Probiotic activity of Lactobacillus delbrueckii subsp. bulgaricus in the oral cavity

Yogurt consumption has been related to longevity of some populations living on the Balkans. Yogurt starter L. delbrueckii subsp. bulgaricus and Str. thermophilus have been recognized as probiotics with verified beneficial health effects. The oral cavity emerges as a arget for probiotic applications. Probiotics have demonstrated promising results in controlling dental diseases and oral yeast infections. However, L. bulgaricus despite its broad availability in dairy products has not been evaluated for probiotic activity in the mouth. These series of studies investigated in vitro properties of L. bulgaricus to outline its potential as an oral probiotic. Prerequisite probiotic properties in the mouth are resistance to oral defense mechanisms, adherence to saliva-coated surfaces, and inhibition of oral pathogens. L. bulgaricus strains showed a strain-dependent inhibition of oral streptococci and Aggregatibacter actinomycetemcomitans, whereas none of the dairy starter strains could affect growth of Porphyromonas gingivalis and Fusobacterium nucleatum. Adhesion is a factor contributing to colonization of the species at the target site. Radiolabeled L. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated surfaces. The effects of lysozyme on adhesion and adhesion of Streptococcus sanguinis after lactobacilli pretreatment were also assessed. Adhesion of L. bulgaricus remained lower in comparison to L. rhamnosus GG. One L. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment significantly increased Lactobacillus adhesion. Low gelatinolytic activity was observed for all strains and no conversion of proMMP-9 to its active form was induced by L. bulgaricus. Safety assessment ruled out deleterious effects of L. bulgaricus on extracellular matrix structures. Cytokine response of oral epithelial cells was assessed by measuring IL-8 and TNF-α in cell culture supernatants. The effect of P. gingivalis on cytokine secretion after lactobacilli pretreatment was also assessed. A strain- and time-dependent induction of IL-8 was observed with live bacteria inducing the highest levels of cytokine secretion. Levels of TNF-α were low and only one of ten L. bulgaricus strains stimulated TNF-α secretion similar to positive control. The addition of P. gingivalis produced immediate reduction of cytokine levels within the first hours of incubation irrespective of lactobacilli strains co-cultured with epithelial cells. According to these studies strains among the L. delbrueckii subsp. bulgaricus species may have beneficial probiotic properties in the mouth. Their potential in prevention and management of common oral infectious diseases needs to be further studied.

Interaction of alcohol and smoking in the pathogenesis of upper digestive tract cancers - possible chemoprevention with cysteine

Background: Alcohol consumption and smoking are the main causes of upper digestive tract cancers. These risk factors account for over 75% of all cases in developed countries. Epidemiological studies have shown that alcohol and tobacco interact in a multiplicative way to the cancer risk, but the pathogenetic mechanism behind this is poorly understood. Strong experimental and human genetic linkage data suggest that acetaldehyde is one of the major factors behind the carcinogenic effect. In the digestive tract, acetaldehyde is mainly formed by microbial metabolism of ethanol. Acetaldehyde is also a major constituent of tobacco smoke. Thus, acetaldehyde from both of these sources may have an interacting carcinogenic effect in the human upper digestive tract. Aims: The first aim of this thesis was to investigate acetaldehyde production and exposure in the human mouth resulting from alcohol ingestion and tobacco smoking in vivo. Secondly, specific L-cysteine products were prepared to examine their efficacy in the binding of salivary acetaldehyde in order to reduce the exposure of the upper digestive tract to acetaldehyde. Methods: Acetaldehyde levels in saliva were measured from human volunteers during alcohol metabolism, during tobacco smoking and during the combined use of alcohol and tobacco. The ability of L-cysteine to eliminate acetaldehyde during alcohol metabolism and tobacco smoking was also investigated with specifically developed tablets. Also the acetaldehyde production of Escherichia coli - an important member of the human microbiota - was measured in different conditions prevailing in the digestive tract. Results and conclusions: These studies established that smokers have significantly increased acetaldehyde exposure during ethanol consumption even when not actively smoking. Acetaldehyde exposure was dramatically further increased during active tobacco smoking. Thus, the elevated aerodigestive tract cancer risk observed in smokers and drinkers may be the result of the increased acetaldehyde exposure. Acetaldehyde produced in the oral cavity during ethanol challenge was significantly decreased by a buccal L-cysteine -releasing tablet. Also smoking-derived acetaldehyde could be totally removed by using a tablet containing L-cysteine. In conclusion, this thesis confirms the essential role of acetaldehyde in the pathogenesis of alcohol- and smoking-induced cancers. This thesis presents a novel experimental approach to decrease the local acetaldehyde exposure of the upper digestive tract with L-cysteine, with the eventual goal of reducting the prevalence of upper digestive tract cancers.

