Dicarbonyl Electrophiles Mediate Inflammation-Induced Gastrointestinal Carcinogenesis.

BACKGROUND & AIMS: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis. METHODS: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing. RESULTS: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity. CONCLUSIONS: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.

MNPmApp: An image analysis tool to quantify mononuclear phagocyte distribution in mucosal tissues.

Mononuclear phagocytes (MNPs) such as dendritic cells and macrophages perform key sentinel functions in mucosal tissues and are responsible for inducing and maintaining adaptive immune responses to mucosal pathogens. Positioning of MNPs at the epithelial interface facilitates their access to luminally-derived antigens and regulates MNP function through soluble mediators or surface receptor interactions. Therefore, accurately quantifying the distribution of MNPs within mucosal tissues as well as their spatial relationship with other cells is important to infer functional cellular interactions in health and disease. In this study, we developed and validated a MATLAB-based tissue cytometry platform, termed "MNP mapping application" (MNPmApp), that performs high throughput analyses of MNP density and distribution in the gastrointestinal mucosa based on digital multicolor fluorescence microscopy images and that integrates a Monte Carlo modeling feature to assess randomness of MNP distribution. MNPmApp identified MNPs in tissue sections of the human gastric mucosa with 98 ± 2% specificity and 76 ± 15% sensitivity for HLA-DR+ MNPs and 98 ± 1% specificity and 85 ± 12% sensitivity for CD11c+ MNPs. Monte Carlo modeling revealed that mean MNP-MNP distances for both HLA-DR+ and CD11c+ MNPs were significantly lower than anticipated based on random cell placement, whereas MNP-epithelial distances were similar to randomly placed cells. Surprisingly, H. pylori infection had no significant impact on the number of HLA-DR and CD11c MNPs or their distribution within the gastric lamina propria. However, our study demonstrated that MNPmApp is a reliable and user-friendly tool for unbiased quantitation of MNPs and their distribution at mucosal sites.

Helicobacter pylori infection downregulates the DNA glycosylase NEIL2, resulting in increased genome damage and inflammation in gastric epithelial cells.

Infection with the Gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and oxidative DNA damage in gastric epithelial cells that can lead to gastric cancer (GC). However, the underlying pathogenic mechanism is largely unclear. Here, we report that the suppression of Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase that specifically removes oxidized bases, is one mechanism through which H. pylori infection may fuel the accumulation of DNA damage leading to GC. Using cultured cell lines, gastric biopsy specimens, primary cells, and human enteroid-derived monolayers from healthy human stomach, we show that H. pylori infection greatly reduces NEIL2 expression. The H. pylori infection-induced downregulation of NEIL2 was specific, as Campylobacter jejuni had no such effect. Using gastric organoids isolated from the murine stomach in coculture experiments with live bacteria mimicking the infected stomach lining, we found that H. pylori infection is associated with the production of various inflammatory cytokines. This response was more pronounced in Neil2 knockout (KO) mouse cells than in WT cells, suggesting that NEIL2 suppresses inflammation under physiological conditions. Notably, the H. pylori-infected Neil2-KO murine stomach exhibited more DNA damage than the WT. Furthermore, H. pylori-infected Neil2-KO mice had greater inflammation and more epithelial cell damage. Computational analysis of gene expression profiles of DNA glycosylases in gastric specimens linked the reduced Neil2 level to GC progression. Our results suggest that NEIL2 downregulation is a plausible mechanism by which H. pylori infection impairs DNA damage repair, amplifies the inflammatory response, and initiates GC.

Functional Properties of Helicobacter pylori VacA Toxin m1 and m2 Variants.

Helicobacter pylori colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated H. pylori strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).

p38 MAPK signaling mediates retinoic acid-induced CD103 expression in human dendritic cells.

