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PubMed2005

Immunohistochemical characterization of the distribution of galectin-4 in porcine small intestine.

Galectins are an evolutionarily conserved family of 15 different lectins found in various combinations in virtually every type of animal cell. One of the primary galectins expressed in intestinal epithelium is galectin-4, a tandem-repeat galectin with two carbohydrate-recognition domains in a single polypeptide chain. In the current study, we produced an anti-galectin-4 monoclonal antibody (MAb) for determining the distribution of galectin-4 in porcine small intestine to enhance our understanding of where galectin-4 performs its functions in the small intestine. In immunohistochemistry studies, this MAb detected galectin-4 primarily in the cytoplasm of absorptive epithelial cells lining intestinal villi. Mature epithelial cells at the villous tips stained the most intensely with this MAb, with progressively less intense staining observed along the sides of villi and into the crypts. In addition to its cytoplasmic localization, galectin-4 was also associated with nuclei in villous tip cells, indicating that some galectin-4 may migrate to the nucleus during terminal maturation of these cells. In intestinal crypts, a specific subset of cells, which may be enteroendocrine cells, expressed galectin-4 at a relatively high level. Galectin-4 distribution patterns were similar in all three regions (duodenum, jejunum, and ileum) of porcine small intestine.

Animals↗
PubMed2005

Effects of nicotine on proliferation, cell cycle, and differentiation in immortalized and malignant oral keratinocytes.

BACKGROUND: Numerous epidemiological studies have reported that tobacco smoking is a major risk factor for oral cancer, but relatively little is known about the effect of nicotine, a major product of cigarette smoking, on immortalized oral keratinocytes and cancer cells. METHODS: We investigated the effects of nicotine on the growth and differentiation of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12), and human skin keratinocytes (HaCaT), in the monolayer and in the three-dimensional (3D) raft cultures using the MTT assay, Western blotting, and cell cycle analysis. RESULTS: Nicotine inhibited the proliferation of immortalized and malignant keratinocytes in dose- and time-dependent manners as determined by MTT assay. The 3D organotypic culture showed that nicotine at high concentration (300 microM) inhibits epithelial maturation, surface keratinization, and decreased epithelial thickness. Flow cytometry showed that nicotine inhibited cell cycle progression by inducing G(0)/G(1) arrest of HaCaT, IHOK, HN4, and HN12 cells without causing apoptosis. Nicotine treatment increased p21 expression in immortalized cells (HaCaT, IHOK) and oral cancer cells (HN4, HN12), but decreased pRb and p53 expression in oral cancer cells. Moreover, after high-dose nicotine treatment, the involucrin expression increased markedly in immortalized cells, but not in oral cancer cells. CONCLUSIONS: We demonstrated that nicotine inhibits growth through cell cycle arrest at G(0)/G(1) phase probably by increasing the expression of p21(WAF1/CIP1). Nicotine also affects epithelial differentiation in immortalized and malignant oral keratinocytes. Malignant oral keratinocytes appear to be more resistant to the effects of nicotine on epithelial growth and differentiation as compared to the immortalized cells.

Carcinoma, Squamous Cell↗
PubMed2026

From immature to mature epithelium: unveiling structural dynamics and transcriptional programs in rainbow trout intestinal barrier.

