Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing.

Background: The bactericidal effect of disinfectants against biofilms is essential to reduce potential endoscopy-related infections caused by contamination. Here, we investigated the bactericidal effect of a high-level disinfectant, peracetic acid (PAA), against Staphylococcus aureus and Pseudomonas aeruginosa biofilm models in vitro. Methods: S. aureus and P. aeruginosa biofilms were cultured at 35 °C for 7 days with catheter tubes. The following high-level disinfectants (HLDs) were tested: 0.3% PAA, 0.55% ortho-phthalaldehyde (OPA), and 2.0% alkaline-buffered glutaraldehyde (GA). Biofilms were exposed to these agents for 1-60 min and observed after 5 min and 30 min by transmission and scanning electron microscopy. A Student's t test was performed to compare the exposure time required for bactericidal effectiveness of the disinfectants. Results: PAA and GA were active within 1 min and 5 min, respectively, against S. aureus and P. aeruginosa biofilms. OPA took longer than 10 min and 30 min to act against S. aureus and P. aeruginosa biofilms, respectively (p < 0.01). Treatment with PAA elicited changes in cell shape after 5 min and structural damage after 30 min. Conclusions: Amongst the HLDs investigated, PAA elicited the most rapid bactericidal effects against both biofilms. Additionally, treatment with PAA induced morphological alterations in the in vitro biofilm models, suggesting that PAA exerts fast-acting bactericidal effects against biofilms associated with endoscopy-related infections. These findings indicate that the exposure time for bactericidal effectiveness of HLDs for endoscope reprocessing in healthcare settings should be reconsidered.

Altered fibroblast proliferation and apoptosis in diabetic gingival wounds.

Although it is known that diabetes impairs oral wound healing, relatively little is known about the cellular parameters affected, particularly in connective tissue. This study investigated the hypothesis that diabetes impairs connective tissue formation in healing gingiva, and that impaired healing is associated with factors that decrease fibroblast numbers. Full-thickness wounds were created in the palatal gingiva of type 1 and type 2 diabetic and normoglycemic mice. Five days after wounding, diabetic mice had less epithelial wound coverage, less new connective tissue formation, and reduced fibroblast density (p < 0.05). This occurred with increased numbers of caspase-3- and TUNEL-positive fibroblasts, decreased fibroblast proliferation, increased nuclear translocation of the pro-apoptotic transcription factor FOXO1, and increased numbers of polymorphonuclear leukocytes, all of which were significant (p < 0.05). The results suggest that diabetes may decrease fibroblast numbers through increased apoptosis and reduced proliferation, both of which may be mediated through increased activation of FOXO1.

Impaired wound healing in mouse models of diabetes is mediated by TNF-alpha dysregulation and associated with enhanced activation of forkhead box O1 (FOXO1).

AIMS/HYPOTHESIS: The role of TNF-alpha in impaired wound healing in diabetes was examined by focusing on fibroblasts. METHODS: Small excisional wounds were created in the db/db mice model of type 2 diabetes and normoglycaemic littermates, and in a streptozotocin-induced type 1 diabetes mouse model and control mice. Fibroblast apoptosis was measured by the TUNEL assay, proliferation by detection of proliferating cell nuclear antigen, and forkhead box O1 (FOXO1) activity by DNA binding and nuclear translocation. TNF-alpha was specifically inhibited by pegsunercept. RESULTS: Diabetic wounds had increased TNF-alpha, fibroblast apoptosis, caspase-3/7 activity and activation of the pro-apoptotic transcription factor FOXO1, and decreased proliferating cell nuclear antigen positive fibroblasts (p < 0.05). TNF-alpha inhibition improved healing in the diabetic mice and increased fibroblast density. This may be explained by a decrease in fibroblast apoptosis and increased proliferation when TNF-alpha was blocked (p < 0.05). Although decreased fibroblast proliferation and enhanced FOXO1 activity were investigated in type 2 diabetes, they may also be implicated in type 1 diabetes. In vitro, TNF-alpha enhanced mRNA levels of gene sets related to apoptosis and Akt and p53 but not mitochondrial or cell-cycle pathways. FOXO1 small interfering RNA reduced gene sets that regulate apoptosis, Akt, mitochondrial and cell-cycle pathways. TNF-alpha also increased genes involved in inflammation, cytokine, Toll-like receptor and nuclear factor-kB pathways, which were significantly reduced by FOXO1 knockdown. CONCLUSIONS/INTERPRETATION: These studies indicate that TNF-alpha dysregulation in diabetic wounds impairs healing, which may involve enhanced fibroblast apoptosis and decreased proliferation. In vitro, TNF-alpha induced gene sets through FOXO1 that regulate a number of pathways that could influence inflammation and apoptosis.

