Altered Mitochondrial Anatomy of Trigeminal Ganglia Neurons in Diabetes.

Neurons from sensory ganglia are exposed to oxidative attack in diabetes. Altered mitochondrial morphologies are due to impaired dynamics (fusion, fission) and to cristae remodeling. This study aimed to evaluate using transmission electron microscopy mitochondrial changes in diabetic trigeminal ganglia suggestive for ignition of apoptosis, in absence of "classical" morphological signs of apoptosis. We used samples of trigeminal ganglia (from six type 2 diabetes human donors and five streptozotocin (STZ)-induced diabetic rats). In human diabetic samples we found three main distributions of mitochondria: (a) small "dark" normal mitochondria, seemingly resulted from fission processes; (b) small "dark" damaged mitochondria, with side-vesiculations (single- and double-coated), large matrix vesicles and cytosolic leakage of reactive species, mixed with larger "light" mitochondria, swollen, and with crystolysis; (c) prevailing "light" mitochondria. In STZ-treated rats a type (c) distribution prevailed, except for nociceptive neurons where we found a different distribution: large and giant mitochondria, suggestive for impaired mitochondrial fission, mitochondrial fenestrations, matrix vesicles interconnected by lamellar cristae, and mitochondrial leakage into the cytosol. Thus, the ultrastructural pattern of mitochondria damage in diabetic samples of sensory neurons may provide clues on the initiation of intrinsic apoptosis, even if the classical morphological signs of apoptosis are not present. Further studies, combining use of biochemical and ultrastructural techniques, may allow a better quantification of the degree in which mitochondrial damage, with membrane alterations and cytosolic leaks, may be used as morphological signs suggesting the point-of-no return for apoptosis. Anat Rec, 299:1561-1570, 2016. © 2016 Wiley Periodicals, Inc.

Accurate Diagnosis of Sigmoid Colon Endometriosis by Immunohistochemistry and Transmission Electron Microscopy - A Case Report.

Endometriosis is described as the presence of functioning endometrial tissue at sites outside the uterus. Up to 15% ofwomen in their reproductive period are affected by this condition. Endometriosis is mostly foundon the uterosacral ligaments, inside the rectovaginalseptum or vagina, in the rectosigmoid area, ovarianfossa, pelvic peritoneum, ureters, and bladder, causinga distortion of the pelvic anatomy. Colonic involvement is rare but is usually found at the level of the rectum or the sigmoid colon. Acute presentation with intestinal obstruction or perforation is rare. While malignant transformation of endometrial lesions is rare, findings of dysplasia on pathology sections can give rise to questions of management. Immunohistochemistry and electron microscopy can help decision making. We present the case of a 38 year old woman with intestinal obstruction caused by sigmoid colon endometriosis with moderate dysplasia in which transmission electron microscopy was used for postoperative diagnosis. Detailed analysis of these cases, while logistically difficult, can prove useful in understanding the etiology and pathophysiology of the disease.

Infrastructure of the telocytes from tumor stroma in the skin basal and squamous cell carcinomas.

In this paper, we focus our interest on the ultrastructure of telocytes (TCs) present inside of tumor-stroma in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Tumor-stroma cooperation is necessary for tumor growth, invasive behavior and ectopic development of microtumors. There is a plethora of reports about the role of different stromal cell types in tumor evolution in the human body. In this line, almost nothing is known about the recently identified interstitial cell type called telocyte (TC). To our best knowledge, this is the first study to publish TCs in malignant tumors, namely BCC and SCC. Here, we described the infrastructural aspects of TCs as well as their relationships with other tumor stroma components. TC from the tumor stroma has cell body where the nucleus is located and exhibits two (rarely more) very long cell extensions of tens (over 60-100 µm) termed telopodes. A telopode appears as an alternation of very thin segments called podomers and dilated segments called podomes, which accommodate mitochondria, rough endoplasmic reticulum, cytoskeleton, caveolae, as well as coated vesicles. TCs establish homocellular junctions leading to a 3-D network inside of peritumoral stroma. TCs may play an important role in intercellular signaling via stromal synapses and shed microvesicle transfer. Comparative evaluation with normal dermal skin showed that telocytes from tumor stroma have a very restraint number of heterocellular junctions. The limitation of TCs heterocellular junctions suggests a possible involvement in induction of cell-cell communication alterations into the peritumoral stroma and, consequently, into the whole tumor mass.

