Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms.

BACKGROUND & AIMS: The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism. METHODS: We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites. RESULTS: Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells. CONCLUSIONS: Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Influence of Binary Toxin Gene Detection and Decreased Susceptibility to Antibiotics among Clostridioides difficile Strains on Disease Severity: a Single-Center Study.

Clostridioides difficile infection (CDI) is the fifth leading cause of death from nonmalignant gastrointestinal disease in the United States. The contribution of resistance to C. difficile-active antibiotics to the outcomes of CDI is unclear. We evaluated the antimicrobial susceptibility of C. difficile isolates in a U.S. hospital and determined associations of clinical variables and binary toxin positivity with antibiotic resistance. C. difficile spores were cultured from fecal specimens of adult patients with CDI for genotyping and antimicrobial susceptibility assay (for clindamycin [CLI], fidaxomicin [FDX], metronidazole [MTZ], moxifloxacin [MXF], tigecycline [TGC], and vancomycin [VAN]). Electronic medical records were reviewed for clinical data extraction. Ninety-seven of 130 (75%) fecal samples grew toxigenic C. difficile in culture. Most of the isolates were tcdA+ tcdB+ cdtB- (80.4%), and 18.6% and 1% were tcdA+ tcdB+ cdtB+ and tcdA-tcdB+ cdtB+, respectively. Susceptibility to VAN, MTZ, FDX, TGC, MXF, and CLI was 96%, 94%, 100%, 100%, 8%, and 79%, respectively. Six isolates, all cdtB positive and belonging to the 027 ribotype, were resistant to VAN and/or MTZ. Higher MICs were found in isolates with a mutation in the VAN-related resistance gene vanR, but not vanS. In addition, cdtB+ isolates exhibited higher MICs of VAN, MTZ, TGC, CLI, and MXF compared to cdtB- strains. Patients with greater intestinal inflammation or severe disease were more likely to be infected with cdtB+ strains. Decreased susceptibility to antibiotics is not directly associated with either severe or recurrent CDI. However, antimicrobial susceptibility of C. difficile is decreased in strains positive for the binary toxin gene.

A novel mouse model of Campylobacter jejuni enteropathy and diarrhea.

Campylobacter infections are among the leading bacterial causes of diarrhea and of 'environmental enteropathy' (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease with Campylobacter has been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche for Campylobacter infection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to test specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress.

Cross-modulation of pathogen-specific pathways enhances malnutrition during enteric co-infection with Giardia lamblia and enteroaggregative Escherichia coli.

Diverse enteropathogen exposures associate with childhood malnutrition. To elucidate mechanistic pathways whereby enteric microbes interact during malnutrition, we used protein deficiency in mice to develop a new model of co-enteropathogen enteropathy. Focusing on common enteropathogens in malnourished children, Giardia lamblia and enteroaggregative Escherichia coli (EAEC), we provide new insights into intersecting pathogen-specific mechanisms that enhance malnutrition. We show for the first time that during protein malnutrition, the intestinal microbiota permits persistent Giardia colonization and simultaneously contributes to growth impairment. Despite signals of intestinal injury, such as IL1α, Giardia-infected mice lack pro-inflammatory intestinal responses, similar to endemic pediatric Giardia infections. Rather, Giardia perturbs microbial host co-metabolites of proteolysis during growth impairment, whereas host nicotinamide utilization adaptations that correspond with growth recovery increase. EAEC promotes intestinal inflammation and markers of myeloid cell activation. During co-infection, intestinal inflammatory signaling and cellular recruitment responses to EAEC are preserved together with a Giardia-mediated diminishment in myeloid cell activation. Conversely, EAEC extinguishes markers of host energy expenditure regulatory responses to Giardia, as host metabolic adaptations appear exhausted. Integrating immunologic and metabolic profiles during co-pathogen infection and malnutrition, we develop a working mechanistic model of how cumulative diet-induced and pathogen-triggered microbial perturbations result in an increasingly wasted host.

Innate Immune Response and Outcome of Clostridium difficile Infection Are Dependent on Fecal Bacterial Composition in the Aged Host.

