Protection against lethal sepsis following immunization with Candida species varies by isolate and inversely correlates with bone marrow tissue damage.

Protection against lethal Candida albicans (Ca)/Staphylococcus aureus (Sa) intra-abdominal infection (IAI)-mediated sepsis can be achieved by a novel form of trained innate immunity (TII) involving Gr-1+ myeloid-derived suppressor cells (MDSCs) that are induced by inoculation (immunization) with low virulence Candida species [i.e., Candida dubliniensis (Cd)] that infiltrate the bone marrow (BM). In contrast, more virulent Candida species (i.e., C. albicans), even at sub-lethal inocula, fail to induce similar levels of protection. The purpose of the present study was to test the hypothesis that the level of TII-mediated protection induced by Ca strains inversely correlates with damage in the BM as a reflection of virulence. Mice were immunized by intraperitoneal inoculation with several parental and mutant strains of C. albicans deficient in virulence factors (hyphal formation and candidalysin production), followed by an intraperitoneal Ca/Sa challenge 14 d later and monitored for sepsis and mortality. Whole femur bones were collected 24 h and 13 d after immunization and assessed for BM tissue/cellular damage via ferroptosis and histology. While immunization with standard but not sub-lethal inocula of most wild-type C. albicans strains resulted in considerable mortality, protection against lethal Ca/Sa IAI challenge varied by strain was usually less than that for C. dubliniensis, with no differences observed between parental and corresponding mutants. Finally, levels of protection afforded by the Ca strains were inversely correlated with BM tissue damage (R 2 = -0.773). TII-mediated protection against lethal Ca/Sa sepsis induced by Candida strain immunization inversely correlates with BM tissue/cellular damage as a reflection of localized virulence.

Spectrum of Trained Innate Immunity Induced by Low-Virulence Candida Species against Lethal Polymicrobial Intra-abdominal Infection.

Polymicrobial intra-abdominal infections (IAI) are clinically prevalent and cause significant morbidity and mortality, especially those involving fungi. Our laboratory developed a mouse model of polymicrobial IAI and demonstrated that coinfection with Candida albicans and Staphylococcus aureus (C. albicans/S. aureus) results in 80 to 90% mortality in 48 to 72 h due to robust local and systemic inflammation. Surprisingly, inoculation with Candida dubliniensis and S. aureus resulted in minimal mortality, and rechallenge of mice with lethal C. albicans/S. aureus conferred >90% protection up to 60 days postinoculation. Protection was mediated by Gr-1+ polymorphonuclear leukocytes, indicating a novel form of trained innate immunity (TII). The purpose of this study was to determine the microbial requirements and spectrum of innate-mediated protection. In addition to Candida dubliniensis, several other low-virulence Candida species (C. glabrata, C. auris, and C. albicansefg1Δ/Δ cph1Δ/Δ) and Saccharomyces cerevisiae conferred significant protection with or without S. aureus For C. dubliniensis-mediated protection, hyphal formation was not required, with protection conferred as early as 7 days after primary challenge but not at 120 days, and also following multiple lethal C. albicans/S. aureus rechallenges. This protection also extended to a lethal intravenous (i.v.) C. albicans challenge but had no effect in the C. albicans vaginitis model. Finally, studies revealed the ability of the low-virulence Candida species that conferred protection to invade the bone marrow by 24 h post-primary challenge, with a positive correlation between femoral bone marrow fungal infiltration at 48 h and protection upon rechallenge. These results support and further extend the characterization of this novel TII in protection against lethal fungal-bacterial IAI and sepsis.