Retinoic acid (RA) is an active derivative of vitamin A and a key regulator of immune cell function. In dendritic cells (DCs), RA drives the expression of CD103 (integrin αE ), a functionally relevant DC subset marker. In this study, we analyzed the cell type specificity and the molecular mechanisms involved in RA-induced CD103 expression. We show that RA treatment caused a significant up-regulation of CD103 in differentiated monocyte-derived DCs and blood DCs, but not in differentiated monocyte-derived macrophages or T cells. DC treatment with an RA receptor α (RARα) agonist led to an increase in CD103 expression similar to that in RA treatment, whereas RARA gene silencing with small interfering RNA blocked RA-induced up-regulation of CD103, pointing to a major role of RARα in the regulation of CD103 expression. To elucidate RA-induced signaling downstream of RARα, we used Western blot analysis of RA-treated DCs and showed a significant increase of p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, DCs cultured with RA and a p38 MAPK inhibitor had a significantly reduced expression of CD103 compared with DCs cultured with RA only, indicating that p38 MAPK is involved in CD103 regulation. In summary, these findings suggest that the RA-induced expression of CD103 is specific to DCs, is mediated primarily through RARα and involves p38 MAPK signaling.

Slug and Snail have differential effects in directing colonic epithelial wound healing and partially mediate the restitutive effects of butyrate.

Restitution of wounds in colonic epithelium is essential in the maintenance of health. Microbial products, such as the short-chain fatty acid butyrate, can have positive effects on wound healing. We used an in vitro model of T84 colonic epithelial cells to determine if the Snail genes Slug (SNAI2) and Snail (SNAI1), implemented in keratinocyte monolayer healing, are involved in butyrate-enhanced colonic epithelial wound healing. Using shRNA-mediated Slug/Snail knockdown, we found that knockdown of Slug (Slug-KD), but not Snail (Snail-KD), impairs wound healing in scratch assays with and without butyrate. Slug and Snail had differential effects on T84 monolayer barrier integrity, measured by transepithelial resistance, as Snail-KD impaired the barrier (with or without butyrate), whereas Slug-KD enhanced the barrier, again with or without butyrate. Targeted transcriptional analysis demonstrated differential expression of several tight junction genes, as well as focal adhesion genes. This included altered regulation of Annexin A2 and ITGB1 in Slug-KD, which was reflected in confocal microscopy, showing increased accumulation of B1-integrin protein in Slug-KD cells, which was previously shown to impair wound healing. Transcriptional analysis also indicated altered expression of genes associated with epithelial terminal differentiation, such that Slug-KD cells skewed toward overexpression of secretory cell pathway-associated genes. This included trefoil factors TFF1 and TFF3, which were expressed at lower than control levels in Snail-KD cells. Since TFFs can enhance the barrier in epithelial cells, this points to a potential mechanism of differential modulation by Snail genes. Although Snail genes are crucial in epithelial wound restitution, butyrate responses are mediated by other pathways as well.NEW & NOTEWORTHY Although butyrate can promote colonic mucosal healing, not all of its downstream pathways are understood. We show that the Snail genes Snail and Slug are mediators of butyrate responses. Furthermore, these genes, and Slug in particular, are necessary for efficient restitution of wounds and barriers in T84 epithelial cells even in the absence of butyrate. These effects are achieved in part through effects on regulation of ß1 integrin and cellular differentiation state.

Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events.

Three-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids have become increasingly popular for studies of gastrointestinal development, mucosal immunology and epithelial infection. However, little is known about the behavior of these complex cultures in their three-dimensional culture matrix. Therefore, we performed extended time-lapse imaging analysis (up to 4 days) of human gastric epithelial spheroids generated from adult tissue samples in order to visualize the dynamics of the spheroids in detail. Human gastric epithelial spheroids cultured in our laboratory grew to an average diameter of 443.9 ± 34.6 µm after 12 days, with the largest spheroids reaching diameters of >1000 µm. Live imaging analysis revealed that spheroid growth was associated with cyclic rupture of the epithelial shell at a frequency of 0.32 ± 0.1/day, which led to the release of luminal contents. Spheroid rupture usually resulted in an initial collapse, followed by spontaneous re-formation of the spheres. Moreover, spheroids frequently rotated around their axes within the Matrigel matrix, possibly propelled by basolateral pseudopodia-like formations of the epithelial cells. Interestingly, adjacent spheroids occasionally underwent luminal fusion, as visualized by injection of individual spheroids with FITC-Dextran (4 kDa). In summary, our analysis revealed unexpected dynamics in human gastric spheroids that challenge our current view of cultured epithelia as static entities and that may need to be considered when performing spheroid infection experiments.

