Tunable nanometer electrode gaps by MeV ion irradiation.

We report the use of MeV ion-irradiation-induced plastic deformation of amorphous materials to fabricate electrodes with nanometer-sized gaps. Plastic deformation of the amorphous metal Pd(80)Si(20) is induced by 4.64 MeV O(2+) ion irradiation, allowing the complete closing of a sub-micrometer gap. We measure the evolving gap size in situ by monitoring the field emission current-voltage (I-V) characteristics between electrodes. The I-V behavior is consistent with Fowler-Nordheim tunneling. We show that using feedback control on this signal permits gap size fabrication with atomic-scale precision. We expect this approach to nanogap fabrication will enable the practical realization of single molecule controlled devices and sensors.

[Perianal fistulas in Crohn's disease--biologicals and surgery: is it worthwhile?]. / Perianales Fistelleiden bei Morbus Crohn - Biologika und Chirurgie: Ein lohnendes Konzept?

Perianal fistulas and abscesses are a common manifestation in Crohn's disease (CD), seen in about 30 - 40 % of the patients. Often they are combined with CD of the anal canal and occur as a complex system of fistulas. The evaluation of these fistulas can be done with endoscopic ultrasound or magnetic resonance imaging, the conceptual accuracy of both methods is high. There are accepted therapeutic concepts for surgery and for the conventional drug therapy according to the classification of the fistulas. In contrast, the therapeutic regimens for a complex perianal fistulising CD are not convincing, especially not for maintainance therapy. However, several studies about therapy with anti-TNF-alpha antibodies have shown good results while long-time results with other recent anti-TNF-alpha antibodies apart from infliximab are still lacking. In this review article we analyse the current literature and develop a stage-adapted therapy for the use of biologicals and surgery in fistulising perianal CD.

[Vacuum-assisted fascial closure in the management of diffuse peritonitis]. / Die V.A.C.-Therapie im Behandlungskonzept der diffusen Peritonitis.

The surgical treatment of patients suffering from peritonitis with an open abdomen is commonplace. However scheduled as well as required dressing changes and peritoneal lavages mean a time- and costs-intensive challenge for the nursing and medical personnel. Since the introduction of the abdominal dressing-kit (KCI, Walluf, Germany) in the management of diffuse peritonitis one expects to influence the patients' recovery in a positive way due to the continuous evacuation of intraabdominal infectious fluids, augmentation of tissue's granulation, prevention of fascial retraction and prevention of an abdominal compartment syndrome. First on trial we established 2004 in our surgical department the use of the abdominal dressing-kit as a routine procedure in the management of diffuse peritonitis.

[Neuroendocrine tumors of the gastroenteropancreatic system--reevaluation using the Capella classification]. / Neuroendokrine Tumoren des Gastroenteropankreatischen Systems--Reevaluierung nach der Capella Klassifikation.

INTRODUCTION: Neuroendocrine tumors of the gastroenteropancreatic system (GEP) are heterogeneous regarding hormone production, localisation and biological behaviour making the prognostic evaluation of these rare tumors difficult. Capella et al. proposed a new classification that combines for the first time both biological and prognostic characteristics. This study aimed to evaluate the prognostic value of Capella's classification in the patients treated at our hospital. METHODS: 86 from 1975-1999 surgically treated and histologically confirmed neuroendocrine tumors were retrospectively classified as benign (B), uncertain behavior (UB), low grade malignant (LG) or high grade malignant (HG) following the Capella classification. These data were correlated with the long-term outcome of the patients 8.5 (range: 1-24) years after surgery. RESULTS: 43 % of tumors were localised in the pancreas, the others were equally distributed in the remaining GEP. Most tumors were classified as B (42 %) or LG (41 %), few were UB (10 %) or HG (7 %). 61 patients survived free of disease, 25 patients died, 13 of them not tumor related. Tumor related deaths were only observed in 6 patients with inoperable LG- and 6 patients with HG-neuroendocrine tumors within 3 years after surgery. CONCLUSION: The Capella classification reliably reflects the heterogeneity and the biological behaviour of GEP-neuroendocrine tumors and can therefore be recommended for clinical use.

