Human Buccal Epithelial Cells Research Articles

Comparison of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis adhesive properties and pathogenicity

Retrospective studies indicate that Candida metapsilosis and Candida orthopsilosis each represents 1–10% of the infections/colonisations attributed to C. parapsilosis by conventional biochemical tests. Little is known on the virulence properties of these fungi and on their role in the establishment/progression of the infection. In this study, the adhesive properties of clinical isolates belonging to the ‘psilosis’ species were assessed in an in vitro model of co-incubation with human buccal epithelial cells (HBECs). Ectophosphatase activity was also measured for all isolates, since the activity of this enzyme has previously been linked to adhesion properties in C. parapsilosis. The results indicate that whilst C. parapsilosis and C. orthopsilosis strains showed similar adhesion abilities, C. metapsilosis isolates displayed a significantly lower ability to adhere to HBECs (P<0.05). No evidence of a correlation between ectophosphatase activity and adhesion was observed, and this finding was also confirmed by phosphatase inhibition experiments. Experimental vaginal candidiasis induced in oestrogen-treated mice with representative isolates of the 3 species indicated that mice infected with C. metapsilosis displayed a reduced vaginal fungal burden, especially in the early stages of the infection. The overall findings confirm that C. orthopsilosis has a comparable behaviour to C. parapsilosis, whilst C. metapsilosis seems to possess a reduced virulence potential.

EFFECT OF SUBMINIMAL INHIBITORY CONCENTRATION OF GENTAMICIN ON THE PATHOGENICITY OF ESCHERICHIA COLI CAUSING INFANTILE DIARRHOEA

One hundred patient infant complaining from infantile diarrhoea were studied, the percentage of E. coli infection was 24%. The in vitro adherence test was done to determine the adhesion capacity of bacteria to Human Buccal Epithelial Cells (HBECs). The result shows, increasing in the mean adhesion of E. coli as compared with the control group. Minimal Inhibitory Concentration (MIC) and sub Minimal Inhibitory Concentration (subMIC) of gentamicin was done to study the effect of subMIC of this antibiotic on the adhesion , the result shows that the using of gentamicin in lower doses (subMIC) decreases the adhesion capacity of bacteria .EPEC still mediclly important as a danger pathogen especially in poorly hyagiene communities such as Iraqi community.

ADHESION OF UROPATHOGENIC AND ENTEROPATHOGENIC Escherichia coli TO HUMAN BUCCAL EPITHELIAL CELLS

Fifty urine samples were collected from patients with urinary tract infections and a 50 stool specimens from diarrhoeal patients . These samples were investigated bacteriologically. The invitro adherence test of Enteropathogenic and Uropathogenic E. coli to healthy human buccal epithelial cells was carried out. The results show no significant differences in the mean adhesion of these bacteria (two sources) on the epithelial cells as compard with the control . but there is a little variation among EPEC isolates and among UPEC isolates too as compared with the control.

Occurrence of the nan1 gene and adhesion of Pseudomonas aeruginosa isolates to human buccal epithelial cells

Abstract This study shows an association between the frequency of the nan1 gene (encoding neuraminidase) among 62 clinical Pseudomonas aeruginosa isolates and adhesion of these bacteria to human buccal epithelial cells. The 52 strains in which the gene was present (83.9%) were characterized by a higher adhesiveness (the mean number of adhering bacteria was 23.51 per cell) than strains in which the gene was not detected (16.23 per cell) and the difference was significant (P = 0.009, Mann-Whitney U test). Thus we found that the nan1 gene may play a role in the binding of clinical P. aeruginosa strains to buccal cells.

Micronucleus investigation in human buccal epithelial cells of gutkha users.

Background:Gutkha is a cheap and convenient betel quid substitute, which is popular among all age groups. Various studies reveal its carcinogenic nature that leads to oral submucosus fibrosis and increases the chances of oral cancer. The micronucleus (MN) assay in exfoliated mucosal cells is a useful method for observing genetic damage in humans.Aim:To observe the genotoxic effect of gutkha on human buccal epithelial cells.Materials and Methods:The MN assay was performed to assess the frequency of MN in human buccal epithelial cells. The study comprises 60 individuals of which 30 individuals were gutkha chewers and another 30 were nonusers (control). The MN frequency was scored to estimate the genotoxic damage.Results:In gutkha users, the frequency of MN was highly significant (17.4 ± 0.944) as compared with nonusers (control) groups (4.53 ± 0.331) (P < 0.001).Conclusions:The MN assay in human buccal epithelial cells is a useful and minimally invasive method for monitoring genetic damage in humans. Asignificantly higher frequency of micronucleated cells are found among gutkha users.

