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Background Diagnosis of invasive candidiasis (IC) relies on insensitive cultures; the relative utility of fungal biomarkers in children is unclear. Methods This multinational observational cohort study enrolled patients aged >120 days and Results Five hundred participants were enrolled at 22 centers in 3 countries, and IC was diagnosed in 13 (2.6%). Thirteen additional blood specimens were collected and successfully spiked with Candida species, to achieve a 5.0% event rate. Valid T2Candida, Fungitell, Platelia Candida Ag Plus, and Platelia Candida Ab Plus assay results were available for 438, 467, 473, and 473 specimens, respectively. Operating characteristics for T2Candida were most optimal for detecting IC due to any Candida species, with results as follows: sensitivity, 80.0% (95% confidence interval, 59.3%–93.2%), specificity 97.1% (95.0%–98.5%), positive predictive value, 62.5% (43.7%–78.9%), and negative predictive value, 98.8% (97.2%–99.6%). Only T2Candida and Platelia Candida Ag Plus assays met the threshold for combination testing. Positive result for either yielded the following results: sensitivity, 86.4% (95% confidence interval, 65.1%– 97.1%); specificity, 94.7% (92.0%–96.7%); positive predictive value, 47.5% (31.5%–63.9%); and negative predictive value, 99.2% (97.7%–99.8%). Conclusions T2Candida alone or in combination with Platelia Candida Ag Plus may be beneficial for rapid detection of Candida species in children with concern for IC. Clinical Trials Registration NCT02220790.

Background Decisions to discontinue antibiotics in infants undergoing evaluation for central nervous system (CNS) infection can be difficult. Previous reports have associated the use of the meningoencephalitis (ME) PCR panel with decreased hospital stay and antimicrobial use in pediatric and adult patients. However, limited focus has been on infants ≤60-days-old with temperature instability (TI) who undergo evaluation for serious bacterial infection based on age and risk-stratification. We hypothesize use of the ME panel will decrease the duration of antimicrobial use in infants with TI and negative bacterial cultures undergoing evaluation for CNS infection. Methods The electronic medical record was queried for patients that met inclusion criteria. Infants ≤60-days-old evaluated for CNS infection due to TI between 1/1/12 – 1/31/22 were included. Exclusion criteria were history of intracranial (IC) devices, previous IC surgery, IC structural or neurovascular abnormalities, or head trauma. A retrospective chart review was performed. We compared length of stay and antimicrobial duration for the groups of patients with the ME panel done versus not done. Results A total of 1808 patients with negative bacterial cultures were identified, 96 of whom had the ME panel performed. Patient age, sex, and race were similar between groups. The median hospital length of stay for the patients that had the ME panel done was 64.7 hours as compared to 47.8 hours for those that did not have the ME panel done. The duration of antibacterial therapy was similar when the ME panel was done versus not done. The median duration of therapy for acyclovir was 8.5 when the ME panel was done versus 14.8 hours when it was not done. Conclusion When including all patients ≤60-days-old evaluated for TI, use of the ME panel did not lead to a shorter length of hospital stay or duration of empiric antibiotics. However, use of the ME panel decreased duration of therapy in the subset of patients empirically started on acyclovir. Further data analysis will seek to identify patient populations that may most benefit from the use of the ME panel. Disclosures All Authors: No reported disclosures.

Antimicrobial peptides (AMPs) are a class of molecules which generally kill pathogens via preferential cell membrane disruption. Chemokines are a family of signaling proteins that direct immune cell migration and share a conserved α–β tertiary structure. Recently, it was found that a subset of chemokines can also function as AMPs, including CCL20, CXCL4, and XCL1. It is therefore surprising that machine learning based analysis predicts that CCL20 and CXCL4’s α-helices are membrane disruptive, while XCL1’s helix is not. XCL1, however, is the only chemokine known to be a metamorphic protein which can interconvert reversibly between two distinct native structures (a β-sheet dimer and the α–β chemokine structure). Here, we investigate XCL1’s antimicrobial mechanism of action with a focus on the role of metamorphic folding. We demonstrate that XCL1 is a molecular “Swiss army knife” that can refold into different structures for distinct context-dependent functions: whereas the α–β chemokine structure controls cell migration by binding to G-Protein Coupled Receptors (GPCRs), we find using small angle X-ray scattering (SAXS) that only the β-sheet and unfolded XCL1 structures can induce negative Gaussian curvature (NGC) in membranes, the type of curvature topologically required for membrane permeation. Moreover, the membrane remodeling activity of XCL1’s β-sheet structure is strongly dependent on membrane composition: XCL1 selectively remodels bacterial model membranes but not mammalian model membranes. Interestingly, XCL1 also permeates fungal model membranes and exhibits anti-Candida activity in vitro, in contrast to the usual mode of antifungal defense which requires Th17 mediated cell-based responses. These observations suggest that metamorphic XCL1 is capable of a versatile multimodal form of antimicrobial defense.

