Downregulation of CX3CR1 ameliorates experimental colitis: evidence for CX3CL1-CX3CR1-mediated immune cell recruitment.

PURPOSE: Inflammatory conditions like inflammatory bowel diseases (IBD) are characterized by increased immune cell infiltration. The chemokine ligand CX3CL1 and its receptor CX3CR1 have been shown to be involved in leukocyte adhesion, transendothelial recruitment, and chemotaxis. Therefore, the objective of this study was to describe CX3CL1-CX3CR1-mediated signaling in the induction of immune cell recruitment during experimental murine colitis. METHODS: Acute colitis was induced by dextran sodium sulfate (DSS), and sepsis was induced by injection of lipopolysaccharide (LPS). Serum concentrations of CX3CR1 and CX3CL1 were measured by ELISA. Wild-type and CX3CR1-/- mice were challenged with DSS, and on day 6, intravital microscopy was performed to monitor colonic leukocyte and platelet recruitment. Intestinal inflammation was assessed by disease activity, histopathology, and neutrophil infiltration. RESULTS: CX3CR1 was upregulated in DSS colitis and LPS-induced sepsis. CX3CR1-/- mice were protected from disease severity and intestinal injury in DSS colitis, and CX3CR1 deficiency resulted in reduced rolling of leukocytes and platelets. CONCLUSIONS: In the present study, we provide evidence for a crucial role of CX3CL1-CX3CR1 in experimental colitis, in particular for intestinal leukocyte recruitment during murine colitis. Our findings suggest that CX3CR1 blockade represents a potential therapeutic strategy for treatment of IBD.

One-year-survey with multicenter data of more than 4,500 patients with degenerative rheumatic diseases treated with therapeutic nuclear magnetic resonance.

BACKGROUND AND OBJECTIVES: Nuclear magnetic resonance (NMR) has been shown to stimulate repair processes and cartilage and to influence pain signalling. It represents an alternative therapy for patients suffering from osteoarthritis (OA). To prove the clinical success of this new therapeutical method, validated measuring parameters are important that are convincing for pain and function in a one-year-follow-up. METHODS: During the course of its application over the last 10 years, over 4,500 protocols of a one-year-follow-up have been collected to record the outcome of NMR therapy. This report reflects the outcome of NMR therapy on patients with the following degenerative rheumatic diseases: OA of the knee (n = 2.770), OA of the hip (n = 673), OA of the ankle joint (n = 420) and chronic low back pain (n = 655). Data were collected at baseline, 6­8 weeks and 6 and 12 months following NMR treatment. RESULTS: Pain was reduced significantly 6 weeks after NMR treatment in the cases of all four examined indications and stayed measurably reduced up to 6 and 12 months. The improvements in all three forms of pain (pain on load, pain on motion, pain at rest) following NMR treatment were around 21­50% on average. CONCLUSIONS: Following therapy with NMR, patients with OA of all four types experienced a distinct improvement in their ability in functional parameters. Overall, the 10 years of a one-year-survey with multicenter data gathered on the effect of NMR therapy on patients verifiably proved its efficacy amongst patients with degenerative rheumatic diseases.

Gender and strain-specific differences in the development of steatosis in rats.

