A critical role for Fas ligand in the active suppression of systemic immune responses by ultraviolet radiation. (25/5658)

Induction of antigen-specific suppression elicited by environmental insults, such as ultraviolet (UV)-B radiation in sunlight, can inhibit an effective immune response in vivo and may contribute to the outgrowth of UV-induced skin cancer. Although UV-induced DNA damage is known to be an initiating event in the immune suppression of most antigen responses, the underlying mechanism(s) of such suppression remain undefined. In this report, we document that Fas ligand (FasL) is critical for UV-induced systemic immune suppression. Normal mice acutely exposed to UV exhibit a profound suppression of both contact hypersensitivity and delayed type hypersensitivity (DTH) reactions and the development of transferable antigen-specific suppressor cells. FasL-deficient mice exposed to UV lack both transferable suppressor cell activity and primary suppression to all antigens tested, with the exception of the DTH response to allogeneic spleen cells. Interestingly, suppression of this response is also known to occur independently of UV-induced DNA damage. Delivery of alloantigen as protein, rather than intact cells, restored the requirement for FasL in UV-induced immune suppression of this response. These results substantiate that FasL/Fas interactions are essential for systemic UV-induced suppression of immune responses that involve host antigen presentation and suggest an interrelationship between UV-induced DNA damage and FasL in this phenomenon. Collectively, our results suggest a model whereby UV-induced DNA damage disarms the immune system in a manner similar to that observed in immunologically privileged sites.  (+info)

Characterization of a haemolytic factor from Candida albicans. (26/5658)

The culture supernatant of Candida albicans promoted the disruption of human red blood cells (RBCs). The haemolytic activity was detected in a sugar-rich fraction (about 200 kDa) from Sephacryl S-100 chromatography. As the haemolytic activity was adsorbed by concanavalin A-Sepharose, the haemolytic factor may be a mannoprotein. The activity was inactivated by periodate oxidation, indicating that the sugar moiety of the mannoprotein played an important role in the haemolysis. The structure of the sugar moiety of the mannoprotein was identified as a cell-wall mannan by 1H-NMR analysis, and purified C. albicans mannan promoted the disruption of RBCs. The binding of mannan to RBCs was demonstrated by flow cytometric analysis and was inhibited by the addition of band 3 protein inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The haemolysis caused by mannan was inhibited by DIDS, SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) and bis(sulfosuccinimidyl) suberate, but not by pyridoxal 5-phosphate. These results indicated that a mannoprotein released from C. albicans bound to the band 3 protein on RBCs, thereby promoting their disruption.  (+info)

Comparisons of the effects of fungicidal and fungistatic antifungal agents on the morphogenetic transformation of Candida albicans. (27/5658)

Eleven different antifungal agents were compared, and their ability to inhibit the morphogenetic transformation of Candida albicans was examined together with their ability to inhibit growth, as measured by MIC methodology. The fungicidal potential of each agent was also determined. Of the antifungal agents tested, only amphotericin B, mulundocandin and aculeacin inhibited the transformation at sub-MIC values; all three agents showed fungicidal activity at concentrations close to the MIC. All other agents were fungicidal only at concentrations much higher than the MIC and inhibited the morphogenetic transformation only at concentrations above the MIC. These data suggest that fungicidal antifungal agents are more likely to act by inhibiting the morphogenetic transformation of C. albicans while fungistatic agents are unable to do so and are more likely to block growth by budding.  (+info)

Assessment of the effect of amphotericin B on the vitality of Candida albicans. (28/5658)

