The use of retinoids as probes for analyzing morphogenesis of glands from epithelial tissues.

Thirty-five years ago Honor Fell and Edward Mellanby were studying effects of high doses of vitamin A on skeletal development in chick embryos when they noticed that a piece of epidermis, accidentally included in an organ culture, had undergone mucous metaplasia. Further studies by Fell and others eventually led to an understanding of the important role of vitamin A in modulating epithelia in vivo. Fifteen years later another organ culture experiment showed me that excess vitamin A could also initiate the morphogenesis of branching and mucus-secreting glands from developing vibrissa follicles in upper lip skin of embryonic mice. Since then our group has shown that induction of this novel structure by naturally occurring retinoids resembles a normal embryonic induction in that it is stage-dependent, time-dependent, and irreversible. Tissue separation and recombination studies showed that isolated upper lip epidermis can form these glands when combined with retinoid-treated upper lip dermis. Untreated mouse epidermis can form similar glands after combination with chick dermis containing higher retinoid levels. The hamster cheek pouch, normally devoid of glandular structures, can also form mucous glands when treated with a retinoid, either in vivo or in vitro. Recombination studies in organ culture have now shown that mesenchyme exposed to retinoid is essential for gland morphogenesis from pouch epithelium. Evidence is accumulating that retinoic acid may even be the active morphogen in some normally developing systems.

Periarteriolar localization of mast cells promotes oriented interstitial migration of leukocytes in the hamster cheek pouch.

As studied by intravital microscopy, mast cell-dependent inflammatory reactions evoked by antigen or compound 48/80 in the hamster cheek pouch involved leakage of plasma and emigration of leukocytes exclusively from the venules. The leukocyte diapedesis and subsequent tissue migration induced by antigen or compound 48/80 were oriented from the venules towards adjacent arterioles. In contrast, leukocyte emigration induced by a mast cell-independent stimulus, leukotriene B4, did not show preferential orientation towards arterioles. Moreover, mast cells were abundant in the hamster cheek pouch, and they were localized predominantly along arterioles, rather than along venules. Because mast cells are considered to be the source of the chemotactic mediators causing the leukocyte emigration, the periarteriolar mast cell localization may be of functional significance by creating chemotactic gradients between arterioles and venules, thereby promoting oriented and effective interstitial migration of leukocytes. Whether or not a similar mechanism is operative in other species and tissues remains to be established, however, arteriolar predominance of mast cells was observed also in rat calvarial periosteum and in mouse skin.

Adherence of alpha-hemolytic streptococci to human endocardial, endothelial and buccal cells.

Human buccal, endothelial and endocardial cells were prepared and the adherence of different bacteria to these cells was tested in vitro. Buccal cells were scraped off immediately before use in the adherence tests. Endothelial and endocardial cells were prepared from human umbilical vein and human heart valves by using collagenase, and cultured in cell culture medium. Seventeen different bacteria were used in the adherence tests; ten strains of alpha-hemolytic streptococci, five from children with infective endocarditis (IE) and five from healthy carriers, two S. aureus, two N. meningitidis, two N. gonorrhoeae and one E. coli. The five alpha-hemolytic streptococcal strains from patients with IE showed significantly higher adherence values than those from healthy carries as well as in comparison with the remaining seven bacteria. The difference in adherence might be an expression for different bacteria surface properties. These differences might be important in explaining the infective mechanism in infective endocarditis.

Role of pili in adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells.

The ability of pili from Pseudomonas aeruginosa K (PAK) to act as an adhesin to human respiratory epithelial cells was examined using an in vitro adhesion assay. Equilibrium analysis of PAK binding to human buccal epithelial cells (BECs) and tracheal epithelial cells (TECs) by means of a Langmuir adsorption isotherm revealed that the maximum numbers of binding sites per epithelial cell (N) were 255 for BECs and 236 for TECs, with apparent association constants (Ka) of 2.8 x 10(-9) and 5.8 x 10(-9) ml/CFU, respectively. Trypsinization of the BECs before the binding assay increased N to 605 and decreased the Ka to 1.7 x 10(-9) ml/CFU. Addition of homologous pili to the binding assay with BECs or TECs or the addition of anti-pilus Fab fragments inhibited PAK adherence. Binding of purified pili to BECs was shown to reach saturation. Purified pili and PAK competed for the same receptor on the BEC surface. Further, by using peptide fragments of PAK pilin (derived from the native pili or produced synthetically) in the binding assay for PAK to BECs, we have presumptively identified the pilus binding domain in the C-terminal region of the pilin and shown that the C-terminal disulfide bridge is important in maintaining the functionality of the binding domain.

