Abstract Necrotizing enterocolitis (NEC) is a devastating intestinal disease primarily affecting preterm neonates and causing high morbidity, high mortality, and huge costs for the family and society. The treatment and the outcome of the disease have not changed in recent decades. Emerging evidence has shown that stimulating the Wnt/β-catenin pathway and enhancing intestinal regeneration are beneficial in experimental NEC, and that they could potentially be used as a novel treatment. Amniotic fluid stem cells (AFSC) and AFSC-derived extracellular vesicles (EV) can be used to improve intestinal injury in experimental NEC. However, the mechanisms by which they affect the Wnt/β-catenin pathway and intestinal regeneration are unknown. In our current study, we demonstrated that AFSC and EV attenuate NEC intestinal injury by activating the Wnt signaling pathway. AFSC and EV stimulate intestinal recovery from NEC by increasing cellular proliferation, reducing inflammation and ultimately regenerating a normal intestinal epithelium. EV administration has a rescuing effect on intestinal injury when given during NEC induction; however, it failed to prevent injury when given prior to NEC induction. AFSC-derived EV administration is thus a potential emergent novel treatment strategy for NEC.
Abstract Prebiotics are non-digestible oligosaccharides that promote the growth of beneficial gut microbes, but it is unclear whether they also have direct effects on the intestinal mucosal barrier. Here we demonstrate two commercial prebiotics, inulin and short-chain fructo-oligosaccharide (scFOS), when applied onto intestinal epithelia in the absence of microbes, directly promote barrier integrity to prevent pathogen-induced barrier disruptions. We further show that these effects involve the induction of select tight junction (TJ) proteins through a protein kinase C (PKC) δ-dependent mechanism. These results suggest that in the absence of microbiota, prebiotics can directly exert barrier protective effects by activating host cell signaling in the intestinal epithelium, which represents a novel alternative mechanism of action of prebiotics.
Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrPC). Species barriers to transmission also reflect PrPC sequence differences. Defining the structure-activity relationship of PrPC/PrPSc with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrPC corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrPSc-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrPSc-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrPSc-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.
Johne's disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts to control JD through traditional animal management practices are complicated by MAP's ability to cause long-term environmental contamination as well as difficulties associated with diagnosis of JD in the pre-clinical stages. As such, there is particular emphasis on the development of an effective vaccine. This is a daunting challenge, in large part due to MAP's ability to subvert protective host immune responses. Accordingly, there is a priority to understand MAP's interaction with the bovine host: this may inform rational targets and approaches for therapeutic intervention. Here we review the early host defenses encountered by MAP and the strategies employed by the pathogen to avert or subvert these responses, during the critical period between ingestion and the establishment of persistent infection in macrophages.
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrP(C)) into an infectious, pathological conformation (PrP(Sc)). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrP(Sc)-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrP(C) associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrP(C) that is associated with early onset of familial dementia. With nanopore analysis, under non-denaturing conditions, we observed binding of the SN6b antibody to bPrP(T194A). This interaction was confirmed through ELISAs as well as immunoprecipitation of the recombinant and cellularly expressed forms of bPrP(T194A). This interaction did not promote formation of a protease resistant conformation of PrP in vitro. Collectively, these findings support the disease-specific approach for immunotherapy of prion diseases but also suggest that the concept of conformation-specific immunotherapy may be complicated in individuals who are genetically predisposed to PrP(C) misfolding.
Scope Marine‐derived n ‐3 PUFAs may ameliorate inflammation associated with inflammatory bowel diseases. Plant‐derived n ‐3 PUFAs are thought to be inferior owing to shorter chain lengths. The aim of this study is to compare the impact of plant‐ and fish‐derived PUFAs on murine colitis. Methods and results C57BL/6 mice are fed high fat (36% kcal) diets with either 2.5% w/w sunflower oil (SO), flaxseed oil (FSO), ahiflower oil (AO), or fish oil (FO). After 4 weeks, mice are orogastrically challenged with Citrobacter rodentium (10 8 CFU) or sham gavaged. Fecal shedding is assayed at 2, 7, 10, and 14 days post infection (PI), and fecal microbiota at 14 days PI. Colonic inflammation and lipid mediators are measured. Supplementation regulates intestinal inflammation with crypt lengths being 66, 73, and 62 ±17 µm shorter (compared to SO) for FSO, AO, and FO respectively, p < 0.01. FSO blunts pathogen shedding at the peak of infection and FSO and AO both enhance fecal microbial diversity. FO attenuates levels of lipoxin and leukotriene B 4 while plant oils increase pro‐resolving mediator concentrations including D, E, and T‐series resolvins. Conclusion Plant and fish n ‐3 PUFAs attenuate colitis‐induced inflammation while exhibiting characteristic pro‐resolving lipid mediator metabolomes. Plant oils additionally promote microbial diversity.
Prebiotics are non-digestible food ingredients that enhance the growth of certain microbes within the gut microbiota. Prebiotic consumption generates immune-modulatory effects that are traditionally thought to reflect microbial interactions within the gut. However, recent evidence suggests they may also impart direct microbe-independent effects on the host, though the mechanisms of which are currently unclear.Kinome arrays were used to profile the host intestinal signaling responses to prebiotic exposures in the absence of microbes. Identified pathways were functionally validated in Caco-2Bbe1 intestinal cell line and in vivo model of murine endotoxemia.We found that prebiotics directly regulate host mucosal signaling to alter response to bacterial infection. Intestinal epithelial cells (IECs) exposed to prebiotics are hyporesponsive to pathogen-induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activations, and have a kinome profile distinct from non-treated cells pertaining to multiple innate immune signaling pathways. Consistent with this finding, mice orally gavaged with prebiotics showed dampened inflammatory response to lipopolysaccharide (LPS) without alterations in the gut microbiota.These findings provide molecular mechanisms of direct host-prebiotic interactions to support prebiotics as potent modulators of host inflammation.
Scope Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants, occurring more often in formula‐fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC, but the mechanism underlying such protection is currently unclear. Methods and Results By extracting HMOs from pooled human breastmilk, the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results, the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO‐gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO‐treated cells have increased Muc2 expression, decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins, it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. Conclusions Taken together, the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins.
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