Christopher A. Squier

The Permeability of Human Oral Mucosa and Skin to Water

Specimens from fbur regions of oral mucosa (palate, buccal mucosa, lateral border of the tongue, and the floor of the mouth) and of abdominal skin were taken from 58 individuals at autopsy, for determination of permeability constants (Kp) to tritium-labeled water. Comparisons between fresh specimens and those stored at -80°C revealed no significant effect on Kp as a result of freezing; similar results were found with use of specimens from corresponding regions of the pig. Values for Kp were significantly different for all of the tissue regions examined and ranged from 44 ± 4 × 10-7 cm/min for skin to 973 ± 33 x 10-7 cmlmin for the floor of the mouth, which was the most penneable region. Similar differences were evident among corresponding regions of porcine oral mucosa and skin. Moreover, the Kp values obtained for human tissues were not significantly different from those of the pig, except for the floor of the mouth, which was more permeable in human than in pig tissue. The results reveal interesting differences in the permeability of human oral mucosa that might be related to susceptibility to mucosal disease in those conditions where local extrinsic etiological agents are implicated.

The Permeability of Oral Mucosa

In discussing permeability, we are describing one of the fundamental barrier functions of oral mucosa. Despite assumptions to the contrary, the oral mucosa is not a uniformly, highly permeable tissue like gut, but shows regional variation. The keratinized areas, such as gingiva and hard palate, are least permeable and nonkeratinized lining areas are most permeable. This variation appears to reflect differences in the types of lipid making up the intercellular permeability barrier in the superficial layers of the epithelium. Differences in permeability may be related to regional differences in the prevalence of certain mucosal diseases and can be utilized to advantage for local and systemic drug delivery.

Cytochemical identification of ATPase-positive Langerhans cells in EDTA-separated sheets of mouse epidermis

Sheets of epidermis for incubation to demonstrate ATPase activity were obtained from specimens of mouse footpad using EDTA as the separation medium. The use of EDTA in place of the NaBr method previously described, resulted in a greatly reduced incubation time, precise localization of reaction product and preservation of ultrastructural detail. A population afclosely and regularly spaced ATPasepositive dendritic cells was demonstrated by light microscopy. Electron microscopy demonstrated that, with short incubation times, reaction product was found only in the extracellular space adjacent to dendritic cells, the majority of which possessed the typical ultrastructural features of Langerhans cells.

Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy

Epidermis and oral epithelia provide permeability barriers that limit penetration of potentially harmful agents. Barrier function is determined by lipids in the superficial epithelial layers and varies regionally by more than 10-fold. The purpose of this study was to determine whether differences in lipid content, composition or organization could account for this variation in barrier function. Stratum corneum from skin, gingiva and palate and superficial layers from buccal regions and the floor of the mouth were isolated, and lipids were extracted and analysed by thin-layer chromatography. Tissue from each region was examined by electron microscopy. There was an inverse correlation between permeability and ceramide content and a direct correlation with triglyceride content. Electron microscopy revealed that the intercellular space in epidermal stratum corneum contained multiple lipid lamellae displaying an alternating broad-narrow-broad spacing. In palatal and gingival stratum corneum, uniformly spaced lamellae were present at the periphery of dilations of the intercellular space, but the interiors of the dilations contained disorganized lamellae and electron-dense material. In the non-keratinized barriers, there was a single, broad lamella at the cell periphery and occasional short stacks of lamellae traversing the intercellular space. These intercellular lamellae may be derived from a population of membrane-coating granules that contain internal lamellae. The results suggest that ceramides may be important barrier components, even in non-keratinizing epithelia where they are very minor components. Regional differences in the physical organization of barrier lipids may also contribute to differences in barrier function.

Delivery of bioactive peptides and proteins across oral (Buccal) mucosa

The identification of an increasing array of highly potent, endogenous peptide and protein factors termed cytokines, that can be efficiently synthesized using recombinant DNA technology, offers exciting new approaches for drug therapy. However, the physico-chemical and biological properties of these agents impose limitations in formulation and development of optimum drug delivery systems as well as on the routes of delivery. Oral mucosa, including the lining of the cheek (buccal mucosa), floor of mouth and underside of tongue (sublingual mucosa) and gingival mucosa, has received much attention in the last decade because it offers excellent accessibility, is not easily traumatized and avoids degradation of proteins and peptides that occurs as a result of oral administration, gastrointestinal absorption and first-pass hepatic metabolism. Peptide absorption occurs across oral mucosa by passive diffusion and it is unlikely that there is a carrier-mediated transport mechanism. The principal pathway is probably via the intercellular route where the major permeability barrier is represented by organized array of neutral lipids in the superficial layers of the epithelium. The relative role of aqueous as opposed to the lipid pathway in drug transport is still under investigation; penetration is not necessarily enhanced by simply increasing lipophilicity, for other effects, such as charge and molecular size, also play an important role in absorption of peptide and protein drugs. Depending on the pharmacodynamics of the peptides, various oral mucosal delivery systems can be designed. Delivery of peptide/protein drugs by conventional means such as solutions has some limitations. The possibility of excluding a major part of drug from absorption by involuntary swallowing and the continuous dilution due to salivary flow limits a controlled release. However these limitations can be overcome by adhesive dosage forms such as gels, films, tablets, and patches. They can localize the formulation and improve the contact with the mucosal surface to improve absorption of peptides and proteins. Addition of absorption promoters/permeabilizers in bioadhesive dosage forms will be essential for a successful peptide/protein delivery system.

