Louis Ercolani

Properties of cultured endothelium from adult human vessels.

Endothelium was isolated from samples of aorta and vena cava obtained from cadaver donors at the time kidneys were harvested for transplantation. Digestion with collagenase and gentle swabbing were used to free the cells from the Intlmal surface. Low density seeding permitted isolation of individual colonies with typical endothellal morphology. Modified Medium 199 supplemented with 10%-20% human plasma-derived serum and an extract from the bovine hypothalamus (500 μg/ml) enabled subcultured colonies to grow to confluency when culture surfaces were coated with fibronectin (1 μg/cm2). The presence of Factor VIII antigen was demonstrated using an indirect immunofluorescence technique. A monoclonal antibody to cultured umbilical vein endothelium, specific for endothelium, reacted with the subcultured cells from the aorta and vena cava. Type IV procollagen, flbronectln, and thrombospondln were identified as labeled proteins secreted by cultures of adult endothelium that had been Incubated with 3H-prollne and 3H-glycine. When the cultured endothelium was used In a sodlum-m-periodate stimulated T lymphocyte mltogenic culture system, the endothelium exhibited accessory cell function. Prostacyclln production stimulated by incubation with arachldonlc acid and PGH2 was variable from vessel to vessel. However, average values were lower than normally seen with cultured primary umbilical vein endothelium.

Rapid Communication Isolation and Characterization of the Promoter of the Human GABAA Receptor α1 Subunit Gene

Abstract: The GABAA receptor, as assessed by ligand binding and chloride flux measurement in vivo and in vitro, is down‐regulated in response to chronic benzodiazepine exposure. The mRNA levels of the α1 and γ2 subunits of the receptor are also reduced. We have isolated the promoter of the gene encoding the α1 subunit of the GABAA receptor to elucidate the regulatory mechanism of its expression. A DNA segment 650 bp long has been Isolated that includes 151 bp of untranslated 5’end of the cDNA sequence and 500 bp of potential promoter‐enhancer region. The transcriptional activity of this DNA segment linked to the firefly luciferase gene showed a strong orientation specificity. The promoter activity was localized to a 60‐bp segment by deletion mapping. Mobility shift binding assay results suggest that this segment may interact with one or more factors in HeLa cell nuclear extracts to form a transcriptional complex. Primary cultures of embryonic chick cortical cells transfected with the promoter‐luciferase construct were treated chronically with lorazepam. Transcriptional activity of this promoter construct was strongly repressed by chronic administration of lorazepam.

Insulin-induced Desensitization at the Receptor and Postreceptor Level in Mitogen-activated Human T-Lymphocytes

Human T-lymphocytes activated by phytohemagglutin acquire insulin receptors in culture. Saturation analysis of insulin-binding activity in the presence of competing ligand revealed curvilinear Scatchard plots. Insulin receptorswere not regulated by insulin before mitogen activation and culture of T-lymphocytes. However, insulin-induced downregulation of insulin receptors was: (1) demonstrable in receptor-positive cells, (2) dependent on insulin concentration, (3) temporally unrelated to insulin internalization, and (4) prevented by culture at 4°C but not by cycloheximide at 37°C. Recovery of insulin receptors required further culture of cells in media depleted of insulin for 24 h. Scatchard analysis revealed loss of receptor number without changes in receptor affinity. Insulin-induced increases in glucose transport and oxidation were demonstrable in receptor-positive cells but not in receptor-negative cells. However, these effects were extremely time-dependent. After a 2-h exposure of cells to 10−8 M insulin, increases in glucose transport were no longer demonstrable. Elution of bound insulin from these cells followed by re-exposure to insulin depressed glucose transport in them. Recovery from this hyporesponsive, desensitized state required a 6-h culture in insulin-depleted media. Glucose oxidation of desensitized cells couldbe stimulated by spermihe but not by insulin. These studies demonstrate the activated human T-lymphocyte is an insulinsensitive tissue that is capable of limiting its physiologic response to insulin by receptor- and postreceptor-mediated mechanisms.

Recurrent thrombotic microangiopathy in a renal allograft. Case report and review of the literature

Thrombotic microangiopathy in a renal allograft may either reflect a recurrence of the patients original disease, i.e., thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome, or more commonly may be a manifestation of allograft rejection. This report describes a patient in whom irreversible renal failure developed during thrombotic thrombocytopenic purpura. Two years later while her condition was in clinical remission, she received a 2 DR-matched cadaveric allograft. Nineteen days following transplantation, thrombotic microangiopathy developed in the graft with eventual loss of allograft function despite vigorous plasmapheresis therapy. Multiple factors in addition to possible recurrent disease that may have contributed to this event were identified. The literature on thrombotic microangiopathy and renal transplantation is reviewed.

Demonstration of receptors for insulin-like growth factor-II on human T-lymphocytes

Primary human T-lymphocytes that have been mitogen activated in chemically defined medium demonstrate cell surface receptor for insulin-like growth factor-II (IGF-II). In contrast resting T-lymphocytes demonstrate little or no IGF-II receptor. Receptors appear within 24 hours of mitogen activation with maximal binding occurring at 72 hours. After this point IGF-II binding declines. Receptor binding of IGF-II to T-lymphocytes does not show a sharp pH dependence but is maximal above pH 7. Insulin does not compete for IGF-II binding sites and proinsulin competes only weakly, suggesting that this is a type 2 IGF receptor and not an insulin receptor. Furthermore, anti-insulin antibodies do not inhibit IGF-II from binding to activated T-lymphocytes indicating divergent binding domains on the two peptide hormones. IGF-II demonstrates stimulating action on T-lymphocyte proliferation probably mediated by binding of IGF-II to this receptor.

Tunicamycin blocks the emergence and maintenance of insulin receptors on mitogen-activated human T lymphocytes☆

Treatment of phytohemagglutinin (PHA) activated human T lymphocytes with tunicamycin, an antibiotic that specifically inhibits asparagine-linked N-glycosylation of proteins, totally blocked the normal emergence of insulin receptors on these lymphocytes and their cellular proliferation during culture in a dose-dependent manner. Carbohydrate incorporation into protein was inhibited 82% by 0.5 microgram/mL while leucine incorporation was unaffected. Tunicamycin exposure of activated T lymphocytes, which had acquired insulin receptors during culture, reduced cellular insulin binding by 35% to 84% and reduced PHA binding to 40% of control levels within 24 hours. Scatchard analysis revealed decreases in insulin binding capacity but not affinity. Similar treatment with cycloheximide only decreased insulin binding by 12%. These findings suggest N-glycosylation of proteins is a necessary biochemical event (1) for the emergence and maintenance of insulin receptors on mitogen activated T lymphocytes, and (2) for mitogen activated T lymphocytes to undergo cell division.