Current patient perspectives of vulvovaginal candidiasis: incidence, symptoms, management and post-treatment outcomes.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common infection affecting women worldwide. Reports of patterns/risk factors/trends for episodic/recurrent VVC (RVVC) are largely outdated. The purpose of this study was to obtain current patient perspectives of several aspects of VVC/RVVC. METHODS: Business cards containing on-line survey information were distributed to healthy volunteers and patients seeking standard, elective, or referral gynecologic care in university-affiliated Obstetrics/Gynecology clinics. The internet-based questionnaire was completed by 284 non-pregnant women (78% Caucasian, 14% African American, 8% Asian). RESULTS: The majority of the participants (78%) indicated a history of VVC with 34% defined as having RVVC. The most common signs/symptoms experienced were itching, burning and redness with similar ranking of symptoms among VVC and RVVC patients. Among risk factors, antibiotic use ranked highest followed by intercourse, humid weather and use of feminine hygiene products. A high number of respondents noted 'no known cause' (idiopathic episodes) that was surprisingly similar among women with a history of either VVC or RVVC. VVC/RVVC episodes reported were primarily physician-diagnosed (73%) with the remainder mostly reporting self-diagnosis and treating with over-the-counter (OTC) medications. Most physician-diagnosed attacks utilized a combination of pelvic examination and laboratory tests followed by prescribed antifungals. Physician-treated cases achieved a higher level of symptom relief (84%) compared to those who self-medicated (57%). The majority of women with RVVC (71%) required continual or long-term antifungal medication as maintenance therapy to control symptoms. CONCLUSIONS: Current patient perspectives closely reflect historically documented estimates of VVC/RVVC prevalence and trends regarding symptomatology, disease management and post-treatment outcomes.

Novel Mechanism behind the Immunopathogenesis of Vulvovaginal Candidiasis: "Neutrophil Anergy".

For over 3 decades, investigators have studied the pathogenesis of vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC) through clinical studies and animal models. While there was considerable consensus that susceptibility was not associated with any apparent deficiencies in adaptive immunity, protective immune mechanisms and the role of innate immunity remained elusive. It was not until an innovative live-challenge design was conducted in women that a fuller understanding of the natural history of infection/disease was achieved. These studies revealed that symptomatic infection is associated with recruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen. Subsequent studies in the established mouse model demonstrated that infiltrating PMNs were incapable of reducing the fungal burden, which supported the hypothesis that VVC/RVVC was an immunopathology, whereby Candida and the host response drive symptomatic disease. Further studies in mice revealed the requirement for C. albicans hyphae and identified pattern recognition receptors (PRRs) and proinflammatory mediators responsible for the PMN response, all of which are critical pieces of the immunopathogenesis. However, a mechanism explaining PMN dysfunction at the vaginal mucosa remained an enigma. Ultimately, by employing mouse strains resistant or susceptible to chronic VVC, it was determined that heparan sulfate (HS) in the vaginal environment of susceptible mice serves as a competitive ligand for Mac-1 on PMNs, which effectively renders the PMNs incapable of binding to Candida to initiate killing. Hence, the outcome of symptomatic VVC/RVVC is postulated to be dependent on a PMN-mediated immunopathogenic response involving HS that effectively places the neutrophils in a state of functional anergy.

Identification of Specific Components of the Eicosanoid Biosynthetic and Signaling Pathway Involved in Pathological Inflammation during Intra-abdominal Infection with Candida albicans and Staphylococcus aureus.

Polymicrobial intra-abdominal infections (IAIs) are a significant cause of morbidity and mortality, particularly when fungal pathogens are involved. Our experimental murine model of IAI involving intraperitoneal inoculation of Candida albicans and Staphylococcus aureus results in synergistic lethality (∼80%) due to exacerbated inflammation. Monomicrobial infection results in no mortality, despite a microbial burden and dissemination similar to those in a coinfection. In the coinfection model, the immunomodulatory eicosanoid prostaglandin E2 (PGE2) was determined to be necessary and sufficient to induce mortality, implicating PGE2 as the central mediator of the amplified inflammatory response. The aim of this study was to identify key components of the PGE2 biosynthetic and signaling pathway involved in the inflammatory response and explore whether these can be targeted to prevent or reduce mortality. Using selective pharmacological inhibitors of cyclooxygenases (COX) or PGE2 receptor antagonists in the C. albicans-S. aureus IAI mouse model, we found that inhibition of COX and/or blocking of PGE2 receptor 1 (EP1) or PGE2 receptor 3 (EP3) signaling reduced proinflammatory cytokine production, promoted interleukin-10 production, reduced cellular damage in the peritoneal cavity, and, most importantly, significantly improved survival. The greatest effect on survival was obtained by the simultaneous inhibition of COX-1 activity and EP1 and EP3 receptor signaling. Importantly, early inhibition of PGE2 pathways dramatically improved the survival of fluconazole-treated mice compared with that achieved with fluconazole treatment alone. These findings indicate that COX-1 and the EP1 and EP3 receptors mediate the downstream pathological effects of PGE2 during polymicrobial IAI and may serve as effective therapeutic targets.

