Mechanisms of azole resistance and carcinogenic acetaldehyde production in chronic mucosal candidosis

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS1) is an autoimmune disease caused by a loss-of function mutation in the autoregulator gene (AIRE). Patients with APECED suffer from chronic mucocutaneous candidosis (CMC) of the oral cavity and oesophagus often since early childhood. The patients are mainly colonized with Candida albicans and decades of exposure to antifungal agents have lead to the development of clinical and microbiological resistance in the treatment of CMC in the APECED patient population in Finland. A high incidence of oral squamous cell carcinoma is associated with oral CMC lesions in the APECED patients over the age of 25. The overall aim of this study was firstly, to investigate the effect of long-term azole exposure on the metabolism of oral C. albicans isolates from APECED patients with CMC and secondly, to analyse the specific molecular mechanisms that are responsible for these changes. The aim of the first study was to examine C. albicans strains from APECED patients and the level of cross-resistance to miconazole, the recommended topical compound for the treatment of oral candidosis. A total of 16% of the strains had decreased susceptibility to miconazole and all of these isolates had decreased susceptibility to fluconazole. Miconazole MICs also correlated with MICs to voriconazole and posaconazole. A significant positive correlation between the years of miconazole exposure and the MICs to azole antifungal agents was also found. These included azoles the patients had not been exposed to. The aim of our second study was to determine if the APECED patients are continuously colonized with the same C. albicans strains despite extensive antifungal treatment and to gain a deeper insight into the genetic changes leading to azole resistance. The strains were typed using MLST and our results confirmed that all patients were persistently colonized with the same or a genetically related strain despite antifungal treatment between isolations. No epidemic strains were found. mRNA expression was analysed by Northern blotting, protein level by western blotting, and TAC1 and ERG11 genes were sequenced. The main molecular mechanisms resulting in azole resistance were gain-of-function mutations in TAC1 leading to over expression of CDR1 and CDR2, genes linked to azole resistance. Several strains had also developed point mutations in ERG11, another gene linked to azole resistance. In the third study we used gas chromatography to test whether the level of carcinogenic acetaldehyde produced by C. albicans strains isolated from APECED patients were different from the levels produced by strains isolated from healthy controls and oral carcinoma patients. Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast, acetaldehyde is a by-product of the pyruvate bypass that converts pyruvate into acetyl-CoA during fermentation. Our results showed that strains isolated from APECED patients produced mutagenic levels of acetaldehyde in the presence of glucose (100mM, 18g/l) and the levels produced were significantly higher than those from strains isolated from controls and oral carcinoma patients. All strains in the study, however, were found to produce mutagenic levels of acetaldehyde in the presence of ethanol (11mM). The glucose and ethanol levels used in this study are equivalent to those found in food and beverages and our results highlight the role of dietary sugars and ethanol on carcinogenesis. The aims of our fourth study were to research the effect of growth conditions in the levels of acetaldehyde produced by C. albicans and to gain deeper insight into the role of different genes in the pyruvate-bypass in the production of high acetaldehyde levels. Acetaldehyde production in the presence of glucose increased by 17-fold under moderately hypoxic conditions compared to the levels produced under normoxic conditions. Under moderately hypoxic conditions acetaldehyde levels did not correlate with the expression of ADH1 and ADH2, genes catalyzing the oxidation of ethanol to acetaldehyde, or PDC11, the gene catalyzing the oxidation of pyruvate to acetaldehyde but correlated with the expression of down-stream genes ALD6 and ACS1. Our results highlight a problem where indiscriminate use of azoles may influence azole susceptibility and lead to the development of cross-resistance. Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations that occur in strains may lead to the development of azole-resistant isolates and metabolic changes leading to increased production of carcinogenic acetaldehyde.

Periodontal and dental health and oral self-care among adults with diabetes mellitus.

