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.

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.

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.

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.

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.

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.

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.

Enhanced gut barrier integrity sensitizes colon cancer to immune therapy.

Oral IL-10 suppressed tumor growth in the APCmin/+ mouse/Bacteroides fragilis colon cancer model while a similar formulation of IL-12 exacerbated disease. In contrast, combined treatment with IL-10 and IL-12 resulted in near-complete tumor eradication and a significant extension of survival. The cytokines mediated distinct immunological effects in the gut, i.e. IL-10 diminished Th17 cell prevalence whereas IL-12 induced IFNγ and enhanced CD8 + T-cell activity. Loss-of-function studies demonstrated that IL-12-driven CD8 + T-cell expansion was only partially responsible for the synergy, and that both the detrimental and the beneficial activities of IL-12 required IFNγ. Examination of colon physiology in mice receiving single vs dual treatment revealed that exacerbation of disease by IL-12 monotherapy was associated with compromised gut barrier integrity whereas combined treatment reversed this effect, uncovering additional activity by the cytokines on the stroma. Further analysis showed that the stromal effects of IL-12 included a 6-fold increase in IL-10RA expression in the colon epithelium, linking the epithelial activity of the cytokines. Finally, dual but not monotherapy induced a 3-fold increase in tight junction protein levels in the colon, identifying the mechanism by which IL-10 blocked the detrimental effect of the IL-12-IFNγ axis on barrier function without interfering with its beneficial immunological activity. These findings establish that the synergy between IL-12 and IL-10 was mediated by pleiotropic effects on the immune and the non-immune compartments and that the latter activity was critical to therapeutic outcome.

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.

Intravaginal Administration of Interleukin 12 during Genital Gonococcal Infection in Mice Induces Immunity to Heterologous Strains of Neisseria gonorrhoeae.

It has previously been shown that genital tract infection with Neisseria gonorrhoeae in mice does not induce a state of protective immunity against reinfection but instead suppresses the development of adaptive immune responses against N. gonorrhoeae dependent on transforming growth factor beta (TGF-ß) and interleukin 10 (IL-10). Intravaginal administration during gonococcal infection of IL-12 encapsulated in biodegradable microspheres (IL-12/ms) reverses the immunosuppression and promotes the production of gamma interferon (IFN-γ) and of specific antibodies in serum and genital secretions and accelerates clearance of the infection. In this study, microspheres were shown to remain largely within the genital tract lumen and to release IL-12 over the course of 4 days. Antigonococcal IgA and IgG antibodies induced by IL-12/ms treatment reacted with antigenically different strains of N. gonorrhoeae and led to resistance to reinfection with heterologous and homologous strains. Immune resistance to reinfection persisted for at least 6 months after clearance of the primary infection. Experiments performed with immunodeficient strains of mice lacking either IFN-γ or B cells demonstrated that both IFN-γ and B cells were necessary for the IL-12-induced generation of immune responses to N. gonorrhoeae and the resulting accelerated clearance of the infection. It is therefore concluded that intravaginally administered IL-12/ms achieves its effect by the sustained release of IL-12 that promotes Th1-driven adaptive immune responses, including the production of specific antigonococcal antibodies that cross-react with multiple strains of N. gonorrhoeae. IL-12-enhanced immunity to N. gonorrhoeae can be recalled against reinfection after prolonged intervals and is dependent upon both IFN-γ and antibody production by B cells. IMPORTANCE Genital infection with Neisseria gonorrhoeae (gonorrhea) is a significant cause of reproductive tract morbidity in women, leading to pelvic inflammatory disease, tubal factor infertility, and increased risk for ectopic pregnancy. WHO estimates that 78 million new infections occur annually worldwide. In the United States, >350,000 cases are reported annually, but the true incidence is probably >800,000 cases/year. Increasing resistance to currently available antibiotics raises concern that gonorrhea might become untreatable. Infection does not induce a state of immune protection against reinfection. Previous studies have shown that N. gonorrhoeae suppresses the development of adaptive immune responses by mechanisms dependent on the regulatory cytokines TGF-ß and IL-10. This study shows that intravaginal treatment of gonococcal infection in female mice with microencapsulated IL-12 induces persisting anamnestic immunity against reinfection with N. gonorrhoeae, even of antigenically diverse strains, dependent on T-cell production of IFN-γ and B-cell production of antibodies.