Non-alcoholic fatty liver disease (NAFLD) is a common problem with a wide variety of phenotypes. While its pathogenesis is still not fully understood, several risk factors for disease progression have been identified. Therefore, defining adequate animal models may serve to unreveal the pathogenesis in NAFLD. We studied Lewis and Sprague-Dawley rats of both genders (n = 6) fed standard (Std) or high-fat (HF) diet for three weeks. Disease stage was assessed by haematoxylin-eosin, Azan Heidenheim and Oil-Red staining, apoptosis by single-stranded DNA (ssDNA) detection and liver regeneration by Ki-67 staining. Serum markers of liver injury and lipid metabolism including adipocytokines were analysed. Livers of both strains and genders fed with HF diet demonstrated evidence of steatosis. Lewis rats developed microvesicular steatosis whereas Sprague-Dawley rats presented macrovesicular steatosis accompanied by pronounced fibrosis. Female gender of both strains was associated with lower steatosis grade and higher proliferation rate (P < 0.05). Gender-specific differences were most prominent in Lewis rats on a HF diet, where females showed lower alkaline phosphatase, cholesterol, triglyceride and leptin levels and a more favourable low-density lipoprotein/high-density lipoprotein ratio than males (P < 0.05). Reverse transcriptase-polymerase chain reaction analysis was performed to demonstrate changes in expression of various genes important for liver regeneration, fibrosis and steatosis. HF diet induced downregulation of proangiogenic genes such as vascular endothelial growth factor receptor 1 and 2 (P < 0.05) in males was not present in females. In conclusion, strain and gender served major roles in disease progression. These differences should be considered when designing studies and may offer new ways to advance therapeutic strategies.

Affinity purification of a framework 1 engineered mouse/human chimeric IgA2 antibody from tobacco.

Complex multimeric proteins such as dimeric and secretory immunoglobulin A (IgA) can be difficult to produce in heterologous systems, although this has been achieved using several platforms including plants. As well as topical mucosal applications, dimeric IgA (dIgA), and secretory IgA (sIgA) can be used in tumor and anti-viral therapy, where their more potent cell-killing properties may increase their efficacy compared to current drugs based on IgG. However, the development of therapeutic IgA formats is hampered by the need to co-express four different polypeptides, and the inability to purify such molecules using conventional protein A or protein G affinity chromatography. The light chain (LC)-specific affinity ligand protein L is a potential alternative, but it only recognizes certain kappa light chain (LC(κ)) subtypes. To overcome these limitations, we have adapted a framework-grafting approach to introduce LCs that bind protein L into any IgA. As a model, we used the chimeric anti-human chorionic gonadotropin (hCG) antibody cPIPP, since this contains a murine LC((κ)) subtype that does not bind protein L. Grafting was achieved by replacing selected framework region 1 (FR1) residues in the cPIPP LC(κ) variable domain with corresponding residues from LC(κ) subtypes that can bind protein L. The grafted antibody variants were successfully purified by protein L affinity chromatography. These modifications affected neither their antigen-binding properties nor the yields achieved by transient expression in tobacco plants. Our results therefore show that LC FR1 grafting can be used as generic strategy for the purification of IgA molecules.

Hepatic ischaemia-reperfusion injury from bench to bedside.

BACKGROUND: Vascular occlusion to prevent haemorrhage during liver resection causes ischaemia-reperfusion (IR) injury. Insights into the mechanisms of IR injury gathered from experimental models have contributed to the development of therapeutic approaches, some of which have already been tested in randomized clinical trials. METHODS: The review was based on a PubMed search using the terms 'ischemia AND hepatectomy', 'ischemia AND liver', 'hepatectomy AND drug treatment', 'liver AND intermittent clamping' and 'liver AND ischemic preconditioning'; only randomized controlled trials (RCTs) were included. RESULTS: Twelve RCTs reported on ischaemic preconditioning and intermittent clamping. Both strategies seem to confer protection and allow extension of ischaemia time. Fourteen RCTs evaluating pharmacological interventions, including antioxidants, anti-inflammatory drugs, vasodilators, pharmacological preconditioning and glucose infusion, were identified. CONCLUSION: Several strategies to prevent hepatic IR have been developed, but few have been incorporated into clinical practice. Although some pharmacological strategies showed promising results with improved clinical outcome there is not sufficient evidence to recommend them.

[Legally effective consent with minors and incompetent patients]. / Rechtswirksame Einwilligung bei minderjährigen und willensunfähigen Patienten.

