The middle cranial fossa approach: an anatomical study

Hearing preservation surgery has become an option for an increasing number of patients with vestibular schwannomas due to diagnosis at an earlier stage. The middle cranial fossa approach represents one such surgical approach for resection of vestibular schwannomas with hearing preservation. We have undertaken an anatomical study of the middle cranial fossa approach to the internal auditory meatus using 20 fresh temporal bones. By simulating the surgical approach it was possible to analyze critically two of the main recognized subapproaches to the internal acoustic meatus. The results confirmed that the angle subtended by the facial nerve and "blue-lined" semicircular canal was much less than 60 degrees but equally important was the degree of individual variability. Furthermore the roof of the geniculate fossa was not infrequently dehiscent. The distance measured from the inner table of the craniotomy to the superior semicircular canal was on average 22 mm, similar to previous reports and utilized by some in their approach in this challenging surgery. From this anatomical study it appears that safe dissection of this area is facilitated by observing the more acute angle between the facial nerve and superior semicircular canal and by taking advantage of the relationship between the inner table and important landmarks.     

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Phorbol myristate acetate and calcium ionophore A23187-stimulated human T cells do not express high-affinity IL-2 receptors

Phorbol myristate acetage (PMA) and Ca2+ ionophore A23187 mimic early signal transduction pathways and activate purified human T cells to secrete large quantities of interleukin-2 (IL-2) and to proliferate. Despite producing 50-100-fold more IL-2 than phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL), PMA/A23187-stimulated human T cells proliferate less than cells activated by PHA. Washing the cells to remove PMA/A23187 was found to increase cellular proliferation two to five-fold. High-affinity IL-2R (HA-IL-2R) were found to be expressed by human T cells that had been washed 24 hr after PMA/A23187 stimulation and recultured without stimulus for an additional 48 hr, but not by T cells constantly exposed to PMA/A23187 for 72 hr. Radioligand binding studies with [125I]IL-2 demonstrated that while the alpha (p55) and beta (p70-75) subunits of HA-IL-2R were both present on the constant PMA/A23187-stimulated T cells, they did not appear to associate to form functional HA-IL-2R. This defect in the expression of bio-active HA-IL-2R on constant PMA/A23187-stimulated human T cells seems to account for their low proliferative response.     

Cholera toxin induces synthesis of phospholipase A2-activating protein.

The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated. CT caused rapid in vitro synthesis of prostaglandin E2 (PGE2) in murine smooth muscle-like cells (BC3H1), reaching maximal levels within 3 to 4 min. In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE2 synthesis. CT-induced PGE2 synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis. Northern blot analysis of total RNA from BC3H1 cells revealed that exposure to CT resulted in an increase in abundance of mRNA encoding phospholipase A2 (PLA2)-activating protein (PLAP). PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), which in turn causes increased hydrolysis of arachidonate from membrane phospholipids. Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithelial) cells in vitro, but the responses were more delayed than that of BC3H1 cells. A protein band of approximately 35 kDa, which corresponded to the size of PLAP, was observed in sodium dodecyl sulfate extracts of Caco-2 cells by Western blot (immunoblot) analysis using affinity-purified antibodies to PLAP synthetic peptides. Synthesis of PLAP protein was increased after 2 h of exposure to CT. Exposure of mouse intestinal loops to either CT or live Salmonella typhimurium for 3 h increased mucosal PLAP mRNA levels. The role of PLAP in CT-induced PGE2 synthesis provides an attractive explanation for the reported suppression of CT-induced intestinal secretion by inhibitors of protein synthesis.     

Reduction of adhesion formation in rabbits by intraperitoneal administration of lazaroid formulations

Adhesion formation is a major source of postoperative morbidity and mortality. In this study, the ability of a variety of lazaroid formulations [the antioxidant 21-aminosteroid PNU74006F (tirilazad) and the non-steroidal 2-methylaminochroman derivative PNU83,836E] to reduce i.p. adhesion formation in three rabbit models was examined. In initial studies, PNU83836E was administered via Alzet miniosmotic pump to the site of injury. In the sidewall and double uterine horn models, PNU83,836E was administered via Alzet miniosmotic pump for the entire postoperative interval. In the sidewall model, there was a dose- dependent reduction in the area of the sidewall injury that was involved in adhesions. In the double uterine horn model, PNU83,836E was administered via Alzet miniosmotic pump to the area of injury for 1, 2, 3 or 7 days. Administration for as little as 24 h after surgery significantly reduced the extent of adhesion formation and the reduction was increased if it was administered for longer. Further studies were conducted in which various lazaroid formulations were administered as a bolus at the end of surgery. In both the sidewall and double uterine horn models, administration of either PNU83,386E (in citrate buffer) or PNU74006F (in cyclodextrin or lipid emulsion vehicles) at the end of surgery reduced adhesion formation. Administration of a bolus of PNU74006F 10 min prior to initiation of surgery with or without additional treatment at the end of surgery further increased its efficacy in the reduction of adhesion formation. Administration of a minimum of 1.5 mg before and after surgery (3 mg total) was required for maximal efficacy. These studies demonstrate that pre- and postoperative administration of either a steroidal (PNU74006F) or non-steroidal (PNU83,836E) lazaroid intraperitoneally reduced the formation and reformation of postoperative adhesions in three animal models.