Cutaneous porphyrias : Clinical and histopathological study

The prevalence of variegate porphyria (VP) (2.1:100 000, in 2006 n=108) was higher in Finland than elsewhere in European countries due to a founder effect (R152C). The incidence of VP was estimated at 0.2:1 000 000 based on the number of new symptomatic patients yearly. The prevalence of porphyria cutanea tarda (PCT) was 1.2:100 000 (in 2006 n=63), which is only one fourth of the numbers reported from other European countries. The estimated incidence of PCT was 0.5:1 000 000. Based on measurements of the uroporphyrinogen decarboxylase activity in erythrocytes, the proportion of familial PCT was 49% of the cases. The prevalence of erythropoietic protoporphyria (EPP) was at 0.8:100 000 (in 2006 n=39) including asymptomatic carriers of a mutation in the ferrochelatase (FECH) gene. The incidence of EPP was estimated at 0.1:1 000 000. After 1980 the penetrance was 37% among patients with VP. Of the mutation carriers (n=57) 30% manifested with skin symptoms. Frequency of skin symptom as only clinical sign was stable before or after 1980 (22% vs. 21%), but acute attacks became infrequent (29% vs. 7%). Of the symptomatic patients 30% had both acute attacks and skin symptoms and 80% had skin symptoms. Fragility (95%) and blistering (46%) of the skin in the backs of the hands were the most common skin symptoms. Transient correction of porphyrin metabolism using eight haem arginate infusions within five weeks had no effect on the skin symptoms in three of four patients with VP. In one case skin symptoms disappeared transiently. One patient with homozygous VP had severe photosensitivity since birth. Sensory polyneuropathy, glaucoma and renal failure developed during the 25-year follow-up without the presence of acute attacks. The I12T mutation was detected in both of his alleles in the protoporphyrinogen oxidase gene. Lack of skin symptoms and infrequency of acute attacks (1/9) in the patients with I12T mutation at the heterozygous stage indicate a mild phenotype (the penetrance 11%). Four mutations (751delGAGAA, 1122delT, C286T, C343T) in the FECH gene were characterised in four of 15 families with EPP. Burning pain (96%) and swelling (92%) of the sun-exposed skin were the major skin symptoms. Hepatopathy appeared in one of 25 symptomatic patients (4%). Clinical manifestations and associated factors of PCT were similar in the sporadic and familial types of PCT. The majority of the patients with PCT had one to three precipitating factors: alcohol intake (78%), mutations in hemochromatosis associated gene (50%), use of oestrogen (25% of women) and hepatitis B or C infections (25 %). Fatty liver disease (67%) and siderosis (67%) were commonly found in their liver biopsies. The major histopathological change of the sun-exposed skin in the patients with VP (n=20), EPP (n=8) and PCT (n=5) was thickening of the vessel walls of the upper dermis suggesting that the vessel wall is the primary site of the phototoxic reaction in each type of porphyria. The fine structure of the vessel walls was similar in VP, EPP and PCT consisting of the multilayered basement membrane and excess of finely granular substance between the layers which were surrounded by the band of homogenous material. EPP was characterised by amorphous perivascular deposits extending also to the extravascular space. In direct immunofluorescence study homogenous IgG deposits in the vessel walls of the upper dermis of the sun-exposed skin were demonstrated in each type of porphyria. In EPP the excess material around vessel walls consisted of other proteins such as serum amyloid protein, and kappa and lambda light chains in addition to the basement membrane constituents such as collagen IV and laminin. These results suggest that the alterations of the vessel walls are a consequence of the repeated damage and the repairing process in the vessel wall. The microscopic alterations could be demonstrated even in the normal looking but sun-exposed skin of the patients with EPP during the symptom-free phase suggesting that vascular change can be chronic. The stability of vascular changes in the patients with PCT after treatment indicates that circulating porphyrins are not important for the maintenance of the changes.

