In Vivo Regulation of Active Matrix Metalloproteinase-8 (aMMP-8) in Periodontitis: From Transcriptomics to Real-Time Online Diagnostics and Treatment Monitoring.

BACKGROUND: This study investigated in vivo regulation and levels of active matrix metalloproteinase-8 (aMMP-8), a major collagenolytic protease, in periodontitis. METHODS: Twenty-seven adults with chronic periodontitis (CP) and 30 periodontally healthy controls (HC) were enrolled in immunohistochemistry and transcriptomics analytics in order to assess Treponema denticola (Td) dentilisin and MMP-8 immunoexpression, mRNA expression of MMP-8 and its regulators (IL-1ß, MMP-2, MMP-7, TIMP-1). Furthermore, the periodontal anti-infective treatment effect was monitored by four different MMP-8 assays (aMMP-8-IFMA, aMMP-8-Oralyzer, MMP-8-activity [RFU/minute], and total MMP-8 by ELISA) among 12 CP (compared to 25 HC). RESULTS: Immunohistochemistry revealed significantly more Td-dentilisin and MMP-8 immunoreactivities in CP vs. HC. Transcriptomics revealed significantly elevated IL-1ß and MMP-7 RNA expressions, and MMP-2 RNA was slightly reduced. No significant differences were recorded in the relatively low or barely detectable levels of MMP-8 mRNAs. Periodontal treatment significantly decreased all MMP-8 assay levels accompanied by the assessed clinical indices (periodontal probing depths, bleeding-on-probing, and visual plaque levels). However, active but not total MMP-8 levels persisted higher in CP than in periodontally healthy controls. CONCLUSION: In periodontal health, there are low aMMP-8 levels. The presence of Td-dentilisin in CP gingivae is associated with elevated aMMP-8 levels, potentially contributing to a higher risk of active periodontal tissue collagenolysis and progression of periodontitis. This can be detected by aMMP-8-specific assays and online/real-time aMMP-8 chair-side testing.

Fibrous dysplasia of the head and neck in Southern Finland: a retrospective study on clinical characteristics, diagnostics, and treatment.

PURPOSE: Fibrous dysplasia (FD) is a rare genetic disease with benign bone tumors. FD can affect one (monostotic FD) or multiple bones (polyostotic FD), with craniofacial lesions being common. Because of its rarity, there are only few clinical reports on FD in the head and neck region and its clinical characteristics remain incompletely defined. This study aimed to determine patient demographics, symptoms, diagnostics, and given treatment in patients with FD of the head and neck in a Finnish population. METHODS: A retrospective review on all patients diagnosed with or treated for FD of the head and neck at the Helsinki University Hospital during 2005-2020. RESULTS: In total 74 patients were identified; 54% were male and the mean age 45 years. Overall 95% had monostotic FD. Mandibula and maxilla were the most common anatomic sites. Majority of patients had symptoms, most commonly pain and lesion growth, and 49% had extra-skeletal symptoms. For all, diagnosis was primarily based on imaging findings, biopsies were obtained from 41%. Altogether 54 patients (73%) were managed by observation only, 20 patients (27%) received treatment; ten bisphosphonates, six surgery and four both. CONCLUSION: Although highly variable in its clinical manifestations, head and neck FD lesions are often symptomatic and impose risk for extra-skeletal complications. Treatment is often conservative but should be individually tailored. Future studies are encouraged to better define the disease characteristics and hopefully offer new treatment possibilities.

Treponema denticola chymotrypsin-like protease as associated with HPV-negative oropharyngeal squamous cell carcinoma.

