Dematiaceous molds are found ubiquitously in the environment and cause a wide spectrum of human disease, including infections associated with high rates of mortality. Despite this, the mechanism of ...the innate immune response has been less well studied, although it is key in the clearance of fungal pathogens. Here, we focus on
, a dematiaceous mold that caused 753 infections during a multistate outbreak due to injection of contaminated methylprednisolone. We show that macrophages are incapable of phagocytosing
Despite a lack of phagocytosis, macrophage production of tumor necrosis factor alpha is triggered by hyphae but not spores and depends upon Dectin-1, a C-type lectin receptor. Dectin-1 is specifically recruited to the macrophage-hyphal interface but not the macrophage-spore interface due to differences in carbohydrate antigen expression between these two fungal forms. Corticosteroid and antifungal therapy perturb this response, resulting in decreased cytokine production.
soft tissue infection in wild-type mice demonstrated that
provokes robust neutrophilic and granulomatous inflammation capable of thwarting fungal growth. However, coadministration of methylprednisolone acetate results in robust hyphal tissue invasion and a significant reduction in immune cell recruitment. Our results suggest that Dectin-1 is crucial for macrophage recognition and the macrophage response to
and that corticosteroids potently attenuate the immune response to this pathogen.
Candida spp. are the fourth leading cause of nosocomial blood stream infections in North America. Candida glabrata is the second most frequently isolated species, and rapid development of antifungal ...resistance has made treatment a challenge. In this study, we investigate the therapeutic potential of metformin, a biguanide with well-established action for diabetes, as an antifungal agent against C. glabrata. Both wild type and antifungal-resistant isolates of C. glabrata were subjected to biguanide and biguanide-antifungal combination treatment. Metformin, as well as other members of the biguanide family, were found to have antifungal activity against C. glabrata, with MIC
50
of 9.34 ± 0.16 mg/mL, 2.09 ± 0.04 mg/mL and 1.87 ± 0.05 mg/mL for metformin, phenformin and buformin, respectively. We demonstrate that biguanides enhance the activity of several antifungal drugs, including voriconazole, fluconazole, and amphotericin, but not micafungin. The biguanide-antifungal combinations allowed for additional antifungal effects, with fraction inhibition concentration indexes ranging from 0.5 to 1. Furthermore, metformin was able to lower antifungal MIC
50
in voriconazole and fluconazole-resistant clinical isolates of C. glabrata. We also observed growth reduction of C. glabrata with rapamycin and an FIC of 0.84 ± 0.09 when combined with metformin, suggesting biguanide action in C. glabrata may be related to inhibition of the mTOR complex. We conclude that the biguanide class has direct antifungal therapeutic potential and enhances the activity of select antifungals in the treatment of resistant C. glabrata isolates. These data support the further investigation of biguanides in the combination treatment of serious fungal infections.
The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune ...responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.
Nanoarrays for DNA detection were fabricated on etched nanofiber bundles based on recently developed techniques for microscale arrays. Two different-sized nanoarrays were created: one with 700nm ...feature sizes and a 1μm center-to-center pitch (∼1×106 array elements/mm2) and one with 300nm feature sizes and a 500nm center-to-center pitch (4.6×106 array elements/mm2). A random, multiplexed array composed of oligonucleotide-functionalized nanospheres was constructed and used for parallel detection and analysis of fluorescently labeled DNA targets. We have used these arrays to detect a variety of target sequences including Bacillus thuringiensis kurstaki and vaccina virus sequences, two potential biowarfare agents, as well as interleukin-2 sequences, an immune system modulator that has been used for the diagnosis of HIV.
Abstract
The field of infectious diseases currently takes a reactive approach and treats infections as they present in patients. Although certain populations are known to be at greater risk of ...developing infection (eg, immunocompromised), we lack a systems approach to define the true risk of future infection for a patient. Guided by impressive gains in “omics” technologies, future strategies to infectious diseases should take a precision approach to infection through identification of patients at intermediate and high-risk of infection and deploy targeted preventative measures (ie, prophylaxis). The advances of high-throughput immune profiling by multiomics approaches (ie, transcriptomics, epigenomics, metabolomics, proteomics) hold the promise to identify patients at increased risk of infection and enable risk-stratifying approaches to be applied in the clinic. Integration of patient-specific data using machine learning improves the effectiveness of prediction, providing the necessary technologies needed to propel the field of infectious diseases medicine into the era of personalized medicine.
Translational systems immunology approaches to infectious diseases will enable the switch from reactive to precision treatment of patients, which will improve clinical outcomes while reducing the use of prophylactic antibiotics and incidence of infection in high-risk individuals.