Importance of COMT in the regulation of prefrontal dopamine

The prefrontal cortex (PFC), located in the anterior region of the frontal lobe, is considered to have several key roles in higher cognitive and executive functions. In general, the PFC can be seen as a coordinator of thought and action allowing subjects to behave in a goal-directed manner. Due to its anatomical connections with a variety of cortical and subcortical structures, several neurotransmitters, including dopamine, are involved in the regulation of PFC activity. In general, the majority of released dopamine is cleared by the dopamine transporter (DAT). In the PFC however, the number of presynaptic DAT is diminished, emphasizing the relative importance of catechol-O-methyltransferase (COMT) in dopamine metabolism. As a result, the role of COMT in the etiology of psychotic disorders is under constant debate. The present study investigated the role of COMT in prefrontal cortical dopamine metabolism by different neurochemical methods in COMT knockout (COMT-KO) mice. Pharmacological tools to inhibit other dopamine clearing mechanisms were also used for a more comprehensive and collective picture. In addition, this study investigated how a lack of the soluble (S-) COMT isoform affects the total COMT activity as well as the pharmacokinetics of orally administered L-dopa using mutant mice expressing only the membrane-bound (MB-) COMT isoform. Also the role of COMT in striatal and accumbal dopamine turnover during Δ9-tetrahydrocannabinol (THC) challenge was studied. We found markedly increased basal dopamine concentrations in the PFC, but not the striatum or nucleus accumbens (NAcc), of mice lacking COMT. Pharmacological inhibition of the noradrenaline transporter (NET) and monoamine oxidase (MAO) elevated prefrontal cortical dopamine levels several-fold, whereas inhibition of DAT did not. The lack of COMT doubled the dopamine raising effects of NET and MAO inhibition. No compensatory expression of either DAT or NET was found in the COMT-KO mice. The lack of S-COMT decreased the total COMT activity by 50-70 % and modified dopamine transmission and the pharmacokinetics of exogenous Ldopa in a sex and tissue specific manner. Finally, we found that subsequent tolcapone and THC increased dopamine levels in the NAcc, but not in the striatum. Conclusively, this study presents neurochemical evidence for the important role of COMT in the PFC and shows that COMT is responsible for about half of prefrontal cortical dopamine metabolism. This study also highlights the previously underestimated proportional role of MB-COMT and supports the clinical evidence of a gene x environment interaction between COMT and cannabis.

Effects of oral calcium sensitizers on experimental heart failure

Suun kautta annosteltava kalsiumherkistäjä parantaa sydämen vajaatoimintaan liittyvää pumppausvajetta kokeellisissa sydämen vajaatoimintamalleissa Huolimatta viime vuosikymmenien lääketieteellisestä kehityksestä krooninen sydämen vajaatoiminta on silti edelleen vakava, elämänlaatua voimakkaasti rajoittava sairaus. Kalsiumherkistäjät ovat uusi, sydämen pumppausvoimaa lisäävä lääkeryhmä. Levosimendaani, kotimaista alkuperää oleva kalsiumherkistäjä, on kliinisessä käytössä akuutin vajaatoiminnan hoitoon suonensisäisesti ja lyhytaikaisesti annosteltavana valmisteena. Levosimendaanilla on aktiivinen metaboliitti, OR-1896, jonka oletetaan olevan vuorokauden mittaisen levosimendaani-infuusion jälkeen havaittujen useita päiviä kestävien hyödyllisisten vaikutuksisten takana. Levosimendaanin kroonisen, suun kautta tapahtuvan annostelun vaikutuksista tieto on vähäisempää, mutta sillä näyttää olevan positiivisia vaikutuksia potilaiden raportoimana. FM Marjut Louhelainen on selvittänyt väitöskirjassaan suun kautta annosteltavan levosimendaanin ja sen pitkäkestoisen aktiivisen metaboliitin vaikutuksia kroonisen vajaatoiminnan hoidossa käyttämällä sekä hypertensiivisen sydäntaudin että 2 tyypin diabeteksen komplisoimaan sydäninfarktin kokeellisia malleja. Tutkimuksessa selvitettiin lisäksi vajaatoimintaan johtavia molekyylitason tapahtumia sydänlihaksessa. Tutkimuksessa osoitettiin, että krooninen suun kautta annosteltu hoito sekä kalsiumherkistäjä levosimendaanilla että sen aktiivisella metaboliitilla estää hypertensiiviseen sydämen vajaatoiminnan aikaasaamaa sydämen uudelleenmuovaantumista ja siihen liittyvää kuolleisuutta. Nämä vaikutukset välittyivät vähentyneen sydänlihassoluhypertrofian, solukuolleisuuden ja neurohumaraalisen aktivaation kautta. Levosimendaanin ja OR-1896:n osoitettiin myös parantavan sydämen pumppausfunktiota tyyppi 2 diabeteksen komplisoimassa sydäninfarktissa. Ei-diabeettiseen tilanteeseen verrattuna diabetekseen liittyvä infarktin jälkeinen vajaatoiminnan kehitys oli yhteydessä lisääntyneeseen tulehdukseen, fibroosiin, solukuolemaan, neurohumoraaliseen aktivaatioon ja ennenaikaiseen kudoksen vanhenemiseen. Sekä levosimendaani, että OR-1869 vähensivät tulehduksen, fibroosin ja solukuoleman merkkejä ja vaimensi neurohumoraalista aktivaatiota. OR-1896 myös vähensi solujen vanhenemiseen liittyvien merkkiaineiden ilmentymistä. Väitöskirjassa todettiin, että suun kautta annosteltuna sekä levosimendaani, että sen aktiivinen metaboliitti OR-1896, omaavat terapeuttista potentiaalia sekä hypertensiivisen sydäntaudin hoitoon että sydäninfarktin jälkeisen vajaatoiminnan estoon. FM Marjut Louhelaisen farmakologian alaan kuuluva väitöskirja Effects of oral calcium sensitizers on experimental heart failure tarkastetaan Helsingin yliopiston Lääketieteellisessä tiedekunnassa perjantaina 29.01.2010 klo 12 (Biomedicum Helsinki, luentosali 2, Haartmaninkatu 8, Helsinki). Vastaväittäjänä toimii professori Raimo Tuominen, Helsingin yliopiston Farmasian tiedekunnasta ja kustoksena professori Eero Mervaala Helsingin yliopiston Lääketieteellisestä tiedekunnasta.