ABSTRACT Bakhshandeh, Soheila. Periodontal and dental health and oral self-care among adults with diabetes mellitus. Department of Oral Public Health, Institute of Dentistry, Faculty of Medicine, University of Helsinki, Helsinki, Finland. 2011. 49 pp. ISBN 978-952-10-7193-5(paperback). The aim of the present study was to assess oral health and treatment needs among Iranian adults with diabetes according to socio-demographic status, oral hygiene, diabetes related factors, and to investigate the relation between these determinants and oral health. Moreover, the effect of an educational oral health promotion intervention on their oral health and periodontal treatment needs was studied. The target population comprised adults with diabetes in Tehran, Iran. 299 dentate patients with diabetes, who were regular attendants to a diabetic clinic, were selected as the study subjects. Data collection was performed through a clinical dental examination and self-administered structured questionnaire. The questionnaire covered information of the subject s social background, medical history, oral health behaviour and smoking. The clinical dental examinations covered the registration of caries experience (DMFT), community periodontal index (CPI) and plaque index (PI). The intervention provided the adults with diabetes dental health education through a booklet. Reduction in periodontal treatment needs one year after the baseline examination was used as the main outcome. A high prevalence of periodontal pockets among the study population was found; 52% of the participants had periodontal pockets with a pocket depth of 4 to 5 mm and 35% had periodontal pockets with pocket depth of 6 mm or more. The mean of the DMFT index was 12.9 (SD=6.1), being dominated by filled teeth (mean 6.5) and missing teeth (mean 5.0). Oral self-care among adults with diabetes was inadequate and poor oral hygiene was observed in more than 80% of the subjects. The educational oral health promotion decreased periodontal treatment needs more in the study groups than in the control group. The poor periodontal health, poor oral hygiene and insufficient oral self-care observed in this study call for oral health promotion among adult with diabetes. An educational intervention showed that it is possible to promote oral health behaviour and to reduce periodontal treatment needs among adults with diabetes. The simplicity of the model used in this study allows it to be integrated to diabetes programmes in particular in countries with a developing health care system.

Oral health and kidney disease with emphasis on diabetic nephropathy

Chronic kidney disease (CKD) is a worldwide health problem, with adverse outcomes of cardiovascular disease and premature death. The ageing of populations along with the growing prevalence of chronic diseases such as diabetes and hypertension is leading to worldwide increase in the number of CKD patients. It has become evident that inflammation plays an important role in the pathogenesis of atherosclerosis complications. CKD patients also have an increased risk of atherosclerosis complications (including myocardial infarction, sudden death to cardiac arrhythmia, cerebrovascular accidents, and peripheral vascular disease). In line with this, oral and dental problems can be an important source of systemic inflammation. A decline in oral health may potentially act as an early marker of systemic disease progression. This series of studies examined oral health of CKD patients from predialysis, to dialysis and kidney transplantation in a 10-year follow-up study and in a cross-sectional study of predialysis CKD patients. Patients had clinical and radiographic oral and dental examination, resting and stimulated saliva flow rates were measured, whilst the biochemical and microbiological composition of saliva was analyzed. Lifestyle and oral symptoms were recorded using a questionnaire, and blood parameters were collected from the hospital records. The hypothesis was that the oral health status, symptoms, sensations, salivary flow rates and salivary composition vary in different renal failure stages and depend on the etiology of the kidney disease. No statistically significant difference were seen in the longitudinal study in the clinical parameters. However, some saliva parameters after renal transplantation were significantly improved compared to levels at the predialysis stage. The urea concentration of saliva was high in all stages. The salivary and plasma urea concentrations followed a similar trend, showing the lowest values in kidney transplant patients. Levels of immunoglobulin (Ig) A, G and M all decreased significantly after kidney transplantation. Increased concentrations of IgA, IgG and IgM may reflect disintegration of the oral epithelium and are usually markers of poor general oral condition. In the cross-sectional investigation of predialysis CKD patients we compared oral health findings of diabetic nephropathy patients to those with other kidney disease than diabetes. The results showed eg. more dental caries and lower stimulated salivary flow rates in the diabetic patients. HbA1C values of the diabetic patients were significantly higher than those in the other kidney disease group. A statistically significant difference was observed in the number of drugs used daily in the diabetic nephropathy group than in the other kidney disease group. In the logistic regression analyses, age was the principal explanatory factor for high salivary total protein concentration, and for low unstimulated salivary flow. Poor dental health, severity of periodontal disease seemed to be an explanatory factor for high salivary albumin concentrations. Salivary urea levels were significantly linked with diabetic nephropathy and with serum urea concentrations. Contrary to our expectation, however, diabetic nephropathy did not seem to affect periodontal health more severely than the other kidney diseases. Although diabetes is known to associate with xerostomia and other oral symptoms, it did not seem to increase the prevalence of oral discomfort. In summary, this series of studies has provided new information regarding the oral health of CKD patients. As expected, the commencement of renal disease reflects in oral symptoms and signs. Diabetic nephropathy, in particular, appears to impart a requirement for special attention in the oral health care of patients suffering from this disease.

