Serological relationships of Cryptococcus spp.: distribution of antigenic factors in Cryptococcus and intraspecies diversity.
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The antigenic formulas of 34 species in the genus Cryptococcus were determined by using type strains and eight factor sera prepared from adsorption experiments with Cryptococcus neoformans serotypes. These antigenic factors were shared by 19 species. The strains used could be divided into eight serological groups. The patterns of groups 1, 2, 3, 5, and 6 were the same as the patterns of C. neoformans serotypes A, D, A-D, B, and C, respectively. The species belonging to group 4 reacted to factor sera 1, 2, and 3. Group 7 contained one species that reacted only to factor serum 1. The 15 species in group 8 did not react to any of the factor sera used. Compared to the reported molecular phylogenetic tree, the serological and phylogenetic data were correlated in the Filobasidium lineage. All the members of the albidus clade in the Filobasidium lineage had antigens 1, 2, and 3, and all the strains in the magnus clade belonged to serogroup 8. Moreover, intraspecies diversity was examined using strains of C. curvatus, C. humicolus, and C. laurentii. Serological heterogeneity was observed in the species C. humicolus and C. laurentii, as well as in phylogenetic relationships previously published. Using serological features, similarities and differences between Cryptococcus species were demonstrated. Our study contributes to a better description of the genus Cryptococcus and related species phenotypically and phylogenetically. (+info)
Protective antigens and mechanisms of anti-Candida immunity.
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Life threatening fungal diseases are now frequent in a substantial fraction of the immunocompromised host population. The toxicity and the relative scarcity of efficacious antifungal drugs highlight the need for developing alternative or integrative immunoprophylactic and therapeutic tools; among them the need to develop prophylactic or therapeutic vaccines against candidiasis, a widespread mucosal or deep-seated infection caused primarily by the fungus Candida albicans, are of clear priority. Vaccination is a highly beneficial medical practice, and probably the most cost-effective measure against disease onset and progression. It is based on the use of microbial antigens capable of conferring protection in a susceptible target host. To date, only a handful of Candida albicans antigens have been produced and very few of them have been thoroughly investigated for immunogenicity and protection in experimental models of candidiasis. Thus, approaches to the molecular, biochemical and functional characterization of novel C. albicans encoded molecules are most welcome to improve the perspective of developing in the near future an effective vaccine against C. albicans. Identification of anti-Candida vaccine candidates must take into account the diversity of Candida diseases, the various underlying mechanisms of protection as well as the major immune dysfunctions observed as predisposing factors for disease. Antigens to be considered possible vaccine candidates include members of the aspartyl proteinase (Sap2) family and the 65kDa mannoprotein (MP65) antigen. An additional molecule of C. albicans which has not yet been identified but deserves great consideration as a vaccine candidate is the yeast-killer toxin receptor (KTR). Initial experimental evidence strongly suggests that the above antigens are able to elicit protective immunity against mucosal and/or systemic candidiasis. A series of molecular, biochemical and immunological studies aimed at validating and strengthening this initial evidence are in progress, with the ultimate goal of producing recombinant or natural antigens that can be assessed for their ability to elicit a protective immunity in animal models and the mechanisms whereby protection is achieved, with emphasis on determination of immune correlates of protection. (+info)
Murine eotaxin-2: a constitutive eosinophil chemokine induced by allergen challenge and IL-4 overexpression.
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The generation of tissue eosinophilia is governed in part by chemokines; initial investigation has identified three chemokines in the human genome with eosinophil selectivity, referred to as eotaxin-1, -2, and -3. Elucidation of the role of these chemokines is dependent in part upon analysis of murine homologues; however, only one murine homologue, eotaxin-1, has been identified. We now report the characterization of the murine eotaxin-2 cDNA, gene and protein. The eotaxin-2 cDNA contains an open reading frame that encodes for a 119-amino acid protein. The mature protein, which is predicted to contain 93 amino acids, is most homologous to human eotaxin-2 (59.1% identity), but is only 38.9% identical with murine eotaxin-1. Northern blot analysis reveals three predominant mRNA species and highest constitutive expression in the jejunum and spleen. Additionally, allergen challenge in the lung with Aspergillus fumigatus or OVA revealed marked induction of eotaxin-2 mRNA. Furthermore, eotaxin-2 mRNA was strongly induced by both transgenic over-expression of IL-4 in the lung and administration of intranasal IL-4. Analysis of eotaxin-2 mRNA expression in mice transgenic for IL-4 but genetically deficient in STAT-6 revealed that the IL-4-induced expression was STAT-6 dependent. Recombinant eotaxin-2 protein induced dose-dependent chemotactic responses on murine eosinophils at concentrations between 1-1000 ng/ml, whereas no activity was displayed on murine macrophages or neutrophils. Functional analysis of recombinant protein variants revealed a critical role for the amino terminus. Thus, murine eotaxin-2 is a constitutively expressed eosinophil chemokine likely to be involved in homeostatic, allergen-induced, and IL-4-associated immune responses. (+info)
Decontamination of gnotobiotic mice experimentally monoassociated with Candida albicans.