Epithelial-mesenchymal transition in oral squamous carcinoma cells

In epithelial-mesenchymal transition (EMT), epithelial cells acquire traits typical for mesenchymal cells, dissociate their cell-cell junctions and gain the ability to migrate. EMT is essential during embryogenesis, but may also mediate cancer progression. Basement membranes are sheets of extracellular matrix that support epithelial cells. They have a major role in maintaining the epithelial phenotype and, in cancer, preventing cell migration, invasion and metastasis. Laminins are the main components of basement membranes and may actively contribute to malignancy. We first evaluated the differences between cell lines obtained from oral squamous cell carcinoma and its recurrence. As the results indicated a change from epithelial to fibroblastoid morphology, E-cadherin to N-cadherin switch, and change in expression of cytokeratins to vimentin intermediate filaments, we concluded that these cells had undergone EMT. We further induced EMT in primary tumour cells to gain knowledge of the effects of transcription factor Snail in this cell model. The E-cadherin repressors responsible for the EMT in these cells were ZEB-1, ZEB-2 and Snail, and ectopic expression of Snail was able to augment the levels of ZEB-1 and ZEB-2. We produced and characterized two monoclonal antibodies that specifically recognized Snail in cell lines and patient samples. By immunohistochemistry, Snail protein was found in mesenchymal tissues during mouse embryonal development, in fibroblastoid cells of healing skin wounds and in fibromatosis and sarcoma specimens. Furthermore, Snail localized to the stroma and borders of tumour cell islands in colon adenocarcinoma, and in laryngeal and cervical squamous cell carcinomas. Immunofluorescence labellings, immunoprecipitations and Northern and Western blots showed that EMT induced a progressive downregulation of laminin-332 and laminin-511 and, on the other hand, an induction of mesenchymal laminin-411. Chromatin immunoprecipitation revealed that Snail could directly bind upstream to the transcription start sites of both laminin α5 and α4 chain genes, thus regulating their expression. The levels of integrin α6β4, a receptor for laminin-332, as well as the hemidesmosomal complex proteins HD1/plectin and BP180 were downregulated in EMT-experienced cells. The expression of Lutheran glycoprotein, a specific receptor for laminin-511, was diminished, whereas the levels of integrins α6β1 and α1β1 and integrin-linked kinase were increased. In quantitative cell adhesion assays, the cells adhered potently to laminin-511 and fibronectin, but only marginally to laminin-411. Western blots and immunoprecipitations indicated that laminin-411 bound to fibronectin and could compromise cell adhesion to fibronectin in a dose-dependent manner. EMT induced a highly migratory and invasive tendency in oral squamous carcinoma cells. Actin-based adhesion and invasion structures, podosomes and invadopodia, were detected in the basal cell membranes of primary tumour and spontaneously transformed cancer cells, respectively. Immunofluorescence labellings showed marked differences in their morphology, as podosomes organized a ring structure with HD1/plectin, αII-spectrin, talin, focal adhesion kinase and pacsin 2 around the core filled with actin, cortactin, vinculin and filamin A. Invadopodia had no division between ring and core and failed to organize the ring proteins, but instead assembled tail-like, narrow actin cables that showed a talin-tensin switch. Time-lapse live-cell imaging indicated that both podosomes and invadopodia were long-lived entities, but the tails of invadopodia vigorously propelled in the cytoplasm and were occasionally released from the cell membrane. Invadopodia could also be externalized outside the cytoplasm, where they still retained the ability to degrade matrix. In 3D confocal imaging combined with in situ gelatin zymography, the podosomes of primary tumour cells were large, cylindrical structures that increased in time, whereas the invadopodia in EMT-driven cells were smaller, but more numerous and degraded the underlying matrix in significantly larger amounts. Fluorescence recovery after photobleaching revealed that the substructures of podosomes were replenished more rapidly with new molecules than those of invadopodia. Overall, our results indicate that EMT has a major effect on the transcription and synthesis of both intra- and extracellular proteins, including laminins and their receptors, and on the structure and dynamics of oral squamous carcinoma cells.

