Sentinel node procedure of the sigmoid using indocyanine green: feasibility study in a goat model.

BACKGROUND: The sentinel lymph node (SLN) procedure alter the strategy for the treatment of patients with colon cancer. New techniques emerge that may provide the surgeon with a tool for accurate intraoperative detection of the SLNs. METHODS: An SLN procedure of the sigmoid was used in six goats. During laparoscopy, the near-infrared dye indocyanine green (ICG) was injected into the subserosa of the sigmoid via a percutaneously inserted needle during four experiments and in the submucosa during colonoscopy in two experiments. After injection, the near-infrared features of a newly developed laparoscope were used to detect the lymph vessels and SLNs. At the end of the procedure, 2 h after injection, all the goats were killed, and autopsy was performed. During postmortem laparotomy, the sigmoid was removed and used for confirmation of ICG node uptake. RESULTS: In all the procedures, the lymph vessels were easily detected by their bright fluorescent emission. In the first two experiments, no lymph nodes were detected. In the subsequent four experiments, human serum albumin was added to the ICG solution before injection to enable better lymph node entrapment. In all four experiments, at least one bright fluorescent lymph node was found after the lymph vessels had been tracked by their fluorescent guidance. The mean time between injection and SLN identification was 10 min. In two cases, the SLNs were located up to 5 mm into the fat tissue of the mesentery and were not seen by regular vision of the laparoscope. By switching on the near-infrared features of the scope, a clear bright dot became visible, which increased in intensity after opening of the mesentery. CONCLUSION: The SLN procedure for the sigmoid using near-infrared laparoscopy in the goat is a very promising technique. Achievements described in this report justify a clinical trial on the feasibility of ICG-guided SLN detection in humans.

The N-terminal segment of endothelin-converting enzyme (ECE)-1b contains a di-leucine motif that can redirect neprilysin to an intracellular compartment in Madin-Darby canine kidney (MDCK) cells.

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metallopeptidase of the neprilysin (NEP) family. ECE-1 is responsible for the conversion of inactive big-endothelins into active endothelins. Three different isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) have been identified. They differ in their N-terminal cytosolic regions, have distinct tissue distribution and intracellular localization. ECE-1a and ECE-1c are both located at the cell surface whereas ECE-1b is targeted to an intracellular compartment. To better understand the nature of the signal responsible for the targeting of ECE-1b to the intracellular compartment, we have constructed several ECE/NEP chimaeric proteins and expressed them by transfection into Madin-Darby canine kidney (MDCK) cells. This allowed us to identify a nine amino acid segment in the cytosolic tail of ECE-1b that is sufficient to relocate NEP from the cell surface to an intracellular compartment. Site-directed mutagenesis on these chimaeras led to the identification of two leucine residues as part of the intracellular retention signal.

Role of the glycosyl-phosphatidylinositol anchor in the intracellular transport of a transmembrane protein in Madin-Darby canine kidney cells.

In order to compare the trafficking of proteins with different membrane anchors, we have constructed and expressed three different recombinant forms of neutral endopeptidase (NEP) in MDCK cells. The wild type form of NEP (WT-NEP) is attached to the plasma membrane by a single N-terminal membrane spanning domain, whereas the glycosylphosphatidylinositol-anchored form of the protein (GPI-NEP) contains a C-terminal GPI anchor. A double anchored form of NEP (DA-NEP) was also constructed, that contains both the original N-terminal membrane spanning domain and a C-terminal GPI anchor. We show here that WT-NEP, GPI-NEP and DA-NEP, which are all apically targeted in MDCK cells, behave differently when subjected to Triton X-100 solubilisation: despite the presence of the transmembrane anchor DA-NEP behaves as a GPI-anchored protein. This suggests that the GPI anchor of DA-NEP is dominant over the transmembrane anchor of the native protein to determine its pattern of solubility in Triton X-100.