γ134.5-deleted HSV-1-expressing human cytomegalovirus IRS1 gene kills human glioblastoma cells as efficiently as wild-type HSV-1 in normoxia or hypoxia.

Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however, the efficacy of γ(1)34.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric human cytolomegalovirus (HCMV)/HSV-1 virus, which expresses the human CMV protein kinase R evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared with the γ(1)34.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia. Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

Metabolism and binding of benzo(a)pyrene and 2-acetylaminofluorene by short-term organ cultures of human and rat bladder.

The ability of organ cultures of normal human and rat bladder to metabolize the polycyclic hydrocarbon, benzo(a)pyrene (BP), and the arylamine, 2-acetylaminofluorene, has been studied. Cultures were maintained for 0 to 6 days in a chemically defined medium before incubation with [3H]BP (0.3 to 0.5 microM) or 2-[14C]acetylaminofluorene (18 to 25 microM) for 24 hr. Ethyl acetate-soluble and water-soluble metabolites were produced from both compounds by both species. The ethyl acetate extracts from [3H]BP-treated human cultures contained 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, and 3-hydroxybenzo(a)pyrene. Rat bladder cultures produced similar metabolites but in slightly different proportions. Ethyl acetate-soluble products of 2-[14C]acetylaminofluorene from human cultures contained 7-hydroxy-2-acetylaminofluorene, 9-hydroxy-2-acetylaminofluorene, 2-aminofluorene, and N-hydroxy-2-aminofluorene. Rat bladder cultures produced similar metabolites, but 2-aminofluorene was found in relatively higher proportion. Hydrolysis by beta-glucuronidase of the water-soluble products produced from both carcinogens gave ethyl acetate-extractable derivatives. These hydrolyzable glucuronide conjugates were relatively more abundant following metabolism of the carcinogens by the rat than by the human cultures. Covalent binding to DNA occurred with [3H]BP in both human (19.7 +/- 13 pmol/mg DNA) and rat cultures (22.8 +/- 8.6 pmol/mg DNA). As with other human tissues, considerable variation (50-fold) was observed between individuals. The results demonstrate that both human and rat bladder epithelium can metabolize known potent carcinogens and, in the case of BP, can effect covalent binding between the products of metabolism and the urothelial cell DNA. In theory, carcinogenesis in the urinary bladder could thus be initiated by carcinogens produced or excreted in the urine without the necessity for their prior metabolism elsewhere in the body.

Operative stabilization of nonpenetrating chest injuries.

Since 1958, a series of 112 patients with severe or moderately severe chest injuries have been treated. An aggressive policy has been adopted toward correcting or preventing major paradoxical chest wall movement by intramedullary pinning of ribs, costal cartilages, and the sternum. Whenever possible, positive-pressure mechanical ventilation and tracheostomy have been avoided. Fifty patients underwent stabilizing operations. The surgical approach was anterolateral in 12 (average 3.3 pins), posterolateral in 35 (average 6.8 pins), and midsternal in 3. Tracheostomy was performed in 8 of these 50 patients. Three died, on the first, third, and twenty-fifth days after injury. The tracheostomy was used only for aspiration of secretions in 3 others and for postoperative intermittent positive-pressure ventilation in 2 others. The duration of intermittent positive-pressure ventilation was 14 days and 1 day, respectively, Orotracheal intubation with positive-pressure mechanical ventilation after operation was required for more than a few hours in 3 patients, 1 of whom died. The 2 survivors were ventilated for 1 and 5 days. There was a total of 11 hospital deaths in these 50 cases. However, in 2 patients the severity of the initial injuries was thought to make death inevitable. Three of the patients who died were over 70 years of age. Operative stabilization permits avoidance or reduction in the duration of tracheostomy and mechanical ventilation. Permanent chest wall deformity is lessened or avoided.

Parathyroid hamartoma: report of two cases and review of the literature.

Parathyroid hamartomas are rare. Only four cases, two of which are associated with clinical hyperparathyroidism, have been reported in the literature. This is a report of two additional cases of functioning parathyroid hamartomas, one accompanied by proliferating fat and the other by a myxomatous, fibrillar stroma.

Sensitivity of routine blood bank methods for the detection of anti-D as determined during proficiency testing.

A proficiency testing program in immunohematology, involving over 240 laboratories, was used to assess the detection of anti-D in six concentrations ranging from 11 to 8,500 ng/mL. Using the indirect antiglobulin test, more than 98% of laboratories reporting detected anti-D at all concentrations. Enzyme and albumin antiglobulin methods as routinely practiced did not clearly increase sensitivity, and the direct agglutination methods used were much less sensitive than indirect antiglobulin methods. If proficiency testing truly reflects performance in practice in Ontario, Canada, the sensitivity of manual indirect antiglobulin methods in routine use for the detection of anti-D appears to meet reasonable expectations of these technics.

The non-covalent binding of benzo[a]pyrene and its hydroxylated metabolites to intracellular proteins and lipid bilayers.

The non-covalent interactions of benzo[a]pyrene (BP) and several of its hydroxylated metabolites with ligandin, aminoazodye-binding protein A (Z-protein, fatty acid binding protein) and lecithin bilayers have been studied by equilibrium dialysis, an adsorption technique and fluorescence spectroscopy. Binding affinities expressed as v/c (where v = moles of BP or BP metabolite bound per mole of protein or lipid and c = unbound concentration), were measured at concentrations sufficiently low that there was no self-association of the unbound compounds as judged by their fluorescence characteristics. 3-Hydroxybenzo[a]pyrene (BP-3-phenol), 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (BP-4,5-dihydrodiol) and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-dihydrodiol) bind more strongly (v/c = 10(5)-5 x 10(5) l x mol-1) to all three binders than does BP itself (v/c = 10(4)-7 x 10(4) l x mol-1). 9,10-Dihydro-9,10-dihydroxybenzo[a]pyrene (BP-9,10-dihydrodiol) binds to ligandin with an affinity similar to those of the other BP metabolites studied here, but binds much less strongly to both protein A and lecithin (v/c = 10(4) and 3 x 10(4) x mol-1, respectively). The low affinity of BP-9,10-dihydrodiol for lecithin would account for earlier findings that on incubation of BP with isolated rat hepatocytes, this metabolite egressed from the cells to the extracellular medium much more readily than either BP-4,5-dihydrodiol or BP-7,8-dihydrodiol. Calculations based on these results suggest that within hepatocytes BP and its metabolites, including BP-9,10-dihydrodiol, will be found almost exclusively associated (> 98%) with lipid membranes.