Impact of Candida auris Infection in a Neutropenic Murine Model.

Candida auris has become a global public health threat due to its multidrug resistance and persistence. Currently, there are limited murine models to study C. auris infection. Those models use a combination of cyclophosphamide and cortisone acetate, suppressing both innate and adaptive immunity. Here, we compare C. auris infection in two neutrophil-depleted murine models in which innate immunity is targeted using the monoclonal antibodies 1A8 and RB6-8C5.

Dectin-2-mediated signaling triggered by the cell wall polysaccharides of Cryptococcus neoformans.

Cryptococcus neoformans is rich in polysaccharides of the cell wall and capsule. Dectin-2 recognizes high-mannose polysaccharides and plays a central role in the immune response to fungal pathogens. Previously, we demonstrated Dectin-2 was involved in the activation of dendritic cells upon stimulation with C. neoformans, suggesting the existence of a ligand recognized by Dectin-2. In the present study, we examined the cell wall structures of C. neoformans contributing to the Dectin-2-mediated activation of immune cells. In a NFAT-GFP reporter assay of the reported cells expressing Dectin-2, the lysates, but not the whole yeast cells, of an acapsular strain of C. neoformans (Cap67) delivered Dectin-2-mediated signaling. This activity was detected in the supernatant of ß-glucanase-treated Cap67 and more strongly in the semi-purified polysaccharides of this supernatant using ConA-affinity chromatography (ConA-bound fraction), in which a large amount of saccharides, but not protein, were detected. Treatment of this supernatant with periodic acid and the addition of excessive mannose, but not glucose or galactose, strongly inhibited this activity. The ConA-bound fraction of the ß-glucanase-treated Cap67 supernatant was bound to Dectin-2-Fc fusion protein in a dose-dependent manner and strongly induced the production of interleukin-12p40 and tumour necrosis factor-α by dendritic cells; this was abrogated under the Dectin-2-deficient condition. Finally, 98 kDa mannoprotein (MP98) derived from C. neoformans showed activation of the reporter cells expressing Dectin-2. These results suggested that a ligand with mannose moieties may exist in the cell walls and play a critical role in the activation of dendritic cells during infection with C. neoformans.

Candida albicans Elicits Pro-Inflammatory Differential Gene Expression in Intestinal Peyer's Patches.

The details of how gut-associated lymphoid tissues such as Peyer's patches (PPs) in the small intestine play a role in immune surveillance, microbial differentiation and the mucosal barrier protection in response to fungal organisms such as Candida albicans are still unclear. We particularly focus on PPs as they are the immune sensors and inductive sites of the gut that influence inflammation and tolerance. We have previously demonstrated that CD11c+ phagocytes that include dendritic cells and macrophages are located in the sub-epithelial dome within PPs sample C. albicans. To gain insight on how specific cells within PPs sense and respond to the sampling of fungi, we gavaged naïve mice with C. albicans strains ATCC 18804 and SC5314 as well as Saccharomyces cerevisiae. We measured the differential gene expression of sorted CD45+ B220+ B-cells, CD3+ T-cells and CD11c+ DCs within the first 24 h post-gavage using nanostring nCounter® technology. The results reveal that at 24 h, PP phagocytes were the cell type that displayed differential gene expression. These phagocytes were able to sample C. albicans and discriminate between strains. In particular, strain ATCC 18804 upregulated fungal-specific pro-inflammatory genes pertaining to innate and adaptive immune responses. Interestingly, PP CD11c+ phagocytes also differentially expressed genes in response to C. albicans that were important in the protection of the mucosal barrier. These results highlight that the mucosal barrier not only responds to C. albicans, but also aids in the protection of the host.

Assessment of environmental and occupational exposure while working with multidrug resistant (MDR) fungus Candida auris in an animal facility.

