Constantine Haidaris - Academia.edu (original) (raw)
Papers by Constantine Haidaris
J Invest Dermatol, 2000
The human CD80 costimulatory molecule is an important signal between professional antigen-present... more 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.
Biochemistry, 2001
A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human path... more A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human pathogen Candida albicans. Both the precursor and ribozyme are functional as determined from in vitro assays. Comparisons of dissociation constants for oligonucleotide binding to the ribozyme and to a hexanucleotide mimic of its internal guide sequence lead to a model for recognition of the 5′ exon substrate by this intron. In particular, tertiary contacts with the P1 helix that help align the splice site include three 2′-hydroxyl groups, a G‚U pair that occurs at the intron's splice junction, and a G‚A pair. The free energy contribution that each interaction contributes to tertiary binding is determined. When the G‚A pair is replaced with a G-C pair, tertiary interactions to 5′ exon mimic 2′-hydroxyl groups are significantly weakened. When the G‚A pair is replaced with a G‚U pair, tertiary interactions are retained and binding is 10-fold tighter. These results expand our knowledge of substrate recognition by group I introns, and also provide a basis for rational design of oligonucleotide-based therapeutics for targeting group I introns by binding enhancement by tertiary interactions and suicide inhibition strategies.
Journal of Eukaryotic Microbiology, 1994
Using RNA: RNA in situ hybridization, the intracellular location of a transcript encoded by and s... more Using RNA: RNA in situ hybridization, the intracellular location of a transcript encoded by and spanning the entire length of a Trypanosoma cruzi kinetoplast DNA minicircle was determined. In axenically cultured T. cruzi epimastigotes, the hybridization signal was restricted to the kinetoplast, which was situated in the perinuclear region of the cell. Following conversion of epimastigotes to culture-derived metacyclic trypomastigotes, the kinetoplast moved to an acentric position in the metacyclic trypomastigote. Again, the hybridization signal co-localized with the position of the kinetoplast. These results suggested that the transcript remained closely associated with the T. cruzi kinetoplast within the mitochondrion in each of the morphological forms. Using specific oligonucleotide probes derived from a cDNA encoding the transcript, the entire native kDNA minicircle encoding the transcript was cloned and its nucleotide sequence was determined. The nucleotide sequence of the intact native minicircle was identical to that of the full-length cDNA corresponding to the minicircle transcript, indicating that the transcript was not modified prior to the time of cDNA synthesis and cloning.
Biochemistry, 1997
The recent increase in the population of immunocompromised patients has led to an insurgence of o... more The recent increase in the population of immunocompromised patients has led to an insurgence of opportunistic human fungal infections. The lack of effective treatments against some of these pathogens makes it important to develop new therapeutic strategies. One such strategy is to target key RNAs with antisense compounds. We report the development of a model system for studying the potential for antisense targeting of group I self-splicing introns in fungal pathogens. The group I intron from the large ribosomal subunit RNA of mouse-derived Pneumocystis carinii has been isolated and characterized. This intron self-splices in Vitro. A catalytically active ribozyme, P-8/4x, has been constructed from this intron to allow measurement of dissociation constants for potential antisense agents. At 37°C, in 50 mM Hepes (25 mM Na + ), 15 mM MgCl 2 , and 135 mM KCl at pH 7.5, the exogenous 5′ exon mimic r(AUGACU) binds about 60 000 times more tightly to this ribozyme than to r(GGUCAU), a mimic of its complementary binding site on the ribozyme. This enhanced binding is due to tertiary interactions. This tertiary stabilization is increased by single deoxynucleotide substitutions in the exon mimic at every position except for the internal A, which is essentially unchanged. Thus 2′ OH groups of the 5′ exon mimic do not form stabilizing tertiary interactions with the P-8/4x ribozyme, in contrast to the Tetrahymena L-21 ScaI ribozyme. Furthermore, at 37°C, the exogenous 5′ exon mimic d(ATGACT) binds nearly 32 000 times more tightly to the P-8/4x ribozyme than to r(GGUCAU). Therefore, oligonucleotides without 2′ OH groups can exploit tertiary stabilization to bind dramatically more tightly and with more specificity than possible from base pairing. These results suggest a new paradigm for antisense targeting: targeting the tertiary interactions of structural RNAs with short antisense oligonucleotides.
