Role of IL-17 in Morphogenesis and Dissemination of Cryptococcus neoformans during Murine Infection.

Cryptococcus neoformans is a pathogenic yeast that can form Titan cells in the lungs, which are fungal cells of abnormally large size. The factors that regulate Titan cell formation in vivo are still unknown, although an increased proportion of these fungal cells of infected mice correlates with induction of Th2-type responses. Here, we focused on the role played by the cytokine IL-17 in the formation of cryptococcal Titan cells using Il17a-/- knockout mice. We found that after 9 days of infection, there was a lower proportion of Titan cells in Il17a-/- mice compared to the fungal cells found in wild-type animals. Dissemination to the brain occurred earlier in Il17a-/- mice, which correlated with the lower proportion of Titan cells in the lungs. Furthermore, knockout-infected mice increased brain size more than WT mice. We also determined the profile of cytokines accumulated in the brain, and we found significant differences between both mouse strains. We found that in Il17a-/-, there was a modest increase in the concentrations of the Th1 cytokine TNF-α. To validate if the increase in this cytokine had any role in cryptococcal morphogenesis, we injected wild-type mice with TNF-α t and observed that fungal cell size was significantly reduced in mice treated with this cytokine. Our results suggest a compensatory production of cytokines in Il17a-/- mice that influences both cryptococcal morphology and dissemination.

Initial In Vivo Evaluation of a Novel Amikacin-Deoxycholate Hydrophobic Salt Delivers New Insights on Amikacin Partition in Blood and Tissues.

In this study, an initial in vivo evaluation of a new amikacin-deoxycholate hydrophobic salt aimed at potentiating amikacin action against hard-to-treat lung infections was undertaken by quantifying, for the first time, amikacin in whole blood. Pharmacokinetic evaluation after intranasal administration in a murine model showed higher drug retention in the lungs compared to blood, with no significant differences between the salt and the free drug. Upon repeated administrations, the two treatments resulted in nonsignificant tissue damage and mild higher inflammation for the hydrophobic salt. Whole-blood analysis highlighted an unreported high partition of amikacin in blood components up to 48 h, while significant lung levels were measured up to 72 h. Such a new observation was considered responsible for the nearly overlapping pharmacokinetic profiles of the two treatments. To overcome such an issue, a dry powder in an inhalable form may be best suited. Moreover, if confirmed in humans, and considering the current once-a-day regimen for amikacin aerosols, important yet-to-be-explored clinical implications may be postulated for such amikacin persistence in the organism.

Optimized Extraction of Amikacin from Murine Whole Blood.

Amikacin (Amk) analysis and quantitation, for pharmacokinetics studies and other types of investigations, is conventionally performed after extraction from plasma. No report exists so far regarding drug extraction from whole blood (WB). This can represent an issue since quantification in plasma does not account for drug partitioning to the blood cell compartment, significantly underrating the drug fraction reaching the blood circulation. In the present work, the optimization of an extraction method of Amk from murine WB has been described. The extraction yield was measured by RP-HPLC-UV after derivatization with 1-fluoro-2,4-dinitrobenzene, which produced an appreciably stable derivative with a favorable UV/vis absorption. Several extraction conditions were tested: spiked Amk disulfate solution/acetonitrile/WB ratio; presence of organic acids and/or ammonium hydroxide and/or ammonium acetate in the extraction mixture; re-dissolution of the supernatant in water after a drying process under vacuum; treatment of the supernatant with a solution of inorganic salts. The use of 5% (by volume) of ammonium hydroxide in a hydro-organic solution with acetonitrile, allowed the almost quantitative (95%) extraction of the drug from WB.

Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA) Impair Neutrophil Candidacidal Activity and Are Increased in the Cellular Fraction of Vaginal Samples from Women with Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) is primarily caused by Candida albicans and affects 75% of childbearing age women. Although C. albicans can colonize asymptomatically, disease is associated with an increased Candida burden, a loss of epithelial tolerance and a breakdown in vaginal microbiota homeostasis. VVC symptoms have been ascribed to a powerful inflammatory response associated with the infiltration of non-protective neutrophils (PMN). Here, we compared the immunological characteristics of vaginal fluids and cellular protein extracts obtained from 28 VVC women and from 23 healthy women colonized by Candida spp. We measured the levels of antibodies against fungal antigens and human autoantigens (anti-Saccharomyces cerevisiae antibodies (ASCA), C. albicans germ tube antibodies (CAGTAs) and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA)), in addition to other immunological markers. Our results show that the pANCA levels detected in the cellular protein extracts from the vaginal fluids of symptomatic women were significantly higher than those obtained from healthy colonized women. Consistent with a potential physiologically relevant role for this pANCA, we found that specific anti-myeloperoxidase antibodies could completely neutralize the ex vivo killing capacity of polymorphonuclear cells. Collectively, this preliminary study suggests for the first time that pANCA are found in the pathogenic vaginal environment and can promptly impair neutrophil function against Candida, potentially preventing a protective response.

Anti-Biofilm Properties of Saccharomyces cerevisiae CNCM I-3856 and Lacticaseibacillus rhamnosus ATCC 53103 Probiotics against G. vaginalis.

Bacterial vaginosis (BV) is characterized by the presence of a polymicrobial biofilm where Gardnerella vaginalis plays a key role. Previously, we demonstrated that Saccharomyces cerevisiae CNCM (French National Collection of Cultures of Microorganisms) I-3856 is helpful in resolving experimental simulated BV in mice. In this study, we analyzed its capacity to affect G. vaginalis biofilms and to potentiate the activity of standard antimicrobial agents. We also investigated the anti-biofilm activity of Lacticaseibacillus rhamnosus GG (ATCC 53103), a well-known strain for its intestinal healthy benefits. Biofilm biomass was assessed by crystal violet staining, and G. vaginalis viability was assessed by a colony forming unit (CFU) assay. Here, for the first time, we demonstrated that S. cerevisiae CNCM I-3856 as well as L. rhamnosus GG were able (i) to significantly inhibit G. vaginalis biofilm formation, (ii) to markedly reduce G. vaginalis viability among the biomass constituting the biofilm, (iii) to induce disaggregation of preformed biofilm, and (iv) to kill a consistent amount of bacterial cells in a G. vaginalis preformed biofilm. Furthermore, S. cerevisiae CNCM I-3856 strongly potentiates the metronidazole effect on G. vaginalis biofilm viability. These results suggest that S. cerevisiae CNCM I-3856 as well as L. rhamnosus GG could be potential novel therapeutic agents against bacterial vaginosis.

Apoptosis of vaginal epithelial cells in clinical samples from women with diagnosed bacterial vaginosis.

Bacterial vaginosis (BV) is one of the most common vaginal infections among women of childbearing age. Gardnerella vaginalis (G. vaginalis) is a keystone microorganism present in more than 95% of all BV cases. The first step of the infection process in BV is mediated by interaction of microorganisms with epithelial cells (ECs). However, the role of these cells in BV pathogenesis is largely unknown. The present study aimed to investigate the vaginal EC response during BV. Twenty healthy women and 34 women with BV were enrolled in this study. The number of ECs in the vaginal swab was counted and analyzed for intracellular signals and apoptosis by flow cytometry. Cell damage was evaluated by lactate dehydrogenase assay. Compared to that in healthy donors, the percentage of exfoliated vaginal ECs was increased in women with BV, and an absence of neutrophils was observed in both groups. Activation signals, such as p-IκBα and c-Fos were unmodulated in the vaginal ECs of women with BV. Moreover, EC damage and apoptosis were significantly increased in patients with BV. Apoptosis was related to caspase-3 activation and the presence of G. vaginalis. This study provides the first evidence of a direct involvement of G. vaginalis in the apoptotic process of vaginal ECs during BV. This effect was mediated by caspase-3 activation, and G. vaginalis appeared to be one of causes for inducing EC apoptosis in BV. Hence, our findings suggest a possible explanation for the increased exfoliation of ECs in the vagina during BV.

