bromochloroacetic-acid and Cadaver

The appearance and function of human skin are dramatically altered with aging, resulting in higher rates of severe xerosis and other skin complaints. The outermost layer of the epidermis, the stratum corneum (SC), is responsible for the biomechanical barrier function of skin and is also adversely transformed with age. With age the keratin filaments within the corneocytes are prone to crosslinking, the amount of intercellular lipids decreases resulting in fewer lipid bilayers, and the rate of corneocyte turnover decreases.. The effect of these structural changes on the mechanical properties of the SC has not been determined. Here we determine how several aspects of the SC's mechanical properties are dramatically degraded with age.. We performed a range of biomechanical experiments, including micro-tension, bulge, double cantilever beam, and substrate curvature testing on abdominal stratum corneum from cadaveric female donors ranging in age from 29 to 93 years old.. We found that the SC stiffens with age, indicating that the keratin fibers stiffen, similarly to collagen fibers in the dermis. The cellular cohesion also increases with age, a result of the altered intercellular lipid structure. The kinetics of water movement through the SC is also decreased.. Our results indicate that the combination of structural and mechanical property changes that occur with age are quite significant and may contribute to the prevalence of skin disorders among the elderly.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Aging; Biomechanical Phenomena; Cadaver; Elasticity; Epidermis; Female; Humans; Keratins; Kinetics; Lipid Metabolism; Middle Aged; Permeability; Skin Aging; Water

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial (HCE) cells. Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell-attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favour the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

Topics: Animals; Biocompatible Materials; Bombyx; Cadaver; Cell Adhesion; Cells, Cultured; Cornea; Corneal Transplantation; Epithelial Cells; Eye; Fibroins; Humans; Keratins; Light; Microscopy, Electron, Scanning; Permeability; Phenotype; Stress, Mechanical; Tissue Engineering

To investigate the morphology of the human eyelid margin and the presence of different cytokeratins, mucins and stem cell markers within the skin epithelium, mucocutaneous junction (MCJ) and palpebral conjunctiva.. Eyelids of body donors were investigated histologically and ultrastructurally as well as by immunohistochemical methods using antibodies to cytokeratins 1, 4, 7, 8, 10, 13, 14, 15, and 19; mucins MUC1, MUC4, and MUC5AC and potential stem cell markers K15, BCRP/ABCG2, integrin β1, and N-cadherin.. The expression pattern of cytokeratins, mucins and stem cell markers varied across the different epithelia of the human eyelid. Within the MCJ, CK7, 15 and 19 were absent, whereas the epithelium reacted positive to antibodies to CK1, 4, 8, 10, 13 and 14. Reactivity was also observed for MUC1 and MUC4, but not for MUC5AC. No reactivity was determined for K15, BCRP/ABCG2 and integrin β1 in the area of the MCJ epithelium but a strong reactivity was present for N-cadherin.. The present immunohistochemical findings lead to a better characterization of the MCJ. Additionally, the knowledge of distribution of biomarkers like cytokeratins, mucins and stem cells can be useful in the investigation of MCJ disturbances which occur in several disorders of the meibomian glands and the lid epithelium in the course of dry eye syndrome and especially meibomian gland dysfunction.

Topics: Aged; Biomarkers; Cadaver; Conjunctiva; Eyelid Diseases; Eyelids; Female; Hair Follicle; Humans; Immunohistochemistry; Keratins; Male; Meibomian Glands; Microscopy, Electron; Middle Aged; Mucins; Mucous Membrane; Skin Physiological Phenomena; Stem Cells; Tissue Fixation

Cytokeratin-positive primitive hepatocytes or hepatic progenitor cells have been described in the fetal ductal plate, as well as in the adult canals of Hering. We examined the fate of ductal plate cells in the hilar region of the liver.. Using liver sections from 10 fetuses and 15 elderly cadavers, we performed immunohistochemistry for cytokeratins 14 and 19, smooth muscle actin, vimentin and CD34.. At 18 weeks of gestation, cytokeratin-positive cells were evident in the ductal plate and liver parenchyma, which were separated by a narrow space. At 25 weeks, most of these positive cells had disappeared, but the remnant cells were aligned along the parenchymal margins facing the hilar portal pedicle in addition to the canals of Hering in the peripheral portal pedicle. The gallbladder bed did not contain cytokeratin 19-positive cells. Notably, even livers in the elderly contained such marginal positive cells in the hilar region. These cells were negative for smooth muscle actin and CD34, but tended to be positive for vimentin.. Cytokeratin-positive hepatic progenitor cells are likely to exist along the hilar portal pedicle even in adults. These hilar marginal hepatocytes seem to be derived not from the fetal ductal plate, but from the liver parenchyma.