Prostaglandin E2 Receptor Antagonist with Antimicrobial Activity against Methicillin-Resistant Staphylococcus aureus.

Polymicrobial intra-abdominal infections (IAI) involving Candida albicans and Staphylococcus aureus are associated with severe morbidity and mortality (∼80%). Our laboratory discovered that the immunomodulatory eicosanoid prostaglandin E2 (PGE2) plays a key role in the lethal inflammatory response during polymicrobial IAI using a mouse model of infection. In studies designed to uncover key PGE2 biosynthesis/signaling components involved in the response, selective eicosanoid enzyme inhibitors and receptor antagonists were selected and prescreened for antimicrobial activity against C. albicans or S. aureus Unexpectedly, we found that the EP4 receptor antagonist L-161,982 had direct growth-inhibitory effects on S. aureusin vitro at the physiological concentration required to block the PGE2 interaction with EP4 This antimicrobial activity was observed with methicillin-sensitive S. aureus and methicillin-resistant S. aureus (MRSA) strains, with the MIC and minimum bactericidal concentration values for planktonic cells being 50 µg/ml and 100 µg/ml, respectively. In addition, L-161,982 inhibited S. aureus biofilm formation and had activity against preformed mature biofilms. More importantly, treatment of mice with L-161,982 following intraperitoneal inoculation with a lethal dose of MRSA significantly reduced the bioburden and enhanced survival. Furthermore, L-161,982 protected mice against the synergistic lethality induced by coinfection with C. albicans and S. aureus The antimicrobial activity of L-161,982 is independent of EP4 receptor inhibitory activity; an alternative EP4 receptor antagonist exerted no antimicrobial or protective effects. Taken together, these findings demonstrate that L-161,982 has potent antimicrobial activity against MRSA and may represent a significant therapeutic alternative in improving the prognosis of mono- or polymicrobial infections involving MRSA.

Synthesis, Antifungal Activity, and Biocompatibility of Novel 1,4-Diazabicyclo[2.2.2]Octane (DABCO) Compounds and DABCO-Containing Denture Base Resins.

The fungal pathogen Candida albicans causes a variety of oral infections, including denture stomatitis, which is characterized by inflammation of the oral mucosa in direct contact with dentures and affects a significant number of otherwise healthy denture wearers. While antifungal treatment reduces symptoms, infections are often recurrent. One strategy to address this problem is to incorporate compounds with fungicidal activities into denture materials to prevent colonization. Our laboratory synthesized novel derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO), which is an organic compound typically used as a catalyst in polymerization reactions. DABCO derivatives with different aliphatic chain lengths (DC16, DC16F, DC18, and C6DC16), as well as methacrylate monomers conjugated to DABCO compounds (DC11MAF and C2DC11MAF), were synthesized and tested for antimicrobial activity. All the compounds exhibited fungicidal activity against several Candida species at concentrations ranging between 2 and 4 µg/ml. Moreover, acrylic denture base resins fabricated to contain 1, 2, or 4 wt% DABCO compounds inhibited surface C. albicans biofilm formation, as well as fungal growth, in disc diffusion assays. Remarkably, discs (4 wt%) aged for 2 months also exhibited approximately 100% growth-inhibitory activity. While some DABCO compounds exerted intermediate to high cytotoxicity against mammalian oral cell types, DC11MAF and denture base resin discs containing 2 or 4 wt% C2DC11MAF exhibited relatively low cytotoxicity against periodontal ligament (PDL) cell and gingival fibroblast (GF) lines, as well as primary oral epithelial cells. These studies demonstrate that DABCO derivatives can be incorporated into denture materials and exert fungicidal activity with minimal cytotoxicity to mammalian cells. DC11MAF and C2DC11MAF are considered strong candidates as therapeutic or preventive alternatives against Candida-associated denture stomatitis.