Legal consent to medical treatment requires comprehensive clarification and the patient's capability to consent. Minors under 14 years are usually not capable of consent -- the right to decide rests with the parents. With persons over 14 years the doctor must test for capability to consent. With adults incapable of consent the court-appointed guardian decides. In acute cases the doctor may act first and obtain permission afterwards. Contractual capability is decisive for a treatment contract to be effective and the doctor's claim for remuneration. Minors up to 7 years are absolutely contractually incapable. Since minors under 18 years are only limitedly contractually capable, the approval of the statutory guardian suffices. With contractually incapable adults the court-appointed guardian or in serious cases the Guardianship Court decides. The legal position is explained, using three sample cases.

[Indications for surgery from the viewpoints of surgeon and juror]. / Die Indikation zur Operation aus chirurgischer und juristischer Sicht.

Whether an operation is indicated or not is a question that is a frequent subject of discussion between physicians and the legal front. As the state has the duty to protect its citizens, any physical surgical operation is legally seen as a personal injury. Only if the patient completely agrees to the surgery after being carefully informed about it is the element of criminal offense (personal injury) revoked. The obligation to disclose medical information on the surgery applies to information on the operation itself and on the possible consequences to the patient in his/her physical and mental social environment. In particular, the patient must be given all information about the risks that could arise during and after the surgery. The legislative aim of this is not to treat a list of questions and to mention all possible risks, but the legislator wants to oblige physicians to give patients who have reached the age of majority full information on diagnosis and therapy and to enable them to consider the pros and cons of the surgery carefully and then to agree to the operation or to refuse it. Besides the obligation to disclose medical information in emergency cases, the obligation to disclose medical information to minors also makes heavy demands on the physician. Examples of contraindications are given.

Experimental models of temporary normothermic liver ischemia.

Hepatic ischemia/reperfusion injury has so far been investigated in various experimental models. A clinical transfer of experimental results is, however, problematic because of anatomical and physiological differences and also the inevitable simplification of experimental work. The choice of model must therefore be adapted to the clinical question to be answered. The simplest procedure for inducing normothermic ischemia is to clamp the hepatoduodenal ligament. Models that do not avert portal congestion are regarded as unsuitable. Our current understanding of the pathogenesis of ischemia/reperfusion injury depends mainly on studies whose authors have investigated either global liver ischemia with a portocaval shunt, spleen transposition and in the isolated perfused system, or partial ischemia. This review is a critical examination of various approaches to the study of normothermic hepatic ischemia in experimental animals.

Four-dimensional imaging of transvacuolar strand dynamics in tobacco BY-2 cells.

The vacuole is a characteristic organelle of plant cells and fulfills several important functions related to metabolism and growth of the cell. To shed light on the details of vacuolar structural changes in plant cells, we explored the three-dimensional organization and dynamics of living Nicotiana tabacum L. cv. Bright Yellow 2 cell vacuoles by real-time confocal time-lapse imaging. For imaging, the cells were pulse-labeled with the amphipathic styryl dye FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), which is delivered to the plant vacuole by endocytic uptake and then incubated overnight. Imaging of the membrane-labeled vacuole revealed a complex vacuole morphology underlaid by constant remodeling. The vacuole is traversed by multiple transvacuolar strands which move along each other and fuse in multiple manners. New strands were created by fission of large membrane sheets. Endocytic vesicle trafficking was followed within the dynamic transvacuolar strands. The movement occurred in a stop-and-go fashion with an average vesicle velocity of 0.46 microm/s and a peak velocity of 0.82 microm/s. Transvacuolar-strand reduction and creation is a characteristic event observed during mitosis. Here we propose a mechanistic model for the alteration of the number of transvacuolar strands, on the basis of their fusion and fission.

Therapy and prognosis of tumors of the genitourinary tract after kidney transplantation.

