Are free radicals responsible for endothelial cell killing of Staphylococcus aureus?

We have recently demonstrated that endothelial cells cultured on Gelfoam blocks, but not monolayer matrices can phagocytose and kill Staphylococcus aureus. Experiments determined that penicillin G, included in the endothelial cell growth medium, induces these cells to exhibit the observed bactericidal activity. In this communication, we report on studies aimed at elucidating the mechanism by which penicillin G-induced endothelial cells, cultured on Gelfoam blocks, kill S. aureus. Despite the fact that there is a substantial literature that demonstrates neutrophilic killing of bacteria can be mediated through free radical-dependent and free radical-independent mechanisms, considerably less is known about pathways by which endothelial cells can catalyze similar microbicidal activities. Studies described herein point to the fact that superoxide and products derived from this free radical were not responsible for endothelial killing of S. aureus. Likewise, a possible role for nitric oxide in bacterial killing was explored. As part of this inquiry, we stably transduced a NOS-2 encoding retrovirus into endothelial cells cultured on Gelfoam blocks in the absence of penicillin G. Even though these cells secreted nitric oxide at a rate of 0.5 microM/h per 1 x 10(6) cells, similar to what has been reported for murine macrophages induced with gamma-interferon, in our model, nitric oxide was not found to kill S. aureus. Data presented demonstrate that the microbicidal activity of endothelial cells is mediated through free radical-independent pathways.

Penicillin G-induced microbicidal activity of endothelial cells cultured on gelfoam blocks.

A body of evidence has surfaced documenting the ability of endothelial cells cultured on monolayers to phagocytose but not kill bacteria. Several years ago, a new three-dimensional endothelial cell culturing model was developed, which simulated the morphology of the endothelium in small vessels and capillaries. Given that endothelial cells may be derived from the same pluripotent stem cells as macrophages, the question of whether endothelial cells might phagocytose and kill bacteria was explored. Endothelial cells grown on Gelfoam blocks exhibited bactericidal activity towards Staphylococcus aureus, reaching maximal killing of > 90% after 2 h. Evidence documents the involvement of bacterial adherence to the plasma membrane of the endothelial cell. This is followed by phagocytosis of S. aureus, leading to intracellular killing. Penicillin G, included in the endothelial cell growth medium, was found to be a critical factor in the bactericidal activity demonstrated by Gelfoam blocks laden with endothelial cells.

Physiological tests to predict long-term outcome of total abdominal colectomy for intractable constipation.

OBJECTIVE: Total abdominal colectomy (TAC) for intractable constipation has a variable reported success rate that decreases to 50% beyond 2 yr. We hypothesize that this inconsistent outcome can be explained by a more extensive intestinal involvement in some patients. DESIGN: A consecutive sample of patients with intractable constipation had preoperative evaluations that included both upper and lower GI studies. Stool frequency, constipation, diarrhea, abdominal pain, and laxative or enema requirements were compared before and after operation. The study took place in an academic referral center and included 37 consecutive referred patients with severe intractable constipation and colonic dysmotility documented by radiopaque marker studies. INTERVENTIONS: TAC, with ileoproctostomy in 34 patients and ileostomy in three. MAIN OUTCOME MEASURES: Patients with motility abnormalities only of the lower GI tract were diagnosed as having colonic inertia (CI). Those with motility disorders of both the upper and the lower GI tracts were considered to have generalized intestinal dysmotility (GID) with colon predominance. RESULTS: Twenty-one patients had CI, and 16 had GID. Ninety percent of CI patients undergoing TAC had a successful outcome with a mean of 23 bowel movements (BMs)/wk at a mean follow-up of 7.5 yr. Although 88% of GID patients had initial improvement, with a mean of 19 BMs/wk at 6 months, only 13% had prolonged relief. After 2 yr, nine of the GID patients had recurrent constipation, and three had severe diarrhea. CONCLUSIONS: This study has identified two distinct types of colonic dysmotility, CI and GID. It has demonstrated the long-term success of TAC for CI and the importance of upper GI physiological studies to identify colon-predominant GID, which has a poor long-term response to TAC.

Culture of bovine pulmonary artery endothelial cells on Gelfoam blocks.

Conventional methods of endothelial cell culture on monolayers and beads require enzymatic digestion, traumatic scraping, or centrifugation to transfer cells to other experimental systems. Gelfoam, a porous gelatin block, not only supports the growth of bovine pulmonary artery endothelial cells but also allows the rapid transfer of cell-laden blocks from one experimental system to another with minimal intervention. This property has been shown to be especially useful for the rapid fixation of endothelial cells for microscopy using standard histologic methods. Histology confirmed that the trabecular nature of the substrate allows endothelial cells to line the interstices of the sponge matrix and grow in a configuration that simulates the appearance of the endothelium in small vessels and capillaries. The inoculation of 1 x 10(5) endothelial cells on 7.5 mg Gelfoam (24 x 8 x 2 mm blocks) was enhanced by fibroblast growth factor and resulted in cell attachment by day 2 with a cell doubling time of 1.7 days. In addition, endothelial cells completely infiltrated 1, 5 and 7.5 mg Gelfoam blocks, as verified by histology. Assays to quantify cell number and protein were easily performed. To facilitate cell counting, the Gelfoam matrix was rapidly removed by the addition of 0.05 mg/ml collagenase, a concentration that interfered minimally with the assay for cellular protein concentration. The data demonstrate that Gelfoam is a suitable support growth matrix for the in vitro culture of bovine pulmonary artery endothelial cells.

