Epithelial-mesenchymal transition in oral cancer cells induced by prolonged and persistent Fusobacterium nucleatum stimulation.

OBJECTIVES: Several studies have reported the effects of Fusobacterium nucleatum stimulation on oral cancer cells. However, given that these studies typically span a stimulation period of three days to eight days, the in vitro studies conducted to date may not fully mimic the oral cancer environment, which involves constant exposure to oral commensal bacteria. This study aimed to elucidate the effects of prolonged and persistent Fusobacterium nucleatum infection on oral cancer cells. METHODS: Human tongue squamous cell carcinoma (SCC) cells were continuously stimulated with Fusobacterium nucleatum for two or four weeks, then experimentally evaluated. RESULTS: Prolonged, persistent Fusobacterium nucleatum stimulation increased the cells' proliferative, invasive, and migratory capacities, decreased their expression of epithelial markers, and increased their expression of mesenchymal markers progressively with time. The cells also adopted a spindle-shaped morphology and cell-to-cell contact dependence was progressively lost, suggesting time-dependent occurrence of epithelial-mesenchymal transition. Furthermore, mRNA levels of CD44, a cancer stem cell marker, were time-dependently upregulated. When SCC cells were stimulated with Fusobacterium nucleatum for four weeks in the presence of dexamethasone, Fusobacterium nucleatum induced epithelial-mesenchymal transition was inhibited. CONCLUSIONS: Epithelial-mesenchymal transition in human tongue SCC cells was time-dependently induced by prolonged, persistent Fusobacterium nucleatum stimulation and inhibited by dexamethasone. Routine decontamination of the oral cavity may be crucial for controlling tumor invasion and metastasis.

Aggregatibacter actinomycetemcomitans infection causes DNA double-strand breaks in host cells.

Periodontal disease, an inflammatory disease, is caused by infection with periodontal pathogens. Long-term periodontal disease increases the risk of oral carcinogenesis. Similar to other peptic cancers, oral carcinogenesis also requires multiple genome instabilities; however, the risk factors related to the accumulation of genome instabilities are poorly understood. Here, we suggested that specific periodontal pathogens may increase the risk of genome instability. Accordingly, we screened several periodontal pathogens based on the ability to induce DNA double-strand breaks (DSBs) in host cells. We found that Aggregatibacter actinomycetemcomitans Y4 infection induced DSB formation in host cells. To assess whether DSB formation induced by infection with A. actinomycetemcomitans occurred through apoptotic chromosome fragmentation, cells were treated with a caspase inhibitor, Z-VAD-FMK. DSB accumulation induced by infection with A. actinomycetemcomitans was observed, even in the presence of Z-VAD-FMK, suggesting that this breakage occurred independently of apoptosis. These results suggested that some periodontal pathogens can increase the risk of genome instabilities in host cells and subsequently increase the risk of carcinogenesis.

[The adhesive and antagonistic activity of microflora of oral cavity in patients with malignant neoplasms of tongue].

The data concerning the degree of adhesion and antagonism of microorganisms of oral cavity in patients with malignant neoplasms of tongue is presented. it is established that pathogenic and conditionally pathogenic microflora has more mainly high and less infrequent degree of adhesion. the normal microflora has average and low degree of adhesion. About 90% of lactobacilla of oral cavity manifest no antagonism concerning pathogenic and conditionally pathogenic microorganisms. The antagonism of enterobacteria, staphylococcii and lactobacilla is absent. The staphylococci, including producing ß-lactamase and methicillin-resistant ones, in 95% of cases are antagonists to streptococcii. The clinical isolates Candida albicans show antagonistic activity related to 90% of lactobacilli and 20°% of streptococci.

Mycoplasma salivarium as a dominant coloniser of Fanconi anaemia associated oral carcinoma.