The intestinal epithelium is crucial for nutrient absorption, immune defense, and barrier function in farmed fish. However, the molecular mechanisms underlying its development and maturation in salmonids remain poorly characterized, hindering our ability to address pervasive gut health challenges in aquaculture. In this study, we use the RTgutGC cell line to implement an epithelial maturation model with the aim of characterizing the global transcriptional program in rainbow trout (Oncorhynchus mykiss). We evaluated in vitro culture conditions to generate a polarized epithelial barrier with high transepithelial electrical resistance (TEER = 75.8 Ω × cm2), low permeability (6.2 × 10-6 cm/s), and well-defined apical specializations, including microvilli-like structures and clusters of these structures (brush border). Comparative transcriptomic profiling between immature (7 days post-seeding, dps) and mature (28 dps) epithelia revealed 3,817 differentially expressed genes (DEGs). Functional enrichment analysis showed that maturation was characterized by the downregulation of proliferative and ribosomal pathways and the concerted upregulation of processes critical for barrier function, including transmembrane transport, proteolysis, cell adhesion, extracellular matrix organization, and tight junction assembly. We identified a core set of 60 genes indicators of epithelial maturation, encompassing solute transporters (slc26a6, slc43a2), tight junction proteins (tjp1, cldn1, cldn3, cldn5, cldn18, among others), and structural components essential for microvilli formation and polarization (cdhr5b, pard6a). By integrating ultrastructural, functional, and transcriptomic data, this study established a framework for future mechanistic investigations into gut development and maturation in vitro. This set of mature epithelium indicators has diverse applications, such as the design of nutritional and pharmacological interventions to improve gut health and resilience in farmed fish.

Animals↗
PubMed2001

Association of mesenchymal cells and immunoglobulins with differentiating epithelial cells.

BACKGROUND: Mesenchymal-epithelial interactions play an important role in the physiology and pathology of epithelial tissues. Mesenchymal cells either associate with epithelium basement membrane [pericytes and perivascular monocyte-derived cells (MDC)] or reside within epithelium (MDC and T cells). Although intraepithelial mesenchymal cells were suggested to contribute to the epithelium physiology, their association with particular steps in differentiation of epithelial cells, interactions among themselves, and their fate remain unclear. We studied epitopes of mesenchymal cells and their products (immunoglobulins) in stratified epithelium of uterine ectocervix, which is one of the prototypes of complete cellular differentiation from stem into the aged cells. RESULTS: Perivascular CD14 primitive MDC associated with basal (stem) epithelial cells. Thy-1 pericytes of microvasculature secreted intercellular vesicles, which associated with Ki67 postmitotic epithelial cells expressing MHC class I. Intraepithelial T cells showed an association with veiled type MDC [dendritic cell (DC) precursors] among parabasal cells, and exhibited fragmentation after entering intermediate (mature) epithelial layers. Mature DC secreted CD68 and exhibited fragmentation after reaching mid intermediate layers. Binding of IgM was detected at the top of each layer: in the upper parabasal, upper intermediate, and most surface epithelial cells. IgG was confined to the entire superficial layer. CONCLUSIONS: These data suggest that the phylogenetically and ontogenetically developed hierarchy of mesenchymal cells (MDC, pericytes, T cells) and immunoglobulins (IgM, IgG) accompanies differentiation of epithelial cells from immature into the mature and aged phenotype. Further studies of an involvement of mesenchymal cells in the regulation of tissue homeostasis may bring novel approaches to the prevention and therapy of tissue dysfunctions characterized by permanent tissue immaturity (muscular dystrophy) or accelerated aging (degenerative diseases).

Apoptosis↗
PubMed2006

Iron chelator-induced growth arrest and cytochrome c-dependent apoptosis in immortalized and malignant oral keratinocytes.

BACKGROUND: Many studies have shown the anti-proliferative effects of iron deprivation on cancer cells, but the effects of iron-chelators on oral cancer have not been clearly elucidated. METHODS: To investigate the effects of an iron chelator, desferrioxamine (DFO), on the growth of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12) and human skin keratinocytes (HaCaT) in the MTT assay, three-dimensional (3D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression for apoptosis signaling pathway were used. RESULTS: Desferrioxamine inhibited the growth of immortalized IHOK and HaCaT and malignant HN4 and HN12 keratinocytes in a time- and dose-dependent manner according to the MTT assay. The 3D organotypic culture also revealed that DFO-treated cells showed less epithelial maturation, less surface keratinization and decreased epithelial thickness. The major mechanism of growth inhibition with the micromolar DFO treatment was by the induction of apoptosis, which was supported by nuclear DAPI staining, DNA fragmentation analysis and flow cytometric analysis for sub-G(1) phase arrest and Annexin V-FITC (fluorescein isothiocyanate) staining. Furthermore, Bax expression increased together with p53 and p21(WAF1/CIP1), while the Bcl-2 expression decreased in the immortalized and malignant keratinocytes treated with DFO. Time-dependent cytochrome c from mitochondria was observed in DFO-treated IHOK and oral cancer cells and was accompanied by the activation of caspase-3 in IHOK cells. CONCLUSION: These results demonstrate that DFO has growth inhibitory effects on immortalized and malignant oral keratinocytes through the induction of apoptosis and suggest that further evaluation of DFO as a potential therapeutic agent for human oral precancerous lesions is warranted.