Background Invasive candidiasis is the most common invasive fungal disease in children and adolescents, but there are limited pediatric-specific antifungal effectiveness data. We compared the effectiveness of echinocandins to triazoles or amphotericin B formulations (triazole/amphotericin B) as initial directed therapy for invasive candidiasis. Methods This multinational observational cohort study enrolled patients aged >120 days and Results Seven-hundred and fifty invasive candidiasis episodes were identified. After exclusions, 541 participants (235 in the echinocandin group and 306 in the triazole/amphotericin B group) remained. Crude failure rates at 14 days for echinocandin and triazole/amphotericin B groups were 9.8% (95% confidence intervals [CI]: 6.0% to 13.6%) and 13.1% (95% CI: 9.3% to 16.8%), respectively. The adjusted 14-day risk difference between echinocandin and triazole/amphotericin B groups was −7.1% points (95% CI: −13.1% to −2.4%), favoring echinocandins. The risk difference was −0.4% (95% CI: −7.5% to 6.7%) at 30 days. Conclusions In children with invasive candidiasis, initial directed therapy with an echinocandin was associated with reduced failure rate at 14 days but not 30 days. These results may support echinocandins as initial directed therapy for invasive candidiasis in children and adolescents. Clinical Trials Registration NCT01869829.

Candida species cause serious infections requiring prolonged and sometimes toxic therapy. Antimicrobial proteins, such as chemokines, hold great interest as potential additions to the small number of available antifungal drugs. Metamorphic proteins reversibly switch between multiple different folded structures. XCL1 is a metamorphic, antimicrobial chemokine that interconverts between the conserved chemokine fold (an α–β monomer) and an alternate fold (an all-β dimer). Previous work has shown that human XCL1 kills C. albicans but has not assessed whether one or both XCL1 folds perform this activity. Here, we use structurally locked engineered XCL1 variants and Candida killing assays, adenylate kinase release assays, and propidium iodide uptake assays to demonstrate that both XCL1 folds kill Candida, but they do so via different mechanisms. Our results suggest that the alternate fold kills via membrane disruption, consistent with previous work, and the chemokine fold does not. XCL1 fold-switching thus provides a mechanism to regulate the XCL1 mode of antifungal killing, which could protect surrounding tissue from damage associated with fungal membrane disruption and could allow XCL1 to overcome candidal resistance by switching folds. This work provides inspiration for the future design of switchable, multifunctional antifungal therapeutics.

BACKGROUND Blastomycosis, an endemic mycosis of immunocompetent individuals, is typically seen after exposure to waterways within rural wooded regions. It is not considered a disease of urban environments. Infection can be solely pneumonic or disseminate to skin, bone or central nervous system. Unknown factors influence disease acquisition and severity in children. METHODS We analyzed acquisition risks and disease characteristics of blastomycosis in children seen at a tertiary care center from 1998 to 2018 to identify potential exposure sources, measure disease severity and assess the effect of race upon disease severity. RESULTS Of 64 infected children, mean age was 12.9 years, with median time to diagnosis 38.5 days. About 72% were male, 38% resided in urban counties and 50% had typical environmental exposure. Isolated pulmonary infection occurred in 33 (52%). The remainder had evidence of dissemination to skin (N = 13), bone (N = 16; 7 clinically silent) and cranium (N = 7; 3 clinically silent). Infection was moderate/severe in 19 (30%). Two children (3%) died. About 79% of children with moderate/severe disease (P = 0.008) and 71% of urban children (P = 0.007) lacked typical environmental exposure. Comparing children from urban counties to other residences, 63% versus 5% were black (P < 0.001) and 71% versus 35% developed extrapulmonary dissemination (P = 0.006). Moderate/severe disease was seen in 7/17 (42%) black children but only 12/47 (26%) children of other races (P = 0.23). CONCLUSIONS Blastomycosis, can be endemic in urban children in the absence of typical exposure history, have frequent, sometimes clinically silent, extrapulmonary dissemination and possibly produces more severe disease in black children.

OBJECTIVES A quality improvement approach was used to increase pediatric liver transplant recipient live and inactive vaccination rates by assessing titers and recommending vaccinations. METHODS A new screening and immunization process for both live and inactive vaccines was discussed with families at their annual visit. Antibody titers for varicella, measles, mumps, rubella, Haemophilus influenzae type B, hepatitis A, and hepatitis B were obtained. Specific criteria were developed for live virus vaccination candidacy. Vaccines were recommended based on patient titers and vaccination candidacy criteria. Surveillance for adverse effects to live vaccines was performed. Repeat titers were obtained approximately 1-month post-vaccine administration. RESULTS After PDSA cycle 1, 99% (71/72) of pediatric liver transplant patients had titers obtained. Live vaccines were recommended for 32 patients and 16 (50%) were vaccinated. Inactive vaccines were recommended to 64 patients, and 31 (48%) were vaccinated. Eight of 13 (62%) patients with follow-up titers achieved immunity for inactive vaccines. Zero patients had an adverse reaction to any live vaccine. Ten of 12 (83%) patients with follow-up titers achieved immunity from live vaccines. The most common barriers to receive live vaccines included not scheduling appointment with primary care provider (n = 3) and "non-vaccinators" (n = 3). CONCLUSIONS Administering live and inactive vaccines to select pediatric liver transplant patients appears to be safe and effective in our studied population. For PDSA cycle 2, we will continue our current practice and consider offering vaccines in transplant clinic, since this was a barrier to vaccination identified during PDSA cycle 1.

