Immunohistochemical Localization of Cytokeratins in the Junctional Region of Ectoderm and Endoderm

Although tridermic species have two junctional regions of ectoderm and endoderm between their epidermis and digestive tract, we actually know little about these particular boundaries. Cytokeratins are the major intermediate filaments of epithelial cells and show a high degree of tissue specificity. Therefore, to characterize the epithelial cells in the junctional region of ectoderm and endoderm, we immunohistochemically examined the localization of cytokeratins 5, 7/17, 14, 18, Sox17, and alpha‐fetoprotein (AFP) in the oropharyngeal and anorectal regions during the mouse gastrulation process. At embryonic day (E) 9.5, cytokeratins 5, 7/17, 14, and 18 were detected in all epithelial cells of the oropharyngeal region. At E12.5, cytokeratin 5‐positive cells were not observed in the middle area of the oral cavity; however, the immunoreactivity was strong in the anterior and posterior areas. The immunoreaction of cytokeratins 18 was seen only in the middle and posterior areas of the oral mucosa. Cytokeratins 7/17 and 14 were localized in all areas of the oropharyngeal region. Sox17 and AFP, which are endodermal markers, were detected in the middle and posterior areas of the oral mucosa, but not in the anterior area. Moreover, this same localization pattern of cytokeratins also existed in the anorectal region of the E12.5 embryo, suggesting that the localization of cytokeratins and endodermal markers might give an implication for the boundary between ectoderm and endoderm. These results also suggest that these cytokeratins are useful molecules for monitoring the epithelial cell differentiation in the junctional region of the germ layers. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Ultrastructure of intramural ganglia in the striated muscle portions of the guinea pig oesophagus

The ultrastructure of the myenteric plexus located in the striated muscle portion of the guinea pig oesophagus was examined and compared with that of the plexus associated with the smooth muscle portion of the rest of the digestive tract. The oesophageal ganglia had essentially the same architecture as those of the smooth muscle portion, such as a compact neuropil without the intervention of connective tissue and blood vessels. Some features, however, were particular to the striated muscle part of the oesophagus. It was clearly demonstrated that myelinated fibres, probably sensory terminals of vagal origin, join the myenteric ganglia. Synapses and terminal varicosities are sparsely distributed within the ganglia and fewer morphological types of axon varicosities could be distinguished compared with other regions. Glial cells are well developed in the oesophageal myenteric ganglia. These cells outnumber the ganglion cells, having a higher ratio than in the lower digestive tract, and form numerous cytoplasmic lamellar processes. The lamellar processes, located at the surface of the ganglia, considerably reduce the area of neuronal membrane which directly contacts the basal lamina. The role of these lamellar processes in the oesophageal ganglia is discussed.     

Skin antigens in the steady state are trafficked to regional lymph nodes by transforming growth factor-beta1-dependent cells

Antigen capturing in the skin and antigen trafficking into regional lymph nodes (LN) initiate immune responses. In this study, employing melanin granule (MG) as an easily traceable antigen in two mouse strains that carried steel factor or hepatocyte growth factor transgenes and had melanocytosis in the epidermis or in the dermis respectively, we investigated the mechanism of antigen trafficking from the skin. MG captured in the epidermis or dermis accumulated in the regional LN, but not other tissues. Only in alymphoplastic mice did MG-laden cells pass through the lymphatics and reached many tissues. Since inflammatory regions were not observed in the skin of either type of transgenic mouse, our developmental system enables us to investigate constitutive capturing and trafficking of insoluble antigens in the steady state. Both dendritic cells and macrophages were laden with MG in the regional LN. To determine which cells traffic antigens to the LN, we prepared double mutants that carried the transgenes and lacked transforming growth factor (TGF)-beta1, since mice lacking TGF-beta1 are reported to be deficient of Langerhans cells. Few MG were observed in the regional LN of these double-mutant mice. We also showed that signaling via macrophage colony stimulating factor receptor or Flt3/Flk2 is not essential for development of the cells for this antigen trafficking. These results indicate that antigens in the epidermis and dermis in the steady state are trafficked into regional LN only by TGF-beta1-dependent cells, which may be a dendritic cell lineage.     