Oral malodour – background and diagnostics

Bad breath or oral malodour can be related to gingival diseases, trimethylaminuria, various inflammation diseases of upper respiratory tract, foreign bodies in nasal cavity etc. Bad breath is usually, in 85 % to 95 % of cases, inflicted by gram negative anaerobic bacteria in tongue coating. These bacteria have a tendency of producing foul-smelling sulphur containing gases called volatile sulphur compounds or VSC. Main cause of bad breath is parodontitis or postnasal drip into posterior part of the tongue. Detecting bad breath is most efficiently done by organoleptic method. By skilled analyser the reason for oral malodour can be determined with great accuracy. For scientific study the most effective method is gas chromatography (GC) with flame photometric detector (FPD). With it almost every component of exhaled air can be detected both quantitative and qualitative. Effective chairside methods include portable sulphur monitors and saliva tests.

Identification of collagenolytic MMP networks as potential biomarkers in progressive chronic periodontitis subjects and MMP-8 null allele model

Chronic periodontitis results from a complex aetiology, including the formation of a subgingival biofilm and the elicitation of the host s immune and inflammatory response. The hallmark of chronic periodontitis is alveolar bone loss and soft periodontal tissue destruction. Evidence supports that periodontitis progresses in dynamic states of exacerbation and remission or quiescence. The major clinical approach to identify disease progression is the tolerance method, based on sequential probing. Collagen degradation is one of the key events in periodontal destructive lesions. Matrix metalloproteinase (MMP)-8 and MMP-13 are the primary collagenolytic MMPs that are associated with the severity of periodontal inflammation and disease, either by a direct breakdown of the collagenised matrix or by the processing of non-matrix bioactive substrates. Despite the numerous host mediators that have been proposed as potential biomarkers for chronic periodontitis, they reflect inflammation rather than the loss of periodontal attachment. The aim of the present study was to determine the key molecular MMP-8 and -13 interactions in gingival crevicular fluid (GCF) and gingival tissue from progressive periodontitis lesions and MMP-8 null allele mouse model. In study (I), GCF and gingival biopsies from active and inactive sites of chronic periodontitis patients, which were determined clinically by the tolerance method, and healthy GCF were analysed for MMP-13 and tissue inhibitor of matrix metalloproteinases (TIMP)-1. Chronic periodontitis was characterised by increased MMP-13 levels and the active sites showed a tendency of decreased TIMP-1 levels associated with increments of MMP-13 and total protein concentration compared to inactive sites. In study (II), we investigated whether MMP-13 activity was associated with TIMP-1, bone collagen breakdown through ICTP levels, as well as the activation rate of MMP-9 in destructive lesions. The active sites demonstrated increased GCF ICTP levels as well as lowered TIMP-1 detection along with elevated MMP-13 activity. MMP-9 activation rate was enhanced by MMP-13 in diseased gingival tissue. In study (III), we analysed the potential association between the levels, molecular forms, isoenzyme distribution and degree of activation of MMP-8, MMP-14, MPO and the inhibitor TIMP-1 in GCF from periodontitis progressive patients at baseline and after periodontal therapy. A positive correlation was found for MPO/MMP-8 and their levels associated with progression episodes and treatment response. Because MMP-8 is activated by hypochlorous acid in vitro, our results suggested an interaction between the MPO oxidative pathway and MMP-8 activation in GCF. Finally, in study (IV), on the basis of the previous finding that MMP-8-deficient mice showed impaired neutrophil responses and severe alveolar bone loss, we aimed to characterise the detection patterns of LIX/CXCL5, SDF-1/CXCL12 and RANKL in P. gingivalis-induced experimental periodontitis and in the MMP-8-/- murine model. The detection of neutrophil-chemoattractant LIX/CXCL5 was restricted to the oral-periodontal interface and its levels were reduced in infected MMP-8 null mice vs. wild type mice, whereas the detection of SDF-1/CXCL12 and RANKL in periodontal tissues increased in experimentally-induced periodontitis, irrespectively from the genotype. Accordingly, MMP-8 might regulate LIX/CXCL5 levels by undetermined mechanisms, and SDF-1/CXCL12 and RANKL might promote the development and/or progression of periodontitis.