Candida albicans is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of Candida infections is limited by the paucity of antifungal drug classes. Naturally occurring antimicrobial peptides are promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has in vitro antimicrobial activity. In this study, we examine the in vivo Candida killing capacity of CCL28 in oropharyngeal candidiasis as well as the spectrum and mechanism of anti-Candida activity. In the mouse model of oropharyngeal candidiasis, application of wild-type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. CCL28 is effective against multiple clinical strains of C. albicans. Polyamine protein transporters are not required for CCL28 anti-Candida activity. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to that of clinical antifungal agents. Application of wild-type CCL28 to C. albicans results in membrane disruption as measured by solute movement, enzyme leakage, and induction of negative Gaussian curvature on model membranes. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results strongly suggest that CCL28 can exert antifungal activity in part via membrane permeation and has potential for development as an anti-Candida therapeutic agent without inflammatory side effects.

Invasive fungal infections are a serious cause of morbidity and mortality in patients with hematologic malignancies. Conidiobolus species are molds within the order Entomophthorales and may disseminate to become rapidly fatal in immunocompromised individuals. This species of fungal infections are often multidrug resistant (MDR) and present unique therapeutic challenges. Reports of Conidiobolus infections are rare in pediatric oncology. We report the successful treatment of an adolescent male with B cell lymphoblastic leukemia and MDR invasive sinopulmonary Conidiobolus infection with emphasis on early and aggressive neutrophil support with surgical debridement. The strategies described could be applied to other MDR fungal infections.

The chemokine CCL28 is constitutively expressed in mucosal tissues and is abundant in low-salt mucosal secretions. Beyond its traditional role as a chemoattractant, CCL28 has been shown to act as a potent and broad-spectrum antimicrobial agent with particular efficacy against the commensal fungus and opportunistic pathogen Candida albicans. However, the structural features that allow CCL28 to perform its chemotactic and antimicrobial functions remain unknown. Here, we report the structure of CCL28, solved using nuclear magnetic resonance spectroscopy. CCL28 adopts the canonical chemokine tertiary fold, but also has a disordered C-terminal domain that is partially tethered to the core by a non-conserved disulfide bond. Structure-function analysis reveals that removal of the C-terminal tail reduces the antifungal activity of CCL28 without disrupting its structural integrity. Conversely, removal of the non-conserved disulfide bond destabilizes the tertiary fold of CCL28 without altering its antifungal effects. Moreover, we report that CCL28 unfolds in response to low pH but is stabilized by the presence of salt. To explore the physiologic relevance of the observed structural lability of CCL28, we investigated the effects of pH and salt on the antifungal activity of CCL28 in vitro. We found that low pH enhances the antifungal potency of CCL28, but also that this pH effect is independent of CCL28's tertiary fold. Given its dual role as a chemoattractant and antimicrobial agent, our results suggest that changes in the salt concentration or pH at mucosal sites may fine-tune CCL28's functional repertoire by adjusting the thermostability of its structure.

Pediatric deep neck space infections (DNSI) are common in children. If unrecognized, DNSIs can rapidly progress with the potential for developing serious complications. Children fair better than their adult counterparts in the face of complications such as mediastinitis, but early recognition and aggressive management is key to prevent mortality and limit morbidity. Knowledge of the anatomy related to the deep neck spaces helps in anticipating and managing the potential complications. In this article, we review the clinical presentation, complications, and management in pediatric DNSI from the point of view of the pediatric infectious disease physician and the otolaryngologist. We discuss the best management based on current available evidence and the experience of the authors.

Purpose The development and implementation of an antimicrobial lock therapy guideline at a large pediatric hospital are described. Summary Central venous access devices (CVADs) are essential in the medical management of patients requiring long-term total parenteral nutrition, chemotherapy, or hemodialysis. However, the use of a CVAD carries a significant risk of the development of central line–associated bloodstream infection (CLABSI). Antimicrobial lock therapy is indicated for patients with CLABSIs who have no signs of exit site or tunnel infection and for whom catheter salvage is a goal. An antimicrobial lock therapy guideline was developed and implemented at a large pediatric hospital by an interprofessional team consisting of providers specializing in CVAD care. Development and implementation of the guideline included a needs assessment, a literature review, determination of patient selection criteria, addition of compounding formulations, development of administration techniques, and education. In all 10 instances of lock therapy in the 18 months after guideline implementation, the criteria for use were met; in 60% of those instances, patients received care from an infectious diseases physician. Each of the available lock solutions was used at least once during the 18-month period. Conclusion Overall, implementation of a guideline on administration of antimicrobial lock therapy was successful. Nine patients received antimicrobial lock therapy in the 18 months after policy implementation; although 2 had their line removed (1 due to repeated line infections), neither required line replacement.