Effects of oral commensal and pathogenic bacteria on human dendritic cells.

BACKGROUND/AIMS: The oral cavity harbors a diverse and complex microbial community. Bacteria accumulate on both the hard and soft oral tissues in sessile biofilms and engage the host in an intricate cellular dialog, which normally constrains the bacteria to a state of commensal harmony. Dendritic cells (DCs) are likely to balance tolerance and active immunity to commensal microorganisms as part of chronic inflammatory responses. While the role played by DCs in maintaining intestinal homeostasis has been investigated extensively, relatively little is known about DC responses to oral bacteria. METHODS: In this study, we pulsed human monocyte-derived immature DCs (iDCs) with cell wall extracts from pathogenic and commensal gram-positive or gram-negative oral bacteria. RESULTS: Although all bacterial extracts tested induced iDCs to mature and produce cytokines/chemokines including interleukin-12p40, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 (MCP-1), the most important factor for programming DCs by oral bacteria was whether they were gram-positive or gram-negative, not whether they were commensal or pathogenic. In general, gram-negative oral bacteria, except for periodontopathic Porphyromonas gingivalis, stimulated DC maturation and cytokine production at lower concentrations than gram-positive oral bacteria. The threshold of bacteria needed to stimulate chemokine production was 100-fold to 1000-fold lower than that needed to induce cytokines. In addition, very low doses of oral commensal bacteria triggered monocytes to migrate toward DC-derived MCP-1. CONCLUSION: Oral commensal and pathogenic bacteria do not differ qualitatively in how they program DCs. DC-derived MCP-1 induced in response to oral commensal bacteria may play a role, at least in part, in the maintenance of oral tissue integrity by attracting monocytes.

Immunocytochemical localization of aromatic L-amino acid decarboxylase and catechol-O-methyltransferase in blood vessel wall of the human dental pulp.

Relative large amounts of DOPA as compared with the concentrations of norepinephrine are found in human dental pulp. AADC and COMT are localized in blood vessel walls of human dental pulp. This localization suggests a functional relationship between COMT and AADC with regard to the metabolism of DOPA.

Inhibition by streptococcal immunopotentiator OK432 of lymph-node metastasis in hamster cheek-pouch carcinoma with enhancement of tumour necrosis factor-alpha and interleukin-6 in serum.

A well-known metastic model of human oral cancer employs 9,10-dimethyl-1,2-benzanthracene (DMBA) to induce hamster cheek-pouch carcinoma. Streptococcal immunopotentiator OK432 was studied here for its inhibitory effect on lymph-node metastasis in that model. The intraperitoneal administration of OK432, after excision of cheek-pouch tumours induced by DMBA, resulted in a marked reduction in the incidence of cervical lymph-node metastasis to 7%, a significant decrease beneath the rates observed for control animals not receiving OK432 (40%). OK432 also caused an increased in serum levels of tumour necrosis factor-alpha and interleukin-6 in tumor-bearing hamsters. These results suggest that the immune response may play an important part in the antimetastatic effects of OK432.

Hypertriglyceridemia associated with tumor necrosis factor-alpha in hamster cheek-pouch carcinogenesis.

