Fungal chitin is not an independent mediator of allergic fungal asthma severity.

Chitin, a polysaccharide found in the fungal cell wall and the exoskeletons of house dust mites and cockroaches, has garnered attention as a potential immunoreactive allergen. Mammals have evolved to express chitin-degrading chitinases (acidic mammalian chitinase/AMCase and chitotriosidase) that may modulate immune responses to chitin. We have previously reported that mice deficient in AMCase (Chia-/-) demonstrated better lung function during allergic fungal asthma. As expected, we show that mice overexpressing AMCase (SPAM mice) had worse airway hyperreactivity (AHR) during allergic fungal asthma. We further demonstrate that chitin-positive Aspergillus fumigatus conidia are detectable in the allergic lung during chronic exposure. Lung function in Chia-/- and SPAM mice directly correlated with the level of chitinase activity during chronic fungal exposure (Chia-/- mice, negligible chitinase activity, lower AHR; SPAM mice, heightened chitinase activity, higher AHR), suggesting that the breakdown of chitin promoted AHR. However, chronic exposure of normal mice to purified A. fumigatus chitin resulted in only moderate inflammatory changes in the lung which were not sufficient to induce AHR. Moreover, despite having dramatic differences in chitinase activity, chronic exposure of Chia-/- and SPAM mice to purified A. fumigatus chitin likewise did not modulate AHR. Collectively, these results indicate that chronic exposure to fungal chitin alone is incapable of driving AHR. Furthermore, our data suggests that the chitinase-mediated degradation of chitin associated with A. fumigatus conidia may facilitate unmasking and/or liberation of other fungal cell wall components that drive inflammatory responses which contribute to AHR.

Impact of Intravenous Fat Emulsion Choice on Candida Biofilm, Hyphal Growth, and Catheter-Related Bloodstream Infections in Pediatric Patients.

BACKGROUND: Use of mixed-oil (MO) intravenous fat emulsion (IFE) was shown to inhibit Candida albicans biofilm formation and overall rate of catheter-related bloodstream infections (CR-BSIs) compared with soybean-oil (SO) IFE). We aimed to delineate this inhibitory mechanism and impact of IFE choice on distribution of fungal CR-BSIs. METHODS: Transcriptional profiling was conducted on C. albicans grown in SO-IFE, MO-IFE, or SO-IFE with capric acid. Overexpression strains of shared down-regulated genes were constructed using a tetracycline-off system to assess hypha and biofilm formation in IFEs. A 5-year retrospective multicenter cohort study was performed to assess differences in CR-BSIs caused by Candida species based on the IFE formulation received in pediatric patients. RESULTS: Genes significantly down-regulated in MO-IFE and SO-IFE with capric acid included CDC11, HGC1, and UME6. Overexpression of HGC1 or UME6 enabled filamentation in capric acid and MO-IFE. Interestingly, only overexpression of UME6 was sufficient to rescue biofilm growth in MO-IFE. MO-IFE administration was associated with a higher proportion of non-albicans Candida versus C. albicans CR-BSIs (42% vs 33%; odds ratio, 1.22 [95% confidence interval, .46-3.26]). CONCLUSIONS: MO-IFE affects C. albicans biofilm formation and hyphal growth via a UME6-dependent mechanism. A numerical but not statistically significant difference in distribution of Candida spp. among CR-BSIs was observed.

Delivery of carbohydrates, amino acids, and lipids via intravenous catheters is necessary for some patients to supply daily caloric needs. These nutrient-dense parenteral solutions can promote microbial biofilm growth on the catheter surface, which may seed subsequent catheter-related bloodstream infection (CR-BSI). In fact, receipt of parenteral nutrition is an established risk factor for CR-BSI caused by the polymorphic fungal pathogen Candida albicans. New intravenous fat emulsions (IFEs) have gained market share and IFEs containing capric acid (mixed-oil [MO] IFE) compared with those without (soybean-oil [SO] IFE) impair the C. albicans yeast-to-hypha switch­a trait strongly associated with pathogenicity and biofilm formation. In this study, we found that MO-IFE and capric acid reduced expression of a transcriptional regulator involved in hyphal extension (UME6) and down-regulated genes involved in cell partitioning (HGC1). Overexpression of these genes enabled hyphal growth in MO-IFE. Secondly, we sought to determine whether the type of IFE administered was associated with the clinical incidence of CR-BSIs caused by C. albicans or other common non-albicans Candida species. There was a nonsignificant numerical reduction in C. albicans infections in patients administered MO-IFE compared with SO-IFE. Collectively, this work shows that IFEs differentially affect Candida biology with potential infectious consequences for the patient.