BACKGROUND: An opportunistic oral pathogen, Treponema denticola (Td), has been linked to orodigestive carcinogenesis, but its role in oropharyngeal squamous cell carcinoma (OPSCC) has remained open. We evaluated the presence of Td chymotrypsin-like protease (Td-CTLP) in a series of 201 unselected consecutive OPSCC patients, and the relation of the Td-CTLP to human papillomavirus (HPV) status, to expression of toll-like receptors (TLR) 5, 7, and 9, and to clinical parameters and patient outcome. METHODS: Clinicopathological data came from hospital registries. The expression of cell surface-bound Td-CTLP was evaluated by immunohistochemistry. Immunoexpression of TLRs 5, 7, and 9, and HPV status we studied earlier in this patient series. RESULTS: We detected Td-CTLP in 81% of the OPSCC, and especially in HPV-negative tumours (48% of all OPSCCs). Among the HPV-positive tumours (52% of all OPSCCs), low Td-CTLP expression associated with low TLR 5 and high TLR 7 expression. Among those HPV-negative, higher TLR 5 and lower TLR 7 expression associated with high Td-CTLP expression. Strong Td-CTLP expression associated with poor disease-specific survival, but no similar association among HPV-positive and HPV-negative subgroups emerged. CONCLUSIONS: Td-CTLP was highly expressed in OPSCC and was associated with the HPV status of tumour tissue.

Treponema denticola chymotrypsin-like proteinase is present in early-stage mobile tongue squamous cell carcinoma and related to the clinicopathological features.

BACKGROUND: Certain periodontopathogenic bacteria have been linked to cancers. Treponema denticola (Td) is associated with severe periodontitis. Chymotrypsin-like proteinase (CTLP), a major virulence factor of Td, can degrade various host proteins and peptides, and modulate inflammatory responses. However, the role of Td in the tongue carcinogenesis remains unknown. This study aimed to investigate the presence of Td-CTLP in early-stage mobile tongue squamous cell carcinoma (MTSCC) and its relation to clinical and pathological characteristics. METHODS: The immunopositivity of Td-CTLP was assessed in samples obtained from 60 patients with MTSCC and associated with their clinicopathological data. Additionally, Td-CTLP expression was compared with immunoexpression of matrix metalloproteinases (MMP-8 and MMP-9), toll-like receptors (TLR-2, TLR-4, TLR-7 and TLR-9), c-Myc, Ki-67, Bmi-1 and Snail. RESULTS: Treponema denticola-chymotrypsin-like proteinase was present in 95% of MTSCC tumours of which many (40.4%) showed high immunopositivity. Td-CTLP positivity was significantly associated with invasion depth, tumour diameter and the expression of TLR-7, TLR-9 and c-Myc. High Td-CTLP immunopositivity in younger patients (≤ 60 years old) predicted early relapse. CONCLUSION: Our data indicate that Td and its CTLP are present in early-stage MTSCC carcinoma and may contribute to carcinogenesis, and therefore provide novel perspectives into intervention and therapeutic measures of MTSCC.

Local Acetaldehyde-An Essential Role in Alcohol-Related Upper Gastrointestinal Tract Carcinogenesis.

The resident microbiome plays a key role in exposure of the upper gastrointestinal (GI) tract mucosa to acetaldehyde (ACH), a carcinogenic metabolite of ethanol. Poor oral health is a significant risk factor for oral and esophageal carcinogenesis and is characterized by a dysbiotic microbiome. Dysbiosis leads to increased growth of opportunistic pathogens (such as Candida yeasts) and may cause an up to 100% increase in the local ACH production, which is further modified by organ-specific expression and gene polymorphisms of ethanol-metabolizing and ACH-metabolizing enzymes. A point mutation in the aldehyde dehydrogenase 2 gene has randomized millions of alcohol consumers to markedly increased local ACH exposure via saliva and gastric juice, which is associated with a manifold risk for upper GI tract cancers. This human cancer model proves conclusively the causal relationship between ACH and upper GI tract carcinogenesis and provides novel possibilities for the quantitative assessment of ACH carcinogenicity in the human oropharynx. ACH formed from ethanol present in "non-alcoholic" beverages, fermented food, or added during food preparation forms a significant epidemiologic bias in cancer epidemiology. The same also concerns "free" ACH present in mutagenic concentrations in multiple beverages and foodstuffs. Local exposure to ACH is cumulative and can be reduced markedly both at the population and individual level. At best, a person would never consume tobacco, alcohol, or both. However, even smoking cessation and moderation of alcohol consumption are associated with a marked decrease in local ACH exposure and cancer risk, especially among established risk groups.