Background: Clostridium difficile infection (CDI) is a serious threat for an aging population. Using an aged mouse model, we evaluated the effect of age and the roles of innate immunity and intestinal microbiota. Methods: Aged (18 months) and young (8 weeks) mice were infected with C difficile, and disease severity, immune response, and intestinal microbiome were compared. The same experiment was repeated with intestinal microbiota exchange between aged and young mice before infection. Results: Higher mortality was observed in aged mice with weaker neutrophilic mobilization in blood and intestinal tissue and depressed proinflammatory cytokines in early infection. Microbiota exchange improved survival and early immune response in aged mice. Microbiome analysis revealed that aged mice have significant deficiencies in Bacteroidetes phylum and, specifically, Bacteroides, Alistipes, and rc4-4 genera, which were replenished by cage switching. Conclusions: Microbiota-dependent alteration in innate immune response early on during infection may explain poor outcome in aged host with CDI.

Amixicile Reduces Severity of Cryptosporidiosis but Does Not Have In Vitro Activity against Cryptosporidium.

Cryptosporidium species cause significant morbidity in malnourished children. Nitazoxanide (NTZ) is the only approved treatment for cryptosporidiosis, but NTZ has diminished effectiveness during malnutrition. Here, we show that amixicile, a highly selective water-soluble derivative of NTZ diminishes Cryptosporidium infection severity in a malnourished mouse model despite a lack of direct anticryptosporidial activity. We suggest that amixicile, by tamping down anaerobes associated with intestinal inflammation, reverses weight loss and indirectly mitigates infection-associated pathology.

Increased Urinary Trimethylamine N-Oxide Following Cryptosporidium Infection and Protein Malnutrition Independent of Microbiome Effects.

Cryptosporidium infections have been associated with growth stunting, even in the absence of diarrhea. Having previously detailed the effects of protein deficiency on both microbiome and metabolome in this model, we now describe the specific gut microbial and biochemical effects of Cryptosporidium infection. Protein-deficient mice were infected with Cryptosporidium parvum oocysts for 6-13 days and compared with uninfected controls. Following infection, there was an increase in the urinary excretion of choline- and amino-acid-derived metabolites. Conversely, infection reduced the excretion of the microbial-host cometabolite (3-hydroxyphenyl)propionate-sulfate and disrupted metabolites involved in the tricarboxylic acid (TCA) cycle. Correlation analysis of microbial and biochemical profiles resulted in associations between various microbiota members and TCA cycle metabolites, as well as some microbial-specific degradation products. However, no correlation was observed between the majority of the infection-associated metabolites and the fecal bacteria, suggesting that these biochemical perturbations are independent of concurrent changes in the relative abundance of members of the microbiota. We conclude that cryptosporidial infection in protein-deficient mice can mimic some metabolic changes seen in malnourished children and may help elucidate our understanding of long-term metabolic consequences of early childhood enteric infections.

Vancomycin Treatment Alters Humoral Immunity and Intestinal Microbiota in an Aged Mouse Model of Clostridium difficile Infection.

BACKGROUND: The elderly host is highly susceptible to severe disease and treatment failure in Clostridium difficile infection (CDI). We investigated how treatment with vancomycin in the aged host influences systemic and intestinal humoral responses and select intestinal microbiota. METHODS: Young (age, 2 months) and aged (age, 18 months) C57BL/6 mice were infected with VPI 10463 after exposure to broad-spectrum antibiotics. Vancomycin was given 24 hours after infection, and treatment was continued for 5 days. At select time points, specimens of serum and intestinal tissue and contents were collected for histopathologic analysis, to measure antibody levels and the pathogen burden, and to determine the presence and levels of select intestinal microbiota and C. difficile toxin. RESULTS: Levels of disease severity, relapse, and mortality were increased, and recovery from infection was slower in aged mice compared to young mice. Serum levels of immunoglobulin M, immunoglobulin A, and immunoglobulin G against C. difficile toxin A were depressed in aged mice, and vancomycin treatment reduced antibody responses in both age groups. While baseline levels of total bacterial load, Bacteroidetes, Firmicutes, and Enterobacteriaceae were mostly similar, aged mice had a significant change in the Firmicutes to Bacteroidetes ratio with vancomycin treatment. CONCLUSIONS: Vancomycin treatment decreases the systemic humoral response to CDI. Increased mortality from and recurrence of CDI in the aged host are associated with an impaired humoral response and a greater susceptibility to vancomycin-induced alteration of intestinal microbiota.

Myeloperoxidase as a biomarker for intestinal-brain axis dysfunction induced by malnutrition and Cryptosporidium infection in weanling mice.

Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle, necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.

Adenosine receptors differentially mediate enteric glial cell death induced by Clostridioides difficile Toxins A and B.

