Ultrastructural localization of histidine-labelled interkeratin matrix during keratinization of amphibian epidermis

Interkeratin histidine-rich proteins (filaggrins) play a functional role in aggregation of keratin filaments into dense bundles during terminal differentiation of mammalian keratinocytes when forming the dense matrix of the stratum corneum. The origin of the stratum corneum during adaptation to land in amphibious vertebrate progenitors was probably linked with the synthesis of matrix proteins. However, whether similar proteins are present in living amphibians is unknown. The possible involvement of interkeratin matrix molecules rich in basic amino acids such as histidine during keratinization of amphibian epidermis has been evaluated in the present study by ultrastructural autoradiography after administration of tritiated histidine. At 4 and 8 h post-injection, labelling was mainly localized over electron-dense amorphous material or irregular granules in between keratin filaments in cells of the upper intermedium, replacement, and immature corneous layers. Nuclear material incorporating tritiated histidine was also present in the maturing corneous layer. Small mucous-like granules did not take up tritiated histidine and X-ray microanalysis indicated that the latter granules contained sulphur. The present study suggests that small amounts of histidine-rich molecules which were not sufficient to form microscopically-visible keratohyalin granules were present in ancestral amphibian epidermis. However, this material was sufficient to promote aggregation of keratin filaments in the cytoplasm of amphibian differentiating keratinocytes, especially near the external corneous cell envelope. Electron-dense material associated with the corneous cell envelope also contained sulphur as indicated by X-ray microanalysis. It is unknown whether sulphur is derived from either sulphated mucins, or disulphide bonds in aggregated keratins, or specific sulphur-rich proteins.     

Light and electron microscopical localization of filagrin-like immunoreactivity in normal and regenerating epidermis of the lizard Podarcis muralis

In the stratum granulosum of mammalian epidermis, keratohyalin contains the histidine-rich protein filagrin which determines the aggregation of keratin bundles in terminally differentiating keratinocytes. Among reptiles, the lizard epidermis possesses keratohyalin-like granules during a period preceding molting. Tritiated histidine is taken up at 3-22 h post-injection mainly in keratohyalin-like granules of the clear layer, and less intensely in oberhautchen cells, in pre-keratinizing cells of wound epidermis and in the alpha-layer. No labelling was observed in beta-cells and mesos cells. Single and double immunogold localization of filagrin and keratin, using mammalian antibodies, show that some filagrin-like immunoreactivity is localized in 0.15-0.40 microm diameter roundish keratohyalin-like granules in cells of wound epidermis, and lacunar and clear layers (soft alpha-layers). Pro-filagrin was not immuno-localized in these layers with the mammalian antibodies. The small granules merge with keratin bundles into mature keratinocytes where immunopositive keratin filaments predominate and the filagrin-like immunolabelling rapidly disappears. Little or no labelling is observed in the large keratohyaline-like granules of the clear layer. This may be due to lack of filagrin-like immunoreactivity but may also be due to epitope-masking or chemical degradation of filagrin-like molecules. No immunoreactivity is present in the beta-layer and mesos-layer but the immunolabelling reappears in the maturing alpha-layer and lacunar layer. This study suggests that histidin-rich protein with some filagrin-like immunoreactivity is initially present in those alpha-layers of lizard epidermis where keratohyalin-like granules are present, such as lacunar and clear cells, and that a filagrin-like molecule is degraded or altered in mature keratinocytes.     

Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia

Historically, the term ‘keratin’ stood for all of the proteins extracted from skin modifications, such as horns, claws and hooves. Subsequently, it was realized that this keratin is actually a mixture of keratins, keratin filament‐associated proteins and other proteins, such as enzymes. Keratins were then defined as certain filament‐forming proteins with specific physicochemical properties and extracted from the cornified layer of the epidermis, whereas those filament‐forming proteins that were extracted from the living layers of the epidermis were grouped as ‘prekeratins’ or ‘cytokeratins’. Currently, the term ‘keratin’ covers all intermediate filament‐forming proteins with specific physicochemical properties and produced in any vertebrate epithelia. Similarly, the nomenclature of epithelia as cornified, keratinized or non‐keratinized is based historically on the notion that only the epidermis of skin modifications such as horns, claws and hooves is cornified, that the non‐modified epidermis is a keratinized stratified epithelium, and that all other stratified and non‐stratified epithelia are non‐keratinized epithelia. At this point in time, the concepts of keratins and of keratinized or cornified epithelia need clarification and revision concerning the structure and function of keratin and keratin filaments in various epithelia of different species, as well as of keratin genes and their modifications, in view of recent research, such as the sequencing of keratin proteins and their genes, cell culture, transfection of epithelial cells, immunohistochemistry and immunoblotting. Recently, new functions of keratins and keratin filaments in cell signaling and intracellular vesicle transport have been discovered. It is currently understood that all stratified epithelia are keratinized and that some of these keratinized stratified epithelia cornify by forming a Stratum corneum. The processes of keratinization and cornification in skin modifications are different especially with respect to the keratins that are produced. Future research in keratins will provide a better understanding of the processes of keratinization and cornification of stratified epithelia, including those of skin modifications, of the adaptability of epithelia in general, of skin diseases, and of the changes in structure and function of epithelia in the course of evolution. This review focuses on keratins and keratin filaments in mammalian tissue but keratins in the tissues of some other vertebrates are also considered.     

Immunohistochemical localization of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta)

The present immunohistochemical study provides the first evidence of the presence of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta) a lower invertebrate. The entire epidermis was labelled for calmodulin which is in agreement with its ubiquitous occurrence. Immunopositivity for calbindin D28K was limited to mucous cells, while that for S-100 protein was present only in neuroendocrine-like small granular cells. Finally, labelling for parvalbumin was specifically present in the subcutaneous nerve plexus. S-100 protein is considered to be a marker of neuroendocrine cells, at least in lower invertebrates such as Annelida. Although calbindin D28K is considered to be a marker of these cells in vertebrates, the same function cannot be attributed in Lumbricus terrestris. However, we can conclude that S-100 protein, as a regulatory protein, is phylogenetically older than calbindin D28K. We assume that the latter has an autoregulatory function in secretory processes. In agreement with previous data, we suggest that small granular cells exert a paracrine action in osmoregulatory and secretory processes.     

Immunocytochemical characterization of 10 pancreatic tumours, associated with the glucagonoma syndrome, using antibodies to separate regions of the pro-glucagon molecule and other neuroendocrine markers

Histological diagnosis of neuroendocrine tumours can be hampered by their lack of peptide or amine immunoreactivity. In order to assess the usefulness of a range of specific and general markers of neuroendocrine differentiation, 10 pancreatic endocrine tumours, associated with high levels of circulating glucagon, were studied using histology, histochemistry, immunocytochemistry and electron microscopy. All cases showed immunoreactivity for one or other of the peptides derived from pro-glucagon, although only seven were found to contain immunoreactive pancreatic glucagon. The presence of secretory granules in eight of the tumours was demonstrated by electron microscopy, argyrophilia or chromogranin immunoreactivity. Not only was neuron specific enolase positively immunostained in all the tumours, thereby revealing their neuroendocrine nature, but also the intensity of the immunostain was higher in four of the five malignant ones than in the rest of the cases. Pancreatic polypeptide was present in non-glucagon cells in six out of 10 cases. Our results emphasize the importance of the use, not only of general histochemical and immunocytochemical tests but also antibodies to all possible derivatives of the precursor form of the active tumour product in the diagnosis of possible endocrine tumours. In this way, any abnormal molecular forms of the peptide synthesized by tumour cells with altered synthetic and secretory mechanisms may be detected.     

Immunocytochemical localization of putative cholinergic neurons in the goldfish retina

The presence of putative cholinergic neurons in goldfish retina was demonstrated by immunocytochemical localization of choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Four populations of ChAT-immunoreactive neurons were localized: two with cell bodies in the inner nuclear layer and two with cell bodies in the ganglion cell layer. The processes of these neurons ramified in lamina 2 and/or 4 (of 5) in the inner plexiform layer. These cell populations are comparable to populations of putative cholinergic neurons that have been identified by [3H]choline uptake [3, 10].     