A simple two-dye basic stain facilitating recognition of mitosis in plastic embedded tissue sections.

Semithin sections of buccal and palatal mucosa fixed in 2.5% glutaraldehyde followed by 1% osmium and embedded in Durcupan (an araldite-based resin) were stained with 2% malachite green in 50% ethanol at 80 C and poststained in 0.05% crystal violet in Sorensen's phosphate buffer (pH 6.4) at 45 C. Nuclear envelopes and chromatin stain vivid purple in contrast to the surrounding green cytoplasm and cell borders. Chromosomes of dividing cells stain bluish violet. Nucleoli, depending on their level in the epithelium, stain differing shades of greenish blue. The distinct and differential staining of each of these components facilitates recognition of mitoses in oral epithelium, where the small size and crowding of cells in the proliferative compartment renders more conventional stains for plastic sections inadequate.

Initial lymphatics are present in the loose areolar connective tissue of the golden hamster's cheek pouch.

The immunologically privileged status of the hamster cheek pouch has been attributed to both the loose, areolar connective tissue that lines the pouch and to the lack of lymphatics in it. Although early reports described an absence of demonstrable lymphatics by classical histologic and lymphangiographic methods, issue has been taken recently as to whether the cheek pouch is alymphatic. We became interested in this issue accidentally during a study of microembolic microvascular injury. Our results, which were obtained by coordinated use of intravital, light, and transmission electron microscopy, showed that a small population of initial lymphatics are present in the loose, areolar connective tissue. The areal density of initial lymphatics in 12 cheek pouches averaged 1.5 lymphatics/cm2 of pouch connective tissue. We conclude that the hamster cheek pouch is drained by a small population of initial lymphatics, the general paucity of which probably contributes to the delayed egress of antigens to regional lymph nodes.

Phorbol ester binding to oral epithelial cells in the presence of retinoic acid or N-nitrosonornicotine.

Although the specific mechanisms by which phorbol ester tumour promoters exert their various effects are not known, their actions are mediated by cell membrane receptors which contain lipids as major components of the receptor complex. Since cell modulators such as retinoic acid (RA) and nitrosonornicotine (NNN) can alter cell lipids, the binding of a phorbol ester to cells was examined at time intervals when lipid changes mediated by these modulators occur. Epithelial cells prepared from hamster cheek pouches were treated with all-trans RA or NNN for varying periods of time, then specific binding of phorbol esters was investigated. Cells treated with RA for intervals up to 24 h showed decreased binding when compared with untreated cells. Those treated with NNN for up to 168 h showed increased specific binding. The results suggest that alterations in the cell lipids may affect the specific binding of phorbol esters to cells.

[The buccal adipose body (Bichat's fat-pad). Morphological study]. / Le corpus adiposum buccae (boule de Bichat). Etude morphologique.

Bichat's fat-pad was examined using dissections of newborns and adults, and histological sections of embryos and fetuses. A main part or body with six extensions (masseteric, superficial temporal, deep temporal, pterygomandibular, sphenopalatine and inferior orbital) can be recognized.

Modificaciones de los índices de exfoliación celular en la mucosa del paladar y carrillo de fumadores de tabacos (puros) / Modifications of cell exfoliation indexes in the mucosa of palate and cheek of cigar smokers

Con el propósito de determinar la influencia del hábito de fumar tabaco (puros) sobre los ritmos de maduración celular de la mucosa del paladar y el carrillo, se toman muestras citológicas de estas regiones a 57 individuos, fumadores desde hacía más de 5 años, mayores de 40 años y que al examen clínico no presentaban evidencias de algún tipo de lesión, los cuales se compararon con un grupo control de individuos de iguales características pero no fumadores. Las muestras fueron obtenidas mediante raspado de los carrillos y el paladar, con una espátula de madera, fijada en alcohol-éter y coloreadas con la técnica de Papanicolaou. Las observaciones se realizan en un microscopio de luz y se seleccionan para el recuento celular los campos microscópicos al azar. Se evaluaron más de 300 células por paciente, las que se consideraron atendiendo a su morfología y características de afinidad tintorial. Las medias de las células queratinizadas sin núcleos, tanto en el paladar como en los carrillos de fumadores de tabaco, fueron significativamente superiores al grupo control, lo que se interpreta como modificaciones de adaptación ante la agresión de los diferentes factores químico-físicos que interactúan en la acción de fumar

The morphological details of globular keratohyalin granules.