In less than a decade since its identification in 2009, the emerging fungal pathogen Candida auris has become a major public health threat due to its multidrug resistant (MDR) phenotype, high transmissibility, and high mortality. Unlike other Candida species, C. auris has acquired high levels of resistance to an already limited arsenal of antifungals. As an emerging pathogen, there are currently a limited number of documented murine models of C. auris infection. These animal models use inoculums as high as 107-108 cells per mouse, and the environmental and occupational exposure of working with these models has not been clearly defined. Using real-time quantitative polymerase chain reaction (PCR) and culture, we monitored the animal holding room as well as the procedure room for up to 6 months while working with an intravenous model of C. auris infection. This study determined that shedding of the organism is dose-dependent, as detectable levels of C. auris were detected in the cage bedding when mice were infected with 107 and 108 cells, but not with doses of 105 and 106 cells. Autoclaving bedding in closed micro-isolator cages was found to be an effective way to minimize exposure for animal caretakers. We found that tissue necropsies of infected mice were also an important source of potential source exposure to C. auris. To mitigate these potential exposures, we implemented a rigorous "buddy system" workflow and a disinfection protocol that uses 10% bleach followed by 70% ethanol and can be used in any animal facility when using small animal models of C. auris infection.

Green Fluorescent Protein Expression in Pseudogymnoascus destructans to Study Its Abiotic and Biotic Lifestyles.

Pseudogymnoascus destructans (Pd) is the etiologic agent of bat White-nose syndrome, a disease that has caused the unprecedented reduction in the hibernating bat populations across eastern North America. The Pd pathogenesis appears to be a complex adaptation of fungus in its abiotic (caves and mines) and biotic (bats) environments. There is a general lack of experimental tools for the study of Pd biology. We described the successful expression of codon-optimized synthetic green fluorescent protein sGFP in Pd. The sGFP(S65T) gene was first fused in frame with the Aspergillus nidulans promoter in the tumor-inducing plasmid pRF-HUE, and the resulting plasmid pHUE-sGFP(S65T) was transformed into Pd by Agrobacterium tumefaciens-mediated transformation system. The integration of sGFP(S65T) in Pd genome was analyzed by PCR, and single integration frequency of approximately 66% was confirmed by Southern hybridization. Fluorescent microscopy and flow cytometric analyses of two randomly selected transformants with single integration revealed high expression of sGFP in both spores and hyphal structures. The biology of mutants as judged by sporulation, growth rate, and urease production was not altered indicating sGFP is not toxic to Pd. Thus, we have generated a valuable tool that will facilitate the elucidation of Pd biology, ecology, and pathogenicity in real time.

Rapid effects of a protective O-polysaccharide-specific monoclonal IgA on Vibrio cholerae agglutination, motility, and surface morphology.

2D6 is a dimeric monoclonal immunoglobulin A (IgA) specific for the nonreducing terminal residue of Ogawa O-polysaccharide (OPS) of Vibrio cholerae. It was previously demonstrated that 2D6 IgA is sufficient to passively protect suckling mice from oral challenge with virulent V. cholerae O395. In this study, we sought to define the mechanism by which 2D6 IgA antibody protects the intestinal epithelium from V. cholerae infection. In a mouse ligated-ileal-loop assay, 2D6 IgA promoted V. cholerae agglutination in the intestinal lumen and limited the ability of the bacteria to associate with the epithelium, particularly within the crypt regions. In vitro fluorescence digital video microscopy analysis of antibody-treated V. cholerae in liquid medium revealed that 2D6 IgA not only induced the rapid (5- to 10-min) onset of agglutination but was an equally potent inhibitor of bacterial motility. Scanning electron microscopy showed that 2D6 IgA promoted flagellum-flagellum cross-linking, as well as flagellar entanglement with bacterial bodies, suggesting that motility arrest may be a consequence of flagellar tethering. However, monovalent 2D6 Fab fragments also inhibited V. cholerae motility, demonstrating that antibody-mediated agglutination and motility arrest are separate phenomena. While 2D6 IgA is neither bactericidal nor bacteriostatic, exposure of V. cholerae to 2D6 IgA (or Fab fragments) resulted in a 5-fold increase in surface-associated blebs, as well an onset of a wrinkled surface morphotype. We propose that the protective immunity conferred by 2D6 IgA is the result of multifactorial effects on V. cholerae, including agglutination, motility arrest, and possibly outer membrane stress.