Mycoplasma salivarium belongs to the class of the smallest self-replicating Tenericutes and is predominantly found in the oral cavity of humans. In general it is considered as a non-pathogenic commensal. However, some reports point to an association with human diseases. M. salivarium was found e.g. as causative agent of a submasseteric abscess, in necrotic dental pulp, in brain abscess and clogged biliary stent. Here we describe the detection of M. salivarium on the surface of a squamous cell carcinoma of the tongue of a patient with Fanconi anaemia (FA). FA is an inherited bone marrow failure syndrome based on defective DNA-repair that increases the risk of carcinomas especially oral squamous cell carcinoma. Employing high coverage, massive parallel Roche/454-next-generation-sequencing of 16S rRNA gene amplicons we analysed the oral microbiome of this FA patient in comparison to that of an FA patient with a benign leukoplakia and five healthy individuals. The microbiota of the FA patient with leukoplakia correlated well with that of the healthy controls. A dominance of Streptococcus, Veillonella and Neisseria species was typically observed. In contrast, the microbiome of the cancer bearing FA patient was dominated by Pseudomonas aeruginosa at the healthy sites, which changed to a predominance of 98% M. salivarium on the tumour surface. Quantification of the mycoplasma load in five healthy, two tumour- and two leukoplakia-FA patients by TaqMan-PCR confirmed the prevalence of M. salivarium at the tumour sites. These new findings suggest that this mycoplasma species with its reduced coding capacity found ideal breeding grounds at the tumour sites. Interestingly, the oral cavity of all FA patients and especially samples at the tumour sites were in addition positive for Candida albicans. It remains to be elucidated in further studies whether M. salivarium can be used as a predictive biomarker for tumour development in these patients.

Cornu cutaneum-like HPV 45 positive subungual squamous cell carcinoma.

A cornu cutaneum (cutaneous horn) is a clinical descriptive term referring to a skin tumor having the appearance of an animal horn. Histopathologically, the lesion may be associated with several different tumors. Along with some benign entities such as warts or seborrheic keratoses, a cutaneous horn may be also associated with actinic keratoses, Bowen disease and squamous cell carcinoma. When an underlying malignancy is present, the entire tumor must be removed with careful control of margins. We report a case of a 68-year-old man with a cornu cutaneum-like HPV 45 positive sub-ungual squamous cell carcinoma on the left second toe.

Using attenuated Salmonella typhi as tumor targeting vector for MDR1 siRNA delivery.

OBJECTIVE: To investigate the feasibility of using attenuated Salmonella typhi as an in vivo delivery vector for multidrug-resistance gene (MDR1) small interference RNA (siRNA) in a mouse model bearing human tongue squamous cell cancer. This technique may represent a novel and effective route for the in vivo administration of siRNA against malignant tumors. METHODS: The cisplatin (DDP)-resistant human tongue squamous cell carcinoma cell line Tca8113/DDP, which highly expresses the MDR1 gene, was established by exposure to gradually increasing concentrations of cisplatin. A plasmid MDR1 siRNAwas constructed and transformed into attenuated Salmonella typhi strain SL7207. Tca8113/ DDP cells were infected with recombinant salmonella and expression of the MDR1 gene encoding P-glycoprotein (P-gp) product was detected. Tca8113/DDP tumor-bearing nude mice were established by inoculation by gavage administration of recombinant salmonella and were simultaneously injected intraperitoneally with cisplatin. Tumor growth was observed. RESULTS: Recombinant salmonella-bearing MDR1 siRNA expression plasmids can infect Tca8113/DDP cells in vitro and suppress P-gp expression and reverse DDP tolerance in Tca8113/DDP cells. Oral administration of recombinant salmonella in tumor-bearing nude mice can suppress tumor proliferation and enhance the therapeutic effect of DDP. CONCLUSION: Attenuated Salmonella typhi is expected to act as an in vivo targeting delivery vector for siRNA in tumor tissues.

Human squamous cell carcinoma of the tongue and colon undergoes apoptosis upon phagocytosis of Saccharomyces cerevisiae, the baker's yeast, in vitro.