Mucosal Infection with Unmasked Candida albicans Cells Impacts Disease Progression in a Host Niche-Specific Manner.

Shielding the immunogenic cell wall epitope ß(1, 3)-glucan under an outer layer of mannosylated glycoproteins is an essential virulence factor deployed by Candida albicans during systemic infection. Accordingly, mutants with increased ß(1, 3)-glucan exposure (unmasking) display increased immunostimulatory capabilities in vitro and attenuated virulence during systemic infection in mice. However, little work has been done to assess the impact of increased unmasking during the two most common manifestations of candidiasis, namely, oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis (VVC). We have shown previously that the expression of a single hyperactive allele of the MAP3K STE11ΔN467 induces unmasking via the Cek1 MAPK pathway, attenuates fungal burden, and prolongs survival during systemic infection in mice. Here, we expand on these findings and show that infection with an unmasked STE11ΔN467 mutant also impacts disease progression during OPC and VVC murine infection models. Male mice sublingually infected with the STE11ΔN467 mutant showed a significant reduction in tongue fungal burden at 2 days postinfection and a modest reduction at 5 days postinfection. However, we find that selection for STE11ΔN467 suppressor mutants that no longer display increased unmasking occurs within the oral cavity and is likely responsible for the restoration of fungal burden trends to wild-type levels later in the infection. In the VVC infection model, no attenuation in fungal burden was observed. However, polymorphonuclear cell recruitment and interleukin-1ß (IL-1ß) levels within the vaginal lumen, markers of immunopathogenesis, were increased in mice infected with unmasked STE11ΔN467 cells. Thus, our data suggest a niche-specific impact for unmasking on disease progression.

Immunogenicity and protective efficacy of a pan-fungal vaccine in preclinical models of aspergillosis, candidiasis, and pneumocystosis.

Invasive fungal infections cause over 1.5 million deaths worldwide. Despite increases in fungal infections as well as the numbers of individuals at risk, there are no clinically approved fungal vaccines. We produced a "pan-fungal" peptide, NXT-2, based on a previously identified vaccine candidate and homologous sequences from Pneumocystis, Aspergillus,Candida, and Cryptococcus. We evaluated the immunogenicity and protective capacity of NXT-2 in murine and nonhuman primate models of invasive aspergillosis, systemic candidiasis, and pneumocystosis. NXT-2 was highly immunogenic and immunized animals had decreased mortality and morbidity compared to nonvaccinated animals following induction of immunosuppression and challenge with Aspergillus, Candida, or Pneumocystis. Data in multiple animal models support the concept that immunization with a pan-fungal vaccine prior to immunosuppression induces broad, cross-protective antifungal immunity in at-risk individuals.

A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

Exogenous Reproductive Hormones nor Candida albicans Colonization Alter the Near Neutral Mouse Vaginal pH.

While human vaginal pH in childbearing-age women is conclusively acidic, the mouse vaginal pH is reported as being near neutral. However, this information appears to be somewhat anecdotal with respect to vulvovaginal candidiasis, as such claims in the literature frequently lack citations of studies that specifically address this physiological factor. Given the disparate pH between mice and humans, the role of exogenous hormones and colonization by the fungal pathogen Candida albicans in shaping vaginal pH was assessed. Use of a convenient modified vaginal lavage technique with the pH indicator dye phenol red demonstrated that indeed vaginal pH was near neutral (7.2 ± 0.24) and was not altered by delivery of progesterone or estrogen in C57BL/6 mice. These trends were conserved in DBA/2 and CD-1 mouse backgrounds, commonly used in the mouse model of vaginitis. It was also determined that vaginal colonization with C. albicans did not alter the globally neutral vaginal pH over the course of one week. Construction and validation of a C. albicans reporter strain expressing GFPy, driven by the pH-responsive PHR1 promoter, confirmed the murine vaginal pH to be at least ≥6.0. Collectively, our data convincingly demonstrate a stable and conserved near neutrality of the mouse vaginal pH during vulvovaginal candidiasis and should serve as a definitive source for future reference. Implications and rationale for disparate pH in this model system are also discussed.

Vulvovaginal Candidiasis: A Current Understanding and Burning Questions.