Topics: Aged; Aging; Cadaver; Cells, Cultured; Female; Hepatocytes; Humans; Keratins; Liver; Male; Tissue Distribution

The diffusivity of water in human nail at 32 degrees C was determined for cadaveric, human finger nails having water contents ranging from 0.536 g H(2)O/g dry nail to 0.035 g H(2)O/g dry nail by measuring the desorption of tritiated water from nails suspended in water or in the vapor phase above salt solutions yielding a range of relative humidities (RH). Diffusivity increased with increasing RH from (7.7 +/- 1.3) x 10(-10) cm(2) s(-1) at 15% RH to (3.2 +/- 1.1) x 10(-7) cm(2) s(-1) in the liquid phase study at 100% RH, a more than 400-fold increase. The diffusivity values, which may be understood in terms of the equilibrium water content of the nail and a free volume theory for diffusion in hydrophilic polymers, were consistent with water diffusivities measured in other keratinized tissues including wool, horn and the corneocyte phase of stratum corneum. Analysis of the tritium desorption data was complicated by a tritium exchange process between (3)H(2)O and nail keratin, the kinetics of which are presented in part. The combination of the concentration-dependent water diffusivity with the natural water activity gradient in nail in vivo leads to the prediction of a nonlinear steady-state water concentration profile in human nail in vivo which, in turn, has implications for ungual drug delivery.

Topics: Cadaver; Diffusion; Humans; Humidity; In Vitro Techniques; Keratins; Kinetics; Models, Biological; Nails; Temperature; Tritium; Volatilization; Water

Previous studies have shown that ameloblast-like cells can be selectively cultured from the enamel organ in a serum-free medium with low calcium concentrations. The purpose of this study was to further characterize this culture system to identify differentiated ameloblast-lineage cells. Tooth organs from 19-24-wk-old fetal cadavers were either frozen and cryosectioned for immunostaining, or digested in collagenase/dispase for cell culture. The cells were grown in keratinocyte media supplemented with 0.05 mM calcium, and characterized by morphology and immunofluorescence. Epithelial clones with two distinct morphologies, including smaller cobblestone-shaped cells and larger (5-15 times in size) rounded cells, began to form between day 8 and day 12 after culture. The cobblestone-shaped cells continued to proliferate in culture, while the larger cells proliferated slowly or not at all. These larger cells formed filopodia, usually had two or more nuclei and a radiating cytoplasm at the cell margin, and were more abundant with increasing time in culture. Both cell types stained for cytokeratin 14, and the larger cells appeared more differentiated, showing stronger staining for amelogenin and ameloblastin. Immunofluorescence of the tooth bud sections showed staining for these matrix proteins as ameloblasts differentiated from the inner enamel epithelium. These results show the successful culture of differentiating ameloblast-lineage cells, and lay a foundation for use of these cells to further understand ameloblast biology with application to tooth enamel tissue engineering.

Topics: Ameloblasts; Amelogenin; Cadaver; Calcium; Cell Differentiation; Cell Lineage; Cell Membrane; Cell Nucleus; Cell Proliferation; Cell Shape; Cells, Cultured; Clone Cells; Culture Media, Serum-Free; Cytoplasm; Dental Enamel; Dental Enamel Proteins; Enamel Organ; Epithelial Cells; Fetus; Humans; Keratins; Pseudopodia; Tooth Germ