There is an increased incidence of tumors of the genitourinary tract among kidney graft recipients. From 1979 to 2001, all patients who received kidney transplants had records of both their underlying diseases and their initial immunosuppression. Patients who developed a genitourinary tract malignancy were evaluated for tumor type, location, stage, tumor therapy and clinical course. During this period, 1804 patients underwent 2068 kidney transplantations. Thirty-four patients had 39 tumors of genitourinary origin. One patient was lost to follow-up. There were 15 patients with 18 renal cell carcinomas (one of them multifocal): six had seven transitional cell carcinomas; six, prostatic carcinoma; six, tumor of the female genital tract (one also had a renal cell carcinoma); and two, a seminoma. Most tumors were diagnosed in their early stages (< or = pT3, N0, M0; n = 31 tumors) and thus accessible to curative therapy, achieving good long-term results: 1- and 5-year survival rates of 100% and 91%, which were better than those obtained in advanced stages (N+, M+; n = 7 tumors), namely both 1- and 5-year survival rates of 38% (P < .05). Death was caused by tumor growth in nine patients (27%) and by other causes in three patients (9%). With appropriate treatment genitourinary tumors at early stage show a good prognosis. New immunosuppressants with supposed antiproliferative effects may help to decrease the incidence of malignancies. The most important factor is risk-adapted screening to identify malignancies early and to initiate appropriate therapy.

Acute liver failure: from bench to bedside.

Acute liver failure constitutes a challenge to clinicians and scientists alike. The course of the disease, usually unpredictable and polarizing, is associated with a high mortality unless liver transplantation is feasible, but can end in a spontaneous restitution. It poses many scientific questions regarding the mechanisms of liver cell damage and regeneration and the possibility of new therapeutic approaches. However, the performance of clinical studies in patients in acute liver failure presents problems because of the varied etiology, the small number of cases, and furthermore due to ethical and logistical difficulties. For this reason experimental investigations have gained a special importance. Arising from the improved understanding of the mechanisms of liver cell damage in acute liver failure, which may be primarily due not to the initial noxious agent alone but may also be triggered secondarily by the release of proinflammatory mediators, there are numerous options for liver cell protection, some of which have already proved successful in experimental studies. New insights into the mechanisms of regulation of liver regeneration and the physiological liver mass, gathered in particular from experimental models of partial hepatectomy and by the use of gene-manipulated animals, have contributed to the development of new therapeutic approaches for the stimulation of liver cell regeneration. Temporary liver support systems have already been successfully employed in some cases under clinical conditions. Although the systematic experimental investigation of many of the questions of acute liver failure has significantly contributed to a better understanding of liver cell damage and regeneration, the application of this new knowledge to clinical practice is to some extent made difficult by the artificial simplification that experimental studies inevitably entail and needs to be validated by controlled clinical studies.

Development and evaluation of an experimental model for investigating the pathogenesis and therapeutic strategies of acute liver failure.

Because of the various etiologies of acute liver failure (ALF) a clinically relevant model must fulfill four criteria--reversibility, reproducibility, ALF-induced death, and a sufficient time interval for diagnosis and therapy between induction and death. In this study an experimental model was evaluated for these criteria. A total of 49 rats were randomized into seven groups: First, a pilot study was performed regarding the survival rate after different treatments: In group I, animals underwent a 70% liver resection. In group II, 70% liver resection was combined with ascending doses of postoperative endotoxin administration up to 400 microg/kg (group IIc). In group III, animals only underwent liver mobilization. In group IV, ALF was induced according to the protocol of group IIc, but with additional treatment of an endothelin-A-receptor (ETAR) antagonist. Animals in group V received only 400 microg endotoxin. After induction of ALF, all animals died within the first day, showing significantly elevated bilirubin and ammonium levels and severe damage to hepatocellular integrity. Application of the ETAR antagonist resulted in the survival of 6/7 animals until the 14th day; the biochemical and histomorphological changes were reversible. All other animals survived to the 14th day. A clinically relevant model of ALF in rats can be created by the combination of 70% liver resection and endotoxin application to produce an inflammatory component.

[A new irrigation-suction boring system facilities low-pressure intramedullary boring of isolated swine femurs]. / Ein neues Spül-Saug-Bohrssystem ermöglicht die druckarme Markraumbohrung an isolierten Schweinefemora.