We demonstrated for the first time that 9,10-dimethyl-1,2-benzanthracene (DMBA)-treated hamsters showed hypertriglyceridemia followed by cachexia. Hypertriglyceridemia is believed to be caused in part by the decreased lipoprotein lipase (LPL) activity, and by cytokines such as tumor necrosis factor (TNF)-alpha. In addition, TNF-alpha action is associated with the LPL activity. Therefore, we determined the content of triglyceride (TG), LPL, and TNF-alpha in the serum from DMBA-treated hamsters. Elevated TG concentration in the serum of tumor-bearing hamsters was more remarkable and preceded the increase in other lipids, whereas the activity of LPL, the key enzyme of TG metabolism in vivo, was drastically reduced. TNF-alpha, known as an endogenous inhibitor of LPL activity, was detected in both the sera and the extract of tumors from DMBA-treated hamsters, whereas it was not detectable in any control samples. Pre-incubation of control sera with exogenous recombinant human TNF-alpha resulted in a potent inhibition of endogenous LPL activity in a dose-dependent manner in vitro. Therefore, the presence of TNF-alpha might lead to the increase in plasma TG mediated by LPL in tumor-bearing hamsters.

Salivary beta 2-microglobulin in NZB/WF1 mice.

Unstimulated parotid and submandibular/sublingual saliva from New Zealand black/WF1 (NZB/WF1) mice as collected by microcapillary (1 microliter), and the content of beta 2-microglobulin (beta 2-MG) determined by a sandwich enzyme immunoassay. Salivary beta 2-MG contents of control C57BL/6 and NZB/WF1 mice (45 weeks of age) were 0.68 +/- 0.18 micrograms/ml and 1.42 +/- 0.21 micrograms/ml (mean +/- SD), respectively, from the parotid gland and 0.62 +/- 0.17 micrograms/ml and 1.34 +/- 0.21 microliters/ml, respectively, from the submandibular sublingual glands. However, the concentration of beta 2-MG was not increased in the NZB/WF1 mice at 5 and 20 weeks of age. Saliva from NZB/WF1 mice (45 weeks old) was fractionated by micro two-dimensional gel electrophoresis; it exhibited both qualitative and quantitative changes in protein composition in comparison to the two-dimensional at 5 weeks of age. These observations parallel those in saliva from patients with Sjögren's syndrome.

Increased beta 2-microglobulin in both parotid and submandibular/sublingual saliva from patients with Sjögren's syndrome.

Samples of unstimulated saliva from patients with sialoadenopathy were collected by microcapillary tube (1 microliter), and their beta 2-microglobulin (B2-MG) content determined by a sandwich enzyme immunoassay. A higher than normal (control) concentration of the globulin was present in both parotid and submandibular/sublingual saliva from the patients with Sjögren's syndrome but not in the samples from the patients with sialoadenitis or diabetes mellitus. The increase in B2-MG in saliva from patients with Sjögren's syndrome may reflect that immunolopathological events are important in the degeneration of both glands in this disease. Therefore, the determination of B2-MG in saliva may be a simple, non-invasive technique for confirming the diagnosis of Sjögren's syndrome as an autoimmune disease.

Infiltrating lipoma of the mental region: report of a case.

Lipoma is an uncommon tumour in the oral cavity. Infiltrating lipoma is extremely rare. An infiltrating lipoma of the mental region in a 48-year old woman is reported. Wide myectomy was performed, and follow-up showed excellent healing without recurrence.

Two-dimensional electrophoresis of human salivary proteins from patients with sialoadenopathy.

Unstimulated saliva was fractionated by micro two-dimensional gel electrophoresis, and the proteins visualized by silver staining and immunostaining. The subjects with Sjögren's syndrome exhibited both quantitative and qualitative alterations in the protein composition of the saliva not only from the parotid gland but also from the submandibular/sublingual glands.

Hyperkeratosis and atypical proliferation appearing in the lining epithelium of a radicular cyst. Report of a case.