Genotoxic effects of banding procedure with different orthodontic cements on human oral mucosa cells

To assess the genotoxic and cytotoxic effects of banding procedure with 5 different orthodontic cements on human oral buccal epithelium cells. Materials and methods: Fifty healthy volunteers (mean age: 14.54 ± 2.37 years) were randomly divided into 5 groups of 10. Preformed stainless steel molar bands (3M Unitek) were cemented to the upper and lower first molar teeth with 5 different orthodontic cements (Durelon, GC Fuji Ortho Band LC Paste Pak, Meron, Ultra Band Lok, and 3M Unitek Multi Cure) in each group. Genotoxic effects of the banding procedure for a 1-month period were evaluated using the micronucleus test (MNT). To monitor cytotoxic effects, binucleated cells (BNs), karyolysis (KL), and karyorrhexis (KR) were also evaluated in this setting. Results: Analysis of micronuclei in buccal epithelial cells revealed a significant increase in chromosomal damage in all groups (P < 0.01). Significant differences were found in the number of BNs in the groups receiving treatment with Meron, Ultra Band Lok, Durelon, and 3M Multi Cure (P < 0.01). Banding with GC Fuji and Durelon significantly elevated KL frequencies (P < 0.05). Durelon had a significant effect on KR (P < 0.01). Conclusion: Band cementation with conventional glass ionomer cement (Meron) showed the least genotoxic effects. The highest cytotoxic effects on human oral buccal epithelium cells were found in the polycarboxylate cement (Durelon) group.

Effects of sub-minimum inhibitory concentrations of gatifloxacin on the inhibition of Staphylococcus aureus and Escherichia coli adherence.

The aim of this study was to investigate the capacity of subinhibitory concentrations of the newly developed fluoroquinolone antibiotic gatifloxacin (CAS 160738-57-8) to interfere with the mechanism of bacterial adhesion. Human buccal epithelial cells were incubated with different strains of both Staphylococcus aureus and Escherichia coli, grown in the presence of subinhibitory gatifloxacin concentrations varying from 1/2 MIC (minimum inhibitory concentration) to 1/128 MIC. A significant decrease was observed in the adherence of S. aureus and E. coli to buccal cells at drug MICs of 1/2 to 1/32 and 1/2 to 1/64, respectively. A large number of filamentous forms of different lengths and shapes were observed in the case of E. coli, whereas there was an abnormal increase in the diameter of the cells in the case of S. aureus. The interpolation of these pharmacodynamic findings with the pharmacokinetic curve indicate that the effect of subinhibitory concentrations of gatifloxacin can prolong its antimicrobial effects against S. aureus and E. coli for as long as 30 h and 37 h, respectively, after the MIC values have been reached.

Effect of Permeation Enhancers on Buccal Absorption

This paper reports an investigation on matrices based on the mucoadhesive polymers hydroxypropylmethylcellulose and sodium alginate, intended for sublingual administration of 1 mg lorazepam (LZ, 7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one, CAS 846-49-1). The effect of different amounts of three permeation enhancers, cetylpyridinium chloride (CPC), polyethoxylated castor oil (PCO) and polyethylene glycol dodecyl ether (PGDE) on LZ permeation from the matrices was investigated using three models: (a) cultured monolayer of human buccal epithelial cells; (b) hamster cheek pouch mucosa in vitro, and (c) buccal administration to rabbits in vivo. In the first two models the presence of promoters, except when present at the higher concentrations, increased the drug permeation rate. The permeation-reducing effect was rationalized on the basis of micellar complexation of the drug. In the living rabbit (c) model, only CPC at the highest tested concentration was moderately active, while in the cultured cell model activity differences among the enhancers were less evident. Different effects of the promoters in the ex vivo and in vivo models were tentatively explained on the basis of the structural characteristics of the absorbing membranes. The present study, while confirming the efficacy of CPC as promoter in models involving biological membranes, does not provide conclusive data on the validity of the cultured cells model for assessment of buccal drug absorption.

Differentiation-Promoting Culture of Competent and Noncompetent Keratinocytes Identifies Biomarkers for Head and Neck Cancer

Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

IL-1 receptor regulates S100A8/A9-dependent keratinocyte resistance to bacterial invasion.

