ABSTRACT
Abstract Objective Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen family. Although its expression has been found in chronic oral inflammatory epithelium, this study aimed to know whether CEACAM1 in oral keratinocytes participates in host immune response against Candida albicans . Methodology We investigated CEACAM1 expression in oral keratinocytes induced by C. albicans as well as by Candida cell wall component β-glucan particles (β-GPs). Furthermore, the effects of CEACAM1 on β-GPs-induced heme oxygenase-1 (HO-1) expression and its related signals were examined. Results Fluorescence staining showed CEACAM1 expression in oral keratinocytes (RT7) cells, whereas quantitative reverse transcription (RT)-PCR indicated that both live and heat-killed C. albicans increased CEACAM1 mRNA expression in RT7 cells. Examinations using quantitative RT-PCR and western blotting indicated that CEACAM1 expression was also increased by β-GPs derived from C. albicans . Specific siRNA for CEACAM1 decreased HO-1 expression induced by β-GPs from C. albicans as well as the budding yeast microorganism Saccharomyces cerevisiae . Moreover, knockdown of CEACAM1 decreased β-GPs-induced ROS activity in the early phase and translocation of Nrf2 into the nucleus. Conclusion CEACAM1 in oral keratinocytes may have a critical role in regulation of HO-1 for host immune defense during Candida infection.
ABSTRACT
Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/ HTLV-1-Associated Myelopathy (HAM/TSP) patients and 11 asymptomatic carrier individuals (AC) coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14 percent (8/14) HAM/TSP patients and 27.28 percent (3/11) AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.
Subject(s)
Humans , Deltaretrovirus Antibodies , HIV , Immunohistochemistry , Immunophenotyping , In Vitro Techniques , Keratinocytes , Polymerase Chain Reaction , Reticuloendotheliosis virus , Tumor Virus Infections , Methods , PatientsABSTRACT
Objective To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes by being seeded on bladder acellular matrix graft(BAMG). Methods Twenty-four male rabbits were randomly divided into 2 groups:experimental group and control group.A length of 2.0 cm and width of 0.8 cm penile urethral mucosal defect was induced in the anterior urethra 2.0 cm awav from the urinary meatus in all the rabbits.Oral keratinocytes were isolated from a small buecal mucosa(1.0 cm×0.4 cm)of the 12 rabbits in experimental group and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3).Passage 2 oraI keratinocytes cuItured with i3T3 were expanded and seeded onto sterilized BAMG(2.2 cm×1.0 cm)to repair the de-fects of the urethra.Urethroplasty was performed with BAMG with no cell seeding in the controlgroup.Catheter examination and retrograde urethI ography was done in 1,2 and 6 months after graft-ing.The urethral grafts were harvested and analyzed by histological staining. Results Oral kerati-nocytes seeded onto a feeder layer of i3T3 had better morphous and amplification capability.The corn-patibility of the compound graft was assessed by HE staining and scanning electron microscope.Twelve rabbits in the experimental group could void without difficulty.Catheter examination and ret-rograde urethrogram revealed that a wide urethral caliber was maintained with no sign of strictures and fistula formation.Histological examination showed the grafted urethra was covered with smooth mu- cosa with no strictures at 1,2 and 6 months.The oral keratinocytes of the graft still existed even 6 months after grafting.On contrast,gross and urethrogram demonstrated urethral stricture in the con-trol group.And inflammatory reactions could be found in the area of the stricture through light micro-scope. Conclusions Oral keratinocytes have good compatibility with BAMG and the compound graft could be used for urethral reconstruction in the animal model.
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Objective To explore the cultural method of oral keratinocytes and make preparations for further investigation in using oral keratinocytes as a new choice of seed cells for the reconstruction of tissue-engineered urethra. Methods Oral keratinocytes of rabbits were isolated in vitro and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3) or a culture dish without i3T3 respectively. Cell morphology was observed and cell growth was detected at intervals.Meanwhile,oral keratinocytes obtained from in vitro culture were performed immunofluorescence staining with broad-spectrum keratin antibody(AE1/AE3) and keratin 19 antibody(K19).The percentage of positive cells of passage 2 reactive to AE1/AE3 was assessed by flow cytometry. ResultsOral keratinocytes seeded onto a feeder layer of i3T3 exhibited finer morphous,better amplification capability,and could be passed for 7 or 8 generations.However,those cultured without i3T3 took on various morphous and could only be subcultured 2 generations before ageing.It was indicated by immunofluorescence staining that oral keratinocytes obtained from in vitro culture were positive for AE1/AE3 staining and 40% were positive for K19 staining.The result of flow cytometry revealed that the amout of positive keratinocytes reactive to AE1/AE3 was more than 95% of total cellular score. Conclusion Oral keratinocytes of rabbits can be cocultured with i3T3 in vitro and magnitude quantity can be attained,laying a favourable foundation for oral keratinocytes as a new choice of seed cells for urethral reconstruction with tissue engineering.
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The purposes of this study were to develop an in vitro co-culture model of epithelial tissue with dermal equivalent, cultured at an air-liquid interface, and to evaluate the effects of extracellular matrix and concentration of calcium and fetal bovine serum in medium to find optimized culture condition. Oral keratinizing epithelial cells in monolayer culture were grown in Mitomycin-treated 3T3 feeder. Primary cultured oral epithelial cells were reconstituted onto the dermal equivalents consisting of 3T3 fibroblast and type I collagen, and co-culture was grown at the air-liquid interface. The histomorphological development of reconstituted oral epithelium in vitro for 21 days revealed 10~12 layered statified epithelium, closely similar to the parakeratinized gingival epithelium. Neither laminin nor type IV collagen was able to induce keratinocyte differentiation. But a mixture of laminin and type IV collagen induced well-polarized keratinizing tissue with anchoring structure of basal cells. When the reconstituted oral epithelium was incubated in 1.0% and 0.5% serum-containing medium, the granular cell layers with orthokeratinization developed. The reconstituted epidermis generated in serum-free keratinocyte growth medium (KGM)-containing pituitary extract showed features of incomplete differentiation. The present study shows that the dermal equivalents containing fibroblasts will support epidermal morphogenesis and differentiation. And these results suggest that extracellular matrix and calcium concentration are important factors during the reconstitution of keratinizing epithelium in vitro.