Three-dimensional reconstruction of murine Peyer's patches from immunostained cryosections.

Peyer's patches, macroscopic aggregates of lymphoid follicles present throughout the small intestines of humans and other mammals, are considered the gateway through which luminal dietary antigens and microbes are sampled by the mucosal immune system. The cellular make-up of Peyer's patch lymphoid follicles is not only complex, but highly dynamic, as there are at least four major cell types that are known to migrate in response to antigenic stimulation. In an effort to capture the complexity and dynamic nature of this specialized tissue, here we report the three-dimensional (3D) reconstruction of immunofluorescent-labeled mouse Peyer's patch cryosections. The technology that enabled the stacking and linear blending of serial cryosections was a novel macro for Fiji, the open source image-processing package based on ImageJ. By simultaneously labeling cryosections for surface markers CD45R, CD3, and CD11c, we provide a 3D image as well as quantitative measures of B-cell, T-cell, and dendritic cell populations at steady state and following exposure to the mucosal adjuvant cholera toxin.

Pathobiology of Candida auris infection analyzed by multiplexed imaging and single cell analysis.

Fungal organisms contribute to significant human morbidity and mortality and Candida auris (C. auris) infections are of utmost concern due to multi-drug resistant strains and persistence in critical care and hospital settings. Pathogenesis and pathology of C. auris is still poorly understood and in this study, we demonstrate how the use of multiplex immunofluorescent imaging (MxIF) and single-cell analysis can contribute to a deeper understanding of fungal infections within organs. We used two different neutrophil depletion murine models (treated with either 1A8-an anti-Ly6G antibody, or RB6-8C5-an anti-Ly6G/Ly6C antibody; both 1A8 and RB6-8C5 antibodies have been shown to deplete neutrophils) and compared to wildtype, non-neutropenic mice. Following pathologist assessment, fixed samples underwent MxIF imaging using a C. albicans antibody (shown to be cross-reactive to C. auris) and immune cell biomarkers-CD3 (T cells), CD68 (macrophages), B220 (B cells), CD45 (monocytes), and Ly6G (neutrophils) to quantify organ specific immune niches. MxIF analysis highlighted the heterogenous distribution of C. auris infection within heart, kidney, and brain 7 days post-infection. Size and number of fungal abscesses was greatest in the heart and lowest in brain. Infected mice had an increased count of CD3+, CD68+, B220+, and CD45+ immune cells, concentrated around C. auris abscesses. CD68+ cells were predominant in wildtype (non-neutropenic mice) and CD3+/CD45+ cells were predominant in neutropenic mice, with B cells being the least abundant. These findings suggest a Th2 driven immune response in neutropenic C. auris infection mice models. This study demonstrates the value of MxIF to broaden understanding of C. auris pathobiology, and mechanistic understanding of fungal infections.

The PAX LC Trial: A Decentralized, Phase 2, Randomized, Double-Blind Study of Nirmatrelvir/Ritonavir Compared with Placebo/Ritonavir for Long COVID.

