PCR for diagnosis of paracoccidioidomycosis. (65/1360)

A PCR assay based on oligonucleotide primers derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis DNA in sputa. In the standardized conditions, it could detect 10 cells/ml of sputum, providing sufficient accuracy to be useful for diagnosis of paracoccidioidomycosis.  (+info)

CD4(+)-T-Cell-mediated resistance to systemic murine candidiasis induced by a membrane fraction of Candida albicans. (66/1360)

We induced resistance to systemic Candida albicans infection through CD4(+)-cell-mediated immunity in mice by immunization with subcutaneous injections of live C. albicans cells emulsified in incomplete Freund adjuvant. Using the resistant mice, we tested subcellular fractions of C. albicans cells for antigenicity. The fractions were derived from digested surface cell walls, insoluble membranes, or soluble and insoluble cytoplasmic materials, which were prepared by treatment with cell wall-digesting enzymes followed by lysis of the consequent protoplasts. Interestingly, the live-cell-immunized mice showed strong cell-mediated immune responses to the membrane fraction (C. albicans membrane antigen [CMA]). In addition, immunization with CMA induced resistance to systemic candidiasis, which disappeared upon administration of anti-CD4 monoclonal antibody. Infusion of splenocytes from the CMA-immunized mice conferred resistance on SCID mice, whereas infusion of CD4(+)-T-cell-depleted splenocytes was unable to induce resistance, indicating the importance of CD4(+) lymphocytes for resistance. These results suggest a potential for the membrane fraction to act as an antigen conferring resistance to systemic candidiasis in place of live cells and also as a source for the isolation of a new antigen.  (+info)

Disruption of the gene which encodes a serodiagnostic antigen and chitinase of the human fungal pathogen Coccidioides immitis. (67/1360)

Disruption of genes in medically important fungi has proved to be a powerful tool for evaluation of putative virulence factors and identification of potential protein targets for novel antifungal drugs. Chitinase has been suggested to play a pivotal role in autolysis of the parasitic cell wall of Coccidioides immitis during the asexual reproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (CTS1 and CTS2) of C. immitis have been cloned. Preliminary evidence has suggested that expression of CTS1 is markedly increased during endospore formation. The secreted CTS1 chitinase has also been shown to react with patient anti-Coccidioides complement-fixing (CF) antibody and is a valuable aid in the serodiagnosis of coccidioidomycosis. To examine the role of CTS1 in the morphogenesis of parasitic cells, the CTS1 gene was disrupted by a single, locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construct that contained a 1.1-kb fragment of the chitinase gene into the chromosomal DNA of C. immitis. Results of Southern hybridizations, immunoblot analyses of culture filtrates using both CTS1-specific murine antiserum and serum from a patient with confirmed coccidioidal infection, an immunodiffusion test for CF antigenicity, and substrate gel electrophoresis assays of chitinase activity confirmed that the CTS1 gene was disrupted and nonfunctional. This is the first report of a successful targeted gene disruption in C. immitis. However, loss of CTS1 function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in the parental and Deltacts1 strains suggested that the absence of a functional CTS1 gene can be compensated for by elevated expression of the CTS2 gene. Current investigations are focused on disruption of CTS2 in the Deltacts1 host to further evaluate the significance of chitinase activity in the parasitic cycle of C. immitis.  (+info)

Utilization of exocellular mannan from Rhodotorula glutinis as an immunoreactive antigen in diagnosis of leptospirosis. (68/1360)

Previously, Rhodotorula glutinis was reported to produce a large amount of exocellular mannan, having a repeating unit of -->3)-D-Manp-(1-->4)-D-Manp-(1-->. Recently, we found that antigenic polysaccharides of Leptospira biflexa serovar patoc strain Patoc I have the same repeating unit and cross-react with antisera raised against extended strains of other leptospires (K. Matsuo, E. Isogai, and Y. Araki, Carbohydr. Res., in press). This structural identity and the difficulty of producing and isolating antigens led us to confirm the usefulness of Rhodotorula mannan as an immunoreactive antigen in a serological diagnosis of leptospirosis. In the present investigation, we confirmed the structural identity of an exocellular mannan isolated from R. glutinis AHU 3479 and tried to use it as an immunoreactive antigen in a serological diagnosis of leptospirosis. From its chemical analysis and (1)H- and (13)C-labeled nuclear magnetic resonance spectrometry, the Rhodotorula mannan was confirmed to consist of the same disaccharide units. Furthermore, such a preparation was shown to immunoreact to various sera from patients suffering with leptospirosis as well as to most rabbit antiserum preparations obtained from immunization with various strains of pathogenic leptospires. Therefore, the Rhodotorula mannan preparation is useful as an immunoreactive antigen in the serological diagnosis for leptospirosis.  (+info)

