Demonstration of substances of innate immunity in the integument of the Malayan pangolin (Manis javanica)

Using immunohistochemistry, the study demonstrates substances of the innate immunity in the skin of the Malayan pangolin (Manis javanica), referring mainly to the epidermis. The results obtained showed clear reaction differences between the dorsolateral body region with its strong cover of hard horny scales and the abdominal body part with a thick soft stratum corneum and a dense cover of fine hairs. Regarding pathogen recognition receptors, positive reactions for Toll-like receptors were generally weak for TLR2, in contrast to TLR4, that exhibited strong reactions in the epidermis of both body regions, with increasing staining intensities towards the stratum corneum; ß-glucan receptors showed positive reactions only for l-ficolin and mannose-binding lectin, but not for dectin-1, and only at the abdomen. A generally positive staining for both body regions was obtained for all cationic antimicrobial peptides tested, whereby cathelicidin exhibited strong reaction intensities in all epidermal layers, ß-defensin 2 staining was often limited to the stratum basale and the stratum spinosum, and positive reactions for ß-defensin 3 appeared distinctly only in the epidermis of the abdomen; for these peptides, positive reaction staining could also be found in the outer epithelial root sheath of hair follicles, their glandular annexes, and free cells of the dermis. Lysozyme was found in the vital epidermis of both body regions studied, with strong staining limited to the dorsum; strong reactions were also visible in the hair follicles.     

Hydrolysis of native proteins by keratinolytic protease of Doratomyces microsporus

A keratinolytic protease from the fungus Doratomyces microsporus was investigated for its ability to hydrolyse different native proteins. The purified enzyme was incubated for up to 24 h with keratinous substrates as well as with non-keratinous proteins. The results showed that the enzyme was broad specific since it hydrolysed various globular and fibrillar proteins. The hydrolysis of keratinous substrates decreased in the following order: skin keratins > nail keratins > hair keratins. With non-keratinous substrates, the order was: casein > BSA > elastin. Feather keratin and collagen could not be hydrolysed. Comparison of the enzyme with some known proteolytic enzymes showed that on keratin from stratum corneum the activity of the keratinase was comparable to that of proteinase K, other enzymes were less active. Hydrolysis of porcine skin with the keratinase revealed the degradation of the epidermis while dermis was not damaged.     

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula

The distribution of three anti-cytokeratin (alpha-keratin) antibodies (AE1, AE2, AE3) in the epidermis of a lizard has been studied by immunocytochemistry at light and electron microscope and by immunoblot analysis. This study shows the expression of different keratins in the resting stage epidermis of the lizard Podarcis sicula. In this stage the epidermis has an external beta-layer, an underlying alpha-layer, some layers of living suprabasal cells and a basal stratum germinativum. The AE1 antibody is localized in the basal and suprabasal cells only in the outer scale surface, but is absent from the inner surface, the hinge region and from the keratinized beta- and alpha-layers. The AE2 antibody is mainly localized at the level of the hinge region and of the alpha-layer and gives a lower reaction in the beta-layer. The AE3 antibody is mainly localized in basal and suprabasal cells, lower in the alpha-layer, and absent from the beta-layer. The electron microscope shows that all the three antibodies immunolabel cytoplasmic fibrillar structures in the deep alpha-layers and that AE2 and AE3 antibodies label small electron-dense areas in the external dense beta-layer within the electron-lucid matrix. Immunoblot analysis of the keratins extracted and separated by gel electrophoresis demonstrates the presence of a band of high molecular weight (67-68 kDa) positive to all three antibodies. In addition AE1 antibody recognizes a 44-45 kDa band and a 57-58 kDa band, AE2 recognizes a 60-61 kDa band, and AE3 recognizes a 47 kDa and a 56-57 kDa band. The localization of the keratins identified by immunoblot analysis in the epithelial layers is discussed taking in account the immunolabeling at light and electron microscope. The present study suggests that also in the normal epidermis of this reptiles, in both the alpha- and the beta-layer, the molecular masses of keratins increase from the basal to the keratinized layers, a phenomenon which is generalized to adult and embryonic amniotes epidermis.     

