Intestinal M-cells Research Articles

Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression.

Microfold cells (M cells) are specific intestinal epithelial cells for monitoring and transcytosis of antigens, microorganisms, and pathogens in the intestine. However, the mechanism for M-cell development remained elusive. Real-time polymerase chain reaction, immunofluorescence, and western blotting were performed to analyze the effect of sorbitol-regulated M-cell differentiation in vivo and in vitro, and luciferase and chromatin Immunoprecipitation were used to reveal the mechanism through which sorbitol-modulated M-cell differentiation. Herein, in comparison to the mannitol group (control group), we found that intestinal M-cell development was inhibited in response to sorbitol treatment as evidenced by impaired enteroids accompanying with decreased early differentiation marker Annexin 5, Marcksl1, Spib, sox8, and mature M-cell marker glycoprotein 2 expression, which was attributed to downregulation of receptor activator of nuclear factor kappa-В ligand (RANKL) expression in vivo and in vitro. Mechanically, in the M-cell model, sorbitol stimulation caused a significant upregulation of phosphodiesterase 4 (PDE4) phosphorylation, leading to decreased protein kinase A (PKA)/cAMP-response element binding protein (CREB) activation, which further resulted in CREB retention in cytosolic and attenuated CREB binds to RANKL promoter to inhibit RANKL expression. Interestingly, endogenous PKA interacted with CREB, and this interaction was destroyed by sorbitol stimulation. Most importantly, inhibition of PDE4 by dipyridamole could rescue the inhibitory effect of sorbitol on intestinal enteroids and M-cell differentiation and mature in vivo and in vitro. These findings suggested that sorbitol suppressed intestinal enteroids and M-cell differentiation and matured through PDE4-mediated RANKL expression; targeting to inhibit PDE4 was sufficient to induce M-cell development.

Оценка эффективности сукросомальной формы железа при лечении пациенток с железодефицитной анемией 1–2-й степени тяжести на фоне обильных менструальных кровотечений

Aim: to evaluate the clinical efficacy of Sucrosomial® iron (SiderAL® Forte) in the treatment of stage 1–2 iron deficiency anemia (IDA) in patients with menorrhagia in the setting of ovulation disorders. Patients and Methods: a prospective randomized multicenter clinical case study of 110 patients with stage 1–2 IDA developed in the setting of menorrhagia as a result of ovulation disorders was conducted. Female patients of the main group (n=74) received Sucrosomial® iron 30 mg 2 times a day for 60 days. In the control group (n=36), a liposomal iron preparation of 30 mg 2 times a day was also prescribed for 60 days. Results: during Sucrosomial® iron ferrotherapy, an increase in Hb levels by more than 20 g/L was observed after 30 days of administration in 65 (87%) female patients, after 60 days in 73 (98.6%) female patients, which demonstrated higher therapeutic efficacy versus liposomal iron preparation (69.4 and 88.8%, respectively, p=0.0001). There was also a greater adherence to therapy (98.6%) during Sucrosomial® iron intake versus liposomal iron intake (86.1%, p=0.0001), due to fewer (1.4%) adverse events compared with the control group (5.5%, p=0.0002). Conclusion: oral ferrotherapy with Sucrosomial® iron is a reasonable option for the IDA treatment in patients with menorrhagia due to its higher efficacy versus the liposomal iron. The effect occurs after 30 days of drug intake with a minimum number of adverse events, due to the unique structural, physico-chemical and pharmacokinetic characteristics of the sucrosomial iron, thanks to which it is protected from the gastric juice action, does not contact with the gastrointestinal mucosa and is absorbed through special intestinal M-cells with further iron release in liver cells. KEYWORDS: iron deficiency anemia, menorrhagia, ovulation disorders, Sucrosomial® iron, women, therapy. FOR CITATION: Kononova I.N., Dobrokhotova Yu.E., Kareva E.N., Shmakova N.A., Graban I.V., Ogurtsova T.A. Evaluation of the sucrosomial iron efficacy in the patient treatment with stage 1–2 iron deficiency anemia during heavy menstrual bleeding. Russian Journal of Woman and Child Health. 2023;6(4):332–339 (in Russ.). DOI: 10.32364/2618-8430-2023-6-4-2.