BACKGROUND: Individuals with long COVID lack evidence-based treatments and have difficulty participating in traditional site-based trials. Our digital, decentralized trial investigates the efficacy and safety of nirmatrelvir/ritonavir, targeting viral persistence as a potential cause of long COVID. METHODS: The PAX LC trial (NCT05668091) is a Phase 2, 1:1 randomized, double-blind, superiority, placebo-controlled trial in 100 community-dwelling, highly symptomatic adult participants with long COVID residing in the 48 contiguous US states to determine the efficacy, safety, and tolerability of 15 days of nirmatrelvir/ritonavir compared with placebo/ritonavir. Participants are recruited via patient groups, cultural ambassadors, and social media platforms. Medical records are reviewed through a platform facilitating participant-mediated data acquisition from electronic health records nationwide. During the drug treatment, participants complete daily digital diaries using a web-based application. Blood draws for eligibility and safety assessments are conducted at or near participants' homes. The study drug is shipped directly to participants' homes. The primary endpoint is the PROMIS-29 Physical Health Summary Score difference between baseline and Day 28, evaluated by a mixed model repeated measure analysis. Secondary endpoints include PROMIS-29 (Mental Health Summary Score and all items), Modified GSQ-30 with supplemental symptoms questionnaire, COVID Core Outcome Measures for Recovery, EQ-5D-5L (Utility Score and all items), PGIS 1 and 2, PGIC 1 and 2, and healthcare utilization. The trial incorporates immunophenotyping to identify long COVID biomarkers and treatment responders. CONCLUSION: The PAX LC trial uses a novel decentralized design and a participant-centric approach to test a 15-day regimen of nirmatrelvir/ritonavir for long COVID.

Nirmatrelvir-Ritonavir and Symptoms in Adults With Postacute Sequelae of SARS-CoV-2 Infection: The STOP-PASC Randomized Clinical Trial.

Importance: There is an urgent need to identify treatments for postacute sequelae of SARS-CoV-2 infection (PASC). Objective: To assess the efficacy of a 15-day course of nirmatrelvir-ritonavir in reducing the severity of select PASC symptoms. Design, Setting, and Participants: This was a 15-week blinded, placebo-controlled, randomized clinical trial conducted from November 2022 to September 2023 at Stanford University (California). The participants were adults with moderate to severe PASC symptoms of 3 months or longer duration. Interventions: Participants were randomized 2:1 to treatment with oral nirmatrelvir-ritonavir (NMV/r, 300 mg and 100 mg) or with placebo-ritonavir (PBO/r) twice daily for 15 days. Main Outcomes and Measures: Primary outcome was a pooled severity of 6 PASC symptoms (fatigue, brain fog, shortness of breath, body aches, gastrointestinal symptoms, and cardiovascular symptoms) based on a Likert scale score at 10 weeks. Secondary outcomes included symptom severity at different time points, symptom burden and relief, patient global measures, Patient-Reported Outcomes Measurement Information System (PROMIS) measures, orthostatic vital signs, and sit-to-stand test change from baseline. Results: Of the 155 participants (median [IQR] age, 43 [34-54] years; 92 [59%] females), 102 were randomized to the NMV/r group and 53 to the PBO/r group. Nearly all participants (n = 153) had received the primary series for COVID-19 vaccination. Mean (SD) time between index SARS-CoV-2 infection and randomization was 17.5 (9.1) months. There was no statistically significant difference in the model-derived severity outcome pooled across the 6 core symptoms at 10 weeks between the NMV/r and PBO/r groups. No statistically significant between-group differences were found at 10 weeks in the Patient Global Impression of Severity or Patient Global Impression of Change scores, summative symptom scores, and change from baseline to 10 weeks in PROMIS fatigue, dyspnea, cognitive function, and physical function measures. Adverse event rates were similar in NMV/r and PBO/r groups and mostly of low grade. Conclusions and Relevance: The results of this randomized clinical trial showed that a 15-day course of NMV/r in a population of patients with PASC was generally safe but did not demonstrate a significant benefit for improving select PASC symptoms in a mostly vaccinated cohort with protracted symptom duration. Further studies are needed to determine the role of antivirals in the treatment of PASC. Trial Registration: ClinicalTrials.gov Identifier: NCT05576662.

Macrophage autophagy in immunity to Cryptococcus neoformans and Candida albicans.

Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans, two important opportunistic fungal pathogens. The autophagosome marker LC3 (microtubule-associated protein 1 light chain 3 alpha) was present in most macrophage vacuoles containing C. albicans. In contrast, LC3 was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans, depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans, increased interleukin-6 secretion, and decreased gamma interferon-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans, as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.