Apoptosis↗
PubMed1983

Exfoliative cytology and ultrastructure of superficial epithelial cells from the normal human oral mucosa.

Superficial, mature epithelial cells were collected from clinically normal hard palate, cheek and lip of 5 volunteers. These cells were stained using 3 different exfoliative cytology methods and prepared for electron microscopy. In total, 209 individual cells were examined both by the light and electron microscopes and classified as orange, red or blue cells exhibiting an oral keratin or a filament pattern. The combination of data sets revealed that type and degree of terminal differentiation are not related to the staining characteristics. Independent of the staining methods, cells displaying an oral keratin pattern stained either orange, red or blue, and cells displaying a filament pattern stained either red or blue. It is concluded that the staining characteristics of superficial oral epithelial cells do not reflect their status of terminal differentiation as seen ultrastructurally.

Adult↗
PubMed1978

Difference between mammary epithelial cells from mature virgin and primiparous mice.

Mammary epithelial cells from mature virgin mice are similar to those from primiparous mice in several respects. However, there is one known difference. The cells from the mature virgin must traverse the cell cycle in order to become competent to make casein and enzymatically active alpha-lactalbumin in vitro; those from the primiparous animal can make these proteins without first traversing the cycle. In this regard, cells from human placental lactogen- and prolactin-treated mature virgins are, after involution, similar to those from primiparous mice. The developemental block in the cells from the mature virgin, imposed by preventing cell cycle traversal, has been partially delineated. It does not appear to reside at the levels of ultrastructural maturation or the formation of casein messenger RNA. Rather, the lesion is postranscriptional and may be at the level of translation, or posttranslational modification, or both.

Animals↗
PubMed2000

Characterization of an axonemal dynein heavy chain expressed early in airway epithelial ciliogenesis.

The most conspicuous evidence of airway epithelial maturation and vitality is the presence of motile cilia. In an effort to generate genetic and antigenic markers of airway maturation, injury, and repair, we characterized airway epithelial expression of a gene identified by two human expressed sequence tags that encoded peptides with sequence similarity to an invertebrate ciliary dynein heavy chain (DHC). Molecular analyses showed that the gene has a very large RNA transcript that encodes a very high molecular weight polypeptide with biochemical properties that are characteristic of a dynein heavy chain. Expression of the gene transcript correlated with the presence of ciliated cells in tissues, and immunohistochemical localization of the gene product confirmed its presence in the cilia of mature airway epithelium. In epithelium undergoing ciliogenesis ex vivo, expression of the gene transcript preceded ciliation of the epithelium and the gene product was present in the cytoplasm and at the apical border of nonciliated cells. These data suggested that the gene encodes an axonemal DHC that is expressed early during ciliogenesis, before the appearance of cilia.

Amino Acid Sequence↗
PubMed1990

Cyclic changes in conjunctival smears from menstruating females.