Vitamin adeficiency and keratin biosynthesis in cultured hamster trachea

Summary Tracheas from vitamin A-deficient hamsters in organ culture in vitamin A-free medium developed squamous metaplasia. Addition of retinyl acetate to the medium prevented squamous metaplasia and a mucociliary epithelium was maintained. Indirect immunofluorescent staining with antikeratin antibodies AE1 and AE3 indicated positive reactions with epithelium of tracheas either cultured in vitamin A-free or retinyl acetate (RAc)-containing medium. The “stratum corneum”-like squames in metaplastic tracheas were strongly stained by AE3. Immunoprecipitation of cytoskeletal extracts from [³⁵S]methionine labeled tracheas with a multivalent keratin antiserum indicated that the concentration of keratins synthesized in tracheas cultured in vitamin A-free medium was greater than that observed in tracheas cultured in the presence of RAc. In addition, new species of keratin were expressed in tracheas cultured in RAc-free medium. Alterations in the program of keratin synthesis were clearly detectable after 1 d in vitamin A-free medium, even though squamous metaplasia was not yet obvious. Squamous tracheas were shown by immunoblot analysis to contain keratins of 50, 48, 46.5, and 45 kilodalton (kd) detected with AE1; and 58, 56, and 52 kd detected with AE3. Immunoblot analysis with monospecific antimouse keratin sera also demonstrated the presence of 60, 55, and 50 kd keratins in the metaplastic tracheas. All these various species of keratins were either absent or present in much reduced quantity in mucociliary tracheas in RAc-containing medium. Interestingly, the induction of squamous metaplasia in tracheal epithelium did not result in the expression of the 59 and 67 kd keratins which are characteristically expressed in the differentiated layers of the epidermis. Therefore, this study shows that squamous metaplasia of tracheas due to vitamin A-free cultivation is accompanied by an increase in keratin synthesis as well as by the appearance of keratin species not normally present in mucociliary tracheal epithelium.     

Structural features and sites of expression of a new murine 65 kD and 48 kD hair-related keratin pair, associated with a special type of parakeratotic epithelial differentiation

In the course of studies on local keratin phenotypes in the epidermis of the adult mouse, we have identified a new 65 kD and 48 kD keratin pair. In mouse skin, this keratin pair is only expressed in suprabasal cells of adult mouse tail scale epidermis which is characterized by the complete absence of a granular layer and the formation of a remarkably compact stratum corneum. A second site in which the 65 kD and 48 kD keratin pair is suprabasally expressed and whose morphology corresponds to that of tail scale epidermis is found in the posterior unit of the complex filiform papillae of mouse tongue. The causal relationship of the expression of the 65 kD and 48 kD keratins with this particular type of a non-pathological epithelial parakeratosis is emphasized by the suppression of the mRNA synthesis of the two keratins during retinoic acid mediated orthokeratotic conversion of tail scale epidermis. Apart from tail scale epidermis and the posterior unit of the filiform papillae, the 65 kD and 48 kD keratin pair is, however, also coexpressed with "hard" alpha keratins in suprabulbar cells of hair follicles and in suprabasal cells of the central core unit of the lingual filiform papillae. The non alpha-helical domains of the two new keratins are rich in cysteine and proline residues and lack the typical subdomains into which epithelial keratins of both types can be divided. This structural resemblance of the 65 kD and 48 kD keratins to "hard" alpha keratins is supported by comparative flexibility predictions for their non alpha-helical domains. Phylogenetic investigations then show that the 65 kD and 48 kD keratin pair has evolved together with hair keratins, but has diverged from these during evolution to constitute an independent branch of a pair of hair-related keratins. In view of this exceptional position of the 65 kD and 48 kD keratins within the keratin multigene family, their expression has apparently been adopted by rare anatomical sites in which an orthokeratinized stratum corneum would be too soft and a hard keratinized structure would be too rigid to meet the functional requirement of the respective epithelia.     