Patterns of cytokeratins and vimentin in guinea pig and mouse eye tissue: evidence for regional variations in intermediate filament expression in limbal epithelium.

The anatomical distribution of different individual cytokeratin polypeptides and of vimentin was investigated by means of immunofluorescence with 41 monoclonal antibodies in guinea pig and mouse eyes. Simple epithelial type cytokeratins 7, 8, 18, and 19 selectively decorated conjunctival goblet cell clusters in mouse specimens and a continuous superficial cell layer of the corresponding part of guinea pig conjunctiva. A changed pattern of squamous epithelial type cytokeratins was found in the limbal region of the guinea pig eye as compared to the corneal epithelium. Cytokertains 3 and 17, which stained the entire corneal epithelium, were not detected, whereas cytokeratin 4, 5 and 13 were expressed. A focal vimentin and cytokeratin coexpression in the limbus of guinea pig is interpreted as indicating corneal stem cells. Similar patterns of expressions were found in the mouse ocular surface. In both species, a cytokeratin 4 staining of basal conjunctival epithelial cells could be detected. The neuroectodermally derived epithelia of the eye such as the retinal pigment epithelium and the ciliary body epithelia expressed solely the cytokeratin pair 8/18.     

Immunohistochemical study of skin lesions in herpes zoster

Summary Thirty-seven biopsy skin tissues of herpes zoster taken from 27 patients were analysed immunohistochemically using two monoclonal antibodies detecting either nucleocapsid or glycoproteins of varicella-zoster virus (VZV) on paraffin sections of formalin fixed tissues. Skin lesions of herpes zoster were divided clinically into four stages: erythematous, vesicular, pustular and ulcerative. In the erythematous stage, VZV antigens, if detected, were found only within ballooning cells in the lower epidermis or follicular epithelium. In the vesicular stage, antigens were detected in the cells around and within the intraepidermal vesicles and in histiocytes or fibrocytes of the dermis in all cases and in the endothelial or perineural cells in 10 of 14 cases. In the pustular stage, the antigens were observed in degenerated or necrotic keratinocytes and multinucleated giant cells within pustules and some necrotic cells in the dermis. In the ulcerative stage, the viral antigens were detected only at the ulcer margin and around the hair shaft in 2 of 7 cases. These results suggest that VZV initially involves the epidermis in the erythematous stage, subsequently invades the dermis in the vesicular stage, and disappears in the early ulcerative stage.     

Perivascular dendritic cells of the human dental pulp

The morphological and phenotypical features of the class II molecule (HLA-DR) expressing cells in human dental pulps were compared with those of previously characterized perivascular dendritic cells in the dermis. We have further investigated how these pulpal cells are structurally related to the vascular system. Double-immunofluorescence staining revealed that a substantial portion of the pulpal HLA-DR expressing cells also expressed factor XIIIa, a marker for dendritic cells. The cells usually had a highly dendritic appearance and formed a reticular network in the pulpal connective tissue. The majority of cells also expressed macrophage-related antigens (CD14 and CD68). A small but distinct population of pulpal cells, representing approximately 13% of the class II molecule expressing cells, was devoid of a typical macrophage phenotype. This subpopulation of pulpal cells may be similar to dendritic cells present in the dermis. Confocal laser scanning microscopy showed that highly dendritic cells, found in close relation to the endothelium, had dendritic processes which were found to be in contact with the peripheral cell membrane of the endothelial cells. These cells formed a three-dimensional structure around the microvessel resembling a cellular conduit. We conclude that the human dental pulp is equipped with class II molecule-expressing perivascular dendritic cells composed of a heterogeneous cell population.     

