The production of TNF/cachectin by human B cell lines and tonsillar B cells was examined. Of the 15 B cell lines examined, 9 cell lines synthesize TNF mRNA constitutively. PMA stimulated most cell lines to accumulate increased amounts of TNF. SeD, 8866P, 32al, RPMI 1788, and four bone marrow-derived EBV-transformed cell lines accumulated high levels of TNF mRNA when stimulated by PMA. TNF production by these cell lines was examined. RPMI 1788 and WIH8 produced little TNF constitutively, but synthesized 5-7 ng/ml TNF when stimulated by PMA. A pre-B cell line, Nalm-6, did not synthesize any detectable amount of TNF mRNA, even with PMA stimulation. Tonsillar B cells could also be stimulated to produce TNF. PMA or Staphylococcus aureus Cowan I strain (SAC) alone stimulated some TNF mRNA accumulation, whereas B cell growth factor (BCGF) or anti-mu did not. This accumulation was synergistically elevated by the combinations of PMA and SAC, or PMA and anti-mu. BCGF increased PMA-, SAC-, PMA plus SAC-, or PMA plus anti-mu-induced TNF mRNA accumulations about twofold. The accumulation of TNF mRNA in tonsillar B cells stimulated by PMA plus SAC was between 32 and 48 h, the same peak interval as the accumulation of TNF and IL-2 mRNA in tonsillar T cells. This is in contrast to PMA or PMA plus A23187-stimulated RPMI 1788 cells in which TNF mRNA accumulation was maximal at 1-2 h. TNF activities found in tonsillar B cell supernatants correlated with the TNF mRNA levels in the cells. However, more TNF activity was found on the second-day than the third-day supernatants, indicating active TNF uptake by the B cells. Cyclosporin A (CsA) inhibited SAC and anti-mu responses in B cells in much the same way as the anti-CD3 responses in T cells. SAC-, PMA plus SAC-, and PMA plus anti-mu-stimulated, but not PMA-stimulated, increases in TNF mRNA accumulations in tonsillar B cells were inhibited by CsA. TNF production seems to increase in parallel with B cell proliferation, but the relationship of these two functions needs to be further examined.

SummaryThe protein phosphatase 1 and 2A inhibitor, okadaic acid, has been shown to stimulate many cellular functions by increasing the phosphorylation state of phosphoproteins. In human monocytes, okadaic acid by itself stimulates tumor necrosis factor c~ (TNF-c~) mRNA accumulation and TNF-c~ synthesis. Calyculin A, a more potent inhibitor of phosphatase 1, has similar effects. TNF-ol mRNA accumulation in okadaic acid-treated monocytes is due to increased TNF-cr mRNA stability and transcription rate. The increase in TNF-c~ mRNA stability is more remarkable in okadaic acid-treated monocytes than the mRNA stability of other cytokines, such as interleukin lot (Iblc~), IL-lf3, and IL-6. Gel retardation studies show the stimulation of AP-1, AP-2, and NF-~cB binding activities in okadaic acid-stimulated monocytes. This increase may correlate with the increase in TNF-ot mRNA transcription rate. In addition to the stimulation of TNF-ot secretion by monocytes, okadaic acid appears to modulate TNF-c~ precursor processing, as indicated by a marked increase in the cell-associated 26-kD precursor. These results suggest that active basal phosphorylation/dephosphorylation occurs in monocytes, and that protein phosphatase 1 or 2A is important in regulating TNF-c~ gene transcription, translation, and posttranslational modification.

The tumor promoter PMA has been shown to induce accessory cell-independent T cell proliferation in conjunction with mAbs specific for CD3, CD28, and CD2 (1-6) . In these systems, PMA induces IL-2-R expression while the presence of these mAbs is synergistic for the maximal expression of IL-2-R and IL-2 production.Recently, we have identified the early activation antigen, EA 1, by the generation of mAb specific for PMA-activated T cells (7). EA 1 expression is seen as early as 30 min after the addition ofPMA. EA 1 expression induced by PMA and mitogens precedes that of IL-2-R . Our further studies (8) show that protein kinase C activation is the primary pathway for the induction of EA 1 expression and that calciumdependent pathways appear to have a secondary role . The EA 1 antigen is a series ofdisulfide-linked dimers (32 kD/32 kD, 32 kD/28 kD, and 28 kD/28 kD), with subunits ofan identical 24-kD core protein that is phosphorylated and differentially glycosylated . Recently, EA 1 has been shown to be expressed very early by fetal thymocytes during ontogeny (Jung, L . K. L., B. F. Haynes, S. Pahwa, and S. M. Fu, manuscript submitted for publication) .In the initial study (7), mAb EA 1 was shown not to inhibit or to stimulate T or B cell proliferation. Because of its early expression during T cell ontogeny and activation and its molecular structure, the possibility that EA 1 plays an important role in either cellular interaction or interaction with a yet-to-be-defined growth factor was suggested. To explore this further, mAbs were generated against affinity-purified EA 1. In this study, a new mAb against EA 1 was shown in collaboration with PMA to induce T cells to express IL-2-R, to secrete IL-2, and to proliferate. This process appeared to be independent of monocytes. Although this mAb by itself did not induce an increase in [Ca" ]i by PMA-treated T cells, crosslinking of EA 1 molecules caused a marked increase in [Ca2+ ];.

Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing.

The expression of lymphotoxin (LT)