Transdermal Delivery of a Tetrapeptide: Evaluation of Passive Diffusion

Skin penetration of the tetrapeptide Ac-Ala-Ala-Pro-Val-NH₂ was assessed. This peptide sequence fits the P-P₁ subsites of elastase and inhibits human neutrophil elastase competitively. Consequently this peptide may be therapeutically useful in a variety of inflammatory disorders, including psoriasis, in which elevated levels of human neutrophil elastase have been reported. Peptide penetration was assessed across whole human skin, whole skin with the stratum corneum removed by tape stripping and epidermis, which had been removed from the dermis by heat separation. The influence of 75 aqueous ethanol as a potential penetration enhancer of the tetrapeptide across epidermis was also assessed. The tetrapeptide did not penetrate whole human skin or epidermis, even under the influence of 75 aqueous ethanol. However, when the stratum corneum was removed tetrapeptide flux of 73.39 μg cm² h⁻¹ was achieved. The study demonstrates that the stratum corneum is the main barrier to tetrapeptide skin penetration and must be overcome if therapeutically relevant amounts of tetrapeptide are to be delivered to the skin.     

Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins

Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co- expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.     

Immunocytochemical observations on the cornification of soft and hard epidermis in the turtle Chrysemys picta

The process of cornification in the shell and non-shelled areas of the epidermis of the turtle Chrysemys picta was analyzed by light and ultrastructural immunohistochemistry for keratins, filaggrin and loricrin. Beta-keratin (hard keratin) was only present in the corneus layer of the plastron and carapace. The use of a beta-keratin antibody, developed against a specific chick scale beta-keratin, demonstrated that avian and reptilian hard keratins share common amino acid sequences. In both, shelled and non-shelled epidermis, acidic alpha keratin (AE1 positive) was limited to tonofilament bundles of the basal and suprabasal layer, while basic keratin (AE3 positive) was present in basal, suprabasal, and less intensely, pre-corneus layers, but tended to disappear in the corneus layer. The AE2 antibody, which in mammalian epidermis recognizes specific keratins of cornification, did not stain turtle shell but only the corneus layer of non-shelled (soft) epidermis. Two and four hours after an injection of tritiated histidine, the labelling was evenly distributed over the whole epidermis of both shelled and non-shelled areas, but was absent from the stratum corneum. In the areas of growth at the margin of the scutes of the shell, the labelling increased in precorneus layers. This suggests that histidine uptake is only related to shell growth and not to the production of a histidine-rich protein involved in keratinization. No filaggrin-like and loricrin-like immunoreactivity was seen in the carapace or plastron epidermis. However, in both proteins, some immunoreactivity was found in the transitional layer and in the lower level of the corneus layer of non-shelled areas. Loricrin- and filaggrin-like labelling was seen in small organelles (0.05-0.3 mum) among keratin bundles, identified with mucous-like granules and vesicular bodies. These organelles, present only in non-shelled epidermis, were more frequent along the border with the corneus layer, and labelling was low to absent in mature keratinocytes. This may be due to epitope masking or degradation. The immunolabelling for filaggrin was seen instead in the extracellular space among mature keratinocytes, over a material previously identified as mucus. The possibility that this labelling identified some epitopes derived from degraded portions of a filaggrin-like molecule is discussed. The present study suggests that proteins with some filaggrin- and loricrin-immunoreactivity are present in alpha-keratinocytes but not in beta-keratin cells of the shell.     

The epidermis and feather follicles of the king penguin (Aptenodytes patagonica) (aves)

The King penguin epidermis has a stratum corneum of flattened solidly keratinized cells without basophilic nuclear remnants. It contains keratin bound substances and is without an underlying stratum granulosum. The insunken feather follicles and the thick phospholipid-rich cornified layer appear to be adaptations for aquatic life.     

Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication

Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2), a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin’s antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials.     

Isolation and characterization of microsatellite markers in pangolins (Mammalia,Pholidota, Manis spp.)

