A spectrophotometric method to precisely determine endpoint titers in complement fixation assays.

Since the early 20th century, complement fixation (CF) testing has been used to quantify the humoral response to various pathogens. The qualification of a positive result is based on a subjective determination of 30% lysis of sheep red blood cells, which can lead to variability in the analysis. A spectrophotometric reading of a standard with a known 30% lysis was used to standardize the currently used CF method and tested with controls and patient sera for various fungal assays. By utilizing this method a precise and non-subjective determination of endpoint titers was achieved.

IgE sensitization to fungi mirrors fungal phylogenetic systematics.

BACKGROUND: Fungal allergy is an elusive disease, and little progress has been made in this field during recent years. Moreover, because of the complexity of the organisms, it is difficult to categorize fungi systematically on the basis of morphologic characterization. However, recent molecular phylogenetics studies have substantially improved fungal categorization. In parallel, new approaches to analyze large IgE antibody datasets enable identification and visualization of IgE sensitization patterns. OBJECTIVE: To study whether molecular phylogenetic relationships of fungal species, commonly used in allergy diagnosis, also are reflected in IgE sensitization profiles of individuals sensitized to fungi. METHODS: A dataset was compiled of recorded serum IgE antibody levels to 17 different fungal species from 668 individuals sensitized to at least 1 of the 17 species. By applying a clustering method to this dataset, the fungal species were grouped into a hierarchical organization. Finally, the resulting organization was compared with recently published fungal systematics. RESULTS: The hierarchical structure of fungi, based on the presence of IgE antibodies in sensitized individuals, very well reflected phylogenetic relationships. Examples include the distinct separation of basal fungi from the subkingdom Dikarya as well as individual cluster formations of fungi belonging to the subphylum Saccharomycotina and order Pleosporales. CONCLUSION: To our knowledge, this is the first in-depth study that demonstrates a close relationship between molecular fungal systematics and IgE sensitization to fungal species. Because close evolutionary organisms typically have a higher degree of protein similarity, IgE cross-reactivity is likely the main reason for obtained organization.

IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans.

In the murine model of Cryptococcus neoformans infection Th1 (IL-12/IFN-gamma) and Th17 (IL-23/IL-17) responses are associated with protection, whereas an IL-4-dependent Th2 response exacerbates disease. To investigate the role of the Th2 cytokine IL-13 during pulmonary infection with C. neoformans, IL-13-overexpressing transgenic (IL-13Tg(+)), IL-13-deficient (IL-13(-/-)), and wild-type (WT) mice were infected intranasally. Susceptibility to C. neoformans infection was found when IL-13 was induced in WT mice or overproduced in IL-13Tg(+) mice. Infected IL-13Tg(+) mice had a reduced survival time and higher pulmonary fungal load as compared with WT mice. In contrast, infected IL-13(-/-) mice were resistant and 89% of these mice survived the entire period of the experiment. Ag-specific production of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with a significant type 2 cytokine shift but only minor changes in IFN-gamma production. Consistent with enhanced type 2 cytokine production, high levels of serum IgE and low ratios of serum IgG2a/IgG1 were detected in susceptible WT and IL-13Tg(+) mice. Interestingly, expression of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with reduced IL-17 production. IL-13 was found to induce formation of alternatively activated macrophages expressing arginase-1, macrophage mannose receptor (CD206), and YM1. In addition, IL-13 production led to lung eosinophilia, goblet cell metaplasia and elevated mucus production, and enhanced airway hyperreactivity. This indicates that IL-13 contributes to fatal allergic inflammation during C. neoformans infection.

Unexpected diversity in the fine specificity of monoclonal antibodies that use the same V region gene to glucuronoxylomannan of Cryptococcus neoformans.