Previously, we reported that epithelial cells respond to exogenous interleukin (IL)-1α by increasing expression of several genes involved in the host response to microbes, including the antimicrobial protein complex calprotectin (S100A8/A9). Given that S100A8/A9 protects epithelial cells against invading bacteria, we studied whether IL-1α augments S100A8/A9-dependent resistance to bacterial invasion of oral keratinocytes. When inoculated with Listeria monocytogenes, human buccal epithelial (TR146) cells expressed and released IL-1α. Subsequently, IL-1α-containing media from Listeria-infected cells increased S100A8/A9 gene expression in naïve TR146 cells an IL-1 receptor (IL-1R)-dependent manner. Incubation with exogenous IL-1α decreased Listeria invasion into TR146 cells, whereas invasion increased with IL-1R antagonist. Conversely, when S100A8/A9 genes were knocked down using short hairpin RNA (shRNA), TR146 cells responded to exogenous IL-1α with increased intracellular bacteria. These data strongly suggest that infected epithelial cells release IL-1α to signal neighboring keratinocytes in a paracrine manner, promoting S100A8/A9-dependent resistance to invasive L. monocytogenes.

Adult human buccal epithelial stem cells: identification, ex-vivo expansion, and transplantation for corneal surface reconstruction

To identify adult human buccal epithelial stem cells (SCs) on the basis of two parameters (high p63 expression and greater nucleus/cytoplasmic (N/C) ratio) and to evaluate clinical efficacy of ex-vivo expanded autologous epithelium in bilateral limbal SC-deficient (LSCD) patients. The epithelial cells were isolated from buccal biopsy and cultured on human amnion in culture inserts with 3T3 feeder layer. The SCs were identified on the basis of two-parameter analysis using confocal microscopy, surface markers, and colony-forming efficiency (CFE). The cultured epithelium was transplanted in 10 LSCD patients followed by penetrating keratoplasty in 4 patients. The clinical outcome was followed up to 3 years. A distinct population (3.0±1.7%) of small cells expressing high levels of p63 with greater N/C ratio was observed in buccal epithelium. The N/C ratio was found to be more appropriate than cell diameter for two-parameter analysis. These cells located in the basal layer were negative for connexin-43 and positive for melanoma-associated chondroitin sulfate proteoglycan, containing holoclones with 0.2% CFE, thus representing the SC population. After transplantation of cultured epithelium with increased (sixfold) SC content, anatomical and visual improvement was observed at 13-34 months in 3/10 LSCD patients. The two-parameter SC marker is useful to identify and quantify buccal epithelial SCs. The transplantation of bioengineered SC-rich buccal epithelium is a strategy for corneal surface reconstruction in bilateral LSCD. However, further studies are required to optimize the culture conditions and to look for other sources of adult SCs for better visual outcome.

Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis

Tannerella forsythia, a Gram-negative anaerobe implicated in periodontitis, has been detected within human buccal epithelial cells and shown to invade oral epithelial cells in vitro. We have previously shown that this bacterium triggers host tyrosine kinase-dependent phosphorylation and actin-dependent cytoskeleton reorganization for invasion. On the bacterial side, the leucine-rich repeat cell-surface BspA protein is important for entry. The present study was undertaken to identify host signalling molecules during T. forsythia entry into human oral and cervical epithelial cells. Specifically, the roles of phosphatidylinositol 3-kinase (PI3K), Rho-family GTPases, cholesterol-rich membrane microdomains and the endocytic protein clathrin were investigated. For this purpose, cell lines were pretreated with chemical inhibitors or small interfering RNAs (siRNAs) that target PI3Ks, Rho GTPases, clathrin and cholesterol (a critical component of 'lipid rafts'), and the resulting effects on T. forsythia uptake were determined. Our studies revealed that T. forsythia entry is dependent on host PI3K signalling, and that purified BspA protein causes activation of this lipid kinase. Bacterial entry also requires the cooperation of host Rac1 GTPase. Finally, our findings indicate an important role for clathrin and cholesterol-rich lipid microdomains in the internalization process.

The influence of a non-ionic surfactant on the adherence of Candida albicans to human buccal epithelial cells

The influence of a non-ionic surfactant on the adherence of <i>Candida albicans</i> to human buccal epithelial cells

In Situ Cell-by-Cell Imaging and Analysis of Small Cell Populations by Mass Spectrometry

