In silico genome wide identification of long non-coding RNAs differentially expressed during Candida auris host pathogenesis.

Candida auris is an invasive fungal pathogen of high concern due to acquired drug tolerance against antifungals used in clinics. The prolonged persistence on biotic and abiotic surfaces can result in onset of hospital outbreaks causing serious health threat. An in depth understanding of pathology of C. auris is highly desirable for development of efficient therapeutics. Non-coding RNAs play crucial role in fungal pathology. However, the information about ncRNAs is scanty to be utilized. Herein our aim is to identify long noncoding RNAs with potent role in pathobiology of C. auris. Thereby, we analyzed the transcriptomics data of C. auris infection in blood for identification of potential lncRNAs with regulatory role in determining invasion, survival or drug tolerance under infection conditions. Interestingly, we found 275 lncRNAs, out of which 253 matched with lncRNAs reported in Candidamine, corroborating for our accurate data analysis pipeline. Nevertheless, we obtained 23 novel lncRNAs not reported earlier. Three lncRNAs were found to be under expressed throughout the course of infection, in the transcriptomics data. 16 of potent lncRNAs were found to be coexpressed with coding genes, emphasizing for their functional role. Noteworthy, these ncRNAs are expressed from intergenic regions of the genes associated with transporters, metabolism, cell wall biogenesis. This study recommends for possible association between lncRNA expression and C. auris pathogenesis.

Identification of the Most Immunoreactive Antigens of Candida auris to IgGs from Systemic Infections in Mice.

The delay in making a correct diagnosis of Candida auris causes concern in the healthcare system setting, and immunoproteomics studies are important to identify immunoreactive proteins for new diagnostic strategies. In this study, immunocompetent murine systemic infections caused by non-aggregative and aggregative phenotypes of C. auris and by Candida albicans and Candida haemulonii were carried out, and the obtained sera were used to study their immunoreactivity against C. auris proteins. The results showed higher virulence, in terms of infection signs, weight loss, and histopathological damage, of the non-aggregative isolate. Moreover, C. auris was less virulent than C. albicans but more than C. haemulonii. Regarding the immunoproteomics study, 13 spots recognized by sera from mice infected with both C. auris phenotypes and analyzed by mass spectrometry corresponded to enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase. These four proteins were also recognized by sera obtained from human patients with disseminated C. auris infection but not by sera obtained from mice infected with C. albicans or Aspergillus fumigatus. Spot identification data are available via ProteomeXchange with the identifier PXD049077. In conclusion, this study showed that the identified proteins could be potential candidates to be studied as new diagnostic or even therapeutic targets for C. auris.

Prevalence of biofilms in Candida spp. bloodstream infections: A meta-analysis.

CONTEXT: Candida-related infections are nowadays a serious Public Health Problem emerging multidrug-resistant strains. Candida biofilm also leads bloodstream infections to invasive systemic infections. OBJECTIVE: The present meta-analysis aimed to analyze Candida biofilm rate, type, and antifungal resistance among hospitalized patients between 1995 and 2020. DATA SOURCES: Web of Science, Scopus, PubMed, and Google Scholar databases were searched for English papers using the following medical subject heading terms (MESH): "invasive candidiasis"; "bloodstream infections"; "biofilm formation"; "biofilm-related infections"; "mortality"; and "prevalence". STUDY SELECTION: The major inclusion criteria included reporting the rate of biofilm formation and the prevalence of biofilm-related to Candida species, including observational studies (more exactly, cohort, retrospective, and case-control studies). Furthermore, data regarding the mortality rate, the geographical location of the study set, and the use of anti-fungal agents in clinical isolates were also extracted from the studies. DATA EXTRACTION: Independent extraction of articles by 2 authors using predefined data fields, including study quality indicators. DATA SYNTHESIS: A total of 31 studies from publicly available databases met our inclusion criteria. The biofilm formation in the data set varied greatly from 16 to 100% in blood samples. Most of the studies belonged to Europe (17/31) and Asia (9/31). Forest plot showed a pooled rate of biofilm formation of 80.0% (CI: 67-90), with high heterogeneity (Q = 2567.45, I2 = 98.83, τ2 = 0.150) in random effects model (p < 0.001). The funnel plot and Egger's linear regression test failed to find publication bias (p = 0.896). The mortality rate in Candida-related bloodstream infections was 37.9% of which 70.0% were from biofilm-associated infections. Furthermore, Candida isolates were also characterized in low, intermediate, or high biofilm formers through their level of biofilm mass (crystal violet staining or XTT assays) after a 24h growth. When comparing between countries, statistical differences were obtained (p = 0.0074), showing the lower and higher biofilm prevalence values in Italy and Spain, respectively. The prevalence of low, intermediate, and high biofilms were 36.2, 18.9, and 35.0% (p < 0.0001), respectively. C. tropicalis was the prevalent species in high biofilm formation (67.5%) showing statistically significant differences when compared to other Candida species, except for C. krusei and C. glabrata. Finally, the rates of antifungal resistance to fluconazole, voriconazole, and caspofungin related to biofilm were 70.5, 67.9 and 72.8% (p < 0.001), respectively. CONCLUSIONS: Early detection of biofilms and a better characterization of Candida spp. bloodstream infections should be considered, which eventually will help preserve public health resources and ultimately diminish mortality among patients.