Mycobacteria and Candida species include significant human pathogens that can cause localized or disseminated infections. Although these organisms may appear to have little in common, several shared pathways of immune recognition and response are important for both control and infection-related pathology. In this article, we compare and contrast the innate and adaptive components of the immune system that pertain to these infections in humans and animal models. We also explore a relatively new concept in the mycobacterial field: biological commensalism. Similar to the well-established model of Candida infection, Mycobacterial species colonize their human hosts in equilibrium with the immune response. Perturbations in the immune response permit the progression to pathologic disease at the expense of the host. Understanding the immune factors required to maintain commensalism may aid with the development of diagnostic and treatment strategies for both categories of pathogens.

Nontuberculous mycobacteria (NTM) infect children with increasing frequency worldwide. Using blood and lymph node tissue from children with NTM lymphadenitis, and uninfected lymph node tissue from community controls, we evaluated helper T (TH) cells in functional assays of TH1/TH17 differentiation and measured the concentration of their associated cytokines at the site of infection. Circulating TH cells from infected children were attenuated in their TH1/TH17 differentiation capacity and expressed less interferon γ and interleukin 17 after polyclonal stimulation. Similar differences were observed at the site of infection, where most cytokine concentrations were unchanged relative to controls. Our data are consistent with a model wherein TH1/TH17 differentiation is attenuated in NTM-infected children.

Background Blastomycosis is an endemic mycosis of immunocompetent individuals, typically seen after exposure to wooded areas near rivers, lakes, and streams in rural locations, and often not considered a disease of urban environments. Disease can be isolated to lung, or disseminate to skin, bone, or central nervous system. Factors influencing disease acquisition and severity in children are unknown. Methods We analyzed acquisition risk factors and disease characteristics of blastomycosis in children treated at a tertiary care center from 1998–2018 to identify exposure source and measure disease severity, to identify cases without “typical exposure”, and to measure the effect of race on disease severity. Results Of 64 children, median age was 13.3 years, 72% were male, 38% resided in urban counties, and 50% had typical environmental exposure. Isolated pulmonary infection occurred in 33 (52%). The remaining children had evidence of dissemination including skin (N=13), bone (N=16; 7 clinically silent), and central nervous system (N=7; 3 clinically silent). Infection was moderate/severe in 19 (30%). Two children (3%) died. 79% of children with moderate/severe disease (p=0.008) and 71% of urban children (p=0.007) had no typical environmental exposure. Comparing children from urban counties to other residences, 63% versus 5% were black (p Conclusions Blastomycosis, endemic in urban children in the absence of typical exposure history, has frequent, sometimes clinically silent, extrapulmonary dissemination, with a trend toward more severe disease in black children.

Candida albicans is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of these infections is limited by the paucity of anti-fungal drug classes. Antimicrobial peptides are a component of the immune response to fungi and promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has in vitro antimicrobial activity. In this study, we examine the spectrum of in vitro antimicrobial activity and the in vivo Candida killing capacity of CCL28 in oropharyngeal candidiasis (OPC), as well as the mechanism of anti-Candida activity. CCL28 is effective against multiple clinical strains of C. albicans, but less effective against C. glabrata. In the mouse model of OPC, application of wild type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to clinical anti-fungal agents. Polyamine protein transporters are not required for CCL28 anti-Candida activity. Application of wild type CCL28 to C. albicans results in membrane disruption as measured by solute movement and enzyme leakage. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results indicate CCL28 functions in the immune response to disrupt fungal membranes and has potential for development as an anti-Candida therapeutic agent without inflammatory side effects.

Background Osteomyelitis and septic arthritis are common pediatric musculoskeletal infections with potential to cause significant morbidity. Empiric antibiotic selection is made prior to the availability of microbiologic data. The aim of this study was to compare the epidemiology of osteoarticular infections (OAI) to antibiotic regimens and local antibiograms. Methods A retrospective study was performed on patients aged 6 months to 18 years with a diagnosis of septic arthritis or acute hematogenous osteomyelitis in a large, free-standing children’s hospital between July 2012 and July 2017. Exclusion criteria were chronic osteomyelitis, prior trauma or surgery at the site, noninfectious arthritis, and Lyme arthritis. Data collected from the electronic medical record included demographics, initial and discharge antibiotic therapy, and microbiologic results. Data were compared with the local antibiogram during the same time period. Results A total of 207 patients were included: 66 patients Conclusion Our study revealed high rates of clindamycin-susceptible S. aureus in older children and K. kingae and culture-negative infection in children < 4 years with OAI. Antibiotic susceptibilities differing from our institutional antibiogram suggest that disease-specific antibiograms will aid with empiric treatment decisions. Disclosures All authors: No reported disclosures.