Molecular imaging by mass spectrometry (MS) is emerging as a tool to determine the distribution of proteins, lipids, and metabolites in tissues. The existing imaging methods, however, mostly rely on predefined rectangular grids for sampling that ignore the natural cellular organization of the tissue. Here we demonstrate that laser ablation electrospray ionization (LAESI) MS can be utilized for in situ cell-by-cell imaging of plant tissues. The cell-by-cell molecular image of the metabolite cyanidin, the ion responsible for purple pigmentation in onion (Allium cepa) epidermal cells, correlated well with the color of cells in the tissue. Chemical imaging using single-cells as voxels reflects the spatial distribution of biochemical differences within a tissue without the distortion stemming from sampling multiple cells within the laser focal spot. Microsampling by laser ablation also has the benefit of enabling the analysis of very small cell populations for biochemical heterogeneity. For example, with a ∼30 μm ablation spot we were able to analyze 3-4 achlorophyllous cells within an oil gland on a sour orange (Citrus aurantium) leaf. To explore cell-to-cell variations within and between tissues, multivariate statistical analysis on LAESI-MS data from epidermal cells of an A. cepa bulb and a C. aurantium leaf and from human buccal epithelial cell populations was performed using the method of orthogonal projections to latent structures discriminant analysis (OPLS-DA). The OPLS-DA analysis of mass spectra, containing over 300 peaks each, provided guidance in identifying a small number of metabolites most responsible for the variance between the cell populations. These metabolites can be viewed as promising candidates for biomarkers that, however, require further verification.

Pathogenic potential of Staphylococcus aureus strains isolated from various origins

Staphylococcus aureus isolates from various origins were investigated for their ability to produce different potential virulence factors. The highest prevalence of resistance among all the S. aureus isolates was shown for ciprofloxacin, clindamicin, and erythromycin. The supernatant from almost all isolates exhibited greater than 40% cytotoxicity. Staphylococcus aureus strains isolated from water had significantly greater ability to adhere to abiotic surfaces of clinical, food industry and medical device origin. The overall ability of S. aureus isolates to adhere to Vero cells was less than their adhesion to human buccal epithelial cells. Most of the strains tested were classified as highly hydrophobic and displayed hemolytic activity. Of the 27 S. aureus isolates tested, 17 were regarded as positive for phospholipase production. Staphylococcus aureus isolates from different sources were able to elaborate virulence factors; the incidence of virulence properties did not differ statistically among the various sources.

Effect of Phyllanthus emblica Linn. on candida adhesion to oral epithelium and denture acrylic

Objective To investigate the effect of Phyllanthus emblica (P. emblica) Linn. ethanolic extract on the adhesion of Candida albicans (C. albicans) to human buccal epithelial cells (BECs) and denture acrylic surfaces. Methods Human BECs and transparent acrylic strips were pretreated with ethanolic extract solution of P. emblica fruits at concentration ranged from 18.7 to 300 mg/mL. After washing BECs and the strips were inoculated with three strains of C. albicans (ATCC 10281 and two clinical isolates) (10 7 cells/mL). Normal saline solution (NSS) and 0.2% chlorhexidine gluconate were used as negative and positive controls, respectively. BECs were harvested on 12 μm-polycarbonate filters (Millipore, USA). The membrane filters and the strips were stained with Gram stain. Adherent yeast cells on 100 randomly selected epithelial cells and 20 randomly selected fields on each strip were counted under microscope. The statistical significance was calculated by Kruskal-Wallis and Tukey tests at a significant level of P< 0.05. Results Significant lower numbers of all strains of yeasts adhering to BECs and acrylic strips were observed after exposure to 75–300 mg/mL of plant extract compared with NSS. Conclusions The present study demonstrates that P. emblica ethanolic extract interferes with the adhesion of C. albicans to BECs and denture acrylic surfaces in vitro.

A naturally occurring isoform inhibits parathyroid hormone receptor trafficking and signaling

Parathyroid hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive receptor (PTHR). We describe here a PTHR isoform harboring an in-frame 42-bp deletion of exon 14 (Δe14-PTHR) that encodes transmembrane domain 7. Δe14-PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C-terminus of the Δe14-PTHR is extracellular, and cell surface expression is strikingly reduced compared with the PTHR. Δe14-PTHR displayed impaired trafficking and accumulated in endoplasmic reticulum. Signaling and activation of cAMP and ERK by Δe14-PTHR was decreased significantly compared with PTHR. Δe14-PTHR acts as a functional dominant-negative by suppressing the action of PTHR. Cells cotransfected with both receptors exhibit markedly reduced PTHR cell membrane expression, colocalization with Δe14-PTHR in endoplasmic reticulum, and diminished cAMP activation and ERK phosphorylation in response to challenge with PTH. Δe14-PTHR forms heterodimers with PTHR, which may account for cytoplasmic retention of PTHR in the presence of Δe14-PTHR. Analysis of the PTHR heteronuclear RNA suggests that base-pair complementarity in introns surrounding exon 14 causes exon skipping and accounts for generation of the Δe14-PTHR isoform. Thus Δe14-PTHR is a poorly functional receptor that acts as a dominant-negative of PTHR trafficking and signaling and may contribute to PTH resistance. © 2011 American Society for Bone and Mineral Research.