Immunohistochemical and electron microscopic studies of Langerhans cells in a case of multiple eccrine spiradenomas

Multiple eccrine spiradenomas are rare. In the present study, a detailed investigation of eccrine spiradenoma was performed, focusing in particular on the presence of Langerhans cells (LCs) in the tumor, and their immunohistochemical and ultrastructural characterization. The patient was a woman in her mid-forties who underwent resection of two tumors of the head that were 2.0 and 0.7 cm in size. They were diagnosed as eccrine spiradenoma and were composed of small and large tumor cells with a dense fibrous capsule in the dermis. Immunohistochemically, staining by antibodies to cytokeratins (AE1/AE3, CAM5.2) and CK 5/6 was diffusely positive in all tumor cells, although not in intermingled LCs, which harbored interdigitated nuclei. The cytoplasm of LCs was positive for S-100 protein and CD1a, and their nuclei were also occasionally positive for S-100 protein. Antibody to epithelial membrane antigen was positive for the surface of both intracytoplasmic and true glandular lumina. Fine structural examination revealed the presence of LCs among the tumor cells, extending fine irregular processes among the tumor cells. Birbeck granules were clearly demonstrated in the cytoplasm of LCs. Other fine structural findings included intracytoplasmic lumina with microvilli on their surfaces in some tumor cells. In these examinations of eccrine spiradenoma, LCs, approximately 15/HPF in the tumor, were distinctly detected even at light microscopic level as negative for various types of cytokeratin stains, although they were positive for S-100 protein and CD1a, whereas on ultrastructural examination Birbeck granules were demonstrated in their cytoplasm. Determination of the significance of these LCs in eccrine spiradenoma requires further investigation of a larger number of cases.     

The lamellar cells in human Meissner corpuscles express TrkB

Cutaneous Meissner corpuscles depend for development and survival exclusively on the NT system TrkB/BDNF/NT-4 unlike other types of sensory corpuscles and nerve endings, which have very complex neuronal and growth factor dependence. However, the pattern of expression of TrkB in human Meissner corpuscles is not known. The experiments in these studies were designed to pursue further findings that suggest that BDNF and NT-4 have critical roles in the development and maintenance of Meissner corpuscles by analyzing the pattern of expression of TrkB, their high-affinity receptor, in human glabrous skin. These experiments showed that TrkB is expressed in different patterns by the lamellar cells of Meissner corpuscles and not by the axon. The studies also show that while the percentage of Meissner corpuscles that express TrkB remains constant from birth till 50-year old cases, it decreases ∼3-fold in subjects older than 50 years. These results are important since the study of Meissner corpuscles from cutaneous biopsies to diagnose some neurological diseases has rapidly become of high interest and therefore the proteins expressed in these corpuscles are potential diagnostic tools.     

Dermis isolated adult stem cells for cartilage tissue engineering

Adult stem cells from the dermal layer of skin are an attractive alternative to primary cells for meniscus engineering, as they may be easily obtained and used autologously. Recently, chondroinducible dermis cells from caprine skin have shown promising characteristics for cartilage tissue engineering. In this study, their multilineage differentiation capacity is determined, and methods of expanding and tissue engineering these cells are investigated. It was found that these cells could differentiate along adipogenic, osteogenic, and chondrogenic lineages, allowing them to be termed dermis isolated adult stem cells (DIAS cells). Focusing on cartilage tissue engineering, it was found that passaging these cells in chondrogenic medium and forming them into self-assembled tissue engineered constructs caused upregulation of collagen type II and COMP gene expression. Further investigation showed that applying transforming growth factor β1 (TGF-β1) or bone morphogenetic protein 2 (BMP-2) to DIAS constructs caused increased sulfated glycosaminoglycan content. Additionally, TGF-β1 treatment caused significant increases in compressive properties and construct contraction. In contrast, BMP-2 treatment resulted in the largest constructs, but did not increase compressive properties. These results show that DIAS cells can be easily manipulated for cartilage tissue engineering strategies, and may also be a useful cell source for other mesenchymal tissues.     

Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity

The transformation of mechanical energy into electrical signals is the first step in mechanotransduction in the peripheral sensory nervous system and relies on the presence of mechanically gated ion channels within specialized sensory organs called mechanoreceptors. Piezo2 is a vertebrate stretch‐gated ion channel necessary for mechanosensitive channels in mammalian cells. Functionally, it is related to light touch, which has been detected in murine cutaneous Merkel cell–neurite complexes, Meissner‐like corpuscles and lanceolate nerve endings. To the best of our knowledge, the occurrence of Piezo2 in human cutaneous mechanoreceptors has never been investigated. Here, we used simple and double immunohistochemistry to investigate the occurrence of Piezo2 in human digital glabrous skin. Piezo2 immunoreactivity was detected in approximately 80% of morphologically and immunohistochemically characterized (cytokeratin 20⁺, chromogranin A⁺ and synaptophisin⁺) Merkel cells. Most of them were in close contact with Piezo2⁻ nerve fibre profiles. Moreover, the axon, but not the lamellar cells, of Meissner's corpuscles was also Piezo2⁺, but other mechanoreceptors, i.e. Pacinian or Ruffini's corpuscles, were devoid of immunoreactivity. Piezo2 was also observed in non‐nervous tissue, especially the basal keratinocytes, endothelial cells and sweat glands. The present results demonstrate the occurrence of Piezo2 in cutaneous sensory nerve formations that functionally work as slowly adapting (Merkel cells) and rapidly adapting (Meissner's corpuscles) low‐threshold mechanoreceptors and are related to fine and discriminative touch but not to vibration or hard touch. These data offer additional insight into the molecular basis of mechanosensing in humans.     

Macrophages,but not dendritic cells,present collagen to T cells

Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen-specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen-presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15-mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen α chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony-stimulating factor-expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell-deficient mice could prime a type II collagen-reactive T cell response, thus excluding B cells as obligatory antigen-presenting cells for the priming of collagen-reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen-presenting cells in the immune response towards type II collagen.     

Isolation and characterization of migratory human skin dendritic cells.

A method is described to isolate and characterize human skin dendritic cells (DC). This method is based on the migratory capacities of these cells. The cells migrated 'spontaneously' out of split-skin explants into the medium during a 24-h culture period and contained up to 75% CD1a+ cells. After removal of co-migrated T cells and macrophages, the highly enriched (> 95% CD1a+) DC showed potent allo-antigen-presenting capacities. About 25% of the CD1a+ cells were also positive for the dermal DC marker CD1b, whereas only 15-20% of the cells contained Birbeck granules, the characteristic cell organelle of the epidermal Langerhans cell. Before culture, CD1a+ DC were observed on cryostat sections not only in the epidermis but also in the dermis. After culture, the number of CD1a+ cells in both epidermis and dermis had decreased. Not all the cells had migrated during the culture period; some CD1a+ cells could still be detected in the epidermis and dermis after culture. Thus, using this method, potent allo-stimulating CD1a+ cells, migrating from both epidermis and dermis, can be obtained without the use of enzymes.     

Ultrastructure of Merkel corpuscles and so-called “transitional” cells in the white leghorn chicken

In the chicken Merkel corpuscles are located in the dermis and consist of specialized Merkel cells, discoid nerve endings and lamellar cells. Merkel cells contain characteristic membrane-bound dense-core granules and bundles of microfilaments. Asymmetric junctions, synapse like, with thickened membranes and clusters of dense-core vesicles were observed between the Merkel cells and the nerve endings. The nerve ending is derived from myelinated nerves and sometimes contains clusters of clear vesicles. A laminar system formed by lamellar cells of the Schwann cell type encloses the Merkel cells and the nerve endings. So called “transitional” cells, showing some of the morphological features of both keratinocytes and Merkel cells, were observed in the basal layer of the epidermis. One was located partly in the epidermis and partly in the dermis. The structure of Merkel corpuscles is compared with that of Merkel cells in other tetrapods. The developmental significance of “transitional” cells and the origin of Merkel cells are discussed.     