Tooth enamel is formed by ameloblasts, which are derived from the epithelial cells of the enamel organ.. The purpose of this study was to grow human ameloblast-like epithelial cells in culture.. Human fetal tooth organs were isolated, and the cells were separated by digestion in collagenase/dispase. The cells were cultured in KGM-2 media with and without serum and at different calcium concentrations. The expression of enamel matrix proteins was analyzed by RT-PCR and cytokeratin 14 was detected by immunohistochemistry. The cells were further characterized by osteogenesis/odontogenesis-related DNA array.. Cells isolated from the tooth organs grown in KGM-2 media containing 2-10% serum, were mixture of cobblestone and spindle shaped cells. Culturing these cells in KGM-2 with 0.05 mM calcium was selective for cobblestone ameloblasts-like cells (CAB), which were immunopositive for cytokeratin 14. Amelogenin, ameloblastin, enamelin, MMP-20 and KLK-4 were detected in CAB cells by RT-PCR. Osteogenesis SuperArray analyses could not detect the presence of typical molecules related to mesenchymal odontoblast or osteoblast lineage cells in these cultures.. These studies showed that cobblestone-shaped ameloblast-like cells are selected from the tooth organ cells, by culture in KGM-2 media with 0.05 mM calcium.

Topics: Ameloblasts; Amelogenin; Cadaver; Calcium; Cell Differentiation; Cell Division; Cell Membrane; Cells, Cultured; Dental Enamel Proteins; Enamel Organ; Epithelial Cells; Humans; Immunohistochemistry; Kallikreins; Keratins; Matrix Metalloproteinase 20; Matrix Metalloproteinases; Membrane Proteins; Odontogenesis; Phenotype; Tooth Germ

Recently, the expression profiles of the members of the complex hair keratin family have been determined in the human anagen hair follicle. In contrast, the details of hair keratin expression in the human nail unit are poorly known.. In order to fill this gap, we have performed an immunohistochemical study of the adult human nail unit by means of specific antibodies against nine hair keratins of both types (hHa2, hHb2, hHa5, hHb5, hHa1, hHb1, hHb6, hHa4 and hHa8) as well as three epithelial keratins (K5, K17 and K10).. Formalin-fixed paraffin sections of adult nails were examined using monoclonal and polyclonal keratin antibodies, respectively. Longitudinal as well as transverse sections were investigated.. Our study revealed two types of epithelial tissue compartments in the nail unit. The first comprised the eponychium and hyponychium and the nail bed, which expressed only epithelial keratins. While keratins K5, K17 (basal) and K10 (suprabasal) were found in the orthokeratinizing eponychium and hyponychium, throughout, the nail bed epithelium expressed only K5 and K17. The second type comprised the apical and ventral matrix which exhibited a mixed pattern of epithelial and hair keratin expression. Thus, K5 and K17 were expressed in the entire multilayered basal cell compartment of the apical and ventral matrix; however, in the latter, K5 and K17 also occurred in the lowermost layers of the overlying keratogenous zone. The hair matrix keratin hHb5, but not its type II partner hHa5, was seen in the entire keratogenous zone of the apical and ventral matrix, but was also located in the uppermost cell layers of the basal compartment of the ventral matrix, where it overlapped with K5 and K17. Similar to their sequential expression in the hair follicle cortex, hair keratins hHa1, hHb1, hHb6 and hHa4 were consecutively expressed in the keratogenous zone of both the ventral and, albeit less distinctly, apical matrix, with hHa1 initiating in the lowermost cell layers. The expression of hHa8 in only single cortex cells of the hair follicle was also preserved in cells of the keratogenous zone. In the region of the so-called dorsal matrix, we observed two histologically and histochemically distinct types of epithelia: (i) a dominant type, histologically similar to the eponychium and an associated K5, K17 and K10 keratin pattern which clearly extended into the apical matrix, and (ii) a minor type, histologically resembling the postulated dorsal matrix without a granular layer and a cuticle, and exhibiting extended K5 expression as well as hair keratin expression in superficial cells.. The coexpression of hHb5 with K5 and K17 in the uppermost cell layers of the basal compartment and the lowermost layers of the keratogenous zone of the ventral matrix prompts us to designate this region the prekeratogenous zone of the ventral matrix. The two alternating types of histology and keratin expression in the dorsal matrix identify this region as a transitional zone between the eponychium and the apical matrix. Finally, our data clearly show that the ventral matrix is the main source of the nail plate. In addition, the mixed scenario of hair and epithelial keratins, including demonstrable amounts of K10, in superficial cells of the apical matrix, lends support to the notion that the dorsal portion of the nail is generated by the apical matrix.