Infection and Immunity, 2007
Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a bi... more 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
Infection and Immunity, 2003
Specific antibody opsonization significantly enhances the level of phagocytosis of Candida in the... more Specific antibody opsonization significantly enhances the level of phagocytosis of Candida in the absence of complement. Furthermore, we have described a system using a recombinant human antibody single-chain variable fragment that allows a comparative study of phagocytosis of multiple Candida species opsonized via a common antigen.
Dna Research, 1998
Since the mouse offers an easily manipulated experimental animal model for the study of the im- m... more Since the mouse offers an easily manipulated experimental animal model for the study of the im- munopathogenesis of pneumonia caused by the opportunist Pneumocystis carinii, we cloned and character- ized cDNAs encoding an abundant, immunogenic surface antigen termed glycoprotein A (gpA) from mouse P. carinii. A cDNA library was constructed in bacteriophage Agtll from P. carinii -infected mouse lung poly(A+)
The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clin... more 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 con- firmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concen- tration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin
Inaseries offive experiments, weattempted totransmit Pneumocystis carinii fromferrets toSCIDmiceb... more Inaseries offive experiments, weattempted totransmit Pneumocystis carinii fromferrets toSCIDmiceby intratracheal inoculation. Using highly specific andsensitive assay techniques, wecould notdocument infection ofSCIDmicebyP.carinii isolated fromferrets. Incontrast, underidentical inoculation conditions, P.carinii waseasily transmissible fromoneSCIDmousetoanother. Theseresults indicate that P.carinii organisms, at least those isolated fromferrets, havearestricted hostrange. Thefinding ofrestricted transmission ofP.carinii isconsistent withtheincreasing evidence forhostspecies-specific antigenic variation amongisolates ofP. carinii. Ifrestricted hostrange isaconsistent biological
Lung Biology in Health and Disease, 2004
To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage... more To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain
The objective of the study was to determine whether vestibular fibroblasts from vulvar vestibulit... more The objective of the study was to determine whether vestibular fibroblasts from vulvar vestibulitis (VVS) patients produce higher proinflammatory cytokine levels when provoked with Candida albicans (yeast) and alpha-melanocyte-stimulating hormone (␣-MSH) in vitro.
American Journal of Obstetrics and Gynecology, 2015
Our goal was to gain a better understanding of the inflammatory pathways affected during localize... more Our goal was to gain a better understanding of the inflammatory pathways affected during localized vulvodynia, a poorly understood, common, and debilitating condition characterized by chronic pain of the vulvar vestibule. In a control matched study, primary human fibroblast strains were generated from biopsies collected from localized provoked vulvodynia (LPV) cases and from age- and race-matched controls. We then examined intracellular mechanisms by which these fibroblasts recognize pathogenic Candida albicans; >70% of vulvodynia patients report the occurrence of prior chronic Candida infections, which is accompanied by localized inflammation and elevated production of proinflammatory/pain-associated interleukin (IL)-6 and prostaglandin E2 (PGE2). We focused on examining the signaling pathways involved in recognition of yeast components that are present and abundant during chronic infection. Dectin-1, a surface receptor that binds C albicans cell wall glucan, was significantly elevated in vestibular vs external vulvar cells (from areas without pain) in both cases and controls, while its abundance was highest in LPV cases. Blocking Dectin-1 signaling significantly reduced pain-associated IL-6 and PGE2 production during the response to C albicans. Furthermore, LPV patient vestibular cells produced inflammatory mediators in response to low numbers of C albicans cells, while external vulvar fibroblasts were nonresponsive. Inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (proinflammatory transcription factor) nearly abrogated IL-6 and PGE2 production induced by C albicans, in keeping with observations that Dectin-1 signals through the nuclear factor kappa-light-chain-enhancer of activated B cells pathway. These findings implicate that a fibroblast-mediated proinflammatory response to C albicans contributes to the induction of pain in LPV cases. Targeting this response may be an ideal strategy for the development of new vulvodynia therapies.
Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during ... more Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during wound repair and tissue remodeling. Thus, we sought to determine whether A549 lung epithelial type II-like cells would endocytose insoluble, surface-bound FBG as a potential mechanism of alveolar matrix remodeling. Surface-bound FBG was endocytosed into either lysosomes or late endosomes by A549 cells through arg-gly- asp-dependent binding to
Antimicrobial Agents and Chemotherapy, 2005
Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents... more Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents and light, termed photodynamic therapy (PDT), offers an alternative to conventional treatments. Initial studies using the clinically approved photosensitizer Photofrin demonstrated the susceptibility of C. albicans to its photodynamic effects. In the present study, we have further refined parameters for Photofrin-mediated pho- todynamic action against C. albicans and examined
Journal of Immunological Methods, 2001
A combinatorial phage display library expressing human immunoglobulin heavy and light chain varia... more A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were
Infection and Immunity, 2006
Mice immunized with recombinant mouse Pneumocystis carinii antigen A12-thiredoxin fusion protein ... more 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 A12thioredoxin had significantly reduced lung P. carinii burdens after CD4 ؉ T-cell depletion and challenge with P. carinii.
Photochemistry and Photobiology, 2008
Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease p... more Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease problem and are often difficult to eradicate because of the high frequency of resistance to conventional antifungal agents. Photodynamic treatment (PDT) offers an attractive therapeutic alternative. Previous studies demonstrated that filamentous forms and biofilms of Candida albicans were sensitive to PDT using Photofrin as a photosensitizer. However, early stationary phase yeast forms of C. albicans and Candida glabrata were not adversely affected by treatment. We report that the cationic porphyrin photosensitizer meso-tetra (N-methyl-4pyridyl) porphine tetra tosylate (TMP-1363) is effective in PDT against yeast forms of C. albicans and C. glabrata. Respiratory-deficient (RD) strains of C. albicans and C. glabrata display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, the salivary antimicrobial peptides histatins and other types of toxic stresses. In contrast to this pattern, RD mutants of both C. albicans and C. glabrata were significantly more sensitive to PDT compared to parental strains. These data suggest that intact mitochondrial function may provide a basal level of anti-oxidant defense against PDT-induced phototoxicity in Candida, and reveals pathways of resistance to oxidative stress that can potentially be targeted to increase the efficacy of PDT against this pathogenic fungus.
Molecular Microbiology, 1993
Lasers in Surgery and Medicine, 2013
The primary therapy for deep tissue abscesses is drainage accompanied by systemic antimicrobial t... more The primary therapy for deep tissue abscesses is drainage accompanied by systemic antimicrobial treatment. However, the long antibiotic course required increases the probability of acquired resistance, and the high incidence of polymicrobial infections in abscesses complicates treatment choices. Photodynamic therapy (PDT) is effective against multiple classes of organisms, including those displaying drug resistance, and may serve as a useful adjunct to the standard of care by reduction of abscess microbial burden following drainage. Aspirates were obtained from 32 patients who underwent image-guided percutaneous drainage of the abscess cavity. The majority of the specimens (24/32) were abdominal, with the remainder from liver and lung. Conventional microbiological techniques and nucleotide sequence analysis of rRNA gene fragments were used to characterize microbial populations from abscess aspirates. We evaluated the sensitivity of microorganisms to methylene blue-sensitized PDT in vitro both within the context of an abscess aspirate and as individual isolates. Most isolates were bacterial, with the fungus Candida tropicalis also isolated from two specimens. We examined the sensitivity of these microorganisms to methylene blue-PDT. Complete elimination of culturable microorganisms was achieved in three different aspirates, and significant killing (P < 0.0001) was observed in all individual microbial isolates tested compared to controls. These results and the technical feasibility of advancing optical fibers through catheters at the time of drainage motivate further work on including PDT as a therapeutic option during abscess treatment.