The Innate Immune System Is Activated by Stimulation of Vaginal Epithelial Cells with Staphylococcus aureus and Toxic Shock Syndrome Toxin 1

ABSTRACT Despite knowledge of the effects of toxic shock syndrome (TSS) toxin 1 (TSST-1) on the adaptive immune system, little is known about stimulation of the innate immune system, particularly epithelial cells. This study investigated the interactions of TSS Staphylococcus aureus and TSST-1 with human vaginal epithelial cells (HVECs) and porcine mucosal surfaces. When cocultured with HVECs for 6 h, TSS S. aureus MN8 proliferated, formed aggregates on the HVEC surfaces, and produced exotoxins. Receptor binding studies showed that 35S-TSST-1 bound to 5 × 104 receptors per HVEC, with saturation at 15 min. Affymetrix Human GeneChip U133A microarray analysis determined S. aureus MNSM (100 bacteria/HVEC) caused at least twofold up- or down-regulation of 410 HVEC genes by 6 h; these data were also confirmed with S. aureus MN8. TSST-1 (100 μg/ml) caused up- or down-regulation of 2,386 HVEC genes by 6 h. In response to S. aureus, the HVEC genes most up-regulated compared to those in controls were those coding for chemokines or cytokines—MIP-3α, 478-fold; GRO-α, 26-fold; GRO-β, 14-fold; and GRO-γ, 30-fold—suggesting activation of innate immunity. TSST-1 also caused up-regulation of chemokine/cytokine genes. Chemokine/cytokine gene up-regulation was confirmed by enzyme-linked immunosorbent assays measuring the corresponding proteins induced by S. aureus and TSST-1. S. aureus MN8, when incubated with porcine vaginal tissue, increased the flux of 35S-TSST-1 across the mucosal surface. This was accompanied by influx of lymphocytes into the upper layers of the tissue. These data suggest innate immune system activation through epithelial cells, reflected in chemokine/cytokine production and influx of lymphocytes, may cause changes in vaginal mucosa permeability, facilitating TSST-1 penetration.

Membrane coating granules in nonkeratinizing oral epithelium.

Examination of a variety of nonkeratinized oral epithelia with the electron microscope reveals the presence of a population of small cytoplasmic granules, approximately 0.2 μm in diameter, that appear in the Golgi region of the prickle cell layers, migrate to the superficial aspect of cells in the midepithelial region and then apparently fuse with the cell membrane in the third quarter of the epithelium. Serial sections cut through the granules show that they are autonomous structures and not infoldings of the cell membrane. These granules are similar in size, distribution, and behavior to the “membrane coating granules” of keratinized oral epithelium and epidermis and may represent an homologous organelle in nonkeratinized epithelium.

Regional variation in the structure and permeability of oral mucosa and skin

Abstract Molecules of low to moderate size are capable of passage through the skin and oral mucosa by simple diffusion. The diffusional resistance of the tissue is determined primarily by lipids in the intercellular spaces of the outer layers of the epithelium. In the skin, palate, and gingiva, this barrier function is provided by a stratum corneum, the intercellular spaces of which contain mainly ceramides, cholesterol and fatty acids. The more flexible regions of the oral mucosa are lined by a non-keratinizing epithelium, the outer half to one-third of which provides a permeability barrier. The barrier region of the non-keratinized epithelia contains phospholipids, cholesterol and monohexosylceramides as major constituents. Differences in the composition and organization of lipids in the epithelial barriers result in regional variation in permeability.

Ultrastructural Quantitation of Connective Tissue Changes in Phenytoin-induced Gingival Overgrowth in the Ferret

Ultrastructural quantitation has been used to determine both the effect of phenytoin (PHT) on gingival connective tissue components and the cellular basis for these changes. The results suggest that the amount of ground substance increases, and this may be due to decreased degradation within fibroblasts.

The effect of stretching on formation of myofibroblasts in mouse skin

SummaryWound contraction results from the contractile activity of modified fibroblasts, termed myofibroblasts, which are present in the granulation tissue of the healing wound. This study examines the relative role of mechanical tension (stretching) and wound healing as events capable of stimulating the formation of myofibroblasts in mouse skin. The skin of hairless mice was subjected to mechanical stretching and to a small incisional wound either separately or in combination. Animals were killed at intervals between 1 and 6 days and the dermis examined with the electron microscope. Stretching alone produced little evidence of inflammation at any time interval but cells with the ultrastructural characteristics of myofibroblasts were present at 4 days and abundant at 6 days. Skin that had been both stretched and wounded showed a marked inflammatory response and also contained myofibroblasts, but they were less frequent than in the skin subjected to stretching alone. Very few myofibroblasts were evident in skin that had only been wounded. It is suggested that the effect of mechanical tension alone may initiate formation of myofibroblasts in a tissue.