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.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.

Oral Health among Iranian Preadolescents : A School-Based Health Education Intervention

The present study assessed oral health and its determinants among Iranian preadolescents, and evaluated a school-based health education programme aimed to promote their oral health. The target population of this study comprised a random sample of the third-grade school children (n = 459) of all public primary schools in 19 areas of Tehran city. The data came from a clinical examination of the children and two self-administered questionnaires: one for children, and one for mothers. The clinical dental examination was performed for recording children's oral health. The mothers' questionnaires covered background factors, oral self-care (OSC) behaviours and oral health-related knowledge and attitude statements. After baseline data collection, a community trial was designed as a 3-month school-based intervention study. For the intervention trial, the third-grade classes as the clusters were randomly assigned to the intervention and control groups. Three kinds of intervention were implemented, one in class, one via the parents, and one as a combination of these. One group served as controls with no intervention. The outcome measures of the study were changes in plaque and bleeding scores recorded. The results showed that mean dmft was 3.75 (SD = 2.8) for the primary teeth and mean DMFT was 0.4 (SD = 0.9) for the permanent teeth. All children had plaque on at least one index tooth and bleeding on probing in at least one index tooth occurred in 81%. About one-third (34%) of the children reported favourable OSC and less than half (46%) of the children reported brushing their teeth at least twice daily. Girls reported favourable OSC (OR = 2.0), had decay-free teeth (OR = 1.8) and treated permanent teeth (OR = 3.3) more than did boys. Mother's oral health-related aspects, i.e., mother's favourable OSC, high knowledge levels of and positive attitudes towards oral health, and active supervision of the child's tooth brushing had a positive effect on all aspects of children's oral health status and behaviours (ORs from 1.3 to 1.9). After the intervention, the results showed a strong intervention effect on healthy gingiva in both groups where parents were involved: the parental-aid group (OR = 7.7, 95% CI 2.2-27.7) and combined group (OR = 6.6, 95% CI 2.0-22.1). To improve children's oral health, community school-based oral health educational programmes should be established to include all primary schools. These programmes should benefit from the common risk factor approach and a multi-sectored approach to employ for communication between the community, the school, and the family. Oral health interventions should empower the parents' ability to improve their own oral health behaviour and then to transfer that healthy behaviour to their children.