Autophagy plays an important role in maintaining cell homeostasis by providing nutrients during periods of starvation and removing damaged organelles from the cytoplasm. A marker in the autophagic ...process is the reversible conjugation of LC3, a membrane scaffolding protein, to double membrane autophagosomes. Recently, a role for LC3 in the elimination of pathogenic bacteria and fungi, including Candida albicans (C. albicans), was demonstrated, but these organisms reside in single membrane phagosomes. This process is distinct from autophagy and is termed LC3-associated phagocytosis (LAP). This review will detail the hallmarks of LAP that distinguish it from classical autophagy and review the role of autophagy proteins in host response to C. albicans and other pathogenic fungi.
The host type I interferon (IFN) pathway is a major signature of inflammation induced by the human fungal pathogen, Candida albicans. However, the molecular mechanism for activating this pathway in ...the host defence against C. albicans remains unknown. Here we reveal that mice lacking cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway components had improved survival following an intravenous challenge by C. albicans. Biofilm-associated C. albicans DNA packaged in extracellular vesicles triggers the cGAS-STING pathway as determined by induction of interferon-stimulated genes, IFNβ production, and phosphorylation of IFN regulatory factor 3 and TANK-binding kinase 1. Extracellular vesicle-induced activation of type I IFNs was independent of the Dectin-1/Card9 pathway and did not require toll-like receptor 9. Single nucleotide polymorphisms in cGAS and STING potently altered inflammatory cytokine production in human monocytes challenged by C. albicans. These studies provide insights into the early innate immune response induced by a clinically significant fungal pathogen.
Beyond its well-documented role in reproduction, embryogenesis and maintenance of body tissues, vitamin A has attracted considerable attention due to its immunomodulatory effects on both the innate ...and the adaptive immune responses. In infectious diseases, vitamin A has been shown to have a host-protective effect in infections of bacterial, viral or protozoan origin. Nevertheless, its impact in fungal infections remains unknown. Meanwhile, the frequency of invasive mycoses keeps on growing, with
Candida albicans
being the major opportunistic fungal pathogen and associated with high mortality. In the present work, we explored the impact of all-trans retinoic acid (atRA), the most active metabolite of vitamin A, on the innate immune response against
C.
albicans
in human monocytes. Our results show a strong immunomodulatory role for atRA, leading to a significant down-regulation of the fungi-induced expression and secretion of the pro-inflammatory cytokines TNFα, IL6 and IL12. Moreover, atRA significantly suppressed the expression of Dectin-1, a major fungal pattern recognition receptor, as well as the Dectin-1-dependent cytokine production. Both RAR-dependent and RAR-independent mechanisms seem to play a role in the atRA-mediated immunomodulation. Our findings open a new direction to elucidate the role of vitamin A on the immune function during fungal infections.
The number of life-threatening fungal infections has risen in immunocompromised patients, and identification of the rules that govern an appropriate immune response is essential to develop better ...diagnostics and targeted therapeutics. The outer cell wall component on pathogenic fungi consists of -1,3-glucan, and Dectin-1, a pattern recognition receptor present on the cell surface of innate immune cells, binds specifically to this carbohydrate. A barrier in understanding the exact immunological response to pathogen-derived carbohydrate epitopes is the presence of multiple types of carbohydrate moieties on fungal cell walls. To dissect the immunological mechanisms used to recognize pathogens, a system of fungal like particles was developed that consisted of polystyrene beads, which mimicked the three dimensional shape of the fungus, coated covalently with purified -1,3-glucan derived from
Saccharomyces cerevisiae.
The morphology of the -1,3-glucan layer was examined by immunofluorescence, flow cytometery, and immuno-transmission electron microscopy. The covalent linkages of the -1,3-glucan to the polystyrene surface were stable after subjecting the beads to detergents. By pre-treating -1,3-glucan beads with laminarinase, a specific -1,3-gluconase, the reactivity of the anti--1,3-glucan antibody was abrogated in comparison to treatment with proteinase K indicating that the coating of these beads was predominantly -1,3-glucan. TNF- was also measured by stimulating bone-marrow derived macrophages with the -1,3-glucan beads, and showed a dose dependent response compared to soluble -glucan, insoluble -1,3-glucan, uncoated beads, and soluble -1,3-glucan mixed with uncoated beads. Finally, -1,3-glucan beads were incubated with GFP-Dectin-1 expressing macrophages and imaged using confocal microscopy. -1,3-beads were taken up within minutes and retained Dectin-1 recruitment to the phagosome as compared to uncoated beads. These data describe a unique fungal-like particle system that will permit immunologists to probe the critical steps in early recognition of pathogen-derived fungal carbohydrate antigens by innate immune cells.
We describe a technique to create fungal-like particles, consisting of carbohydrates covalently coated to polymer spheres, to probe immunological responses.
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