It is widely accepted that radicular cysts (apical periodontal cysts) are commonly lined with stratified squamous epithelium without keratin formation. However, we identified a case of maxillary radicular cyst with remarkable keratinization and atypical proliferation of the lining epithelium among the 207 radicular cyst cases seen at our department. Histopathological and clinical findings of these cysts were reviewed.

Enhancement by cyclosporin A of metastasis from hamster cheek pouch carcinoma.

Daily intravenous administration of 50 mg/kg cyclosporin A (CsA), after excision of cheek pouch tumors induced with 9,10-dimethyl-1,2-benzanthracene (DMBA), enhanced the incidence of cervical lymph node metastasis to 93%, a significant increase over the rates observed for animals receiving a dosage of 25 mg/kg (43%), and for control animals receiving no CsA (40%). No significant effect was evident on the growth rates of the metastasized tumour cells in the lymph node.

Lung and cervical lymph-node metastasis after cortisone enhancement in hamster cheek-pouch carcinogenesis.

The administration of hydrocortisone after excision of DMBA-induced tumours in hamster cheek pouch produced cervical lymph-node metastases in 9 out of 13 animals (69 per cent), and resulted in lung metastasis in 1 animal. In the control group, which received no cortisone but had tumours cut from the pouch, 4 out of 9 animals had cervical lymph-node metastases (44 per cent).

Regional lymph node metastasis created by partial excision of carcinomas induced in hamster cheek pouch with 9,10-dimethyl-1,2-benzanthracene.

The feasibility of using the hamster cheek pouch/dimethyl-benzanthracene (DMBA) system as an experimental model of lymphatic metastasis was investigated. Forty male Syrian golden hamsters treated with DMBA were divided into two equal groups--one with surgical excision of their tumors and a control group without tumor excision. In the excision group, the animals received three applications/week to the left cheek pouch of 0.3% DMBA in acetone for 14 weeks. Following a three-week observation period, the tumors in the pouch were excised at their base, and the animals were killed after four weeks of further observation. In the control group, the animals were treated for 14 weeks in a manner similar to that used for the excision group, left for seven weeks without treatment, and then killed. Cheek pouches with tumors and cervical lymph nodes were processed for histological examination. All of the animals, both with and without metastasis, had borne squamous cell carcinomas (SCC) in their treated cheek pouches. Histologically, seven out of 16 animals in the excision group showed metastatic deposits of SCC confined to the left cervical lymph nodes, while in the control group, metastasis was not found in any of the 19 animals with SCC in their cheek pouches. The results demonstrate that surgical excision of the hamster cheek pouch carcinoma is efficient in producing unequivocal lymph node metastasis.

A transmission electron microscopic study of two cases of oral smooth muscle neoplasm.

Two cases of smooth muscle tumors that had appeared in the oral regions were examined by means of histopathology and electron microscopy. One was a case of angiomyoma that appeared in the lip of a 33-year-old man, and the other was a case of leiomyosarcoma in the maxilla of a 63-year-old woman. The results of the examination of both cases were as follows: the benign tumor (angiomyoma) was composed of mainly mature smooth muscle cells having dark cytoplasm, and the malignant tumor (leiomyosarcoma) consisted mainly of two types of cells, undifferentiated mesenchymal cells or fibroblast-like cells (type I) and myofibroblast-like cells (type II). Based upon these results, the relationship between the myogenous differentiation and the component cell types, and biological behavior of these smooth muscle tumors was discussed.

Analysis and identification of human parotid salivary proteins by micro two-dimensional electrophoresis and Western-blot techniques.

Using this method, without denaturing agents, over 60 protein components in 2 microliter (about 4 micrograms of protein) of unconcentrated parotid saliva could be detected. After two-dimensional separation, the spots of albumin, secretory IgA, IgG, acid phosphatase, amylase and plasma-originating protein were identified by antibodies with the Western-blot technique.