Laparoscopic ultrasonographic appearance of the common bile duct mucosa: a predictor of choledocholithiasis.

Previously we reported on the use of laparoscopic ultrasonography in detecting common bile duct stones during laparoscopic cholecystectomy. The aim of this study is to describe the laparoscopic ultrasonographic appearance of the common bile duct mucosa in patients with choledocholithiasis. Medical records of 44 patients with an increased risk for common bile duct stones undergoing laparoscopic cholecystectomy between 1993 and 1998 were reviewed. In the operating room, the laparoscopic ultrasonographic appearance of the common bile duct mucosa was scored in real time as normal, mild changes (hyperechoic mucosa), or severe changes (hyperechoic with mucosal thickening). Of the 31 patients (70%) with stones or sludge in the biliary tree, 29 (94%) had either severe (58%) or mild (36%) hyperechoic and 2 (6%) had normal-appearing common bile duct mucosa on laparoscopic ultrasonography. Of the 13 patients (30%) with no documented stones or sludge, 11 (85%) had normal and 2 (15%) had mild hyperechoic common bile duct mucosa on laparoscopic ultrasonography. Both of these patients had laboratory values indicating recent passage of common bile duct stones. The association between common bile duct stones and the presence of hyperechoic common bile duct mucosa was statistically significant (P < .0001, Fisher's exact test). This is the first report of hyperechoic common bile duct mucosa demonstrated by laparoscopic ultrasonography as a predictor of common bile duct stones. This finding is evident in the majority of patients with common bile duct stones and also may be associated with recent passage of a stone into the duodenum.

Use of CT Hounsfield unit density to identify ablated tumor after laparoscopic radiofrequency ablation of hepatic tumors.

BACKGROUND: When attempting to interpret CT scans after radiofrequency thermal ablation (RFA) of liver tumors, it is sometimes difficult to distinguish ablated from viable tumor tissue. Identification of the two types of tissue is specially problematic for lesions that are hypodense before ablation. The aim of this study was to determine whether quantitative Hounsfield unit (HU) density measurements can be used to document the lack of tumor perfusion and thereby identify ablated tissue. METHODS: Liver spiral CT scans of 13 patients with 51 lesions undergoing laparoscopic RFA for metastatic liver tumors within a 2-year time period were reviewed. HU density of the lesions as well as normal liver were measured pre- and postoperatively in each CT phase (noncontrast, arterial, portovenous). Statistical analyses were performed using Student's paired t-test and ANOVA. RESULTS: Normal liver parenchyma, which was used as a control, showed a similar increase with contrast injection in both pre- and postprocedure CT scans (56.4 +/- 2.4 vs 57.1 +/- 2.4 HU, respectively; p = 0.3). In contrast, ablated liver lesions showed a preablation increase of 45.7 +/- 3.4 HU but only a minimal postablation increase of 6.6 +/- 0.7 HU (p < 0.0001). This was true for highly vascular tumors (neuroendocrine) as well as hypovascular ones (adenocarcinoma). CONCLUSIONS: This is the first study to define quantitative radiological criteria using HU density for the evaluation of ablated tissues. A lack of increase in HU density with contrast injection indicates necrotic tissue, whereas perfused tissue shows an increase in HU density. This technique can be used in the evaluation of patients undergoing RFA.

Laparoscopic ultrasound vs triphasic computed tomography for detecting liver tumors.

