No abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disorder. Systemic treatment of IBD patients with anti-tumor necrosis factor (TNF)-alpha antibodies has proven to be a highly promising approach, but several drawbacks remain, including side effects related to systemic administration and high cost of treatment. Lactococcus lactis was engineered to secrete monovalent and bivalent murine (m)TNF-neutralizing Nanobodies as therapeutic proteins. These therapeutic proteins are derived from fragments of heavy-chain camelid antibodies and are more stable than conventional antibodies. L. lactis-secreted anti-mTNF Nanobodies neutralized mTNF in vitro. Daily oral administration of Nanobody-secreting L. lactis resulted in local delivery of anti-mTNF Nanobodies at the colon and significantly reduced inflammation in mice with dextran sulfate sodium (DSS)-induced chronic colitis. In addition, this approach was also successful in improving established enterocolitis in interleukin 10 (IL10)(-/-) mice. Finally, L. lactis-secreted anti-mTNF Nanobodies did not interfere with systemic Salmonella infection in colitic IL10(-/-) mice.In conclusion, this report details a new therapeutic approach for treatment of chronic colitis, involving in situ secretion of anti-mTNF Nanobodies by orally administered L. lactis bacteria. Therapeutic application of these engineered bacteria could eventually lead to more effective and safer management of IBD in humans.

LLR significantly reduced time to oral intake, hospital stay, and incisional hernias compared to OS. Bleeding is a major risk and should be carefully considered when resecting benign tumors. In the hands of expert surgeons, LLR may become the gold standard for the resection of benign liver tumors located in the anterior and lateral sectors and for minor hepatic resections.

SummaryThis intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl 4 ) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and a-smooth muscle actin (a-SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl 4 and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl 4 mice, CV were enlarged, with marked sinusoidal-free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and a-SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl 4 and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.

Angiogenesis has recently been described as a component of inflammatory bowel disease. Placental growth factor (PlGF), a vascular endothelial growth factor (VEGF) homologue, establishes its angiogenic capacity under pathophysiological conditions. We investigated the function of PlGF in experimental models of acute colitis. Acute colonic damage was induced in PlGF knock-out (-/-) mice and PlGF wild-type (+/+) mice by dextran sodium sulfate (DSS) and trinitrobenzenesulfonic acid (TNBS). The concentrations of PlGF and VEGF were measured in distal colonic lysates using an enzyme-linked immunosorbent assay. Colonic injury was evaluated by assessing colon length, colonocyte apoptosis (by terminal dUTP nick-end labeling), colonic cytokine production and histological score. Infiltration of polymorphonuclear cells was determined by assaying myeloperoxidase (MPO) activity. In a separate experiment, recombinant PlGF was administered to PlGF -/- mice by adenoviral transfer before DSS administration. Mucosal vascularization was quantified by computerized morphometric analysis of CD31-stained distal colonic sections. Colonic mucosal hypoxia was visualized by pimonidazole staining. Both VEGF and PlGF were upregulated during acute colitis. In addition, compared with PlGF +/+ controls, PlGF -/- mice showed a significant increase in weight loss and colonic shortening during both DSS and TNBS colitis. This correlated with enhanced colonocyte apoptosis, elevated colonic cytokine levels and increased histological damage score, but not with enhanced inflammatory cell infiltration (MPO activity). The increased morbidity of PlGF -/- mice during DSS colitis was preventable by adenovirus (Ad)-mediated overexpression of PlGF. After the administration of DSS, strongly reduced mucosal angiogenesis was observed in PlGF -/- mice compared with PlGF +/+ mice. This was associated with an early increase in intestinal epithelial pimonidazole accumulation in PlGF -/- mice, suggesting a function of enhanced epithelial hypoxia in the observed differences between the two groups. In summary, our data show that the absence of PlGF strongly inhibits mucosal intestinal angiogenesis in acute colitis, which is associated with an early increase in intestinal epithelial hypoxia and aggravation of the course of the disease