Morphology-Independent Virulence of Candida Species during Polymicrobial Intra-abdominal Infections with Staphylococcus aureus.

Intra-abdominal polymicrobial infections cause significant morbidity and mortality. An experimental mouse model of Candida albicans-Staphylococcus aureus intra-abdominal infection (IAI) results in 100% mortality by 48 to 72 h postinoculation, while monomicrobial infections are avirulent. Mortality is associated with robust local and systemic inflammation without a requirement for C. albicans morphogenesis. However, the contribution of virulence factors coregulated during the yeast-to-hypha transition is unknown. This also raised the question of whether other Candida species that are unable to form hyphae are as virulent as C. albicans during polymicrobial IAI. Therefore, the purpose of this study was to evaluate the ability of non-albicans Candida (NAC) species with various morphologies and C. albicans transcription factor mutants (efg1/efg1 and cph1/cph1) to induce synergistic mortality and the accompanying inflammation. Results showed that S. aureus coinoculated with C. krusei or C. tropicalis was highly lethal, similar to C. albicans, while S. aureus-C. dubliniensis, S. aureus-C. parapsilosis, and S. aureus-C. glabrata coinoculations resulted in little to no mortality. Local and systemic interleukin-6 (IL-6) and prostaglandin E2 (PGE2) levels were significantly elevated during symptomatic and/or lethal coinfections, and hypothermia strongly correlated with mortality. Coinoculation with C. albicans strains deficient in the transcription factor Efg1 but not Cph1 reversed the lethal outcome. These results support previous findings and demonstrate that select Candida species, without reference to any morphological requirement, induce synergistic mortality, with IL-6 and PGE2 acting as key inflammatory factors. Mechanistically, signaling pathways controlled by Efg1 are critical for the ability of C. albicans to induce mortality from an intra-abdominal polymicrobial infection.

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.

Morphogenesis is not required for Candida albicans-Staphylococcus aureus intra-abdominal infection-mediated dissemination and lethal sepsis.

Intra-abdominal polymicrobial infections cause significant morbidity and mortality. An established experimental mouse model of Staphylococcus aureus-Candida albicans intra-abdominal infection results in ∼60% mortality within 48 h postinoculation, concomitant with amplified local inflammatory responses, while monomicrobial infections are avirulent. The purpose of this study was to characterize early local and systemic innate responses during coinfection and determine the role of C. albicans morphogenesis in lethality, a trait involved in virulence and physical interaction with S. aureus. Local and systemic proinflammatory cytokines were significantly elevated during coinfection at early time points (4 to 12 h) compared to those in monoinfection. In contrast, microbial burdens in the organs and peritoneal lavage fluid were similar between mono- and coinfected animals through 24 h, as was peritoneal neutrophil infiltration. After optimizing the model for 100% mortality within 48 h, using 3.5 × 10(7) C. albicans (5× increase), coinfection with C. albicans yeast-locked or hypha-locked mutants showed similar mortality, dissemination, and local and systemic inflammation to the isogenic control. However, coinfection with the yeast-locked C. albicans mutant given intravenously (i.v.) and S. aureus given intraperitoneally (i.p.) failed to induce mortality. These results suggest a unique intra-abdominal interaction between the host and C. albicans-S. aureus that results in strong inflammatory responses, dissemination, and lethal sepsis, independent of C. albicans morphogenesis.