Cytomegalovirus enhances macrophage TLR expression and MyD88-mediated signal transduction to potentiate inducible inflammatory responses.

Circulating monocytes carrying human CMV (HCMV) migrate into tissues, where they differentiate into HCMV-infected resident macrophages that upon interaction with bacterial products may potentiate tissue inflammation. In this study, we investigated the mechanism by which HCMV promotes macrophage-orchestrated inflammation using a clinical isolate of HCMV (TR) and macrophages derived from primary human monocytes. HCMV infection of the macrophages, which was associated with viral DNA replication, significantly enhanced TNF-α, IL-6, and IL-8 gene expression and protein production in response to TLR4 ligand (LPS) stimulation compared with mock-infected LPS-stimulated macrophages during a 6-d in vitro infection. HCMV infection also potentiated TLR5 ligand-stimulated cytokine production. To elucidate the mechanism by which HCMV infection potentiated inducible macrophage responses, we show that infection by HCMV promoted the maintenance of surface CD14 and TLR4 and TLR5, which declined over time in mock-infected macrophages, and enhanced both the intracellular expression of adaptor protein MyD88 and the inducible phosphorylation of IκBα and NF-κB. These findings provide additional information toward elucidating the mechanism by which HCMV potentiates bacteria-induced NF-κB-mediated macrophage inflammatory responses, thereby enhancing organ inflammation in HCMV-infected tissues.

HIV-1 envelope glycan moieties modulate HIV-1 transmission.

UNLABELLED: The HIV-1 envelope protein (Env) is heavily glycosylated, with approximately 50% of the Env molecular mass being contributed by N-glycans. HIV-1 Env N-glycans shield the protein backbone and have been shown to play key roles in determining Env structure, surface exposure, and, consequently, antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. Also, the role of Env glycan moieties on HIV-1 transmission has not been systematically defined. Using viruses with modified Env glycan content and heterogeneity, we examined the effects of Env glycan moieties on the major events of HIV-1 transmission. Compared to viruses with less oligomannose and more complex Env glycans, viruses with more oligomannose and less complex glycans more efficiently (i) transcytosed across an epithelial cell monolayer, (ii) attached to monocyte-derived macrophages (MDMs), (iii) bound monocyte-derived dendritic cells (MoDCs), and (iv) trans-infected primary lymphocytes via MoDCs. However, viruses with more oligomannose and less complex glycans displayed impaired infectivity in TZMbl cells, MDMs, primary lymphocytes, and fresh human intestinal tissue. Thus, N-linked Env glycans display discordant effects on the major events of HIV-1 transmission, with mature oligosaccharide structures on Env playing a crucial role in HIV-1 infection. Env glycosylation should be taken into consideration in the development of vaccine strategies to interdict HIV-1 transmission. IMPORTANCE: HIV-1 Env N-glycans shield the protein backbone and play key roles in determining Env structure and surface exposure, thereby impacting Env antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. In the study described in this report, we investigated systematically the role of Env glycan moieties on HIV-1 transmission. We show that N-linked Env glycans display discordant effects on the major events of HIV-1 transmission. These data indicate that Env glycan moieties impact HIV-1 transmission and that modulation of Env glycan moieties offers a potential strategy for the development of therapeutic or prophylactic vaccines against HIV-1.

Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells.

After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti-inflammatory effects.