The present study was carried out to evaluate the effect of phagocytosis of killed yeast on the induction of apoptosis in two human solid tumors of the gastrointestinal (GI): the squamous cell carcinomas of the tongue (SCCA) are (SCC-4 and SCC-9) and the adenocarcinomas of the colon (ADENOCA) are (Caco-2 and DLD-1). Cancer cells were cultured with heat killed Saccharomyces cerevisiae, baker's and brewer's yeast, at ratio of yeast to cancer cells = 10:1. The percentage of tumor cells that had attached/phagocytosed yeast and oxidative burst was determined by using oxidative sensitive dye (DCFH-DA) and flow cytometry. SCC-4 and colon Caco-2 cells demonstrated initial high levels of phagocytosis that peaked (35.8-52.8%) at 2 hr. The oral SCC-9 and colon DLD-1 cells demonstrated low phagocytic activity (7-12%). Phagocytosis was not associated with oxidative burst. Upon phagocytosis of yeast, cancer cells underwent apoptosis that was maximized at 4 hr. Yeast-induced apoptosis was significant in SCC-4 (45%), as compared with SCC-9 cells (17%), and Caco-2 (76%), as compared with DLD cells (12%). Apoptosis in cancer cells was inhibited by caspase inhibitor, Z-VAD-FMK; this suggests that caspases may be involved in apoptosis of the GI cancers. This data may have clinical implications for the treatment of solid tumors.

Ultrastructural findings in mucocutaneous infections of patients seropositive to HIV.

Tissue samples from 19 HIV-seropositive immunocompromised patients suffering from oral hairy leukoplakia, chronic vesicular or ulcerative herpes simplex, chronic nonmetameric herpes zoster, secondary syphilis, condylomata acuminata, molluscum contagiosum, or disseminated cutaneous mycobacteriosis were examined ultrastructurally in order to better define the fine structure of the causative organisms in parasitic conditions and to clarify the host-parasite relationships. Taking into account the few data in the literature regarding the same disorders in immunocompetent subjects, no striking differences in the morphology of the infectious agents or in the types of parasitism were found. Nevertheless, isolated herpesvirus and papillomavirus virions were found outside the infected cells, and this observation, if confirmed in a larger series of cases, could suggest a persistent infectivity of the lesions in immunocompromised patients. Moreover, electron microscopy proved to be useful for diagnostic purposes; in one case of disseminated cutaneous mycobacteriosis, repeated cultures failed to grow the organism.

Epstein-Barr virus in lingual epithelium of liver transplant patients.

Thirty-three liver transplant patients and 20 controls were examined for the presence of EBV in epithelium of the lateral border of the tongue by negative staining electron microscopy. Five of the specimens (15%) showed particles typical of the herpes virus family, while all controls were negative for EBV. In 3 of the 5 patients moderate oral hairy leukoplakia was observed clinically. Our results indicate that EBV may be expressed at the lateral border of the tongue of liver transplant patients, who in some cases show oral hairy leukoplakia clinically.

Detection of Epstein-Barr virus and human papillomavirus type 16 DNA in hairy leukoplakia by in situ hybridisation and the polymerase chain reaction.

Demonstration of Epstein-Barr virus (EBV) is considered desirable for the accurate diagnosis of hairy leukoplakia (HL). Previous studies have reported possible associations with human papillomavirus (HPV) infection although this is not a universal finding. Presence of EBV and HPV 16 was examined in biopsy specimens from 18 cases of HL and ten control specimens by in situ hybridisation using digoxigenin-labelled synthetic oligonucleotide probes and by the polymerase chain reaction (PCR). The presence of EBV was demonstrated in 12 cases by both techniques. Of the remaining six cases EBV could be detected in three by in situ hybridisation but not by PCR; EBV was not detected by either method in a further three cases. All samples were negative for HPV 16 by both techniques under conditions of high stringency, although when stringency of in situ hybridisation was reduced, four samples appeared to harbour HPV DNA sequences. This study provides further evidence to support the role of EBV in the pathogenesis of HL and suggests that HPV 16 is not regularly encountered.

Human papillomavirus in oral verrucal-papillary lesions. A comparative histological, clinical and immunohistochemical study.