IL-17 protects against pathogens by acting on nonhematopoietic cells to induce neutrophil recruitment through upregulation of chemokines and G-CSF. IL-17- and Th17-deficient humans and mice are susceptible to mucosal Candida albicans infections, linked to impaired neutrophil responses. IL-17 production is traditionally associated with CD4+ Th17 cells. However, IL-17 is also expressed during innate responses to facilitate rapid pathogen clearance. Innate IL-17-expressing cells include various lymphocyte-type subsets, including ILC3, NKT, γδ-T and “natural” Th17 (nTh17) cells. Some reports suggest that neutrophils can express IL-17 during fungal infections. Here, we asked whether neutrophils serve as a source of IL-17 during acute oropharyngeal candidiasis (OPC) using an IL-17A fate-tracking reporter mouse. Mice were subjected to OPC for two days, and oral tissue was analyzed by flow cytometry. IL-17A was expressed by γδ-T cells and TCRβ+ natural Th17 (nTh17) cells, as recently reported. Although infiltrating neutrophils were recruited to the tongue following infection, they did not express the IL-17A reporter. Moreover, neutrophil-depleted mice exhibited normal transcription of both Il17a and downstream IL-17-dependent gene targets after Candida challenge. Thus, in acute OPC, neutrophils are not a measurable source of IL-17 production, nor are they necessary to trigger IL-17-dependent gene expression, although they are essential for ultimate pathogen control.

Candida albicans is a dimorphic commensal fungus that causes severe oral infections in immunodeficient patients. Invasion of C. albicans hyphae into oral epithelium is an essential virulence trait. Interleukin-17 (IL-17) signaling is required for both innate and adaptive immunity to C. albicans. During the innate response, IL-17 is produced by γδ T cells and a poorly understood population of innate-acting CD4+ αβ T cell receptor (TCRαβ)+ cells, but only the TCRαβ+ cells expand during acute infection. Confirming the innate nature of these cells, the TCR was not detectably activated during the primary response, as evidenced by Nur77eGFP mice that report antigen-specific signaling through the TCR. Rather, the expansion of innate TCRαβ+ cells was driven by both intrinsic and extrinsic IL-1R signaling. Unexpectedly, there was no requirement for CCR6/CCL20-dependent recruitment or prototypical fungal pattern recognition receptors. However, C. albicans mutants that cannot switch from yeast to hyphae showed impaired TCRαβ+ cell proliferation and Il17a expression. This prompted us to assess the role of candidalysin, a hyphal-associated peptide that damages oral epithelial cells and triggers production of inflammatory cytokines including IL-1. Candidalysin-deficient strains failed to up-regulate Il17a or drive the proliferation of innate TCRαβ+ cells. Moreover, candidalysin signaled synergistically with IL-17, which further augmented the expression of IL-1α/β and other cytokines. Thus, IL-17 and C. albicans, via secreted candidalysin, amplify inflammation in a self-reinforcing feed-forward loop. These findings challenge the paradigm that hyphal formation per se is required for the oral innate response and demonstrate that establishment of IL-1– and IL-17–dependent innate immunity is induced by tissue-damaging hyphae.

Invasive fungal diseases are important clinical problems that are often complicated by severe illness and therefore the inability to use invasive measures to definitively diagnose the disease. Tests for a range of fungal biomarkers that do not require an invasive sample-collection procedure have been incorporated into adult clinical practice, but pediatric data and pediatric-specific recommendations for some of these diagnostic tools are lacking. In this review, we summarize the published literature and contemporary strategies for using the biomarkers galactomannan, (1→3)-β-d-glucan, Candida mannan antigen and anti-mannan antibody, and fungal polymerase chain reaction for diagnosing invasive fungal disease in children. Data on biomarker use in neonates and children with cancer, history of hematopoietic stem cell transplant, or primary immunodeficiency are included. Fungal biomarker tests performed on blood, other body fluids, or tissue specimens represent promising adjuncts to the diagnostic armamentarium in populations with a high prevalence of invasive fungal disease, but substantial gaps exist in the correct use and interpretation of these diagnostic tools in pediatric patients.

Oropharyngeal candidiasis (OPC [thrush]) is an opportunistic infection caused by the commensal fungus Candida albicans . OPC is common in individuals with HIV/AIDS, infants, patients on chemotherapy, and individuals with congenital immune defects. Immunity to OPC is strongly dependent on the interleukin-23 (IL-23)/IL-17R axis, as mice and humans with defects in IL-17R signaling ( IL17F , ACT1 , IL-17RA ) or in genes that direct Th17 differentiation ( STAT3 , STAT1 , CARD9 ) are prone to mucocutaneous candidiasis. Conventional Th17 cells are induced in response to C. albicans infection via signals from C-type lectin receptors, which signal through the adaptor CARD9, leading to production of Th17-inducing cytokines such as IL-6, IL-1β, and IL-23. Recent data indicate that IL-17 can also be made by numerous innate cell subsets. These innate “type 17” cells resemble conventional Th17 cells, but they can be activated without need for prior antigen exposure. Because C. albicans is not a commensal organism in rodents and mice are thus naive to this fungus, we had the opportunity to assess the role of CARD9 in innate versus adaptive responses using an OPC infection model. As expected, CARD9 −/− mice failed to mount an adaptive Th17 response following oral Candida infection. Surprisingly, however, CARD9 −/− mice had preserved innate IL-17-dependent responses to Candida and were almost fully resistant to OPC. Thus, CARD9 is important primarily for adaptive immunity to C. albicans , whereas alternate recognition systems appear to be needed for effective innate responses.