Complications due to gallstones lost during laparoscopic cholecystectomy.

BACKGROUND: The aim of this study was to identify predisposing factors for complications after gallstone spillage during laparoscopic cholecystectomy (LC). METHODS: Papers derived from Medline search and papers from reference lists within these papers were studied. Ninety-one reports on complications caused by lost gallstones published between 1991 and 1998 were analyzed. These patients were compared with cases in published series on LC in general. RESULTS: Gallbladder perforation (20%) and stone spillage (9%) were the two most common complications of LC which occurred during the dissection (75%) and removal (25%) of the gallbladder. Predisposing factors for developing complications after stone spillage were: older age, male sex, acute cholecystitis, spillage of pigment stones, number of stones (>15) or size of the stone (Ø > 1.5 cm), and perihepatic localization of lost stones. CT-scan and ultrasound examination proved best for the recognition of complications caused by lost stones. Explorative laparotomy and surgical removal of the stones was the most frequently used therapy. CONCLUSIONS: Gallbladder perforation and stone spillage might cause hazardous complications. In cases with loss of numerous or large pigment stones which cannot be retrieved by laparoscopy, intraoperative conversion to open surgery can be justified.

Role of appendix and spleen in experimental colitis.

There is growing clinical evidence suggesting that certain secondary lymphoid tissues (e.g., appendix and spleen) contribute to the initiation and/or perpetuation of ulcerative colitis. In this study, the importance of secondary lymphoid tissues in inducing colitis was assessed experimentally by removing the spleen and/or appendix (or sham operation) prior to inducing colitis in mice. Feeding 2.5% dextran sulphate sodium (DSS) in drinking water over 7 days induced colitis. Clinical disease activity was assessed based on weight loss, stool consistency, and presence of blood in stools. Additional measurements included white blood cell count and hematocrit, and myeloperoxidase activity (MPO) in colon samples. Colonic injury was assessed by histology and computerized image analysis. DSS treatment in sham-operated mice produced colitis associated with weight loss, bloody diarrhea, and mucosal ulceration. Clinical assessment of DSS-treated mice subjected to appendectomy or combined appendectomy/splenectomy exhibited a delayed onset and course of disease activity. Histomorphologic examination revealed significantly lower damage scores and a reduction in ulcerated mucosal surface area. Colonic MPO activity, which correlated with tissue injury and disease activity, was lowest in appendectomized mice. No beneficial effects of splenectomy were observed after 7 days of colitis. These findings support the hypothesis that appendicular lymphoid tissue, but not the spleen, contributes to the development of colitis.

Disulfide-mediated dimerization of L1 Ig domains.

The neural cell adhesion molecule L1 contains immunoglobulin-like (Ig) domains in its extracellular region that mediate homophilic binding, neurite outgrowth and other activities relevant to CNS development. To correlate conformations of these domains to biological function, several L1-Fc fusion proteins whose bioactivities were previously characterized were analyzed by rotary shadowing electron microscopy. We found that bioactive L1-Fcs containing Ig domains 1-4 or 1-6 exhibited extended, branched structures. In contrast, inactive L1-Fcs containing only the first two or three Ig domains assumed compact shapes that suggested interactions between the L1 arms of these proteins. Analysis of an untagged L1 fragment composed of Ig domains 1-3 demonstrated a mixture of monomeric and dimeric forms. Surprisingly, these dimers were stabilized by intermolecular disulfide bonds. Finally, cell surface L1-GFP fusion proteins containing only the first two or three Ig domains in the extracellular region also engaged in disulfide-mediated dimerization. These results suggest a novel mechanism by which mutations in L1 could interfere with its biological functioning.

Contacts with fibrils containing collagen I, but not collagens II, IX, and XI, can destabilize the cartilage phenotype of chondrocytes.