The purpose of this study was to determine the presence of Human Papillomavirus (HPV) virions in the verrucal-papillary (OVP) lesions by clinical, histological and immunohistochemical [peroxidase-antiperoxidase (PAP) features]. Thirty seven positive cases were compared with 47 negative cases. Considerable overlapping of clinical and histological features can be seen among all these lesions. The study provides evidence that there are no clinical or histologic features with taken alone are strongly correlated with the presence of virions in oral lesions.

Human papillomavirus type 16 DNA in oral white sponge nevus.

White sponge nevus (WSN) is a benign hereditary lesion of the mucous membranes. DNA extracted from a biopsy specimen of oral WSN was assayed for the presence of DNA sequences homologous to human papillomavirus (HPV) types 1, 2, 4, 6, 11, 13, 16, and 18 by Southern blot hybridization. Only HPV-16 homologous DNA sequences were detected at a copy number of approximately 200 to 250 genome copies per diploid cell. The viral DNA sequences did not appear to be integrated into the host cell chromosome. The finding of HPV-16 in an inherited lesion such as WSN indicates that caution must be exercised in ascribing a causal association in relation to the demonstration of HPV in other mucosal disorders.

Immunocytochemical detection of herpes viruses in oral smears of HIV-infected patients.

Cytologic smears (CS) were taken from the lateral border of the tongue of HIV-seropositive patients (HIV+) (n = 39) and of seronegative controls (HIV-) (n = 19) and examined by immunocytochemistry (APAAP) and in situ hybridization (ISH) (biotinylated DNA probes) for the presence of viral antigens/DNA of EBV and CMV. While none of the HIV controls showed positive results for EBV antigen, 61% (APAAP) resp. 79% (ISH) of oral epithelial cells in the group of HIV+ patients were EBV-positive. While all CS taken from areas with the clinical diagnosis of hairy leukoplakia (HL) were EBV positive (APAAP and/or ISH), the detection of EBV in CS from uninvolved oral mucosa seemed to be associated with the later development of HL. In the group of HIV+ patients the detection rate for CMV was about five times (APAAP) resp. three times (ISH) higher than in HIV- persons. This non-invasive technique seems to be a valuable tool to screen for viral antigens/genomes.

Detection of human papillomavirus DNA sequences in tongue squamous-cell carcinoma utilizing the polymerase chain reaction method.

Twenty-four cases of tongue squamous-cell carcinoma (SCC) were analyzed for human papillomavirus (HPV) DNAs by the polymerase chain reaction (PCR) method and the dot-blot hybridization technique. HPV DNAs were detected in 8 cases. One specimen histopathologically diagnosed as poorly differentiated grade-III SCC contained both HPV-16 and HPV-18 DNA, and 7 other cases contained HPV-16 DNA.

Application of molecular techniques in the rapid diagnosis of EBV-associated oral hairy leukoplakia.

A rapid method for the detection of EBV-DNA in paraffin sections of lesions of oral hairy leukoplakia (OHL) is described. The method makes use of advances in molecular technology, including the use of synthetic oligonucleotides with digoxigenin labelling in an in situ hybridisation (ISH) reaction, which can be completed in 24 h. Using this method, sections from 15 of 17 patients clinically diagnosed as having OHL contained readily detectable EBV-DNA in small foci along the upper layers of the stratum spinosum. The sections examined from the two remaining patients appeared to be EBV-DNA negative but both patients were on AZT therapy and one was in addition receiving acyclovir.

Diagnosis of oral hairy leukoplakia by ultrastructural examination of exfoliative cytologic specimens.