P053 The intestinal complement system and its role in dysregulation of the intestinal B-cell compartment in Crohn’s Disease patients

Abstract Background For several decades, there is growing evidence that a hyperactive B-cell compartment may be involved in the disturbed mucosal immune response in Crohn’s disease (CD) patients. This is reflected by approximately one third of all CD patients displaying an excessive production of autoantibodies against the intestinal M-cell expressed FimH receptor glycoprotein 2 (GP2) which inhibits induction of adaptive immunity against adherent-invasive flagellated bacteria. However, underlying mechanisms of an imbalanced B-cell compartment in CD patients still remain elusive. Given that the complement system plays a critical role in the detection and clearance of bacteria and promotes B-cell immunity, we asked if the complement system contributes to a dysregulated B-cell response in CD. Methods Systematical expression analyses of the intestinal complement components were performed in IBD patients, including CD and ulcerative colitis (UC) patients, versus non-IBD controls. Additionally, activity of the three activation pathways of complement system was studied in serum samples of CD patients and controls. Further, immunoglobulin levels in intestinal biopsies, sera and fecal samples were analyzed. Results CD patients, but no UC patients, showed increased expression of the early classical complement pathway components C1QA, C1QB, C1S, C2, C3, C4A, CR1 and CR2 in sigmoidal biopsy samples that does not differ significantly between remission and active disease state. This could be verified by immunohistochemistry, displaying an over-representation of mucosal C1q, C3 and B-cell expressed CR2 (also CD21) in colon biopsies of CD patients in remission. In line with this, CD patients in remission present with enhanced activity of the classical as well as alternative pathway of complement activation in serum compared to non-IBD controls. Accordingly, high protein levels of C3 cleavage fragments were detected in fecal samples from CD patients in contrast to non-IBD controls. While there were no differences in systemic immunoglobulin levels in serum, intestinal IgM was increased in CD patients in remission. Notably, fecal IgA levels were strongly diminished in these patients, while expression of the polymeric immunoglobulin receptor was not affected. Conclusion In summary, our findings demonstrate an augmented activation of the intestinal complement system that may potentially be involved in the etiology of CD. Expansion of mucosal IgM+/CR2+ B cells together with a reduced fecal IgA level, points to a locally disturbed induction of intestinal IgA secreting plasma cells in CD patients.

Characterization of M Cells in Tear Duct-Associated Lymphoid Tissue of Mice: A Potential Role in Immunosurveillance on the Ocular Surface.

The ocular mucosal tissues are exposed to potentially harmful foreign antigens in the air and tear fluid. The tear duct-associated lymphoid tissue (TALT) may contribute to immune surveillance in the eye region. Follicle-associated epithelium (FAE) of TALTs is classified as stratified squamous epithelium and consists of squamous epithelial cells arranged in layers on the basement membrane. In contrast, most mucosa-associated lymphoid tissue is covered by a monolayer of epithelium containing microfold (M) cells. Therefore, antigen uptake and the presence of M cells in TALT are not fully understood. The present study found that a small population of FAE cells in the TALT expressed intestinal M-cell markers, namely Sox8, Tnfaip2, GP2, and OPG. This cell population was identified as functional M cells because of their uptake capacity of luminal nanoparticles. In addition, RANKL, which is essential for M-cell differentiation, was expressed by stroma-like cells at the subepithelial region and its receptor RANK by the FAE in the TALT. The administration of RANKL markedly increased the number of Sox8+ M cells. In contrast, deficiency in OPG, an endogenous inhibitor of RANKL, increased the number of M cells in the TALT. These data demonstrate that the RANKL-RANK axis is essential for M-cell differentiation in the TALT. Furthermore, immunization via eye drops elicited the production of antigen-specific antibodies in tears, which was enhanced by RANKL administration. Thus, TALT M cells play an important role in the immunosurveillance of the eye region.

Uptake and effects of orally ingested polystyrene microplastic particles in vitro and in vivo.

Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1µm (4.55 × 107 particles), 4µm (4.55 × 107particles) and 10µm (1.49 × 106 particles) microplastics at a volume of 10mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.