Skin telocytes.

A distinctive stromal cell-type, the telocyte (TC), has recently been described to send specific long prolongations (telopodes) alternating thin segments (podomers) with dilations (podoms). Even though one would expect TCs to be identified in various stromal tissues, there were not yet reported evidence of skin TCs. We aimed to check for the presence of TCs in human skin dermis. Transmission electron microscopy revealed the presence in dermis of TCs projecting specific telopodes. Skin TCs were closely related to or contacting fibroblasts, mast cells, adipocytes, and connective fiber bundles (collagenous and elastic). As it appears, skin TCs exist and are related to other stromal cells. The structural association of TCs to elastic fibers deserves further investigation.

Tracheal telocytes.

Recently, a novel type of stromal cell - the telocytes (TC) - was identified in mouse trachea. These cells are known to possess the ultrastructural characteristics, which support their role in intercellular signaling. We found TC in all stromal compartments of the tracheal wall. TC with long prolongations (telopodes, Tp) were lining longitudinally the collagen bundles, and were serially arranged (end-to-end connections of Tp were found). Noteworthy, Tp frequently establish stromal synapses with mast cells (MC). Primary cilia were also identified in TC. In conclusion, tracheal TC could be involved in the tracheal regulation (e.g. secretion, contractility). The tandem TC-MC deserves further investigations.

The human trigeminal ganglion: c-kit positive neurons and interstitial cells.

OBJECTIVES: The presence of c-kit positive neurons in sensory ganglia has been verified in various species but not in humans. Our aim has been to identify whether human primary trigeminal neurons label with c-kit/CD117 and thus, whether data gathered in animal studies can be extrapolated to humans. We also intended to establish whether, and which non-neuronal cells also label with c-kit in the trigeminal ganglion. METHODS: Human adult trigeminal ganglia from eight cadavers were processed for immunohistochemistry on paraffin embedded samples using monoclonal antibodies for CD117/c-kit, and three additional trigeminal ganglia were used for transmission electron microscopy (TEM). To evaluate which neuronal type (A or B) was labeled with c-kit, we evaluated the same neurons on adjacent sections labeled with antibodies for neurofilaments (NF). RESULTS: c-kit has labeled trigeminal neurons (TNs), mast cells and interstitial cells (ICs) within the trigeminal ganglion. c-kit+TNs were NF-and thus were strongly presumed to be nociceptive, as such neurons are known to be NF-poor. c-kit+ICs with long and moniliform processes intermingled with the satellite glial cells (SGCs) of the neuronal envelopes. TEM evaluations confirmed this mixed composition of the neuronal envelopes and demonstrated that the perineuronal ICs are in fact interstitial Cajal-like cells (ICLCs) and/or telocytes. CONCLUSIONS: c-kit+TNs were objectified in humans and strongly presumed to be nociceptive. TNs envelopes mostly consist of SGCs, but are also combined with ICLCs/telocytes.

Defects of basement membrane and hemidesmosome structure correlate with malignant phenotype and stromal interactions in HaCaT-Ras xenografts.

