feed with OVA by fluorescence and electron microscopy. RESULTS: Ten minutes after oral administration, OVA was transported to the proximity of MHC class II antigens within LAMP-1-positive vacuoles and to the basolateral membrane of enterocytes from BALB/c strain mice. However, in SCID mice, OVA reached the paracellular spaces during the same time period through LAMP-1-negative vacuoles of enterocytes, which lacked MHC class II antigens. CONCLUSIONS: Orally administered OVA is rapidly targeted to late endosomes containing LAMP-1 and MHC class II antigens in enterocytes of BALB/c mice but not in SCID mice bred on a BALB/c background. We suggest that this targeting process within the enterocytes is one of the requirements for the induction of oral tolerance.

The egghead gene product influences oocyte differentiation by follicle cell-germ cell interactions in Drosophila melanogaster.

Oogenesis in Drosophila is a useful model for studying cell differentiation. We have analyzed the role of the egh gene in these processes with the aid of a newly isolated viable but female sterile allele. This mutation results in diverse variable defects in oogenesis. The most frequent defect being follicles that have either more or less than the normal number of 16 germ cells. This is caused by erroneous splitting and/or fusion of correct clusters of 16 cystocytes. The entire follicle has a rather flexible structure in this allele, most obvious by a highly variable position of the oocyte within the follicle. Moreover, a second oocyte can also develop in egh clusters. This is exclusively observed in aberrant follicles that are generated by the aforementioned splitting/fusion process. Surprisingly, even a germ cell which is distinct from the two pro-oocytes can differentiate into an oocyte under these circumstances. Hence, determination of the oocyte is definitely not fixed when germ cell clusters are enveloped by prefollicular cells, and interactions between follicle cells and germ cells must play an important role in oocyte specification. Molecular analysis proves that the oocyte-specific transcript of the egh gene is drastically reduced in this viable allele.