Analysis of mechanisms involved in the prevention of gamma irradiation-induced apoptosis by hGM-CSF.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) induces proliferation and sustains viability of the mouse interleukin (IL)-3 dependent lymphoid cell line BA/F3 expressing the hGM-CSF receptor. Caspase-3 like enzyme activity and DNA fragmentation were augmented by depletion of this factor from the cell, and exposure to gamma irradiation accelerated kinetics of these events. Anti gamma irradiation-induced apoptosis occurred through various mutant GM-CSF receptors and only the box1 region was essential while the C terminal region, including tyrosine residues which are required for MAPK cascade activation, was dispensable. Consistent with this notion, the addition of PD98059 had no effect on this activity thereby indicating that activation of MAPK is not essential for the activity. As expected, gamma irradiation increased p53 protein and bax mRNA levels and the presence of hGM-CSF dramatically modulated bax/bcl-X(L) ratio. The PI-3K specific inhibitor wortmannin did not affect hGM-CSF dependent anti gamma irradiation induced apoptosis nor bcl-X(L) induction, thus bcl-X(L) but not PI-3K pathway seems to be involved in hGM-CSF dependent anti gamma irradiation-induced apoptosis. It is well documented that the boxl region is essential for GM-CSF dependent activation of JAK2 and JAK2 specific inhibitor AG490 suppressed anti gamma, irradiation-induced apoptosis by hGM-CSF. An artificial JAK2 activating molecule in which extracellular and the transmembrane of beta(c) fused with whole JAK2 can sustain BA/F3 cells survival and proliferation mIL-3 independently, but these cells are susceptible to gamma irradiation. Furthermore GyrB/Jak2, which can activate STAT5 but not the MAPK cascade nor survival of BA/F3 cells, also could not prevent gamma irradiation-induced apoptosis. Although JAK2 is essential for hGM-CSF dependent anti gamma irradiation-induced apoptosis, it appeared that JAK2 does not seem sufficient for the activity.

Activation and functional analysis of Janus kinase 2 in BA/F3 cells using the coumermycin/gyrase B system.

Janus kinase 2 (Jak2) protein tyrosine kinase plays an important role in interleukin-3- or granulocyte-macrophage colony-stimulating factor-mediated signal transduction pathways leading to cell proliferation, activation of early response genes, and inhibition of apoptosis. However, it is unclear whether Jak2 can activate these signaling pathways directly without the involvement of cytokine receptor phosphorylation. To investigate the specific role of Jak2 in the regulation of signal transduction pathways, we generated gyrase B (GyrB)-Jak2 fusion proteins, dimerized through the addition of coumermycin. Coumermycin induced autophosphorylation of GyrB-Jak2 fusion proteins, thus bypassing receptor activation. Using different types of chimeric Jak2 molecules, we observed that although the kinase domain of Jak2 is sufficient for autophosphorylation, the N-terminal regions are essential for the phosphorylation of Stat5 and for the induction of short-term cell proliferation. Moreover, coumermycin-induced activation of Jak2 can also lead to increased levels of c-myc and CIS mRNAs in BA/F3 cells stably expressing the Jak2 fusion protein with the intact N-terminal region. Conversely, activation of the chimeric Jak2 induced neither phosphorylation of Shc or SHP-2 nor activation of the c-fos promoter. Here, we showed that the GyrB-Jak2 system can serve as an excellent model to dissect signals of receptor-dependent and -independent events. We also obtained evidence indicating a role for the N-terminal region of Jak2 in downstream signaling events.

Monoclonal antibodies to the enterotoxin of Bacteroides fragilis: production, characterization, and immunodiagnostic application.

A monoclonal antibody, ICT11, specific for the toxin of enterotoxigenic Bacteroides fragilis (ETBF) neutralized the cytotoxic effect of the toxin on human colonic cell line HT-29/C1. In an evaluation using 115 diarrheal stool specimens and culture as the "gold standard," the assay showed a sensitivity of 85% and a specificity of 100%. An ICT11-based sandwich enzyme-linked immunosorbent assay showed a sensitivity of 100% and a specificity of 98% for direct detection of toxin from stool samples compared with those of culture. Thus, ICT11-based assays will be useful for screening for ETBF.

Production, characterization and immunodiagnostic application of a monoclonal antibody to Shiga toxin.

A mouse monoclonal antibody (MAb ICT7) that is specific for Shiga toxin was produced. The MAb neutralises the cytotoxic effects of both purified Shiga toxin and culture extracts of Shigella dysenteriae type 1 in HeLa cells. Using MAb ICT7 and polyclonal rabbit antiserum, a sandwich ELISA was developed. This test detects Shiga toxin in both S. dysenteriae type 1 bacterial extracts and in stools of patients with S. dysenteriae type 1 infection. The ELISA also detects toxin in enterohaemorrhagic Escherichia coli (EHEC) strains positive for Shiga-like toxin I. The test could detect a minimum of 100 pg of purified Shiga toxin. Furthermore, the ELISA did not detect toxin in non-S. dysenteriae type 1 Shigella species or Shiga-like toxin II produced by EHEC strains.