Studies on Matrix Metalloproteinase (MMP-2, -9 and -14) and β2 Integrin Targeting as Potential Anticancer Therapeutics

Proteolytic enzymes, such as matrix metalloproteinases (MMP), are associated to the progression of several cancers. They degrade extracellular components, which helps tumors to expand and cancer cells to escape from the primary site. Of all MMPs, gelatinases (MMP-2 and -9) and membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), in particular, are often associated to more aggressive types of head and neck carcinomas as well as to a poorer outcome in patient survival. Although therapies during the last decades have advanced, the mortality of the disease is still rather high and adjuvant therapies are searched for continuously. MMP-9 and MT1-MMP are also involved in neo-angiogenesis, which is necessary for tumor expansion. For this reason, we have identified synthetic peptides-targeting gelatinases and MT1-MMP, and have also evaluated their anticancer effects in vitro and in vivo. Antigelatinolytic peptides effectively inhibited tongue-carcinoma cell invasion and reduced the growth of xenografted tumors. In tumor samples of mice that were treated with antigelatinolytic peptides, the micro-vessel density was significantly reduced. We also identified a novel MT1-MMP targeting peptide and demonstrated that it exerted anticancer effects against several malignant cell lines in vitro. The effects of MT1-MMP inhibition on tongue-squamous cell carcinomas were evaluated by using xenograft tumors, which it effectively inhibited. Tranexamic acid was also demonstrated to inhibit tongue-squamous cell carcinoma invasion, most probably due to its ability to prevent the plasmin-mediated activation of proMMP-9. Leukocyte β2 integrins are another interesting option when evaluating targets for the therapeutic intervention of inflammatory conditions or malignancies of hematopoietic origin, since β2 integrins are expressed mainly by leukocytes. We identified a novel technique for screening small-molecule libraries against β2 integrins, and by using this technique we identified a novel αMβ2 integrin-binding chemical (IMB-10). IMB-10 significantly enhances leukocyte adhesion and inhibits their motility. We also demonstrated that IMB-10 can be used to inhibit inflammation and lymphoma growth in vivo. Interestingly, IMB-10 also reduced leukocyte tumor infiltration and inhibited tumor invasion.

Oral Health among Young Adults and the Middle-aged in Iran

The aim of the present study was to assess oral health and treatment needs among adult Iranians according to socio-demographic status, smoking, and oral hygiene, and to investigate the relationships between these determinants and oral health. Data for 4448 young adult (aged 18) and 8301 middle-aged (aged 35 to 44) Iranians were collected in 2002 as part of a national survey using the World Health Organization (WHO) criteria for sampling and clinical diagnoses, across 28 provinces by 33 calibrated examiners. Gender, age, place of residence, and level of education served as socio-demographic information, smoking as behavioural and modified plaque index (PI) as the biological risk indicator for oral hygiene. Number of teeth, decayed teeth (DT), filled teeth (FT), decayed, missing, filled teeth (DMFT), community periodontal index (CPI), and prosthodontic rehabilitation served as outcome variables of oral health. Mean number of DMFT was 4.3 (Standard deviation (SD) = 3.7) in young adults and 11.0 (SD = 6.4) among middle-aged individuals. Among young adults the D-component (DT = 70%), and among middle-aged individuals the M-component (60%) dominated in the DMFT index. Among young adults, visible plaque was found in nearly all subjects. Maximum (max) PI was associated with higher mean number of DT, and higher periodontal treatment needs. A healthy periodontium was a rare condition, with 8% of young adults and 1% of middle-aged individuals having a max CPI = 0. The majority of the CPI findings among young adults consisted of calculus (48%) and deepened periodontal pockets (21%). Respective values for middle-aged individuals were 40% and 53%. Having a deep pocket (max CPI = 4) was more likely among young adults with a low level of education (Odds ratio (OR) = 2.7, 95% Confidence interval (CI) = 1.9–4.0) than it was among well-educated individuals. Among middle-aged individuals, having calculus or a periodontal pocket was more likely in men (OR = 1.8, 95% CI = 1.6–2.0) and in illiterate subjects (OR = 6.3, 95% CI = 5.1–7.8) than it was for their counterparts. Among young adults, having 28 teeth was more (p < 0.05) prevalent among men (72% vs. 68% for women), urban residents (71% vs. 67% for rural residents), and those with a high level of education (73% vs. 60% for those with a low level). Among middle-aged individuals, having a functional dentition was associated with younger age (OR = 2.0, 95% CI = 1.7−2.5) and higher level of education (OR = 1.8, 95% CI = 1.6−2.1). Of middle-aged individuals, 2% of 35- to 39-year-olds and 5% of those aged 40 to 44 were edentulous. Among the dentate subjects (n = 7,925), prosthodontic rehabilitation was more prevalent (p < 0.001) among women, urban residents, and those with a high level of education than it was among their counterparts. Among those having 1 to 19 teeth, a removable denture was the most common type of prosthodontic rehabilitation. Middle-aged individuals lacking a functional dentition were more likely (OR = 6.0, 95% CI = 4.8−7.6) to have prosthodontic rehabilitation than were those having a functional dentition. In total, 81% of all reported being non-smokers, and 32% of men and 5% of women were current smokers. Heavy smokers were the most likely to have deepened periodontal pockets (max CPI ≥ 3, OR = 2.9, 95% CI = 1.8−4.7) and to have less than 20 teeth (OR = 2.3, 95% CI = 1.5−3.6). The findings indicate impaired oral health status in adult Iranians, particularly those of low socio-economic status and educational level. The high prevalence of dental plaque and calculus and considerable unmet treatment needs call for a preventive population strategy with special emphasis on the improvement of oral self-care and smoking cessation to tackle the underlying risk factors for oral diseases in the Iranian adult population.

