Chronic smoke exposure is associated with autophagy in murine Peyer's patches

INTRODUCTION: Cigarette smoke causes oxidative stress, leading to smoke-induced autophagy in several organs. Autophagy is a homeostatic process regulating the turnover of proteins and cytoplasmatic organelles. However, recently it has also been associated with many autoimmune and inflammatory disorders, among which Crohn’s disease. The purpose of the present study was to investigate whether cigarette smoke exposure is associated with increased autophagy in Peyer’s patches and its epithelium. AIMS & METHODS: C57BL/6 mice were exposed to cigarette smoke or air. After 24 weeks, the animals were sacrificied and Peyer’s patches were collected. m RNA expression of autophagy-related genes was determined by RT-PCR. Transmission electron microscopy (TEM) was used to evaluate the presence of autophagic vesicles in the follicleassociated epithelium of Peyer’s patches. RESULTS: Expression of Beclin-1, a protein involved in the nucleation of autophagosomes, and of Atg5 and Atg7, which both play a role in the autophagosome vesicle elongation and completion, increased after chronic smoke exposure. Furthermore, electron microscopy of the follicle-associated epithelium demonstrated that the mean area of autophagic vesicles per epithelial cell increased considerably from 1.1 μm2 ± 0.4 μm2 in the air group to 2.4 μm2 ± 0.4 μm2 in the smoke group (p < 0.05). Epithelial cells had a significantly higher number of autophagic vesicles after smoke exposure (1.1 ± 0.1 after smoke exposure versus 0.5 ± 0.1 vesicles per cell after air exposure, p < 0.05), but the size of the vesicles did not differ between both groups. CONCLUSION: Here we provide the first evidence that chronic exposure to cigarette smoke is associated with autophagy in murine Peyer’s patches, and more in particular in the follicle-associated epithelium covering Peyer’s patches. Our findings can help to understand the role of smoking in the pathogenesis of inflammatory bowel disease, such as Crohn’s disease

Translational research into the effects of cigarette smoke on inflammatory mediators and epithelial TRPV1 in Crohn’s disease

Crohn's disease is a pathological condition of the gastro-intestinal tract, causing severe transmural inflammation in the ileum and/or colon. Cigarette smoking is one of the best known environmental risk factors for the development of Crohn's disease. Nevertheless, very little is known about the effect of prolonged cigarette smoke exposure on inflammatory modulators in the gut. We examined the effect of cigarette smoke on cytokine profiles in the healthy and inflamed gut of human subjects and in the trinitrobenzene sulphonic acid mouse model, which mimics distal Crohn-like colitis. In addition, the effect of cigarette smoke on epithelial expression of transient receptor potential channels and their concurrent increase with cigarette smoke-augmented cytokine production was investigated. Active smoking was associated with increasedIL-8transcription in ileum of controls (p < 0,001; n = 18-20/group). In the ileum, TRPV1 mRNA levels were decreased in never smoking Crohn's disease patients compared to healthy subjects (p <0,001; n = 20/group). In the colon, TRPV1 mRNA levels were decreased (p = 0,046) in smoking healthy controls (n = 20/group). Likewise, healthy mice chronically exposed to cigarette smoke (n = 10/group) showed elevated ilealCxcl2(p = 0,0075) and colonicKcmRNA levels (p = 0,0186), whereas TRPV1 mRNA and protein levels were elevated in the ileum (p = 0,0315). Although cigarette smoke exposure prior to trinitrobenzene sulphonic acid administration did not alter disease activity, increased pro-inflammatory cytokine production was observed in the distal colon (Kc: p = 0,0273; Cxcl2: p = 0,104; Il1-beta: p = 0,0796), in parallel with the increase ofTrpv1mRNA (p < 0,001). We infer that CS affects pro-inflammatory cytokine expression in healthy and inflamed gut, and that the simultaneous modulation of TRPV1 may point to a potential involvement of TRPV1 in cigarette smoke-induced production of inflammatory mediators

The effect of cigarette smoke exposure on the development of inflammation in lungs, gut and joints of TNFΔARE mice

The inflammatory cytokine TNF-alpha is a central mediator in many immune-mediated diseases, such as Crohn's disease (CD), spondyloarthritis (SpA) and chronic obstructive pulmonary disease (COPD). Epidemiologic studies have shown that cigarette smoking (CS) is a prominent common risk factor in these TNF-dependent diseases. We exposed TNF Delta ARE mice; in which a systemic TNF-alpha overexpression leads to the development of inflammation; to 2 or 4 weeks of air or CS. We investigated the effect of deregulated TNF expression on CS-induced pulmonary inflammation and the effect of CS exposure on the initiation and progression of gut and joint inflammation. Upon 2 weeks of CS exposure, inflammation in lungs of TNF Delta ARE mice was significantly aggravated. However, upon 4 weeks of CS-exposure, this aggravation was no longer observed. TNF Delta ARE mice have no increases in CD4+ and CD8+ T cells and a diminished neutrophil response in the lungs after 4 weeks of CS exposure. In the gut and joints of TNF Delta ARE mice, 2 or 4 weeks of CS exposure did not modulate the development of inflammation. In conclusion, CS exposure does not modulate gut and joint inflammation in TNF Delta ARE mice. The lung responses towards CS in TNF Delta ARE mice however depend on the duration of CS exposure

