A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function.

Studies of brain cell function and physiology are hampered by the limited availability of immortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. In this study, we demonstrate the application of recombinant DNA vectors expressing SV40 T antigen for the development of immortal human cell cultures, with morphological, growth, and functional properties of astrocytes. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. One of these cultures gave rise to an immortal cell line, designated A735. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Immunohistochemical studies and Western blot analysis of the intermediate filament proteins and glutamine synthetase demonstrated a differentiated but immature astrocyte phenotype. Transport of gamma-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines' retaining aspects of normal glial function. We conclude that methods based on the use of SV40 T antigen can successfully immortalize human astrocytes, retaining key astrocyte functions, but T antigen-induced proliferation appeared to interfere with expression of glial fibrillary acidic protein. We believe A735 is the first documented nontumor-derived human glial cell line which is immortal.

Mutation of p53 is not a prerequisite for immortalization of human fibroblasts by SV40 T antigen.

The in vitro life span of human cells is under genetic control and limited. Immortalized cells, however, can be obtained at a low frequency following expression of the SV40 T antigen gene though the steps that lead to immortality are not well understood. p53 has been implicated in cell cycle regulation and evidence suggests it may have a role in controlling life span in rodent and human cells. In this study, we investigated whether allelic loss or mutation of p53 was an essential step during SV40 immortalization leading to the appearance of immortal cell lines. The gross structure of the p53 gene was examined in a primary fibroblast cell strain (1BR.3) and two SV40-immortalized derivatives, 1BRMT1 and 1BRgn2. There was no evidence for allelic loss of the p53 gene during immortalization. The primary cells and the immortal derivatives all expressed authentic p53 mRNAs, though the immortal cell lines had higher levels of expression. Sequence analysis of exons 5-8 did not detect mutations associated with the immortal phenotype. These data are consistent with SV40 immortalization being independent of genetic changes in p53.

Cell cycle progression, morphology and contact inhibition are regulated by the amount of SV40 T antigen in immortal human cells.

Expression of Simian Virus 40 (SV40) T antigen in human dermal fibroblasts over-rides the normal controls on cell division leading to changes in cellular proliferation and life span. These changes are accompanied by other changes in cell morphology, expression of cell specific functions, and altered cell-cell interactions. In this study, we have examined the effects of different amounts of T antigen on cell cycle progression, life span and morphology in human dermal fibroblasts and demonstrated T antigen to be a concentration dependent regulator of the cell cycle. Using a novel, metal inducible episomal expression vector (p735.6) which produces low basal levels of protein but high (greater than 100-fold) levels of induction, we have compared the effects of low and high levels of T antigen expression in a precrisis and immortalised human line (1BRMT1). The presence of inducing agent led to maximal levels of T antigen expression and resulted in cultures with a high rate of proliferation, an extended in vitro life span, a loss of contact inhibition of growth and a morphology characteristic of SV40-transformed cells. In the absence of inducing agent, read-through of the T antigen gene resulted in low but detectable levels of protein. The reduction in T antigen levels was accompanied by a 50% or greater reduction in the proliferative rate and restoration of cell morphology and contact inhibition similar to that found in non-transfected cells. The results presented here demonstrate that low amounts of T antigen are sufficient to maintain cell viability and prevent the re-expression of the senescent phenotype seen in the absence of T antigen. Similarly, the ability of T antigen to extend the in vitro life span is not dependent on high level expression of T antigen. In contrast, the rate of proliferation of human cells as well as the cell morphology and contact inhibition are dependent on the amount of T antigen present. Many of the cellular effects can be minimised or reversed by reducing T antigen expression.

Laboratory diagnosis of peritonitis in patients on continuous ambulatory peritoneal dialysis.

The clinical course and laboratory diagnosis of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis was studied in 32 consecutive episodes. Peritonitis was associated with a failure in aseptic technique in eight episodes and with an exit-site infection in four episodes. Intraperitoneal vancomycin and ceftazidime were safe, effective, and convenient. Most patients administered their antibiotics at home, and symptoms usually resolved by day 4. Culture of the deposit obtained by centrifugation of 50 ml of effluent after leukocyte lysis provided the best rate of recovery (84% culture positive) but was technically demanding. Filtration of the same volume without leukocyte lysis was simple to perform and almost as effective. Enrichment was less satisfactory (65% culture positive) owing to the presence of antibiotic or infection with fastidious microorganisms. Culture of 50 ml of effluent after concentration by a commonly used laboratory technique, centrifugation without leukocyte lysis, performed poorly (59% culture positive at 48 h), as this method caused sequestration and death of microorganisms within the leukocytes. Culture of nearly 1 liter of effluent from 33 asymptomatic patients by the same techniques yielded no microorganisms.