Clinical features of invasive fungal disease in children with no underlying disease.

There is limited research into Invasive fungal disease (IFD) in children with no underlying disease. We undertook a retrospective study of children with IFD who did not suffer from another underlying disease, from June 2010 to March 2018 in Changsha, China. Nine children were identified. Eosinophil counts were elevated in six cases. The level of procalcitonin (PCT) was elevated in six cases. Fungal culture was positive in all patients, including eight cases of Cryptococcus neoformans and one case of Candida parapsilosis. 8.33 days following antifungal treatment, the body temperature of the eight patients affected by cryptococcal disease had returned to normal. Our study indicates that the primary pathogen in IFD was Cryptococcus neoformans in children who had no other underlying disease. Eosinophils can be considered to be indicators of cryptococcal infection. IFD in children with no other underlying disease has a satisfactory prognosis.

False-positive HIV serology, Candida lusitaniae pneumonia, and a novel mutation in the CYBB gene.

BACKGROUND: Chronic granulomatous disease (CGD) presents with a myriad of clinical manifestations pertaining to both immunodeficiency and hyperinflammation. Although Candida infection is a signature organism for patients with CGD, C. lusitaniae pneumonia in CGD has rarely been reported. C. lusitaniae is a ubiquitous ascomycete predominantly infecting immunocompromised hosts and has the potential to rapidly develop multi-drug resistance during therapy. Additionally, C. lusitaniae is recognized for its variable resistance against amphotericin B. To date, C. lusitaniae infections in patients with CGD have not been reviewed in detail. False-positive HIV serology, resulting from polyclonal hypergammaglobulinemia, has been reported in association with several infections, auto-immune diseases, and malignancies. Although CGD is often associated with hypergammaglobulinemia, a false-positive HIV serology in CGD has not been reported previously. PROCEDURE: We report a combination of unique findings in a child with CGD - a false-positive HIV serology, Candida lusitaniae pneumonia, and a novel CYBB mutation. We also provide a detailed review of C. lusitaniae infections in patients with CGD. RESULTS: In patients with CGD, C. lusitaniae has been reported to cause lymphadenitis (cervical, abdominal), fungemia, meningoencephalitis, or abscesses in the liver and spleen. Many CGD patients with C. lusitaniae infection have associated inflammatory complications of the gut (inflammatory bowel disease, colitis). Additionally, almost all C. lusitaniae infections in CGD have been reported in young infants or in patients receiving long-term immunosuppressive therapy. This reflects that further immunocompromise (in addition to the underlying immune deficiency in CGD) may specifically predispose to C. lusitaniae infection (unlike other candidal infections). Most of the CGD patients with documented C. lusitaniae infection have X-linked form of the disease which generally has been postulated to have a more severe clinical phenotype than the autosomal recessive forms of the disease. CONCLUSIONS: HIV serology may be positive in patients with CGD and other inborn errors of immunity as a result of hypergammaglobulinemia. C. lusitaniae, which may have peculiar and evolving antimicrobial sensitivity patterns, needs to be considered in patients with CGD and pneumonia. Lastly, to reiterate, CGD should to be considered in patients with proven C. lusitaniae infection.