Diagnostic particle agglutination using ultrasound: a new technology to rejuvenate old microbiological methods. (69/1360)

Microbial antigen in clinical specimens can be detected rapidly by commercial test-card latex agglutination, but poor sensitivity is a potential difficulty. Antigen detection by immuno-agglutination of coated latex micro-particles can be enhanced in comparison with the conventional test-card method in both rate and sensitivity by the application of a non-cavitating ultrasonic standing wave. Antibody-coated micro-particles suspended in the acoustic field are subjected to physical forces that promote the formation of agglutinates by increasing particle-particle contact. This report reviews the application of ultrasound to immuno-agglutination testing with several commercial antibody-coated diagnostic micro-particles. This technique is more sensitive than commercial card-based agglutination tests by a factor of up to 500 for fungal cell-wall antigen, 64 for bacterial polysaccharide and 16 for viral antigen (in buffer). The detection sensitivity of meningococcal capsular polysaccharide in patient serum or CSF has been increased to a stage where serotyping by ultrasound-enhanced agglutination is comparable to that achievable with the PCR, but is available more rapidly. Serum antigen concentration as measured by ultrasonic agglutination has prognostic value. Increasing the sensitivity of antigen detection by increasing the acoustic forces that act on suspended particles is considered. Employing turbidimetry to measure agglutination as part of an integrated ultrasonic system would enable the turnover of large numbers of specimens. Ultrasound-enhanced latex agglutination offers a rapid, economical alternative to molecular diagnostic methods and may be useful in situations where microbiological and molecular methods are impracticable.  (+info)

Distinct human T cell repertoires mediate immediate and delayed-type hypersensitivity to the Trichophyton antigen, Tri r 2. (70/1360)

The 29-kDa subtilase homologue, Tri r 2, derived from the dermatophyte fungus Trichophyton rubrum, exhibits unique immunologic characteristics in its ability to elicit immediate (IH) and delayed-type (DTH) hypersensitivity skin tests in different individuals. Thus, Tri r 2 provides a model for comparing the T cell repertoire in subjects with distinct immune responses to a single Ag. Recombinant Tri r 2 produced as a GST fusion protein in Escherichia coli stimulated strong in vitro lymphoproliferative responses in 10 IH and 10 DTH responders. Patterns of T cell epitope recognition were compared between skin test groups using 28 overlapping peptides (each in 12 replicate wells) derived from Tri r 2 to stimulate T lymphocyte proliferation in vitro. Peptide 5 (P5; aa 41-60) induced the strongest response in DTH subjects and showed the largest difference between DTH and IH responders in proliferation (mean standardized index, 2.22 and 0.82, respectively; p = 0.0047) and number of positive wells (81 vs 12). Responses to P5 were associated with diverse HLA haplotypes. These results showed that P5 contains an immunodominant epitope specifically associated with DTH and that this peptide is recognized in a permissive manner. Cross-validated linear discriminant analysis using T cell proliferative responses to two regions of Tri r 2 (aa 51-90 and 231-270) gave a 95% predictive accuracy for classification of subjects into IH or DTH groups. We conclude that different immune responses to Trichophyton are mediated by distinct T cell repertoires between individuals with IH and DTH reactions to Tri r 2.  (+info)

The Cryptococcus neoformans gene DHA1 encodes an antigen that elicits a delayed-type hypersensitivity reaction in immune mice. (71/1360)

When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5' rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.  (+info)

Polymorphism in the gene coding for the immunodominant antigen gp43 from the pathogenic fungus Paracoccidioides brasiliensis. (72/1360)

The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM). It is protective against murine PCM and is a putative virulence factor. The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp. Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points. In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P. brasiliensis isolates after sequencing two PCR products from each fungal sample. The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp. Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein. The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms. The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots. The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine.  (+info)