Benign and malignant HaCaT-ras clones, derived from immortalized HaCaT cells were grown as nude mouse surface transplants rendering a human tumor progression model. Searching for malignancy-related alterations, the deposition, localization and mRNA of basement membrane and hemidesmosome components were analysed by immunofluorescence, in situ hybridization and electron microscopy. Initially, at 1 week epithelia of benign and malignant cells revealed a similarly low polarity and an enlarged 'activated basal' compartment, reflected by partial dislocation and extended pericellular staining of the hemidesmosome constituent integrin alpha 6 beta 4 seen by immunofluorescence. Whereas benign grafts eventually normalized, closely resembling grafts of HaCaT cells, malignant growth was correlated with a persisting epithelial activation state and continuing higher expression of alpha 6 (by immunofluorescence and in situ hybridization). The basement membrane components bullous pemphigoid antigen 1, laminin-5 and collagen IV exhibited a largely linear distribution at 1 week. However, in the malignant cell transplants initially minor basement membrane discontinuities became more severe at around 2 weeks, associated with close stromal cell contacts, angiogenesis and invasion. Most striking were basement membrane alterations seen by electron microscopy. At 1 week stretches of basement membrane had developed in malignant transplants, though to a much lesser extent than in benign specimens. With invasion these basement membrane structures mostly disappeared despite persistent although variable immunofluorescence, suggesting high turnover without ultrastructural assembly. The hemidesmosome structures were defective throughout, completely lacking anchoring plaques with keratin filaments, whereas they were still associated with basement membrane deposits. Thus, malignant HaCaT-ras transplants, while initially resembling regenerating wounds, revealed an increasing loss of tissue polarity and basement membrane structures, which seemed to be accelerated upon stromal cell contacts.

Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation.

Skin equivalents formed by keratinocytes cocultured with fibroblasts embedded in collagen lattices represent promising tools for mechanistic studies of skin physiology, for pharmacotoxicologic testing, and for the use as skin substitutes in wound treatment. Such cultures would be superior in defined media to avoid interference with components of serum or tissue extracts. Here we demonstrate that a defined medium (supplemented keratinocyte defined medium) supports epidermal morphogenesis in organotypic cocultures equally well as serum-containing medium (mixture of Ham's F12 and Dulbecco's modified Eagle's medium), as documented by hallmarks of the epidermal phenotype studied by immunofluorescence and electron microscopy. In both cases regularly structured, orthokeratinized epithelia evolved with similar kinetics. Morphology in mixture of Ham's F12 and Dulbecco's modified Eagle's medium was slightly hyperplastic, and keratins 1 and 10 synthesis less co-ordinated than in supplemented keratinocyte defined medium, but a consistently inverted sequence of expression of keratins 1 and 10 was found in either medium. The late differentiation markers filaggrin, involucrin, keratin 2e, and transglutaminase 1 corresponded in their typical distribution in upper suprabasal layers. Keratin 16 persisted under both conditions indicating the activated epidermal state. Keratinocyte proliferation was comparable in both media, whereas fibroblast multiplication and proliferation was delayed and reduced in supplemented keratinocyte defined medium. In both media, ultrastructural features of epidermal differentiation as well as reconstitution of a basement membrane occurred similarly. Immature lamellar bodies and cytoplasmatic vacuoles, however, indicated an impaired lipid metabolism in supplemented keratinocyte defined medium. Nevertheless, these defined organotypic cocultures provide a suitable basis for in vitro skin models to study molecular mechanisms of tissue homeostasis and for use in pharmacotoxicologic testing.

Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts.

The immortal human keratinocyte line HaCaT is frequently used as a paradigm for skin keratinocytes in vitro because of its highly preserved differentiation capacity. HaCaT cells form a nearly regular epidermal architecture when transplanted onto subcutaneous tissue of athymic mice. In order to analyze further their differentiation capacity in vitro, HaCaT cells were studied in organotypic cocultures on top of collagen gels containing human dermal fibroblasts. Within 1 wk HaCaT cells formed a still dysplastic epithelium, the thickness of which correlated with the number of fibroblasts in the collagen gel. With further culture time of up to 3 wk a remarkably well structured and differentiated squamous epithelium developed. After 1 wk, keratins 10 and 16, involucrin, and transglutaminase I were expressed in suprabasal layers, whereas filaggrin, keratin 2e, and loricrin appeared after 2-3 wk. Within this time, a nearly complete basement membrane had formed including hemidesmosomes and anchoring fibrils. Epithelial cell proliferation became restricted to the basal layer after 2 and 3 wk. Using the TdT-mediated dUTP nick end labeling assay, fragmentation of DNA was detectable in nuclei of the parakeratotic stratum corneum. Ultrastructurally, many features of keratinization accumulated after 2 and 3 wk, though an orthokeratotic keratinization was not achieved, in contrast to HaCaT transplants. This differentiation deficiency - as compared with normal keratinocytes -- might be due to a lack of paracrine factors important for keratinocyte differentiation or to a reduced sensitivity of these cells. Nevertheless, this high degree of differentiation under organotypic conditions qualifies this cell line as an appropriate model for elucidation of the molecular mechanisms regulating keratinocyte growth and differentiation and for use in pharmacotoxicology.