Increased risk of intestinal dysfunction has been reported in patients after Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.

Development of spirulina for the manufacture and oral delivery of protein therapeutics.

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter-a major cause of infant mortality in the developing world-prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

G2A deficiency in mice promotes macrophage activation and atherosclerosis.

G2A is a stress-inducible G protein-coupled receptor that is expressed on several cell types within atherosclerotic lesions. We demonstrated previously that G2A deficiency in mice increased aortic monocyte recruitment and increased monocyte:endothelial interactions. To investigate the impact of G2A deficiency in macrophages, we isolated peritoneal macrophages from G2A(+/+)ApoE(-/-) and G2A(-/-)ApoE(-/-) mice. G2A(-/-)ApoE(-/-) macrophages had significantly lower apoptosis than control macrophages. The prosurvival genes BCL-2, BCL-xL, and cFLIP were increased in G2A(-/-)ApoE(-/-) macrophages. Macrophages from G2A(-/-)ApoE(-/-) mice also had increased proinflammatory status that was indicative of a M1 macrophage phenotype. This was indicated by significantly increased nuclear translocation of nuclear factor kappaB, as well as production of interleukin-12p40, tumor necrosis factor alpha, and interleukin-6, and reduced expression of arginase-I. Moreover, G2A(-/-)ApoE(-/-) macrophages had reduced ability to engulf apoptotic cells in vitro. We examined atherosclerosis in mice fed a Western diet for 10 weeks and found that G2A deficiency increased lesion size in the aortic root by 50%. Plasma lipid levels were not changed in G2A(-/-)ApoE(-/-) mice. However, we found that absence of G2A increased the number of aortic macrophages and attenuated apoptosis in this cell type. Moreover, bone marrow transplantation studies indicated that deficiency of G2A in marrow-derived cells significantly contributed to atherosclerosis development. In the absence of G2A, increased macrophage activation and decreased apoptosis is associated with accumulation of macrophages in the aorta and increased atherosclerosis.

ABCG1 deficiency in mice promotes endothelial activation and monocyte-endothelial interactions.

OBJECTIVE: Activated endothelium and increased monocyte-endothelial interactions in the vessel wall are key early events in atherogenesis. ATP binding cassette (ABC) transporters play important roles in regulating sterol homeostasis in many cell types. Endothelial cells (EC) have a high capacity to efflux sterols and express the ABC transporter, ABCG1. Here, we define the role of ABCG1 in the regulation of lipid homeostasis and inflammation in aortic EC. METHODS AND RESULTS: Using EC isolated from ABCG1-deficient mice (ABCG1 KO), we observed reduced cholesterol efflux to high-density lipoprotein compared to C57BL/6 (B6) EC. However, total cholesteryl ester levels were not changed in ABCG1 KO EC. Secretions of KC, MCP-1, and IL-6 by ABCG1 KO EC were significantly increased, and surface expressions of intercellular adhesion molecule-1 and E-selectin were increased several-fold on ABCG1 KO EC. Concomitant with these findings, we observed a 4-fold increase in monocyte adhesion to the intact aortic endothelium of ABCG1 KO mice ex vivo and to isolated aortic EC from these mice in vitro. In a gain-of-function study in vitro, restoration of ABCG1 expression in ABCG1 KO EC reduced monocyte-endothelial interactions. Utilizing pharmacological inhibitors for STAT3 and the IL-6 receptor, we found that blockade of STAT3 and IL-6 receptor signaling in ABCG1 KO EC completely abrogated monocyte adhesion to ABCG1 KO endothelium. CONCLUSIONS: ABCG1 deficiency in aortic endothelial cells activates endothelial IL-6-IL-6 receptor-STAT3 signaling, thereby increasing monocyte-endothelial interactions and vascular inflammation.

Modeling Enteropathy or Diarrhea with the Top Bacterial and Protozoal Pathogens: Differential Determinants of Outcomes.