Thirty‐four polymorphic dinucleotide microsatellite loci were developed in the Malayan pangolin Manis javanica. Of the 34 markers, 32 and 18 were also amplified, respectively, in the Chinese pangolin (Manis pentadactyla) and the African tree pangolin (Manis tricuspis). Analysis of 24 Malayan, 12 Chinese and 2 African tree pangolins showed high levels of variability (heterozygosity ranging from 0.321 to 0.708). These are the first available microsatellite markers in Pholidota and will be an invaluable tool for evolutionary and conservation genetic studies in pangolins.     

CELL JUNCTIONS IN AMPHIBIAN SKIN

Cell junctions have been investigated in the amphibian epidermis, a stratified squamous epithelium, and compared to those described previously in simple columnar epithelia of mammalian cavitary organs. In adult frogs and toads, and in larvae approaching metamorphosis, belts of membrane fusion or zonulae occludentes of considerable depth are regularly found between adjoining cells of the outermost layer of the stratum corneum, binding the cells together into a continuous, uninterrupted sheet. Another set of occluding zonules appears in the second cornified layer (when such a layer is present), and a third set usually occurs in the outermost layer of the stratum granulosum. Specialized elements described as "modified" and "composite" desmosomes are encountered along the lateral and basal aspects, respectively, of the cornified cells; ordinary desmosomes and maculae occludentes (i.e., spots of membrane fusion) are found in all other strata. The usual 200 A intercellular gap is generally maintained between the cells of the stratum germinativum at the basal ends of the intercellular spaces. Hence, the intercellular spaces of the epidermis form a largely continuous network, closed to the external medium and open to the dermal interstitia. The situation is comparable to that found in columnar epithelia, except that the intercellular spaces are much more extensive, and an extracellular subcompartment (or two) apparently exists in the stratum corneum and between the latter and the stratum granulosum. The last subcompartment is usually filled with a dense substance, probably derived from discharged secretory granules. The tripartite junctional complex characteristic of lumen-lining epithelia (i.e., a zonula occludens followed by a zonula adhaerens, and desmosome) is seen only in early larvae; in adults and in larvae approaching metamorphosis, the occluding zonule is followed directly by a series of modified desmosomes. Interpreted in the light of current physiological data, these findings suggest that the diffusion of water, ions, and small, water-soluble molecules is impeded along the intercellular spaces of the epidermis by zonulae occludentes while it is facilitated from cell to cell within the epidermis by zonulae and maculae occludentes.     

Femoral glands of the lizard, Crotaphytus collaris

The anatomy of the femoral glands in an iguanid lizard, Crotaphytus collaris collaris (Say), is described. The 48 lizards (including three embryos) from which glands were examined were obtained throughout their season of activity at one locality in Kansas. In animals of both sexes the glands lie in a linear series on the ventral aspect of each thigh. They are composed of branching tubes and tubules of epidermal and dermal origin. The row of femoral pores is the only external manifestation of the glands. Post hatching, the glands of males increase in size and complexity; little onto-genetic change occurs in the glands of females. The relative length of the glands appears to vary seasonally in adult males suggesting variation in their activity. The greatest relative sizes occur in the breeding season. At times a stratum corneum, continuous with the stratum corneum of the skin, occurs in the duct of the gland internal to part of the secretion plug. Formation of the stratum corneum seems to be initiated in the autumn prior to hibernation, and the stratum corneum removes the outer part of the secretion plug in the next ecdysis; meanwhile, production of a new secretion plug is initiated. The anatomy of the femoral glands in Crotaphytus is similar to that of the described glands of other species of lizards.     

Immunocytochemical and electrophoretic distribution of cytokeratins in the regenerating epidermis of the lizard Podarcis muralis