BackgroundLaparoscopic left lateral sectionectomy (LLS) has gained popularity in its use for benign and malignant tumors. This report describes the evolution of the authors’ experience using laparoscopic LLS for different indications including living liver donation.MethodsBetween January 2004 and January 2009, 37 consecutive patients underwent laparoscopic LLS for benign, primary, and metastatic liver diseases, and for one case of living liver donation. Resection of malignant tumors was indicated for 19 (51%) of the 37 patients.ResultsAll but three patients (deceased due to metastatic cancer disease) are alive and well after a median follow-up period of 20 months (range, 8–46 months). Liver cell adenomas (72%) were the main indication among benign tumors, and colorectal liver metastases (84%) were the first indication of malignancy. One case of live liver donation was performed. Whereas 16 patients (43%) had undergone a previous abdominal surgery, 3 patients (8%) had LLS combined with bowel resection. The median operation time was of 195 min (range, 115–300 min), and the median blood loss was of 50 ml (range, 0–500 ml). Mild to severe steatosis was noted in 7 patients (19%) and aspecific portal inflammation in 11 patients (30%). A median free margin of 5 mm (range, 5–27 mm) was achieved for all cancer patients. The overall recurrence rate for colorectal liver metastases was of 44% (7 patients), but none recurred at the surgical margin. No conversion to laparotomy was recorded, and the overall morbidity rate was 8.1% (1 grade 1 and 2 grade 2 complications). The median hospital stay was 6 days (range, 2–10 days).ConclusionsLaparoscopic LLS without portal clamping can be performed safely for cases of benign and malignant liver disease with minimal blood loss and overall morbidity, free resection margins, and a favorable outcome. As the ultimate step of the learning curve, laparoscopic LLS could be routinely proposed, potentially increasing the donor pool for living-related liver transplantation.

Although portal venous supply is considered essential to preserve hepatic integrity, in this study, effects of portal arterialization on liver regeneration were evaluated in a rat model of partial hepatectomy (PH). Ninety-six Lewis rats were randomly assigned to four groups of 24 rats each: PH only (group 1), PH with either venous or arterialized portal supply (groups 2 and 3, respectively), and PH without portal supply (group 4). Liver regeneration rate (LRR), 5-bromo-2-deoxyuridine (BrdU) labeling index, and liver biological characteristics were assessed on days 1, 2, 3, and 7. Compared with group 1, all tested rats had a marked body weight loss after surgery, and only rats in group 4 showed no signs of recovery on day 7. With maintained portal inflow (groups 1, 2, and 3), LRRs increased steadily to day-7 values of 89.2% ؎ 11.8%, 81.4% ؎ 8%, and 77.4% ؎ 9.4%, respectively (P ‫؍‬ not significant), and 24-hour peak values of BrdU labeling index were 159 ؎ 26, 157 ؎ 42, and 149 ؎ 48, respectively (P ‫؍‬ not significant). Conversely, rats deprived of portal supply (group 4) showed profound inhibition of these two parameters (14 ؎ 13; P < .01; 32.1% ؎ 7.7%; P < .001, respectively). These results indicate that proper portal blood supply is essential to initiate and maintain liver regeneration after PH. With an equivalent portal inflow rate of either venous or arterial source, the hepatic regeneration response can be sustained. (Liver Transpl 2002;8:146-152.)

A Belgian ring trial for pan-TRK immunohistochemistry (IHC) staining was organised to harmonise pan-TRK IHC staining protocols and interpretation. As a reference method, the VENTANA pan-TRK Assay (clone EPR17341) on the Benchmark Ultra platform was selected. Six samples were selected: 2 negative, 2 fusion positive and 2 samples with wild-type endogenous TRK expression. Each participating laboratory stained the slides using their routine pan-TRK IHC and reported their results. In addition, they were asked to return one TRK-stained slide from each case. The coordinating lab evaluated these slides, compared them with the reference method and scored them. Two clones were used during the ring trial: A7H6R (Cell Signaling) and EPR17341 (Abcam/Ventana). Seven protocols achieved a sufficient performance mark, and three labs were advised to further optimise the protocol. Interpretation of pan-TRK IHC proved to be challenging in cases with physiological TRK expression. In addition, depending on the NTRK fusion partner, the staining can vary strongly in both intensity and staining pattern. Labs using the Ventana ready-to-use system based on the EPR17341 clone and using the recommended protocol settings scored best. However, given some small optimisation, all labs scored well on the technical staining and the succeeding evaluation.