Measurement of Serum Bactericidal Activity Specific forHaemophilus influenzaeType b by Using a Chromogenic and Fluorescent Metabolic Indicator

Abstract

Degradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal ␤-subunits (␤ 1 , ␤ 2 , and ␤ 5 ) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2D b molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways.

Evaluation of Serum Bactericidal Antibody Assays forHaemophilus influenzaeSerotype a

“…The many similarities between H. influenzae type a and type b, including their epidemiology, capsular polysaccharides (type-specific or serotype antigens), and the pathology of the systemic infections they cause, are the basis for our SBA assay results can be characterized with viability, fluorometric, or colorimetric endpoints. Previous work in Hib demonstrated that the best correlation for Hib SBA assay was obtained between the viability count endpoint and the fluorometric endpoint for titer determination (15). Therefore, we chose to compare only SBA assay with viability colony counts and fluorometric endpoints and not to assess the colorimetric endpoint.…”

“…The complement source was sterile serum from 3-to 4-week-old baby rabbits (Pel-Freez, Brown Deer, WI) previously used in the Hib SBA (15). Although the concentration of cross-reactive antibodies was not measured in the complement lots, three different complement lots were qualified for use on both assays by testing active and heat-inactivated complements.…”

“…The alamarBlue buffer consisted of 16% alamarBlue, 64% Hanks buffer (containing Ca , and 2% Fildes enrichment), and 20% BHI broth (BBL). alamarBlue was found to be stable under SBA assay conditions and did not result in bacterial death in the absence of antibodies (15). After a 6-h incubation period at 37°C in 5% CO 2 , assay plates were read in a fluorometer (model FL 600 synergy; BIO-TEK Instruments Inc., Winooski, VT).…”

“…SBA assays (with a viability endpoint [CFU counts] and with a fluorometric endpoint) were performed as described by Romero-Steiner et al (15) for Hib, which is a modification of the method described by Schlesinger and Granoff (17).…”

Time-suppression test using a colorimetric probe (alamarBlue) that measures bacterial metabolic activity

“…Since the reduced AlaB is stable and photometrically distinguishable from unreduced AlaB, cell viability can be assessed by photometry. Some investigators have applied this feature of AlaB to bacterial drug-susceptibility testing (2,3,8). Here we report its application to antibacterial time-suppression testing for estimating the mode of antibacterial action.…”