Depletion of the Microbiota Has a Modest but Important Impact on the Fungal Burden of the Heart and Lungs during Early Systemic Candida auris Infection in Neutropenic Mice.

The progression and systemic pathobiology of C. auris in the absence of a microbiota have not been described. Here, we describe the influence of the microbiota during the first 5 days of C. auris infection in germ-free or antibiotic-depleted mice. Depletion of the bacterial microbiota in both germ-free and antibiotic-depleted models results in a modest but important increase in the early stages of C. auris infection. Particularly the heart and lungs, followed by the cecum, uterus, and stomach, of intravenously (i.v.) infected neutropenic mice showed significant fungal organ burden. Understanding disease progression and pathobiology of C. auris in individuals with a depleted microbiota could potentially help in the development of care protocols that incorporate supplementation or restoration of the microbiota before invasive procedures, such as transplantation surgeries.

Involvement of glycoreceptors in galactoxylomannan-induced T cell death.

The major virulence factor of Cryptococcus neoformans is its capsular polysaccharide, which is also released into tissues. The shed polysaccharide is composed of glucuronoxylomannan, galactoxylomannan (GalXM), and mannoproteins. In a previous study, we demonstrated a direct interaction of purified soluble GalXM with T cells that induced their apoptosis. In this study, we focus on the mechanisms involved in the apoptotic effect of GalXM. In our experimental system, we analyzed the effect of GalXM on purified human T cells and Jurkat cells, a T cell line routinely used for apoptotic studies. Our results reveal that GalXM activates the extrinsic and intrinsic apoptotic pathways through the cleavage and recruitment of caspase-8. Caspase-8 elicits the downstream executioner caspase-3, caspase-6, and caspase-7 both directly and indirectly, via Bid cleavage and caspase-9 activation. These effects appeared to be primarily mediated by the interaction of GalXM with the glycoreceptors, which differed in human T and Jurkat cells. CD45 was primarily involved in Jurkat cells apoptosis while CD7 and CD43 mediated human T cell apoptosis. Our results highlight a new mechanism by which a microbial product can contribute to virulence through direct interaction with T cell glycoreceptors, thereby triggering lymphocyte apoptosis.

Galactoxylomannan-mediated immunological paralysis results from specific B cell depletion in the context of widespread immune system damage.

The mechanisms responsible for polysaccharide-induced immunological paralysis have remained unexplained almost a century after this phenomenon was first described. Cryptococcus neoformans capsular polysaccharides glucuronoxylomannan and galactoxylomannan (GalXM) elicit little or no Ab responses. This study investigates the immunological and biological effects of GalXM in mice. GalXM immunization elicits a state of immunological paralysis in mice characterized by the disappearance of Ab-producing cells in the spleen. Immunological paralysis and lack of immunogenicity could not be overcome by immunization with GalXM conjugated to a protein carrier, Bacillus anthracis protective Ag. Additionally, immunization with GalXM in either complete or IFA was associated with spleen enlargement in BALB/c mice. TUNEL and flow cytometry revealed widespread apoptosis in the spleen after GalXM administration. Administration of a cocktail of caspase-3 inhibitor Z-DEVD-FMK and general caspase inhibitor Z-VAD-FMK or Fas-deficient mice abrogated the complete disappearance of Ab-producing cells. Analysis of spleen cytokine expression in response to GalXM systemic injection revealed that GalXM down-regulated the production of inflammatory cytokines. Hence, we conclude that GalXM-induced immune paralysis is a result of specific B cell depletion mediated by its proapoptotic properties in the context of widespread dysregulation of immune function.

Galactoxylomannans from Cryptococcus neoformans varieties neoformans and grubii are structurally and antigenically variable.

