Oral Immune Defense against Chronic Hyperplastic Candidosis

Candida yeast species are widespread opportunistic microbes, which are usually innocent opportunists unless the systemic or local defense system of the host becomes compromised. When they adhere on a fertile substrate such as moist and warm, protein-rich human mucosal membrane or biomaterial surface, they become activated and start to grow pseudo and real hyphae. Their growth is intricately guided by their ability to detect surface defects (providing secure hiding , thigmotropism) and nutrients (source of energy, chemotropism). The hypothesis of this work was that body mobilizes both non-specific and specific host defense against invading candidal cells and that these interactions involve resident epithelial cells, rapidly responding non-specific protector neutrophils and mast cells as well as the antigen presenting and responding den-dritic cell lymphocyte plasma cell system. It is supposed that Candida albicans, as a result of dar-winistic pressure, has developed or is utilizing strategies to evade these host defense reactions by e.g. adhering to biomaterial surfaces and biofilms. The aim of the study was to assess the host defense by taking such key molecules of the anti-candidal defense into focus, which are also more or less characteristic for the main cellular players in candida-host cell interactions. As a model for candidal-host interaction, sections of chronic hyperplastic candidosis were used and compared with sections of non-infected leukoplakia and healthy tissue. In this thesis work, neutrophil-derived anti-candidal α-defensin was found in the epithelium, not only diffusely all over in the epithelium, but as a strong α-defensin-rich superficial front probably able to slow down or prevent penetration of candida into the epithelium. Neutrophil represents the main host defence cell in the epithelium, to which it can rapidly transmigrate from the circulation and where it forms organized multicellular units known as microabscesses (study I). Neutrophil chemotactic inter-leukin-8 (IL-8) and its receptor (IL-8R) were studied and were surprisingly also found in the candidal cells, probably helping the candida to keep away from IL-8- and neutrophil-rich danger zones (study IV). Both leukocytes and resident epithelial cells contained TLR2, TLR4 and TLR6 receptors able to recognize candidal structures via utilization of receptors similar to the Toll of the banana fly. It seems that candida can avoid host defence via stimulation of the candida permissive TLR2 instead of the can-dida injurious TLR4 (study V). TLR also provides the danger signal to the immune system without which it will not be activated to specifically respond against candidal antigens. Indeed, diseased sites contained receptor activator of nuclear factor kappa B ligand (RANKL; II study), which is important for the antigen capturing, processing and presenting dendritic cells and for the T lymphocyte activation (study III). Chronic hyperplastic candidosis provides a disease model that is very useful to study local and sys-temic host factors, which under normal circumstances restrain C. albicans to a harmless commensal state, but failure of which in e.g. HIV infection, cancer and aging may lead to chronic infection.

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

Cavity-enhanced laser spectroscopic studies of vibrational overtones of acetylene

This thesis contains five experimental spectroscopic studies that probe the vibration-rotation energy level structure of acetylene and some of its isotopologues. The emphasis is on the development of laser spectroscopic methods for high-resolution molecular spectroscopy. Three of the experiments use cavity ringdown spectroscopy. One is a standard setup that employs a non-frequency stabilised continuous wave laser as a source. In the other two experiments, the same laser is actively frequency stabilised to the ringdown cavity. This development allows for increased repetition rate of the experimental signal and thus the spectroscopic sensitivity of the method is improved. These setups are applied to the recording of several vibration-rotation overtone bands of both H(12)C(12)CH and H(13)C(13)CH. An intra-cavity laser absorption spectroscopy setup that uses a commercial continuous wave ring laser and a Fourier transform interferometer is presented. The configuration of the laser is found to be sub-optimal for high-sensitivity work but the spectroscopic results are good and show the viability of this type of approach. Several ro-vibrational bands of carbon-13 substituted acetylenes are recorded and analysed. Compared with earlier work, the signal-to-noise ratio of a laser-induced dispersed infrared fluorescence experiment is enhanced by more than one order of magnitude by exploiting the geometric characteristics of the setup. The higher sensitivity of the spectrometer leads to the observation of two new symmetric vibrational states of H(12)C(12)CH. The precision of the spectroscopic parameters of some previously published symmetric states is also improved. An interesting collisional energy transfer process is observed for the excited vibrational states and this phenomenon is explained by a simple step-down model.

Production of F4 Fimbrial Adhesin in Plants: a Model for Oral Porcine Vaccine against Enterotoxigenic Escherichia coli