Candida albicans, along with other closely related Candida species, are the primary causative agents of vulvovaginal candidiasis (VVC)-a multifactorial infectious disease of the lower female reproductive tract resulting in pathologic inflammation. Unlike other forms of candidiasis, VVC is a disease of immunocompetent and otherwise healthy women, most predominant during their child-bearing years. While VVC is non-lethal, its high global incidence and profound negative impact on quality-of-life necessitates further understanding of the host and fungal factors that drive disease pathogenesis. In this review, we cover the current state of our understanding of the epidemiology, host response, fungal pathogenicity mechanisms, impact of the microbiome, and novel approaches to treatment of this most prevalent human candidal infection. We also offer insight into the latest advancements in the VVC field and identify important questions that still remain.

The Interleukin (IL) 17R/IL-22R Signaling Axis Is Dispensable for Vulvovaginal Candidiasis Regardless of Estrogen Status.

Candida albicans, a ubiquitous commensal fungus that colonizes human mucosal tissues and skin, can become pathogenic, clinically manifesting most commonly as oropharyngeal candidiasis and vulvovaginal candidiasis (VVC). Studies in mice and humans convincingly show that T-helper 17 (Th17)/interleukin 17 (IL-17)-driven immunity is essential to control oral and dermal candidiasis. However, the role of the IL-17 pathway during VVC remains controversial, with conflicting reports from human data and mouse models. Like others, we observed induction of a strong IL-17-related gene signature in the vagina during estrogen-dependent murine VVC. As estrogen increases susceptibility to vaginal colonization and resulting immunopathology, we asked whether estrogen use in the standard VVC model masks a role for the Th17/IL-17 axis. We demonstrate that mice lacking IL-17RA, Act1, or interleukin 22 showed no evidence for altered VVC susceptibility or immunopathology, regardless of estrogen administration. Hence, these data support the emerging consensus that Th17/IL-17 axis signaling is dispensable for the immunopathogenesis of VVC.

The Host Immune System Facilitates Disseminated Staphylococcus aureus Disease Due to Phagocytic Attraction to Candida albicans during Coinfection: a Case of Bait and Switch.

Invasive Staphylococcus aureus infections account for 15 to 50% of fatal bloodstream infections annually. These disseminated infections often arise without a defined portal of entry into the host but cause high rates of mortality. The fungus Candida albicans and the Gram-positive bacterium S. aureus can form polymicrobial biofilms on epithelial tissue, facilitated by the C. albicans adhesin encoded by ALS3 While a bacterium-fungus interaction is required for systemic infection, the mechanism by which bacteria disseminate from the epithelium to internal organs is unclear. In this study, we show that highly immunogenic C. albicans hyphae attract phagocytic cells, which rapidly engulf adherent S. aureus and subsequently migrate to cervical lymph nodes. Following S. aureus-loaded phagocyte translocation from the mucosal surface, S. aureus produces systemic disease with accompanying morbidity and mortality. Our results suggest a novel role for the host in facilitating a bacterium-fungus infectious synergy, leading to disseminated staphylococcal disease.

Disparate Candida albicans Biofilm Formation in Clinical Lipid Emulsions Due to Capric Acid-Mediated Inhibition.

Receipt of parenteral nutrition (PN) remains an independent risk factor for developing catheter-related bloodstream infections (CR-BSI) caused by fungi, including by the polymorphic fungus Candida albicans, which is notoriously adept at forming drug-resistant biofilm structures. Among a variety of macronutrients, PN solutions contain lipid emulsions to supply daily essential fats and are often delivered via central venous catheters (CVCs). Therefore, using an in vitro biofilm model system, we sought to determine whether various clinical lipid emulsions differentially impacted biofilm growth in C. albicans We observed that the lipid emulsions Intralipid and Omegaven both stimulated C. albicans biofilm formation during growth in minimal medium or a macronutrient PN solution. Conversely, Smoflipid inhibited C. albicans biofilm formation by approximately 50%. Follow-up studies revealed that while Smoflipid did not impair C. albicans growth, it did significantly inhibit hypha formation and hyphal elongation. Moreover, growth inhibition could be recapitulated in Intralipid when supplemented with capric acid-a fatty acid present in Smoflipid but absent in Intralipid. Capric acid was also found to dose dependently inhibit C. albicans biofilm formation in PN solutions. This is the first study to directly compare different clinical lipid emulsions for their capacity to affect C. albicans biofilm growth. Results derived from this study necessitate further research regarding different lipid emulsions and rates of fungus-associated CR-BSIs.

Comparative Analysis of the Capacity of the Candida Species To Elicit Vaginal Immunopathology.