Dissection of the anti-Candida albicans mannan immune response using synthetic oligomannosides reveals unique properties of ß-1,2 mannotriose protective epitopes.

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and ß anomery complemented with a synthetic ß-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or ß linkages, excepted for MAb B6.1 (protective epitope) reacting with ß-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients' sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing ß- and α-Man epitopes and detection of antibodies against ß-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and ß-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/ß ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

Candidaemia in Central Slovenia: A 12-year retrospective survey.

BACKGROUND: Candida bloodstream infections (BSI) became an important invasive disease in the late 20th century, in particular among immunocompromised patients. Although considerable progress has been made in the management of patients with invasive mycoses, Candida BSI are still widespread among hospitalised patients and are associated with relatively high mortality. OBJECTIVES: We conducted a retrospective study to evaluate patient characteristics, incidence, species distribution and antifungal susceptibility of BSI isolates of Candida spp. as well as outcomes of Candida BSI from 2001 to 2012, before the widespread use of echinocandins. This is the first epidemiological study of Candida BSI in Slovenia so far. METHODS: All documented candidaemia cases from 2001 to 2012 in two major hospitals-University Medical Centre and Institute of Oncology in Ljubljana, Slovenia-were taken into consideration. Candida BSI were identified in 422 patients (250 male, 172 female). Laboratory and clinical data of these patients were retrospectively analysed. Mann-Whitney U test was used to compare continuous variables and Fisher's exact test or chi-squared test for categorical variables. RESULTS AND CONCLUSIONS: The average incidence of Candida BSI was 0.524/10.000 patient-days (0,317/1000 admissions); 16/422 were younger than 1 year and 251/422 patients were over 60 years old. The most commonly isolated species were Candida albicans and Candida glabrata, followed by Candida parapsilosis. Majority of the patients had a single episode of Candida BSI, multiple episodes of Candida BSI occurred in 18/434 patients (4.1%); in 25/434 patients (5.8%) mixed Candida BSI were observed. Crude 30-day case-fatality rate was 55.4%.

Interplay between Humoral and CLA+ T Cell Response against Candida albicans in Psoriasis.

Candida albicans (CA) infections have been associated with psoriasis onset or disease flares. However, the integrated immune response against this fungus is still poorly characterized in psoriasis. We studied specific immunoglobulins in plasma and the CA response in cocultures of circulating memory CD45RA- cutaneous lymphocyte antigen (CLA)+/- T cell with autologous epidermal cells from plaque and guttate psoriasis patients (cohort 1, n = 52), and also healthy individuals (n = 17). A complete proteomic profile was also evaluated in plaque psoriasis patients (cohort 2, n = 114) regarding their anti-CA IgA levels. Increased anti-CA IgA and IgG levels are present in the plasma from plaque but not guttate psoriasis compared to healthy controls. CA cellular response is confined to CLA+ T cells and is primarily Th17. The levels of anti-CA IgA are directly associated with CLA+ Th17 response in plaque psoriasis. Proteomic analysis revealed distinct profiles in psoriasis patients with high anti-CA IgA. C-C motif chemokine ligand 18, chitinase-3-like protein 1 and azurocidin were significantly elevated in the plasma from plaque psoriasis patients with high anti-CA levels and severe disease. Our results indicate a mechanism by which Candida albicans exposure can trigger a clinically relevant IL-17 response in psoriasis. Assessing anti-CA IgA levels may be useful in order to evaluate chronic psoriasis patients.

Optimization of Micafungin Dosage for Chinese Patients with Sepsis in the Intensive Care Unit Based on a Population Pharmacokinetic-Pharmacodynamic Analysis.