Most mAbs to the capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans are generated from the same VH and VL gene families. Prior Ab studies have assessed protective efficacy, Id structure and binding to capsular polysaccharides, and peptide mimetics. These data have been interpreted as indicating that most mAbs to GXM have the same specificity. A new approach to Ab specificity analysis was investigated that uses genetic manipulation to generate C. neoformans variants with structurally different capsules. C. neoformans mutants expressing GXM with defective O-acetylation were isolated and complemented by the C. neoformans gene CAS1, which is necessary for the O-acetylation of GXM. The mAbs exhibited differences in their binding to the GXM from these mutant strains, indicating previously unsuspected differences in specificity. Analysis of three closely related IgMs revealed that one (mAb 12A1) bound to an epitope that did not require O-acetylation, another (mAb 21D2) was inhibited by O-acetylation, and the third (mAb 13F1) recognized an O-acetylation-dependent conformational epitope. Furthermore, an IgG Ab (mAb 18B7) in clinical development retained binding to de-O-acetylated polysaccharide; however, greater binding was observed to O-acetylated GXM. Our findings suggest that microbial genetic techniques can provide a new approach for epitope mapping of polysaccharide-binding Abs and suggest that this method may applicable for studying the antigenic complexity of polysaccharide Ags in other capsulated microorganisms.

[Occurrence of Pneumocystis carinii DNA and anti-Pneumocystis antibodies in the sera of infants during a period of of physiologic decreased immune response]. / Wystepowanie DNA Pneumocystis carinii i przeciwcial pneumocystozowych w surowicach niemowlat w okresie fizjologicznego spadku odpornosci.

The polymerase chain reaction (PCR) and indirect immunofluorescent test (IF) were used for examination of serum samples obtained from infants with respiratory tract infections. Sixty (11.9%) out of the 503 examined infant samples were positive for anti-P. carinii IgM and 354 (70.4%) contained anti-Pneumocystis IgG. P. carinii DNA was found in 6 (6.7%) sera from 90 of infected infants. Five out these 6 samples were for anti-Pneumocystis antibodies positive; 4 contained both IgG and IgM classes and one had only IgG. The sixth sample had neither IgG nor IgM, despite of P. carinii DNA presence. The results of the studies indicated that for diagnosis Pneumocystis carinii pneumonia (PCP) in infants on serum specimens detection of antibodies by IF test is of greater value than Pneumocystis DNA amplification by PCR method.

IgG subclasses of anti Saccharomyces cerevisiae antibody in inflammatory bowel disease.

Elevation of serum anti Saccharomyces cerevisiae antibody (ASCA) has been reported in patients with Crohn's disease. We analysed the subclasses of Immunoglobulin (Ig) G reaction in ASCA in sera from patients with inflammatory bowel disease, healthy controls, and patients with intestinal Behçet's disease. Serum samples were obtained from 29 patients with Crohn's disease, 30 patients with ulcerative colitis, 7 patients with intestinal Behçet's disease, and 12 healthy controls. Serum IgG subclasses IgG1, IgG2, IgG3, and IgG4 of ASCA were analysed using ELISA. IgG4 ASCA was significantly increased in patients with inflammatory bowel disease. In patients with intestinal Behçet's disease, IgG1, IgG3, and IgG4 ASCA were increased. Differential responses, in terms of subclasses in ASCA, were found in patients with inflammatory bowel disease and patients with intestinal Behçet's disease, which may represent different pathophysiologies of these intestinal inflammatory diseases.

A new method of antibiotyping yeasts for subspecies discrimination and distribution in human clinical specimens.

