Mucosal Tissue Surfaces Research Articles

The Direct Exposure of Cortisol Does Not Modulate the Expression of Immune-Related Genes on Tissue Explants of Mucosal Surfaces in Rainbow Trout (Oncorhynchus mykiss) Nor in Gilthead Sea Bream (Sparus aurata)

The stress response in fish is characterized by the activation of the HPI axis resulting in the release of cortisol. Previous studies in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) have shown that an acute stressor modulates the expression of immune- and stress-related genes in mucosal-associated lymphoid tissues (MALTs), particularly in the skin (SALT), gills (GIALT), and gut (GALT). However, there are no antecedents on whether the modulation on the mucosal transcriptomic profile is coordinated through the local presence of cortisol in the mucosal tissue surface. Thus, the aim of this study was to evaluate the effect of cortisol upon the modulation of a set of immune- (il-1β, il-6, tnf-α, and cox-2) and stress-related (hsp70, gr1) genes. For this purpose, tissue explants cultures were incubated with cortisol (100 ng/100 mg tissue) for 2-, 4-, and 24 h and the gene expression profile was evaluated at each time-point by real-time PCR. No differences were found in the gene expression between cortisol-incubated tissue explants and mock-incubated tissues in any of the time-points tested for both species. These results suggest that the quick modulation of the gene expression during the first 24 h after the exposure to stressor challenge reported in previous studies, is probably coordinated and mediated through a systemic-dependent mechanism but not through a peripheral/local response on mucosal tissue surfaces.

Clarifying and Imaging Candida albicans Biofilms.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 µm biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Mucus-penetrating phage-displayed peptides for improved transport across a mucus-like model

The objective of this work is to use phage display libraries as a screening tool to identify peptides that facilitate transport across the mucus barrier. Mucus is a complex selective barrier to particles and molecules, limiting penetration to the epithelial surface of mucosal tissues. In mucus-associated diseases such as cystic fibrosis (CF), mucus has increased viscoelasticity and a higher concentration of covalent and non-covalent physical entanglements compared to healthy tissues, which greatly hinders permeability and transport of drugs and particles across the mucosae for therapeutic delivery. Treatment of CF lung diseases and associated infections must overcome this abnormal mucosal barrier. Critical bottlenecks hindering effective drug penetration remain and while recent studies have shown hydrophilic, net-neutral charge polymers can improve the transport of nanoparticles and minimize interactions with mucus, there is a dearth of alternative carriers available. We hypothesized that the screening of a phage peptide library against a CF mucus model would lead to the identification of phage-displayed peptide sequences able to improve transport in mucus. These combinatorial libraries possess a large diversity of peptide-based formulations (108–109) to achieve unprecedented screening for potential mucus-penetrating peptides. Here, phage clones displaying discovered peptides were shown to have up to 2.6-fold enhanced diffusivity in the CF mucus model. In addition, we demonstrate reduced binding affinities to mucin compared to wild-type control. These findings suggest that phage display libraries can be used as a strategy to improve transmucosal delivery.

Chemical modification of drug molecules as strategy to reduce interactions with mucus.

Many drug molecules possess inadequate physical-chemical characteristics that prevent to surpass the viscous mucus layer present in the surface of mucosal tissues. Due to mucus protective role and its fast turnover, these drug molecules end up being removed from the body before being absorbed and, thus, before exerting any physiologic affect. Envisaging a better pharmacokinetics profile, chemical modifications, to render drug a more mucopenetrating character, have been introduced to drug molecules backbone towards more effective therapies. Mucus penetration increases when drug molecules are provided with net-neutral charge, when they are conjugated with mucolytic agents and through modifications that makes them resistant to enzymes present in mucus, with the overall increase of their hydrophilicity and the decrease of their molecular weight. All of these characteristics act as a whole and influence each other so they must be well thought when drug molecules are being designed for mucosal delivery.