Cytohormonal evaluation has become an accurate procedure for the assessment of estrogenic effect on the vaginal and buccal mucosa. Both show a cyclical variation in the maturation index (an "estrogenic value" based on epithelial maturation). Conjunctival smears were taken from the temporal conjunctiva of 11 premenopausal and 7 postmenopausal females on a near-daily basis. No females had evidence of ocular surface disease, including keratoconjunctivitis sicca. After fixation and Papanicolaou staining, maturation indices for each specimen based on the differential morphology were red in a single blind fashion by a Papanicolaou cytologist. Cyclical variations paralleling the menstrual cycle in eight of nine premenopausal women were noted with maturation index values ranging from 30 (low estrogen effect) to 82 (high estrogen effect), peaking on day 11 or 21 of the menstrual cycle. No cyclical change was found in postmenopausal smears and smears of subjects with endocrine abnormalities. Maturation index values ranged from 30 to 38. Conjunctiva appears to be an estrogen-sensitive epithelium.

Cell Division↗
PubMedDate not supplied

Human papillomavirus infection in atrophic smears. A case report.

BACKGROUND: Human papillomavirus (HPV) infection in atrophic smears can be misleading and may produce the diagnosis of cervical intraepithelial neoplasia. CASE: A routine cervical smear in a 62-year-old female revealed an atrophic smear with nuclear changes suggestive of a high grade squamous intraepithelial lesion (HSIL). An estrogen cream for topical vaginal use was prescribed. A new smear was collected seven days later and revealed koilocytosis but no evidence of HSIL. CONCLUSION: Koilocytosis is a cellular finding of mature epithelial cells. The use of estrogen produces maturation of HPV-infected basal cells, allowing a correct diagnosis of this disease in patients with atrophic smears.

Atrophy↗
PubMed1990

Changes in the number and distribution of Langerhans cells in the hydantoin hyperplasic gingiva as compared with the clinically normal one.

A study was made of the number of Langerhan's cells (LCs) per mm2 of section which express the antigens T6 and/or HLA-DR in seriated gingival sections of diphenylhydantoine-induced hyperplasia (HG) and clinically normal gingivae (NG). NG showed histological correlation with its macroscopic appearance. In HG the classical histopathological findings were verified, as well as the epithelial maturation irregularities, conductive to the development of epithelial gaps. In the immunostained samples, LCs appear amply distributed in the epithelium in greater numbers than in NG and more branched except in the immature areas, where they mostly express HLA-DR. In HG keratinocytes, HLA-DR+ are observed in the basal layer, except in developing epithelial gap zones. The Wilcoxon test for the NG-T6/NG-DR and HG-T6/HG-DR was not significant; but the Mann Whitney test for NG-T6/HG-T6 and NG-DR/HG-DR was significant to p less than 0.05. It is understood that the increase in LC numbers in HG is a manifestation of their active participation in local immune reactions. The presence of DR+/T6- LCs in the less keratinized areas seems to indicate the relationship of LCs with epithelial proliferation and/or differentiation.

Adult↗
PubMed1989

Immunology of atopic dermatitis.

Atopic dermatitis is a chronic eczematous skin disease which characteristically starts early in life and later tends to disappear. Genetic predisposition seems important for the development of the disease. The immune system is involved through a lymphocyte-mediated inflammation in the skin creating the eczema, and an increased incidence of type I and possibly type IV allergies induced by environmental allergens. Recent findings of a changed concentration of some interleukins in atopic patients support the evidence for an increased T-lymphocyte activation and may explain the increased amount of polyclonal IgE in many of the patients. Hypothetically, atopic dermatitis can be considered to be due to an inborn error of the maturation of epithelial tissue. Maturation of epithelial tissue is essential for both the appearance of normal skin and for correct maturation of the cell-mediated immune system. The immune deviation is later more or less corrected through maturation which explains why the disease in most patients disappears in childhood. However, it still leaves a certain increased capacity for inflammation of the atopic persons.

Adult↗
PubMed2005

Toll-like receptors-2, -3 and -4 expression patterns on human colon and their regulation by mucosal-associated bacteria.