The human fungal pathogen Candida albicans is the major etiological agent of vulvovaginal candidiasis (VVC). Despite this fact, other non-albicans Candida (NAC) species have frequently been reported, as well. Despite their presence in the vaginal environment, little is known about their capacities to elicit immune responses classically associated with C. albicans-mediated immunopathology, including neutrophil recruitment and proinflammatory cytokine signaling. Therefore, using a combination of in vitro and in vivo approaches, we undertook a comparative analysis to determine whether a representative panel of NAC species could colonize, induce immunopathological markers, or cause damage at the vaginal mucosa. Using a murine model of VVC, C. albicans was found to induce robust immunopathology (neutrophils and interleukin 1ß [IL-1ß]) and elicit mucosal damage. However, all the NAC species tested (including C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. glabrata, and C. auris) induced significantly less damage and neutrophil recruitment than C. albicans, despite achieving similar early colonization levels. These results largely correlated with a notable lack of ability by the NAC species (including C. dubliniensis and C. tropicalis) to form hyphae both in vitro and in vivo Furthermore, both C. dubliniensis and C. tropicalis induced significantly less expression of the ECE1 gene encoding candidalysin, a key fungal virulence determinant driving VVC immunopathology. In order to determine the relative capacities of these species to elicit inflammasome-dependent IL-1ß release, both wild-type and NLRP3-/- THP-1 cells were challenged in vitro While most species tested elicited only modest amounts of IL-1ß, challenge with C. albicans led to significantly elevated levels that were largely NLRP3 dependent. Collectively, our findings demonstrate that although NAC species are increasingly reported as causative agents of VVC, C. albicans appears to be exceedingly vaginopathogenic, exhibiting robust immunopathology, hypha formation, and candidalysin expression. Thus, this study provides mechanistic insight into why C. albicans is overwhelmingly the major pathogen reported during VVC.

Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity.

Inactivation of sterol Δ5,6-desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3-deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicanserg3Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistance.IMPORTANCE While conferring resistance to the azole antifungals in vitro, loss of sterol Δ5,6-desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted.

Candidalysin Drives Epithelial Signaling, Neutrophil Recruitment, and Immunopathology at the Vaginal Mucosa.

Unlike other forms of candidiasis, vulvovaginal candidiasis, caused primarily by the fungal pathogen Candida albicans, is a disease of immunocompetent and otherwise healthy women. Despite its prevalence, the fungal factors responsible for initiating symptomatic infection remain poorly understood. One of the hallmarks of vaginal candidiasis is the robust recruitment of neutrophils to the site of infection, which seemingly do not clear the fungus, but rather exacerbate disease symptomatology. Candidalysin, a newly discovered peptide toxin secreted by C. albicans hyphae during invasion, drives epithelial damage, immune activation, and phagocyte attraction. Therefore, we hypothesized that Candidalysin is crucial for vulvovaginal candidiasis immunopathology. Anti-Candida immune responses are anatomical-site specific, as effective gastrointestinal, oral, and vaginal immunities are uniquely compartmentalized. Thus, we aimed to identify the immunopathologic role of Candidalysin and downstream signaling events at the vaginal mucosa. Microarray analysis of C. albicans-infected human vaginal epithelium in vitro revealed signaling pathways involved in epithelial damage responses, barrier repair, and leukocyte activation. Moreover, treatment of A431 vaginal epithelial cells with Candidalysin induced dose-dependent proinflammatory cytokine responses (including interleukin 1α [IL-1α], IL-1ß, and IL-8), damage, and activation of c-Fos and mitogen-activated protein kinase (MAPK) signaling, consistent with fungal challenge. Mice intravaginally challenged with C. albicans strains deficient in Candidalysin exhibited no differences in colonization compared to isogenic controls. However, significant decreases in neutrophil recruitment, damage, and proinflammatory cytokine expression were observed with these strains. Our findings demonstrate that Candidalysin is a key hypha-associated virulence determinant responsible for the immunopathogenesis of C. albicans vaginitis.

Diet and Exercise: a Match Made in Bone.

