Franch Chillida

Luigi Nibali
email: l.nibali@eastman.ucl.ac.uk

Genotype Associated with A.actinomycetemcomitans in Indians

Keywords: Genetics, Periodontitis, Interleukin 6, Bacteria, A.actinomycetemcomitans Periodontitis, Interleukin-6, Genetic, Bacteria, A.actinomycetemcomitans

AIM: Genetic factors have recently been associated with presence of Aggregatibacter actinomycetemcomitans subgingivally in populations living in industrialised countries. The aim of this study was to analyze associations between Interleukin-6 (IL6) single nucleotide polymorphisms and presence and levels of A.actinomycetemcomitans and other subgingival microbes in a rural Indian population. SUBJECTS AND METHODS: 251 individuals from a rural village in India with a periodontal phenotype ranging from healthy to severe periodontitis were included. Checkerboard DNA-DNA analysis was performed to detect 40 periodontal taxa in subgingival plaque samples. Genomic DNA was extracted to genotype five polymorphisms in the IL6 promoter region. RESULTS: The IL6 -174 GG genotype was associated with high (above median) counts of A.actinomycetemcomitans (both in all subjects and in periodontally healthy only) and with presence and counts of Capnocytophaga sputigena. Differences in detection of several other bacteria were noted between periodontitis and healthy subjects. CONCLUSION: These findings support the influence of genetic factors on the subgingival microbiota.

INTRODUCTION

The oral cavity may contain as many as 19,000 bacterial phylotypes (Kejiser et al., 2008). We have recently introduced the concept of periodontal infectogenomics, which studies the effect host genetic factors have on the composition of the subgingival microbiota of each individual. This is based on the assumption that genetic factors in the host seem to play a major role in deciding which bacteria (commensal and pathogenic) are able to colonise the host (Nibali et al., 2009a).

Most studies on subgingival microbes with a putative influence on periodontally-relevant immune responses have been carried out in industrialised communities with routine access to dental care [START_REF] Hardie | Bacterial-Flora of Dental Plaque[END_REF][START_REF] Papapanou | An Analysis of the Subgingival Microflora in Randomly Selected Subjects[END_REF][START_REF] Umeda | Risk indicators for harboring periodontal pathogens[END_REF]. Analysis of rural populations not subjected to dental care allows the study of possible effects of host genetic factors in the composition of the subgingival microbiota in almost pristine conditions, undisturbed by routine tooth scaling and use of antibiotics.

We have recently shown an association between IL6 genetic factors and clinical status in non smokers living in a rural Indian village (Franch et al., 2010). The main aim of this analysis was to study the association between IL6 genetic variants and the presence and levels (counts) of A.actinomycetemcomitans in subgingival plaque samples from the same population. Secondary aims were to explore the association between IL6 genetic factors and the detection and counts of other bacteria, and the association between periodontal status and the detection and levels of other

MATERIALS AND METHODS

Subject Selection

The population reported here has also been described elsewhere (Franch et al., 2010). The study took place in the village of Dokur, Andhra Pradesh, where the Institute for Rural Health Studies (IRHS), a local Non-Governmental Organization (NGO), runs a rural clinic. All adult inhabitants between the ages of 18 and 70 were invited to attend a screening visit by means of local advertising within the village. A group of 500 villagers attended and were initially screened based on the following exclusion criteria: i) presence of serious systemic conditions (malaria, tuberculosis or cardiovascular disease), ii) presence of infectious or parasitic diseases, iii) pregnancy or breastfeeding, iv) antibiotic or anti-inflammatory medications within the last 3 months, v) fewer than 16 standing teeth.

Two hundred and fifty one suitable subjects (10% of the total village population) were identified and included in the study.

Ethical Considerations

The study was conducted in full accordance with ethics principles, including the World Medical Association Declaration of Helsinki (version 2002). Ethical approval for the study was obtained education, social status, type of toothbrushing, alcohol consumption and smoking habit were recorded (Franch et al., 2010).

Clinical Data Collection

Full mouth periodontal examination of probing pocket depth (PPD) and clinical attachment level (CAL) was carried out for each subject with an EN-15 probe on 6 sites per tooth. The clinical examination was performed by one single operator (IM), calibrated to an exact kappa level of 0.85 for PPD. Patients were divided into 2 groups (periodontitis/ healthy) based on their periodontal status. Both definitions suggested by the European Workshop on Periodontitis (EWP) (Tonetti and Claffey, 2005) were used:

• Presence of proximal attachment loss of ≥ 3mm in ≥2 non-adjacent teeth (EWP1)

• Presence of proximal attachment loss of ≥ 5mm in ≥30% of teeth present (EWP2)

Microbiological analysis

Dental plaque samples were taken from the mesio-buccal aspects of the upper right and lower left first molar teeth, unless a deeper pocket had been found at another site, in which case this site was used in preference. Subgingival plaque was removed from each site isolated with cotton rolls, following removal of supragingival plaque, using sterile 13/14 double-ended Columbia curettes.

