Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

OBJECTIVE: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Liver transplantation for hepatocellular carcinoma.

Twenty-nine patients with hepatocellular carcinoma (HCC) underwent orthotopic liver transplantation (OLTx) at the University of Toronto. Four patients did not have cirrhosis. Of the 25 patients with cirrhosis, 19 had known or suspected HCC before OLTx. Eleven patients tested positive for the hepatitis B surface antigen (HBsAg). No patients received adjuvant chemotherapy. None of the patients have developed recurrent HCC in a follow-up of 9 to 87 months (mean: 33 months). The actuarial post-transplant survival of all patients at 3 months, 1 year and 3 years was 75%, 61%, and 46%, respectively. The survival of HBsAg-negative patients was 69% at 3 years, whereas HBsAg-negative patients had a 3-year survival of 18% (p = 0.045). These results suggest that OLTx for carefully selected patients with otherwise unresectable HCC is associated with a low risk of recurrence. HBsAg-positive patients with HCC have a high mortality, suggesting that they make poor candidates for OLTx.

Subglottic tracheal resection and synchronous laryngeal reconstruction.

Postintubation injury of the upper airway commonly results in stenotic lesions of the larynx, subglottis, and adjacent trachea. The traditional approach to surgical correction is laryngofissure for the laryngeal component and staged plastic reconstruction of the subglottic stenosis. Reported results are variable and unpredictable, and permanent extubation is impossible in a significant number of patients. We report experience with 15 patients with combined laryngeal, subglottic, and tracheal stenosis who were managed by a one-stage operation: circumferential resection of the subglottis and trachea with primary thyrotracheal anastomosis, combined with laryngofissure and laryngeal reconstruction. These procedures required the collaboration of the Departments of Otolaryngology and Thoracic Surgery of the Toronto General Hospital. Between 1972 and 1991, our thoracic surgical division did 53 circumferential subglottic tracheal resections with primary thyrotracheal anastomosis for benign disease. There were no operative deaths and 51 of 53 patients were successfully extubated. In 15 of these patients, a concomitant laryngofissure for laryngeal reconstruction was required. Laryngeal repair included excision or incision of interarytenoid scar (n = 13), interarytenoid mucosal graft (n = 6), or mobilization of cricoarytenoid joint (n = 3). A temporary laryngotracheal stent (usually a Montgomery T tube) was maintained after the operation in all cases (duration 3 to 42 months). Thirteen of these 15 patients are now permanently extubated and none has functionally significant restenosis. Vocal function is satisfactory to good in these patients. The approach described for these combined laryngotracheal lesions provides better results than those reported with traditional staged and plastic techniques of reconstruction. The collaboration of the departments of otolaryngology and thoracic surgery was essential to achieve these results.

Albumin absorption and protein secretion by the gallbladder in man and in the pig.

To study albumin absorption by the gallbladder in man, an in vitro model was first established in the pig and compared with in vivo function in the same species. Water and electrolyte transport and 125I-albumin absorption and protein secretion in vivo and in vitro were compared. Then similar in vitro studies were performed on human gallbladders obtained at surgery. The in vivo study in the pig was performed without disturbing the gallbladder except to tie a cannula in the cystic duct end. The in vitro model was identical in the pig and human gallbladders. Gallbladders were excised using a technique causing minimal injury and anoxia. They were oxygenated on both mucosal and serosal surfaces in a temperature-controlled environment. Luminal and external bath test solutions consisted of modified Ringers bicarbonate with added glucose; luminal solutions also contained 125I-albumin from different species, depending on the study. Active absorption of sodium and water occurred in both types of studies in the pig but in vivo absorption rates were considerably greater than in vitro rates. Albumin absorption in vivo was substantial; although present in vitro, the absorption of albumin was diminished relatively more than electrolyte transport rates. Protein secretion rates into the gallbladder were similar in vitro and in vivo. The results of studies in the human gallbladders in vitro were similar to the pig, except albumin absorption was greater. Some human gallbladders were obtained from control patients and some from patients with cholesterol gallstones. There were no significant differences between the two groups for any of the variables studied; however, the numbers were small and some control gallbladders were not normal gallbladders.

High protein and total lipid concentration are associated with reduced metastability of bile in an early stage of cholesterol gallstone formation.

Previous studies from this laboratory suggested that high gallbladder protein concentrations as well as excessive dehydration of bile might reduce the normal metastability of human gallbladder bile. This study attempted to identify persons in an early stage of stone formation, when there are crystals but no stones, and to determine the composition of bile under these conditions of reduced metastability. Two hundred twenty-seven patients were studied, 96 without gallstones. Twenty-three of 96 control patients had cholesterol crystals in their bile. Total protein concentration, total lipid concentration, and cholesterol saturation index were greater in control patients with crystals in bile. To determine whether or not cholesterol saturation index alone could account for the presence of crystals, control patients with cholesterol saturation index above the median value of 1.04 were studied. In this case there was no difference in cholesterol saturation index between the 19 crystal-positive (1.27) and 29 crystal-negative patients (1.26), but the difference in total protein and lipid concentrations persisted. Total protein and total lipid concentrations were even higher in crystal-positive sediments containing large numbers of crystals. Sludge seen by ultrasonography was more common in patients with crystal-positive sediments. High protein and lipid concentrations are associated with reduced metastability of bile.

Fluorometric assay of protein in native human bile.

Proteins in human native bile samples were determined by a fluorometric assay. Results were compared to biliary proteins quantified by the Lowry and Bradford techniques. The mean protein concentrations in bile as determined by the Lowry, Bradford and fluorometric assays were respectively 7.26 +/- 4.52 (SD), 2.9 +/- 1.42, and 2.12 +/- 1.28 mg/ml (n = 27). Bilirubin was shown to significantly interfere with the Lowry and Bradford assays but not the fluorometric assay. Bile salts remaining in the trichloroacetic acid (TCA) precipitate did not interfere with the fluorometric assay. No cholesterol or phospholipid could be detected in the TCA preparation prior to protein analysis. Proteolytic digestion of proteins in native bile was shown to occur at 37 degrees C and to a lesser extent at 22 degrees C. The fluorometric protein assay is an easy and accurate method to quantitate proteins in native human bile.

Total protein output during rapid reduction of bile salt secretion rates in man.

An investigation was undertaken to study the effect of bile salt secretion on total biliary protein secretion in man. Bile was collected in eight patients from a tube in the bile duct. Collection was started after a meal and continued for six hours, in order to obtain bile salt secretion rates over the entire physiological range. Total protein secretion rates did not vary with change in bile salt secretion or bile flow. The protein pattern assessed by SDS-PAGE did not vary with bile salt secretion. The results indicate that bile salt secretion has little influence on biliary protein secretion under these conditions in man. Changes in bile salt secretion were associated with linear change in bile flow, but there was no relationship between bile flow and protein secretion rates. This argues against convective sieving of plasma proteins into bile.