PURPOSE OF REVIEW: Multiple dietary components have the potential to positively affect bone mineral density in early life and reduce loss of bone mass with aging. In addition, regular weight-bearing physical activity has a strong positive effect on bone through activation of osteocyte signaling. We will explore possible synergistic effects of dietary components and mechanical stimuli for bone health by identifying dietary components that have the potential to alter the response of osteocytes to mechanical loading. RECENT FINDINGS: Several (sub)cellular aspects of osteocytes determine their signaling towards osteoblasts and osteoclasts in response to mechanical stimuli, such as the osteocyte cytoskeleton, estrogen receptor α, the vitamin D receptor, and the architecture of the lacunocanalicular system. Potential modulators of these features include 1,25-dihydroxy vitamin D3, several forms of vitamin K, and the phytoestrogen genistein. Multiple dietary components potentially affect osteocyte function and therefore may have a synergistic effect on bone health when combined with a regime of physical activity.

Overexpression of Candida albicans Secreted Aspartyl Proteinase 2 or 5 Is Not Sufficient for Exacerbation of Immunopathology in a Murine Model of Vaginitis.

The secreted aspartyl proteinases of Candida albicans have long been implicated in virulence at the mucosal surface, including contributions to colonization and immunopathogenesis during vulvovaginal candidiasis. In an effort to disentangle hypha-associated virulence factor regulation from morphological transition, the purpose of this study was to determine if overexpression of SAP2 or SAP5 in an efg1Δ/Δ cph1Δ/Δ mutant could restore the capacity to cause immunopathology during murine vaginitis to this avirulent hypofilamentous strain. Two similar yet distinct genetic approaches were used to construct expression vectors to achieve SAP overexpression, and both genetic and functional assays confirmed elevated SAP activity in transformed strains. Similar to previous findings, intravaginal challenge of C57BL/6 mice with hypha-defective strains attained high levels of mucosal colonization but failed to induce robust vaginal immunopathology (neutrophil recruitment, interleukin-1ß [IL-1ß] secretion, and lactate dehydrogenase release) compared to that with the hypha-competent control. Moreover, constitutive expression of SAP2 or SAP5 in two distinct sets of such strains did not elicit immunopathological markers at levels above those observed during challenge with isogenic empty vector controls. Therefore, these results suggest that the physiological contributions of SAPs to vaginal immunopathology require hypha formation, other hypha-associated factors, or genetic interaction with EFG1 and/or CPH1 to cause symptomatic infection. Additionally, the outlined expression strategy and strain sets will be useful for decoupling other downstream morphogenetic factors from hyphal growth.

Candida albicans in Multispecies Oral Communities; A Keystone Commensal?

The complexity of the oral cavity, in which many hundreds of microbial species interact represents a challenge for modern microbiologists. What are all these species doing there? And why do we accept so many opportunistic pathogens to be part of our health (commensal) microflora? While the role of bacteria are often being studied, the role of fungi in the interactions within the oral cavity are understudied. This is partly because fungi in the oral cavity are generally considered as pathogens and related to diseases. In this chapter we will explore mechanisms of interaction between bacteria and fungi in the oral cavity that are involved in maintenance of oral health. We will argue that fungi in general and C. albicans specifically, should be regarded a keystone commensal in the oral cavity.

Candida-Bacteria Interactions: Their Impact on Human Disease.

Candida species are the most common infectious fungal species in humans; out of the approximately 150 known species, Candida albicans is the leading pathogenic species, largely affecting immunocompromised individuals. Apart from its role as the primary etiology for various types of candidiasis, C. albicans is known to contribute to polymicrobial infections. Polymicrobial interactions, particularly between C. albicans and bacterial species, have gained recent interest in which polymicrobial biofilm virulence mechanisms have been studied including adhesion, invasion, quorum sensing, and development of antimicrobial resistance. These trans-kingdom interactions, either synergistic or antagonistic, may help modulate the virulence and pathogenicity of both Candida and bacteria while uniquely impacting the pathogen-host immune response. As antibiotic and antifungal resistance increases, there is a great need to explore the intermicrobial cross-talk with a focus on the treatment of Candida-associated polymicrobial infections. This article explores the current literature on the interactions between Candida and clinically important bacteria and evaluates these interactions in the context of pathogenesis, diagnosis, and disease management.

In Vitro Models for Candida Biofilm Development.