OBJECTIVE: Cell-matrix interactions are important regulators of cellular functions, including matrix synthesis, proliferation and differentiation. This is well exemplified by the characteristically labile phenotype of chondrocytes that is lost in monolayer culture but is stabilized in suspension under appropriate conditions. We were interested in the role of collagen suprastructures in maintaining or destabilizing the cartilage phenotype of chondrocytes. DESIGN: Primary sternal chondrocytes from 17-day-old chick embryos were cultured in gels of fibrils reconstituted from soluble collagen I from various sources. The culture media either contained or lacked FBS. Cells were cultured for up to 28 days and the evolution of the phenotype of the cells was assessed by their collagen expression (collagens II and X for differentiated chondrocytes and hypertrophic chodrocytes, repectively; collagen I for phenotypically modulated cells), or by their secretion of alkaline phosphatase (hypertrophic cartilage phenotype). RESULTS: The cells often retained their differentiated phenotype only if cultured with serum. Under serum-free conditions, cartilage characteristics were lost. The cells acquired a fibroblast-like shape and, later, synthesized collagen I instead of cartilage collagens. Shape changes were influenced by beta1-integrin-activity, whereas other matrix receptors were important for alterations of collagen patterns. Heterotypic fibrils reconstituted from collagens II, IX, and XI did not provoke this phenotypic instability. CONCLUSIONS: Chondrocytes sensitively recognize the suprastructures of collagen fibrils in their environment. Cellular interactions with fibrils with appropriate molecular organizations, such as that in cartilage fibrils, result in the maintenance of the differentiated cartilage phenotype. However, other suprastructures, e.g. in reconstituted fibrils mainly containing collagen I, lead to cell-matrix interactions incompatible with the cartilage phenotype. The maintenance of the differentiated traits of chondrocytes is pivotal for the normal function of, e.g., articular cartilage. If pathologically altered matrix suprastructures lead to a dysregulation of collagen production also in vivo compromised cartilage functions inevitably will be propagated further.

Increased expression of metallothionein in inflammatory bowel disease.

OBJECTIVE: Metallothioneins (MT) are cytoprotective against the damaging effects of oxygen-derived free radicals. Therefore MT may be involved in defence mechanisms to counter Crohn's disease (CD) and ulcerative colitis (UC). MATERIALS: 107 routinely processed tissue samples from 22 patients with CD and 48 patients with UC were tested with the monoclonal anti-MT antibody E9. METHODS: Immunohistochemistry was used to assess MT staining in a semiquantitative manner. Chi-square test was used for statistical analysis. RESULTS: MT overexpression was found in the fibroblasts of all ulcerative and/or fissural lesions in UC and CD. MT overexpression in intestinal epithelial cells of 40% of UC and CD lesions correlated significantly with the grade of inflammation. CONCLUSIONS: MT-immunoreactivity in fibroblasts supports a protective role for MT in inflammatory bowel disease. It remains unclear whether MT overexpression in epithelial cells is also important in this protection.

[Role of FDG-PET in oncological surgery]. / Die Rolle der FDG-PET in der onkologischen Chirurgie.

Fluorine-18 fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) allows in vivo analysis of tissue metabolic activity. Based on the observation that most malignant tumors display a higher metabolic activity than benign tissues, [18F]FDG-PET offers an interesting option for the diagnosis of primary and recurrent malignant tumors. For the oncological surgeon [18F]FDG-PET is particularly helpful for the diagnosis of tumors of the pancreas, colorectum, lung and esophagus. This short review describes the biological basis of [18F]FDG-PET and gives a critical discussion of its role in oncological surgery.

Cell adhesion molecule L1 in folded (horseshoe) and extended conformations.

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.

Acetyl-11-keto-beta-boswellic acid, a constituent of a herbal medicine from Boswellia serrata resin, attenuates experimental ileitis.