Keratinization and ultrastructure of the epidermis of late embryonic stages in the alligator (Alligator mississippiensis)

Using specific anti-beta keratin and general anti-alpha keratin antibodies, keratins were located in the epidermis of the alligator during the final developmental stages by ultrastructural and immunocytochemical methods. The maturation of the bilayered periderm (= embryonic epidermis) coincides with the disappearance of cell organelles, including the 25-35-nm-thick coarse filaments, and the coalescing of alpha-keratin filaments into a compact mass. The plasmalemma of peridermal cells forms a 15-25-nm-thick electron-dense corneous envelope. These changes start at stage 25, about 3 weeks before hatching, and continue until hatching when the embryonic epidermis is shed. Immature beta-keratogenic cells beneath the embryonic epidermis accumulate immunolabelled beta-filaments which are packed into thin, electron-pale beta-keratogenic cells in the corneous layer. Together, electron-pale and electron-dense materials form a compact 3-4-nm filament pattern of beta-keratin. Melanosomes from epidermal melanocytes, incorporated into beta-cells, give rise to the banded skin pattern of hatchlings. Beta-keratin production is much reduced in the hinge regions, where many alpha-filaments remain packed together with lipid droplets or mucous granules into thinner, more electron-dense, alpha-cells. The keratinaceous material of the alpha-cells is mostly concentrated along the cell membrane, while the lipid/mucous material remains centrally located, as in sebokeratinocytes of the apteric areas of avian skin. Some lipid and mucus is also incorporated into typical beta-cells of the outer scale surface, so that lipids are part of the fully keratinized hard keratin layer of the alligator. Lipids within beta-cells of outer scale surfaces and alpha-cells of the hinge region are probably responsible for limiting water loss and ion movements across the skin. Neither typical mammalian keratohyalin granules nor lepidosaurian keratohyalin-like granules were detected anywhere in alligator epidermis. The combination of anti-beta and anti-alpha keratin antibodies revealed different distributions of beta- and alpha-keratins. In late embryonic stages (25-26 to hatching), beta-keratin occurs only in the upper suprabasal cells, in prekeratinized and keratinized layers, whereas alpha-keratin bundles (tonofilaments) remain only in the lowest layers. The cross-reactivity of the beta-antibody, produced against a chick scale keratin, further shows that avian and crocodilian hard (beta) keratins share common antigenic sites, reflecting a phylogenetic affinity between these taxa.     

Immunocytochemical visualization of taurine: Neuronal localization in the rat cerebellum

A novel technique for immunocytochemical demonstration of taurine (Tau) is presented. Antisera raised against Tau conjugated to protein by glutaraldehyde (GA) react selectively with similar conjugates in model systems and in tissue fixed with GA. In rat cerebellum, Tau-like immunoreactivity is high in the Purkinje cells but low in other cell types, including the stellate cells for which Tau has been proposed as transmitter.     

Immunocytochemical localization of c-erbB-2 protein in transitional cell carcinoma of the urinary bladder

The level of expression and cellular localization of the c-erbB-2 gene product in transitional cell carcinoma of the urinary tract is controversial. Analysis of the c-erbB-2 gene structure and comparison of its expression in the same cells by Southern, Northern and immunoblotting, and by immunocytochemistry minimize the errors of interpretation inherent in one technique. Such a ‘correlative study’ has been performed on tumours from 82 patients, c-erbB-2 gene amplification was detected in 14 per cent of initial tumours and was associated with grade (.P< 0·001). Raised levels of mRNA were seen in those tumours with increased gene copy number and in 13 per cent of the remainder. Immunoblotting detected the expected 185 kD immunoreactive protein and a 155 kD piotein associated with high gene copy number. Immunocytochemistry localized c-erbB-2 immunoreactivity to the cell membrane and cytoplasm, and the latter predominated. Four antibodies to c-erbB-2 (AB-3, 21N, pAb 1, and NCL CB11) were compared on contiguous sections of the same tumour and showed the same pattern of immunoreactivity. Similarly, analyses carried out in three independent laboratories identified the same cellular localization. Membrane and cytoplasmic immunoreactivity was demonstrated in all tumours with gene amplification or increased mRNA levels and in 40 per cent of the remaining tumours. We showed that immunocytochemistry requires careful standardization of techniques and quantitation between different groups. However, despite variations in the intensity of immunoreactivity, the total number of positive cells remained constant. Therefore quantitation must be based on the number of positive cells and, ideally, their immunoreactive content relative to normal and positive tissue controls.     