Regulatory aspects in the pharmaceutical development of nanoparticle drug delivery systems designed to cross the intestinal epithelium and M-cells

This article reviews the field of oral uptake of nanoparticles across the gastrointestinal epithelium for the period 2006–2016. Analysis is conducted from the viewpoint of i) M-cell genetics and model development, ii) drug targeting to Peyer's patches and M-cells, and iii) physicochemical interactions of nanoparticles in the intestinal milieu. In light of these recent developments, regulatory considerations in the development of orally-absorbable nanoparticle drug products are discussed and focused on Module 3.2.P sub-sections of the Common Technical Document. Particular attention is paid to novel excipients, ligands and the non-standard method of manufacture. The novelty of this drug delivery system demands not only a multi-disciplinary scientific and regulatory approach but also a risk-adjusted consideration for a system defined by both processes and specifications. Given the current state of scientific development in the field it is suggested (in the author's personal opinion) that the design of nanoparticulate drug delivery systems should be kept as simple as possible (from a regulatory and manufacturing perspective) and to target the entire gastrointestinal epithelium.

Design and statistical modeling of mannose-decorated dapsone-containing nanoparticles as a strategy of targeting intestinal M-cells.

The aim of the present work was to develop and optimize surface-functionalized solid lipid nanoparticles (SLNs) for improvement of the therapeutic index of dapsone (DAP), with the application of a design of experiments. The formulation was designed to target intestinal microfold (M-cells) as a strategy to increase internalization of the drug by the infected macrophages. DAP-loaded SLNs and mannosylated SLNs (M-SLNs) were successfully developed by hot ultrasonication method employing a three-level, three-factor Box–Behnken design, after the preformulation study was carried out with different lipids. All the formulations were systematically characterized regarding their diameter, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, and loading capacity. They were also subjected to morphological studies using transmission electron microscopy, in vitro release study, infrared analysis (Fourier transform infrared spectroscopy), calorimetry studies (differential scanning calorimetry), and stability studies. The diameter of SLNs, SLN-DAP, M-SLNs, and M-SLN-DAP was approximately 300 nm and the obtained PDI was <0.2, confirming uniform populations. Entrapment efficiency and loading capacity were approximately 50% and 12%, respectively. Transmission electron microscopy showed spherical shape and nonaggregated nanoparticles. Fourier transform infrared spectroscopy was used to confirm the success of mannose coating process though Schiff’s base formation. The variation of the ZP between uncoated (approximately −30 mV) and mannosylated formulations (approximately +60 mV) also confirmed the successful coating process. A decrease in the enthalpy and broadening of the lipid melting peaks of the differential scanning calorimetry thermograms are consistent with the nanostructure of the SLNs. Moreover, the drug release was pH-sensitive, with a faster drug release at acidic pH than at neutral pH. Storage stability for the formulations for at least 8 weeks is expected, since they maintain the original characteristics of diameter, PDI, and ZP. These results pose a strong argument that the developed formulations can be explored as a promising carrier for treating leprosy with an innovative approach to target DAP directly to M-cells.

M-Cells Contribute to the Entry of an Oral Vaccine but Are Not Essential for the Subsequent Induction of Protective Immunity against Francisella tularensis.

M-cells (microfold cells) are thought to be a primary conduit of intestinal antigen trafficking. Using an established neutralizing anti-RANKL (Receptor Activator of NF-κB Ligand) antibody treatment to transiently deplete M-cells in vivo, we sought to determine whether intestinal M-cells were required for the effective induction of protective immunity following oral vaccination with ΔiglB (a defined live attenuated Francisella novicida mutant). M-cell depleted, ΔiglB-vaccinated mice exhibited increased (but not significant) morbidity and mortality following a subsequent homotypic or heterotypic pulmonary F. tularensis challenge. No significant differences in splenic IFN-γ, IL-2, or IL-17 or serum antibody (IgG1, IgG2a, IgA) production were observed compared to non-depleted, ΔiglB-vaccinated animals suggesting complementary mechanisms for ΔiglB entry. Thus, we examined other possible routes of gastrointestinal antigen sampling following oral vaccination and found that ΔiglB co-localized to villus goblet cells and enterocytes. These results provide insight into the role of M-cells and complementary pathways in intestinal antigen trafficking that may be involved in the generation of optimal immunity following oral vaccination.