J Invest Dermatol, 2000
The human CD80 costimulatory molecule is an important signal between professional antigen-present... more 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.
Biochemistry, 2001
A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human path... more A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human pathogen Candida albicans. Both the precursor and ribozyme are functional as determined from in vitro assays. Comparisons of dissociation constants for oligonucleotide binding to the ribozyme and to a hexanucleotide mimic of its internal guide sequence lead to a model for recognition of the 5′ exon substrate by this intron. In particular, tertiary contacts with the P1 helix that help align the splice site include three 2′-hydroxyl groups, a G‚U pair that occurs at the intron's splice junction, and a G‚A pair. The free energy contribution that each interaction contributes to tertiary binding is determined. When the G‚A pair is replaced with a G-C pair, tertiary interactions to 5′ exon mimic 2′-hydroxyl groups are significantly weakened. When the G‚A pair is replaced with a G‚U pair, tertiary interactions are retained and binding is 10-fold tighter. These results expand our knowledge of substrate recognition by group I introns, and also provide a basis for rational design of oligonucleotide-based therapeutics for targeting group I introns by binding enhancement by tertiary interactions and suicide inhibition strategies.
Journal of Eukaryotic Microbiology, 1994
Using RNA: RNA in situ hybridization, the intracellular location of a transcript encoded by and s... more Using RNA: RNA in situ hybridization, the intracellular location of a transcript encoded by and spanning the entire length of a Trypanosoma cruzi kinetoplast DNA minicircle was determined. In axenically cultured T. cruzi epimastigotes, the hybridization signal was restricted to the kinetoplast, which was situated in the perinuclear region of the cell. Following conversion of epimastigotes to culture-derived metacyclic trypomastigotes, the kinetoplast moved to an acentric position in the metacyclic trypomastigote. Again, the hybridization signal co-localized with the position of the kinetoplast. These results suggested that the transcript remained closely associated with the T. cruzi kinetoplast within the mitochondrion in each of the morphological forms. Using specific oligonucleotide probes derived from a cDNA encoding the transcript, the entire native kDNA minicircle encoding the transcript was cloned and its nucleotide sequence was determined. The nucleotide sequence of the intact native minicircle was identical to that of the full-length cDNA corresponding to the minicircle transcript, indicating that the transcript was not modified prior to the time of cDNA synthesis and cloning.
Biochemistry, 1997
The recent increase in the population of immunocompromised patients has led to an insurgence of o... more The recent increase in the population of immunocompromised patients has led to an insurgence of opportunistic human fungal infections. The lack of effective treatments against some of these pathogens makes it important to develop new therapeutic strategies. One such strategy is to target key RNAs with antisense compounds. We report the development of a model system for studying the potential for antisense targeting of group I self-splicing introns in fungal pathogens. The group I intron from the large ribosomal subunit RNA of mouse-derived Pneumocystis carinii has been isolated and characterized. This intron self-splices in Vitro. A catalytically active ribozyme, P-8/4x, has been constructed from this intron to allow measurement of dissociation constants for potential antisense agents. At 37°C, in 50 mM Hepes (25 mM Na + ), 15 mM MgCl 2 , and 135 mM KCl at pH 7.5, the exogenous 5′ exon mimic r(AUGACU) binds about 60 000 times more tightly to this ribozyme than to r(GGUCAU), a mimic of its complementary binding site on the ribozyme. This enhanced binding is due to tertiary interactions. This tertiary stabilization is increased by single deoxynucleotide substitutions in the exon mimic at every position except for the internal A, which is essentially unchanged. Thus 2′ OH groups of the 5′ exon mimic do not form stabilizing tertiary interactions with the P-8/4x ribozyme, in contrast to the Tetrahymena L-21 ScaI ribozyme. Furthermore, at 37°C, the exogenous 5′ exon mimic d(ATGACT) binds nearly 32 000 times more tightly to the P-8/4x ribozyme than to r(GGUCAU). Therefore, oligonucleotides without 2′ OH groups can exploit tertiary stabilization to bind dramatically more tightly and with more specificity than possible from base pairing. These results suggest a new paradigm for antisense targeting: targeting the tertiary interactions of structural RNAs with short antisense oligonucleotides.