Lingual exfoliative cytologic specimens (scrapings) were obtained from 18 patients positive for human immunodeficiency virus with clinical oral hairy leukoplakia. Buccal mucosal scrapings were obtained from 12 of these patients. The specimens were processed for examination by transmission electron microscopy (TEM). Sixteen (89%) of the lingual specimens revealed infection of keratinocytes by herpes-type virus. There was no evidence of virus infection in the 12 buccal mucosal scrapings. Fungal hyphae were seen by TEM in 14 (78%) of the lingual scrapings and two (17%) of the buccal scrapings. One exfoliative specimen and two biopsy specimens were stained for Epstein-Barr virus DNA with a DNA probe. The demonstration of herpes-type virions by TEM in keratinocytes from a lesion clinically suspected to be hairy leukoplakia provides direct, objective diagnosis. Furthermore, use of exfoliative cytologic specimens provides a clinically simple, noninvasive technique.

Oral hairy leukoplakia among HIV-positive intravenous drug abusers: a clinicopathologic and ultrastructural study.

During a prospective investigation of oral lesions of 120 consecutive patients positive for human immunodeficiency virus, belonging to the intravenous drug abuser risk group and other risk categories, we observed hairy leukoplakia (HL) in 23 cases (19%). The median age of the patients was 27 years (range, 20 to 50 years). Twenty patients were men and three were women. All but two of the twenty three patients used intravenous drugs for a median period of 6 years (range, 5 to 18 years) and were involved in several episodes of needle sharing. Eight men were also bisexual, one man was homosexual, and one man was hemophiliac and bisexual. Eleven patients had asymptomatic infection, five had lymphadenopathy syndrome, six had AIDS-related complex, and one had acquired immunodeficiency syndrome. In all patients, HL lesions were localized on the lateral borders of the tongue. In twelve patients, the lesion was unilateral, and in eleven patients, it was bilateral. Microscopically, hyperparakeratosis and the presence of koilocytes were observed in all cases. Surface candidiasis could be detected with staining with periodic acid-Schiff in two thirds of the cases. In four cases, electron microscopy showed the presence of intracellular and extracellular hyphae of Candida albicans in the parakeratin layer associated with coccobacilli in the spaces between surface epithelial cells. The spinous layer included koilocytes, which had a clear cytoplasmic matrix, sparse organelles and tonofilaments, and dispersed chromatin. These cells were found to be infected by a herpes-type virus in all cases examined. There was no ultrastructural evidence of human papillomavirus in the nuclei of the epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural and immunohistochemical findings in oral hairy leukoplakia.

Three cases of HL from the lateral border of the tongue of male homosexual AIDS patients were investigated by thin section electron microscopy. Keratinocytes contained condensed chromatin in their pyknotic nuclei and a few organelles in the oedematous cytoplasm. Chromatin was in close association to the nuclear membrane and showed a punched-out appearance. Particles typical of the herpes virus group were abundant in the upper two thirds of the epithelium in all three cases. Virus particles were seen frequently in the nuclei of the ballooned keratinocytes, but rarely in cells containing Candida albicans. Viral nucleocapsids were observed budding at the inner nuclear membrane, thereby acquiring the prospective viral envelope. Complete, enveloped virions were found in the endoplasmic reticulum and in the extracellular space. These virions were identified immunohistochemically as Epstein-Barr virus (EBV) using two monoclonal antibodies directed against EBV capsid and membrane antigen, respectively. Candida albicans was observed in the stratum corneum and in the upper layer of the stratum spinosum. Special cytoplasmic tubular structures arranged in parallel bundles were found in koilocytotic cells in addition to characteristic membrane structures composed of undulating convoluted membranes. Epithelial basement membranes were always intact.

Oral hairy leukoplakia in a HIV-negative renal transplant patient: a marker for immunosuppression?

We report the case of a 58-year-old renal transplant patient who developed oral hairy leukoplakia. Examination for HIV-1 and HIV-2 infection was negative. Biopsy of the lateral tongue showed ballooned prickle cells and electron microscopy revealed herpes-type viruses. In situ hybridization and examinations with the Southern blot technique yielded Epstein-Barr virus. Serology for Epstein-Barr virus was reactive. Immunological investigation of the patient showed a marked decrease of T-helper and T-suppressor cells as the result of immunosuppressive regimen. Oral hairy leukoplakia may be a marker for severe immunosuppression but is not necessarily associated with HIV infection.