BACKGROUND: Accurate staging of malignant tumors in the liver has major implications in defining prognosis and guiding both surgical and nonsurgical therapy. Intraoperative ultrasound in open surgery compares favorably with computed tomography (CT) in the detection of liver tumors; however, there is little experience with laparoscopic ultrasound (LUS). HYPOTHESIS: Laparoscopic ultrasound is more sensitive than triphasic CT for detecting primary and metastatic liver tumors. DESIGN: Prospective study. SETTING: University hospital. PATIENTS: Fifty-five patients with a total of 222 lesions, including primary and metastatic liver tumors, who underwent both CT examinations and LUS as a part of a tumor ablation procedure. INTERVENTIONS: Triphasic spiral CT scans of the liver were obtained within 1 week before surgery. Liver LUS was performed with a linear 7.5-MHz side-viewing laparoscopic transducer. RESULTS: The LUS detected all 201 tumors seen on preoperative CT and detected 21 additional tumors (9.5%) in 11 patients (20.0%). These tumors missed by CT ranged in size from 0.3 to 2.7 cm. Smaller tumors tended to be missed by CT scan (28.6% of the lesions <1 cm, 15.8% of those 1-2 cm, 4% of those 2-3 cm, and 0% of those >3 cm), as did those in segments III and IV. There was good correlation between the size of lesions imaged by the 2 modalities (Pearson r = 0.86; P<.001). CONCLUSION: Laparoscopic ultrasound offers increased sensitivity over CT for the detection of liver tumors, especially for smaller lesions. This study documents the ability of LUS in detecting liver tumors and argues for more widespread use in laparoscopic staging procedures.

Selective use of tube cholecystostomy with interval laparoscopic cholecystectomy in acute cholecystitis.

HYPOTHESIS: Tube cholecystostomy followed by interval laparoscopic cholecystectomy is a sale and efficacious treatment option in critically ill patients with acute cholecystitis. DESIGN: Retrospective cohort study within a 4 1/2%-year period. SETTING: University hospital. PATIENTS: Of 324 patients who underwent laparoscopic cholecystectomy, 65 (20%) had acute cholecystitis; 15 of these 65 patients (mean age, 75 years) underwent tube cholecystostomy. INTERVENTION: Thirteen patients at high risk for general anesthesia because of underlying medical conditions underwent percutaneous tube cholecystostomy with local anesthesia. Laparoscopic tube cholecystostomy was performed on 2 patients during attempted laparoscopic cholecystectomy because of severe inflammation. Interval laparoscopic cholecystectomy was attempted after an average of 12 weeks. MAIN OUTCOME MEASURES: Technical details and clinical outcome. RESULTS: Prompt clinical response was observed in 13 (87%) of the patients after tube cholecystostomy. Twelve patients (80%) underwent interval cholecystectomy. Laparoscopic cholecystectomy was attempted in 11 patients and was successful in 10 (91%), with 1 conversion to open cholecystectomy. One patient had interval open cholecystectomy during definitive operation for esophageal cancer and another had emergency open cholecystectomy due to tube dislodgment. Two patients (13%) had complications related to tube cholecystostomy and 2 patients died from sepsis before interval operation. One patient died from sepsis after combined esophagectomy and cholecystectomy. Postoperative minor complications developed in 2 patients. At a mean follow-up of 16.7 months (range, 0.5-53 months), all patients were free of biliary symptoms. CONCLUSIONS: Tube cholecystostomy allowed for resolution of sepsis and delay of definitive surgery in selected patients. Interval laparoscopic cholecystectomy was safely performed once sepsis and acute infection had resolved in this patient group at high risk for general anesthesia and conversion to open cholecystectomy. Just as catheter drainage of acute infection with interval appendectomy is accepted in patients with periappendiceal abscess, tube cholecystostomy with interval laparoscopic cholecystectomy should have a role in the management of selected patients with acute cholecystitis.

Laparoscopic management of a posterior mediastinal tumor mimicking an adrenal neoplasm.

BACKGROUND: Rarely, a posterior mediastinal mass may mimic an adrenal tumor on preoperative computed tomography scan. The intraoperative discovery that a mass thought to be associated with the adrenal gland actually is above the diaphragm in the posteroinferior mediastinum poses a challenge for the laparoscopic surgeon. Conversion to a thoracotomy or to videothoracoscopy incurs additional morbidity and risk for the patient. MATERIALS AND METHODS: We describe a technique for the transdiaphragmatic removal of a benign mass from the posterior mediastinum. A posterior mediastinal tumor was detected during a laparoscopic procedure for a suspected right adrenal tumor. Frozen section proved benign, and the mass was resected laparoscopically via transdiaphragmatic access to the posterior mediastinum. RESULTS: No complications were noted during or after surgery. The patient was ready for discharge from the hospital on postoperative day 1. CONCLUSIONS: Transdiaphragmatic resection was done successfully instead of conversion to a thoracotomy or thoracoscopic procedure for a benign posterior mediastinal tumor found incidentally during laparoscopic surgery for a presumed adrenal lesion. This transdiaphragmatic approach can be applied to selected benign mediastinal masses.

Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors.

We compared the desensitization of neurokinin1 and neurokinin2 (NK1 and NK2) receptors expressed in Chinese hamster ovary cells to substance P and neurokinin A, respectively. Substance P and neurokinin A stimulated a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) for both receptors, which was due to release of Ca2+ from intracellular stores. This was followed by a plateau in [Ca2+]i, which was due to influx of extracellular Ca2+, and was more sustained for the NK2 receptor. When Ca2+ was present in the extracellular solution, the Ca2+ response of the NK1 receptor, but not the NK2 receptor, rapidly desensitized and slowly resensitized to two exposures to agonist. In contrast, the [Ca2+]i response, measured in Ca2+-free solution, and inositol triphosphate generation desensitized and resensitized similarly for the NK1 and NK2 receptors. Thus, differences in desensitization between the NK1 receptor and the NK2 receptor may be related to differences in entry of extracellular Ca2+. We compared endocytosis of the NK1 and NK2 receptors to determine whether disparities could account for differences in desensitization. Fluorescent and radiolabeled substance P and neurokinin A were internalized similarly by cells expressing NK1 and NK2 receptors. Thus, disparities in internalization cannot account for differences in desensitization. We used inhibitors to examine the contribution of endocytosis, recycling, and phosphatases to desensitization and resensitization of the NK1 receptor. Desensitization did not require endocytosis. However, resensitization required endocytosis, recycling, and phosphatase activity. This suggests that the NK1 receptor desensitizes by phosphorylation and resensitizes by dephosphorylation in endosomes and recycling.

Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.

Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.

Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.

Direct observation of substance P-induced internalization of neurokinin 1 (NK1) receptors at sites of inflammation.

Substance P (SP) can cause plasma leakage at sites of inflammation by binding to neurokinin type 1 (NK1) receptors on the surface of endothelial cells. Internalization after ligand binding could reduce the number of NK1 receptors on the cell surface and thus participate in the desensitization and resensitization of the inflammatory response to SP. By using an antibody to the receptor, we directly observed SP-induced internalization of NK1 receptors into endosomes in endothelial cells of postcapillary venules in the rat tracheal mucosa. In the absence of SP, an average of 15 immunoreactive endosomes were present per endothelial cell. After an intravenous injection of SP, the number of immunoreactive endosomes peaked at 107 per cell at 3 min and gradually returned to the baseline by 120 min. In parallel experiments we observed that when cultured cells transfected with the NK1 receptor were exposed to rhodamine-SP and an antibody to an extracellular Flag epitope of the NK1 receptor, the SP was internalized with the receptor antibody. Both in the cultured cells and in the endothelial cells of intact animals, the prompt SP-induced internalization was accompanied by rapid, long-lasting desensitization to SP. These studies suggest that internalization of NK1 receptors by endothelial cells may be one of the mechanisms that limit the amount of plasma leakage at sites of inflammation.

M13-oriC cloning vehicles: use for amplification of high copy lethal (HCL) genetic elements.

M13 cloning vehicles have been constructed which contain the Escherichia coli origin for DNA replication (oriC), with and without selectable antibiotic-resistance genes. Since the M13 viral strand origin requires a functional rep gene product, using oriC these vehicles propagate as low-copy-number plasmids in E. coli rep mutants. This property is exploited to amplify cloned "high copy lethal" (HCL) DNA fragments, those containing genetic elements which kill the E. coli host when present at multiple copies in the cell. Following cloning of such fragments in these vehicles and initial selection in E. coli rep cells, the M13-oriC chimeric plasmid DNA is used to transfect appropriate E. coli rep+ cells. The chimeric DNA propagates as M13 viral DNA, yielding double-stranded and single-stranded DNA products and phage particles prior to killing of the host via expression of the HCL element; these events mimic a lytic phage infection. Such amplification will greatly facilitate both DNA "library" constructions (HCL elements are absent a priori from libraries using high-copy-number cloning vehicles) and studies of HCL elements including restriction mapping, DNA sequencing, and physiological studies.

Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.