F4 fimbriae of enterotoxigenic Escherichia coli (ETEC) are highly stable multimeric structures with a capacity to evoke mucosal immune responses. With these characters F4 offer a unique model system to study oral vaccination against ETEC-induced porcine postweaning diarrhea. Postweaning diarrhea is a major problem in piggeries worldwide and results in significant economic losses. No vaccine is currently available to protect weaned piglets against ETEC infections. Transgenic plants provide an economically feasible platform for large-scale production of vaccine antigens for animal health. In this study, the capacity of transgenic plants to produce FaeG protein, the major structural subunit and adhesin of F4 fimbria, was evaluated. Using the model plant tobacco, the optimal subcellular location for FaeG accumulation was examined. Targeting of FaeG into chloroplasts offered a superior accumulation level of 1% of total soluble proteins (TSP) over the other investigated subcellular locations, namely, the endoplasmic reticulum and the apoplast. Moreover, we determined whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, i.e. stability in gastrointestinal conditions, binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. The chloroplast-derived FaeG protein did show resistance against low pH and proteolysis in the simulated gastrointestinal conditions and was able to bind to the F4R, subsequently inhibiting the F4+ ETEC binding in a dose-dependent manner. To investigate the oral immunogenicity of FaeG protein, the edible crop plant alfalfa was transformed with the chloroplast-targeting construct and equally to tobacco plants, a high-yield FaeG accumulation of 1% of TSP was obtained. A similar yield was also obtained in the seeds of barley, a valuable crop plant, when the FaeG-encoding gene was expressed under an endosperm-specific promoter and subcellularly targeted into the endoplasmic reticulum. Furthermore, desiccated alfalfa plants and barley grains were shown to have a capacity to store FaeG protein in a stable form for years. When the transgenic alfalfa plants were administred orally to weaned piglets, slight F4-specific systemic and mucosal immune responses were induced. Co-administration of the transgenic alfalfa and the mucosal adjuvant cholera toxin enhanced the F4-specific immune response; the duration and number of F4+ E. coli excretion following F4+ ETEC challenge were significantly reduced as compared with pigs that had received nontransgenic plant material. In conclusion, the results suggest that transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against porcine F4+ ETEC infections. The findings here thus present new approaches to develop the vaccination strategy against porcine postweaning diarrhea.

Effects of SLCO1B1 polymorphism on the pharmacokinetics of the oral antidiabetic drugs repaglinide, nateglinide, rosiglitazone, and pioglitazone

Organic anion-transporting polypeptide 1B1 (OATP1B1), encoded by the SLCO1B1 gene, is an influx transporter expressed on the sinusoidal membrane of human hepatocytes. The common c.521T>C (p.Val174Ala) single-nucleotide polymorphism (SNP) of the SLCO1B1 gene has been associated with reduced OATP1B1 transport activity in vitro and increased plasma concentrations of several of its substrate drugs in vivo in humans. Another common SNP of the SLCO1B1 gene, c.388A>G (p.Asn130Asp), defining the SLCO1B1*1B (c.388G-c.521T) haplotype, has been associated with increased OATP1B1 transport activity in vitro. The aim of this thesis was to investigate the role of SLCO1B1 polymorphism in the pharmacokinetics of the oral antidiabetic drugs repaglinide, nateglinide, rosiglitazone, and pioglitazone. Furthermore, the effect of the SLCO1B1 c.521T>C SNP on the extent of interaction between gemfibrozil and repaglinide as well as the role of the SLCO1B1 c.521T>C SNP in the potential interaction between atorvastatin and repaglinide were evaluated. Five crossover studies with 2-4 phases were carried out, with 20-32 healthy volunteers in each study. The effects of the SLCO1B1 c.521T>C SNP on single doses of repaglinide, nateglinide, rosiglitazone, and pioglitazone were investigated in Studies I and V. In Study II, the effects of the c.521T>C SNP on repaglinide pharmacokinetics were investigated in a dose-escalation study, with repaglinide doses ranging from 0.25 to 2 mg. The effects of the SLCO1B1*1B/*1B genotype on repaglinide and nateglinide pharmacokinetics were investigated in Study III. In Study IV, the interactions of gemfibrozil and atorvastatin with repaglinide were evaluated in relation to the c.521T>C SNP. Plasma samples were collected for drug concentration determinations. The pharmacodynamics of repaglinide and nateglinide was assessed by measuring blood glucose concentrations. The mean area under the plasma repaglinide concentration-time curve (AUC) was ~70% larger in SLCO1B1 c.521CC participants than in c.521TT participants (P ≤ 0.001), but no differences existed in the pharmacokinetics of nateglinide, rosiglitazone, and pioglitazone between the two genotype groups. In the dose-escalation study, the AUC of repaglinide was 60-110% (P ≤ 0.001) larger in c.521CC participants than in c.521TT participants after different repaglinide doses. Moreover, the AUC of repaglinide increased linearly with repaglinide dose in both genotype groups (r > 0.88, P 0.001). The AUC of repaglinide was ~30% lower in SLCO1B1*1B/*1B participants than in SLCO1B1*1A/*1A (c.388AA-c.521TT) participants (P = 0.007), but no differences existed in the AUC of nateglinide between the two genotype groups. In the drug-drug interaction study, the mean increase in the repaglinide AUC by gemfibrozil was ~50% (P = 0.002) larger in c.521CC participants than in c.521TT participants, but the relative (7-8-fold) increases in the repaglinide AUC did not differ significantly between the genotype groups. In c.521TT participants, atorvastatin increased repaglinide peak plasma concentration and AUC by ~40% (P = 0.001) and ~20% (P = 0.033), respectively. In each study, after repaglinide administration, there was a tendency towards lower blood glucose concentrations in c.521CC participants than in c.521TT participants. In conclusion, the SLCO1B1 c.521CC genotype is associated with increased and the SLCO1B1*1B/*1B genotype with decreased plasma concentrations of repaglinide, consistent with reduced and enhanced hepatic uptake, respectively. Inhibition of OATP1B1 plays a limited role in the interaction between gemfibrozil and repaglinide. Atorvastatin slightly raises plasma repaglinide concentrations, probably by inhibiting OATP1B1. The findings on the effect of SLCO1B1 polymorphism on the pharmacokinetics of the drugs studied suggest that in vivo in humans OATP1B1 significantly contributes to the hepatic uptake of repaglinide, but not to that of nateglinide, rosiglitazone, or pioglitazone. SLCO1B1 polymorphism may be associated with clinically significant differences in blood glucose-lowering response to repaglinide, but probably has no effect on the response to nateglinide, rosiglitazone, or pioglitazone.

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.

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.