RNA isolation from Peyer's patch lymphocytes and mononuclear phagocytes to determine gene expression profiles using NanoString technology.

Sampling and immune surveillance within gut-associated lymphoid tissues such as the intestinal Peyer's patch (PP) occurs by an elegantly orchestrated effort that involves the epithelial barrier, B and T lymphocytes, and an extensive network of mononuclear phagocytes. Although we now understand more about the dynamics of antigen and microbial sampling within PPs, the gene expression changes that occur in individual cell subsets during sampling are not well characterized. This protocol describes the isolation of high-quality RNA from sorted PP, B and T-lymphocytes, and CD11c+ phagocytes for use with nCounter-NanoString technology. This method allows investigators to study gene expression changes within PPs in response to antigens, microbes, and oral vaccine delivery vehicles of interest that are sampled.

Oral IL-10 suppresses colon carcinogenesis via elimination of pathogenicCD4+ T-cells and induction of antitumor CD8+ T-cell activity.

An oral sustained-release formulation of Interleukin-10 suppressed tumor growth and enhanced survival in the APCmin/+/Bacteroides fragilis spontaneous colon cancer model. Therapeutic benefit was associated with a 5-fold reduction in CD4+RORγt+Foxp3-IL-17+ T-helper cell, CD4+RORγt+Foxp3+IL-17+ pathogenic T-regulatory cell and CD4+RORγt-Foxp3+IL-17- conventional T-regulatory cell numbers and a concurrent 2-fold enhancement in CD8+ T-cell activity in the colon. Selective subset depletion and functional blockade studies demonstrated that at steady-state CD4+RORγt+IL-17+ T-cell subsets and CD4+Foxp3+ cTreg supported tumorigenesis, whereas CD8+ cytotoxic T-lymphocytes impeded tumor progression following IL-10 therapy. Suppression of tumor growth by CD8+ T-cells was associated with enhanced tumor infiltration and cytotoxic granule exocytosis. These findings establish the utility of oral IL-10 as a potential new therapeutic in the management of colon cancer and shed light on the cellular mechanisms that underlie its antitumor activity.

Role of Maternal Dietary Peanut Exposure in Development of Food Allergy and Oral Tolerance.

BACKGROUND: The impact of maternal ingestion of peanut during pregnancy and lactation on an offspring's risk for peanut allergy is under debate. OBJECTIVE: To investigate the influence of maternal dietary peanut exposure and breast milk on an offspring's allergy risk. METHODS: Preconceptionally peanut-exposed C3H/HeJ females were either fed or not fed peanut during pregnancy and lactation. The offsprings' responses to peanut sensitization or oral tolerance induction by feeding antigen prior to immunization were assessed. We also assessed the impact of immune murine milk on tolerance induction pre- or post-weaning. For antigen uptake studies, mice were gavaged with fluorescent peanut in the presence or absence of immune murine milk; Peyer's patches were harvested for immunostaining. RESULTS: Preconceptional peanut exposure resulted in the production of varying levels of maternal antibodies in serum (and breast milk), which were transferred to the offspring. Despite this, maternal peanut exposure either preconceptionally or during pregnancy and lactation, when compared to no maternal exposure, had no impact on peanut allergy. When offspring were fed peanut directly, dose-dependent tolerance induction, unaltered by maternal feeding of peanut, was seen. Although peanut uptake into the gut-associated lymphoid tissues was enhanced by immune milk as compared to naïve milk, tolerance induction was not affected by the co-administration of immune milk either pre- or post-weaning. CONCLUSION: Maternal peanut exposure during pregnancy and lactation has no impact on the development of peanut allergy in the offspring. Tolerance to peanut can be induced early, even pre-weaning, by giving moderate amounts of peanut directly to the infant, and this is neither enhanced nor impaired by concurrent exposure to immune milk.