PURPOSE: This study aimed to identify parameters that influence micafungin pharmacokinetics in Chinese patients with sepsis in the intensive care unit and optimize micafungin dosage by determining the probability of reaching pharmacodynamic targets. METHODS: Blood samples were collected from 32 Chinese patients with sepsis who were treated with micafungin. The samples were analyzed and used to build a population pharmacokinetic model. Monte Carlo simulations were performed to estimate the probability of achieving adequate plasma levels of micafungin against Candida species. RESULTS: Alanine aminotransferase and sequential organ failure assessment score were found to significantly influence the clearance and peripheral distribution volume of micafungin, respectively. Monte Carlo simulations based on area under the plasma concentration-time curve over 24 h showed that patients must be administered at least 200 and 250 mg micafungin daily to reach minimum inhibitory concentration breakpoints of 0.032 and 0.064 mg/L for Candida glabrata and Candida tropicalis, respectively. Additionally, a probability of target attainment of ≥ 90% could not be achieved for Candida krusei or Candida parapsilosis with a 300 mg daily dose. CONCLUSIONS: The recommended daily dose of micafungin (100 mg) may produce low clinical success ratios in non-Candida albicans infections; therefore, higher doses should be administered to improve clinical outcomes.

A case-series of bloodstream infections caused by the Meyerozyma guilliermondii species complex at a reference center of oncology in Brazil.

Bloodstream infections (BSI) caused by Candida species are the fourth cause of healthcare associated infections worldwide. Non-albicans Candida species emerged in the last decades as agents of serious diseases. In this study, clinical and microbiological aspects of six patients with BSI due to the Meyerozyma (Candida) guilliermondii species complex from an oncology reference center in Brazil, were evaluated. To describe demographic and clinical characteristics, medical records of the patients were reviewed. Molecular identification of the isolates was performed by ITS1-5.8S-ITS2 region sequencing. Antifungal susceptibility was evaluated by the EUCAST method and the minimal inhibitory concentrations (MIC) assessed according to the epidemiological cutoff values. Virulence associated phenotypes of the isolates were also studied. Ten isolates from the six patients were evaluated. Five of them were identified as Meyerozyma guilliermondii and the others as Meyerozyma caribbica. One patient was infected with two M. caribbica isolates with different genetic backgrounds. High MICs were observed for fluconazole and echinocandins. Non-wild type isolates to voriconazole appeared in one patient previously treated with this azole. Additionally, two patients survived, despite infected with non-wild type strains for fluconazole and treated with this drug. All isolates produced hemolysin, which was not associated with a poor prognosis, and none produced phospholipases. Aspartic proteases, phytase, and esterase were detected in a few isolates. This study shows the reduced antifungal susceptibility and a variable production of virulence-related enzymes by Meyerozyma spp. In addition, it highlights the poor prognosis of neutropenic patients with BSI caused by this emerging species complex. LAY ABSTRACT: Our manuscript describes demographic, clinical and microbiological characteristics of patients with bloodstream infection by the Meyerozyma guilliermondii species complex at a reference center in oncology in Brazil.

Survival Strategies of Pathogenic Candida Species in Human Blood Show Independent and Specific Adaptations.

