Rebecca De Smet

β-Glucan microparticles are good candidates for mucosal antigen delivery in oral vaccination

Continuously improving the developmental process and the efficacy of oral vaccines is essential in the fight against intestinal pathogens. A promising strategy for vaccination applying safe, biodegradable and non-replicating antigen delivery systems has gained increased interest for eliciting cellular and humoral immune responses. The current study evaluates the potential of β-glucan particles (GP) as an oral antigen delivery system and their adjuvant characteristics. GP are efficiently internalized by human intestinal epithelial cell lines (Caco-2 and HT-29 cells), without exerting negative effects on cell viability. GP triggered the expression of pro-inflammatory cytokines IL-23p19, IL-8 and the β-glucan receptors dectin-1 and TLR2 by activated Caco-2 cells, and CCL20 in HT-29 cells. In contrast, the expression level of TGF-β, an important mediator of oral tolerance, was significantly downregulated in HT-29 cells. Additionally, adoptive transfer experiments showed proliferating ovalbumin (OVA)-specific CD4(+) T cells mainly in the spleens of GP-OVA-fed mice. Furthermore, we detected a significantly increased IL-17 and a trend towards increased IFN-γ production in the spleen of GP-OVA-fed mice upon antigen restimulation. Oral administration of GP-OVA induced increased OVA-specific IgA, secretory-IgA (S-IgA) and secretory component (SC) production in intestinal fluids. Our data show that GP vehicles are able to deliver OVA via an oral route allowing efficient antigen presentation alongside adaptive immune activation, resulting in a Th17-biased response and the production of OVA-specific IgA, secretory-IgA and secretory component antibodies.

Chronic cigarette smoke exposure induces microbial and inflammatory shifts and mucin changes in the murine gut

Inflammatory bowel diseases (IBD) are complex multifactorial diseases characterized by an inappropriate host response to an altered commensal microbiome and dysfunctional mucus barrier. Cigarette smoking is the best known environmental risk factor in IBD. Here, we studied the influence of chronic smoke exposure on the gut microbiome, mucus layer composition and immune factors in conventional mice. We compared smoke-exposed with air-exposed mice (n = 12) after a smoke exposure of 24 weeks. Both Illumina sequencing (n = 6) and denaturing gradient gel electrophoresis (n = 12) showed that bacterial activity and community structure were significantly altered in the colon due to smoke exposure. Interestingly, an increase of Lachnospiraceae sp. activity in the colon was observed. Also, the mRNA expression of Muc2 and Muc3 increased in the ileum, whereas Muc4 increased in the distal colon of smoke-exposed mice (n = 6). Furthermore, we observed increased Cxcl2 and decreased Ifn-γ in the ileum, and increased Il-6 and decreased Tgf-β in the proximal colon. Tight junction gene expression remained unchanged. We infer that the modulating role of chronic smoke exposure as a latently present risk factor in the gut may be driven by the altered epithelial mucus profiles and changes in microbiome composition and immune factors.

The effect of smoking on intestinal inflammation: What can be learned from animal models?

Epidemiological evidence demonstrates that smoking is the most important environmental risk factor in Crohns disease while it positively interferes with the disease course of ulcerative colitis. However, the underlying mechanisms through which smoking exerts this divergent effect and affects pathogenesis of inflammatory bowel disease are largely unknown. Animal smoke models are good models to investigate the impact of cigarette smoke on intestinal physiology and inflammation. They enable one to explore the interaction of smoke components and the gut on cellular and molecular level, clarifying how smoking interferes with normal gut function and with disease course in inflammatory conditions. This review describes the currently used animal models for studying the impact of cigarette smoke on the intestinal tract. We first discuss the different methods for simulation of smoking. Furthermore, we focus on the effect of smoke exposure on normal gut physiology and immunology, on experimental (entero)colitis, and on inflammation-induced neoplasia. Based on this current knowledge, a hypothesis is formulated about the mechanisms through which cigarette smoke interferes with the gut in normal and pathological conditions.