Developing effective therapeutics or preventive interventions for important health threats is greatly enhanced whenever accessible models can enable the assessment of clinically important outcomes. While no non-human model is ever perfect, inexpensive in vivo small animal models in such as mice are often of great help in assessing the relevant efficacy of potential interventions. In addition to acute diarrhea, the long-term growth and developmental effects of enteric infections, with or without overt diarrhea, are increasingly recognized. To address these diverse effects, inexpensive animal models are proving to be very helpful. Herein, we review the major clinical concerns with enteric parasitic and bacterial infections that are extremely common worldwide, especially in vulnerable young children living in impoverished areas, and the recently published murine models of these infections and their outcomes. We find that common dietary deficiencies seen in children in developing areas have striking effects on diarrhea and enteropathy outcomes in mice. However, these effects differ with different pathogens. Specifically, the effects of protein or zinc deficiency differ considerably with different major protozoal and bacterial pathogens, suggesting different pathogenetic pathways and intervention effects. The pathogens reviewed are the seven top parasitic and bacterial pathogens seen in children, namely, Cryptosporidium, Giardia, Campylobacter, Shigella, enterotoxigenic Escherichia coli (ETEC), enteroaggregative E. coli (EAEC), and enteropathogenic E. coli (EPEC).

Detecting Glucose Fluctuations in the Campylobacter jejuni N-Glycan Structure.

Campylobacter jejuni is a significant cause of human gastroenteritis worldwide, and all strains express an N-glycan that is added to at least 80 different proteins. We characterized 98 C. jejuni isolates from infants from 7 low- and middle-income countries and identified 4 isolates unreactive with our N-glycan-specific antiserum that was raised against the C. jejuni heptasaccharide composed of GalNAc-GalNAc-GalNAc(Glc)-GalNAc-GalNAc-diNAcBac. Mass spectrometric analyses indicated these isolates express a hexasaccharide lacking the glucose branch. Although all 4 strains encode the PglI glucosyltransferase (GlcTF), one aspartate in the DXDD motif was missing, an alteration also present in ∼4% of all available PglI sequences. Deleting this residue from an active PglI resulted in a nonfunctional GlcTF when the protein glycosylation system was reconstituted in E. coli, while replacement with Glu/Ala was not deleterious. Molecular modeling proposed a mechanism for how the DXDD residues and the structure/length beyond the motif influence activity. Mouse vaccination with an E. coli strain expressing the full-length heptasaccharide produced N-glycan-specific antibodies and a corresponding reduction in Campylobacter colonization and weight loss following challenge. However, the antibodies did not recognize the hexasaccharide and were unable to opsonize C. jejuni isolates lacking glucose, suggesting this should be considered when designing N-glycan-based vaccines to prevent campylobacteriosis.

S100B Inhibition Attenuates Intestinal Damage and Diarrhea Severity During Clostridioides difficile Infection by Modulating Inflammatory Response.

The involvement of the enteric nervous system, which is a source of S100B, in Clostridioides difficile (C. difficile) infection (CDI) is poorly understood although intestinal motility dysfunctions are known to occur following infection. Here, we investigated the role of S100B in CDI and examined the S100B signaling pathways activated in C. difficile toxin A (TcdA)- and B (TcdB)-induced enteric glial cell (EGC) inflammatory response. The expression of S100B was measured in colon tissues and fecal samples of patients with and without CDI, as well as in colon tissues from C. difficile-infected mice. To investigate the role of S100B signaling in IL-6 expression induced by TcdA and TcdB, rat EGCs were used. Increased S100B was found in colonic biopsies from patients with CDI and colon tissues from C. difficile-infected mice. Patients with CDI-promoted diarrhea exhibited higher levels of fecal S100B compared to non-CDI cases. Inhibition of S100B by pentamidine reduced the synthesis of IL-1ß, IL-18, IL-6, GMCSF, TNF-α, IL-17, IL-23, and IL-2 and downregulated a variety of NFκB-related genes, increased the transcription (SOCS2 and Bcl-2) of protective mediators, reduced neutrophil recruitment, and ameliorated intestinal damage and diarrhea severity in mice. In EGCs, TcdA and TcdB upregulated S100B-mediated IL-6 expression via activation of RAGE/PI3K/NFκB. Thus, CDI appears to upregulate colonic S100B signaling in EGCs, which in turn augment inflammatory response. Inhibition of S100B activity attenuates the intestinal injury and diarrhea caused by C. difficile toxins. Our findings provide new insight into the role of S100B in CDI pathogenesis and opens novel avenues for therapeutic interventions.

Investigation of a monoclonal antibody against enterotoxigenic Escherichia coli, expressed as secretory IgA1 and IgA2 in plants.

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68-81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68-61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

Enteropathogenic Escherichia coli Infection Induces Diarrhea, Intestinal Damage, Metabolic Alterations, and Increased Intestinal Permeability in a Murine Model.