Spindling artifact of urothelial cells in post-laser treatment urinary cytology

We reviewed 22 post-laser (Nd:YAG laser) coagulation bladder washes collected immediately after treatment. All washes demonstrated a striking artifact of cellular spindling. These spindled cells occurred singly, in loose clusters, and in lamellar stacks and had elongated nuclei with dense chromatin and bipolar cytoplasm that was fused in the stacks. Concurrent biopsies demonstrated similar cytologic changes. The spindling is a nonspecific epithelial response to heat. Conventionally electrocauterized epithelia show this artifact in biopsies, but since only the base of the lesion and surrounding urothelium are subjected to heat with electrocautery, the relatively few spindled epithelial cells created presumably go undetected in cytology specimens. With laser treatment, however, the whole urothelial surface of the lesion is coagulated, producing a much greater number of spindled cells. It is important to avoid misinterpreting the spindled cells as cells from a mesenchymal neoplasm or a sarcomatoid carcinoma, mistakes that were made in some of our initial cases. Malignancy cannot be evaluated when cells exhibit spindling artifact; this judgement should be made on undistorted cells. Thus, pre-laser and post-laser washes should be submitted for evaluation of malignancy.     

Electron microscopic histochemistry of ATPase and alkaline phosphatase activities in mouse digital corpuscles

Summary ATPase and alkaline phosphatase (ALPase) activities were histochemically examined in the sensory corpuscle of mouse digital pads at light and electron microscopic levels. ATPase activity was found on the plasma membranes of the lamellar cells. Moderate activity was present in the caveolae, but little activity could be demonstrated in the other portions of the plasma membrane. Slight ATPase activity was also demonstrated on the plasma membranes of nerve terminals. ALPase activity was found in the caveolae but not on other portions of the plasma membranes of the lamellar cells. ALPase activity was also observed on the nerve terminals. Neither ATPase nor ALPase activity was found on the pre-terminal portions of the nerve fibres.These findings indicate that the caveolae of the lamellar cells may be involved in a transport function and that the plasma membrane of the nerve terminals may have some permeability properties different from those of the pre-terminal portions of nerve fibres.     

Dual parameter flow cytometry for deoxyribonucleic acid and intermediate filament proteins of residual mature teratoma. All tumor cells are aneuploid

Most testicular germ cell tumors of adults are presumably derived from polyploid carcinoma in situ. Thus, one would expect that even highly differentiated teratoma components are aneuploid and that it is unlikely to find diploid tumor cell (sub)populations. We studied 10 residual mature teratomas (RMTs) using a dual parameter flow cytometry procedure. Nuclear DNA was stained with propidium iodide and cytoplasmic intermediate filament proteins, in particular, cytokeratins, with fluorescein isothiocyanate-labeled specific monoclonal antibodies. Cells in RMTs, immunoreactive with antibodies to cytokeratins were considered to be tumor cells. These were always found to be aneuploid, in agreement with the available cytogenetic data on these tumors. The diploid cells present in RMTs were devoid of cytokeratins; therefore, these cells represent the nonmalignant normal host stromal and inflammatory cells. These results, in accordance with our earlier finding, indicate that diploid testicular germ cell tumors are extremely rare in adults, and that even the histologically benign somatic tissues in RMT after polychemotherapy are aneuploid.     

