A sensitive double-sandwich ELISA for neutrophil elastase. Assay results in unconcentrated bronchoalveolar lavage fluid.

We developed a sensitive double-sandwich ELISA assay for neutrophil elastase (NE) using affinity-purified NE antibody. The assay was capable of detecting NE levels of 0.2 ng/ml and was used to determine NE in bronchoalveolar lavages (BAL) of 12 healthy subjects (6 nonsmokers and 6 smokers) with a mean age of about 27 yr. NE levels in the unconcentrated cell-free supernatant of BAL, subjected to high-speed centrifugation (17,000 x g for 30 min) to sediment subcellular debris, were similar in the smokers who abstained overnight from smoking and in the nonsmokers (24.4 +/- 13.9 versus 23.7 +/- 12.3 ng/mg [SD] albumin). NE levels were significantly higher in lavage fluid not subjected to high-speed centrifugation, reflecting the presence of NE bound to subcellular debris that was sedimented by high-speed centrifugation. Concentration by ultrafiltration through a Millipore CX-10 filter was accompanied by loss of protein with a relatively greater loss of NE than albumin, resulting in lower NE/albumin ratios in concentrated than in unconcentrated lavage. It is therefore recommended that NE levels be determined on unconcentrated BAL after high-speed centrifugation to sediment subcellular debris.

Cells from newborn rat brain established in primary tissue culture will support rubella virus replication.

We have initiated an in vitro study comparing the susceptibility of newborn and adult animals to rubella virus (RV) associated encephalitis. Glial cells from injured adult rat brain (RG cells) have been established in continuous culture and these cells were reported to restrict RV replication. When RG cells were infected, no infectious progeny virus particles were detected in tissue culture media and only five intracellular viral polypeptides could be detected using immune precipitation techniques (p75, p60, VP44, VP41, and VP19). Two polypeptides normally associated with a productive infection. VP24 and p30, could not be detected. In this report we have applied these techniques to an investigation of RV replication in newborn brain cells and have shown that relatively normal yields of all seven polypeptides found in RV-infected cells could be detected. These data indicate that some glia from newborn brain in primary culture are permissive for RV replication unlike the restricting RG cells and that this difference in restriction is critical in determining the outcome of their infection by RV.

Differentiation of glioblasts from adult brain.

Rat glial (RG) cells have established in continuous culture using sections of kainic acid-lesioned rat striata as the primary cell source. Such cultures consistently provided at least 2 morphologically distinguishable cell types. More than 95% of this population was composed of large, flat cells which possessed ill-defined junctions and lacked cellular processes. The second class of cells comprised less than 5% of the population, was smaller and had several processes per cell. Dibutyryl cyclic-AMP (dB-cAMP), which has been shown to promote a maturation-stimulating effect on embryonic glioblasts in vitro, appears to have a similar effect on RG cells. In the presence of 1 mM dB-cAMP virtually all RG cells undergo morphological transformation, becoming smaller and denser, in addition to forming several processes per cell. Furthermore, two cell-type-specific markers could be detected in dB-cAMP-treated cultures: the surface oligodendrocyte-specific galactocerebroside (GC) moiety was demonstrated to be present on less than 10% of the cells, whereas the intracellular astrocyte-specific glial fibrillary acidic (GFA) protein could be readily detected in 80% of the population. These data indicate that glia derived from the striata of injured adult rat brain exhibit fetal characteristics.