RANKL regulates differentiation of microfold cells in mouse nasopharynx-associated lymphoid tissue (NALT)

Murine nasopharynx-associated lymphoid tissue (NALT), located at the base of the nasal cavity, serves as a major site for the induction of mucosal immune responses against airway antigens. The follicle-associated epithelium (FAE) covering the luminal surface of NALT is characterized by the presence of microfold cells (M cells), which take up and transport luminal antigens to lymphocytes. Glycoprotein 2 (GP2) has recently been identified as a reliable marker for M cells in Peyer's patches of the intestine. However, the expression of GP2 and other functional molecules in the M cells of NALT has not yet been examined. We have immunohistochemically detected GP2-expressing cells in the FAE of NALT and the simultaneous expression of other intestinal M-cell markers, namely Tnfaip2, CCL9, and Spi-B. These cells have been further identified as M cells because of their higher uptake capacity of luminal microbeads. Electron microscopic observations have shown that GP2-expressing cells on the FAE display morphological features typical of M cells: they possess short microvilli and microfolds on the luminal surface and are closely associated with intraepithelial lymphocytes. We have also found that the receptor activator of nuclear factor kappa-B ligand (RANKL) is expressed by stromal cells underneath the FAE, which provides its receptor RANK. The administration of RANKL markedly increases the number of GP2(+)Tnfaip2(+) cells on the NALT FAE and that of intestinal M cells. These results suggest that GP2(+)Tnfaip2(+) cells in NALT are equivalent to intestinal M cells, and that RANKL-RANK signaling induces their differentiation.

The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells

Intestinal microfold (M) cells are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses by uptake and transcytosis of luminal antigens. The mechanisms of M-cell differentiation are poorly understood as the rarity of these cells has hampered analysis. Exogenous RANKL administration can synchronously activate M-cell differentiation in mice. Here we show the Ets transcription factor Spi-B was induced early during M-cell differentiation. Absence of Spi-B silenced the expression of multiple M-cell markers and prevented the differentiation of M cells in mice. Oral antigen-specific T cell activation was significantly impaired in the intestine of Spib−/− mice. Our study demonstrates that intestinal M-cell lineage commitment requires Spi-B as a candidate master regulator.

The elucidation of the oral/respiratory axis in inducing protective immunity against Francisella tularensis (161.22)

Abstract Using a Francisella tularensis subspecies novicida live attenuated vaccine strain (U112ΔiglB), we have shown that oral vaccination of C57BL/6 mice induces significant protective immunity against pulmonary challenge with the highly human virulent F. tularensis strain SCHU S4. This protection is primarily mediated by antigen-specific CD4+ T cells and IFN-γ production and is dependent on mucosal IgA expression. We are examining the mechanism(s) by which the oral/respiratory axis induces optimal protective immunity against F. tularensis. Intestinal M-cells play an important role in oral vaccination by trafficking antigens from the lumen to the lamina propria and in presentation of antigens to underlying APCs for the subsequent induction of mucosal immunity. We have shown that mCherry-labeled U112ΔiglB co-localizes to intestinal mouse M-cells by 90 minutes following vaccination. Localization to M-cells was further verified with mouse ileal loops. Translocation of U112ΔiglB has also been demonstrated through M-cell-like-cells in an in vitro transwell co-culture system similar to M-cell trophic enteric bacteria. Current studies are examining the functional importance of intestinal M-cells in induction of mucosal immunity for the rational development of an effective oral vaccine against pulmonary tularemia. These studies will provide information for the general design of oral vaccines using live attenuated bacterial strains.

Targeting of whole killed bacteria to gastrointestinal M-cells induces humoral immunity in the female reproductive tract

Recently, we demonstrated that oral delivery of whole killed bacteria, when agglutinated by an M-cell targeting lectin, resulted in an enhanced systemic and mucosal antibody response, as well as a protective immunity, against the gut pathogens Helicobacter pylori and Campylobacter jejuni. Importantly, this protection was achieved without the addition of exogenous adjuvant. Here, in this addendum, we extend this initial study by reporting on the vaginal antibody response induced by these vaccinations. These data show that the targeting of M-cells within the gastrointestinal tract also induces the secretion of antigen-specific antibodies (IgG and IgA) at a distal mucosal site, namely the vaginal mucosa. This observation raises the possibility that oral delivery of a whole, killed bacteria vaccine that target intestinal M-cells could potentially provide a strategy for inducing protective immunity against pathogenic bacteria that infect mucosal sites outside the gastrointestinal tract.