Major histocompatibility complex and coronary artery disease: Special emphasis on Chlamydia pneumoniae, and periodontitis

Most of the genes in the MHC region are involveed in adaptive and innate immunity, with essential function in inflammatory reactions and in protection against infections. These genes might serve as a candidate region for infection and inflammation associated diseases. CAD is an inflammatory disease. The present set of studies was performed to assess whether the MHC region harbors genetic markers for CAD, and whether these genetic markers explain the CAD risk factors: e.g., C. pneumoniae, periodontitis, and periodontal pathogens. Study I was performed using two separate patient materials and age- and sex-matched healthy controls, categorizing them into two independent studies: the HTx and ACS studies. Both studies consistently showed the HLA-A3– B35– DR1 (35 ancestral haplotype) haplotype as a susceptible MHC genetic marker for CAD. HLA-DR1 alone was associated not only with CAD, but also with CAD risk factor diseases, e.g., diabetes mellitus, and hyperlipidemia. The ACS study further showed the HLA-B*07 and -DRB1*15 -related haplotype as a protective MHC haplotype for CAD. Study II showed that patients with CAD showed signs of chronic C. pneumoniae infection when compared to age- and sex-matched healthy controls. HLA-B*35 or -related haplotypes associated with the C. pneumoniae infection markers. Among these haplotype carriers, males and smokers associated with elevated C. pneumoniae infection markers. Study III showed that CAD patients with periodontitis had elevated serum markers of P. gingivalis and occurrence of the pathogen in saliva. LTA+496C strongly associated with periodontitis, while HLA-DRB1*01 with periodontitis and with the elevated serum antibodies of P. gingivalis. Study IV showed that the increased level of C3/C4 ratio was a new risk factor and was associated with recurrent cardiovascular end-points. The increased C3 and decreased C4 concentrations in serum explained the increased level of the C3/C4 ratio. Both the higher than cut-off value (4.53) and the highest quartile of the C3/C4 ratio were also associated with worst survival, increased end-points, and C4 null alleles. The presence of C4 null alleles associated with decreased serum C4 concentration, and increased C3/C4 ratio. In conclusion, the present studies show that the CAD susceptibility haplotype (HLA-A3− B35− DR1 -related haplotypes, Study I) partially explains the development of CAD in patients possessing several recognized and novel risk factors: diabetes mellitus, increased LDL, smoking, C4B*Q0, C. pneumnoiae, periodontitis, P. gingivalis, and complement C3/C4 ratio (Study II, III, and IV).

Pathological changes at the target tissue level in Sjögren's syndrome and their effect on the exocrine function of the salivary glands