Immunocytochemical localization of prohormone convertase 1/3 and 2 in gastrointestinal carcinoids

Gastrointestinal carcinoids are derived from the diffuse intestinal endocrine system and may produce amines and many peptides, including serotonin, chromogranin A (CGA), and tachykinins. Most peptide hormones are synthesized as bigger prohormones, which are processed to smaller active hormones by prohormone convertases (PCs). A total of 35 cases of gastrointestinal carcinoids, including gastric, duodenal, small intestinal, appendiceal, and large intestinal carcinoids, were immunocytochemically stained for serotonin, CGA, and PC 1/3 and 2, in order to colocalize CGA and PCs in the carcinoids. All carcinoids were positive for CGA and PCs. Carcinoids that stained strongly for CGA were generally weakly stained for PCs and those weakly staining for CGA were more strongly stained for PCs in the majority of the small and large intestinal tumors. Gastrointestinal carcinoids were positive for CGA and PCs, and the presence of PCs may suggest that the conversion of peptide prohormones to smaller peptide hormones occurs in gastrointestinal carcinoids. PCs immunocytochemistry may be added as a new phenotypic characterization for gastrointestinal carcinoids.     

“Immunocytochemical expression of P53, PTEN,FAS (CD95), P16INK4A and HPV L1 major capsid proteins in ThinPrep cervical samples with squamous intraepithelial lesions”

The aim of this study was to further investigate the immunocytochemical expression of p53, PTEN, Fas, p16, and HPV L1 capsid proteins in cervical smears with low and high grade squamous intraepithelial lesions (LSIL and HSIL, respectively). A total of 92 ThinPrep cervical samples, comprising 11 cases of HSIL, 61 cases of LSIL, and 20 negative cases were studied by immunocytochemical methods. The results obtained in LSIL cases were correlated with the available follow up data. Abnormal p53, PTEN, or Fas expression was found in a subset of HSIL cases, while positive expression for p16 was significantly associated with the diagnosis of HSIL (P < 0.0001, P = 0.001, P < 0.0001, and P < 0.0001, respectively). Among cases positive for p16 expression, the staining pattern was weak in 88.9% of LSIL cases and strong in 80% of HSIL cases (P < 0.0001). The p16 negative/L1 positive and p16 positive/L1 negative staining patterns were significantly associated with the presence of LSIL and HSIL, respectively (P < 0.0001). None of these markers had a significant prognostic value in LSIL cases (P > 0.05). Our results suggest that loss of PTEN or Fas expression and p53 overexpression may be involved in the process of neoplastic transformation of the cervical epithelium. Furthermore, negative or weak immunocytochemical staining for p16 in a Pap smear may strongly argue against the presence of a high grade lesion, while the combined p16/L1 staining pattern may be useful as a diagnostic adjunct for differentiating between LSIL and HSIL. Diagn. Cytopathol. 2014;42:465–475. © 2013 Wiley Periodicals, Inc.     

Immunocytochemical observations on pineal organ and retina of the Antarctic teleosts Pagothenia borchgrevinki and Trematomus bernacchii

In spite of the unique conditions they have to operate under, the pineal organs of Antarctic fishes have not previously been examined. We determined immunohistochemically that in the end-vesicles and the pineal stalks of Pagothenia borchgrevinki (a species found directly beneath the sea-ice) as well as Trematomus bernacchii (a species preferring somewhat deeper water than the former) at least two populations of physiologically-different cells occurred that displayed reactions indicative of typical vertebrate photoreceptors. Comparisons with immunocytochemically treated retinal sections from the eyes of the same two species showed that anti-opsin reactivity, characteristic of rods, was particularly strong in the lumina of the pineal stalks of both species. Anti-visinin reactions stained cones in the retinal sections of both fishes and occurred throughout the pineal organs, but in particular in the end vesicles of the pineals of both species. The difference in preferred habitat depth between the two species appears to have had very little influence on both retinal and pineal immunocytochemistry. It is concluded that the pineal organs of both species, at least during the austral summer, exhibit signs of being directly photo-sensitive.     