Transport of chitosan–DNA nanoparticles in human intestinal M-cell model versus normal intestinal enterocytes

Oral vaccination is one of the most promising applications of polymeric nanoparticles. Using two different in vitro cellular models to partially reproduce the characteristics of intestinal enterocytes and M-cells, this study demonstrates that nanoparticle transport through the M-cell co-culture model is 5-fold that of the intestinal epithelial monolayer, with at least 80% of the chitosan–DNA nanoparticles uptaken in the first 30 min. Among the properties of nanoparticles studied, ligand decoration has the most dramatic effect on the transcytosis rate: transferrin modification enhances transport through both models by 3- to 5-fold. The stability of the nanoparticles also affects transport kinetics. Factors which de-stabilize the nanoparticles, such as low charge (N/P) ratio and addition of serum, result in aggregation and in turn decreases transport efficiency. Of these stability factors, luminal pH is of great interest as an increase in pH from 5.5 to 6.4 and 7.4 leads to a 3- and 10-fold drop in nanoparticle transport, respectively. Since soluble chitosan can act as an enhancer to increase paracellular transport by up to 60%, this decrease is partially attributed to the soluble chitosan precipitating near neutral pH. The implication that chitosan–DNA nanoparticles are more stable in the upper regions of the small intestine suggests that higher uptake rates may occur in the duodenum compared to the ileum and the colon.

W1999 Soluble Plant Fibers, Particularly Plantain and Broccoli, Inhibit Translocation of Crohn's Disease Mucosa-Associated Adherent-Invasive Escherichia coli Across Intestinal M-Cells

W1999 Soluble Plant Fibers, Particularly Plantain and Broccoli, Inhibit Translocation of Crohn's Disease Mucosa-Associated Adherent-Invasive Escherichia coli Across Intestinal M-Cells

M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B

The transcytotic capability and expression of distinct carbohydrate receptors on the intestinal M-cells render it a potential portal for the targeted oral vaccine delivery. PLGA nanoparticles loaded with HBsAg were developed and antigen was stabilized by co-encapsulation of trehalose and Mg(OH)2. Additionally, Ulex europaeus 1 (UEA-1) lectin was anchored to the nanoparticles to target them to M-cells of the peye's patches. The developed systems was characterized for shape, size, polydispersity index and loading efficiency. Bovine submaxillary mucin (BSM) was used as a biological model for the in vitro determination of lectin activity and specificity. The targeting potential of the lectinized nanoparticles were determined by Confocal Laser Scanning Microscopy (CLSM) using dual staining technique. The immune stimulating potential was determined by measuring the anti-HBsAg titre in the serum of Balb/c mice orally immunized with various lectinized formulations and immune response was compared with the alum-HBsAg given intramuscularly. Induction of the mucosal immunity was assessed by estimating secretary IgA (sIgA) level in the salivary, intestinal and vaginal secretion. Additionally, cytokines (interleukin-2; IL-2 and interferon-γ; IFN-γ) level in the spleen homogenates was also determined. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of protein stabilizers. The lectinized nanoparticles have demonstrated approximately 4-fold increase in the degree of interaction with the BSM as compared to plain nanoparticles and sugar specificity of the lectinized nanoparticles was also maintained. CLSM showed that lectinized nanoparticles were predominantly associated to M-cells. The serum anti-HBsAg titre obtained after oral immunization with HBsAg loaded stabilized lectinized nanoparticles was comparable with the titre recorded after alum-HBsAg given intramuscularly. The stabilized UEA-1 coupled nanopartilces exhibited enhanced immune response as compared to stabilized non-lectinized nanoparticles. Furthermore, the stabilized lectinized nanoparticles elicited sIgA in the mucosal secretion and IL-2 and IFN-γ in the spleen homogenates. These stabilized lectinized nanoparticles could be a promising carrier-adjuvant for the targeted oral-mucosal immunization.

Bacterial Invasin: Structure, Function, and Implication for Targeted Oral Gene Delivery