Topics: Adult; Antibodies; Antibody Specificity; Cadaver; Epithelium; Hair; Hair Follicle; Humans; Keratin-10; Keratin-5; Keratins; Nails

The etiology of upper airway collapsibility in patients with snoring and obstructive sleep apnea (OSA) remains unclear. Structural mucosal changes could be contributory factors. The objective of this study was to determine whether pathologic changes in the epithelium or the epithelial-connective tissue interface are present in patients with snoring and/or OSA by means of scanning electron microscopy and immunohistochemistry. Uvulae were obtained by uvulopalatopharyngoplasty from three patients with habitual snoring and nine patients with mild to severe OSA, as well as by dissection from 43 nonsnoring body donors. Scanning electron microscopy revealed structural changes in the epithelial-connective tissue boundary that significantly differed from age-related changes in the control subjects. The immunohistochemical staining with antibodies against epithelial cytokeratins showed differences in the expression pattern of cytokeratin 13 between patients and control subjects. No differences were found in the distribution pattern of laminin. Analysis of defense cells revealed a significant diffuse infiltration of leukocytes, mainly T cells, inside the lamina propria of the patient group, which was not observed in the control group. In conclusion, these results support the hypothesis that progressive structural changes in the mucosa caused by the trauma of snoring are a possible contributory factor to upper airway collapsibility.

Topics: Adult; Aged; Aged, 80 and over; Anthropometry; Biopsy; Body Mass Index; Cadaver; Case-Control Studies; Cause of Death; Disease Progression; Humans; Immunohistochemistry; Keratins; Leukocyte Count; Male; Microscopy, Electron, Scanning; Middle Aged; Polysomnography; Respiratory Mucosa; Severity of Illness Index; Sleep Apnea, Obstructive; Snoring; Uvula

The NPHS1 gene product, nephrin, is a crucial component of the glomerular filtration barrier preventing proteinuria and previously assumed to be kidney-specific. The aim of this study was to describe the expression of nephrin mRNA and protein in human pancreas as well as identify the nephrin-expressing cell types.. RNA dot blot, reverse transcriptase-polymerase chain reaction, sequencing, immunoblotting and dual immunofluorescence were used for the characterisation of nephrin in the pancreas.. Except for the kidney, the pancreas was found to be the only tissue expressing nephrin as screened with a human tissue RNA dot blot. The expression was verified with reverse transcriptase-polymerase chain reaction and by sequencing nephrin from a human pancreatic complementary DNA library. Nephrin antibody in immunoblot detected a 165,000 M(r) protein in the pancreas. Dual immunofluorescence showed that nephrin was specifically localised in the beta cells of the islets of Langerhans. There was no overlap with glucagon, somatostatin, or the ductal cell marker cytokeratin 19.. These data show that nephrin is a novel molecule of pancreatic beta cells.

Topics: Cadaver; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Fluorescent Dyes; Gene Expression; Glucagon; Humans; Immunoblotting; Islets of Langerhans; Keratins; Kidney; Kidney Cortex; Membrane Proteins; Molecular Weight; Pancreas; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Somatostatin

Samples from parotid, submaxillary, and von Ebner salivary glands of six chronic alcoholic individuals who had died of alcoholic hepatic cirrhosis were analyzed by topographic and histochemical routine stains and marked for cytokeratins; two normal adult individuals were used as control. Modifications in the acinar cells were found, but the most evident changes were observed in the ductal system: enlargement of major ducts, heterogeneous expression of cytokeratins and athrophy in epithelial cells, desquamated cells and stasis of content, and ductal hyperplasia in von Ebner glands. The lymphoplasmocytic infiltration does not represent the typical lymphocytic focus on Sjögren's syndrome or other connective tissue pathologies. Our findings indicate that functional and structural variations are produced both in serous acini and ducts parotid, submaxilar and von Ebner glands affected by alcoholic sialosis.

Topics: Adult; Aged; Alcoholism; Cadaver; Epithelial Cells; Humans; Keratins; Lymphocytes; Middle Aged; Parotid Gland; Salivary Ducts; Salivary Glands, Minor; Submandibular Gland