Sjögren s syndrome (SS) is a common autoimmune disease affecting the lacrimal and salivary glands. SS is characterized by a considerable female predominance and a late age of onset, commonly at the time of adreno- and menopause. The levels of the androgen prohormone dehydroepiandrosterone-sulphate (DHEA-S) in the serum are lower in patients with SS than in age- and sex-matched healthy control subjects. The eventual systemic effects of low androgen levels in SS are not currently well understood. Basement membranes (BM) are specialized layers of extracellular matrix and are composed of laminin (LM) and type IV collagen matrix networks. BMs deliver messages to epithelial cells via cellular LM-receptors including integrins (Int) and Lutheran blood group antigen (Lu). The composition of BMs and distribution of LM-receptors in labial salivary glands (LSGs) of normal healthy controls and patients with SS was assessed. LMs have complex and highly regulated distribution in LSGs. LMs seem to have specific tasks in the dynamic regulation of acinar cell function. LM-111 is important for the normal acinar cell differentiation and its expression is diminished in SS. Also LM-211 and -411 seem to have some acinar specific functional tasks in LSGs. LM-311, -332 and -511 seem to have more general structure maintaining and supporting roles in LSGs and are relatively intact also in SS. Ints α3β1, α6β1, α6β4 and Lu seem to supply structural basis for the firm attachment of epithelial cells to the BM in LSGs. The expression of Ints α1β1 and α2β1 differed clearly from other LM-receptors in that they were found almost exclusively around the acini and intercalated duct cells in salivons suggesting some type of acinar cell compartment-specific or dominant function. Expression of these integrins was lower in SS compared to healthy controls suggesting that the LM-111 and -211-to-Int α1β1 and α2β1 interactions are defective in SS and are crucial to the maintenance of the acini in LSGs. DHEA/DHEA-S concentration in serum and locally in saliva of patients with SS seems to have effects on the salivary glands. These effects were first detected using the androgen-dependent CRISP-3 protein, the production and secretion of which were clearly diminished in SS. This might be due to the impaired function of the intracrine DHEA prohormone metabolizing machinery, which fails to successfully convert DHEA into its active metabolites in LSGs. The progenitor epithelial cells from the intercalated ductal area of LSGs migrate to the acinar compartment and then undergo a phenotype change into secretory acinar cells. This migration and phenotype change seem to be regulated by the LM-111-to-Int α1β1/Int α2β1 interactions. Lack of these interactions could be one factor limiting the normal remodelling process. Androgens are effective stimulators of Int α1β1 and α2β1 expression in physiologic concentrations. Addition of DHEA to the culture medium had effective stimulating effect on the Int α1β1 and α2β1 expression and its effect may be deficient in the LSGs of patients with SS.

Oral health and menopause

Hormone therapy (HT) is widely used to relieve climacteric symptoms in order to increase the well-being of the women. The benefits as well as side-effects of HT are well documented. The principal menopausal oral symptoms are dry mouth (DM) and sensation of painful mouth (PM) due to various causes. Profile studies have indicated that HT users are more health-conscious than non-users. The hypothesis of the present study was that there are differences in oral health between woman using HT and those not using HT. A questionnaire study of 3173 women of menopausal age (50-58 years old) was done to investigate the prevalence of self-assessed sensations of PM and DM. Of those women participating in the questionnaire study, a random sample of 400 (200 using, 200 not using HT) was examined clinically in a 2-year follow-up study. Oral status was recorded according to WHO methods using DMFT and CPITN indices. The saliva flows were measured, salivary total protein, albumin and immunoglobulin concentrations and selected periodontal micro-organisms were analysed, and panoramic tomography of the jaws was taken. The patients filled in a structured questionnaire on their systemic health, medication and health habits. According to our questionnaire study there was no significant difference in the occurrence of self- assessed PM or DM between the HT users and non-users. According to logistic regression analyses, climacteric complaints significantly correlated with the occurrence of PM (p=0.000) and DM (p=0.000) irrespective of the use of HT, indicating that PM and DM are associated with climacteric symptoms in general. There was no difference between the groups in DMFT index values at follow up. The number of filled teeth (FT) showed a significant (p<0.05) increase in the HT group at follow-up. Periodontitis was diagnosed in 79% of HT users at baseline and in 71% at the follow-up. The values for non-HT users were 80% vs. 76%, respectively (Ns.). The mean numbers of ≥ 6 mm deep periodontal pockets were 0.9 ± 1.7 at baseline vs. 1.1 ± 2.1 two years later in the HT group, and 1.0 ± 1.7 vs. 1.2 ± 1.9, respectively, in the non-HT group. In a large Finnish national health survey, the prevalence of peridontitis of women of this age group was lower, but the prevalence of severe periodontitis seemed to be higher than in our study. Salivary albumin, IgG and IgM concentrations decreased in the HT group during the 2-year follow up (p<0.05), possibly indicating an improvement in epithelial integrity. No difference was found in any other salivary parameters or in the prevalence of the periodontal bacteria between or within the groups. In conclusion, the present findings showed that 50 to 58 year old women living in Helsinki have fairly good oral and dental health. The occurrence of PM and DM seemed to be associated with climacteric symptoms in general, and the use of HT did not affect the oral symptoms studied.

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.

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.