Oropharyngeal candidiasis (OPC), caused by the commensal fungus Candida albicans, is an opportunistic infection associated with infancy, AIDS, and IL-17–related primary immunodeficiencies. The Th17-associated cytokines IL-23 and IL-17 are crucial for immunity to OPC, but the mechanisms by which they mediate immunity are poorly defined. IL-17RA–deficient humans and mice are strongly susceptible to OPC, with reduced levels of CXC chemokines and concomitantly impaired neutrophil recruitment to the oral mucosa. Paradoxically, humans with isolated neutropenia are typically not susceptible to candidiasis. To determine whether immunity to OPC is mediated via neutrophil recruitment, mice lacking CXCR2 were subjected to OPC and were found to be highly susceptible, although there was no dissemination of fungi to peripheral organs. To assess whether the entire neutrophil response is IL-17 dependent, IL-17RA−/− and IL-23−/− mice were administered neutrophil-depleting Abs and subjected to OPC. These mice displayed increased oral fungal burdens compared with IL-17RA−/− or IL-23−/− mice alone, indicating that additional IL-17–independent signals contribute to the neutrophil response. WT mice treated with anti–Gr-1 Abs exhibited a robust infiltrate of CD11b+Ly-6GlowF4/80− cells to the oral mucosa but were nonetheless highly susceptible to OPC, indicating that this monocytic influx is insufficient for host defense. Surprisingly, Ly-6G Ab treatment did not induce the same strong susceptibility to OPC in WT mice. Thus, CXCR2+ and Gr-1+ neutrophils play a vital role in host defense against OPC. Moreover, defects in the IL-23/17 axis cause a potent but incomplete deficiency in the neutrophil response to oral candidiasis.

Conti et al. show that IL-17 is produced by tongue-resident populations of γδ T cells and nTh17 cells in response to oropharyngeal candidiasis in mice., Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα−/−, and Rag1−/− mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1–2 d by tongue-resident populations of γδ T cells and CD3+CD4+CD44hiTCRβ+CCR6+ natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-β−/− and TCR-δ−/− mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-β clonal diversity, and was absent in Rag1−/−, IL-7Rα−/−, and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens.

Oropharyngeal candidiasis (OPC) is an opportunistic infection caused by Candida albicans. Despite its prevalence, little is known about C. albicans-specific immunity in the oral mucosa. Vaccines against Candida generate both T helper type 1 (Th1) and Th17 responses, and considerable evidence implicates interleukin (IL)-17 in immunity to OPC. However, IL-17 is also produced by innate immune cells that are remarkably similar to Th17 cells, expressing the same markers and localizing to similar mucosal sites. To date, the relative contribution(s) of Th1, Th17, and innate IL-17-producing cells in OPC have not been clearly defined. Here, we sought to determine the nature and function of adaptive T-cell responses to OPC, using a new recall infection model. Mice subjected to infection and re-challenge with Candida mounted a robust and stable antigen-specific IL-17 response in CD4+ but not CD8+ T cells. There was little evidence for Th1 or Th1/Th17 responses. The Th17 response promoted accelerated fungal clearance, and Th17 cells could confer protection in Rag1-/- mice upon adoptive transfer. Surprisingly, CD4 deficiency did not cause OPC but was instead associated with compensatory IL-17 production by Tc17 and CD3+CD4-CD8- cells. Therefore, classic CD4+Th17 cells protect from OPC but can be compensated by other IL-17-producing cells in CD4-deficient hosts.

tudy Objective To describe our experience with voriconazole in three patients younger than 2 years using an optimized dosing strategy for voriconazole that incorporates intensive therapeutic drug monitoring (TDM). Design Case series. Setting Large pediatric hospital. Patients Three patients younger than 2 years who received voriconazole therapy and had serum trough concentrations measured between January 1, 2010, and October 31, 2015. Measurements and Main Results A clinical practice guideline developed at our institution was used to standardize initial dosing, appropriate use and timing of TDM, and dosage modifications based on TDM. TDM was used to guide dosing to achieve a target voriconazole serum trough concentration of 2–6 μg/ml. Voriconazole samples were assayed by using a high-performance liquid chromatography analytical method with solid-phase extraction. Initial dosages for the three patients were 9 mg/kg intravenously every 12 hours (one patient) and 9 mg/kg enterally twice/day (two patients). Multiple dose escalations and a more frequent dosing interval were required to achieve trough concentrations within the target range. The final dosages were 12 mg/kg intravenously every 8 hours, 17.7 mg/kg enterally 3 times/day, and 8.5 mg/kg enterally 3 times/day, respectively. In addition to voriconazole trough concentrations, TDM included evaluations for drug toxicities. Visual, neurologic, or hepatic adverse effects were not encountered in the three patients. Conclusion Our data support higher initial doses and perhaps a 3 times/day dosing schedule to achieve voriconazole serum concentrations in the target range for children younger than 2 years. Implementation of a clinical practice guideline with the participation of pharmacists specializing in pharmacokinetics allows for effective use of voriconazole in young children.

Discussion of how mast cells can acquire IL-17 through cellular uptake.