Fungal morphogenetic pathways are required for the hallmark inflammatory response during Candida albicans vaginitis.

Vulvovaginal candidiasis, caused primarily by Candida albicans, presents significant health issues for women of childbearing age. As a polymorphic fungus, the ability of C. albicans to switch between yeast and hyphal morphologies is considered its central virulence attribute. Armed with new criteria for defining vaginitis immunopathology, the purpose of this study was to determine whether the yeast-to-hypha transition is required for the hallmark inflammatory responses previously characterized during murine vaginitis. Kinetic analyses of vaginal infection with C. albicans in C57BL/6 mice demonstrated that fungal burdens remained constant throughout the observation period, while polymorphonuclear leukocyte (PMN), S100A8, and interleukin-1ß levels obtained from vaginal lavage fluid increased by day 3 onward. Lactate dehydrogenase activity was also positively correlated with increased effectors of innate immunity. Additionally, immunodepletion of neutrophils in infected mice confirmed a nonprotective role for PMNs during vaginitis. Determination of the importance of fungal morphogenesis during vaginitis was addressed with a two-pronged approach. Intravaginal inoculation of mice with C. albicans strains deleted for key transcriptional regulators (bcr1Δ/Δ, efg1Δ/Δ, cph1Δ/Δ, and efg1Δ/Δ cph1Δ/Δ) controlling the yeast-to-hypha switch revealed a crucial role for morphogenetic signaling through the Efg1 and, to a lesser extent, the Bcr1 pathways in contributing to vaginitis immunopathology. Furthermore, overexpression of transcription factors NRG1 and UME6, to maintain yeast and hyphal morphologies, respectively, confirmed the importance of morphogenesis in generating innate immune responses in vivo. These results highlight the yeast-to-hypha switch and the associated morphogenetic response as important virulence components for the immunopathogenesis of Candida vaginitis, with implications for transition from benign colonization to symptomatic infection.

A fungal metabolic regulator underlies infectious synergism during Candida albicans-Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

A fungal metabolic regulator underlies infectious synergism during Candida albicans - Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) uncovered synergistic lethality that was driven by Candida -induced upregulation of functional S. aureus ⍺-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken and revealed that zcf13 Δ/Δ failed to drive augmented ⍺-toxin or lethal synergism during co-infection. Using a combination of transcriptional and phenotypic profiling approaches, ZCF13 was shown to regulate genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments revealed that ribose inhibited the staphylococcal agr quorum sensing system and concomitantly repressed toxicity. Unlike wild-type C. albicans , zcf13 Δ/Δ was unable to effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13 Δ/Δ mutant fully restored pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

Candida albicans-Staphylococcus aureus polymicrobial peritonitis modulates host innate immunity.

Despite advances in medical device fabrication and antimicrobial treatment therapies, fungal-bacterial polymicrobial peritonitis remains a serious complication for surgery patients, those on peritoneal dialysis, and the critically ill. Using a murine model of peritonitis, we have demonstrated that monomicrobial infection with Candida albicans or Staphylococcus aureus is nonlethal. However, coinfection with these same doses leads to a 40% mortality rate and increased microbial burden in the spleen and kidney by day 1 postinfection. Using a multiplex enzyme-linked immunosorbent assay, we have also identified a unique subset of innate proinflammatory cytokines (interleukin-6, granulocyte colony-stimulating factor, keratinocyte chemoattractant, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α) that are significantly increased during polymicrobial versus monomicrobial peritonitis, leading to increased inflammatory infiltrate into the peritoneum and target organs. Treatment of coinfected mice with the cyclooxygenase (COX) inhibitor indomethacin reduces the infectious burden, proinflammatory cytokine production, and inflammatory infiltrate while simultaneously preventing any mortality. Further experiments demonstrated that the immunomodulatory eicosanoid prostaglandin E2 (PGE2) is synergistically increased during coinfection compared to monomicrobial infection; indomethacin treatment also decreased elevated PGE2 levels. Furthermore, addition of exogenous PGE2 into the peritoneal cavity during infection overrode the protection provided by indomethacin and restored the increased mortality and microbial burden. Importantly, these studies highlight the ability of fungal-bacterial coinfection to modulate innate inflammatory events with devastating consequences to the host.