Treponema denticola chymotrypsin-like proteinase may contribute to orodigestive carcinogenesis through immunomodulation.

BACKGROUND: Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. METHODS: The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. RESULTS: Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and α-1-antichymotrypsin, as well as complement C1q. CONCLUSIONS: Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.

Matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 predict incident cardiovascular disease events and all-cause mortality in a population-based cohort.

Background Extracellular matrix degrading proteases and their regulators play an important role in atherogenesis and subsequent plaque rupture leading to acute cardiovascular manifestations. Design and methods In this prospective cohort study, we investigated the prognostic value of circulating matrix metalloproteinase-8, tissue inhibitor of matrix metalloproteinase-1 concentrations, the ratio of matrix metalloproteinase-8/ tissue inhibitor of matrix metalloproteinase-1 and, for comparison, myeloperoxidase and C-reactive protein concentrations for incident cardiovascular disease endpoints. The population-based FINRISK97 cohort comprised 7928 persons without cardiovascular disease at baseline. The baseline survey included a clinical examination and blood sampling. During a 13-year follow-up the endpoints were ascertained through national healthcare registers. The associations of measured biomarkers with the endpoints, including cardiovascular disease event, coronary artery disease, acute myocardial infarction, stroke and all-cause death, were analysed using Cox regression models. Discrimination and reclassification models were used to evaluate the clinical implications of the biomarkers. Results Serum tissue inhibitor of matrix metalloproteinase-1 and C-reactive protein concentrations were associated significantly with increased risk for all studied endpoints. Additionally, matrix metalloproteinase-8 concentration was associated with the risk for a coronary artery disease event, myocardial infarction and death, and myeloperoxidase concentration with the risk for cardiovascular disease events, stroke and death. The only significant association for the matrix metalloproteinase-8/ tissue inhibitor of matrix metalloproteinase-1 ratio was observed with the risk for myocardial infarction. Adding tissue inhibitor of matrix metalloproteinase-1 to the established risk profile improved risk discrimination of myocardial infarction ( p=0.039) and death (0.001). Both matrix metalloproteinase-8 (5.2%, p < 0.001) and tissue inhibitor of matrix metalloproteinase-1 (12.9%, p < 0.001) provided significant clinical net reclassification improvement for death. Conclusions Serum matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 can be considered as biomarkers of incident cardiovascular disease events and death.

Low MMP-8/TIMP-1 reflects left ventricle impairment in takotsubo cardiomyopathy and high TIMP-1 may help to differentiate it from acute coronary syndrome.

BACKGROUND: Matrix metalloproteinase 8 (MMP-8) is the most potent type-I collagen protease. Such collagen mainly constitutes the transient fibrosis in takotsubo cardiomyopathy (TTC) endomyocardial biopsies. High MMP-8 and tissue-inhibitor of matrix metalloproteinase-1 (TIMP-1) levels are implicated in acute coronary syndrome (ACS). We compared MMP-8 and TIMP-1 levels in consecutive TTC and ACS patients, and their association to TTC severity. METHODS AND RESULTS: In 45 acute serum samples of TTC, 2072 ACS and 1000 controls, TIMP-1 differed between ACS 146.7ng/mL (115.0-186.3) (median (interquartile range)), TTC 115.7 (94.3-137.7) and controls 80.9 (73.2-90.4), (p<0.0001). MMP-8 levels were similar between ACS and TTC. In receiver-operating characteristics analysis, TIMP-1 differentiated TTC from ACS with an area under the curve (AUC) of 0.679 (p<0.0001) surpassing troponin T (TnT) at 0.522 (p = 0.66). Compared to other differing factors (age, sex, smoking), TIMP-1 improved diagnostic specificity and sensitivity from AUC of 0.821 to 0.844 (p = 0.007). The MMP8/TIMP-1 molar ratio differentiated normal ejection fraction (EF) at 0.27 (0.13-0.51) from decreased EF<50% at 0.08 (0.05-0.20), (p = 0.04) in TTC, but not in ACS. CONCLUSIONS: Even with other differing factors considered, TIMP-1 differentiated TTC from ACS better than TnT. In TTC, the low MMP-8/TIMP-1 molar ratio may reflect decreased proteolysis and increased transient fibrosis, perhaps in part explaining the left-ventricle impairment.