Ultrastructural localization of hair keratins,high sulfur keratin-associated proteins and sulfhydryl oxidase in the human hair

Hardening of the human hair shaft during cornification results from the bonding of keratins and keratin-associated proteins. In situ hybridization and light immunocytochemical studies have shown the general distribution of different keratins and some associated proteins but not determined their ultrastructural localization. I report here the localization of hair keratins, two high-sulfur keratin-associated proteins and sulfhydryl oxidase has been studied under the transmission electron microscope in the cornification zone of the human hair. The ultrastructural study on keratin distribution in general confirms previous light microscopic studies. Sulfur-rich KAP1 is mainly cortical but the labeling disappears in fully cornified cortical cells while a diffuse labeling is also present in differentiating cuticle cells. Sulfur-rich K26 immunolocalization is only detected in the exocuticle and endocuticle. Sparse labeling for sulfhydryl oxidase occurs in differentiating cortical cells but is weak and uneven in cuticle cells and absent in medulla and inner root sheath. Labeling disappears in the upper fully cornified cortex and cuticle. The observations indicate that sulfhydryl oxidase and keratin associated proteins are initially produced in the cytoplasm among keratin bundles accumulating in cortical and cuticle cells but these proteins undergo changes during the following cornification that alter the epitopes tagged by the antibodies.     

Immunocytochemical localization of S-100 protein in interstitial cells of the monkey Macaca irus pineal gland

Pineal interstitial cells of the monkey Macaca irus were shown to react with an anti-human S-100 protein antibody, using the indirect immunoperoxidase technique on sections of paraplast-embedded pineal glands. Immunoreactivity was seen in the cytoplasm of the cells, stellate in shape and intermingled with pinealocytes; the latter did not stain with the antiserum against S-100 protein. Immunoreactivity was also present in the nuclei, as was reported in various other cell types immunostained with anti-S-100 protein antibodies. The present results support the view that interstitial cells of the monkey Macaca irus pineal gland may be of astroglial origin.     

Immunocytochemical localization of neuron specific enolase in small cell carcinomas and carcinoid tumours of the lung

Carcinoid tumours and small cell carcinomas of the lung share many characteristics with normal neuroendocrine cells. While carcinoid tumours contain many dense-cored neurosecretory granules and are frequently argyrophil, small cell carcinomas are poorly granulated and rarely argyrophil, which casts doubt on their neuroendocrine nature. Immunostaining of the enzyme neuron specific enolase (NSE) was recently used to demonstrate the neuroendocrine components of the lung including nerves and neuroendocrine cells. We therefore used NSE immunostaining to investigate neuroendocrine differentiation in 79 lung tumours, including 18 bronchial carcinoids and 31 small cell carcinomas, and compared these results with those obtained with silver stains. Thirteen of the 18 carcinoids were reactive to silver, all other types being negative. NSE-immunoreactivity occurred in 16 carcinoids and 18 small cell carcinomas. None of the squamous cell carcinomas, large cell anaplastic carcinomas and adenocarcinomas examined showed NSE-immunoreactivity. Radioimmunoassay of extractable NSE from 10 fresh lung tumours correlated well with the immunostaining results, demonstrating large amounts in two small cell carcinomas (334 and 517 ng/mg protein) and three carcinoids (152, 908, and 1143 ng/mg protein). Values were much lower for four squamous cell carcinomas (31-44 ng/mg protein) and one large cell anaplastic carcinoma (30 ng/mg protein) and were accounted for by the presence of NSE-positive nerves and neuroendocrine cells in the surrounding lung. NSE immunostaining is a useful marker of neuroendocrine differentiation in lung tumours and should prove particularly valuable in the diagnosis of small cell anaplastic tumours and their metastases.     

Mathematics of non-steady state uptake and release of Na in a frog skin epidermis model.

This study concerns itself with computation of non-steady state flow of tracer Na¹ ions into and out of a multicompartment model membrane. Loading of the membrane from one side (outside) to steady state levels is followed by washout in the opposite (inside) direction. The structure of the model resembles frog skin epidermis which is frequently used in the laboratory to study the flow of Na¹ across a complex biological tissue membrane. The computations yield complete kinetic equations for loading and washout in all individual compartments as well as in the total of membrane compartments. The results are used to critically evaluate the conclusions drawn from laboratory work in regard to the number of compartments, and the pathways of flows of Na⁺ involved in skin epidermis.