Enteropathogenic E. coli (EPEC) are recognized as one of the leading bacterial causes of infantile diarrhea worldwide. Weaned C57BL/6 mice pretreated with antibiotics were challenged orally with wild-type EPEC or escN mutant (lacking type 3 secretion system) to determine colonization, inflammatory responses and clinical outcomes during infection. Antibiotic disruption of intestinal microbiota enabled efficient colonization by wild-type EPEC resulting in growth impairment and diarrhea. Increase in inflammatory biomarkers, chemokines, cellular recruitment and pro-inflammatory cytokines were observed in intestinal tissues. Metabolomic changes were also observed in EPEC infected mice with changes in tricarboxylic acid (TCA) cycle intermediates, increased creatine excretion and shifts in gut microbial metabolite levels. In addition, by 7 days after infection, although weights were recovering, EPEC-infected mice had increased intestinal permeability and decreased colonic claudin-1 levels. The escN mutant colonized the mice with no weight loss or increased inflammatory biomarkers, showing the importance of the T3SS in EPEC virulence in this model. In conclusion, a murine infection model treated with antibiotics has been developed to mimic clinical outcomes seen in children with EPEC infection and to examine potential roles of selected virulence traits. This model can help in further understanding mechanisms involved in the pathogenesis of EPEC infections and potential outcomes and thus assist in the development of potential preventive or therapeutic interventions.

A bivalent vaccine confers immunogenicity and protection against Shigella flexneri and enterotoxigenic Escherichia coli infections in mice.

Vaccine studies for Shigella flexneri and enterotoxigenic Escherichia coli have been impaired by the lack of optimal animal models. We used two murine models to show that a S. flexneri 2a bivalent vaccine (CVD 1208S-122) expressing enterotoxigenic Escherichia coli colonization factor antigen-I (CFA/I) and the binding subunits A2 and B of heat labile-enterotoxin (LTb) is immunogenic and protects against weight loss and diarrhea. These findings document the immunogenicity and pre-clinical efficacy effects of CVD 1208S-122 vaccine and suggest that further work can help elucidate relevant immune responses and ultimately its clinical efficacy in humans.

Sphingosine-1-phosphate inhibits high glucose-mediated ERK1/2 action in endothelium through induction of MAP kinase phosphatase-3.

Endothelial activation is a key early event in vascular complications of Type 1 diabetes. The nonobese diabetic (NOD) mouse is a well-characterized model of Type 1 diabetes. We previously reported that Type 1 diabetic NOD mice have increased endothelial activation, with increased production of monocyte chemoattractant protein (MCP)-1 and IL-6, and a 30% increase of surface VCAM-1 expression leading to a fourfold increase in monocyte adhesion to the endothelium. Sphingosine-1-phosphate (S1P) prevents monocyte:endothelial interactions in these diabetic NOD mice. Incubation of diabetic NOD endothelial cells (EC) with S1P (100 nmol/l) reduced ERK1/2 phosphorylation by 90%, with no significant changes in total ERK1/2 protein. In the current study, we investigated the mechanism of S1P action on ERK1/2 to reduce activation of diabetic endothelium. S1P caused a significant threefold increase in mitogen-activated kinase phosphatase-3 (MKP-3) expression in EC. MKP-3 selectively regulates ERK1/2 activity through dephosphorylation. Incubation of diabetic NOD EC with S1P and the S1P(1)-selective agonist SEW2871 significantly increased expression of MKP-3 and reduced ERK1/2 phosphorylation, while incubation with the S1P(1)/S1P(3) antagonist VPC23019 decreased the expression of MKP-3, both results supporting a role for S1P(1) in MKP-3 regulation. To mimic the S1P-mediated induction of MKP-3 diabetic NOD EC, we overexpressed MKP-3 in human aortic endothelial cells (HAEC) cultured in elevated glucose (25 mmol/l). Overexpression of MKP-3 in glucose-cultured HAEC decreased ERK1/2 phosphorylation and resulted in decreased monocyte:endothelial interactions in a static monocyte adhesion assay. Finally, we used small interfering RNA to MKP-3 and observed increased monocyte adhesion. Moreover, S1P was unable to inhibit monocyte adhesion in the absence of MKP-3. Thus, one mechanism for the anti-inflammatory action of S1P in diabetic EC is inhibition of ERK1/2 phosphorylation through induction of MKP-3 expression via the S1P-S1P(1) receptor axis.