Allergens and Irritants Transcriptionally Upregulate CD80 Gene Expression in Human Keratinocytes

The human CD80 costimulatory molecule is an important signal between professional antigen-presenting cells and T helper cells. The immunobiology of CD80 expression by keratinocytes, especially during allergic and irritant contact dermatitis, however, is less well understood. CD80 cell surface expression and gene transcription by keratinocytes was increased when keratinocytes were exposed to certain allergens (chemicals that induce inflammation via hapten-specific T cells) and irritants (chemicals that are toxic to epidermal cells). Therefore, the human CD80 promoter was cloned and luciferase reporter constructs containing various promoter fragments were engineered. Promoter mapping of these CD80 constructs in transiently transfected keratinocytes showed that a construct containing the proximal 231 bp immediately upstream of the transcription start site of the CD80 promoter was most active in keratinocytes and was inducible to a level ranging from 2- to 10-fold higher in keratinocytes treated with certain allergens and irritants, compared with untreated keratinocytes. This pattern of promoter fragment activity in keratinocytes is identical to that found in professional antigen-presenting cells. This is the first demonstration that the CD80 promoter is active in keratinocytes and that this activity is further increased in keratinocytes treated with certain allergens and irritants. These data suggest that allergens and irritants may, in part, break peripheral tolerance by their direct effects on keratinocyte costimulatory molecule expression, thereby facilitating interactions with epidermotropic T helper cells via the CD80–CD28 or CTLA-4 pathways

Photodynamic treatment of the fungal opportunistic pathogen Candida

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2008.Infection of the mucosal epithelium by the opportunistic fungus Candida results from fungal overgrowth and penetration of epithelium when the body's physical and immunological defenses become compromised. Treatment of Candida infections has become increasingly problematic given the inherent and acquired resistance to antifungals, especially to azoles, of the most commonly seen Candida species, C. albicans and C. glabrata. Meeting the challenge of increased resistance to currently used antifungal agents will require the development of novel strategies for control of the infection. Photodynamic treatment (PDT), the activation of a photosensitizing agent by a specific wavelength of light to kill a target cell via the production of reactive oxygen species, presents a new therapeutic approach for mucocutaneous and cutaneous candidiasis. The present work focused on investigating the efficacy of PDT against Candida employing structurally different photosensitizers, examining the effect of respiratory deficiency and azole resistance as contributing factors to enhanced sensitivity to PDT, and understanding the different pathways involved in respiratory deficiency that might induce hypersensitivity to PDT. We demonstrated the ability to effectively photosensitize Candida using two structurally different porphyrin based photosensitizers, Photofrin® and TMP-1363. We report a differential in PDT susceptibility between the different C. albicans morphologies and Candida species. Our studies revealed that the increased sensitivity to oxidative stress induced by PDT is independent of both inherent and acquired fluconazole resistance of Candida to azoles. In addition, we demonstrate that azole-resistant, respiratory deficient organisms are hypersensitive to photooxidative damage induced by PDT. Furthermore, we investigated specific pathways implicated in innate protection against oxidative stress that, if altered, cause respiratory deficiency and enhanced sensitivity to PDT. We focused on cytochrome c respiratory chain assembly and function, oxidative phosphorylation and mitochondrial redox balance pathways. We demonstrate that alterations in these pathways, in yeasts, induce respiratory deficiency as well as enhanced sensitivity to photooxidative damage induced by PDT. The data presented here reveals the success of effectively photosensitizing Candida independently of its resistance to antifungals, and the importance of intact mitochondrial function to provide a basal level of anti-oxidant defense against PDT-induced phototoxicity in Candida

Active Immunization against Pneumocystis carinii with a Recombinant P. carinii Antigen

Mice immunized with recombinant mouse Pneumocystis carinii antigen A12-thiredoxin fusion protein developed an antibody response that recognized P. carinii antigens, as determined by Western blotting and immunofluorescence analysis. Compared to mice immunized with thioredoxin alone, mice immunized with A12-thioredoxin had significantly reduced lung P. carinii burdens after CD4(+) T-cell depletion and challenge with P. carinii

Passive Intranasal Monoclonal Antibody Prophylaxis against Murine Pneumocystis carinii Pneumonia

Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii

Susceptibility of Candida Species to Photodynamic Effects of Photofrin

The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candida was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicans blastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabrata took up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicans displayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. krusei that is relatively resistant to fluconazole compared to C. albicans was equally sensitive to C. albicans at Photofrin concentrations of ≥3 μg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candida therapeutic modalities, particularly for superficial infections on surfaces amenable to illumination

Nitrate Sensing and Metabolism Modulate Motility, Biofilm Formation, and Virulence in Pseudomonas aeruginosa▿

Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient- and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type P. aeruginosa PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in P. aeruginosa motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for P. aeruginosa pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host Caenorhabditis elegans. Interestingly, the membrane nitrate reductase mutant was avirulent in C. elegans, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in P. aeruginosa pathogenesis