Cigarette smoke and the terminal ileum: increased autophagy in murine follicle-associated epithelium and Peyer’s patches

Cigarette smoke (CS) exposure is associated with increased autophagy in several cell types, such as bronchial epithelial cells. Smoking is also an environmental risk factor in Crohn’s disease, in which impairment of the autophagy-mediated anti-bacterial pathway has been implicated. So far, it is unknown whether CS induces autophagy in the gut. Here, we examined the effect of chronic CS exposure on autophagy in the follicle-associated epithelium (FAE) of murine Peyer’s patches. Transmission electron microscopy revealed that the proportion of cell area occupied by autophagic vesicles significantly increased in the FAE after CS exposure. An increased number of autophagic vesicles was observed in the FAE, whereas the vesicle size remained unaltered. Besides enterocytes, also M-cells contain more autophagic vesicles upon CS exposure. In addition, the mRNA level of the autophagy-related protein Atg7 in the underlying Peyer’s patches is increased after CS exposure, which indicates that the autophagy-inducing effect of CS is not limited to the FAE. In conclusion, our results demonstrate that CS exposure induces autophagy in murine FAE and in the underlying immune cells of Peyer’s patches, suggesting that CS exposure increases the risk for Crohn’s disease by causing epithelial oxidative damage, which needs to be repaired by autophagy.

Spray-dried polyelectrolyte microparticles in oral antigen delivery: stability, biocompatibility, and cellular uptake.

During the past decade, extensive research has undeniably improved the formulation and delivery of oral vaccines. Nevertheless, several factors, such as the harsh gastrointestinal environment together with tolerance induction to exogenous antigens, have thus far impeded the optimal effectiveness and clinical application of oral delivery systems. The current study encompasses an initial evaluation of the stability, biocompatibility, and cellular uptake of two promising candidate systems for oral antigen delivery, that is, calcium carbonate- (CP) and mannitol-templated (MP) porous microspheres. Both spray-dried formulations were efficiently internalized by human intestinal epithelial cells (Caco-2 and HT-29) and degraded into phagolysosomal intracellular compartments. In addition, cellular particle uptake and processing significantly up-regulated the expression of (HLA) class-II and costimulatory molecules on intestinal epithelial cells. Even though the high surface-area-to-volume ratio of the microspheres was expected to favor protease access, antigen release was remarkably limited in simulated intestinal fluid and was even absent under gastric conditions. Finally, neither CP nor MP exerted cytotoxicity upon prolonged in vitro incubation with high antigen concentration. Altogether, these data support the potential of CP and MP for oral antigen delivery and motivate the further development of these promising carrier systems in in vivo studies.

Recent advances in oral vaccine development: yeast-derived β-glucan particles.

Oral vaccination is the most challenging vaccination method due to the administration route. However, oral vaccination has socio-economic benefits and provides the possibility of stimulating both humoral and cellular immune responses at systemic and mucosal sites. Despite the advantages of oral vaccination, only a limited number of oral vaccines are currently approved for human use. During the last decade, extensive research regarding antigen-based oral vaccination methods have improved immunogenicity and induced desired immunological outcomes. Nevertheless, several factors such as the harsh gastro-intestinal environment and oral tolerance impede the clinical application of oral delivery systems. To date, human clinical trials investigating the efficacy of these systems are still lacking. This review addresses the rationale and key biological and physicochemical aspects of oral vaccine design and highlights the use of yeast-derived β-glucan microparticles as an oral vaccine delivery platform.

Platinum Recovery from Synthetic Extreme Environments by Halophilic Bacteria

Metal recycling based on urban mining needs to be established to tackle the increasing supply risk of critical metals such as platinum. Presently, efficient strategies are missing for the recovery of platinum from diluted industrial process streams, often characterized by extremely low pHs and high salt concentrations. In this research, halophilic mixed cultures were employed for the biological recovery of platinum (Pt). Halophilic bacteria were enriched from Artemia cysts, living in salt lakes, in different salt matrices (sea salt mixture and NH4Cl; 20-210 g L(-1) salts) and at low to neutral pH (pH 3-7). The main taxonomic families present in the halophilic cultures were Halomonadaceae, Bacillaceae, and Idiomarinaceae. The halophilic cultures were able to recover >98% Pt(II) and >97% Pt(IV) at pH 2 within 3-21 h (4-453 mg Ptrecovered h(-1) g(-1) biomass). X-ray absorption spectroscopy confirmed the reduction to Pt(0) and transmission electron microscopy revealed both intra- and extracellular Pt precipitates, with median diameters of 9-30 nm and 11-13 nm, for Pt(II) and Pt(IV), respectively. Flow cytometric membrane integrity staining demonstrated the preservation of cell viability during platinum recovery. This study demonstrates the Pt recovery potential of halophilic mixed cultures in acidic saline conditions.

Biological Recovery of Platinum Complexes from Diluted Aqueous Streams by Axenic Cultures.