A Role for Yeast/Pseudohyphal Cells of Candida albicans in the Correlated Expression of NLRP3 Inflammasome Inducers in Women With Acute Vulvovaginal Candidiasis.

In acute vulvovaginal candidiasis (VVC), the fungus Candida albicans activates inflammasome receptors of vaginal epithelial cells through the production of virulence and immuno-inflammatory factors. Here, we show that in VVC patients, genes encoding some of the above factors (SAP2, SAP5, SAP6, ECE1, and HWP1) are expressed in a correlated fashion. Cytological observations pointed out that pseudohyphal filaments with yeast cells are dominant at the acidic vaginal pH, and this is coupled with co-expression, at roughly similar level, of SAP2, a typical yeast and ECE1, a typical hyphae-associated genes. In contrast, vigorous hyphal growth dominated at the neutral vaginal pH of mice experimentally infected with C. albicans isolates from VVC subjects, and this is coupled with a high ratio of ECE1 to SAP2 expression. We suggest that the pseudohyphal rather than true hyphal cells of C. albicans play a critical role in VVC, possibly through the activity of multiple inflammasome inducers.

Vaginal Epithelial Cells Discriminate Between Yeast and Hyphae of Candida albicans in Women Who Are Colonized or Have Vaginal Candidiasis.

BACKGROUND: Vaginal candidiasis is common disease affecting women; however, how Candida albicans shift from commensalism towards a pathogenic status remains poorly understood. The present study investigated the vaginal epithelial cell (EC) response dynamics under various conditions. METHODS: Healthy women, asymptomatic C. albicans carriers, and symptomatic patients with vaginal candidiasis were enrolled in this study. ECs in vaginal swabs were analyzed with cytofluorimetric analysis for pattern recognition receptors and intracellular signals, with lactate dehydrogenase assay performed for cell damage, and an enzyme-linked immunosorbent assay for cytokine expression. RESULTS: The level of toll-like receptor 4 (TLR4), TLR2, and erythropoietin-producing hepatoma A2 (EphA2) expression was significantly higher in ECs from asymptomatic and symptomatic subjects compared to healthy subjects. Activation of transcription factors, nuclear factor-κB (NF-κB) and c-Fos-p-38, was observed in ECs from symptomatic and asymptomatic pseudohyphae/hyphae carriers but not from the asymptomatic yeast carriers. EC damage was only observed in symptomatic patients. CONCLUSIONS: The presence of pseudohyphae/hyphae is required to determine vaginal candidiasis; however, it may be not sufficient to induce the pathologic process associated with neutrophil recruitment and EC damage. This study sheds light on the ambiguous role of the hyphal form during vaginal human commensalism.

Saccharomyces cerevisiae CNCM I-3856 as a New Therapeutic Agent Against Oropharyngeal Candidiasis.

Oropharyngeal candidiasis is a common opportunistic mucosal infection of the oral cavity, mainly caused by an overgrowth of Candida albicans. This infection can inhibit nutritional intakes and strongly affect quality of life. To date, standard therapeutic strategies involving the administration of antifungal drugs can bring several side effects, not least the emergence of drug-resistant strains. The purpose of this study is to investigate the effectiveness of Saccharomyces cerevisiae CNCM I-3856 (live or inactivated cells) against oropharyngeal candidiasis. Our results show that administration of S. cerevisiae CNCM I-3856 (live or inactivated cells) in the oral cavity of C57BL/6J mice resulted in a protective effect against oropharyngeal candidiasis. The strongest effect was obtained with live S. cerevisiae CNCM I-3856. This was related to: (1) a decrease in C. albicans load in the oral cavity, esophagus, stomach, and duodenum; (2) an early resolution of inflammatory process in the tongue; (3) a marked reduction in C. albicans virulence factors; and (4) a consistent increase in neutrophil antimicrobial capacity. These findings suggest that S. cerevisiae products are potentially beneficial in the treatment of oropharyngeal candidiasis.