Large mononuclear Ia-positive veiled cells in Peyer''s patches. I. Isolation and characterization in rat, guinea-pig and pig

The antigen presenting cell system in the skin is extensively studied, and is supposed to consist of Langerhans cells in epidermis and dermis, veiled cells in skin lymph and interdigitating cells in skin lymph nodes. In order to detect whether a similar cell system is present in the gut, we studied Peyer's patch cell suspensions. Mononuclear cells with long actively moving cytoplasmic veils were found in cell suspensions from Peyer's patches of rat, guinea-pig, and pig, and in cell suspensions from small intestinal villi of guinea-pig and pig, but not of rat. These veiled cells are strongly Ia-positive. Because of their Ia positivity, their enzyme cytochemical staining pattern, and their ultrastructure, these cells resemble the antigen presenting cells of the skin, skin lymph and lymph node.     

Expression patterns of male germ cell markers in cryptorchid pig testes

Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.     

Immunohistochemical localization of intermediate filament and S-100 proteins in several non-endocrine cells of the human pituitary gland

The presence and distribution of glial fibrillary acidic protein, vimentin, neurofilament protein, cytokeratins No. 8 (52 Kd), No. 18 (45 Kd) and No. 19 (40 Kd) and S-100 protein in pituicytes, folliculo-stellate cells, the epithelium of the Rathke's cysts and squamous cell nests of the pars tuberalis were investigated immunohistochemically by the peroxidase-antiperoxidase (PAP) method in eleven normal human pituitary glands. An identical immunostaining pattern was expressed by both folliculo-stellate cells and pituicytes. In both cell types the immunostaining for glial fibrillary acidic protein (GFAP), S-100 protein and vimentin was strongly positive. These results indicate the probable glial origin of the folliculo-stellate cell, and enlarge the group of glial cell types expressing vimentin. The co-expression of cytokeratins No. 8 and 19, both characteristic for simple epithelia, and S-100 protein was evident in the epithelial cells lining the Rathke's cysts and the squamous cell nests of the pars tuberalis. Furthermore, some epithelial cells of the Rathke's cysts co-expressed cytokeratins, S-100 protein and GFAP, a fact seldom reported and only in relation to rare neoplasms. The cytokeratin No. 18, characteristic for glandular epithelia, was not clearly demonstrated. Finally, the neurofilament protein was detected only in axons of the neurohypophysis; no immunopositive cells could be found throughout the adenohypophysis. Similarities in the antigenic patterns of these cell populations and the possible relation with their origin and nature are discussed.     

Giant cell tumor of the skin: a morphologic and immunohistochemical study of five cases

Giant cell tumor (GCT) of the skin is a rare entity that possesses similar gross and histologic features to GCT of bone. When located predominantly in the dermis GCT has been mistaken for benign fibrous histiocytoma and atypical fibroxanthoma. We report the clinical, morphologic, and immunohistochemical features of five cases of GCT of the skin. With one exception, all tumors are confined to the dermis. Patients' ages range from 6 to 78 years (median, 73 years) with a male to female ratio of 3:2. Gross and histologic features of the lesions are similar to those of GCT of bone (eg, brown fleshy tumor and a biphasic population of mononuclear cells admixed with osteoclast-like giant cells, respectively). The nuclei of the giant cells are similar to those of the mononuclear cells. A fascicular pattern with focal storiform arrangement of spindle neoplastic cells is noted in two cases. The osteoclast-like giant cells and some of the mononuclear cells are strongly positive for CD68, alpha-1-antitrypsin, and alpha-1-antichymotrypsin. Only the mononuclear cells express smooth muscle actin focally in one case. Both the osteoclast-like giant cells and the mononuclear cells are negative for cytokeratins (AE1/AE3 and CAM5.2) and S-100 protein in all cases. One patient developed lung metastases at presentation and local recurrence 4 months status post surgery. All patients are without evidence of disease 1 month to 12 years status post surgery. Cutaneous GCTs are low-grade sarcomas that can recur locally and infrequently metastasize. These tumors should be distinguished from a variety of cutaneous neoplasms that contain multinucleated giant cells.