The colonic epithelium provides an interface between the host and micro-organisms colonising the gastrointestinal tract. Molecular recognition of bacteria is facilitated through Toll-like receptors (TLR). The colonic epithelium expresses relatively high levels of mRNA for TLR3 and less for TLR2 and -4. Little is known of the expression patterns and mode of induction of expression for these pattern recognition receptors in human colon. The aim of this study was to investigate their localization in the gut and induction of expression in epithelial cell lines by mucosal bacteria. TLR2 and -4 were expressed only in crypt epithelial cells, expression was lost as the cells matured and moved towards the gut lumen. In contrast, TLR3 was only produced in mature epithelial cells. HT29 and CACO-2 had different levels of expression for TLR1-4. Co-culture of HT29 cells with different mucosal isolates showed that they were highly responsive to bacterial challenge, with up-regulation of mRNA for TLR1-4. In contrast, CACO-2 cells were refractive to bacterial challenge, showing little difference in mRNA levels. TLR3 was induced in HT29 only by Gram-positive commensals with up-regulation of both mRNA and protein and an enhancement of the antiviral immune response. This pattern of expression allows induction of responsiveness to bacteria only by the crypt epithelium so that tolerance to commensal organisms can be maintained. In contrast, mature columnar epithelium is able to respond to viral pathogens, which are not part of the normal gut commensal microbiota.

Bacteroides fragilis↗
PubMed1987

Precocious development of lectin (Ulex europaeus agglutinin I) receptors in dome epithelium of gut-associated lymphoid tissues.

Dome epithelium (DE), the tissue covering lymphoid domes of gut-associated lymphoid tissues, was examined in both adult and neonatal rabbit appendix or sacculus rotundus to determine if dome epithelial cells matured earlier than epithelial cells covering adjacent villi. The localization of well-differentiated epithelial cells in rabbit gut-associated lymphoid tissues (GALT) was accomplished histochemically by use of molecular probes: fluorescein isothiocyanate or horseradish peroxidase conjugates of Ulex europaeus agglutinin I (UEA), a lectin specific for terminal L-fucose molecules on certain glycoconjugates. The villus epithelial cells of newborn and 2-, 5-, or 10-day-old rabbits did not bind UEA, but between the twelfth and fifteenth days of postnatal life, UEA receptors were expressed by well-differentiated villus epithelial cells. In contrast to villus epithelium, DE in appendix and sacculus rotundus of neonatal rabbits expressed UEA receptors two days after birth, a feature that distinguished the DE of neonatal GALT for the next two weeks. In adult rabbits, UEA receptors were associated with dome epithelial cells extending from the mouths of glandular crypts to the upper domes; in contrast to the domes, UEA receptors were only present on well-differentiated epithelial cells at the villus tips. Results suggested that in neonatal rabbits most dome epithelial cells developed UEA receptors shortly after birth, reflecting precocious development of DE as compared to villus epithelium. In adult rabbit dome epithelium UEA receptors appeared on dome epithelial cells as they left the glandular crypts, representing accelerated epithelial maturation.

Animals↗
PubMed2006

Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth.

PURPOSE: To determine the kinetics of corneal epithelial maturation during embryonic development and postnatal growth. METHODS: Expression patterns of keratin (K)12 and K14 were determined in mouse embryos (embryonic days [E]15.5-19.5), corneas of postnatal day (P)0 to 10 months, and healing corneas after epithelial debridement in P30 and P90 mice. The expression of alkaline phosphatase (AP) was determined during postnatal growth and healing of epithelial debridement of Krt12(Cre/Cre)/ZAP bitransgenic mice. RESULTS: During embryonic development, K12 expression by corneal peridermal epithelium commenced at E15.5. In the period from E15.5 to P10, the expression of K12 was restricted to the suprabasal and/or superficial cells of the corneal epithelium, whereas the K14 expression was restricted to the basal cells. After P30, K12 expression was sporadically detected in the basal corneal epithelium, and the number of K12-positive basal cells increased as the mice grew older. The number of K14-positive cells that coexpressed K12 increased with age and reached a plateau after P180. Healing of the debrided epithelium facilitated the increase in K14-positive cells that coexpressed K12. Many basal cells of Krt12(Cre/Cre)/ZAP mice remained undifferentiated and expressed LacZ at P15, and they then differentiated to express Cre, which leads to excision of LacZ and AP expression. CONCLUSIONS: In the mouse, the corneal epithelium does not become fully mature until 3 to 6 months after birth, in that a significant number of corneal basal epithelial cells of young mice (<P30), which derive from embryonic surface ectoderm remain undifferentiated and serve as corneal epithelial progenitor cells. These progenitor cells may have some stem cell characteristics.