The widespread use of platinum in high-tech and catalytic applications has led to the production of diverse Pt loaded wastewaters. Effective recovery strategies are needed for the treatment of low concentrated waste streams to prevent pollution and to stimulate recovery of this precious resource. The biological recovery of five common environmental Pt-complexes was studied under acidic conditions; the chloro-complexes PtCl42- and PtCl62-, the amine-complex Pt(NH3)4Cl2 and the pharmaceutical complexes cisplatin and carboplatin. Five bacterial species were screened on their platinum recovery potential; the Gram-negative species Shewanella oneidensis MR-1, Cupriavidus metallidurans CH34, Geobacter metallireducens, and Pseudomonas stutzeri, and the Gram-positive species Bacillus toyonensis. Overall, PtCl42- and PtCl62- were completely recovered by all bacterial species while only S. oneidensis and C. metallidurans were able to recover cisplatin quantitatively (99%), all in the presence of H2 as electron donor at pH 2. Carboplatin was only partly recovered (max. 25% at pH 7), whereas no recovery was observed in the case of the Pt-tetraamine complex. Transmission electron microscopy (TEM) revealed the presence of both intra- and extracellular platinum particles. Flow cytometry based microbial viability assessment demonstrated the decrease in number of intact bacterial cells during platinum reduction and indicated C. metallidurans to be the most resistant species. This study showed the effective and complete biological recovery of three common Pt-complexes, and estimated the fate and transport of the Pt-complexes in wastewater treatment plants and the natural environment.

Transient Receptor Potential Channels in Intestinal Inflammation: What Is the Impact of Cigarette Smoking?

Inflammatory bowel disease (IBD) is characterized by severe gastrointestinal inflammation and results from a complex interplay between genetic and environmental factors. IBD includes two prominent subtypes: Crohns disease (CD) and ulcerative colitis (UC). One of the main risk factors for the development of CD is cigarette smoking, while UC is rather a disease of ex-smokers. To date, many of the mechanisms underlying the immune imbalance in IBD and the involvement of cigarette smoke (CS) are incompletely understood. Transient receptor potential (TRP) proteins are non-selective cation channels that, upon activation, lead to plasma membrane depolarization and, in general, to Ca2+ influx. TRP channels of the ankyrin and vanilloid family, expressed by sensory neurons in the central and enteric nervous systems, have been extensively studied in the context of intestinal inflammation. Moreover, recent advances made on the role of non-neuronal expressed TRP channels shed light on the involvement of epithelial cells in inflammatory processes. This review focuses on how CS may impact TRP channel function in intestinal inflammation. Firstly, we discuss the current knowledge on neuronal TRP channels, known to be linked to IBD, in health, immune homeostasis and intestinal inflammation. Subsequently, we address how TRP channels are activated by CS and its components in other organ systems and also hypothesize on the potential implications for CS-mediated TRP channel activation in gut inflammation.

Hydrocarbonoclastic Alcanivorax Isolates Exhibit Different Physiological and Expression Responses to n-dodecane

Autochthonous microorganisms inhabiting hydrocarbon polluted marine environments play a fundamental role in natural attenuation and constitute promising resources for bioremediation approaches. Alcanivorax spp. members are ubiquitous in contaminated surface waters and are the first to flourish on a wide range of alkanes after an oil-spill. Following oil contamination, a transient community of different Alcanivorax spp. develop, but whether they use a similar physiological, cellular and transcriptomic response to hydrocarbon substrates is unknown. In order to identify which cellular mechanisms are implicated in alkane degradation, we investigated the response of two isolates belonging to different Alcanivorax species, A. dieselolei KS 293 and A. borkumensis SK2 growing on n-dodecane (C12) or on pyruvate. Both strains were equally able to grow on C12 but they activated different strategies to exploit it as carbon and energy source. The membrane morphology and hydrophobicity of SK2 changed remarkably, from neat and hydrophilic on pyruvate to indented and hydrophobic on C12, while no changes were observed in KS 293. In addition, SK2 accumulated a massive amount of intracellular grains when growing on pyruvate, which might constitute a carbon reservoir. Furthermore, SK2 significantly decreased medium surface tension with respect to KS 293 when growing on C12, as a putative result of higher production of biosurfactants. The transcriptomic responses of the two isolates were also highly different. KS 293 changes were relatively balanced when growing on C12 with respect to pyruvate, giving almost the same amount of upregulated (28%), downregulated (37%) and equally regulated (36%) genes, while SK2 transcription was upregulated for most of the genes (81%) when growing on pyruvate when compared to C12. While both strains, having similar genomic background in genes related to hydrocarbon metabolism, retained the same capability to grow on C12, they nevertheless presented very different physiological, cellular and transcriptomic landscapes.