Only four species, Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis, together account for about 90% of all Candida bloodstream infections and are among the most common causes of invasive fungal infections of humans. However, virulence potential varies among these species, and the phylogenetic tree reveals that their pathogenicity may have emerged several times independently during evolution. We therefore tested these four species in a human whole-blood infection model to determine, via comprehensive dual-species RNA-sequencing analyses, which fungal infection strategies are conserved and which are recent evolutionary developments. The ex vivo infection progressed from initial immune cell interactions to nearly complete killing of all fungal cells. During the course of infection, we characterized important parameters of pathogen-host interactions, such as fungal survival, types of interacting immune cells, and cytokine release. On the transcriptional level, we obtained a predominantly uniform and species-independent human response governed by a strong upregulation of proinflammatory processes, which was downregulated at later time points after most of the fungal cells were killed. In stark contrast, we observed that the different fungal species pursued predominantly individual strategies and showed significantly different global transcriptome patterns. Among other findings, our functional analyses revealed that the fungal species relied on different metabolic pathways and virulence factors to survive the host-imposed stress. These data show that adaptation of Candida species as a response to the host is not a phylogenetic trait, but rather has likely evolved independently as a prerequisite to cause human infections.IMPORTANCE To ensure their survival, pathogens have to adapt immediately to new environments in their hosts, for example, during the transition from the gut to the bloodstream. Here, we investigated the basis of this adaptation in a group of fungal species which are among the most common causes of hospital-acquired infections, the Candida species. On the basis of a human whole-blood infection model, we studied which genes and processes are active over the course of an infection in both the host and four different Candida pathogens. Remarkably, we found that, while the human host response during the early phase of infection is predominantly uniform, the pathogens pursue largely individual strategies and each one regulates genes involved in largely disparate processes in the blood. Our results reveal that C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis all have developed individual strategies for survival in the host. This indicates that their pathogenicity in humans has evolved several times independently and that genes which are central for survival in the host for one species may be irrelevant in another.

A designed antifungal peptide with therapeutic potential for clinical drug-resistant Candida albicans.

Due to the increasing drug-resistant of Candida albicans (C. albicans), there is an urgent need to develop a novel therapeutic agent for C. albicans induced inflammatory disease treatment. Antimicrobial peptides (AMPs) are regarded as one of the most promising antifungal drugs. However, most of the designed AMPs showed side-effects. In the present study, 10 novel peptides were designed based on the sequence of frog skin secretions peptide (Ranacyclin AJ). Among them, AKK8 (RWRFKWWKK) exhibited the strongest antifungal effect against both standard and clinically isolated drug-resistant C. albicans. AKK8 killed C. albicans (within 30 min), and the antifungal effect lasted for 24 h, showed an efficient and long lasted antifungal effect against C. albicans. Notably, AKK8 showed low toxicity to human red blood cells and high stability in human serum. Moreover, AKK8 administration showed therapeutic effects on systemic infections mice induced by the clinical drug-resistant C. albicans, in a dose-depended manner. These findings suggested that AKK8 may be a potential candidate for the anti-inflammation treatments for diseases caused by clinical drug-resistant C. albicans.

Assessment of Silver Levels in a Closed-Incision Negative Pressure Therapy Dressing: In Vitro and In Vivo Study.

Objective: In recent years, reticulated open-cell foam-based closed-incision negative pressure therapy (ROCF-ciNPT) has shown effectiveness in management of various postoperative incisions. These dressings consist of a skin interface layer that absorbs fluid from the skin surface and reduces the potential for microbial colonization within the dressing by means of ionic silver. This study examines the ability of silver to reduce the bioburden within the dressing as well as the localized effect due to potential silver mobility. Approach: Ability of silver to reduce bioburden within the ROCF-ciNPT dressing was assessed using Staphylococcus aureus, Pseudomonas aeruginosa, and Candida spp. Furthermore, silver mobility was assessed using an in vitro skin model to study the zone of inhibition along with released silver quantification. Using a porcine model, diffusion of silver into blood and tissue was studied using emission spectrometry and histology. Results: Microbial growth in the ROCF-ciNPT dressing was significantly reduced (∼2.7-4.9 log reduction) compared to a silver-free negative control. No zone of inhibition was observed for microbial colonies for up to 7 days with minimal localized silver release (<5.5 ppm release). In vivo studies demonstrated no measurable concentration (<0.2 µg/g) of silver in the blood, urine, feces, kidney, and liver tissue biopsy. Innovation: This study provides an important insight into silver concentration and mobility within the ROCF-ciNPT dressing, given emerging concerns associated with potential silver cytotoxicity. Conclusion: These results indicate the concentration of silver (0.019% silver by weight) in the ROCF-ciNPT dressings has been adequate to reduce bioburden within the skin interface layer, while severely limiting the amount of silver leaching out.

Molecular Elucidation of Riboflavin Production and Regulation in Candida albicans, toward a Novel Antifungal Drug Target.