Infection and Immunity, 2007
Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a bi... more 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
Infection and Immunity, 2003
Specific antibody opsonization significantly enhances the level of phagocytosis of Candida in the... more Specific antibody opsonization significantly enhances the level of phagocytosis of Candida in the absence of complement. Furthermore, we have described a system using a recombinant human antibody single-chain variable fragment that allows a comparative study of phagocytosis of multiple Candida species opsonized via a common antigen.
Dna Research, 1998
Since the mouse offers an easily manipulated experimental animal model for the study of the im- m... more Since the mouse offers an easily manipulated experimental animal model for the study of the im- munopathogenesis of pneumonia caused by the opportunist Pneumocystis carinii, we cloned and character- ized cDNAs encoding an abundant, immunogenic surface antigen termed glycoprotein A (gpA) from mouse P. carinii. A cDNA library was constructed in bacteriophage Agtll from P. carinii -infected mouse lung poly(A+)
The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clin... more 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 con- firmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concen- tration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin
Inaseries offive experiments, weattempted totransmit Pneumocystis carinii fromferrets toSCIDmiceb... more Inaseries offive experiments, weattempted totransmit Pneumocystis carinii fromferrets toSCIDmiceby intratracheal inoculation. Using highly specific andsensitive assay techniques, wecould notdocument infection ofSCIDmicebyP.carinii isolated fromferrets. Incontrast, underidentical inoculation conditions, P.carinii waseasily transmissible fromoneSCIDmousetoanother. Theseresults indicate that P.carinii organisms, at least those isolated fromferrets, havearestricted hostrange. Thefinding ofrestricted transmission ofP.carinii isconsistent withtheincreasing evidence forhostspecies-specific antigenic variation amongisolates ofP. carinii. Ifrestricted hostrange isaconsistent biological
Lung Biology in Health and Disease, 2004
To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage... more To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain
The objective of the study was to determine whether vestibular fibroblasts from vulvar vestibulit... more The objective of the study was to determine whether vestibular fibroblasts from vulvar vestibulitis (VVS) patients produce higher proinflammatory cytokine levels when provoked with Candida albicans (yeast) and alpha-melanocyte-stimulating hormone (␣-MSH) in vitro.
American Journal of Obstetrics and Gynecology, 2015
Our goal was to gain a better understanding of the inflammatory pathways affected during localize... more Our goal was to gain a better understanding of the inflammatory pathways affected during localized vulvodynia, a poorly understood, common, and debilitating condition characterized by chronic pain of the vulvar vestibule. In a control matched study, primary human fibroblast strains were generated from biopsies collected from localized provoked vulvodynia (LPV) cases and from age- and race-matched controls. We then examined intracellular mechanisms by which these fibroblasts recognize pathogenic Candida albicans; >70% of vulvodynia patients report the occurrence of prior chronic Candida infections, which is accompanied by localized inflammation and elevated production of proinflammatory/pain-associated interleukin (IL)-6 and prostaglandin E2 (PGE2). We focused on examining the signaling pathways involved in recognition of yeast components that are present and abundant during chronic infection. Dectin-1, a surface receptor that binds C albicans cell wall glucan, was significantly elevated in vestibular vs external vulvar cells (from areas without pain) in both cases and controls, while its abundance was highest in LPV cases. Blocking Dectin-1 signaling significantly reduced pain-associated IL-6 and PGE2 production during the response to C albicans. Furthermore, LPV patient vestibular cells produced inflammatory mediators in response to low numbers of C albicans cells, while external vulvar fibroblasts were nonresponsive. Inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (proinflammatory transcription factor) nearly abrogated IL-6 and PGE2 production induced by C albicans, in keeping with observations that Dectin-1 signals through the nuclear factor kappa-light-chain-enhancer of activated B cells pathway. These findings implicate that a fibroblast-mediated proinflammatory response to C albicans contributes to the induction of pain in LPV cases. Targeting this response may be an ideal strategy for the development of new vulvodynia therapies.
Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during ... more Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during wound repair and tissue remodeling. Thus, we sought to determine whether A549 lung epithelial type II-like cells would endocytose insoluble, surface-bound FBG as a potential mechanism of alveolar matrix remodeling. Surface-bound FBG was endocytosed into either lysosomes or late endosomes by A549 cells through arg-gly- asp-dependent binding to
Antimicrobial Agents and Chemotherapy, 2005
Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents... more Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents and light, termed photodynamic therapy (PDT), offers an alternative to conventional treatments. Initial studies using the clinically approved photosensitizer Photofrin demonstrated the susceptibility of C. albicans to its photodynamic effects. In the present study, we have further refined parameters for Photofrin-mediated pho- todynamic action against C. albicans and examined
Journal of Immunological Methods, 2001
A combinatorial phage display library expressing human immunoglobulin heavy and light chain varia... more A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were
Infection and Immunity, 2006
Mice immunized with recombinant mouse Pneumocystis carinii antigen A12-thiredoxin fusion protein ... more 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 A12thioredoxin had significantly reduced lung P. carinii burdens after CD4 ؉ T-cell depletion and challenge with P. carinii.
Photochemistry and Photobiology, 2008
Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease p... more Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease problem and are often difficult to eradicate because of the high frequency of resistance to conventional antifungal agents. Photodynamic treatment (PDT) offers an attractive therapeutic alternative. Previous studies demonstrated that filamentous forms and biofilms of Candida albicans were sensitive to PDT using Photofrin as a photosensitizer. However, early stationary phase yeast forms of C. albicans and Candida glabrata were not adversely affected by treatment. We report that the cationic porphyrin photosensitizer meso-tetra (N-methyl-4pyridyl) porphine tetra tosylate (TMP-1363) is effective in PDT against yeast forms of C. albicans and C. glabrata. Respiratory-deficient (RD) strains of C. albicans and C. glabrata display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, the salivary antimicrobial peptides histatins and other types of toxic stresses. In contrast to this pattern, RD mutants of both C. albicans and C. glabrata were significantly more sensitive to PDT compared to parental strains. These data suggest that intact mitochondrial function may provide a basal level of anti-oxidant defense against PDT-induced phototoxicity in Candida, and reveals pathways of resistance to oxidative stress that can potentially be targeted to increase the efficacy of PDT against this pathogenic fungus.
Molecular Microbiology, 1993
Lasers in Surgery and Medicine, 2013
The primary therapy for deep tissue abscesses is drainage accompanied by systemic antimicrobial t... more The primary therapy for deep tissue abscesses is drainage accompanied by systemic antimicrobial treatment. However, the long antibiotic course required increases the probability of acquired resistance, and the high incidence of polymicrobial infections in abscesses complicates treatment choices. Photodynamic therapy (PDT) is effective against multiple classes of organisms, including those displaying drug resistance, and may serve as a useful adjunct to the standard of care by reduction of abscess microbial burden following drainage. Aspirates were obtained from 32 patients who underwent image-guided percutaneous drainage of the abscess cavity. The majority of the specimens (24/32) were abdominal, with the remainder from liver and lung. Conventional microbiological techniques and nucleotide sequence analysis of rRNA gene fragments were used to characterize microbial populations from abscess aspirates. We evaluated the sensitivity of microorganisms to methylene blue-sensitized PDT in vitro both within the context of an abscess aspirate and as individual isolates. Most isolates were bacterial, with the fungus Candida tropicalis also isolated from two specimens. We examined the sensitivity of these microorganisms to methylene blue-PDT. Complete elimination of culturable microorganisms was achieved in three different aspirates, and significant killing (P < 0.0001) was observed in all individual microbial isolates tested compared to controls. These results and the technical feasibility of advancing optical fibers through catheters at the time of drainage motivate further work on including PDT as a therapeutic option during abscess treatment.