AIM: Intramedullary nailing is the treatment of choice for the stabilization of fractures of long tubular bones. An important disadvantage of this method is the increase in intramedullary pressure and the resulting release of fat into the venous blood system during reaming of the medullary canal. We have developed a new type of rinsing-suction-reamer (SSB) in order to minimize these disadvantages. Trials were initiated to investigate whether it is possible to ream the medullary canal with the SSB without pressure increase in comparison with the standard AO-reamer (AOB). METHODS: Reamed intramedullary nailing was performed in 20 isolated pig femora. The intramedullary pressure was recorded continuously. RESULTS: While stepwise reaming was performed, the pressure only rose above the physiological level in AOB. During insertion of the guide wire and the nail, comparable values were measured for AOB and SSB. CONCLUSION: Our experiments show that reaming of the medullary canal is possible without a pressure increase using the SSB in comparison with AOB.

[Reconstruction of bile duct lesions by an autologous vein graft and a bio-degradable endoluminal stent in an animal model: technique and clinical impact]. / Rekonstruktion von Gallengangsdefekten mit autologem Veneninterponat und resorbierbarem Stent im Tiermodell: Technik und klinische Bedeutung.

INTRODUCTION: In this study a new treatment of bile duct lesions was investigated. A segment of the bile duct was replaced by an autologous venous interponate which had been endoluminally stented with a braided bio-degradable stent. METHODS: A total of 18 pigs (20-28 kg) was divided into three equal groups (I-III). In each group a 2 cm segment of the jugular vein was harvested. The animals in Group I (vein group, n = 6) underwent resection of a 2 cm long segment of the common bile duct which was replaced solely by the venous interponate, in Group II (stent group, n = 6) the venous interponate had been endoluminally stented by a braided bio-degradable stent. Group III (control group, n = 6) underwent only a circular mobilization of the common bile duct. Postoperatively survival rate, general condition as well as the weight were observed and checked for 6 months. During surgery and finally after sacrifice after 6 months blood and tissue samples were taken and semiquantitatively scored concerning grade of inflammation and fibrosis. RESULTS: In the stent and control group all animals survived in good condition. 3 pigs of the vein group died within 3 weeks showing signs of biliary peritonitis, another one died due to a high grade stenosis of the common bile duct with secondary biliary cirrhosis after 4 months. In the stent group all animals survived until sacrifice after 6 months. On examination the venous interponate was laminated with bile duct epithelium showing the diameter of the implanted stent. CONCLUSION: The reconstruction of bile duct lesions by a venous interponate in combination with a bio-degradable stent is easy to perform and represents a clinically interesting alternative to the biliodigestive anastomosis because of the preservation of the sphincter oddi. After 6 months the stent is completely absorbed and the venous interponate is laminated with bile duct epithelium.

Achilles tendon healing: long-term biomechanical effects of postoperative mobilization and immobilization in a new mouse model.

The aim of the study was to investigate the long-term effects of postoperative immobilization as opposed to mobilization on the biomechanical attributes of healing Achilles tendons in a new experimental mouse model. In 114 Balb-C-mice the left Achilles tendon was transected and sutured by the Kirchmayr-Kessler technique. The tendons healed either under postoperative immobilization effected by fixing the upper ankle joint in equinus position or under mobilization through a limited range of movement. The contralateral Achilles tendons served as internal control. All tendons were tested biomechanically at short intervals up to the 112th postoperative day in terms of load to failure [N], tendon deflection [mm] and tendon stiffness [N/mm], and were evaluated histologically after 8 and 112 days. Postoperative mobilization resulted in a continuous and significantly more rapid restoration of load to failure in comparison to the immobilization group. Tendon deflection was decreased by postoperative mobilization, whereas under immobilization it paradoxically increased still further in the later course. After 112 days the tendons of the mobilization group had regained their original tendon stiffness, whereas the tendons after immobilization reached only about half the values seen in the control tendons. Histologically, postoperative mobilization led to increased immigration of inflammatory cells in the early phase. In the late phase, as compared to immobilization, tendon structure was more mature, with fibre bundles arranged in parallel and interposed tendocytes. Tensile loading of the healing tendon by postoperative mobilization leads to fundamental changes in the biological process of tendon healing resulting in accelerated restoration of load to failure and reduced tendon deflection.

