is a pathogenic yeast capable of a unique and intriguing form of cell-to-cell transfer between macrophage cells. The mechanism for cell-to-cell transfer is not understood. In this study, we imaged ...mouse macrophages with CellTracker Green 5-chloromethylfluorescein diacetate-labeled cytosol to ascertain whether cytosol was shared between donor and acceptor macrophages. Analysis of several transfer events detected no transfer of cytosol from donor-to-acceptor mouse macrophages. However, blocking Fc and complement receptors resulted in a major diminution of cell-to-cell transfer events. The timing of cell-to-cell transfer (11.17 min) closely approximated the sum of phagocytosis (4.18 min) and exocytosis (6.71 min) times. We propose that macrophage cell-to-cell transfer represents a nonlytic exocytosis event, followed by phagocytosis into a macrophage that is in close proximity, and name this process Dragotcytosis ("Dragot" is a Greek surname meaning "sentinel"), as it represents sharing of a microbe between two sentinel cells of the innate immune system.
Scientific research output has increased exponentially over the past few decades, but not equally across all fields of study, and we lack clear methods for estimating the size of any given field of ...research. Understanding how fields grow, change, and are organized is essential to understanding how human resources are allocated to the investigation of scientific problems. In this study, we estimated the size of certain biomedical fields from the number of unique author names appearing in field-relevant publications in the PubMed database. Focusing on microbiology, where the size of fields is often associated with those who work on a particular microbe, we find large differences in the size of its subfields. We found that plotting the number of unique investigators as a function of time can show changes consistent with growing or shrinking fields. In general, the number of unique author names associated with a particular microbe correlated with the number of disease cases attributed to that microbe, suggesting that the microbiology field workforce is deployed in a manner consistent with the medical importance of the microbe in question. We propose that unique author counts can be used to measure the size of the workforce in any given field, analyze the overlap of the workforce between fields, and compare how the workforce correlates to available research funds and the public health burden of a field.IMPORTANCEScience and its individual fields are growing at spectacular rates along with the number of papers being generated each year. However, we lack methods to investigate the size of these fields, many times relying on anecdotal knowledge on which fields are "hot topics" or oversaturated. Thus, we developed a bibliometric method analyzing authorship information from PubMed to estimate the size of fields based on unique author counts. Our major findings are that unique author counts serve as an efficient measurement of the size of a given field. Additionally, the size of a biomedical science field correlates to its public health burden when compared to case numbers. This method allows us to compare growth rates, workforce distribution, and the allocation of resources between fields to understand how scientific fields self-regulate. These insights can, in turn, help guide policymaking, for example, in funding allocation, to ensure fields are not neglected.
The US Food and Drug Administration authorized COVID-19 convalescent plasma (CCP) therapy for hospitalized COVID-19 patients via the Expanded Access Program (EAP) and the Emergency Use Authorization ...(EUA), leading to use in about 500,000 patients during the first year of the pandemic for the USA.
We tracked the number of CCP units dispensed to hospitals by blood banking organizations and correlated that usage with hospital admission and mortality data.
CCP usage per admission peaked in Fall 2020, with more than 40% of inpatients estimated to have received CCP between late September and early November 2020. However, after randomized controlled trials failed to show a reduction in mortality, CCP usage per admission declined steadily to a nadir of less than 10% in March 2021. We found a strong inverse correlation (r = -0.52, p=0.002) between CCP usage per hospital admission and deaths occurring 2 weeks after admission, and this finding was robust to examination of deaths taking place 1, 2, or 3 weeks after admission. Changes in the number of hospital admissions, SARS-CoV-2 variants, and age of patients could not explain these findings. The retreat from CCP usage might have resulted in as many as 29,000 excess deaths from mid-November 2020 to February 2021.
A strong inverse correlation between CCP use and mortality per admission in the USA provides population-level evidence consistent with the notion that CCP reduces mortality in COVID-19 and suggests that the recent decline in usage could have resulted in excess deaths.
There was no specific funding for this study. AC was supported in part by RO1 HL059842 and R01 AI1520789; MJJ was supported in part by 5R35HL139854. This project has been funded in whole or in part with Federal funds from the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority under Contract No. 75A50120C00096.
The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and ...transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.
The capsule of Cryptococcus neoformans Casadevall, Arturo; Coelho, Carolina; Cordero, Radames J. B. ...