Production of Carcinogenic Acetaldehyde by Oral Microbiome

Oral cancer is the seventh most common cancer worldwide and its incidence is increasing. The most important risk factors for oral cancer are chronic alcohol consumption and tobacco smoking, up to 80 % of oral carcinomas are estimated to be caused by alcohol and tobacco. They both trigger an increased level of salivary acetaldehyde, during and after consumption, which is believed to lead to carcinogenesis. Acetaldehyde has multiple mutagenic features and it has recently been classified as a Group 1 carcinogen for humans by the International Agency for Research on Cancer. Acetaldehyde is metabolized from ethanol by microbes of oral microbiota. Some oral microbes possess alcohol dehydrogenase enzyme (ADH) activity, which is the main enzyme in acetaldehyde production. Many microbes are also capable of acetaldehyde production via alcohol fermentation from glucose. However, metabolism of ethanol into acetaldehyde leads to production of high levels of this carcinogen. Acetaldehyde is found in saliva during and after alcohol consumption. In fact, rather low ethanol concentrations (2-20mM) derived from blood to saliva are enough for microbial acetaldehyde production. The high acetaldehyde levels in saliva after alcohol challenge are explained by the lack of oral microbiota and mucosa to detoxify acetaldehyde by metabolizing it into acetate and acetyl coenzymeA. The aim of this thesis project was to specify the role of oral microbes in the in vitro production of acetaldehyde in the presence of ethanol. In addition, it was sought to establish whether microbial metabolism could also produce acetaldehyde from glucose. Furthermore, the potential of xylitol to inhibit ethanol metabolism and acetaldehyde production was explored. Isolates of oral microbes were used in the first three studies. Acetaldehyde production was analyzed after ethanol, glucose and fructose incubation with gas chromatography measurement. In studies I and III, the ADH enzyme activity of some microbes was measured by fluorescence. The effect of xylitol was analyzed by incubating microbes with ethanol and xylitol. The fourth study was made ex vivo and microbial samples obtained from different patient groups were analyzed. This work has demonstrated that isolates of oral microbiota are able to produce acetaldehyde in the presence of clinically relevant ethanol and glucose concentrations. Significant differences were found between microbial species and isolates from different patient groups. In particular, the ability of candidal isolates from APECED patients to produce significantly more acetaldehyde in glucose incubation compared to healthy and cancer patient isolates is an interesting observation. Moreover, xylitol was found to reduce their acetaldehyde production significantly. Significant ADH enzyme activity was found in the analyzed high acetaldehyde producing streptococci and candida isolates. In addition, xylitol was found to reduce the ADH enzyme activity of C. albicans. Some results from the ex vivo study were controversial, since acetaldehyde production did not correlate as expected with the amount of microbes in the samples. Nevertheless, the samples isolated from patients did produce significant amounts of acetaldehyde with a clinically relevant ethanol concentration.