In quickly dividing epithelia such as that of the tongue, keratohyalin formation takes place in globular keratohyalin granules (KHG). This is in contrast with the irregular KHG as seen in normal, slowly dividing epidermis. The morphogenesis of the globular KHG is explained in this study. In small KHG, dense aggregates of ribosomes can be seen at the site of blebs. It is suggested that these blebs framed with ribosomes are internalized giving rise to "dense homogeneous deposits" or "single granules". Lipid droplets occur in the upper spinous and horny layer. Globular KHG also contain variable amounts of lipids, and the lipid content seems to be inversely related to the protein content, dependent on the degree of cell differentiation or on the rate of cell turnover. It is suggested that in epithelia with a high cell turnover few rigid keratohyalin components are dispersed in lipids, which maintain a globular shape due to the surface tension.

The influence of antifungal drugs on adherence of Candida albicans to buccal epithelial cells.

The adherence of two strains of Candida albicans serotype A to human epithelial cells was measured after exposure to different concentrations of amphotericin B, 5-fluorocytosine, nystatin, miconazole and ketoconazole. Germ-tube formation after different exposure times to the antifungal drugs as a preliminary test was carried out. Pretreatment of blastospores with minimum inhibitory concentrations (MIC) and sub-MIC (1/2 and 1/4 of MIC values) for 3 and 72 h did not affect adherence for all drugs tested except amphotericin B. This antimycotic agent reduces significantly the adherence either after 3 or 72 h exposure time. The other antifungal drugs interfere with adherence only after 72 h and at the highest concentrations tested, above MIC values. The decrease in adherence by antifungal drugs suggests that some of these drugs would be useful in the prophylaxis of patients at high risk for candidosis.

In vitro modulation of differentiation by calcium in organ cultures of human and murine epithelial tissue.

The differentiation of epithelial tissue in organ cultures of murine buccal mucosa, various human oral mucosa, and human newborn foreskin was found to be dependent on the calcium concentration of the culture media. In low calcium medium (less than or equal to 0.07 mM) epithelial differentiation was inhibited. The original stratifying layers separate and can be removed, producing a destratified explant. Histologically such an explant consists of a dorsal epithelial layer of basal keratinocytes resting on an intact basal lamina with subjacent stroma. At 0.01 mM calcium, the epithelial layer was one to two cells thick whereas at 0.07 mM it could be three or more layers in thickness with the most superficial cells being spread over the underlying cells. In addition to differentiation, keratinocyte migration over the sides of the explant (epiboly) and epithelial proliferation as determined by [3H]thymidine autoradiography were reduced by culture in low calcium medium. Redifferentiation occurs upon return to normal calcium levels (1.8 mM); addition of hydrocortisone to low calcium media was found to facilitate this redifferentiation.

Adherence of Staphylococcus strains to human urinary and buccal epithelial cells.

91 strains of Staphylococci belonging to different species were investigated for their adhesive capacity to urinary and buccal human epithelial cells. Furthermore the adherence of the same bacterial strains was evaluated in relation with their methicillin-sensitive and methicillin-resistant properties. S. aureus showed an high adhesion to buccal and urinary cells; S. saprophyticus and S. epidermidis attached mainly to uroepithelial cells and to buccal cells, respectively. Bacterial strains, either sensitive or resistant to methicillin antibiotic, did not exhibit significant differences of adhesive capacity.

Dietary-induced changes in the fatty acid composition of human cheek cell phospholipids: correlation with changes in the dietary polyunsaturated/saturated fat ratio.

Healthy normotensive volunteers aged 20 to 59 yr were randomly allocated either to a control group or to one of two experimental groups. The control group ate a low P/S ratio diet for 12 wk while the first experimental group ate a high P/S ratio diet for 6 wk followed by a low P/S ratio diet for the next 6 wk. The second experimental group ate a low P/S ratio diet in the first 6 wk followed by a high P/S ratio diet for the next 6 wk. Dietary P/S ratio, plasma linoleic acid (18:2), and cheek cell phospholipid 18:2 levels were compared in each dietary group at the end of the 1st and 2nd 6 wk. On change from a low to a high P/S ratio diet, there was a 36% increase in the proportion of 18:2 in the cheek cell phospholipids in comparison with the proportion existing before the change. This was associated with an increase in the proportion of 18:2 in the plasma lipids of this group. No reduction in the proportion of 18:2 in the cheek cell phospholipids was apparent in the control group or the group which changed from a high to a low P/S ratio diet, although in the latter group there was a reduction in the proportion of 18:2 in the plasma lipids. As the phospholipid fatty acid composition of human cheek cells reflects dietary lipid status under certain conditions, this observation may be useful in dietary and nutritional studies, particularly as human cheek cells can be obtained in a noninvasive manner.