Helicobacter pylori infection inhibits phagocyte clearance of apoptotic gastric epithelial cells.

Increased apoptotic death of gastric epithelial cells is a hallmark of Helicobacter pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells (AECs) in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR(+) mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive AEC material, indicating that gastric phagocytes are involved in AEC clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the AECs by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori-induced macrophage TNF-α, which was expressed at higher levels in H. pylori-infected, compared with uninfected, gastric mucosa. Importantly, we show that H. pylori-infected gastric mucosa contained significantly higher numbers of AECs and higher levels of nonphagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection.

Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes.

The optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This paper presents a highly reproducible method for the precise measurement of pH within the lumina of 3D human gastric organoids via micromanipulator-controlled microelectrodes. The pH microelectrodes are commercially available and consist of beveled glass tips of 25 µm in diameter. For measurements, the pH microelectrode is advanced into the lumen of an organoid (>200 µm) that is suspended in Matrigel, while a reference electrode rests submerged in the surrounding medium in the culture plate. Using such microelectrodes to profile organoids derived from the human gastric body, we demonstrate that luminal pH is relatively consistent within each culture well at ~7.7 ± 0.037 and that continuous measurements can be obtained for a minimum of 15 min. In some larger organoids, the measurements revealed a pH gradient between the epithelial surface and the lumen, suggesting that pH measurements in organoids can be achieved with high spatial resolution. In a previous study, microelectrodes were successfully used to measure luminal oxygen concentrations in organoids, demonstrating the versatility of this method for organoid analyses. In summary, this protocol describes an important tool for the functional characterization of the complex luminal space within 3D organoids.

Human intestinal stromal cells promote homeostasis in normal mucosa but inflammation in Crohn's disease in a retinoic acid-deficient manner.

Intestinal stromal cells (SCs), which synthesize the extracellular matrix that gives the mucosa its structure, are newly appreciated to play a role in mucosal inflammation. Here, we show that human intestinal vimentin+CD90+smooth muscle actin- SCs synthesize retinoic acid (RA) at levels equivalent to intestinal epithelial cells, a function in the human intestine previously attributed exclusively to epithelial cells. Crohn's disease SCs (Crohn's SCs), however, synthesized markedly less RA than SCs from healthy intestine (normal SCs). We also show that microbe-stimulated Crohn's SCs, which are more inflammatory than stimulated normal SCs, induced less RA-regulated differentiation of mucosal dendritic cells (DCs) (circulating pre-DCs and monocyte-derived DCs), leading to the generation of more potent inflammatory interferon-γhi/interleukin-17hi T cells than normal SCs. Explaining these results, Crohn's SCs expressed more DHRS3, a retinaldehyde reductase that inhibits retinol conversion to retinal and, thus, synthesized less RA than normal SCs. These findings uncover a microbe-SC-DC crosstalk in which luminal microbes induce Crohn's disease SCs to initiate and perpetuate inflammation through impaired synthesis of RA.

Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

Stromal regulation of human gastric dendritic cells restricts the Th1 response to Helicobacter pylori.

BACKGROUND & AIMS: Mucosal dendritic cells (DCs) play a key role in initiating the T-helper (Th)1 response to Helicobacter pylori. To further elucidate the mucosal response to H pylori, we examined whether gastric stromal factors condition DCs to support tolerance to H pylori, analogous to intestinal stromal factor-driven macrophage tolerance to commensal bacteria. METHODS: To model mucosal DC development, we isolated and cultured cell-depleted human stroma/extracellular matrix from fresh gastric and intestinal mucosa to generate stroma-conditioned media. We then analyzed the capacity of stroma-conditioned media-treated monocyte-derived DCs and primary human gastric and intestinal DCs pulsed in vitro with H pylori to induce T-cell proliferation and interferon gamma secretion. RESULTS: Stromal factors in gastric mucosa suppressed H pylori-stimulated DC activation and the ability of DCs to drive a Th1 proliferative and cytokine response to H pylori. The ability of gastric stromal factors to down-regulate DC function was similar to that of intestinal stromal factors and was independent of transforming growth factor ß, prostaglandin E2, interleukin (IL)-10, and thymic stromal lymphopoietin. Stroma-conditioned media-induced reduction in DC-stimulated Th1 responses was associated with reduced DC release of IL-12. CONCLUSIONS: Gastric stromal factors down-regulate DC responsiveness to H pylori, resulting in a dampened gastric Th1 response. We speculate that stroma-induced down-regulation of DC function contributes to the permissiveness of both gastric and intestinal mucosa to colonization by persistent residential microbes.