Prior studies have established that the Cryptococcus neoformans capsular polysaccharide component galactoxylomannan (GalXM) manifests serotype-related structural differences that translate into antigenic differences. We analyzed GalXM from acapsular serotype A and D strains by carbohydrate analysis and static and dynamic light scattering to determine mass, effective diameter, polydispersity, and diffusion coefficients. Multiangle laser light scattering showed that GalXM from C. neoformans var. grubii strain cap59 (serotype A) had larger molecular mass (4.21 x 10(6) +/- 0.95 x 10(6) g/mol) and radius of gyration (207 +/- 27 nm) than GalXM from C. neoformans var. neoformans cap67 (serotype D). cap67 GalXM had corresponding values of 0.70 x 10(6) +/- 0.05 x 10(6) g/mol and 120 +/- 22 nm, respectively. The effective diameter for GalXM and polydispersity from the two strains varied depending on temperature and medium growth conditions, indicating that GalXM structure can vary within a strain, depending on its environment. Zeta potential determinations were negative for GalXM from both strains under all conditions, consistent with the recently reported presence of glucuronic acid. These results imply that C. neoformans GalXM, like glucuronoxylomannan, can manifest variety- and growth condition-related variations. Analysis of 16 C. neoformans and 7 Cryptococcus gattii strains with polyclonal antibody to a GalXM strain revealed antigenic similarities among the C. neoformans variety neoformans and grubii strains and no reactivity with C. gattii. As a result of the deleterious effects of GalXM on immune function, structural and antigenic variability between serotypes may translate into differences in immunomodulatory effects.

Identification of linear epitopes in Bacillus anthracis protective antigen bound by neutralizing antibodies.

Protective antigen (PA), the binding subunit of anthrax toxin, is the major component in the current anthrax vaccine, but the fine antigenic structure of PA is not well defined. To identify linear neutralizing epitopes of PA, 145 overlapping peptides covering the entire sequence of the protein were synthesized. Six monoclonal antibodies (mAbs) and antisera from mice specific for PA were tested for their reactivity to the peptides by enzyme-linked immunosorbent assays. Three major linear immunodominant B-cell epitopes were mapped to residues Leu(156) to Ser(170), Val(196) to Ile(210), and Ser(312) to Asn(326) of the PA protein. Two mAbs with toxin-neutralizing activity recognized two different epitopes in close proximity to the furin cleavage site in domain 1. The three-dimensional complex structure of PA and its neutralizing mAbs 7.5G and 19D9 were modeled using the molecular docking method providing models for the interacting epitope and paratope residues. For both mAbs, LeTx neutralization was associated with interference with furin cleavage, but they differed in effectiveness depending on whether they bound on the N- or C-terminal aspect of the cleaved products. The two peptides containing these epitopes that include amino acids Leu(156)-Ser(170) and Val(196)-Ile(210) were immunogenic and elicited neutralizing antibody responses to PA. These results identify the first linear neutralizing epitopes of PA and show that peptides containing epitope sequences can elicit neutralizing antibody responses, a finding that could be exploited for vaccine design.

Capsular localization of the Cryptococcus neoformans polysaccharide component galactoxylomannan.

Cryptococcus neoformans capsular polysaccharide is composed of at least two components, glucuronoxylomannan (GXM) and galactoxylomannans (GalXM). Although GXM has been extensively studied, little is known about the location of GalXM in the C. neoformans capsule, in part because there are no serological reagents specific to this antigen. To circumvent the poor immunogenicity of GalXM, this antigen was conjugated to protective antigen from Bacillus anthracis as a protein carrier. The resulting conjugate elicited antibodies that reacted with GalXM in mice and yielded an immune serum that proved useful for studying GalXM in the polysaccharide capsule. In acapsular cells, immune serum localized GalXM to the cell wall. In capsulated cells, immune serum localized GalXM to discrete pockets near the capsule edge. GalXM was abundant on the nascent capsules of budding daughter cells. The constituent sugars of GalXM were found in vesicle fractions consistent with vesicular transport for this polysaccharide. In addition, we generated a single-chain fraction variable fragment antibody with specificity to oxidized carbohydrates that also produced punctate immunofluorescence on encapsulated cells that partially colocalized with GalXM. The results are interpreted to mean that GalXM is a transient component of the polysaccharide capsule of mature cells during the process of secretion. Hence, the function of GalXM appears to be more consistent with that of an exopolysaccharide than a structural component of the cryptococcal capsule.