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Gnotobiotic AKR mice, experimentally monoassociated with Candida albicans, were successfully decontaminated by oral treatment with amphotericin B incorporated in the drinking water. Germfree mice first were swabbed orally with viable C. albicans and then were allowed to acclimatize for 4 weeks. The log10 of number of C. albicans per gram of organ (with luminal contents) was 7.9 and 7.7 in the stomach and cecum, respectively. Direct fecal smears, as well as impresssion smears of stomach and cecum mucosal surfaces, revealed yeastphase cells, many with germ tubes, but no hyphal forms. No illness or mortality was observed over this period. The mice then were given amphotericin B DISsolved in the drinking water and offered ad libitum. At levels of 0.1 and 0.2 mg/ml, the number of fecal C. albicans was decreased but not eliminated completely. However, 0.3 mg/ml was sufficient to decontaminate the mice completely and return them to the germfree state. Residual amphotericin B was detected in the feces of the mice only while they were receiving the 0.3 mg/ml dose level. These mice remained germfree until the termination of the experiment, 10 weeks after the antibiotic had been discontinued and replaced by plain drinking water. (+info)
Skin test and blastogenic responses to Sporotrichun schenckii.
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In vivo skin testing and in vitro lymphocyte blastogenesis were evaluated in a young adult population as methods for detecting cellular immunity to Sporotrichum schenckii. Similar procedures for Candida albicans and Coccidioides immitis were also investigated. 5 of 143 subjects had positive skin tests and 14 had positive blastogenic responses to S. schenckii. These 14 subjects also exhibited unusually high responses to C. albicans in vitro and 11 of the 14 were female. Data demonstrated a correlation coefficient of 0.89 when comparing the blastogenic assays for S. schenckii and C. albicans, suggesting cross antigenicity. Intact cellular immune mechanisms in combination with exposure to C. albicans may protect the host from systemic infection with S. schenckii. Although a limited number of subjects were studied, as a group, females had more vigorous cellular immune responses to C. albicans than males. The rare occurence of sporothrix infection in females as compared to males may be the result of antigenic stimulation from commonly observed vaginal colonization with C. albicans. The present data indirectly support this hypothesis. (+info)
Generation of a recombinant 65-kilodalton mannoprotein, a major antigen target of cell-mediated immune response to Candida albicans.
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A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogen Candida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designated CaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His(6)-tagged protein (rCaMp65) was expressed in Escherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4(+) T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies. (+info)
A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection.
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Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens. (+info)
Effects of exposure to flax dust in Polish farmers: work-related symptoms and immunologic response to microbial antigens associated with dust.
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Medical examinations were performed in a group of 51 Polish farmers heavily exposed to flax dust during harvesting and scutching (threshing) and in a group of 50 healthy urban dwellers not exposed to organic dusts (controls). The examinations included: interview concerning the occurrence of respiratory disorders and work-related symptoms, physical examination, X-ray examination of chest, lung function tests, oxymetric examinations, determination of the concentration of cytokines (IL-1alpha IL-6, TNFalpha) in blood serum and allergological tests with microbial antigens associated with organic dust, comprising: skin prick test with 4 antigens, agar-gel precipitation test with 12 antigens and test for specific inhibition of leukocyte migration with 4 antigens. As many as 32 farmers (62.7%) reported the occurrence of work-related symptoms during harvesting, transporting and scutching of flax. The most common complaint was general weakness reported by 15 farmers (29.4%), followed by headache reported by 14 (27.5%), blocking of the nose - by 11 (21.6%), dry cough, shivering, and eyes itching - each by 10 (19.8%), chest tightness and hoarseness - each by 9 (17.6%). No control subjects reported these work-related symptoms. The mean spirometric values in the examined group of farmers were within a normal range and did not show a significant post-shift decline. In contrast, a significant post-shift decline of oxymetric values was found among flax farmers. The farmers showed a frequency of the positive early skin reactions to environmental allergens in the range of 0-19.6%, a frequency of positive precipitin reactions in a range of 0-56.9%, and frequency of positive reactions of leukocyte migration inhibition in a range of 7.8-21.6%. The members of the control group responded to the majority of allergens with a significantly lower frequency of positive results compared to the farmers. Elevated concentrations of IL-1alpha and IL-6, but not TNFalpha, were found in blood sera of flax farmers. In conclusion, farmers engaged in harvesting and scutching of flax represent a group of elevated professional risk because of high incidence of work-related symptoms and high frequency of allergic reactions to bacteria and fungi associated with organic dust. (+info)