High-Frequency Ultrasonic Imaging of Growth and Development in Manufactured Engineered Oral Mucosal Tissue Surfaces

This study uses high-resolution ultrasound to examine the growth and development of engineered oral mucosal tissues manufactured under aseptic conditions. The specimens are a commercially available natural tissue scaffold, AlloDerm, and oral keratinocytes seeded onto AlloDerm to form an ex vivo-produced oral mucosal equivalent (EVPOME) suitable for intra-oral grafting. The seeded cells produce a keratinized protective upper layer that smooths out any remaining surface irregularities on the underlying AlloDerm. Two-dimensional acoustic imaging of unseeded AlloDerm and developing EVPOMEs was performed on each day of their growth and development, each tissue specimen being imaged under aseptic conditions (total time from seeding to maturation: 11 d). Ultrasonic monitoring offers us the ability to determine the constituents of the EVPOME that are responsible for changes in its mechanical behavior during the manufacturing process. Ultrasonic monitoring affords us an opportunity to non-invasively assess, in real time, tissue-engineered constructs before release for use in patient care.

Pulmonary delivery of DNA vaccine constructs using deacylated PEI elicits immune responses and protects against viral challenge infection

Vaccination through mucosal surfaces has been shown to elicit antiviral immune responses against a number of mucosal pathogens. Here we demonstrate that both mucosal and systemic immune responses can be elicited against a model HIV-1 CN54gp140 antigen when cation-complexed plasmid DNA vaccines are applied topically to the murine pulmonary mucosa as an immune priming strategy. Furthermore, using an influenza challenge model we show that a plasmid DNA vaccine complexed to a less toxic form of PEI called dPEI (a nearly fully hydrolysed linear PEI with 11% additional free protonatable nitrogen atoms) can provide significant protection against a respiratory challenge infection in mice. Furthermore, we show that dPEI polyplexes have the potential to transfect not only mucosal epithelium, but also to enter deeper into tissues through the modulation of tight junction integrity. Taken together, these results demonstrate that less toxic forms of PEI can be effective delivery vehicles for plasmid DNAs to elicit cellular and humoral protective responses in vivo. Moreover, our observations suggest that these less toxic derivatives of PEI could be utilised for topical plasmid DNA vaccine delivery to human mucosal tissue surfaces, and that this application may permit dissemination of the immune responses through the linked mucosal network thus providing protective immunity at distal portals of pathogen entry.

Changing Views of the Evolution of Immunity

Changing Views of the Evolution of Immunity

Design of a visible-light spectroscopy clinical tissue oximeter

We develop a clinical visible-light spectroscopy (VLS) tissue oximeter. Unlike currently approved near-infrared spectroscopy (NIRS) or pulse oximetry (SpO2%), VLS relies on locally absorbed, shallow-penetrating visible light (475 to 625 nm) for the monitoring of microvascular hemoglobin oxygen saturation (StO2%), allowing incorporation into therapeutic catheters and probes. A range of probes is developed, including noncontact wands, invasive catheters, and penetrating needles with injection ports. Data are collected from: 1. probes, standards, and reference solutions to optimize each component; 2. ex vivo hemoglobin solutions analyzed for StO2% and pO2 during deoxygenation; and 3. human subject skin and mucosal tissue surfaces. Results show that differential VLS allows extraction of features and minimization of scattering effects, in vitro VLS oximetry reproduces the expected sigmoid hemoglobin binding curve, and in vivo VLS spectroscopy of human tissue allows for real-time monitoring (e.g., gastrointestinal mucosal saturation 69+/-4%, n=804; gastrointestinal tumor saturation 45+/-23%, n=14; and p<0.0001), with reproducible values and small standard deviations (SDs) in normal tissues. FDA approved VLS systems began shipping earlier this year. We conclude that VLS is suitable for the real-time collection of spectroscopic and oximetric data from human tissues, and that a VLS oximeter has application to the monitoring of localized subsurface hemoglobin oxygen saturation in the microvascular tissue spaces of human subjects.