Animals↗
PubMedDate not supplied

Biochemical and morphometric analyses in hypoplastic lungs.

We report the results of biochemical and morphometric studies on lungs of infants with bilateral lung hypoplasia either with or without oligohydramnios (OH or NOH) in comparison with findings in normally grown lungs. The OH and NOH lungs were equally hypoplastic in terms of DNA content but OH lungs had a significantly lower disaturated phosphatidylcholine (DSPC) concentration than NOH or normal lungs, apart from a subgroup with gastrointestinal or airway obstruction. Hydroxyproline concentration in OH lungs was higher than that in NOH or normal lungs. Desmosine concentrations did not differ significantly between groups despite the obvious lack of elastin in the septal crests of the OH group on histology. Morphometry revealed low lung volume, low radial alveolar counts, low alveolar numbers, and low alveolar surface area in both OH and NOH groups. Alveoli and alveolar ducts constituted a higher proportion of lung volume in NOH than in OH lungs. The similarity of most morphometric indices in the two groups implies that maturation does not depend on quantitative elaboration of airways and alveoli. The finding of impaired epithelial maturation despite the high hydroxyproline concentration in the OH lungs suggests an abnormality in epithelial-mesenchymal interaction that is not present in the equally small lungs of the NOH group.

DNA↗
PubMed1996

The apical membranes of maturing gut columnar epithelial cells contain the enzymatically active form of a newly identified fyn-related tyrosine kinase.

Recently, we isolated a new src family member from a rat small intestinal cDNA library which by RNase protection analysis is selectively expressed in the columnar epithelium of gut. Complete nucleotide sequencing of the gastrointestinal associated tyrosine kinase (gtk) has revealed that it is a rat homologue of frk/rak-a fyn related human tyrosine kinase. Unlike frk/rak, gtk is myristylated, in vivo. Furthermore, by immunohistochemical analysis, the kinase is concentrated in the brush border membranes of epithelial cells, throughout the maturation axis of the adult small intestine. In vitro analysis revealed that gtk kinase activity is present in intestinal cells throughout their maturation, suggesting that the enzyme might influence signal transduction pathways in both mitotic and post-mitotic states. Gtk is expressed in all regions of the gastrointestinal tract which contain columnar epithelium, but is absent in the stratified epithelium of the esophagus. Moreover, during gestation, the kinase dramatically appears at high levels in plasma membranes, at the time of transition of gut cells from an undifferentiated to a simple columnar phenotype. After solubilization of cellular membranes with Triton X-100, sucrose gradient analysis of gtk revealed that it partitions differently than c-yes, demonstrating that the brush border src kinases associate with different components of the plasma membranes. These findings suggest that gtk plays a specialized role in the growth/differentiation of gut columnar epithelial cells.

3T3 Cells↗
PubMed1999

The kinetics of epithelial cell generation: its relevance to cancer and ageing.

A hierarchical model of epithelial cell generation is proposed, in which even in extreme old age mature epithelial cells in humans are only a limited number of cell divisions from the zygote (60-120). This contrasts with conventional models in which regularly cycling stem cells can be several thousands of cell divisions from the zygote. The hierarchical model is supported by data on the rate of telomere shortening both in vivo and in vitro, and by data on the rate of synonymous substitutions in Y-linked, X-linked and autosomal genes in rodents. Limiting the number of cell generations leads to a vast reduction in the risk of cancer and reduces the rate of ageing. It is suggested that longer-lived animals need stricter control of the hierarchy than do shorter-lived animals and this difference has implications for theories of ageing.

Aging↗