Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant- induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium antagonist lanthanum chloride prevented xanthine- xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis.     

Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section

We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 A membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.     

Radiotherapy for benign disease; assessing the risk of radiation-induced cancer following exposure to intermediate dose radiation

Most radiotherapy (RT) involves the use of high doses (>50 Gy) to treat malignant disease. However, low to intermediate doses (approximately 3–50 Gy) can provide effective control of a number of benign conditions, ranging from inflammatory/proliferative disorders (e.g. Dupuytren''s disease, heterotopic ossification, keloid scarring, pigmented villonodular synovitis) to benign tumours (e.g. glomus tumours or juvenile nasopharyngeal angiofibromas). Current use in UK RT departments is very variable. This review identifies those benign diseases for which RT provides good control of symptoms with, for the most part, minimal side effects. However, exposure to radiation has the potential to cause a radiation-induced cancer (RIC) many years after treatment. The evidence for the magnitude of this risk comes from many disparate sources and is constrained by the small number of long-term studies in relevant clinical cohorts. This review considers the types of evidence available, i.e. theoretical models, phantom studies, epidemiological studies, long-term follow-up of cancer patients and those treated for benign disease, although many of the latter data pertain to treatments that are no longer used. Informative studies are summarized and considered in relation to the potential for development of a RIC in a range of key tissues (skin, brain etc.). Overall, the evidence suggests that the risks of cancer following RT for benign disease for currently advised protocols are small, especially in older patients. However, the balance of risk vs benefit needs to be considered in younger adults and especially if RT is being considered in adolescents or children.     

Dermatologic Surgical Research and the Institutional Review Board

BACKGROUND: Dermatologic surgical techniques have transformed cosmetic surgery over the last two decades. Many of the advances in cutaneous laser surgery, botulinum toxin injections, tumescent liposuction, and the latest filler agents have been evaluated on willing volunteers as part of organized trials under the auspices of an Institutional Review Board (IRB). OBJECTIVES: To review the developmental history of today's IRB and to provide relevance to cosmetic dermatologic research. RESULTS: Although an IRB must evaluate the "voluntariness" of a proposed research protocol, the risk-benefit ratio, particularly of elective cosmetic dermatologic surgical human studies, is of even greater importance. CONCLUSION: As more dermatologic surgeons become involved in clinical research, an understanding of the function and purpose of the IRB has become increasingly important. Research relating to cosmetic dermatologic procedures raises challenging and perplexing problems for an IRB.     

Thrombotic Micro‐Angiopathy with Sirolimus‐Based Immunosuppression: Potentiation of Calcineurin‐Inhibitor‐Induced Endothelial Damage?

Thrombotic microangiopathy is a rare but important finding in the context of organ transplantation. Acute renal insufficiency in the setting of hemolysis and thrombocytopenia, a triad that constitutes 'hemolytic uremic syndrome', can be associated with, or triggered by, conditions such as verocytotoxin-producing Escherichia coli, viral infections, malignant hypertension, scleroderma, allograft rejection, lupus erythematosus, pregnancy, and medications including mitomycin C, calcineurin inhibitors, and oral contraceptives. After renal transplantation, it can occur, as either a de novo episode, or recurrent disease. Calcineurin inhibitors have long been associated with post-transplantation thrombotic microangiopathy. Sirolimus has been used as a primary immunosuppressant in patients transplanted with a history of earlier hemolytic-uremic syndrome, and also as rescue therapy in patients with calcineurin-inhibitor-associated thrombotic microangiopathy. We describe four cases where there was significant thrombotic microangiopathy in the context of contemporaneous or contiguous calcineurin inhibitor and sirolimus usage. As the intrarenal cyclosporin concentration is thought to be significantly elevated when cyclosporin and sirolimus are used together, this may explain these findings, and mandates caution in their co-administration.     

Midterm evaluation of the Tissuemed (Aspire) porcine bioprosthesis: 493 patients, 506 bioprostheses

BACKGROUND: Valve durability has been a major concern with bioprostheses, and the Tissuemed (Aspire) porcine bioprosthesis was designed to provide a solution to structural valve failure. Because bioprostheses tend to fail by 8 years, the aim of our study was to determine its midterm durability and performance. METHODS: We reviewed 506 prostheses that were implanted in 493 patients (287 men; mean age 73 +/- 6 years) between 1991 and 1999. Preoperatively 316 (68%) patients were in New York Heart Association class III or IV. There were 417 (85%) aortic, 61 (12%) mitral, 13 (2.6%) aortic and mitral, and two (0.4%) tricuspid procedures. Concomitant procedures were performed in 163 (33%) patients. Follow-up was complete in 488 (98.9%) patients with a total cumulative follow-up of 1,402 patient-years. RESULTS: The 30-day mortality in this elderly population was 10% (95% confidence interval, 8 to 13), with no early valve-related deaths. Patients' survival at 8 years was 46% +/- 7%. This was influenced by the following factors: (1) the patient's age, being worse for those 70 years or older (p = 0.005); (2) those in New York Heart Association functional class III and IV (p = 0.004); (3) those in atrial fibrillation before the operation (p = 0.006); (4) those with poor left ventricular function (p = 0.009); and (5) those who had a previous cardiac operation (p = 0.003). Valve-related complications (expressed as percent per patient-year) were thromboembolism at 0.9%/patient-year; major hemorrhage at 1.4%/patient-year; bacterial endocarditis at 0.4%/patient-year; nonstructural dysfunction at 0.2%/patient-year, and reoperation at 0.2%/patient-year. At 8 years, freedom from thromboembolism was 93% +/- 7%, major hemorrhage, 90% +/- 4%, nonstructural dysfunction, 99% +/- 1%, structural valve failure, 100%, and reoperation, 99% +/- 1%. At follow-up, 98% of survivors were in New York Heart Association class I or II. CONCLUSIONS: Our study suggests that at 8 years, the Tissuemed (Aspire) porcine bioprosthesis is durable and has satisfactory performance with low complication rates.