U6 RNA is a key component of the catalytic core of the spliceosome. A metal ion essential for the first catalytic step of pre-mRNA splicing binds to the U80 Sp phosphate oxygen within the yeast U6 intramolecular stem-loop (ISL). Here we present the first structural data for U6 RNA, revealing the three-dimensional structure of the highly conserved U6 ISL. The ISL binds metal ion at the U80 site with the same stereo specificity as the intact spliceosome. The metal-binding site is adjacent to a readily protonated C.A wobble pair. Protonation of the C.A pair and metal binding are mutually antagonistic. These results support a ribozyme model for U6 RNA function and suggest a possible mechanism for the regulation of RNA splicing.

Candida albicans is normally a commensal fungus of the human mucosae and skin, but it causes life-threatening systemic infections in hospital settings in the face of predisposing conditions, such as indwelling catheters, abdominal surgery, or antibiotic use. Immunity to C. albicans involves various immune parameters, but the cytokine interleukin-17A (IL-17A) (also known as IL-17) has emerged as a centrally important mediator of immune defense against both mucosal and systemic candidiasis. Conversely, IL-17A has been suggested to enhance the virulence of C. albicans , indicating that it may exert detrimental effects on pathogenesis. In this study, we hypothesized that a C. albicans strain expressing IL-17A would exhibit reduced virulence in vivo . To that end, we created a Candida -optimized expression cassette encoding murine IL-17A, which was transformed into the DAY286 strain of C. albicans . Candida -derived IL-17A was indistinguishable from murine IL-17A in terms of biological activity and detection in standard enzyme-linked immunosorbent assays (ELISAs). Expression of IL-17A did not negatively impact the growth of these strains in vitro . Moreover, the IL-17A-expressing C. albicans strains showed significantly reduced pathogenicity in a systemic model of Candida infection, mainly evident during the early stages of disease. Collectively, these findings suggest that IL-17A mitigates the virulence of C. albicans .

In recent years, proinflammatory cytokines in the nephritic kidney appear to contribute to the pathogenesis of AGN. The complex inflammatory cytokine network that drives renal pathology is poorly understood. IL-17, the signature cytokine of Th17 cells, which promotes autoimmune pathology in a variety of settings, is beginning to be identified in acute and chronic kidney diseases as well. However, the role of IL-17-mediated renal damage in the nephritic kidney has not been elucidated. Here, with the use of a murine model of experimental AGN, we showed that IL-17RA signaling is critical for the development of renal pathology. Despite normal systemic autoantibody response and glomerular immune-complex deposition, IL-17RA−/− mice exhibit a diminished influx of inflammatory cells and kidney-specific expression of IL-17 target genes correlating with disease resistance in AGN. IL-17 enhanced the production of proinflammatory cytokines and chemokines from tECs. Finally, we were able to show that neutralization of IL-17A ameliorated renal pathology in WT mice following AGN. These results clearly demonstrated that IL-17RA signaling significantly contributes to renal tissue injury in experimental AGN and suggest that blocking IL-17RA may be a promising therapeutic strategy for the treatment of proliferative and crescentic glomerulonephritis.

Multiple forms of candidiasis are clinically important in humans. Established murine models of disseminated, oropharyngeal, vaginal, and cutaneous candidiasis caused by Candida albicans are described in this unit. Detailed materials and methods for C. albicans growth and detection are also described.

Oropharyngeal candidiasis (OPC) is an opportunistic infection caused by the commensal fungus Candida albicans. There is an urgent need for new therapeutic candidates to manage mucosal infection and prevent progression to high mortality disseminated candidiasis. The T helper 17 (Th17)-associated cytokines IL-23 and IL-17 are crucial for immunity to OPC. However, our recent work indicates the current model of IL-17-mediated defense is insufficient to completely explain susceptibility to OPC. CCL28 is a CC chemokine with antimicrobial peptide (AMP) activity that has a potential role in oral host defense. CCL28 is most abundant in the salivary gland and has in vitro antimicrobial activity against multiple pathogens including C. albicans. Little is known about the in vivo function or regulation of CCL28 in defense from infection. In the current study, we explore the motifs of CCL28 contributing to AMP activity and the relationship with OPC. We demonstrate that C. albicans killing by CCL28 at physiologic concentrations can be eliminated by truncation of the C-terminus. The N-terminus of CCL28, which is required for receptor binding, is dispensable for anti-fungal activity. In an in vivo model of OPC in mice, application of recombinant CCL28 reduces oral fungal burdens in immunodeficient mice. Ccl28 gene expression in salivary gland is suppressed at baseline in mice susceptible to OPC. Our results indicate that the CCL28 flexible C-terminal domain is an important functional motif for anti-Candida AMP activity, CCL28 treatment reduces mucosal fungal infection in vivo, and CCL28 may be a component of host defense from infection. CCL28 is a promising candidate for mucosal immunity and future development for treatment of Candida infections.

