Evidence for an oncogenic role of AHI-1 in Sezary syndrome, a leukemic variant of human cutaneous T-cell lymphomas.

Ahi-1 (Abelson helper integration site 1) is a novel gene frequently activated by provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in human leukemia cells, particularly in cutaneous T-cell lymphoma cell lines where increases in AHI-1 transcripts of 40-fold are seen. To test directly whether deregulated expression of AHI-1 contributes to their transformed properties, knockdown of AHI-1 expression in Hut78 cells, a cell line derived from a patient with Sezary syndrome (SS), was performed using retroviral-mediated RNA interference. Retroviral-mediated suppression specifically inhibited expression of AHI-1 and its isoforms in transduced cells by 80% and also reduced autocrine production of interleukin (IL)-2, IL-4 and tumor necrosis factor-alpha (TNFalpha) by up to 85%. It further significantly reduced their growth factor independence in vitro and the ability to produce tumors in immunodeficient mice. Interestingly, aberrant expression of AHI-1, particularly truncated isoforms, was present in CD4+CD7- Sezary cells from some patients with SS. Elevated expression of IL-2 and TNFalpha was also found in these cells. These findings provide strong evidence of the oncogenic activity of AHI-1 in human leukemogenesis and demonstrate that its deregulation may contribute to the development of SS.

Immunoassays at a quartz-liquid interface: theory, instrumentation and preliminary application to the fluorescent immunoassay of human immunoglobulin G.

The theoretical basis and instrumental requirements of an optical detection technique for monitoring antibody-antigen reactions at a quartz-liquid interface are described. The antibody is covalently immobilized on the optical surface of a planar, fused-quartz waveguide and reacted with antigen solution. A light beam is internally reflected within the waveguide and penetrates into the solution only a fraction of the wavelength of the incident light. This is the evanescent wave which interacts optically with the growing number of antigen-antibody complexes but minimally with the bulk solution. A two-site immunofluorescent assay for human IgG measurement is described using fluorescein as the label. The assay detection limit is approximately 0.8 micrograms/ml and individual fluorescence measurements are completed within 10 min. It is expected that this evanescent wave immunoassay should have wide applicability in both routine and research fields.

Optical detection of antibody-antigen reactions at a glass-liquid interface.

We describe an optical technique for detecting and monitoring antibody-antigen reactions at a solid-liquid interface. The antibody is covalently immobilized onto the surface of either a planar (microscope slide) or cylindrical (fibre optic) waveguide made of fused quartz. The reaction of immobilized antibody with antigen in solution is detected through use of the evanescent wave component of a light beam, which has a characteristic depth of penetration of a fraction of a wavelength into the aqueous phase, thus optically interacting primarily with substances bound (or located very close) to the interface and only minimally with the bulk solution. This resulting in-situ spatial separation of the antibody-bound from free antigen precludes a formal separation step and allows the reaction to be monitored kinetically. An immunoassay for methotrexate by absorption spectrometry achieved a detection limit of about 270 nmol/L; binding of methotrexate by immobilized antibody was monitored by the decrease in transmittance at 310 nm. A two-site immunofluorometric assay for human IgG could detect as little as 30 nmol/L; binding of fluorescein-labeled antibody was monitored by the increase in signal above 520 nm (lambda ex = 495 nm). With both immunoassays the signal-generating phase was monitored kinetically and was completed within 15 min.