Development of pulmonary infection in mice inoculated with Blastomyces dermatitidis conidia.

Intratracheal injection of Balb/cByJ mice with 10(4) Blastomyces dermatitidis conida produces chronic pulmonary and disseminated blastomycosis characterized by pyogranulomatous inflammation. To study the evolution of the pulmonary infection, mice were killed at varying intervals after inoculation, their lungs cultured and examined histologically. Nodular intraalveolar infiltrates of macrophages (M phi) were seen on Day 1 with occasional admixed polymorphonuclear leukocytes (PMN). Phagocytized yeast forms within M phi were evident by Day 5. By Day 28 pyogranulomas, which developed first as central microabscesses associated with a peripheral zone of M phi and giant cells containing internalized yeast, were a prominent feature of the infection. Lymphocytic and plasmacytic infiltrates, accumulating next to granulomas, formed the major peripheral component of the granuloma by Day 35. Formation of pyogranulomas was coincident with the host's failure to contain fungal growth measured by the sharp rise in colony-forming units recovered from lungs. Antibody against B. dermatitidis was first detected at Day 35 by enzyme immunoassay, but not until Day 63 by double immunodiffusion. During the 4 wk after inoculation, pulmonary lavage fluid contained > 90% M phi and < 3% PMN. On day 28, PMN rose to 17%, reaching 40% on Day 42. These data contribute to our knowledge of this model and help form the basis for investigations into the roles of fungal pathogenic and host defense mechanisms in blastomycosis.

Decline in meningococcal antibody levels in African children 5 years after vaccination and the lack of an effect of booster immunization.

Antibodies to group A meningococcal polysaccharide were measured by hemagglutination (HA) and by ELISA in sera obtained from Gambian children before vaccination and 3 weeks, 2 years, and 5 years after vaccination with a group A + group C meningococcal capsular polysaccharide vaccine. Children were 1-4 years old at the time of vaccination. Most showed a good initial response to vaccination, including those aged 1-2 years. However, antibody titers declined progressively during follow-up, and 5 years after vaccination, antibody titers measured by both HA and ELISA had returned to prevaccination levels. This decline was not influenced significantly by a booster dose of vaccine given 2 years after initial immunization. Administration of malaria chemoprophylaxis reduced the rate at which antibody levels fell after initial immunization. Sustained protection of children against group A meningococcal disease will require the development of vaccines that are immunogenic in infants and that can induce T cell memory.

Structural variability in the glucuronoxylomannan of Cryptococcus neoformans serotype A isolates determined by 13C NMR spectroscopy.

Cryptococcus neoformans, the etiologic agent of cryptococcal meningoencephalitis, produces glucuronoxylomannan (GXM) as the major capsule component. Purified GXMs obtained from eight serotype A isolates of C. neoformans were treated by ultrasonic irradiation and then O-deacetylated prior to their comprehensive chemical analysis by GLC, GLC-MS, and 13C NMR spectroscopy. The average xylose: mannose: glucuronic acid molar ratio of the eight isolates is 1.96 +/- 0.25: 3.00: 0.58 +/- 0.10. Methylation analyses and 13C NMR spectroscopy show a general structure for GXM that is comprised of a linear (1----3)-alpha-D-mannopyranan substituted with beta-D-GlcpA and with beta-D-Xylp at O-2. Variable quantities of unsubstituted (1----3)-alpha-D-Manp were observed between the eight isolates studied. In several isolates some of the (1----3)-alpha-D-Manp residues are disubstituted with beta-D-GlcpA at O-2 and with beta-D-Xylp at O-4; this type of substitution was not previously thought to occur in serotype A isolates. Heterogeneity, between isolates, in the disposition of the substituents along the mannopyranan backbone was revealed by 13C NMR spectroscopy. The eight isolates, and three isolates previously studied, were each assigned to one of four distinct groups based on the 13C NMR chemical shifts of the anomeric carbons. Six of the eleven isolates gave identical spectra (Group I). The six major anomeric resonances from Group I were assigned to specific glycosidic linkages present in GXM. The remaining five isolates gave more complex spectra that are indicative of additional linkages and comprise the remaining three groups. Three of these five isolates contain substantial amounts of linkages previously thought to be distinctive of serotypes B and C, i.e., Manp residues that are 4-O-glycosylated with beta-D-Xylp. Methylation analyses only predicted an average repeating unit, whereas 13C NMR spectroscopy demonstrated that GXM from each isolate may be categorized into four groups by the occurrence of distinct sequences of carbohydrate residues.

Multicenter comparison of levels of antibody to the Neisseria meningitidis group A capsular polysaccharide measured by using an enzyme-linked immunosorbent assay.