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.

Study of sleep and evaluation of sumatriptan and rizatriptan in the acute treatment of migraine in children and adolescents

Migraine is a common disease in children and adolescents, affecting roughly 10% of school-aged children. Recent studies have revealed an increasing incidence of childhood migraine, but migraine remains an underrecognized and undertreated condition in the pediatric population. Migraine attacks are painful and disabling and can affect a child´s life in many ways. Effective drug treatment is usually needed. The new migraine drugs, triptans, were introduced at the beginning of the 1990s and have since been shown to be very effective in the treatment of migraine attacks in adults. Although they are widely used in adults, the acute treatment of migraine in children and adolescents is still based on paracetamol and nonsteroidal anti-inflammatory drugs. Some children can control their attacks satisfactorily with simple analgesics, but at least one-third need more powerful treatments. When this thesis work commenced, hardly any information existed on the efficacy and safety of triptans in children. The study aim of the thesis was to identify more efficient treatments of migraine for children and adolescents by investigating the efficacy of sumatriptan nasal spray and oral rizatriptan compared with placebo in them. Sleep has an impact on migraine in many aspects. Despite the clinical relevance and common manifestation of sleep in the context of migraine in children, very little research data on the true frequency of sleep exist. As sleeping is so often related to childhood migraine, it can be a confounding factor in clinical drug trials of migraine treatments in children and adolescents. How the results of a sleeping child should be analyzed is under continual debate. The aim of the thesis was also to clarify this as well as to evaluate the frequency of sleeping during migraine attacks in children and factors affecting frequency. Both nasal sumatriptan and oral rizatriptan were effective (superior to placebo), and well tolerated in treatment of migraine attacks in children and adolescents aged 8-17 and 6-17 years, respectively. No serous adverse effects were observed. The results of this work suggest that nasal sumatriptan 20 mg and rizatriptan 10 mg can be effectively and safely used to treat migraine attacks in adolescents aged over 12 years if more effective drugs than NSAIDs are needed. No difference was observed in efficacy or safety of nasal sumatriptan and rizatriptan between children aged younger than 12 years and older children, but because the treated number of patients under 12 years is still small, more studies are needed before sumatriptan or rizatriptan can be recommended for use in this population. Sleeping during migraine attacks was very common, and most children at least occasionally slept during an attack. Falling asleep was especially common in children under eight years of age and during the first hour after the onset of attack. Children who were able to sleep soon after attack onset were more likely pain-free at two hours. Sleeping probably both improves recovery from a migraine attack and is a sign of headache relief. Falling asleep should be classified as a sign of headache relief in clinical drug trials when studying migraine treatments in children and adolescents.