Candida albicans is a major cause of fungal infections, both superficial and invasive. The economic costs as well as consequences for patient welfare are substantial. Only a few treatment options are available due to the high resemblance between fungal targets and host molecules, as both are eukaryotes. Riboflavin is a yellow pigment, also termed vitamin B2 Unlike animals, fungi can synthesize this essential component themselves, thereby leading us to appreciate that targeting riboflavin production is a promising novel strategy against fungal infections. Here, we report that the GTP cyclohydrolase encoded by C. albicansRIB1 (CaRIB1) is essential and rate-limiting for production of riboflavin in the fungal pathogen. We confirm the high potential of CaRib1 as an antifungal drug target, as its deletion completely impairs in vivo infectibility by C. albicans in model systems. Furthermore, the stimulating effect of iron deprivation and PKA activation on riboflavin production seems to involve CaRib1 and the upstream transcription factor CaSef1. Gathering insights in the synthesis mechanism of riboflavin in pathogenic fungi, like C. albicans, will allow us to design a novel strategy and specifically target this process to combat fungal infections.IMPORTANCECandida albicans is an important fungal pathogen causing common superficial infections as well as invasive diseases with an extremely high morbidity and mortality. Antifungal therapies are limited in efficiency and availability. In this research, we describe the regulation of riboflavin production in C. albicans Since riboflavin biosynthesis is essential to this organism, we can appreciate that targeting it would be a promising new strategy to combat these fungal infections. We provide evidence that one particular enzyme in the production process, CaRib1, would be most promising as an antifungal drug target, as it plays a central role in regulation and proves to be essential in a mouse model of systemic infection.

Population pharmacokinetics of micafungin over repeated doses in critically ill patients: a need for a loading dose?

OBJECTIVES: To study the population pharmacokinetics of micafungin in critically ill patients, evaluate and optimize dosage regimens. METHODS: An HPLC-fluorescence bioassay for micafungin was developed, fully validated and applied to a pharmacokinetic study conducted in 14 ICU patients. Dense blood sampling was performed from days 1 to 7. A population pharmacokinetic model accounting for interindividual (IIV) and interoccasion variability (IOV) of the PK parameters was developed. Simulations were performed to estimate the probability of target attainment (PTA) for several dosing regimens. KEY FINDINGS: A two-compartment pharmacokinetic model best described the data, with population clearance CL = 1.31 L/h and central volume V1 = 14.2 L. The relatively high IOV observed (45% for CL, 27% for V1) sets limits for the dose individualization in this population. The low PTA on the first day of treatment suggests the need of a loading dose. PTA and CFR estimates show that the current micafungin dosage may be insufficient for the treatment of borderline susceptible Candida strains. CONCLUSIONS: A loading dose of up to 300 mg of micafungin is needed for the treatment of invasive candidiasis in ICU patients while a maintenance dose of up to 200 mg can be considered in empirical antifungal treatment.

Candida Species-Dependent Release of IL-12 by Dendritic Cells Induces Different Levels of NK Cell Stimulation.

BACKGROUND: Candida albicans and Candida glabrata are the 2 most prevalent Candida species causing bloodstream infections. Patterns of innate immune activation triggered by the 2 fungi differ considerably. METHODS: To analyze human natural killer (NK) cell activation by both species, we performed ex vivo whole-blood infection assays and confrontation assays with primary human NK cells. RESULTS: C. albicans was a stronger activator for isolated human NK cells than C. glabrata. In contrast, activation of blood NK cells, characterized by an upregulated surface exposure of early activation antigen CD69 and death receptor ligand TRAIL, as well as interferon-γ (IFN-γ) secretion, was more pronounced during C. glabrata infection. NK cell activation in blood is mediated by humoral mediators released by other immune cells and does not depend on direct activation by fungal cells. Cross-talk between Candida-confronted monocyte-derived dendritic cells (moDC) and NK cells resulted in the same NK activation phenotype as NK cells in human blood. Blocking experiments and cytokine substitution identified interleukin-12 as a critical mediator in regulation of primary NK cells by moDC. CONCLUSIONS: Activation of human NK cells in response to Candida in human blood mainly occurs indirectly by mediators released from monocytic cells.