The major soyabean allergen P34 resists proteolysis in vitro and is transported through intestinal epithelial cells by a caveolae-mediated mechanism.

Soya is considered to be one of the eight most significant food allergens. Among the allergenic soya proteins determined to date, P34 has been identified as one of the immunodominant soya antigens. Sensitisation to a specific food antigen like P34 generally follows the transit of intact antigens across the intestinal barrier and usually occurs in infants, who are most susceptible to food allergies. In the present study, we used the intestinal epithelial cell line IPEC-J2, which was originally derived from the jejunum of a neonatal piglet, to recapitulate the infant intestinal epithelium and study the binding and uptake of P34 protein. P34 was partially resistant to degradation in an in vitro proteolysis assay. IPEC-J2 cells were able to endocytose intact P34, as shown by immunofluorescence and immunoelectronmicroscopy methods. P34 associated with lipid raft microdomains of IPEC-J2 cells, and disruption of caveolae/lipid raft microdomains using methyl-ß-cyclodextrin abolished P34 endocytosis, indicating that the observed endocytosis was mediated by caveolae. Using IPEC-J2 cells grown on Transwell filters, we further demonstrated that P34 is transported through the epithelial monolayer by transcytosis. Piglets frequently show hypersensitivity to soya antigens, and in this study, we show that healthy adult pigs with dietary exposure to soya protein mount an antibody response to soyabean protein P34, suggesting that this protein has entered the body, probably through gastrointestinal uptake. In summary, our data suggest that soya P34 resists proteolysis in the gastrointestinal tract and is transported through the intestinal epithelial barrier, thereby allowing sensitisation of immune cells in the sub-epithelial compartment.

GP41-specific antibody blocks cell-free HIV type 1 transcytosis through human rectal mucosa and model colonic epithelium.

Monostratified epithelial cells translocate HIV type 1 (HIV-1) from the apical to the basolateral surface via vesicular transcytosis. Because acutely transmitted HIV-1 is almost exclusively CCR5-tropic and human intestinal epithelial cells preferentially transcytose CCR5-tropic virus, we established epithelial monolayers using polarized HT-29 cells transduced to express CCR5, and an explant system using normal human rectal mucosa, to characterize biological parameters of epithelial cell transcytosis of HIV-1 and assess antiviral Ab blockade of transcytosis. The amount of cell-free HIV-1 transcytosed through the epithelial monolayer increased linearly in relation to the amount of virus applied to the apical surface, indicating transcytosis efficiency was constant (r(2) = 0.9846; p < 0.0001). The efficiency of HIV-1 transcytosis ranged between 0.05 and 1.21%, depending on the virus strain, producer cell type and gp120 V1-V3 loop signature. Inoculation of HIV-1 neutralizing Abs to the immunodominant region (7B2) or the conserved membrane proximal external region (2F5) of gp41 or to cardiolipin (IS4) onto the apical surface of epithelial monolayers prior to inoculation of virus significantly reduced HIV-1 transcytosis. 2F5 was the most potent of these IgG1 Abs. Dimeric IgA and monomeric IgA, but not polymeric IgM, 2F5 Abs also blocked HIV-1 transcytosis across the epithelium and, importantly, across explanted normal human rectal mucosa, with monomeric IgA substantially more potent than dimeric IgA in effecting transcytosis blockade. These findings underscore the potential role of transcytosis blockade in the prevention of HIV-1 transmission across columnar epithelium such as that of the rectum.