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Taha Toros Arşivi, Dosya No: 220-Kaptanzade Ali Rıza Be

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Microparticles as antigen delivery systems for oral vaccination

Many gastro-intestinal infections are still endemic in large parts of the world causing morbidity and mortality among humans and animals. Mucosal surfaces are the major entrance for infectious pathogens and the induction of local adaptive immune responses are required to combat intestinal pathogens. Furthermore, the gastrointestinal system performs a balancing act in making the decision between immunity and oral tolerance, as a discrimination between harmful and beneficial antigens. Without doubt, the development of vaccines constitutes one of the major breakthroughs of human medicine, allowing us to control and prevent numerous infectious diseases in an efficient and cost-effective manner. Especially in developing countries, oral vaccination provides different social and economic advantages (needle-free, non-invasive administration route, patient compliance, less risk of contamination,…). Current oral vaccines, based on killed or attenuated pathogens, provoke an inefficient immune response or maintain the risk of reversion to virulence and the induction of disease in immunocompromised individuals. Subunit vaccines are a safe alternative, however are less efficacious and require adjuvants. A promising strategy for vaccination with safe, biodegradable non-replicating antigen delivery systems has gained increased interest for eliciting cellular and humoral immune responses. However, the low gastric pH and degradation by proteolytic enzymes present in the gastrointestinal tract, are huge obstacles that inhibit the ability to successfully develop new mucosal vaccines. Microparticulate encapsulation strategies can protect the antigen from degradation, increase the concentration of antigen in the vicinity of mucosal tissue for better absorption and selectively target immune inductive sites, namely the gut-associated lymphoid tissue (GALT), Peyer’s patches (PP) and intestinal antigen presenting cells (APCs), including dendritic cells (DCs). Microbial (bacterial toxins, CpG DNA) and synthetic (microspheres, liposomes, PLGA,…) strategies have been investigated. Contemporary developments in the use of delivery systems and adjuvants have an undeniable value in the oral vaccine development, but a better understanding of the unique properties of mucosal surfaces and the vaccination strategies are indispensable. A first part of this thesis describes an introduction on the intestinal immune system expanding on antigen sampling, presentation and epithelial crosstalk in the small intestine. Furthermore, we summarize current registered human oral vaccines and highlight the importance of animal models in the contribution to the translational research into humans. Lastly, we address the rationale and key biological and physicochemical aspects of oral vaccine design and emphasize the use of yeast-derived β-glucan microparticles as an oral vaccine delivery platform. In a second part of this thesis, we performed a layer-by-layer synthesis of hollow yeast shells through electrostatic interactions using ovalbumin (OVA) as model antigen. Zeta-potential measurements and the quantification of antigen loading gave more information about the OVA-yeast shell interaction and its encapsulation efficiency. Additionally, the kinetics of antigen release and processing is investigated via OVA-DQ-loaded yeast particles in pronase solutions and in in vitro cultured murine BMDCs. In a third part of this thesis, we investigated the particle characteristics of β-glucan microparticles and evaluated their potential as an oral antigen delivery vehicle in human intestinal epithelial cell lines and murine models. A first finding is that β-glucan microparticles are of an ideal size of natural fungal pathogens to be recognized and highly efficient internalized by human Caco-2 and HT-29 cells. Internalization of β-glucan microparticles increased with incubation time and was dose-dependent. Secondly, β-glucan microparticles are capable of reaching the murine Peyer’s patches (specialized lymphoid tissue of the small intestine) via the M cell-mediated transcellular pathway and promoted MHCII–mediated presentation of the model antigen ovalbumin. Additionally, β-glucan microparticles induced intestinal secretory-IgA (S-IgA) and secretory component (SC) antibodies and a mixed Th1/Th17 cellular-mediated immune response against the model antigen ovalbumin. The final part of this thesis, focuses on porous antigen-loaded polyelectrolyte microparticles obtained by the spray-drying technique, namely calcium carbonate- and mannitol-based microspheres. The spray-drying method not only enhances the stability and controls the particle size ranges of the dry formulation, it also avoids the cold-chain-constrained settings, which is a very important aspect for vaccination campaigns in third world countries. Stability tests of CP and MP revealed negligible or no antigen loss in gastric and intestinal conditions. Both particle types are successfully internalized by intestinal epithelial cells and subsequently induced MHC class II and co-stimulatory molecules