OBJECTIVE:The goal was to determine the performance of low-risk criteria for serious bacterial illnesses (SBIs) in febrile infants in prospective studies in which empiric antibiotic treatment was withheld, compared with studies (prospective and retrospective) in which empiric antibiotic treatment was administered.METHODS:A search of the English-language literature was undertaken by using a PubMed database and reference lists of relevant studies of fever, low-risk criteria, and SBIs. Studies of infants >90 days of age, infants with specific infections, or infants with additional risk factors for infection were excluded. Publications were categorized as retrospective, prospective with empiric antibiotic treatment for all patients, or prospective with antibiotics withheld. The relative risk of SBI in high-risk versus low-risk patients was determined for pooled data in each category. The rates of SBIs in low-risk patients in each category were compared.RESULTS:Twenty-one studies met the inclusion criteria. In prospective studies in which patients were cared for without empiric antibiotic treatment, 6 patients assigned to the low-risk category had SBIs; all recovered uneventfully. The rate of SBIs in these low-risk patients was 0.67%. The relative risk of SBIs in high-risk versus low-risk patients in these studies was 30.56 (95% confidence interval: 7.0–68.13). The rate of SBIs in low-risk patients in all studies was 2.23%. The rate of SBIs in low-risk patients in the prospective studies without empiric antibiotic treatment was significantly different from the rate in all other studies (0.67% vs 2.71%; P = .01).CONCLUSIONS:Low-risk criteria perform well in prospective studies in which empiric antibiotic treatment is withheld. These criteria allow ∼30% of young febrile infants to be observed without antibiotic treatment, thus avoiding unnecessary hospitalization, nosocomial infection, injudicious use of antibiotics, and adverse effects of antibiotics.

Huppler, A. R., H. R. Conti, N. Hernandez-Santos, T. Darville, P. S. Biswas, and S. L. Gaffen. 2014. Role of neutrophils in IL-17–dependent immunity to mucosal candidiasis. J. Immunol. 192: [1745–1752][1]. There was an inadvertent error in the calculation of the absolute neutrophil count due

Protein kinase D localizes in the Golgi and regulates protein transport from the Golgi to the plasma membrane. In the present study, we found that PKD3, a novel member of the PKD family, and its fluorescent protein fusions localized in the Golgi and in the vesicular structures that are in part marked by endosome markers. Fluorescent recovery after photobleaching (FRAP) showed that the PKD3-associated vesicular structures were constantly forming and dissolving, reflecting active subcellular structures. FRAP on plasma membrane-located PKD3 indicated a slower recovery of PKD3 fluorescent signal compared to those of PKC isoforms, implying a different targeting mechanism at the plasma membrane. VAMP2, the vesicle-localized v-SNARE, was later identified as a novel binding partner of PKD3 through yeast two-hybrid screening. PKD3 directly interacted with VAMP2 in vitro and in vivo, and colocalized in part with VAMP2 vesicles in cells. PKD3 did not phosphorylate VAMP-GFP and the purified GST-VAMP2 protein in in vitro phosphorylation assays. Rather, PKD3 was found to promote the recruitment of VAMP2 vesicles to the plasma membrane in response to PMA, while the kinase dead PKD3 abolished this effect. Thus, the kinase activity of PKD3 was required for PMA-induced plasma membrane trafficking of VAMP2. In summary, our findings suggest that PKD3 localizes to vesicular structures that are part of the endocytic compartment. The vesicular distribution may be attributed in part to the direct interaction between PKD3 and vesicle-associated membrane protein VAMP2, through which PKD3 may regulate VAMP2 vesicle trafficking by facilitating its recruitment to the target membrane.

IL-17 and related cytokines are direct and indirect targets of selective immunosuppressive agents for the treatment of autoimmune diseases and other diseases of pathologic inflammation. Insights into the potential adverse effects of IL-17 blockade can be drawn from the experience of patients with deficiencies in the IL-17 pathway. A unifying theme of susceptibility to mucocutaneous candidiasis is seen in both mice and humans with a variety of genetic defects that converge on this pathway. Mucocutaneous candidiasis is a superficial infection of mucosal, nail or skin surfaces usually caused by the fungal pathogen Candida albicans. The morbidity of the disease includes significant pain, weight loss and secondary complications, including carcinoma and aneurysms. This review describes the known human diseases associated with chronic mucocutaneous candidiasis (CMC) as well as the known and proposed connections to IL-17 signaling. The human diseases include defects in IL-17 signaling due to autoantibodies (AIRE deficiency), receptor mutations (IL-17 receptor mutations) or mutations in the cytokine genes (IL17F and IL17A). Hyper-IgE syndrome is characterized by elevated serum IgE, dermatitis and recurrent infections, including CMC due to impaired generation of IL-17-producing Th17 cells. Mutations in STAT1, IL12B and IL12RB1 result in CMC secondary to decreased IL-17 production through different mechanisms. Dectin-1 defects and CARD9 defects result in susceptibility to C. albicans because of impaired host recognition of the pathogen and subsequent impaired generation of IL-17-producing T cells. Thus, recent discoveries of genetic predisposition to CMC have driven the recognition of the role of IL-17 in protection from mucosal fungal infection and should guide counseling and management of patients treated with pharmacologic IL-17 blockade.