Transduodenal exploration of the common bile duct in patients with nondilated ducts.

Exploration of the small common bile duct can be technically difficult and is associated with a significant risk of ductal injury or late stricture, or both. Transduodenal common duct exploration after sphincteroplasty (TCDE/S) is an alternative method of duct exploration that avoids choledochotomy. Cholecystectomy followed by TCDE/S was performed upon 28 patients with nondilated ducts and suspected choledocholithiasis. Common duct stones were retrieved in 17 patients. Failure to retrieve stones in the remaining 11 patients was attributed to either false-positive results of cholangiography, forceful passage of stones into the duodenum during the initial insertion of a Fogarty catheter through the cystic duct or a false-negative finding at duct exploration. There was no perioperative mortality. Two patients had asymptomatic postoperative hyperamylasemia. One patient had postoperative pancreatitis, hyperbilirubinemia and cholangitis that resolved with antibiotic therapy by the eighth postoperative day. Other complications included wound infection, delayed gastric emptying, pneumonia and otitis media. The over-all morbidity rate was 28.6 per cent. Long term follow-up was obtained in all 28 patients. All patients in the follow-up group are free of recurrent biliary tract disease. TCDE/S appears to be a safe and effective method of exploring the nondilated common bile duct.

Nonoperative management of splenic injuries in adults: an alternative in selected patients.

Between November 1979 and September 1984, ten adults with documented traumatic injuries of the spleen were treated nonoperatively at The Johns Hopkins Medical Institutions. The mechanisms of injury varied from assaults with a blunt object to a low-velocity motor vehicle accident. Diagnosis was confirmed in all with computed tomography of the abdomen. Seven patients were successfully managed nonoperatively. The hospital course in these seven patients was uncomplicated with a mean hospital stay of 10 days. Three patients underwent splenectomy after failure of nonoperative management; two operations were performed semielectively and one as an emergent procedure. In the latter patient, the diagnosis of splenic rupture was made 7 days after the injury and was not suspected on initial presentation. The limited experience suggests that adults selected for nonoperative management should fulfill certain criteria that include 1) rapid hemodynamic stabilization after fluid resuscitation, 2) lack of other serious intra-abdominal injuries, 3) lack of extra-abdominal trauma that requires a prolonged general anesthetic or that results in an altered state of consciousness, and 4) progressive symptomatic improvement early during the hospitalization. Patients involved in high-speed motor vehicle accidents should not be considered as candidates because of the high prevalence of other serious injuries. From these guidelines, the results support the concept of nonoperative management of selected adults with splenic injury.

The primary localization of free radical generation after anoxia/reoxygenation in isolated endothelial cells.

While free radical-mediated reperfusion injury is clearly important in a variety of disparate organs, the particular cellular source of these radicals is unclear. To address this question, we subjected relatively pure (92% +/- 3% by factor VIII immunoassay) cultures of rat pulmonary artery endothelial cells to 0 to 45 minutes of anoxia (95% N2, 5% CO2), followed by reoxygenation (95% air, 5% CO2), to simulate ischemia/reperfusion. Cell injury was assayed after reoxygenation by the release of previously incorporated 51chromium and/or lactate dehydrogenase, and viability was determined by means of trypan blue exclusion. These three end points correlated closely. Without anoxia, the cells remained viable, with minimal evidence of injury for the entire experimental period, while 45 minutes of hypoxia followed by 30 minutes of reoxygenation produced substantial evidence of cell injury in 71% +/- 6% of the cells. This injury was reduced to 21% +/- 2% by treatment with the highly specific free radical scavengers superoxide dismutase and catalase together, either before anoxia or after anoxia, but just before reoxygenation. Similar protection was provided by xanthine oxidase inhibition with allopurinol. The injury was mimicked (without anoxia) by the exogenous generation of superoxide radicals with xanthine and xanthine oxidase. These experiments establish the essential components of free radical generation at reperfusion to be localized within the isolated endothelial cell in the absence of neutrophils or parenchymal cells.

Free-radical-mediated postischemic reperfusion injury in the kidney.

Acute tubular necrosis is a frequent occurrence following hypovolemic shock and human renal transplantation. Although this postischemic injury was originally thought to result from ischemia alone, it has recently been recognized that significant tissue injury can occur during the period of reperfusion. The demonstration of the oxygen free-radical-mediated postischemic reperfusion injury by Granger, Rutili, and McCord in ischemic cat intestine suggested that this mechanism might also be operative following renal ischemia. In the kidney, postischemic injury results in necrosis of the proximal renal tubule and accumulation of erythrocytes in the outer renal medulla. It has been proposed that the primary event leading to these pathologic changes is a free-radical-mediated injury to the endothelial cells in the inner stripe of the outer medulla. Experimental evidence in animals subjected to warm and cold ischemia supports a free-radical-mediated mechanism. The clinical significance of these findings is demonstrated in preclinical animal studies of renal transplantation in which approximately two-thirds of the injury following cold ischemia could be ablated by superoxide dismutase administered just prior to reperfusion or by allopurinol when administered both at the time of preservation and reperfusion or at the time of preservation alone.