Virulence,
01/2019, Volume:
10, Issue:
1
Journal Article
Peer reviewed
Open access
The capsule of Cryptococcus neoformans is its dominant virulence factor and plays a key role in the biology of this fungus. In this essay, we focus on the capsule as a cellular structure and note the ...limitations inherent in the current methodologies available for its study. Given that no single method can provide the structure of the capsule, our notions of what is the cryptococcal capsule must be arrived at by synthesizing information gathered from very different methodological approaches including microscopy, polysaccharide chemistry and physical chemistry of macromolecules. The emerging picture is one of a carefully regulated dynamic structure that is constantly rearranged as a response to environmental stimulation and cellular replication. In the environment, the capsule protects the fungus against desiccation and phagocytic predators. In animal hosts the capsule functions in both offensive and defensive modes, such that it interferes with immune responses while providing the fungal cell with a defensive shield that is both antiphagocytic and capable of absorbing microbicidal oxidative bursts from phagocytic cells. Finally, we delineate a set of unsolved problems in the cryptococcal capsule field that could provide fertile ground for future investigations.
and
are pathogenic fungi that cause significant morbidity and mortality. Cell surface hydrophobicity (CSH) is a biophysical parameter that influences the adhesion of fungal cells or spores to biotic ...and abiotic surfaces.
is encased by polysaccharide capsule that is highly hydrophilic and is a critical determinant of virulence. In this study, we report large differences in the CSH of some
and
strains. The capsular polysaccharides of
strains differ in repeating motifs and therefore vary in the number of hydroxyl groups, which, along with higher-order structure of the capsule, may contribute to the variation in hydrophobicity that we observed. We found that cell wall composition, in the context of chitin-chitosan content, does not influence CSH. For
, CSH correlated with phagocytosis by natural soil predator
Furthermore, capsular binding of the protective antibody (18B7), but not the nonprotective antibody (13F1), altered the CSH of
strains. Variability in CSH could be an important characteristic in comparing the biological properties of cryptococcal strains.
The interaction of a microbial cell with its environment is influenced by the biophysical properties of a cell. The affinity of the cell surface for water, defined by the cell surface hydrophobicity (CSH), is a biophysical parameter that varies among different strains of
The CSH influences the phagocytosis of the yeast by its natural predator in the soil, the amoeba. Studying variation in biophysical properties like CSH gives us insight into the dynamic host-predator interaction and host-pathogen interaction in a damage-response framework.
is a fungal pathogen with worldwide distribution.
resides within mature phagolysosomes where it often evades killing and replicates.
induces phagolysosomal membrane permeabilization (PMP), but the ...mechanism for this phenomenon and its consequences for macrophage viability are unknown. In this study, we used flow cytometry methodology in combination with cell viability markers and LysoTracker to measure PMP in J774.16 and murine bone marrow-derived macrophages infected with
Our results showed that cells manifesting PMP were positive for apoptotic markers, indicating an association between PMP and apoptosis. We investigated the role of phospholipase B1 in
induction of PMP. Macrophages infected with a
Δplb1 mutant had reduced PMP compared with those infected with wild-type and phospholipase B1-complemented strains, suggesting a mechanism of action for this virulence factor. Capsular enlargement inside macrophages was identified as an additional likely mechanism for phagolysosomal membrane damage. Macrophages undergoing apoptosis did not maintain an acidic phagolysosomal pH. Induction of PMP with ciprofloxacin enhanced macrophages to trigger lytic exocytosis whereas nonlytic exocytosis was common in those without PMP. Our results suggest that modulation of PMP is a critical event in determining the outcome of
macrophage interaction.
A key component of the insect immune response is melanin production, including within nodules, or aggregations of immune cells surrounding microbes. Melanization produces oxidative and toxic ...intermediates that limit microbial infections. However, a direct fungicidal role of melanin during infection has not been demonstrated. We previously reported that the fungus Cryptococcus neoformans is encapsulated with melanin within nodules of Galleria mellonella hosts. Here we developed techniques to study melanin's role during C. neoformans infection in G. mellonella. We provided evidence that in vivo melanin-encapsulation was fungicidal. To further study immune melanization, we applied tissue-clearing techniques to visualize melanized nodules in situ throughout the larvae. Further, we developed a time-lapse microscopy protocol to visualize the melanization kinetics in extracted hemolymph following fungal exposure. Using this technique, we found that cryptococcal melanin and laccase enhance immune melanization. We extended this approach to study the fungal pathogens Candida albicans and Candida auris. We find that the yeast morphologies of these fungi elicited robust melanization responses, while hyphal and pseudohyphal morphologies were melanin-evasive. Approximately 23% of melanin-encapsulated C. albicans yeast can survive and breakthrough the encapsulation. Overall, our results provide direct evidence that immune melanization functions as a direct antifungal mechanism in G. mellonella.
The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements. First, a threshold image is generated based on ...intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet. The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics. The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets. This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.
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