Epidermal differentiation and basement membrane formation by HaCaT cells in surface transplants.

The immortal human keratinocyte line HaCaT has been employed in many studies as paradigm for epidermal keratinocytes. In order to demonstrate its potential to form stable epidermal structures in response to connective tissue, this was challenged in surface transplants on nude mice, where normal keratinocytes rebuild a typical epidermis within two weeks. During the initial regeneration phase (day 1-4) multilayered but poorly organized epithelia formed with proliferating cells in all layers in analogy to normal keratinocytes. Similarly, with tissue consolidation (around day 7) proliferation was reduced and restricted to cells in basal position marked by keratin K14 and beta1-integrin immunostaining. The strong suprabasal reaction for K1 and K10, the appearance of the late markers K2e, filaggrin and loricrin as well as the polarized distribution of alpha2beta1 and alpha3beta1 indicated advancing tissue normalization (day 14). Keratinization further improved at around three weeks switching from the initial parakeratotic to the regular orthokeratotic type which was prominent at six weeks. Accordingly, most ultrastructural features typical for epidermis or normal keratinocyte grafts were detectable including a complete basement membrane (BM) with regular attachment structures. Matrix- and BM-components appeared sequentially with marked linear deposition of laminin-5 (day 4) followed by accumulation of collagen-IV and 'classical' BM-laminin between one and two weeks. With the general codistribution of integrin alpha6beta4 and BM-molecules (day 14) collagen-VII lining of BM became prominent, while epithelium and host connective tissue were still separated by the collagen matrix. In accordance with the delayed orthokeratinization, wound-matrix molecules (fibronectin, tenascin) persisted longer than in normal keratinocyte transplants. Finally, grafts of long-term passaged (no. 310) cells demonstrated a remarkable stability in the expression of epidermal markers. Thus, the immortalized HaCaT cells reveal a generally high competence to realize an epidermal phenotype in a natural environment and appear therefore qualified for in vitro studies on structural and regulatory aspects of keratinocyte physiology and pathology.

Dynamics of basement membrane formation by keratinocyte-fibroblast interactions in organotypic skin culture.

The cutaneous basement membrane zone, composed of numerous macromolecules, plays a multifunctional role in tissue regeneration and maintenance. To elucidate the cellular origin and dynamics of basement membrane formation, de novo synthesis, deposition, and ultrastructural assembly of its components were analyzed in organotypic cultures of adult skin keratinocytes on collagen gels with or without collagen-embedded dermal cells. Collagen IV and laminin-1 deposition occurred only in the presence of mesenchymal cells: patchy at day 4 and continuous after 1 week. Chain-specific mRNA expression started at day 2 in both keratinocytes and fibroblasts. It steadily increased up to day 10, however, with a reciprocal induction pattern, mRNA abundance shifting from keratinocytes to fibroblasts. On the other hand, laminin-5 staining was first observed at day 4, but in keratinocyte both mono- and cocultures. This was followed by nidogen, which was detected in cocultures but also in dermal monocultures. Laminin-5 protein persisted throughout day 21, whereas nidogen steadily increased in intensity. Expression kinetics revealed high levels of laminin-5 transcripts early and in keratinocytes only, whereas nidogen was expressed later and predominantly in fibroblasts. Although basement membrane protein deposition was continuous at day 14, the ultrastructural organization was still fragmentary, eventually normalizing at 3 weeks. These data demonstrate a dynamic interaction and cooperation of epithelial and mesenchymal skin cells in basement membrane formation. This interaction is supposedly mediated via diffusible factors. Our findings further extend the scope of epithelial-mesenchymal interactions stressing that both cell compartments are essential to constitute a tissue-specific extracellular matrix structure.

Integrin and basement membrane normalization in mouse grafts of human keratinocytes--implications for epidermal homeostasis.