A study of the antibiotypes of 764 isolates of the genera Candida and Torulopsis from different clinical specimens is reported. The typing method was based on the susceptibility results obtained by the standardized and partially automated kit ATB-Fungus (API-bioMérieux), giving to each strain a code of six figures, according to these criteria: susceptibility to 5-fluorocytosine, amphotericin B, nystatin, miconazole, econazole, and ketoconazole. Candida albicans serotypes were determined by the Candida Check test (Iatron, Japan). Twenty-six antibiotypes were found in C. albicans (482 isolates), 21 types in serotype A, and 15 in serotype B strains. Candida parapsilosis (115 isolates) was divided into 11 antibiotypes, Torulopsis glabrata (53 isolates) into five, Candida guilliermondii (36 isolates) into 10 and Candida tropicalis (31 isolates) into eight. Depending on the sample origin, 000000 (susceptibility to all the antifungals tested) was the predominant C. albicans antibiotype (92.9% of blood isolates, 41.2% of vaginal isolates, 33.3% of respiratory isolates, isolates, 31.01% or oral and digestive tract isolates, and 25.0% of nail and skin isolates). No predominant antibiotypes were found in strains from respiratory tract, skin ad nails. A reproducibility close to 99% was found with the test. Simplicity and standardization could make this method useful for typing Candida and Torulopsis isolates.

The role of somatic structure of the fungus Paracoccidioides brasiliensis upon B cell activation in experimental paracoccidioidomycosis.

In this study, we report an increase of the number of antibody-secreting cells and the augmentation of antibody production against unrelated antigens in mice infected with the fungus P. brasiliensis, as well as in mice inoculated with cell wall preparation isolated from P. brasiliensis (CW). The immunomodulatory effect of the live fungus and the CW preparation was dose-dependent, and their actions were mainly restricted to the i.v. or i.p. inoculation simultaneously with the sheep erythrocyte challenge by the i.v. route or restricted to i.p. inoculation of CW when bovine serum albumin (BSA) antigen was used. The dependence of antibody production on different routes of CW inoculation was correlated with the number of antigen-specific B cells in the spleen as determined by direct and reverse plaque-forming cell assays. The immunization schedules using CW preparation caused a preferential production of IgM and IgG3 antibodies. The results also showed that the hyperactive humoral immune response of mice induced by i.p. inoculation of CW was devoid of polyclonal B cell activation compared with the effects observed for the lipopolysaccharide (LPS)-treated groups. Paracoccidioides brasiliensis CW components may have potent immunological properties related to the non-specific B cell activation found in paracoccidioidomycosis.

Determination of antigen binding specificities of Cryptococcus neoformans factor sera by enzyme-linked immunosorbent assay.

The competitive binding specificities of glucuronoxylomannan (GXM) and its derivatives to factor sera of Cryptococcus neoformans were studied by enzyme-linked immunosorbent assay. An effort was made to determine the epitope specificity of each factor serum. Despite the presence of antigenic factor 1 on all serotypes of C. neoformans, variations in inhibition ability were observed with different GXMs. The panspecific component of factor serum 1 (antibody 1) appeared to be due to the presence of more than one antibody component. The activity was dependent on the 6-O-acetyl substituent. GXMs of serotypes A and D inhibited factor serum 2 equally well, indicating a low titer for the antibody 7 component. Serotype B GXM was a poor inhibitor, and serotype C GXM did not inhibit factor serum 2. The activity of factor serum 2 was 6-O-acetyl dependent. GXMs from typical serotype A and serotype D isolates were excellent inhibitors of factor serum 3. GXMs from serotype B were poorly inhibitory and serotype C did not inhibit factor serum 3. The activity of factor serum 3 was 6-O-acetyl dependent. The activity of factor serum 4 was due predominantly to antibody component 6. The activity of factor 4 was directed mainly against serotype C, and it was independent of 6-O-acetyl substitution Factor serum 5 was specific for serotype B GXMs. The inhibitory effect was independent of 6-O-acetyl substitution, but the effect was diminished by reduction of the glucuronic acid. The GXMs with a typical serotype C structure inhibited antibody 6. O deacetylation of the GXMs did not affect their inhibitory activity. However, reduction of glucuronic acid reduced factor serum 6 binding. Factor serum 8 was specific to serotype D; native GXMs of serotype A were slightly inhibitory. O deacetylation of the serotype D GXMs abrogated the inhibitory effect. O deacetylation alone abrogates the activity of antibody components 1, 2, 3, and 8. Reduction of glucuronic acid reduces the inhibitory activity of the GXM to antibody components 4, 5, and 6. Partial GXM structures and methyl glycosides did not effectively inhibit the activity of any of the factor sera.

