Effect of pharmacologic blockade of the autonomic nervous system on electrophysiologic properties of the accessory pathway and atrioventricular node in patients with Wolff-Parkinson-White syndrome

In 17 patients aged 21-61 (mean 32) with WPW syndrome the transoseophageal stimulation was performed before and after intravenous administration of propranolol (0.1 mg/kg b.w.) and atropine (0.02 mg/kg b.w.). Pharmacological blockade of the autonomic nervous system resulted in statistically significant increase of heart rate (from 81 to 111/min), shortening of a-v nodal and atrial refraction (from 298 to 272 ms) as well as in shortening of stimuli cycle length revealing Wenckebach's point (from 324 to 291 ms). The Kent bundle refraction did not at the average change (333 and 324 ms), while in individuals great differences were observed. Generally, pharmacological blockade of the autonomic nervous system improves a-v nodal conduction, but in patients with WPW syndrome does not significantly effect on the accessory pathway.     

Absence of IFNA and IFNB genes from human malignant glioma cell lines and lack of correlation with cellular sensitivity to interferons

We report that 5 of 19 human malignant glioma cell lines have neither interferon alpha (IFNA) nor interferon beta (IFNB) genes that are detectable by Southern blotting. Of 5 other of these malignant glioma lines that have a single IFNB gene copy, 3 lack the IFNA genes entirely and two have one copy. One of the lines that lacks the IFNA genes entirely but has one copy of the IFNB gene has a rearrangement near the IFNB gene that is most easily interpreted as an insertion of a large segment of DNA (at least 50 kilobases) the 3' end of which is less than 1.3 kilobases 5' to the known regulatory sequences of the IFNB gene. In spite of the rearrangement, IFNB-specific RNA is highly inducible in this line by poly(I)-poly(C). The ability of interferon alpha or interferon beta to inhibit cell growth does not depend upon the presence or absence of the respective gene. This finding adds solid tumors to those tumor cell lines (acute lymphocytic leukemia, chronic myelogeneous leukemia) previously determined to lack the IFNA and IFNB genes (Diaz et al., Proc. Natl. Acad. Sci. USA, 85:5259-5263, 1988).     

Improved Technique for Establishing Short Term Human Brain Tumor Cultures

Culturing human central nervous system tumors has been difficult compared to other neoplasms. We report improved success rates for establishing short term human brain tumor cultures using a modified tissue processing technique. Eighty-seven brain tumor specimens (56 glioblastomas, 8 mid grade astrocytomas, 8 oligodendrogliomas, 15 other) were obtained from June 1988 to March 1997. The first twenty-three samples were processed by dissection, partial enzyme dissociation, and filtration through a tissue culture sieve. Subsequent samples were processed identically except tumor cells were centrifuged on a density gradient prior to plating. Successful cultures were defined as those surviving greater than three passages in tissue culture and growing to sufficient numbers (>10⁶ cells) to allow freezing. Success rate was 42% (10/23) using standard processing methods and 86% (55/64) with the addition of density gradient centrifugation. Glial fibrillary acidic protein (GFAP) and vimentin staining, karyotypes, and growth curves were obtained for representative glioma cultures. All cultures tested were positive for vimentin (29/29) while 62% (18/29) were positive for GFAP. Of four cultures karyotyped (two glioblastomas, two oligodendrogliomas), all but one oligodendroglioma culture exhibited clonal cytogenetic abnormalities. These immunohistochemical and karyotypic results are consistent with the malignant glial origin of these cells. Of note, low passage human glioma cultures grew slower and exhibited more contact inhibition than immortalized human glioblastoma cell lines. Nevertheless, this simple method for establishing short term human brain tumor cultures should aid in further developing human brain tumor pre-clinical models as well as enhancing clinical applications dependent on in vitro human brain tumor cell growth adjust.     

Gene Gun Transfection of Human Glioma and Melanoma Cell Lines with Genes Encoding Human IL-12 and GM-CSF

We used particle-mediated gene transfer by a custom-built gene gun to transfect two well-established human glioma (D54MG and U251) and melanoma (SK mel 28 and Ed 141) cell lines, as well as two glioma lines locally established from primary patient tumors (Ed 147 and Ed 149). Using -galactosidase as a reporter gene, D54MG, U251, Ed 141 and SK mel 28 showed an average transfection efficiency of 15–40%, whereas Ed 147 and Ed 149 had mean transfection efficiencies of 3% and 5% respectively. Twenty-four hours after transfection with the gene encoding human interleukin-12 (IL-12), ELISA was performed on cell supernatants (mean of n=12 for each cell line). IL-12 expression was extremely variable between the different cell lines, ranging from 52 to 1151pg/10⁶ cells/24h. Results were very similar when cells were exposed to 20,000rads of gamma irradiation 2h after transfection. When the cell lines were transfected with human granulocyte-macrophage colony-stimulating factor, 24h levels were: 13.0 (Ed 147), 17.8 (Ed 149), 18.6 (Ed 141), 27.4 (D54MG) and 27.7ng/10⁶ cells/24h (U251). SK mel 28 produced 88.1ng/10⁶ cells/24h. We conclude that the gene gun can efficiently transfect a variety of immortalized, well-established and locally-established glioma and melanoma cell lines. High dose gamma irradiation does not adversely affect the expression of the foreign gene (IL-12) at 24h. Significantly, transfected cell lines show different levels of expression depending on the particular gene/plasmid introduced. Therefore, each cell line has to be assessed individually for the level of expression of each introduced gene.