Rate of incorporation of radiolabelled nucleosides does not necessarily reflect the metabolic state of cells in culture: effects of latent mycoplasma contamination.

In response to cell-free conditioned medium derived from the human bladder carcinoma line T24 (T24 SN), we found greatly reduced incorporation of tritiated thymidine and uridine ([3H]TdR, [3H]UR) by the human carcinoma lines UCHNCu (small-cell lung carcinoma) and LS174T (colon carcinoma). The effect was not due to an excess of nucleosides or cytokines known to be present in T24 SN. Cell-cycle distribution, increase in cell numbers, and de novo nucleoside synthesis in the indicator cells were only slightly altered. This was in contrast to the gross reduction in [3H]TdR/[3H]UR incorporation and seemed to indicate selective downregulation of pyrimidine-salvage pathways, despite ongoing polynucleotide synthesis. Spontaneous [3H]TdR uptake remained low for several passages in vitro but was readily restored by pharmacological inhibition of de novo pathways with 5-fluoro-deoxy-uridine (5-FUdR). This suggested a stable but reversible regulatory effect of T24 SN on the pyrimidine metabolism of the indicator cells. Further investigation showed degradation of [3H]TdR by a particle-bound activity in T24 SN. Mycoplasma contamination of T24 had not been detectable using standard cultural and staining methods, but became apparent when T24-cell lysates were hybridized with a recently described DNA probe (Goebel and Stanbridge, 1984). We conclude that latent mycoplasma contamination can stimulate changes in cellular pyrimidine metabolism. Our results provide an example for latent mycoplasma infection mimicking metabolic changes in cultured cells by direct interference of a microbial enzyme with the assay system. We describe a rapid and simple bioassay to detect and distinguish particle-associated and soluble phosphorylase activity by [3H]TdR degradation. It may be a useful screening assay for mycoplasma contamination in tissue culture

B cell growth and differentiation induced by supernatants of transformed epithelial cell lines.

Growth and differentiation of B cells is thought to be regulated by soluble factors derived from T cells. However, human T cell lines and hybridomas have proved to be notoriously unreliable and unstable sources of such factors. We report here that three stable human bladder carcinoma cell lines T24, RT4 and 5637 produce, in a constitutive fashion, factors which promote growth and differentiation of human B cells

Effects of long-term oral treatment with leflunomide on allergic sensitization, lymphocyte activation, and airway inflammation in a rat model of asthma

Background Short-term treatment with leflunomide is effective in suppressing antigen-specific antibody production and allergen-induced bronchoconstriction after sensitization. This agent may thus have a role in future primary prevention strategies in allergic disease

Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: Covert responses are unmasked by serum-free medium

Background It is widely held that in vitro T cell responses to allergens are more prominent in atopic than in normal individuals, though this conclusion is based upon culture techniques which fail to detect proliferative responses in a significant minority of atopics and many normals