Experimental histoplasmosis in the mouse. Immunoglobulin class response, total immunoglobulin A levels, immune complex formation, and occurrence of antigen in serum and urine.

Biotin-avidin enzyme-linked immunosorbent assays (ELISA) were developed to study the appearance of free antibody of the IgM, G, and A classes of immune complexes (IC) containing those classes and of antigen in the serum and urine of mice with experimentally induced, disseminated histoplasmosis (histo) over a period ranging from 4 to 64 days after infection. Free IgM was detected 4 days after infection, remained at low levels, and disappeared on Day 64, whereas free IgG was first detected on Day 7 and rose to very high levels before declining on Day 64. Free IgA was detected only once, on Day 21. IC containing IgM were seen first on Day 4, remained at low levels, and became undetectable on Day 64. IC containing IgM were detected on Day 7, peaked on Day 14, and declined through Day 64. IC containing IgA wee seen at low levels on Days 7, 14, and 21. Estimation of total IgA levels, done by single radial immunodiffusion, showed a statistically significant decrease on Day 14, followed by a slightly significant increase on Days 21 and 30. Antigen was detected in as much as 80% of serum specimens and 100% of urine samples during the first 2 wk postinfection but rarely afterwards. We conclude that ELISA provides a highly sensitive way to study antibody, IC, and antigen in host body fluids during histo infection and that IgM and antigen detection can be very early indices of infection. Measurements of IC and total IgA seem to be of relatively less importance in detection of infection.

Immunoglobulin A1 and A2 subclass of salivary antibodies to Candida albicans in patients with oral candidosis.

Immunoglobulin A antibody titres to a cytoplasmic protein extract of Candida albicans were determined by ELISA in saliva from 20 patients with oral candidosis and 21 controls. Patients had significantly increased levels of salivary IgA anti-Candida antibodies when compared with controls (P less than 0.001). Antibody levels were associated with IgA1 subclass in 90% of the patients; in contrast, IgA2 subclass was predominant in 67% of the controls. Antifungal therapy resulted in significantly decreased IgA1 titres (P less than 0.05) whilst the mean IgA2 antibody titre remained unchanged. The results indicate that Candida infection may change the subclass pattern of salivary IgA antibodies.

Immunoglobulin class of anti-Candida antibodies in patients with vaginal candidiasis.

Studies of the immunoglobulin class of circulating anti-Candida antibodies in patients with vaginal candidiasis were undertaken with the aim of answering the question of whether these antibodies are predominantly IgA or IgG. Earlier work resulted in conflicting data that we felt would be clarified by the use of a quantitative technique, FIAX fluoroimmunoassay, which could objectively measure the relative amounts of antibody of each class. Results using this assay and the indirect immunofluorescence assay on whole Candida cells demonstrate that the immunoglobulin class distribution of the circulating anti-Candida antibodies of these patients is similar to that seen in other forms of Candida infection, the predominant antibody class being IgG.

Antibodies against Aspergillus fumigatus II. Identification and quantification by means of crossed immunoelectrophoresis.

Individual antigenic components of Aspergillus fumigatus were identified by crossed immunoelectrophoresis (CIE) using pooled rabbit hyperimmune antiserum. Patients' sera with enhanced precipitins against A. fumigatus showed antibodies against identical antigenic components in CIE. Identification of the antigenic components by the intermediate gel technique showed high concentrations of antibodies against a small group of antigenic components. No differences were found in the individual antigenic components involved in the precipitation reactions between groups of sera with enhanced and normal IgE levels against A. fumigatus. Crossed immunoelectrofocusing was used to determine the isoelectric point of particular components that were identified by CIE.