Conjugation of Fluorochromes to Monoclonal Antibodies.

Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is the most widely used application of flow cytometry. Here, we present protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, we provide a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation.

Dendritic cells Trigger IFN-γ secretion by NK cells independent of IL-12 and IL-18.

It is commonly believed that IL-12 produced by DCs in response to pathogens is the first signal that stimulates the production of IFN-γ by NK cells. However, IL-12 production by DCs in response to bacterial LPS depends on either engagement of CD40 by CD40L on activated T cells or IFN-γ from NK cells. This suggests that during the primary immune response, NK cells produce IFN-γ before IL-12 production by DCs.

Free IL-12p40 monomer is a polyfunctional adaptor for generating novel IL-12-like heterodimers extracellularly.

IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23, respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines.

Anti-IFN-gamma autoantibodies in disseminated nontuberculous mycobacterial infections.

Although many patients with disseminated nontuberculous mycobacterial disease have molecular defects in the IFN-gamma/IL-12 axis, recent case reports have shown autoantibodies against IFN-gamma associated with severe nontuberculous mycobacterial infections. To check this finding in an independent population, we screened 35 patients with either disseminated or pulmonary nontuberculous mycobacterial infections for whom no molecular defect was known. We identified high-titer-neutralizing anti-IFN-gamma IgG in the plasma of six patients.