ICAM-1 and VCAM-1 antisense oligonucleotides attenuate in vivo leucocyte adherence and inflammation in rat inflammatory bowel disease.

BACKGROUND: Recruitment of circulating cells to the inflamed intestine is modulated by adhesion molecules expressed on the surface of both leucocytes and endothelial cells. AIMS: The objective of this study was to test whether 2'-O-methoxyethyl chimeric antisense oligonucleotides directed against endothelial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) can downregulate leucocyte-endothelial interactions and thereby attenuate inflammation in rat experimental ileitis. METHODS: Indomethacin (7.5 mg/kg ) was injected subcutaneously into Sprague-Dawley rats 48 and 24 hours prior to intravital microscopy. Animals were treated with either ICAM-1 (ISIS 17470), VCAM-1 (ISIS 18155), or scrambled control antisense oligonucleotides administered subcutaneously or intravenously in parallel with indomethacin. Leucocyte trafficking was observed in ileal submucosal collecting venules. Macroscopic and histological grades of inflammation were measured 48 hours after the first indomethacin application. ICAM-1 and VCAM-1 expression in ileal submucosal venules was detected by immunohistochemistry. RESULTS: Intravenous administration of ICAM-1 oligonucleotides 2 mg/kg (rolling leucocytes 5.7 (2.4)/0.01 mm(2) endothelial surface, adherent leucocytes 0.8 (1.1)) and VCAM-1 oligonucleotides 8 mg/kg (9.2 (4.4), 0.6 (0.8)) significantly reduced leucocyte adhesion compared with diseased controls (27.8 (5.3), 14 (4.4)) in a dose dependent manner whereas subcutaneous treatment did not. Correspondingly, macroscopic and histological inflammation was significantly decreased. ICAM-1 oligonucleotides markedly reduced endothelial ICAM-1 expression while VCAM-1 oligonucleotides clearly diminished endothelial VCAM-1 expression. CONCLUSIONS: Both ICAM-1 and VCAM-1 2'-O-methoxyethyl chimeric antisense oligonucleotides attenuate rat ileitis by downregulation of leucocyte adherence and thus are potential candidates for anti-inflammatory treatment in inflammatory bowel disease.

Auxiliary partial orthotopic liver transplantation: treatment of acute liver failure in a new rat model.

BACKGROUND AND AIMS: Liver regeneration and functional interaction between native liver and graft after auxiliary partial orthotopic liver transplantation (APOLT) are not entirely understood and, therefore, require an experimental model simulating the clinical features of acute liver failure (ALF) and the surgical technique of APOLT. MATERIALS AND METHODS: ALF was induced by subtotal hepatectomy in 50 Lewis rats (200-250 g). Sham operation (I), ALF without treatment (II), ALF with portocaval shunt for decreasing blood flow of the remnant liver (III), and ALF treated by APOLT (IV) were performed. The auxiliary graft represented a left donor liver lobe which was orthotopically implanted using a microsurgical technique including reconstruction of the graft artery and internal biliary drainage. Operative outcomes, serum chemistry and histopathological findings were examined up to the 14th day. RESULTS: ALF without treatment (groups II and III) led to a small droplet fatty degeneration in the hepatocytes and a significant increase of liver parameters until the death of the animals within the first two postoperative days ( P<0.05). After APOLT (group IV), 80% of the animals survived up to the 14th day, revealing significantly decreased liver parameters ( P<0.05), a well-perfused graft and an up to five times increased native liver size with normal architecture. CONCLUSION: This new rat model simulates the clinical features of an ALF treated by APOLT and is especially interesting for further basic research on the interaction between native liver and auxiliary graft after APOLT.