Development of Candida spp. biofilms on medical devices such as catheters and voice prosthesis has been recognized as an increasing clinical problem. Different in vitro models are presented with increasing complexity. Each model system can be utilized for analysis of new active compounds to prevent or treat Candida biofilms as well as to study molecular processes involved in biofilm formation. Susceptibility studies of clinical isolates are generally performed in a simple 96-well model system similar to the CLSI standard. In the present chapter, optimized conditions that promote biofilm formation within individual wells of microtiter plates are described. In addition, the method has proven useful in preparing C. albicans biofilms for investigation by a variety of microscopic and molecular techniques. A more realistic and more complex biofilm system is presented by the Amsterdam Active Attachment (AAA) model. In this 24-well model all crucial steps of biofilm formation: adhesion, proliferation, and maturation, can be simulated on various surfaces, while still allowing a medium throughput approach. This model has been applied to study susceptibility, complex molecular mechanisms as well as interspecies (Candida-bacterium) interactions. Finally, a realistic microfluidics channel system is presented to follow dynamic processes in biofilm formation. In this Bioflux-based system, molecular mechanisms as well as dynamic processes can be studied at a high time-resolution.

VDR dependent and independent effects of 1,25-dihydroxyvitamin D3 on nitric oxide production by osteoblasts.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) strongly mediates bone mass. Mechanical stimulation also affects bone mass, partly via enhancing nitric oxide (NO) production by osteoblasts. We aimed to determine whether 1,25(OH)(2)D(3) affects NO production by osteoblasts in the presence or absence of mechanical stimulation. We hypothesised that 1,25(OH)(2)D(3) stimulates NO production via nuclear actions of the vitamin D receptor (VDR), which requires hours of incubation with 1,25(OH)(2)D(3) to occur. MC3T3-E1 osteoblasts and long-bone osteoblasts of adult wildtype and VDR(-/-) mice were pre-incubated for 24h with or without 1,25(OH)(2)D(3) (10(-13)-10(-9)M), followed by 30 min pulsating fluid flow (PFF; 0.7±0.3 Pa, 5 Hz) or static culture with or without 1,25(OH)(2)D(3). NO production and NO synthase (NOS) expression were quantified. 10(-11)M 1,25(OH)(2)D(3) for 24h, but not 30 min, stimulated NO production by MC3T3-E1 osteoblasts (eightfold). 1,25(OH)(2)D(3) for 24h increased inducible-NOS gene-expression (twofold), suggesting that 1,25(OH)(2)D(3) stimulated NO production via activation of NOS gene transcription. PFF rapidly increased NO production by MC3T3-E1 osteoblasts, wildtype osteoblasts, and VDR(-/-) osteoblasts. This PFF effect was abolished after incubation with 1,25(OH)(2)D(3) for 24h, or during PFF only. Our results suggest that 1,25(OH)(2)D(3) stimulates inducible-NOS expression and NO production by osteoblasts in the absence of mechanical stimulation, likely via genomic VDR action. In contrast, 1,25(OH)(2)D(3) may affect mechanical loading-induced NO production independent of genomic VDR action, since 1,25(OH)(2)D(3) diminished PFF-induced NO production in VDR(-/-) bone cells. In conclusion, 1,25(OH)(2)D(3) and mechanical loading interact at the level of mechanotransduction, whereby 1,25(OH)(2)D(3) seems to act independently of VDR genomic mechanism.

Mechanical loading stimulates BMP7, but not BMP2, production by osteocytes.

Bone mechanical adaptation is a cellular process that allows bones to adapt their mass and structure to mechanical loading. This process is governed by the osteocytes, which in response to mechanical loading produce signaling molecules that affect osteoblasts and osteoclasts. Bone morphogenic proteins (BMPs) are excellent candidates as signaling molecules, but it is unknown whether mechanically stimulated osteocytes affect bone adaptation through BMP production. Therefore, the aim of this study was to assess whether osteocytes produce BMPs in response to mechanical loading. In addition, since BMP7 has a vitamin D receptor (VDR) response element in the promoter region, we also investigated whether VDR is involved in the BMP7 response to mechanical loading. Human or VDR(-/-) mouse primary bone cells were submitted in vitro to 1 h pulsating fluid flow (PFF) and postincubated without PFF (PI) for 1-24 h, and gene and protein expression of BMP2 and BMP7 were quantified. In human bone cells, PFF did not change BMP2 gene expression, but it upregulated BMP7 gene expression by 4.4- to 5.6-fold at 1-3 h PI and stimulated BMP7 protein expression by 2.4-fold at 6 h PI. PFF did not stimulate BMP7 gene expression in VDR(-/-) mouse bone cells. These results show for the first time that mechanical loading upregulates BMP7, likely via the VDR, but not BMP2, gene and protein expression in osteocytes in vitro. Since BMP7 plays a major role in bone development and remodeling, these data might contribute to a better understanding of the mechanism leading to the mechanical adaptation of bone.