Granular Matrigel: restructuring a trusted extracellular matrix material for improved permeability.

Matrigel is a polymeric extracellular matrix material produced by mouse cancer cells. Over the past four decades, Matrigel has been shown to support a wide variety of two- and three-dimensional cell and tissue culture applications including organoids. Despite widespread use, transport of molecules, cells, and colloidal particles through Matrigel can be limited. These limitations restrict cell growth, viability, and function and limit Matrigel applications. A strategy to improve transport through a hydrogel without modifying the chemistry or composition of the gel is to physically restructure the material into microscopic microgels and then pack them together to form a porous material. These 'granular' hydrogels have been created using a variety of synthetic hydrogels, but granular hydrogels composed of Matrigel have not yet been reported. Here we present a drop-based microfluidics approach for structuring Matrigel into a three-dimensional, mesoporous material composed of packed Matrigel microgels, which we call granular Matrigel. We show that restructuring Matrigel in this manner enhances the transport of colloidal particles and human dendritic cells (DCs) through the gel while providing sufficient mechanical support for culture of human gastric organoids (HGOs) and co-culture of human DCs with HGOs.

Transfer and persistence of bovine immunoglobulins in lambs fed a colostrum replacer.

BACKGROUND: Colostrum-derived antibodies are crucial for the protection of newborn lambs from infectious diseases. Several colostrum replacer products that contain bovine antibodies are on the market. We investigated the absorption and persistence of bovine antibodies from a powdered colostrum replacer in newborn lambs. METHODS: We tested a lamb colostrum replacer containing bovine serum in lambs that were separated from their dams at birth. Immunoglobulin G (IgG) uptake was analysed by ELISA, and the persistence of antigen-specific antibodies was analysed by parainfluenza 3 virus (PI-3) neutralisation assay. RESULTS: Serum antibody ELISA performed on days 1 and 14 revealed IgG levels of 17.9 ± 2.8 and 27.5 ± 2.5 mg/ml, respectively. PI-3 antibodies derived from the colostrum replacer were present for 86.3 ± 10.6 days. CONCLUSIONS: Antibodies derived from bovine serum protein delivered to lambs via a commercial colostrum replacer are readily absorbed and persist for months, suggesting that these products may offer adequate protection.

Pathophysiology of Influenza D Virus Infection in Specific-Pathogen-Free Lambs with or without Prior Mycoplasma ovipneumoniae Exposure.

Polymicrobial pneumonias occur frequently in cattle, swine, and sheep, resulting in major economic losses. Individual pathogens comprising these complex infections may be mild on their own but can instead exhibit synergism or increase host susceptibility. Two examples of such pathogens, Mycoplasma ovipneumoniae (M. ovipneumoniae) and influenza D viruses (IDVs), naturally infect domestic sheep. In sheep, the role of M. ovipneumoniae in chronic nonprogressive pneumonia is well-established, but the pathogenesis of IDV infection has not previously been studied. We utilized a specific-pathogen-free sheep flock to study the clinical response to IDV infection in naïve vs. M. ovipneumoniae-exposed lambs. Lambs were inoculated intranasally with M. ovipneumoniae or mock infection, followed after four weeks by infection with IDV. Pathogen shedding was tracked, and immunological responses were evaluated by measuring acute phase response and IDV-neutralizing antibody titers. While lamb health statuses remained subclinical, M. ovipneumoniae-exposed lambs had significantly elevated body temperatures during IDV infection compared to M. ovipneumoniae-naïve, IDV-infected lambs. Moreover, we found a positive correlation between prior M. ovipneumoniae burden, early-infection IDV shedding, and IDV-neutralizing antibody response. Our findings suggest that IDV infection may not induce clinical symptoms in domestic sheep, but previous M. ovipneumoniae exposure may promote mild IDV-associated inflammation.