Changes in mammalian copper homeostasis during microbial infection.

Animals carefully control homeostasis of Cu, a metal that is both potentially toxic and an essential nutrient. During infection, various shifts in Cu homeostasis can ensue. In mice infected with Candida albicans, serum Cu progressively rises and at late stages of infection, liver Cu rises, while kidney Cu declines. The basis for these changes in Cu homeostasis was poorly understood. We report here that the progressive rise in serum Cu is attributable to liver production of the multicopper oxidase ceruloplasmin (Cp). Through studies using Cp-/- mice, we find this elevated Cp helps recover serum Fe levels at late stages of infection, consistent with a role for Cp in loading transferrin with Fe. Cp also accounts for the elevation in liver Cu seen during infection, but not for the fluctuations in kidney Cu. The Cu exporting ATPase ATP7B is one candidate for kidney Cu control, but we find no change in the pattern of kidney Cu loss during infection of Atp7b-/- mice, implying alternative mechanisms. To test whether fungal infiltration of kidney tissue was required for kidney Cu loss, we explored other paradigms of infection. Infection with the intravascular malaria parasite Plasmodium berghei caused a rise in serum Cu and decrease in kidney Cu similar to that seen with C. albicans. Thus, dynamics in kidney Cu homeostasis appear to be a common feature among vastly different infection paradigms. The implications for such Cu homeostasis control in immunity are discussed.

Significant Differences in Host-Pathogen Interactions Between Murine and Human Whole Blood.

Murine infection models are widely used to study systemic candidiasis caused by C. albicans. Whole-blood models can help to elucidate host-pathogens interactions and have been used for several Candida species in human blood. We adapted the human whole-blood model to murine blood. Unlike human blood, murine blood was unable to reduce fungal burden and more substantial filamentation of C. albicans was observed. This coincided with less fungal association with leukocytes, especially neutrophils. The lower neutrophil number in murine blood only partially explains insufficient infection and filamentation control, as spiking with murine neutrophils had only limited effects on fungal killing. Furthermore, increased fungal survival is not mediated by enhanced filamentation, as a filament-deficient mutant was likewise not eliminated. We also observed host-dependent differences for interaction of platelets with C. albicans, showing enhanced platelet aggregation, adhesion and activation in murine blood. For human blood, opsonization was shown to decrease platelet interaction suggesting that complement factors interfere with fungus-to-platelet binding. Our results reveal substantial differences between murine and human whole-blood models infected with C. albicans and thereby demonstrate limitations in the translatability of this ex vivo model between hosts.

Initial Immune Response in Escherichia coli, Staphylococcus aureus, and Candida albicans Bacteremia.

Sepsis remains a leading cause of mortality worldwide and is characterized by sustained inflammatory responses, reflected as changes in the expression profile of cytokines with time. The aim of the present study was to investigate the dynamic changes in complete blood count, serum chemistry, procalcitonin (PCT), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) in Escherichia coli, Staphylococcus aureus, and Candida albicans bacteremia. Study subjects were 32 healthy male Landrace-Large White pigs, aged 10-15 weeks and of average weight 19 ± 2 kg. Bacteremia was induced by continuous intravenous infusion of microbial suspensions during a period of 8 h. E. coli and S. aureus bacteremia were associated with a significant gradual decrease in white blood cells and platelets, respectively (p = 0.002 and p = 0.004), while candidemia was characterized by a significant gradual decrease in lymphocytes (p = 0.009). Serum PCT levels were either undetectable or very low, with no significant changes with time in all groups. E. coli bacteremia elicited a strong pro-inflammatory response, characterized by a significant increase in TNF-α expression from the onset of bacteremia (p = 0.042). C. albicans exhibited a different profile with an early, moderate increase in TNF-α followed by a subsequent marked increase in IL-6 levels (p = 0.03). The differential regulation of inflammatory and hematological responses depending on the pathogenic agent can reveal differences in the underlying inflammatory mechanisms, which may assist in the ongoing quest for the identification of a panel of circulating biomarkers during bacteremia.