The gum resin extract from Boswellia serrata (H15), an herbal product, was recently shown to have positive therapeutic effects in inflammatory bowel disease (IBD). However, the mechanisms and constituents responsible for these effects are poorly understood. This study examined the effect of the Boswellia extract and its single constituent acetyl-11-keto-beta-boswellic acid (AKBA) on leukocyte-endothelial cell interactions in an experimental model of IBD. Ileitis was induced by two subcutaneous injections of indomethacin (7.5 mg/kg) in Sprague-Dawley rats 24 h apart. Rats also received oral treatment with the Boswellia extract (H15) or AKBA at two different doses (low and high) equivalent to recommendations in human disease over 2 days. Controls received only the carriers NaHCO3 (subcutaneously) and tylose (orally). Effects of treatment were assessed by intravital microscopy in ileal submucosal venules for changes in the number of rolling and adherent leukocytes and by macroscopic and histological scoring. Increased leukocyte-endothelial cell adhesive interactions and severe tissue injury accompanied indomethacin-induced ileitis. Treatment with the Boswellia extract or AKBA resulted in a dose-dependent decrease in rolling (up to 90%) and adherent (up to 98%) leukocytes. High-dose Boswellia extract as well as both low- and high-dose AKBA significantly attenuated tissue injury scores. Oral therapy with the Boswellia extract or AKBA significantly reduces macroscopic and microcirculatory inflammatory features normally associated with indomethacin administration, indicating that the anti-inflammatory actions of the Boswellia extract in IBD may be due in part to boswellic acids such as AKBA.

Near-field optical microscopy based on microfabricated probes.

We demonstrate high resolution imaging with microfabricated, cantilevered probes, consisting of solid quartz tips on silicon levers. The tips are covered by a 60-nm thick layer of aluminium, which appears to be closed at the apex when investigated by transmission electron microscopy. An instrument specifically built for cantilever probes was used to record images of latex bead projection patterns in transmission as well as single molecule fluorescence. All images were recorded in constant height mode and show optical resolutions down to 32 nm.

Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-gamma) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells.

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll-Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-gamma and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-gamma-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-gamma+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial-lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD-even in an inactive state of disease-exert an increased capacity for IFN-gamma induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-gamma-producing CD8+ lymphocytes.

Fabrication and characterization of a silicon cantilever probe with an integrated quartz-glass (fused-silica) tip for scanning near-field optical microscopy.

A cantilever-based probe is introduced for use in scanning near-field optical microscopy (SNOM) combined with scanning atomic-force microscopy (AFM). The probes consist of silicon cantilevers with integrated 25-mum-high fused-silica tips. The probes are batch fabricated by microfabrication technology. Transmission electron microscopy reveals that the transparent quartz tips are completely covered with an opaque aluminum layer before the SNOM measurement. Static and dynamic AFM imaging was performed. SNOM imaging in transmission mode of single fluorescent molecules shows an optical resolution better than 32 nm.

Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo.

The pro-inflammatory cytokine interleukin (IL)-6 (refs. 1-5) can bind to cells lacking the IL-6 receptor (IL-6R) when it forms a complex with the soluble IL-6R (sIL-6R) (trans signaling). Here, we have assessed the contribution of this system to the increased resistance of mucosal T cells against apoptosis in Crohn disease (CD), a chronic inflammatory disease of the gastrointestinal tract. A neutralizing antibody against IL-6R suppressed established experimental colitis in various animal models of CD mediated by type 1 T-helper cells, by inducing apoptosis of lamina propria T cells. Similarly, specific neutralization of sIL-6R in vivo by a newly designed gp130-Fc fusion protein caused suppression of colitis activity and induction of apoptosis, indicating that sIL-6R prevents mucosal T-cell apoptosis. In patients with CD, mucosal T cells showed strong evidence for IL-6 trans signaling, with activation of signal transducer and activator of transcription 3, bcl-2 and bcl-xl. Blockade of IL-6 trans signaling caused T-cell apoptosis, indicating that the IL-6-sIL-6R system mediates the resistance of T cells to apoptosis in CD. These data indicate that a pathway of T-cell activation driven by IL-6-sIL-6R contributes to the perpetuation of chronic intestinal inflammation. Specific targeting of this pathway may be a promising new approach for the treatment of CD.

Microfabrication of a combined AFM-SNOM sensor

The objective of this work is to fabricate a scanning probe sensor that combines the well-established method for atomic force microscopy, employing a micro-machined Si cantilever and integrated tip, with a probe for the optical near field. A photosensitive pn-junction is integrated into the tip for that purpose and an Al coating is applied to the tip. It comprises an aperture of 50-70 nm in diameter at the apex of the tip in order to spatially limit the interaction of the tip to the optical near field of the sample. Characterization of the tip and first results of simultaneously recorded force and photon images are presented.