Sampling of Candida albicans and Candida tropicalis by Langerin-positive dendritic cells in mouse Peyer's patches.

Members of the Candida genus, including C. albicans and C. tropicalis are opportunistic fungal pathogens that are increasingly associated with gastrointestinal infections and inflammatory bowel diseases. In healthy populations, however, C. albicans and C. tropicalis are considered benign members of the mycobiome, and are presumably kept in check by the mucosal immune system. In this study, we demonstrate in mice that C. albicans and C. tropicalis are sampled by Peyer's patch (PP) dendritic cells (DCs). Uptake into gut-associated lymphoid tissues occurred rapidly and was at least partly M cell-dependent. C. albicans and C. tropicalis preferentially localized in (and persisted within) a recently identified sub- population of Peyer's patch DCs distinguished by their expression of the C-type lectin receptor, Langerin. This study is the first to identify a subset of PP DCs capable of sampling Candida species.

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.

Oral interleukin-10 alleviates polyposis via neutralization of pathogenic T-regulatory cells.

Immune dysregulation drives the pathogenesis of chronic inflammatory, autoimmune, and dysplastic disorders. While often intended to address localized pathology, most immune modulatory therapies are administered systemically and carry inherent risk of multiorgan toxicities. Here, we demonstrate, in a murine model of spontaneous gastrointestinal polyposis, that site-specific uptake of orally administered IL10 microparticles ameliorates local and systemic disease to enhance survival. Mechanistic investigations showed that the therapeutic benefit of this treatment derived from neutralization of disease-promoting FoxP3(+)RoRγt(+)IL17(+) pathogenic T-regulatory cells (pgTreg), with a concomitant restoration of FoxP3(+)RoRγt(-)IL17(-) conventional T-regulatory cells (Treg). These findings provide a proof-of-principle for the ability of an oral biologic to restore immune homeostasis at the intestinal surface. Furthermore, they implicate local manipulation of IL10 as a tractable therapeutic strategy to address the inflammatory sequelae associated with mucosal premalignancy.

A population of Langerin-positive dendritic cells in murine Peyer's patches involved in sampling ß-glucan microparticles.

Glucan particles (GPs) are 2-4 µm hollow, porous shells composed of 1,3-ß-D-glucan that have been effectively used for oral targeted-delivery of a wide range of payloads, including small molecules, siRNA, DNA, and protein antigens. While it has been demonstrated that the transepithelial transport of GPs is mediated by Peyer's patch M cells, the fate of the GPs once within gut-associated lymphoid tissue (GALT) is not known. Here we report that fluorescently labeled GPs administered to mice by gavage accumulate in CD11c+ DCs situated in Peyer's patch sub-epithelial dome (SED) regions. GPs appeared in DCs within minutes after gavage and remained within the SED for days afterwards. The co-administration or sequential administration of GPs with differentially labeled GPs or poly(lactic-co-glycolic acid) nanoparticles demonstrated that the SED DC subpopulation in question was capable of internalizing particles of different sizes and material compositions. Phenotypic analysis identified the GP-containing DCs as being CD8α- and CD11blo/-, suggesting they are the so-called myeloid and/or double negative (DN) subset(s) of PP DCs. A survey of C-type lectin receptors (CLRs) known to be expressed by leukocytes within the intestinal mucosa revealed that GP-containing SED DCs were positive for Langerin (CD207), a CLR with specificity for ß-D-glucan and that has been shown to mediate the internalization of a wide range of microbial pathogens, including bacteria, viruses and fungi. The presence of Langerin+ DCs in the SED as determined by immunofluorescence was confirmed using Langerin E-GFP transgenic mice. In summary, our results demonstrate that following M cell-mediated transepithelial transport, GPs (and other micro/nanoparticles) are sampled by a population of SED DCs distinguished from other Peyer's patch DC subsets by their expression of Langerin. Future studies will be aimed at defining the role of Langerin in antigen sampling and antigen presentation within the context of the GALT.