Ethanol impairs intestinal barrier defense by modulation of immunoglobulin A transport

Background. Increased epithelial permeability may contribute to septic complications after alcohol intoxication. Prior ethanol (EtOH) exposure leads to structural and functional effects on cytoskeletal components important in maintaining barrier integrity. The cytoskeleton is also important in the transcellular transport of proteins including the polymeric immunoglobulin receptor (pIgR) mediated transport of immunoglobulin A (IgA) to the luminal surface of mucosal tissues. We (1) investigated the effects of acute EtOH exposure on cell monolayer barrier integrity and IgA transport and (2) assessed the importance of microtubule function on these functions in vitro. Methods. Confluent Madin-Darby canine kidney (MDCK) epithelial cells transfected with pIgR cDNA were exposed to 1% or 5% EtOH, and transcytosis of dimeric IgA was measured. Paclitaxel was used to pretreat a subset of MDCK cells. Results. EtOH exposure resulted in a concentration and time dependent decrease in IgA transcytosis. This was associated with physical derangements in the integrity of the cell monolayer. Pretreatment with paclitaxel completely abrogated these effects. Conclusions. EtOH causes both physical and immune derangement of epithelial cell barrier function. Decreased secretory IgA at mucosal surfaces may initiate septic complications after EtOH intoxication. (Surgery 2002;132:573-81.)

From sticky stuff to sweet receptors--achievements, limits and novel approaches to bioadhesion.

About 10 years ago, the concept of bioadhesion was introduced into the pharmaceutical literature and has since stimulated much research and development both in academia and in industry. The first generation of bioadhesive drug delivery systems (BBDS) were based on so-called mucoadhesive polymers, i.e. natural or synthetic macromolecules, often already well accepted and used as pharmaceutical excipients for other purposes, which show the remarkable ability to 'stick' to humid or wet mucosal tissue surfaces. While these novel dosage forms were mainly expected to allow for a possible prolongation, better localization or intensified contact to mucosal tissue surfaces, it had to be realized that these goals were often not so easily accomplished, at least not by means of such relatively straightforward technology. However, although not always convincing as a 'pharmaceutical glue', some of the mucoadhesive polymers were found to display other, possibly even more important biological activities, namely to inhibit proteolytic enzymes and/or to modulate the permeability of usually tight epithelial tissue barriers. Such features were found to be particularly useful in the context of peptide and protein drug delivery. But still, the interest in realizing 'true' bioadhesion continues: instead of mucoadhesive polymers, plant or bacterial lectins, i.e. adhesion molecules which specifically bind to sugar moieties of the epithelial cell membrane, are now widely being investigated as drug delivery adjuvants. These second-generation bioadhesives not only provide for cellular binding, but also for subsequent endo- and transcytosis. This makes the novel, specifically bioadhesive molecules particularly interesting for the controlled delivery of DNA/RNA molecules in the context of antisense or gene therapy.

Gamma delta T cells down-regulate primary IgE responses in rats to inhaled soluble protein antigens.

The biologic role and repertoire of cells bearing the gamma delta T cell receptor has not been fully defined. However, their tropism for epithelial microenvironments is recognized and suggests an important role for these cells in immune defense at mucosal tissue surfaces. The study presented below utilizes an experimental model in which repeated exposure of Brown Norway rats to OVA by inhalation induces a state of Ag-specific, IgE isotype-specific "tolerance" via immune deviation. This process seems similar to oral tolerance in the gut. This form of tolerance was adoptively transferred to naive syngeneic recipients by i.p. injection of as few as 10(3) positively selected TCR-gamma delta+ cells from OVA-exposed rats. These TCR-gamma delta+ T-cells are demonstrated to produce high levels of INF-gamma in response to OVA stimulation, and this provides a potential mechanism for the inhibition of Th2 cell proliferation, resulting in suppression of IgE production. The unique potency of these cells in selective suppression of IgE Ab production in response to natural "mucosal" Ag exposure suggests a potentially important role in protection against primary allergic sensitization in vivo.