Effect of S53P4 bone substitute on staphylococcal adhesion and biofilm formation on other implant materials in normal and hypoxic conditions.

To study the effect of bioactive glass bone substitute granules (S53P4) on bacterial adhesion and biofilm formation on other simultaneously used implant materials and the role of the hypoxic conditions to the adhesion. Bacterial and biofilm formation were studied on materials used both in middle ear prostheses and in fracture fixtures (titanium, polytetrafluoroethylene, polydimethylsiloxane and bioactive glass plates) in the presence or absence of S53P4 granules. The experiments were done either in normal atmosphere or in hypoxia simulating atmospheric conditions of middle ear, mastoid cavity and sinuses. We used two collection strains of Staphylococcus aureus and Staphylococcus epidermidis. In the presence of bioglass and hypoxic conditions the adhesion of the planktonic bacterial cells was decreased for most of the materials. The biofilm formation was decreased for S. epidermidis on titanium and polydimethylsiloxane in both atmospheric conditions and on bioglass plates in normoxia. For S. aureus the biofilm formation was decreased on bioglass plates and polytetrafluoroethylene in normoxia. Hypoxia produces a decrease in the biofilm formation only for S. aureus on polytetrafluoroethylene and for S. epidermidis on bioglass plates. However, in none of the cases bioactive glass increased the bacterial or biofilm adhesion. The presence of bioglass in normoxic and hypoxic conditions prevents the bacterial and biofilm adhesion on surfaces of several typical prosthesis materials in vitro. This may lead to diminishing postoperative infections, however, further in vivo studies are needed.

Practical implications of novel serum ELISA-assay for matrix metalloproteinase-8 in acute cardiac diagnostics.

Matrix metalloproteinases (MMPs) play a major role in inflammatory processes as they degrade extracellular proteins and modify immune responses. Inflammation is the driving factor in atherogenesis and MMPs, particularly MMP-8, has been linked to atherosclerotic plaque progression. MMP-8 is shown to be strongly associated with cardiovascular diseases (CVDs) and its complications thus providing a potential marker to identify patients at risk. Previously, laborious and expensive immunofluorometric assay (IFMA) was needed to reliably detect MMP-8 levels in serum. In this study, we compared a novel in-house ELISA-assay, dentoELISA, to the standard IFMA in determination of serum MMP-8 concentrations. As a cheaper and non-laborious assay, ELISA proved to be diagnostically as sensitive and specific as the IFMA. ROC statistics showed highly similar areas under the curve for both assays (0.779 versus 0.781). Furthermore, the concentrations measured by ELISA correlated significantly with concentrations determined with IFMA (r = 0.881, P < 0.001). In our study population, MMP-8 levels were significantly higher in the acute coronary syndrome patients (n = 2071) in comparison to reference population without significant coronary artery disease (n = 653). With this background, MMP-8-ELISA could provide interesting new approaches to novel CVD diagnostics.

A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

Alcohol and acetaldehyde in African fermented milk mursik--a possible etiologic factor for high incidence of esophageal cancer in western Kenya.