Routine immunohistochemical analysis of human donor pancreata indicated the frequent occurrence of single insulin-immunoreactive cells. In a quantitative analysis of nine organs consecutively recruited from adult donors, 15 percent of all beta cells were found in units with a diameter less than < 20 microm and without associated glucagon-, somatostatin-, or pancreatic polypeptide cells. These single beta-cell units are located in or along ductules, from which they appear to bud as previously noticed in fetal and neonatal organs. They contain significantly smaller beta cells than endocrine aggregates with a larger diameter. The use of ductal cell markers such as cytokeratin 19, carbonic anhydrase-II and carbohydrate antigen 19.9 identified a close topographical association between ductal cells and budding beta cells; it also indicated that pancreatic lobules are composed of nearly one third ductal cells. The presence of Ki67 proliferation marker-immunoreactive ductal cells (0.05 %) and absence of Ki67-immunoreactive budding beta cells is compatible with the view that beta cell neogenesis depends on ductal cell proliferation and differentiation. The high proportion of budding beta cells in the adult human pancreas suggests the presence of numerous loci with a potential for beta cell neogenesis.

Topics: Adolescent; Adult; Aged; Biomarkers; CA-19-9 Antigen; Cadaver; Carbonic Anhydrases; Cell Communication; Cell Count; Female; Humans; Immunohistochemistry; Islets of Langerhans; Keratins; Ki-67 Antigen; Male; Middle Aged; Pancreas; Pancreatic Ducts

We report a modified method for the isolation and propagation of adult human Müller cells in culture.. The retina of postmortem human eyes was mechanically dissociated and cultured. Using immunocytochemical techniques, these cells were stained with monoclonal antibodies specific for Müller cells, glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase (GS) and keratin. Transmission electron microscopy (TEM) was also performed.. The dissociated and cultured cells expressed vimentin and GS, but not GFAP. At least 85% of these cells stained with a Müller cell-specific monoclonal antibody. Using TEM, flat cells containing 13-nm intermediate filaments and glycogen were identified.. Human retinal Müller cells can be isolated and propagated in culture. Purified cell cultures are required for controlled studies of the normal physiology and pathologic responses of Müller cells.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Cadaver; Cell Culture Techniques; Glial Fibrillary Acidic Protein; Glutamate-Ammonia Ligase; Glycogen; Humans; Immunohistochemistry; Keratins; Microscopy, Electron; Neuroglia; Retina; Vimentin

Recent work carried out at the Ear Pathology Research Laboratory has demonstrated that cerumen plugs consist of a large amount of keratin debris. In this study the site of origin of this keratin was investigated. This was done by histologically examining these cerumen plugs without disturbing the natural relationship between the plug and the surrounding epithelium. We have clearly demonstrated that a cerumen plug consists of keratin arising from the migratory epithelium of the deep external auditory canal and epithelium of the superficial external auditory canal.

Topics: Cadaver; Cerumen; Ear Canal; Epithelium; Humans; Keratins

An adult with burns over 55% of body surface area (80% of which were third degree) was treated with cadaver skin allografts. The allografts were later abraded to remove allogeneic epidermis and resurfaced with autogenous keratinocyte cultures. Complete reconstitution of skin, consisting of epidermal autograft and dermal allograft, was achieved.

Topics: Burns; Cadaver; Cells, Cultured; Epidermal Cells; Epidermis; Humans; Keratins; Male; Middle Aged; Skin Transplantation; Transplantation, Autologous; Transplantation, Homologous

The molecular structures of both native and false hairs as well as the tendon of the female cadaver buried about 2,100 years ago, which was excavated from the Han Tomb, No. 1 at Mawangdui near Changsha, Human Province, China, have been studied with X-ray fiber diffraction. It has been shown that the conformations of alpha-keratin and collagen and the basic longitudinal period of these fibrous protein molecular chains have been preserved in those specimens respectively, but the aggregations of fibrous molecules have somewhat changed. Some exploration was made on the origin where came the cubic mercury sulphide inside the cadaver's hair.

Topics: Cadaver; China; Collagen; Female; Hair; History, Ancient; Humans; Keratins; Mercury; Paleontology; Protein Conformation; Tendons; X-Ray Diffraction

Topics: Amino Acids; Autoanalysis; Cadaver; Callosities; Chemical Precipitation; Chromatography, Gel; Dextrans; Electrophoresis, Polyacrylamide Gel; Humans; Hydrogen-Ion Concentration; Ichthyosis; Keratins; Protein Denaturation; Psoriasis; Skin; Tissue Extracts

Topics: Blood; Cadaver; Cell Nucleus; Culture Media; Histology; Keratins; Organ Culture Techniques; Research Design; Skin; Tissue Culture Techniques