In vitro binding of Candida albicans yeast cells to human fibronectin.

The binding of Candida albicans yeast cells to human fibronectin (Fn), a major glycoprotein of mammalian cells, was studied using an in vitro assay. Adherence was quantitated in microtiter dishes coated with Fn to which radiolabeled yeast cells were added. Under optimum conditions of the assay, i.e., 1 mM CaCl2 and 70 micrograms Fn protein, approximately 40% of the radiolabeled yeast cells adhered to the Fn. Adherence to Fn was greater at 30 degrees C than at 4 degrees C and was greater with viable yeast cells than with heat-killed cells. Candida albicans (two strains) and C. tropicalis adhered to Fn to a greater extent than C. pseudotropicalis, C. krusei, or Saccharomyces cerevisiae. Pretreatment of C. albicans with chymotrypsin, pronase, or papain, but not pepsin, decreased adherence to Fn. Blocking experiments using mannan, sugars, or amino sugars were carried out by preabsorbing the Fn with each of the above-mentioned compounds. Candida mannan blocked adherence of C. albicans to Fn. The mannan effect was dose dependent. However, adherence of C. albicans to Fn was not significantly reduced by mannose, glucose, or several other sugars. The role of FN as a receptor for the binding of C. albicans yeast cells to buccal and vaginal epithelial cells was investigated also using an in vitro assay. We determined, using indirect fluorescent antibody techniques, that both buccal and vaginal epithelial cells possessed Fn. In addition, yeast cells, when pretreated with Fn, showed reduced adherence with buccal and vaginal cells when compared with nontreated cells. These studies may indicate a role for Fn in the adherence of C. albicans to buccal and vaginal epithelial cells.

Human mucosal Langerhans cells: postmortem identification of regional variations in oral mucosa.

Modified ATPase histochemistry was used to identify and count Langerhans cells (LC) in autopsy tissue from 8 oral mucosal sites, 8-20 h postmortem. The specificity of the ATPase method was confirmed on serial sections with indirect immunofluorescence using monoclonal antibodies OKT6 and antihuman Ia. Average LC counts on ATPase-stained epithelial sheets from each of the 8 sites ranged from 160-550 LC/mm2. Nonkeratinized mucosae of the soft palate, ventral tongue, lip, and floor of the mouth had the highest counts (mean +/- SD, 508 +/- 110 LC/mm2, n = 24), and keratinized mucosae of the hard palate and gingiva had the lowest counts (201 +/- 97 LC/mm2; n = 8). LC frequency was variable in 2 sites: In the dorsal tongue, LC occurred on only one side of filiform papillae and were absent from regularly recurring areas of interpapillary epithelium. In the cheek mucosa, LC clustered around connective tissue papillae and their numbers showed marked individual variation (130-650 LC/mm2). The number of LC in nonkeratinized oral mucosa is approximately the same as in skin, but keratinized oral mucosa has fewer LC. The frequency of oral mucosal LC varies inversely with the degree of keratinization. There are regions of the oral mucosa that have no LC.

Identification of human connective tissue in transplant of human oral mucosa in nude mice.

The present study describes a method for identification of connective tissue of human oral mucosal transplants in nude mice. The method was based on the development of a murine antiserum to human fibroblasts. After absorption with murine fibroblasts the antiserum in an immunofluorescence method appeared to react specifically with human connective tissue of frozen sections, whereas the antiserum did not react with murine connective tissue. The antiserum, applied to frozen sections of human oral mucosal transplants in nude mice, could distinguish between human and murine connective tissue in the sections. The ability to distinguish between the two types of tissue was utilized to elucidate a possible relation between epithelial morphology and underlying type of connective tissue. It was found that the formation of rete ridges of transplanted human oral epithelium was dependent on the presence of subepithelial human connective tissue. The method described may be useful for the recognition of human tissue in experimental studies of human transplants to other species.