Bioimmunological activities of Candida glabrata cellular mannan.

Candida glabrata is a second most common human opportunistic pathogen which causes superficial but also life-threatening systemic candidosis. According to the localisation of mannans and mannoproteins in the outermost layer of the cell wall, mannan detection could be one of the first steps in the cell recognition of Candida cells by the host innate immune system. Mannans from the cell wall provide important immunomodulatory activities, comprising stimulation of cytokine production, induction of dendritic cells (DCs) maturation and T-cell immunity. The model of DCs represents a promising tool to study immunomodulatory interventions throughout the vaccine development. Activated DCs induce, activate and polarise T-cell responses by expression of distinct maturation markers and cytokines regulating the adaptive immune responses. In addition, they are uniquely adept at decoding the fungus-associated information and translate it in qualitatively different T helper responses. We find out, that C. glabrata mannan is able to induce proliferation of splenocytes and to increase the production of TNF-α and IL-4. Next, increased the expression of co-stimulatory molecules CD80 and CD86 and the proportion of CD4+CD25+ and CD4+CD28+ T cells during in vitro stimulation of splenocytes. Reported results provide C. glabrata mannan capability to modulate cytokine production, DCs activation and antigen presentation activity, influencing T-cell phenotype in response to stimulation.

GILZ restrains neutrophil activation by inhibiting the MAPK pathway.

Glucocorticoid-induced leucine zipper (GILZ) exerts anti-inflammatory effects on the immune cells. However, less is known about GILZ function in neutrophils. We aimed to define the specific role of GILZ in basal neutrophil activity during an inflammatory response. GILZ knockdown resulted in a persistent activation state of neutrophils, as evidenced by increased phagocytosis, killing activity, and oxidative burst in GILZ-knockout (KO) neutrophils. This enhanced response caused severe disease in a dinitrobenzene sulfonic acid (DNBS)-induced colitis model, where GILZ-KO mice had prominent granulocytic infiltrate and excessive inflammatory state. We used a Candida albicans intraperitoneal infection model to unravel the intracellular pathways affected by GILZ expression in activated neutrophils. GILZ-KO neutrophils had stronger ability to clear the infectious agent than the wild-type (WT) neutrophils, and there was more activation of the NOX2 (NADPH oxidase 2) and p47phox proteins, which are directly involved in oxidative burst. Similarly, the MAPK pathway components, that is, ERK and p38, which are involved in the oxidative burst pathway, were highly phosphorylated in GILZ-KO neutrophils. Evaluation of GILZ expression kinetics during C. albicans infection revealed down-regulation that correlated inversely with the state of neutrophil activation, which was evaluated as oxidative burst. Overall, our findings define GILZ as a regulator of neutrophil functions, as its expression contributes to limiting neutrophil activation by reducing the activation of the signaling pathways that control the basal neutrophil functions. Controlling GILZ expression could help regulate a continuous inflammatory state that can result in chronic inflammatory and autoimmune diseases.

Eighty Years of Mycopathologia: A Retrospective Analysis of Progress Made in Understanding Human and Animal Fungal Pathogens.