Efficacy of ethanol against Candida albicans and Staphylococcus aureus polymicrobial biofilms.

Candida albicans, an opportunistic fungus, and Staphylococcus aureus, a bacterial pathogen, are two clinically relevant biofilm-forming microbes responsible for a majority of catheter-related infections, with such infections often resulting in catheter loss and removal. Not only do these pathogens cause a substantial number of nosocomial infections independently, but also they are frequently found coexisting as polymicrobial biofilms on host and environmental surfaces. Antimicrobial lock therapy is a current strategy to sterilize infected catheters. However, the robustness of this technique against polymicrobial biofilms has remained largely untested. Due to its antimicrobial activity, safety, stability, and affordability, we tested the hypothesis that ethanol (EtOH) could serve as a potentially efficacious catheter lock solution against C. albicans and S. aureus biofilms. Therefore, we optimized the dose and time necessary to achieve killing of both monomicrobial and polymicrobial biofilms formed on polystyrene and silicone surfaces in a static microplate lock therapy model. Treatment with 30% EtOH for a minimum of 4 h was inhibitory for monomicrobial and polymicrobial biofilms, as evidenced by XTT {sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide inner salt} metabolic activity assays and confocal microscopy. Experiments to determine the regrowth of microorganisms on silicone after EtOH treatment were also performed. Importantly, incubation with 30% EtOH for 4 h was sufficient to kill and inhibit the growth of C. albicans, while 50% EtOH was needed to completely inhibit the regrowth of S. aureus. In summary, we have systematically defined the dose and duration of EtOH treatment that are effective against and prevent regrowth of C. albicans and S. aureus monomicrobial and polymicrobial biofilms in an in vitro lock therapy model.

Candida-induced granulocytic myeloid-derived suppressor cells are protective against polymicrobial sepsis.

IMPORTANCE: Polymicrobial intra-abdominal infections are serious clinical infections that can lead to life-threatening sepsis, which is difficult to treat in part due to the complex and dynamic inflammatory responses involved. Our prior studies demonstrated that immunization with low-virulence Candida species can provide strong protection against lethal polymicrobial sepsis challenge in mice. This long-lived protection was found to be mediated by trained Gr-1+ polymorphonuclear leukocytes with features resembling myeloid-derived suppressor cells (MDSCs). Here we definitively characterize these cells as MDSCs and demonstrate that their mechanism of protection involves the abrogation of lethal inflammation, in part through the action of the anti-inflammatory cytokine interleukin (IL)-10. These studies highlight the role of MDSCs and IL-10 in controlling acute lethal inflammation and give support for the utility of trained tolerogenic immune responses in the clinical treatment of sepsis.

Effect of MMR Vaccination to Mitigate Severe Sequelae Associated With COVID-19: Challenges and Lessons Learned.