BACKGROUND: Esophageal cancer is unusually frequent in Western Kenya, despite the low prevalence of classical risk factors such as heavy drinking and tobacco smoking. Among Kenyans consumption of fermented milk is an old tradition. Our hypothesis is that alcohol and acetaldehyde are produced during the fermentation process and that their carcinogenic potential contributes to the high incidence of esophageal cancer. METHODS: Eight samples of mursik milk starter cultures were collected from different Kalenjin families in the Rift Valley province, Western Kenya. A protocol provided by the families was used for milk fermentation. Ethanol and acetaldehyde levels were measured by gas chromatography. The microbial flora in starter cultures was identified by 16S and 18S sequencing. RESULTS: 7/8 starter cultures produced mutagenic (>100 µmol/L) levels of acetaldehyde and 4/8 starter cultures produced more than 1,000 µmol/L of acetaldehyde. The highest alcohol levels (mean 79.4 mmol/L) were detected in the four fermented milks with highest acetaldehyde production. The mean number of microbial species in the starter cultures was 5 (range 2-8). Yeasts were identified in all starter cultures (mean 1.5 species/milk) but their proportion of the total microbial count varied markedly (mean 35%, range 7%-90%). A combination of yeast and lactobacilli, especially Candida krusei with Lactobacillus kefiri, with the exclusion of other species, seemed to correlate with higher acetaldehyde and ethanol levels. CONCLUSIONS: Significant levels of ethanol and acetaldehyde were produced during mursik fermentation. IMPACT: When ingested several times daily the repeated exposure to carcinogenic levels of acetaldehyde may contribute to esophageal carcinogenesis.

Xylitol inhibits carcinogenic acetaldehyde production by Candida species.

Acetaldehyde is a highly toxic and mutagenic product of alcohol fermentation and metabolism which has been classified as a Class I carcinogen for humans by the International Agency for Research on Cancer of the World Health Organisation (WHO). Many Candida species representing oral microbiota have been shown to be capable of marked acetaldehyde production. The aim of our study was to examine the effects of various sugar alcohols and sugars on microbial acetaldehyde production. The study hypothesis was that xylitol could reduce the amount of acetaldehyde produced by Candida. Laboratory and clinical isolates of seven Candida species were selected for the study. The isolates were incubated in 12 mM ethanol and 110 mM glucose, fructose or xylitol at 37°C for 30 min and the formed acetaldehyde was measured by gas chromatography. Xylitol significantly (p < 0.0001) reduced the amount of acetaldehyde produced from ethanol by 84%. In the absence of xylitol, the mean acetaldehyde production in ethanol incubation was 220.5 µM and in ethanol-xylitol incubation 32.8 µM. This was found to be mediated by inhibition of the alcohol dehydrogenase enzyme activity. Coincubation with glucose reduced the amount of produced acetaldehyde by 23% and coincubation with fructose by 29%. At concentrations that are representative of those found in the oral cavity during the intake of proprietary xylitol products, xylitol was found to reduce the production of carcinogenic acetaldehyde from ethanol by Candida below the mutagenic level of 40-100 µM.

Acetaldehyde production from ethanol and glucose by non-Candida albicans yeasts in vitro.

BACKGROUND: Major environmental risk factors for upper digestive tract cancers are tobacco smoking, alcohol intake and poor oral hygiene. They all result in increased acetaldehyde (ACH) levels in saliva which has been shown to be carcinogenic. During alcohol challenge the oral microbiota is the main determinant of the local ACH concentration. Many bacteria and Candida albicans have been shown to be capable of ACH production. Moreover, chronic candidal mucositis can be carcinogenic. The ability of non-C. albicans Candida to produce ACH has not been studied. AIM: The aim of this study was to explore the ability of non-C. albicans Candida species to produce ACH in vitro during ethanol and glucose incubation. METHODS: A total of 30 non-C. albicans Candida isolates and one C. albicans reference strain were used. The cells were exposed to 11 mM of ethanol and to 100mM glucose in vitro. ACH was measured by gas chromatography. RESULTS: All Candida isolates produced significant amounts of ACH in ethanol incubation. C. tropicalis isolates were the highest (252.3 microM) and C. krusei isolates were the lowest (54.6 microM) producers of ACH from ethanol. Only C.glabrata produced significant amounts of ACH by fermentation from glucose. CONCLUSION: Colonization of oral mucosa with a non-C.albicans species such as C. glabrata, capable of producing carcinogenic amounts of ACH from both ethanol and glucose, may contribute to the development of oral cancer.