Using immunocytochemistry at light- and electron-microscope levels, we studied the distribution of three monoclonal antibodies (AE1, AE2, AE3) specific for mammalian alpha-keratins in regenerating lizard epidermis. We also characterized the keratins expressed during this process by immunoblotting after electrophoretic separation. The AE1 antibody is localized in the basal and suprabasal layers of prescaling and scaling epidermis. During the first stages of scale neogenesis, the AE1 antibody also marks the differentiating oberhautchen and beta-layer, but it disappears from these layers as they mature. This antibody does not stain the prekeratinized and keratinized outermost layers in the hinge region. The AE2 antibody labels the superficial wound epidermis, prekeratinizing and keratinized beta- and alpha-layers, but not basal and suprabasal cells. The AE3 antibody labels all living and keratinized epidermal layers, although AE3 immunoreactivity decreases and disappears as the beta-layer matures. The ultrastructural study shows that the AE2 and AE3, but not the AE1, antibodies specifically label small electron-dense areas within the beta-layer, suggesting retention of alpha-keratins. In the stages of tail regeneration examined, immunoblotting with the three antibodies used for the immunolocalization gives a pattern similar to that of the normal epidermis, except distally, where the process of scale differentiation begins. In this region, in addition to the keratin forms discovered in the normal and in proximal regenerating epidermis, an intense low molecular weight band at 40-41 kDa, positive to all three antibodies, is clearly detectable. Furthermore, in the distal region AE1 and AE3 antibodies, but not the AE2, recognize a weak band at 77-78 kDa not present in the normal and proximal epidermis. The localization and the possible role of the different keratins in the regenerating epidermis is discussed.     

Surfactant treatments influence drying mechanics in human stratum corneum

We describe a high-throughput method capable of quantifying the elastic modulus and drying stress of ex vivo samples of human stratum corneum. Spatially resolved drying deformations in circular tissue samples are measured, azimuthally averaged and fitted with a profile based on a linear elastic model. Our approach enables the comparison of the physical effects of different cleansers. We find that cleansing can cause dramatic changes to the mechanical properties of stratum corneum. In some cases, cleansing can lead to an order of magnitude increase in elastic modulus and drying stress. We expect that these mechanical properties have a direct impact on cracking and chapping skin as well as the milder sensation of perceived tightness often experienced after washing. Mechanical drying studies are also combined with drop wetting studies and pyranine staining experiments. This combination of techniques allows one to establish a multidimensional profile of stratum corneum including stiffness, susceptibility to drying, hydrophilicity and barrier functionality.     

Investigation of dorsal/ventral skin and the parotoid region of Lyciasalamandra billae and Lyciasalamandra luschani basoglui (Urodela: Salamandridae)

In the present study, the parotoid region, dorsal and ventral integuments of Lyciasalamandra billae and Lyciasalamandra luschani basoglui were investigated in terms of localization of hyaluronic acid (HA) and histochemical characteristics. HA immunoreactivity was carried out using biotinylated hyaluronic acid binding protein (B-HABP) labelled with streptavidin-fluorescein isothiocyanate (FITC). HA was mainly localized in the stratum spongiosum of L. billae and L. luschani basoglui for water homeostasis and skin functionality. Light microscopic observations revealed that the dorsal and ventral integuments of L. billae and L. luschani basoglui exhibited basic morphological characteristics of other amphibians: the epidermis was composed of a stratified squamous epithelium and the dermis subdivided into stratum spongiosum and stratum compactum. Two different types of dermal glands (mucous and granular glands) were identified in the spongious dermis of the dorsal and ventral integuments whereas in the parotoid region, three different types of glands (mucous, granular and parotoid glands) were examined.     

Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.     

Cytokeratin localization in toe pads of the anuran amphibian Philautus annandalii (Boulenger, 1906)

We have examined cytokeratin distribution and their nature in toe pads of the Himalayan tree-frog Philautus annandalii. Toe pads are expanded tips of digits and show modifications of their ventral epidermis for adhesion. The toe pad epidermal cells, being organized into 3–4 rows, possess keratin bundles, especially in surface nanostructures that are involved in adhesion. Immunohistochemical localization using a pan-cytokeratin antibody revealed that cytokeratin immunoreactivity is the strongest in the mid- to basal cell rows of the epidermis, which parallels our previous ultrastructural observation of dense keratin bundles present in this part of the epidermis. The remainder of the epidermis (i.e., the superficial cell layer) showed little immunoreactivity. Immunoblot analysis revealed that toe-pads possessed keratins prominently in the molecular mass of 50 kDa. Possible presence of keratin 5 in toe pad epidermis has been correlated with its usual distribution pattern in mammalian epidermis.