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

AbstractEpidemiological evidence demonstrates that smoking is the most important environmental risk factor in Crohn's 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

β-glucan microparticles as mucosal delivery system in oral vaccine development

Several enteric pathogens infect the body following oral uptake and cause life-threatening diseases such as cholera, dysentery and typhoid fever. Oral vaccination is essential to generate protective local immunity against intestinal pathogens. However, antigen delivery to the inductive sites for mucosal immunity (the small intestine Peyer’s patches, PP) has proven to be particularly challenging. Recently, there has been a lot of interest in the use of microparticles as antigen delivery systems in the development of more efficient mucosal vaccines. We evaluated the potential of β-glucan microcapsules to deliver antigen transmucosally in the PP regions of the murine small intestine. β-glucan microparticles (2-4 µm) were prepared from Saccharomyces cerevisiae and loaded with FITC or Alexa Fluor 488-conjugated bovine serum albumin. Firstly, the appropriateness of β-glucan particles as antigen delivery systems for oral and intestinal applications was assessed in stability tests. Fluorimetric analysis demonstrated that the BSA concentration within particles is stable during the first 12 hours of simulated gastric or simulated intestinal fluid treatment. Secondly, β-glucan particles were administered to male C57BL/6 mice (8-10 weeks old) via intestinal loops at a particle concentration of 100*106/ml. After one hour of incubation, transmucosal particle transport and uptake in the PP was evaluated by flow cytometry, confocal microscopy and transmission electron microscopy. Using flow cytometry, we could not observe any particle uptake in the main antigen presenting cell population, the dendritic cells, however, a modest particle uptake was repeatedly detected in the B-cell population. Confocal microscopic images showed localization of β-glucan particles in the follicle associated epithelium and B-cell internalization. Moreover, transmission electron microscopy demonstrated transcellular transport of yeast particles in M-cells. In conclusion, stability tests show that antigen concentration of β-glucan particles remains stable in gastric and intestinal environment. This means that the β-glucan particles can be administered orally without prior enteric coating and are suitable for mucosal delivery of antigen in the murine gastro-intestinal tract. Our data suggest that M-cells, but not subepithelial dendritic cells, are crucial for the transmucosal transport of β-glucan particles from the intestinal lumen to the PP and transcytosis of antigen to underlying antigen presenting cells and immune cells

Beta-glucan particles as novel antigen delivery systems: towards oral vaccination

Several enteric pathogens infect the body following oral uptake and cause life-threatening diseases such as cholera, dysentery and typhoid fever. Oral vaccination is essential to generate protective local immunity against intestinal pathogens. However, antigen delivery to the inductive sites for mucosal immunity (the small intestine Peyer’s patches, PP) has proven to be particularly challenging. Recently, there has been a lot of interest in the use of microparticles as antigen delivery systems in the development of more efficient mucosal vaccines. We evaluated the potential of β-glucan microcapsules to deliver antigen transmucosally in the PP regions of the murine small intestine. β-glucan microparticles (2-4 µm) were prepared from Saccharomyces cerevisiae and loaded with FITC or Alexa Fluor 488-conjugated bovine serum albumin. Firstly, the appropriateness of β-glucan particles as antigen delivery systems for oral and intestinal applications was assessed in stability tests. Fluorimetric analysis demonstrated that the BSA concentration within particles is stable during the first 12 hours of simulated gastric or simulated intestinal fluid treatment. Secondly, β-glucan particles were administered to male C57BL/6 mice (8-10 weeks old) via intestinal loops at a particle concentration of 100*106/ml. After one hour of incubation, transmucosal particle transport and uptake in the PP was evaluated by flow cytometry, confocal microscopy and transmission electron microscopy. Using flow cytometry, we could not observe any particle uptake in the main antigen presenting cell population, the dendritic cells, however, a modest particle uptake was repeatedly detected in the B-cell population. Confocal microscopic images showed localization of β-glucan particles in the follicle associated epithelium and B-cell internalization. Moreover, transmission electron microscopy demonstrated transcellular transport of yeast particles in M-cells. In conclusion, stability tests show that antigen concentration of β-glucan particles remains stable in gastric and intestinal environment. This means that the β-glucan particles can be administered orally without prior enteric coating and are suitable for mucosal delivery of antigen in the murine gastro-intestinal tract. Our data suggest that M-cells, but not subepithelial dendritic cells, are crucial for the transmucosal transport of β-glucan particles from the intestinal lumen to the PP and transcytosis of antigen to underlying antigen presenting cells and immune cells