Mortality in COVID-19 cases was strongly associated with progressive lung inflammation and eventual sepsis. There is mounting evidence that live attenuated vaccines commonly administered during childhood, also provide beneficial non-specific immune effects, including reduced mortality and hospitalization due to unrelated infections. It has been proposed that live attenuated vaccine-associated non-specific effects are a result of inducing trained innate immunity to function more effectively against broader infections. In support of this, our laboratory has reported that immunization with a live attenuated fungal strain induces a novel form of trained innate immunity which provides protection against various inducers of sepsis in mice via myeloid-derived suppressor cells. Accordingly, we initiated a randomized control clinical trial with the live attenuated Measles, Mumps, Rubella (MMR) vaccine in healthcare workers in the greater New Orleans area aimed at preventing/reducing severe lung inflammation/sepsis associated with COVID-19 (ClinicalTrials.gov Identifier: NCT04475081). Included was an outcome to evaluate the myeloid-derived suppressor cell populations in blood between those administered the MMR vaccine vs placebo. The unanticipated emergency approval of several COVID-19 vaccines in the midst of the MMR clinical trials eliminated the ability to examine effects of the MMR vaccine on COVID-19-related health status. Unfortunately, we were also unable to show any impact of the MMR vaccine on peripheral blood myeloid-derived suppressor cells due to several inherent limitations (low percentages of blood leukocytes, small sample size), that also included a collaboration with a similar trial (CROWN CORONATION; ClinicalTrials.gov Identifier: NCT04333732) in St. Louis, MO. In contrast, monitoring the COVID-19 vaccine response in trial participants revealed that high COVID-19 antibody titers occurred more often in those who received the MMR vaccine vs placebo. While the trial was largely inconclusive, lessons learned from addressing several trial-associated challenges may aid future studies that test the non-specific beneficial immune effects of live attenuated vaccines.

Development of a contemporary animal model of Candida albicans-associated denture stomatitis using a novel intraoral denture system.

Denture stomatitis (DS) is a fungal infection characterized by inflammation of the oral mucosa in direct contact with the denture and affects up to 50% of denture wearers. Despite the prevalence, very little is known about the role of fungal or host factors that contribute to pathogenesis. Recently, we developed a novel intraoral denture system for rodent research. This denture system consists of custom-fitted fixed and removable parts to allow repeated sampling and longitudinal studies. The purpose of this study was to use this denture system to develop a clinically relevant animal model of DS. To establish DS, rats were inoculated with pelleted Candida albicans, which resulted in sustained colonization of the denture and palate for 8 weeks postinoculation. Biofilm formation on the denture was observed by week 4 and on the palate by week 6 postinoculation. Rats were monitored for clinical signs of disease by assigning a clinical score after macroscopic examination of the palate tissue according to Newton's method. By week 4 postinoculation, the majority of inoculated rats with dentures exhibited a clinical score of 1 (pinpoint erythema). By week 6 and week 8 postinoculation, increasing percentages of rats exhibited a clinical score of 2 (diffuse erythema/edema). Histological analysis of palate tissue demonstrated progressively increasing inflammatory cell recruitment throughout the time course of the infection. Palatal biofilm formation was commensurate with development of palatal erythema, which suggests a role for biofilm in the inflammatory response.

Cytokines in the host response to Candida vaginitis: Identifying a role for non-classical immune mediators, S100 alarmins.

Vulvovaginal candidiasis (VVC), caused by Candida albicans, affects a significant number of women during their reproductive years. More than two decades of research have been focused on the mechanisms associated with susceptibility or resistance to symptomatic infection. Adaptive immunity by Th1-type CD4(+) T cells and downstream cytokine responses are considered the predominant host defense mechanisms against mucosal Candida infections. However, numerous clinical and animal studies have indicated no or limited protective role of cells and cytokines of the Th1 or Th2 lineage against vaginal infection. The role for Th17 is only now begun to be investigated in-depth for VVC with results already showing significant controversy. On the other hand, a clinical live-challenge study and an established animal model have shown that a symptomatic condition is intimately associated with the vaginal infiltration of polymorphonuclear leukocytes (PMNs) but with no effect on vaginal fungal burden. Subsequent studies identified S100A8 and S100A9 alarmins as key chemotactic mediators of the acute PMN response. These chemotactic danger signals appear to be secreted by vaginal epithelial cells upon interaction and early adherence of Candida. Thus, instead of a putative immunodeficiency against Candida involving classical immune cells and cytokines of the adaptive response, the pathological inflammation in VVC is now considered a consequence of a non-productive innate response initiated by non-classical immune mediators.