There is no standard immunoassay for evaluating immune responses to meningococcal vaccines. We developed an enzyme-linked immunosorbent assay to measure total levels of antibody to Neisseria meningitidis group A capsular polysaccharide. Five laboratories measured the antibody levels in six paired pre- and postvaccination serum samples by using the enzyme-linked immunosorbent assay. Methylated human serum albumin was used to bind native group A polysaccharide to microtiter plate surfaces. The between-laboratory coefficients of variation for pre- and postvaccination sera had ranges of 31 to 91 and 17 to 31, respectively. The mean laboratory coefficients of variation for pre- and postvaccination sera, respectively, were 17 and 11 (Molecular Biology Laboratory, Centers for Disease Control), 12 and 15 (Immunodiagnostic Methods Laboratory, Centers for Disease Control), 22 and 19 (Dana-Farber Cancer Institute), 38 and 38 (Bacterial Polysaccharide Laboratory, U.S. Food and Drug Administration), and 11 and 10 (Praxis Biologics, Inc.). Standardization of this enzyme-linked immunosorbent assay should allow interlaboratory comparison of meningococcal vaccine immunogenicity, thus providing a laboratory-based assessment tool for evaluating meningococcal vaccines.

Comparisons of standard curve-fitting methods to quantitate Neisseria meningitidis group A polysaccharide antibody levels by enzyme-linked immunosorbent assay.

We examined several of the more commonly used models (log-log, two forms of the logit-log, and the four-parameter logistic-log transformations) for forming standard or calibration curves by using a standardized enzyme-linked immunosorbent assay (ELISA). Assay range, accuracy, and error for each function were measured and compared. Antibody levels to Neisseria meningitidis group A polysaccharide were estimated by calculating antibody concentrations of a serially diluted standard reference serum of known concentration. Each function achieved a high squared correlation coefficient (r2 greater than 0.97), indicating a high degree of accuracy in forming the standard curves. However, when predicted antibody concentrations were compared with the known values, the log-log function exhibited the least precision, with extreme percentages of error occurring at several dilutions. A partially specified logit-log transformation performed better than the log-log model over a reduced range of standard dilutions. This indicated that a high r2 alone was not a reliable measure of the accuracy of the standard curve. Of the methods surveyed, the logistic-log and fully specified logit-log functions were the most accurate models for forming standard curves and for interpolating antibody concentrations from the standard curve. The accuracy of the fully specified logit-log function is highly dependent on the precise specification of two unknown quantities, the optical densities at zero and infinite concentrations, prior to fitting the model to a typical set of calibration data. The four-parameter logistic-log function was the preferred choice for quantitating N. meningitidis group A total polysaccharide antibody by using a standardized ELISA. The function does not require prespecification of any parameters before estimating the standard curve, and the four parameters are readily interpretable in terms of identifiable physical quantities. This model also has the advantage that it is easiest to visualize since it does not incorporate complex transformations of the optical density scale.

Glucuronoxylomannan of Cryptococcus neoformans serotype B: structural analysis by gas-liquid chromatography-mass spectrometry and 13C-nuclear magnetic resonance spectroscopy.

The major extracellular polysaccharide (glucuronoxylomannan, GXM) from six strains of Cryptococcus neoformans serotype B was characterized by gas-liquid chromatography (g.l.c.), g.l.c.-mass spectrometry (g.l.c.-m.s.), and nuclear magnetic resonance (n.m.r.) spectroscopy. Ultrasonic irradiation (u.i.) was used to reduce the mol.wt. of native GXM from 9.75 x 10(5) to 1.15 x 10(5) without apparent change in its composition (GXM-S). The Xylp:Manp:GlcpA molar ratio of the GXM and GXM-S from the six strains of C. neoformans serotype B is approximately 3.5:3.0:0.6. GXM-S was O-deacetylated (GXM-D) by treatment with NH4OH. The 13C-n.m.r. analysis of GXM-D gave spectra that served as characteristic fingerprints of the structure and also facilitated the assignment of the anomeric carbon resonances to specific structural moieties present in GXM-D. The GXM-D from each serotype B strain was found to be similar by 13C-n.m.r. spectroscopy. The structure contains a linear (1----3)-alpha-D-Manp backbone substituted with 2-O-beta-GlcpA and 2-O-beta-Xylp. beta-Xylp is also O-4 linked to the Manp substituted with GlcpA. In addition, a model for the disposition of the Xylp and GlcpA side chain substituents along the mannopyranan backbone is proposed, based upon results from the combination of g.l.c.-m.s. and 13C-n.m.r. spectroscopy.