The mucosal surface of the gastrointestinal (GI) tract is the first line of defense against foreign pathogens and toxins ingested orally. The content of the GI tract is constantly being sampled by the immune system through specialized epithelial cells known as M-cells, which are present in the Peyer's patches of the gut, providing a thin covering over lymphoid tissue. In this way, once a harmful entity is found an immune response can be activated to eliminate the threat. Many bacterial pathogens, such as Yersinia, Listeria, Salmonella, and Shigella, have evolved ways of exploiting M-cells to gain entrance to the body. The Yersinia species is of particular interest since its extracellular protein invasin provides one of the most direct and efficient manners of host cell invasion. Invasin binds to a subset of beta1 integrin receptors located on the apical membrane of intestinal M-cells, thereby facilitating the bacteria's entry into the cells and the lymphatic system underneath. This mechanism is highly specific and effective, making the invasin protein a very attractive modality for use in the oral delivery of molecules that include therapeutic genes and gene-based vaccines. This article provides a brief overview of the molecular structure and properties of the Yersinia invasin as related to the protein's ability to facilitate binding and entry into M-cells. Also discussed are several innovative approaches that demonstrate the use of invasin as an effective targeting agent for biological and synthetic gene carrier systems, and the future prospect of developing invasin-based oral gene delivery formulations.

Mucosal targeting of allergen-loaded microspheres by Aleuria aurantia lectin

Murine intestinal M-cells express α- l-fucose residues. We constructed α- l-fucose-targeting particles for oral immunotherapy of IgE-mediated allergy. Poly( d, l-lactic-co-glycolic acid)-microspheres were loaded with birch pollen allergens, and functionalised with the α- l-fucose specific Aleuria aurantia lectin (AAL). The AAL-microspheres had a size of 1–3 μm, protected the entrapped allergens from gastric degradation and released 46.6 ± 1.3% allergen over 21 days in vitro. Oral gavages of AAL-particles to naïve BALB/c mice induced birch pollen-specific IgG2a, but not IgG1 antibodies. We conclude that targeting allergens to α- l-fucose-receptor bearing cells using AAL-microspheres induces specific Th1-antibody responses possibly counteracting Th2-dominated allergy, and therefore provides a potentially useful formulation for oral immunotherapy.

Rho-GTP binding proteins in Yersinia target cell interaction.

Among the more than 10 Yersinia species known three are pathogenic for humans: (i) Y. pestis is the causative agent of plaque, (ii) Y. pseudotuberculosis and (iii) Y. enterocolitica are enteropathogenic. The infection process of enteropathogenic yersiniae involves invasive as well as antiphagocytic and other immunomodulatory components. It starts with yersiniae at ambient temperature becoming ingested with contaminated food. A fraction of the infecting bacteria reaches the gut and attaches to the intestinal cells. At this point the chromosomal gene inv encoding an outer membrane protein (Invasin) that binds with high affinity to integrincontaining extracellular matrix (ECM) receptors is upregulated (Isberg et al., 2000). Binding of invasin to integrins on the surface of intestinal M-cells triggers bacterial translocation and delivery into subepithelial Peyer’s patches. Here inv is down-regulated and a variety of genes on the 70 kb virulence plasmid (called pYV in Y. enterocolitica) are upregulated (Jacobi et al., 1998). It is well established that the antiphagocytic and immunomodulatory functions of the three pathogenic Yersinia species depend on the pYV plasmid which carries genes encoding (i) a protein type III secretion system (TTSS) (ii) a set of at least six effector proteins (Yersinia outer proteins, Yop’s), (iii) regulators for gene expression and Yop-secretion/translocation, and (iv) a Yersinia adhesin (YadA), which among other functions can mediate Yersinia attachment to host cells. Contact

Identification of cytokeratins as accessory mediators of Salmonella entry into eukaryotic cells ☆

Pathogenic Salmonella species initiate infection of a mammalian host by inducing their own uptake into intestinal M-cells. During the uptake process, the bacteria utilize an intrinsic secretion system to release proteins that enter host cells. The secreted invasion-mediating proteins subsequently interact with host cell components that induce alterations in the actin cytoskeleton. To identify potential cellular determinants of invasion, we employed a yeast two-hybrid system using the secreted Salmonella invasion protein (SipC) as the bait protein. This system identified cytokeratins, supportive components of the cytoskeletal matrix, as proteins that may physically interact with SipC. Transfection-based studies revealed an inhibition of Salmonella invasion when a dominant negative cytokeratin-18 was expressed. Immunofluorescent confocal microscopy studies revealed that Salmonella did not enter HEp-2 cells expressing the dominant negative cytokeratin-18. These results suggest that an interaction between SipC and cytokeratin-18 may occur as part of Salmonella invasion.

Transport of Chitosan Microparticles for Mucosal Vaccine Delivery in a Human Intestinal M-cell Model

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14 C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres ®) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres ® (size 0.2 μ m) and chitosan microparticles (size 1.7 μ m) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.