Mycopathologia was founded in 1938 to 'diffuse the understanding of fungal diseases in man and animals among mycologists.' This was an important mission considering that pathogenic fungi for humans and animals represent a tiny minority of the estimated 1.5-5 million fungal inhabitants on Earth. These pathogens have diverged from the usual saprotrophic lifestyles of most fungi to colonize and infect humans and animals. Medical and veterinary mycology is the subdiscipline of microbiology that dwells into the mysteries of parasitic, fungal lifestyles. Among the oldest continuing scientific publications on the subject, Mycopathologia had its share of 'classic papers' since the first issue was published in 1938. An analysis of the eight decades of notable contributions reveals many facets of host-pathogen interactions among 183 volumes comprising about 6885 articles. We have analyzed the impact and relevance of this body of work using a combination of citation tools (Google Scholar and Scopus) since no single citation metric gives an inclusive perspective. Among the highly cited Mycopathologia publications, those on experimental mycology accounted for the major part of the articles (36%), followed by diagnostic mycology (16%), ecology and epidemiology (15%), clinical mycology (14%), taxonomy and classification (10%), and veterinary mycology (9%). The first classic publication, collecting nearly 200 citations, appeared in 1957, while two articles published in 2010 received nearly 150 citations each, which is notable for a journal covering a highly specialized field of study. An empirical analysis of the publication trends suggests continuing interests in novel diagnostics, fungal pathogenesis, review of clinical diseases especially with relevance to the laboratory scientists, taxonomy and classification of fungal pathogens, fungal infections and carriage in pets and wildlife, and changing ecology and epidemiology of fungal diseases around the globe. We anticipate that emerging and re-emerging fungal pathogens will continue to cause significant health burden in the coming decades. It remains vital that scientists and physicians continue to collaborate by learning each other's language for the study of fungal diseases, and Mycopathologia will strive to be their partner in this increasingly important endeavor to its 100th anniversary in 2038 and beyond.

Carbapenemase-producing Enterobacteriaceae isolates resistant to last-line antibiotics in an Italian general hospital.

The global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is of great concern for public health. These bacteria have the potential for rapid dissemination in healthcare settings and cause infections associated with high rates of morbidity and mortality. A total of 221 carbapenem non-susceptible Enterobacteriaceae isolates were collected from patients admitted to an Italian general hospital from January 2016 to March 2017. Among these isolates, 78.3% were carbapenemase producers: 96% were positive for the blaKPC gene and the remainder for the blaVIM gene (allelic variant VIM-1). CPE isolates were mainly Klebsiella pneumoniae, but we also detected carbapenemase enzymes in Citrobacter freundii, Enterobacter cloacae and Escherichia coli. Among CPE isolates, 79.2% exhibited co-resistance to two or more non-b-lactam agents and 38% of these isolates (all KPC-positive) were resistant to colistin. This percentage reached 55% among CPE isolated from the bloodstream. All patients with colistin-resistant CPE isolates recovered from blood samples showed an unfavorable outcome within 7 days from the first positive blood culture. Our data show the dissemination of a high percentage of CPE isolates co-resistant to last-line antibiotics. In addition, we report the first identification in our hospital of CPE isolates harboring the blaVIM gene and Escherichia coli harboring the blaKPC gene. These results underline the need to implement antibiotic stewardship and infection control programs, and emphasize the need for novel antimicrobial agents active against CPE.

Epitope unmasking in vulvovaginal candidiasis is associated with hyphal growth and neutrophilic infiltration.

Vaginal candidiasis is a common disorder in women of childbearing age, caused primarily by the dimorphic fungus Candida albicans. Since C. albicans is a normal commensal of the vaginal mucosa, a long-standing question is how the fungus switches from being a harmless commensal to a virulent pathogen. Work with human subjects and in mouse disease models suggests that host inflammatory processes drive the onset of symptomatic infection. Fungal cell wall molecules can induce inflammation through activation of epithelial and immune receptors that trigger pro-inflammatory cytokines and chemokines, but pathogenic fungi can evade recognition by masking these molecules. Knowledge about which cell wall epitopes are available for immune recognition during human infection could implicate specific ligands and receptors in the symptoms of vaginal candidiasis. To address this important gap, we directly probed the surface of fungi present in fresh vaginal samples obtained both from women with symptomatic Candida vaginitis and from women that are colonized but asymptomatic. We find that the pro-inflammatory cell wall polysaccharide ß-glucan is largely masked from immune recognition, especially on yeast. It is only exposed on a small percentage of hyphal cells, where it tends to co-localize with enhanced levels of chitin. Enhanced ß-glucan availability is only found in symptomatic patients with strong neutrophil infiltration, implicating neutrophils as a possible driver of these cell wall changes. This is especially interesting because neutrophils were recently shown to be necessary and sufficient to provoke enhanced ß-glucan exposure in C. albicans, accompanied by elevated immune responses. Taken together, our data suggest that the architecture of C. albicans cell wall can be altered by environmental stress during vaginal candidiasis.