Inflammatory and coagulation disturbances in acute pancreatitis

Acute pancreatitis (AP), a common cause of acute abdominal pain, is usually a mild, self-limited disease. However, some 20-30% of patients develop a severe disease manifested by pancreatic necrosis, abscesses or pseudocysts, and/or extrapancreatic complications, such as vital organ failure (OF). Patients with AP develop systemic inflammation, which is considered to play a role in the pathogenesis of multiple organ failure (MOF). OF mimics the condition seen in patients with sepsis, which is characterized by an overwhelming production of inflammatory mediators, activation of the complement system and systemic activation of coagulation, as well as the development of disseminated intravascular coagulation (DIC) syndrome. Vital OF is the major cause of mortality in AP, along with infectious complications. About half of the deaths occur within the first week of hospitalization and thus, early identification of patients likely to develop OF is important. The aim of the present study was to investigate inflammatory and coagulation disturbances in AP and to find inflammatory and coagulation markers for predicting severe AP, and development of OF and fatal outcome. This clinical study consists of four parts. All of patients studied had AP when admitted to Helsinki University Central Hospital. In the first study, 31 patients with severe AP were investigated. Their plasma levels of protein C (PC) and activated protein C (APC), and monocyte HLA-DR expression were studied during the treatment period in the intensive care unit; 13 of these patients developed OF. In the second study, the serum levels of complement regulator protein CD59 were studied in 39 patients during the first week of hospitalization; 12 of them developed OF. In the third study, 165 patients were investigated; their plasma levels of soluble form of the receptor for advanced glycation end products (sRAGE) and high mobility group box 1 (HMGB1) protein were studied during the first 12 days of hos-pitalization; 38 developed OF. In the fourth study, 33 patients were studied on admission to hospital for plasma levels of prothrombin fragment F1+2 and tissue factor pathway inhibitor (TFPI), and thrombin formation capacity by calibrated automated thrombogram (CAT); 9 of them developed OF. Our results showed significant PC deficiency and decreased APC generation in patients with severe AP. The PC pathway defects seemed to be associated with the development of OF. In patients who developed OF, the levels of serum CD59 and plasma sRAGE, but not of HMGB1, were significantly higher than in patients who recovered without OF. The high CD59 levels on admission to the hospital seemed to be predictive for severe AP and OF. The median of the highest sRAGE levels was significantly higher in non-survivors than in survivors. No significant difference between the patient groups was found in the F1+2 levels. The thrombograms of all patients were disturbed in their shape, and in 11 patients the exogenous tissue factor did not trigger thrombin generation at all ( flat curve ). All of the patients that died displayed a flat curve. Free TFPI levels and free/total TFPI ratios were significantly higher in patients with a flat curve than in the others, and these levels were also significantly higher in non-survivors than in survivors. The flat curve in combination with free TFPI seemed to be predictive for a fatal outcome in AP.

Neurological manifestations and molecular genetics of acute intermittent porphyria in North Western Russia

Acute intermittent porphyria (AIP, MIM #176000) is an inherited metabolic disease due to a partial deficiency of the third enzyme, hydroxymethylbilane synthase (HMBS, EC: 4.3.1.8), in the haem biosynthesis. Neurological symptoms during an acute attack, which is the major manifestation of AIP, are variable and relatively rare, but may endanger a patient's life. In the present study, 12 Russian and two Finnish AIP patients with severe neurological manifestations during an acute attack were studied prospectively from 1995 to 2006. Autonomic neuropathy manifested as abdominal pain (88%), tachycardia (94%), hypertension (75%) and constipation (88%). The most common neurological sign was acute motor peripheral neuropathy (PNP, 81%) often associated with neuropathic sensory loss (54%) and CNS involvement (85%). Despite heterogeneity of the neurological manifestations in our patients with acute porphyria, the major pattern of PNP associated with abdominal pain, dysautonomia, CNS involvement and mild hepatopathy could be demonstrated. If more strict inclusion criteria for biochemical abnormalities (>10-fold increase in excretion of urinary PBG) are applied, neurological manifestations in an acute attack are probably more homogeneous than described previously, which suggests that some of the neurological patients described previously may not have acute porphyria but rather secondary porphyrinuria. Screening for acute porphyria using urinary PBG is useful in a selected group of neurological patients with acute PNP or encephalopathy and seizures associated with pain and dysautonomia. Clinical manifestations and the outcome of acute attacks were used as a basis for developing a 30-score scale of the severity of an acute attack. This scale can easily be used in clinical practice and to standardise the outcome of an attack. Degree of muscle weakness scored by MRC, prolonged mechanical ventilation, bulbar paralysis, impairment of consciousness and hyponatraemia were important signs of a poor prognosis. Arrhythmia was less important and autonomic dysfunction, severity of pain and mental symptoms did not affect the outcome. The delay in the diagnosis and repeated administrations of precipitating factors were the main cause of proceeding of an acute attack into pareses and severe CNS involvement and a fatal outcome in two patients. Nerve conduction studies and needle EMG were performed in eleven AIP patients during an acute attack and/or in remission. Nine patients had severe PNP and two patients had an acute encephalopathy but no clinically evident PNP. In addition to axonopathy, features suggestive of demyelination could be demonstrated in patients with severe PNP during an acute attack. PNP with a moderate muscle weakness was mainly pure axonal. Sensory involvement was common in acute PNP and could be subclinical. Decreased conduction velocities with normal amplitudes of evoked potentials during acute attacks with no clinically evident PNP indicated subclinical polyneuropathy. Reversible symmetrical lesions comparable with posterior reversible encephalopathy syndrome (PRES) were revealed in two patients' brain CT or MRI during an acute attack. In other five patients brain MRI during or soon after the symptoms was normal. The frequency of reversible brain oedema in AIP is probably under-estimated since it may be short-lasting and often indistinguishable on CT or MRI. In the present study, nine different mutations were identified in the HMBS gene in 11 unrelated Russian AIP patients from North Western Russia and their 32 relatives. AIP was diagnosed in nine symptom-free relatives. The majority of the mutations were family-specific and confirmed allelic heterogeneity also among Russian AIP patients. Three mutations, c.825+5G>C, c.825+3_825+6del and c.770T>C, were novel. Six mutations, c.77G>A (p.R26H), c.517C>T (p.R173W), c.583C>T (p.R195C), c.673C>T (p.R225X), c.739T>C (p.C247R) and c.748G>C (p.E250A), have previously been identified in AIP patients from Western and other Eastern European populations. The effects of novel mutations were studied by amplification and sequencing of the reverse-transcribed total RNA obtained from the patients' lymphoblastoid or fibroblast cell lines. The mutations c.825+5G>C and c.770T>C resulted in varyable amounts of abnormal transcripts, r.822_825del (p.C275fsX2) and [r.770u>c, r.652_771del, r.613_771del (p.L257P, p.G218_L257del, p.I205_L257del)]. All mutations demonstrated low residual activities (0.1-1.3 %) when expressed in COS-1 cells confirming the causality of the mutations and the enzymatic defect of the disease. The clinical outcome, prognosis and correlation between the HMBS genotype and phenotype were studied in 143 Finnish and Russian AIP patients with ten mutations (c.33G>T, c.97delA, InsAlu333, p.R149X, p.R167W, p.R173W, p.R173Q, p.R225G, p.R225X, c.1073delA) and more than six patients in each group. The patients were selected from the pool of 287 Finnish AIP patients presented in a Finnish Porphyria Register (1966-2003) and 23 Russian AIP patients (diagnosed 1995-2003). Patients with the p.R167W and p.R225G mutations showed lower penetrance (19% and 11%) and the recurrence rate (33% and 0%) in comparison to the patients with other mutations (range 36 to 67% and 0 to 66%, respectively), as well as milder biochemical abnormalities [urinary porphobilinogen 47±10 vs. 163±21 mol/L, p<0.001; uroporphyrin 130±40 vs. 942±183 nmol/L, p<0.001] suggesting a milder form of AIP in these patients. Erythrocyte HMBS activity did not correlate with the porphobilinogen excretion in remission or the clinical of the disease. In all AIP severity patients, normal PBG excretion predicted freedom from acute attacks. Urinary PBG excretion together with gender, age at the time of diagnosis and mutation type could predict the likelihood of acute attacks in AIP patients.