Inclusive Science: Inspiring 8th Graders from Underrepresented Groups to Embrace STEM with Microbiology and Immunology.

Inclusivity in STEM requires practices that include transforming the culture in a classroom. This can be done not only by placing value on diversity but also by providing an engaging student experience, instilling a sense of belonging, and encouraging students at all levels to use a critical lens to solve problems. As a way to develop an inclusive science curriculum for students in a community that is among the poorest in New York state, local STEM organization Rise High Inc. partners with experts in STEM fields, K-12 educators, mentors, and community organizations to create sustainable low-cost, high-quality, engaging, and relevant content that sparks curiosity and exploration for this underserved community. The educators and mentors also have a unique opportunity to develop self-awareness about their own pathways and how they can use their experiences to enrich the classroom. An exemplary case is this highly interactive two-part instructional module in Microbiology and Immunology, which targets 8th graders and was designed in partnership with a local expert in these fields. This module offers creative means to learn and apply knowledge in realistic ways, while using easy-to-access materials in classrooms.

The capsule of the fungal pathogen Cryptococcus neoformans.

The capsule of the fungal pathogen Cryptococcus neoformans has been studied extensively in recent decades and a large body of information is now available to the scientific community. Well-known aspects of the capsule include its structure, antigenic properties and its function as a virulence factor. The capsule is composed primarily of two polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), in addition to a smaller proportion of mannoproteins (MPs). Most of the studies on the composition of the capsule have focused on GXM, which comprises more than 90% of the capsule's polysaccharide mass. It is GalXM, however, that is of particular scientific interest because of its immunological properties. The molecular structure of these polysaccharides is very complex and has not yet been fully elucidated. Both GXM and GalXM are high molecular mass polymers with the mass of GXM equaling roughly 10 times that of GalXM. Recent findings suggest, however, that the actual molecular weight might be different to what it has traditionally been thought to be. In addition to their structural roles in the polysaccharide capsule, these molecules have been associated with many deleterious effects on the immune response. Capsular components are therefore considered key virulence determinants in C. neoformans, which has motivated their use in vaccines and made them targets for monoclonal antibody treatments. In this review, we will provide an update on the current knowledge of the C. neoformans capsule, covering aspects related to its structure, synthesis and particularly, its role as a virulence factor.

Polydopamine Coating of Glucan Particles Increases Uptake into Peyer's Patches.

Glucan particles (GPs) are hollow, porous 3-4 µm microspheres derived from the cell walls of Baker's yeast (Saccharomyces cerevisiae). The ß-1,3-D glucan outer shell of GPs provides for receptor-mediated uptake by phagocytic cells expressing ß-glucan receptors. GPs have been used for efficient encapsulation of different types of payloads (DNA, siRNA, proteins, antigens, small molecules), and these payloads have been delivered in vivo by a variety of routes including oral delivery. It is known that GPs are transported across the intestinal epithelium by Peyer's patch M-cells and accumulate in a subset of CD11c+Langerin-positive dendritic cells (DC) in the subepithelial dome (SED). An increase in GP uptake in the intestinal epithelium is needed to improve our efforts to develop GPs for oral delivery of therapeutics and vaccines. In this Article, we report that polydopamine coating of GPs (PDA-GPs) increases transepithelial uptake. Synthesis of PDA-GPs was optimized to allow for encapsulation of payloads inside the hollow cavity of GPs. PDA-GPs and GP controls were orally administered to mice, and PDA-GPs showed a 42% increased uptake in SED phagocytes. PDA-GP uptake by SED phagocytes in control and M-cell-depleted mice demonstrated both M-cell-dependent and -independent mechanisms. In future studies, we will evaluate PDA-GPs for oral vaccine delivery and the use of PDA-functional groups for secondary surface derivatization to generate particles with ligands targeting other intestinal epithelium cell-surface receptors.