The processes involved in cell death are complex, and individual techniques measure specific fractions of the total population. The interaction of Candida albicans with amphotericin B was measured with fluorescent probes with different cellular affinities. These were used to provide qualitative and quantitative information of physiological parameters which contribute to fungal cell viability. SYBR Green I and 5,(6)-carboxyfluorescein were used to assess membrane integrity, and bis-(1,3-dibutylbarbituric acid)trimethine oxonol and 3,3-dihexyloxacarbocyanine iodide were used to evaluate alterations in membrane potential. The fluorescent indicators were compared with replication competency, the conventional indicator of viability. By using these tools, the evaluation of the response of C. albicans to amphotericin B time-kill curves delineated four categories which may represent a continuum between alive and dead. The data showed that replication competency (CFU per milliliter) as determined by conventional antifungal susceptibility techniques provided only an estimate of inhibition. Interpretation of fluorescent staining characteristics indicated that C. albicans cells which were replication incompetent after exposure to greater than 0.5 microgram of amphotericin B per ml still maintained degrees of physiological function.  (+info)

Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450. (29/5658)

The sterol 14-demethylase P450 (CYP51) of a fluconazole-resistant isolate of Candida albicans, DUMC136, showed reduced susceptibility to this azole but with little change in its catalytic activity. Twelve nucleotide substitutions, resulting in four amino acid changes, were identified in the DUMC136 CYP51 gene in comparison with a reported CYP51 sequence from a wild-type, fluconazole-susceptible C. albicans strain. Seven of these substitutions, including all of those causing amino acid changes, were located within a region covering one of the putative substrate recognition sites of the enzyme (SRS-1). Polymorphisms within this region were observed in several C. albicans isolates, and some were found to be CYP51 heterozygotes. Among the amino acid changes occurring in this region, only an alteration of Y132 was common among these fluconazole-resistant isolates, which suggests the importance of this residue to the fluconazole resistance of the target enzyme. DUMC136 and another fluconazole-resistant isolate were homozygotes with respect to CYP51, although the typical wild-type, fluconazole-susceptible C. albicans was a CYP51 heterozygote. These findings suggest that part of the fluconazole-resistant phenotype of C. albicans DUMC136 was acquired through a mutation-prone area of CYP51, an area which might promote the formation of fluconazole-resistant CYP51, along with a mechanism(s) which allows the formation of a homozygote of this altered CYP51 in this diploid pathogenic yeast.  (+info)

Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity. (30/5658)

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic beta-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The beta-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000- and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.  (+info)

Production, characterization, and epitope mapping of a monoclonal antibody against aspartic proteinase of Candida albicans. (31/5658)

A monoclonal antibody (MAb; MAb CAP1) that was reactive with extracellular aspartic proteinase of Candida albicans (CAP) was produced. The MAb showed strong sensitivity and reactivity to CAP but not to the aspartic proteinases of Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus or to human cathepsin D or porcine pepsin. The epitope of the CAP recognized by the MAb was the proteinaseous part of CAP and the putative epitope of the MAb was located in the Asp77 to Gly103 sequence. This antibody could be useful for the characterization of CAP and would be a valuable probe for the detection of CAP antigen in the sera of patients with invasive candidiasis.  (+info)

Antibody response to Cryptococcus neoformans proteins in rodents and humans. (32/5658)

The prevalence and specificity of serum antibodies to Cryptococcus neoformans proteins was studied in mice and rats with experimental infection, in individuals with or without a history of potential laboratory exposure to C. neoformans, human immunodeficiency virus (HIV)-positive individuals who developed cryptococcosis, in matched samples from HIV-positive individuals who did not develop cryptococcosis, and in HIV-negative individuals. Rodents had little or no serum antibody reactive with C. neoformans proteins prior to infection. The intensity and specificity of the rodent antibody response were dependent on the species, the mouse strain, and the viability of the inoculum. All humans had serum antibodies reactive with C. neoformans proteins regardless of the potential exposure, the HIV infection status, or the subsequent development of cryptococcosis. Our results indicate (i) a high prevalence of antibodies reactive with C. neoformans proteins in the sera of rodents after cryptococcal infection and in humans with or without HIV infection; (ii) qualitative and quantitative differences in the antibody profiles of HIV-positive individuals; and (iii) similarities and differences between humans, mice, and rats with respect to the specificity of the antibodies reactive with C. neoformans proteins. The results are consistent with the view that C. neoformans infections are common in human populations, and the results have implications for the development of vaccination strategies against cryptococcosis.  (+info)