Adherence of Candida albicans and Candida parapsilosis to epithelial cells correlates with fungal cell surface carbohydrates

Many studies have described the adherence of Candida albicans to epithelial cells but little is known about Candida parapsilosis adhesion and its role in host cell surface recognition. This study was designed to evaluate the correlation between the adherence of 20 C. albicans and 12 C. parapsilosis strains to human buccal epithelial cells and the expression of fungal cell surface carbohydrates using lectin histochemistry. Adherence assays were carried out by incubating epithelial cells in yeast suspensions (10(7) cells ml(-1) ) and peroxidase conjugated lectins (Con A, WGA, UEA I and PNA at 25 μg ml(-1) ) were used for lectin histochemistry. The results showed that adherence was overall greater for C. albicans than for C. parapsilosis (P < 0.01) and that the individual strain differences correlated with a high content of cell surface α-l-fucose residues as indicated by the UEA I staining pattern. Based on the saccharide specificity of the lectins used, these results suggest that l-fucose residues on cell surface glycoconjugates may represent recognition molecules for interactions between the yeast strain studied and the host (r = 0.6985, P = 0.0045). In addition, our results indicated the presence of α-d-glucose/α-d-mannose, N-acetyl-D-glucosamine/N-acetylneuraminic acid and D-galactose/N-acetyl-D-galactosamine in fungal cell wall.

Comparative study of adherence of oral Candida albicans isolates from HIV sero-positive individuals and HIV sero-negative individuals to human buccal epithelial cells

Candida albicans occurs as a commensal of the gastrointestinal tract. Under predisposing conditions, candida can produce a broad array of infections. HIV seropositive individuals show increased oral colonization compared to the HIV seronegative healthy individuals. C. albicans shows a variety of pathogenic factors. We have studied one such factor here; the adherence property of C. albicans isolated from HIV seropositive individuals and HIV seronegative to Human Buccal Epithelial Cells (HBEC) of normal healthy individuals. Concentrated oral rinse specimen were collected from 50 healthy volunteers (control group) and 25 HIV positive individuals (test group) and used for isolation of C. albicans. Adherence assay was done using C. albicans isolates from both groups on HBEC collected from HIV sero-negative, normal individuals. The adherence assay method described by Kimura and Pearsall was used with minor modification. The results of Adhesion assay were subjected to statistical analysis using student "t" test. C. albicans isolated from both the groups were tested for their adherence property to normal HBEC. The isolates from test group showed more adherence to HBEC compared to those of the control group, with average rate of adherence being 56.6%. The control group showed average adherence rate of 29.1%. This was statistically significant with p value equal to 0.05. C. albicans from HIV infected individuals showed significant rise in degree of adhesion to the buccal epithelial cells than the isolates from healthy controls, suggesting the enhancement of virulence factors such as adherence in the presence of predisposing condition.

Effect of phenotypic switching on expression of virulence factors by Candida albicans causing candidiasis in diabetic patients

A total of 110 strains belonging to seven species of Candida were isolated from various forms of candidiasis in diabetic patients. They were Candida albicans 53 (47%), Candida tropicalis 36 (33%), Candida glabrata 9 (8%), Candida parapsilosis 4 (4%), Candida guilliermondii 2 (2%), Candida krusei 5 (5%) and Candida kefyr 1 (1%). All 53 strains of C. albicans isolated were observed to express virulence factors such as cell surface hydrophobicity (CSH), adherence to human buccal epithelial cell (BEC) and proteinase activity (100%), while phospholipase activity was observed in 52 (98%). Phenotypic switching and its influence on the pathogenicity of C. albicans were studied. Two C. albicans strains isolated from oral and vaginal thrush, respectively, in diabetic individuals, and the control strain C. albicans NCPF 3153A were induced to undergo phenotypic switching by exposure to UV light and the degree of expression of virulence factors by the different morphological forms was determined. Three different morphological forms of C. albicans were obtained, namely Star (S), Wrinkled (W) and Ring (R) types from the original Smooth (O) variety. It was found that proteinase activity was greatest with the W type followed by the R type then the O type. The S type produced the least proteinase. The phospholipase activity was greatest with O type followed by R type. The W and S types produced the least phospholipase. Expression of CSH and adherence was greatest in the O type followed by the R and then the W type and finally the S type. Differential expression of virulence factors occurs with different phenotypic forms of C. albicans and this may provide a particular morphological type with a distinct advantage over other types in causing candidiasis.