Integrin patterns and formation of basement membrane (BM) were investigated in correlation to epidermal growth and differentiation during skin regeneration in human keratinocyte transplants on nude mice. Immuno-fluorescence and transmission electron microscopy (TEM) showed that different stages of tissue reconstruction were characterized by a sequence of coordinated events. Features of the initial tissue activation, with rapid keratinocyte proliferation around day 4, including cells in a suprabasal position, were: (1) a marked increase in and extended distribution of the integrin chains alpha 2, alpha 3, beta 1 and alpha 6, while beta 4 already showed a preferential basal location; (2) de novo expression of alpha 5 and alpha v; and (3) marked deposition of laminin-5 and nidogen but low levels of other BM components. Tissue normalization during the 2nd week, initiated by a drastic decrease in the number of proliferating cells after day 4, now strictly in basal position, was signified: by (1) orthotopic staining for basal-type keratins (K5, K14) together with a regular pericellular alpha 2 beta 1 and alpha 3 beta 1 distribution, (2) linear, balanced deposition of BM components (e.g. laminin-1, type IV collagen) and (3) colocalization of integrin alpha 6 beta 4 and bullous pemphigoid antigen. Simultaneously at 7 days hemidesmosomes and a defined BM had developed (TEM), becoming continuous at 14 days. This coincided with the regular distribution of suprabasal keratins (K1, K10) as well as intermediate (involucrin) and late differentiation markers (filaggrin, loricrin). Type-VII collagen deposition, still irregular at 14 days, became continuous at 22 days together with developing BM-anchoring fibrils indicating final tissue consolidation. This model mimics principal stages of epidermal wound healing in human skin and implies a linkage between BM assembly, integrin distribution and the compartment of proliferation competent cells, which in turn determines the onset of differentiation. Thus, apart from the balance of diffusible growth regulators, this positional control of keratinocytes, largely accomplished by integrin-matrix interactions, seems to be prerequisite to establishment and maintenance of tissue homeostasis.

Cytodifferentiation and dynamics of intercellular relations during early ontogenesis.

The paper presents ultrastructural aspects related to zygotes and embryo cytodifferentiation of Sus scrofa d., used in the biotechnology of embryo transfer, from the first division of segmentation to the stage of preimplantational blastocyst. Together with the intense cellular proliferations, the morphostructural and functional differentiation of cells takes place. First, the blastomeres are similar from an ultrastructural point of view and do not present intercellular junctions to the advanced stage of morula. Gradually, the polarity of the cellular surface is more evident (microvilli occupy only certain domains of the cellular surface and junctions of intercellular solidarity of desmosomal type occur). Ultrastructural modifications of nuclei, mitochondria and cytoskeleton are noticed. Embryoblastic cells as well as those to form the trophoblast are morphostructurally and positionally differentiated. In the stage of preimplantational blastocyst, the nuclei of some embryoblastic cells have a characteristic form of a callotte or biconcave lens with passages of nuclear material to the cytoplasm.

The action of the collagen-Fe2+ complex at tissular level.

The aim of this paper is to know the iron dynamics and action of the collagen-Fe2+ complex at tissular level. The results showed that the collagen-Fe2+ complex is biocompatible at tissular level and the ferrous iron entered the animal organism on the well-known metabolic pathways. When a high dose of the complex was administered, an overloading with hemosiderin of macrophages and hepatocytes was noticed.

Penetration and proliferation of BHK and HeLa cells into a three-dimensional collagen matrix of bone stroma type. Preliminary results.

Some preliminary data regarding the test of penetration and proliferation of BHK and HeLa cells biocompatibility to a three dimensional stroma saturated with phosphorous and calcium salts (a biomaterial replacing bone) are presented. These data suggest that the analysed biomaterial might be used in surgical practices of prosthesis appliance on interfaces of fractured bone.

Particular ultrastructural aspects in some splenectomized cases of hairy cell leukemia.

Some peculiar ultrastructural aspects of hairy cells obtained from the examination with SEM and TEM are presented. Images of erythrocyte rosette-formation around hairy cells in spleen as well as some additional data on the biogenesis of ribosome-lamellae complexes are reported. Some considerations on the origin of hairy cells are added.