Saccharomyces cerevisiae-based probiotic as novel anti-microbial agent for therapy of bacterial vaginosis.

In this study, we demonstrate, for the first time, that Saccharomyces cerevisiae-based probiotic shows an inhibitory effect on Gardnerella vaginalis infection. This effect is likely due to several actions: direct interference with adherence to vaginal tissues, inhibition of sialidase activity, reduction of vaginal epithelial exfoliation. Gardnerella vaginalis does not induce vaginal inflammation and no inflammatory cytokines were, indeed, produced, by the mouse vagina, neither by Gardnerella vaginalis and by the probiotic. Collectively, our data incite to further investigations on Saccharomyces cerevisiae probiotic as a potential prophylactic or therapeutic agent in the vaginosis caused by Gardnerella vaginalis.

NLRP3 inflammasome is a key player in human vulvovaginal disease caused by Candida albicans.

The expression of host inflammatory and Candida albicans putative virulence factors was studied in women with vulvovaginal candidiasis (VVC; twenty) or colonized by the fungus but asymptomatic (carriers; fifteen) or non-colonized asymptomatic (ten subjects). Overexpression of genes encoding NLRP3 and caspase-1 inflammasome components sharply differentiated VVC patients from asymptomatic colonized or non-colonized women. Inflammasome expression was coupled with neutrophils recruitment in the vagina of VVC women and IL-1ß and IL-8 production. Both cytokines were present, though to a lower concentration, also in the vaginal fluid of colonized and non-colonized women. Secretory aspartyl proteinases (SAPs) and hyphae associated genes HWP1 and ECE1 were upregulated in VVC but with some differences among infected women. The most overexpressed SAP gene was SAP2, that correlated with neutrophils accumulation. Our data provide clinical evidence that the intracytoplasmic activation of NLRP3 inflammasome complex plays a critical, pathogenesis-relevant role in human VVC.

Role of the glucocorticoid-induced leucine zipper gene in dexamethasone-induced inhibition of mouse neutrophil migration via control of annexin A1 expression.

The glucocorticoid-induced leucine zipper (GILZ) gene is a pivotal mediator of the anti-inflammatory effects of glucocorticoids (GCs) that are known to regulate the function of both adaptive and innate immunity cells. Our aim was to investigate the role of GILZ in GC-induced inhibition of neutrophil migration, as this role has not been investigated before. We found that GILZ expression was induced by dexamethasone (DEX), a synthetic GC, in neutrophils, and that it regulated migration of these cells into inflamed tissues under DEX treatment. Of note, inhibition of neutrophil migration was not observed in GILZ-knockout mice with peritonitis that were treated by DEX. This was because DEX was unable to up-regulate annexin A1 (Anxa1) expression in the absence of GILZ. Furthermore, we showed that GILZ mediates Anxa1 induction by GCs by transactivating Anxa1 expression at the promoter level via binding with the transcription factor, PU.1. The present findings shed light on the role of GILZ in the mechanism of induction of Anxa1 by GCs. As Anxa1 is an important protein for the resolution of inflammatory response, GILZ may represent a new pharmacologic target for treatment of inflammatory diseases.-Ricci, E., Ronchetti, S., Pericolini, E., Gabrielli, E., Cari, L., Gentili, M., Roselletti, E., Migliorati, G., Vecchiarelli, A., Riccardi, C. Role of the glucocorticoid-induced leucine zipper gene in dexamethasone-induced inhibition of mouse neutrophil migration via control of annexin A1 expression.

Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis.

Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection.