Protein kinase inhibitors and the dynamics of tight junction opening and closing in A6 cell monolayers.

This study focuses, in A6 cell monolayers, on the role of protein kinases in the dynamics of tight junction (TJ) opening and closing. The early events of TJ dynamics were evaluated by the fast Ca++-switch assay (FCSA), which consisted of opening the TJs by removing basolateral Ca++ (Ca++(bl)), and closing them by returning Ca++(bl) to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na+. The FCSA allows the evaluation of the effects of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in response to the FCSA followed single-exponential time courses. A rise of apical Ca++ (Ca++(ap))causes a reduction of TJ opening rate in an FCSA or even a partial recuperation of G, an effect that is interpreted as mediated by Ca++(ap) entering the open TJs. Protein kinase C (PKC) inhibition by H7 at low concentrations caused a reduction of the rate of junction opening in response to Ca++(bl) removal, without affecting junction closing, indicating that PKC in this preparation is a key element in the control of TJ opening dynamics. H7 at 100 microm completely inhibits TJ opening in response to Ca++(bl) withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase, a process that can be halted again by H7 reintroduction into the bathing solution. Differently from the condition in which Ca++ is absent from the apical solution, in which H7 halts the process of G increase in response to a FCSA, when Ca++ is present in the apical solution, addition of H7 during G increase in an FCSA not only induces a halt of the G increase but causes a marked recuperation of the TJ seal, indicated by a drop of G, suggesting a cooperative effect of Ca++ and H7 on the TJ sealing process. Staurosporine, another PKC inhibitor, differently from H7, slowed both G increase and G decrease in an FCSA. Even at high concentrations (400 nm) staurosporine did not completely block the effect of Ca++ withdrawal. These discrepancies between H7 and staurosporine might result from distinct PKC isoforms participating in different steps of TJ dynamics, which might be differently affected by these inhibitors. Immunolocalizations of TJ proteins, carried out in conditions similar to the electrophysiological experiments, show a very nice correlation between ZO-1 and claudin-1 localizations and G alterations induced by Ca++ removal from the basolateral solution, both in the absence and presence of H7.

The effect of a reconstituted basement membrane (matrigel) on a human salivary gland myoepithelioma cell line.

We have already demonstrated that a reconstituted basement membrane (Matrigel) is a key modulator of morphogenetic changes and cytodifferentiation of pleomorphic adenoma cells in culture. Myoepithelioma is considered to be a neoplasm closely related to pleomorphic adenoma and should experience similar induction processes. Thus, the aim of this study was to investigate whether Matrigel would influence myoepithelioma cells. We used a cell line derived from a human salivary gland plasmacytoid myoepithelioma (M1 cells) grown in a three-dimensional preparation of Matrigel. Phenotype differences were assessed using conventional light microscopy technique (haematoxylin and eosin) and phase and differential interference contrast (Nomarski). Immunofluorescence was carried out to detect smooth-muscle actin, laminin and type-IV collagen. M1 cells exhibited all proteins studied, showing a myoepithelial differentiation. M1 cells grown inside Matrigel presented morphological changes and changes in smooth-muscle actin status. By growing M1 cells inside Matrigel, it was possible to reproduce the tumour architecture with no duct-like structures. Based on our findings, we suggest that myoepithelioma would be derived from a cell with a commitment to myoepithelial differentiation. We also suggest that the mechanical properties of the matrix environment will likely regulate smooth-muscle actin expression in myoepithelioma.

Effect of N-CAM on in vitro invasion of human adenoid cystic carcinoma cells.

Adenoid cystic carcinoma of salivary glands is characterised by aggressive behaviour, high rate of local recurrences, neurotropism and late metastasis. In a previous work we demonstrated that adenoid cystic carcinoma cultured cells (CAC2 cells) expressed N-CAM. It was suggested that this expression, modulated by extracellular matrix, would be correlated to cell movement. The aim of our study was to verify whether CAC2 cells presented invasion capacity. Moreover, we tested whether the neural adhesion molecule (N-CAM) would participate in this process. CAC2 cells were either previously treated, or not (control), with a monoclonal antibody against N-CAM. Invasion assays were carried out using a modified Boyden chamber (Transwell chamber). CAC2 cells (10(5)) were dispensed into Transwell upper chamber on the top of Matrigel coated filter. The cells that invaded the filters in the first 8 h were counted under light microscopy, yielding data for the invasion rates (%). Control CAC2 cells presented an invasion rate of 5.28+/-0.04%. The invasion rate raised to 6.53+/-0.2% when N-CAM was blocked with monoclonal antibody. N-CAM impaired the adenoid cystic carcinoma cell invasion in vitro. Therefore, we suggest an anti-invasive role for N-CAM in adenoid cystic carcinoma.

Comparison of the low level laser therapy effects on cultured human gingival fibroblasts proliferation using different irradiance and same fluence.

BACKGROUND AND OBJECTIVE: The low level laser therapy (LLLT) has been used in Dentistry to improve wound healing. In order to analyse the effect of LLLT on the in vitro proliferation of gingival fibroblasts we developed a primary culture of human gingival fibroblasts. STUDY DESIGN/MATERIALS AND METHODS: The cell line named LMF was grown in Dulbecco's Modified Eagle's medium (DME) with either 5% (nutritional deficit) or 10% fetal bovine serum (fbs). Laser irradiation was carried out with diode lasers with the following wavelengths: 670 nm (L1), 780 nm (L2), 692 nm (L3), and 786 nm (L4). The fluence was fixed in 2 J/cm(2). For growth analysis, control (not irradiated) and treated cultures (irradiated) were plated in 60 mm diameter culture dishes for 12 h before the irradiation. RESULTS: We found that cells cultured in nutritional deficit condition grown in medium supplemented by only 5% fbs presented a cell proliferation rate significantly smaller that cell grown in ideal culture conditions (10% fbs). However, when irradiated, cells in nutritional deficit presented cell growth similar or higher than that of control cells grown in ideal culture conditions. Using the same fluence, the infrared laser induced a higher cell proliferation than visible laser when the power outputs were different. However, lasers of equal power output presented similar effect on cell growth independently of their wavelengths. CONCLUSIONS: The LLLT acts by improving the in vitro fibroblast proliferation and a smaller laser exposure time results in higher proliferation.

Verruciform xanthoma of the oral mucosa. Report of four cases and a review of the literature.

We present four new cases of verruciform xanthoma (VX) in the oral mucosa and review the literature. Clinical, histological, and immunohistochemical features of four new cases of VX were analysed together with cases found in a review of the literature. Expression of CD-68 was studied by immunohistochemistry. Only 162 cases were reported in the oral mucosa. Ninety were males (55.5%) and 72 were females (44.5%). Mean age was 44.9 years. The majority of cases occurred in masticatory mucosa (69.7%). Our cases exhibited papillary or verrucous proliferation of squamous epithelium associated with hyperparakeratosis and with numerous foamy cells confined to the lamina propria papillae. Foamy cells were positive to CD-68 antibody, showing a macrophagic nature. VX is a rare benign lesion, and is probably inflammatory. However, its aetiology and pathological mechanisms remain unknown.

Pulp response and cytotoxicity evaluation of 2 dentin bonding agents.

OBJECTIVE: This study evaluated the biocompatibility of two dentin bonding agents (Clearfil Liner Bond 2 and Scotchbond Multi-Purpose) applied in human dental pulps and cell cultures. METHOD AND MATERIALS: In vivo: Twenty human third molars that were scheduled for extraction were used. After cavity preparation, pulp exposure was achieved with a carbide bur. Hemorrhage control was obtained with saline solution. In 16 teeth, adhesive pulp capping was performed and the cavities were sealed with resin composite. In the control group (n = 4), pulps were capped with Ca(OH)2 and the cavities were sealed with IRM. Teeth were extracted 30 or 90 days following treatment and prepared for histological examination and bacterial detection. In vitro: materials were applied in Petri dishes, where NIH-3T3 cells were plated. The cells were counted 2, 4, and 6 days after plating to obtain the growth curves and to determine cell viability. All data were submitted to statistical analysis. RESULTS: In vivo: Dentin bridge formation was seen in all teeth capped with Ca(OH)2, without an inflammatory response. Mild inflammatory responses and dentin bridge formation after 90 days were observed in 50% of specimens treated with Liner Bond 2. Pulps treated with Scotchbond Multi-Purpose presented mild to severe inflammatory response, and no mineralized tissue formation was detected. Bacteria were not disclosed in any specimen. In vitro: The cytotoxicity was similar between the two bonding agents, and both had statistically higher cytotoxic effects (P < 0.002) than Ca(OH)2. CONCLUSION: Ca(OH)2 produced pulp healing in all teeth and exhibited lower cytotoxic effects than both adhesive systems; however, pulp healing was also observed under Liner Bond 2.

Cytotoxic effects of 10% citric acid and EDTA-T used as root canal irrigants: an in vitro analysis.

EDTA-T and 10% citric acid used as root canal irrigants lead to more visible dentinal tubules than 5% sodium hypochlorite associated with 3% hydrogen peroxide. However, these cleansing agents must be compatible with apical periodontal tissue. We analyzed the cytotoxicity of 10% citric acid and EDTA-T in cultured fibroblasts using Trypan blue. The solutions were diluted to 1%, 0.1%, and 0.01% and applied to NIH 3T3 cell cultures. Cells grown on fresh DMEM served as a control. After 0, 6, 12, and 24 h (short-term assay, viability) and 1, 3, 5, and 7 days (long-term assay, survival), the cells were counted using a hemocytometer. In short-term tests, cell viability ranged from 85% to 99% for all experimental groups with no statistical differences when compared with control cultures, except for the group treated with 1% EDTA-T, which caused a progressive decrease in cell viability. In long-term tests, all cultures increased in number from day 1 to the end of the experimental period, showing no inhibition of cell proliferation, except for the cultures treated with 1% EDTA-T, which totally prevented cell growth. All dilutions of 10% citric acid were more biocompatible than EDTA-T. Cultures treated with citric acid had a higher percentage of viable cells in the short-term assays, and the cells retained their self-renewal capacity.

Effect of a ceramic and a non-ceramic hydroxyapatite on cell growth and procollagen synthesis of cultured human gingival fibroblasts.

BACKGROUND: Ceramic hydroxyapatites and non-ceramic hydroxyapatites have been used extensively as alloplastic materials for bone reconstruction. However, different forms of hydroxyapatite induce different types of tissue response. METHODS: Human gingival fibroblasts (FMM1 cells) were used to analyze ceramic and non-ceramic hydroxyapatite biocompatibility. The cells were grown on surfaces covered either by collagen (control group), collagen plus ceramic hydroxyapatite, or collagen plus non-ceramic hydroxyapatite. Scanning electron microscopy, growth and cell viability curves, and procollagen immunoprecipitation were obtained. For the growth and viability curves, 10(4) cells were seeded on 60 mm dishes. Cells from each group were counted, in triplicate, at 1, 3, 4, 5, and 6 days after seeding using the Trypan blue dye exclusion assay. RESULTS: The cells grew in close contact with both types of hydroxyapatite particles. No differences were found in the amount of procollagen synthesis among any experimental group. The cultures treated with ceramic hydroxyapatite had a growth delay for the first 5 days. There was no difference in cell viability between the control group and the non-ceramic hydroxyapatite group. However, cultures treated with ceramic hydroxyapatite showed significantly lower viability percentages than the other groups. CONCLUSIONS: Hydroxyapatite supports cell growth and fibroblast metabolism including collagen production, and hence is biocompatible. Cell viability and structural studies showed that non-ceramic hydroxyapatite has relevant physical and biological properties as an implant material.

The role of basement membrane proteins on the expression of neural cell adhesion molecule (N-CAM) in an adenoid cystic carcinoma cell line.

We investigated the presence of neural cell adhesion molecule (N-CAM) in the adenoid cystic carcinoma cell line CAC2 using immunofluorescence microscopy. Additionally, we analysed the role of laminin and type IV collagen in N-CAM expression. We demonstrated that cultured adenoid cystic carcinoma cells express N-CAM. Control cells presented a scattered N-CAM expression on cell membrane, and type IV collagen had no effect in N-CAM distribution. CAC2 cells grown on laminin-coated coverslips expressed N-CAM concentrated on cell lamellipodia suggesting relationship with migratory activity. Our results showed that cultured adenoid cystic carcinoma cells express N-CAM and this expression is modulated by extracellular matrix.

Expression of cytokeratins in human enamel organ.

Cytokeratin (CK) is a filament which plays a central role in epithelial tissue and, like the polypeptides of intermediate filaments in general, shows a high degree of tissue specificity. The CK expression patterns of odontogenic epithelia are still poorly described. We studied the distribution of individual CK polypeptides in the human enamel organ at bell stage and in remnants of the dental lamina. Our immunohistochemical study showed that epithelial cells stained for CKs 7, 13, 14 and 19 with slight changes in their pattern during the differentiation phase of odontogenesis. There was negative staining for all other CK polypeptides tested (CKs 8, 10, 16, 17 and 18). Most of the CKs in the enamel organ epithelia did not show differences related to the stage-specific state of differentiation, except for CKs 14 and 19 at the inner enamel epithelium. A strong label for CK 14 was present at the inner dental epithelium at early bell stage, and this was substituted by CK 19 at the late bell stage when the ameloblasts were fully differentiated.

Small synthetic peptides homologous to segments of the first external loop of occludin impair tight junction resealing.

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca2+ removal and was characterized by a marked drop of TER. The reintroduction of Ca2+ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca2+ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions.

A collagen substrate enhances the sealing capacity of tight junctions of A6 cell monolayers.

A6 cells, a kidney derived epithelial cell line, when cultured either on a collagen-coated substrate or on polycarbonate substrate without collagen form confluent monolayers that are similar in cell density and overall morphology. However, the transepithelial electrical resistance (TER) of monolayers grown on the collagen-coated substrate is ninefold higher than that of monolayers grown without collagen. A comparative freeze-fracture study showed that this large difference in TER is not related to the length or number of tight junction strands but to differences in the specific conductance of individual strands. This conductance was obtained considering the TER, the linear junctional density and the mean number of tight junction strands. We estimated the specific linear conductance of the tight junction strands to be 2.56 x 10(-7) S/cm for cells grown on collagen and 30.3 x 10(-7) S/cm for the cells grown without collagen. We also examined changes in distribution and phosphorylation states of the zonula occludens associated protein, ZO-1, during monolayer formation. Immunocytochemistry reveals that the distribution of ZO-1 follows a similar time course and pattern independent of the presence or absence of collagen. While the amount of ZO-1 expression is identical in cells grown on both substrates, this protein is phosphorylated to a greater extent during the initial stages of confluence in cells cultured on collagen. We suggest that the phosphorylation levels of ZO-1 in A6 cells at the early stages of monolayer formation may determine the final molecular structure and specific conductance of the tight junctions strands.

Effect of spatial arrangement of the basement membrane on cultured pleomorphic adenoma cells. Study by immunocytochemistry and electron and confocal microscopy.

In a cell line from human pleomorphic adenoma (AP2 cells) we studied the response of these cells to basement membrane proteins. The culture was characterized as myoepithelial-like by transmission electron microscopy and immunocytochemistry. AP2 cells were grown in contact with a reconstituted basement membrane (Matrigel). Cells grown on Matrigel showed conspicuous phenotypic alterations, depending on how the substrate was applied. Cells grown on the top of Matrigel developed a dendritic phenotype, exhibiting thin, long and intercommunicating cytoplasmic extensions resembling normal myoepithelial cells. Cells grown inside Matrigel formed multi-layered clusters. Light, confocal and transmission electron microscopy showed that these clusters were formed by double-layered epithelioid cells delimiting luminal spaces. The cells facing the lumen were cuboidal, showing microvilli at the apical plasmalemmal and junctional complexes. The spatial arrangement of basement membrane is a key modulator of morphogenetic changes and cytodifferentiation of tumour myoepithelial cell lineage in culture.

Morphometric study of nucleolar organiser regions in ameloblastoma and basal cell carcinoma.

Ameloblastoma and basal cell carcinoma share histological similarities. Morphometric analysis of nucleolar organiser regions (NORs) from ameloblastoma and basal cell carcinoma (BCC) was carried out by silver (Ag) staining. Mean counts were lower in ameloblastoma (1.652 +/- 0.032) compared to those in BCC (2.354 +/- 0.054). Ameloblastoma presented one or two NORs per nucleus, in a narrow distribution (one to four NORs per nucleus). In contrast, BCC exhibited two or three NORs per nucleus, in a broad distribution (one to six NORs per nucleus). Perimeter and area measurements of AgNOR dots yielded significantly higher mean values for ameloblastoma. Our data suggest that most BCC cells are in mitosis, showing small and numerous NORs in each nucleus, while ameloblastoma cells are in interphase, showing one or two large NORs in each nucleus. Although ameloblastoma and BCC are neoplasms with similar growth patterns, they have cell populations with statistically significant differences in AgNOR patterns.

Expression of smooth-muscle actin in cultured cells from human plasmacytoid myoepithelioma.

OBJECTIVE: The definition of plasmacytoid myoepithelioma, a neoplasm exhibiting myoepithelial differentiation, has been recently questioned. To better understand the histogenesis of this neoplasm, we searched for myoepithelial markers in histologic sections of plasmacytoid myoepithelioma and in a cell line (M1) derived from this same neoplasm. STUDY DESIGN: Expression of vimentin, pan-keratin (AE-3) and smooth-muscle actin was studied by immunohistochemistry in paraffin-embedded tissue and by immunofluorescence in M1 cells. RESULTS: Plasmacytoid myoepithelioma tumor sections showed vimentin and AE-3 reactivity, but evidence of smooth-muscle actin was not seen. The cell line derived from this tumor also produced vimentin and cytokeratin. In addition, all cultured cells expressed smooth-muscle actin. CONCLUSION: We demonstrated that cells derived from a case of plasmacytoid myoepithelioma appear to show full myoepithelial differentiation in vitro. Thus, they are myoepithelial-like cells in nature. The lack of myogenous differentiation in vivo could be due to an inhibitory process mediated by the extracellular matrix.

Analysis of the interdependent localization of vimentin and microtubules in neoplastic myoepithelial cells.

Salivary gland neoplastic myoepithelial cells in culture form very thin cytoplasmic processes in which the vimentin network is well dispersed. These vimentin filaments can be individually visualized by immunofluorescence. In this study, we have analyzed the role of microtubules in the distension and organization of the vimentin filament network found in these cells. We find that vimentin filaments colocalize along microtubules; however, a significant number of filaments can also be found in microtubule-free domains. Additionally, vimentin filaments are absent from large domains of microtubule-rich domains. Treatment of neoplastic myepithelial cells with the microtubule inhibitor nocodazole did not cause any retraction of the distended vimentin network. This observation suggests that the structural integrity of microtubules is not important for the stability of the vimentin network. Combining procedures for transient disruption of vimentin filaments and microtubules we observed that, in the absence of microtubules, the vimentin network could reassemble in the perinuclear region but was unable to extend toward the cell periphery. The dispersion of vimentin filaments to the peripheral regions of the cytoplasm could only be observed upon microtubule reassembly. This indicates that microtubules are not required for the stability of the vimentin network, but the dispersion of vimentin filaments to the peripheral cytoplasm depends on active interactions with microtubules.

Modulation of tight junction morphology and permeability by an epithelial factor.

We report evidence of a factor secreted at the apical side of epithelial monolayers which modulates tight junction structure and permeability. This activity was detected within 4-7 days of conditioning of the apical medium by MDCK, A6 or Caco-2 epithelial cell lines cultured on permeable membranes in bipartite chambers. Apical conditioned medium (ACM), applied to the basolateral surface of a confluent monolayer, increased the transepithelial electrical resistance (TER), progressively reaching values 12-22% higher than the baseline within 5-10 min. After 40-60 min, the TER returned slowly to the basal value. This phenomenon was not observed either when using preheated ACM or the ACM filtrate obtained through a 30,000 MW cutoff membrane. The ACM maintained its activity even when applied to cell lines from different organs and species, as demonstrated when ACM from MDCK monolayers promoted an increase of 22% in the TER of Caco-2 cells. The increase of TER induced by the ACM treatment is accompanied by a change in the distribution of the number of tight junction strands, from an initial pattern, dominated mostly by junctions with one or two strands, to a new pattern after treatment dominated by junctions with two or three strands. Our results suggest the existence of a mechanism in epithelial cells that could signal leakage of apically secreted components to the basolateral side, thereby modulating the junction structure and permeability.

Feeding pattern in Peromyscus maniculatus: the response to periodic food deprivation.

The prairie deermouse (Peromyscus maniculatus bairdi) is strongly nocturnal on a 12:12 LD-cycle with a bimodal, dusk-dawn feeding pattern. It has been suggested for the rat that the dusk peak is more dependent on the animal's immediate energy needs while the dawn peak has an anticipatory function in storing food. The present study investigates this pattern in terms of the feeding response of deermice following periodic food deprivation. It was found that the response is not uniform within the D-phase; survivability, food intake, and maintenance of body mass were all favored by access to food during the final six hours vs the initial six hours. Deprivation experience enhanced survivability with the early-food, presumably through a reduction in locomotor activity and not by increased food intake. These results suggest a rigid temporal organization of feeding behaviors whereby early-night foraging and hoarding are necessary in allowing for late-night filling in anticipation of the L-phase.