The scraped mass was transferred from the curette to an Eppendorf tube containing 150µl of sterile TE buffer (10mM Tris HCl, 1.0mM EDTA, pH 7.6) and subsequently transferred to the University of Berne. The presence of 40 periodontal pathogens was evaluated using checkerboard DNA-DNA hybridisation technique [START_REF] Socransky | Checkerboard DNA-DNA hybridisation[END_REF]. as previously described (Nibali et al., 2008b).

Statistical Analysis

Comparisons of continuous and categorical data between groups (periodontitis and healthy) were analysed with ANOVA and Chi-square test, respectively. The alpha value was set at 0.05.

The approach to evaluate the association between genetic factors, clinical diagnosis and microbiological outcomes was performed using the SPSS 12.0 package. The main outcome was association between IL6 -174 GG genotype and the presence (at detection threshold 1 x10 4 ) and levels (total counts) of A.actinomycetemcomitans. An exploratory analysis was performed to investigate associations between all studied IL6 genetic variables (and clinical diagnosis) and other taxa and bacterial complexes. The α value for this exploratory analysis was lowered to 0.01 in order to adjust for multiple testing. Microbiological data were available from two sites for all subjects.

Data on detection or not of all 40 studied taxa are reported. For analysis of bacterial counts, negative (no detection) reactions were given an arbitrary value of 0.99 x10 4 , just below detection limit. The mean individual count for each species was obtained as the averaged between the 2 sites in each patient. A priori, median values for each bacterium were calculated and two clusters of patients for each bacterium were identified: high (above median) and low (below median) counts.

Therefore, the microbiological outcomes were: i) detection of each bacterium (at 1 x10 could not be scored. The distribution of genetic polymorphisms has been reported elsewhere and satisfied the chi-squared analysis for Hardy-Weinberg equilibrium on all subjects (Franch et al., 2010).

The detection of the 40 studied taxa ranged from 98% of subjects for Prevotella intermedia to 51%

for Capnocytophaga sputigena. In particular, 93% of subjects were positive for A.

actinomycetemcomitans, 93% for Porphyromonas gingivalis, 90% for Tannerella forsythia, 70%

for Treponema denticola and 60% for Campylobacter rectus. The mean counts ranged from 11.2 x 10 5 for Neisseria mucosa to 0.2 x 10 5 for Propionibacterium acnes. Average counts for A.

actinomycetemcomitans, P. gingivalis and T. forsythia for all subjects were respectively 5.1 x 10 5 , 12.0 x 10 5 and 6.4 x 10 5 . [START_REF] Socransky | Microbial complexes in subgingival plaque[END_REF] were found according to IL6 -174 genotypes (figure 2).

Associations between clinical diagnosis and bacterial detection

Figure 3 shows the frequency of detection of the 40 studied bacteria in periodontitis and healthy individuals according to EWP1 (Tonetti and Claffey, 2005). Statistically significant associations were found between diagnosis of periodontitis (EWP1) and presence of N.mucosa (p=0.008) and

Capnocytophaga ochracea (p=0.001), low (below-median) counts of N.mucosa (p<0.001), and high (above-median) counts of Parvimonas micra (previously Peptostreptococcus micros) (p=0.002).

Periodontitis patients also exhibited lower absolute counts of N.mucosa (p=0.003).

EWP2 diagnosis of periodontitis was associated with absence (no detection) of C.ochracea (p=0.005) and S.noxia (p<0.001) and higher absolute counts of P.micra (p=0.025). When bacteria were grouped in complexes, periodontal status (EWP1) was associated with percentage of bacteria undefined in complexes ('others') (p=0.012).

DISCUSSION

We have previously shown an association between IL6 -174 genotypes and presence of periodontitis in non-smokers living in a rural village in India (Franch et al., 2010). The present study describes associations between IL6 -174 GG genotype and presence of A.actinomycetemcomitans in periodontal pockets in these subjects (with a periodontal phenotype ranging from healthy to severe periodontitis), confirming association previously observed in subjects living in the United Kingdom [START_REF] Nibali | Gene polymorphisms and the prevalence of key periodontal pathogens[END_REF], Nibali et al., 2008a). Among these Indian villagers living in Andra Pradesh, A.actinomycetemcomitans was almost ubiquitous subgingivally, as it was harboured by 93% of participating individuals. However, IL6 -174 GG individuals had higher counts of this bacterium.

