Intact T cell receptor signaling by CD4(+) T cells cultured in the rotating wall-vessel bioreactor.

T lymphocytes fail to proliferate or secrete cytokines in response to T cell receptor (TCR) agonists during culture in spaceflight or ground-based microgravity analogs such as rotating wall-vessel (RWV) bioreactors. In RWVs, these responses can be rescued by co-stimulation with sub-mitogenic doses of the diacyl glycerol (DAG) mimetic phorbol myristate acetate. Based on this result we hypothesized that TCR activation is abrogated in the RWV due to impaired DAG signaling downstream of the TCR. To test this hypothesis we compared TCR-induced signal transduction by primary, human, CD4(+) T cells in RWV, and static culture. Surprisingly, we found little evidence of impaired DAG signaling in the RWV. Upstream of DAG, the tyrosine phosphorylation of several key components of the TCR-proximal signal was not affected by culture in the RWV. Similarly, the phosphorylation and compartmentalization of ERK and the degradation of IkappaB were unchanged by culture in the RWV indicating that RAS- and PKC-mediated signaling downstream of DAG are also unaffected by simulated microgravity. We conclude from these data that TCR signaling through DAG remains intact during culture in the RWV, and that the loss of functional T cell activation in this venue derives from the affect of simulated microgravity on cellular processes that are independent of the canonical TCR pathway.

Dynamic culture in a rotating-wall vessel bioreactor differentially inhibits murine T-lymphocyte activation by mitogenic stimuli upon return to static conditions in a time-dependent manner.

Depressed immune function is a well-documented effect of spaceflight. Both in-flight studies and ground-based studies using microgravity analogs, such as rotating wall vessel (RWV) bioreactors, have demonstrated that mitogen-stimulated T lymphocytes exhibit decreased proliferation, IL-2 secretion, and activation marker expression in true microgravity and the dynamic RWV-culture environment. This study investigates the kinetics of RWV-induced T lymphocyte inhibition by monitoring the ability of Balb/c mouse splenocytes to become activated under static culture conditions after concanavalin A (Con A) stimulation in an RWV. Splenocytes were stimulated with Con A and cultured for up to 24 h in the RWV before being allowed to "recover" under static culture conditions in the continued presence of Con A. The T-lymphocyte fraction of splenocytes was assayed during the recovery period for IL-2 secretion, expansion of the T-lymphocyte population, and expression of the activation marker CD25. Our results indicate that CD25 expression was not affected by any duration of RWV exposure. In contrast, proliferation and IL-2 secretion were inhibited by >8 and 12 h of exposure, respectively. Culture in the RWV for 24 h resulted in a near-complete loss of cellular viability during the recovery period, which was not seen in cells maintained in the RWV for 16 h or less. Taken together, these results indicate that for up to 8 h of RWV culture activation is not significantly impaired upon return to static conditions; longer duration RWV culture results in a gradual loss of activation during the recovery period most likely because of decreased T-cell viability and/or IL-2 production.

Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin.

A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.

Amatoxins and phallotoxins in Amanita species of the northeastern United States.

The amatoxin and phallotoxin content of some American specimens of green A. phalloides and white A. bisporigera, A. verna and A. virosa was determined. The analytical procedure consisted of extracting the toxins from dried mushroom tissue, defatting, fractionating the toxins by adsorption chromatography on Sephadex LH-20, desalting and running thin-layer chromatograms of appropriate fractions along with authentic toxin samples. One amatoxin, amanin, was identified by hydrolysis to its components amino acids. Except for some difference in relative amounts of toxins, American and European varieties of A. phalloides were quite similar. Neither phallotoxins nor amatoxins were present in three out of four collections of A. verna; the fourth contained only a trace of beta-amanitin. Amanin was the sole of amatoxin present in two out of four collections of A. virosa; alpha-amanitin was the chief amatoxin in the other two. None of the white Amanita species contained phallacidin. The taxonomy of the above species is discussed. A literature report that edible A. rubescens contains phallotixins was not confirmed.