The IL6 -174 GG has been associated with presence of periodontitis (Trevilatto et al., 2003, Nibali et al., 2009b). The study reported here confirms the previously reported association between IL6 genotypes and A.actinomycetemcomitans [START_REF] Nibali | Gene polymorphisms and the prevalence of key periodontal pathogens[END_REF], Nibali et al., 2008a), despite being conducted on a different population of different ethnicity and socio-economic status and not subjected to routine dental care. Furthermore, the microbiological analysis consisted of a checkerboard DNA-DNA hybridisation analysis [START_REF] Socransky | Checkerboard DNA-DNA hybridisation[END_REF], as opposed to previously reported culture [START_REF] Nibali | Gene polymorphisms and the prevalence of key periodontal pathogens[END_REF] and PCR (Nibali et al., 2008a) analyses. This association was seen in all subjects, independent from clinical diagnosis. In addition to this, this paper for the first time shows the association between IL6 genotypes and A.actinomycetemcomitans also in nonperiodontitis individuals (according to EWP2 definition). As previously hypothesized, this association may be due to the increased local inflammatory response of -174 GG individuals [START_REF] Fishman | An interleukin-6 promoter polymorphism that influences gene transcription, plasma IL-6 levels and is associated with systemic-onset juvenile chronic arthritis[END_REF][START_REF] Bennermo | Genetic predisposition of the interleukin-6 response to inflammation: Implications for a variety of major diseases?[END_REF], which favours the overgrowth of components of the microbiota that grow well in inflamed areas, such as A.actinomycetemcomitans. This study brings preliminary evidence that other bacteria belonging to the green complex, in particular C.sputigena, may also be associated with increased counts in periodontal pockets of IL6 -174 GG individuals. In other terms, the effect of genetic factors may not be limited to the presence or levels of one or two potentially pathogenic bacteria, but may extend to bacterial complexes or in general to the whole composition of the subgingival biofilm. This is in agreement with the concept of infectogenomics, suggesting that genetic factors in the host seem to play a major role in deciding which bacteria (commensal and pathogenic) are able to colonise the host [START_REF] Kellam | Infectogenomics: Insights from the host genome into infectious diseases[END_REF]Weiss, 2006, Nibali et al., 2009a).

The microbiological analysis of the current subject group showed a population presenting with a complex oral flora, with a breakdown in bacterial complexes quite different from that observed in industrialized populations [START_REF] Haffajee | Subgingival microbiota in healthy, well-maintained elder and periodontitis subjects[END_REF]. Individuals diagnosed with periodontitis had a tendency towards higher percentage of green complex bacteria, and lower percentage of bacteria not defined in complexes ('others'). Overall, there was a predominance of bacteria belonging to the orange complex, which have been associated with established periodontitis in industrialised populations, while no significant differences in percentage of orange or red complex bacteria were noted between periodontitis and non-periodontitis individuals. This was consistent with studies in other untreated populations, which have shown ubiquitous presence of certain bacterial species and a tendency towards a stable, mixed microflora (Timmerman et al., 1998[START_REF] Dowsett | Subgingival microbiota of indigenous Indians of Central America[END_REF][START_REF] Papapanou | Periodontal microbiota and clinical periodontal status in a rural sample in southern Thailand[END_REF][START_REF] Colombo | Subgingival microbiota of Brazilian subjects with untreated chronic periodontitis[END_REF]. N.mucosa, a Gram negative commensal bacterium, was the bacterium found in highest counts, followed by periodontopathogenic bacteria P.gingivalis, T.forsythia and A.actinomycetemcomitans. This shows a considerable shift from the bacterial profile shown in a study with checkerboard DNA-DNA hybridization on a large United States population [START_REF] Haffajee | Association of Eubacterium nodatum and Treponema denticola with human periodontitis lesions[END_REF]. The difference in prevalence and counts of A.actinomycetemcomitans between the study described here and the one by study. Therefore, the average bacterial counts in these cases in not representative of the whole subgingival microbiota. However, even when the sampled site was used as unit for analysis, no differences were detected between bacterial complexes between deep (>6mm) and shallow (<4mm) pockets (data not presented).

Among the drawbacks of this study, we have to acknowledge the limitation of having only two plaque samples sites per patient, the risk for false positive results associated with DNA-DNA checkerboard analysis (van Steenbergen et al., 1996) and the difficulty of defining periodontitis in this population [START_REF] Preshaw | Definitions of periodontal disease in research[END_REF], Franch et al. 2010). The results reported here show an association between IL6 -174 polymorphism and A.actinomycetemcomitans in subjects living in a rural Indian village, bringing further evidence, consistent with previous studies, of the importance of infectogenomics in periodontitis (Nibali et al., 2009a)

  from the Medical Board of IRHS and conformed to the Indian Council of Medical Research Guidelines on Medical Ethics 2000 (Indian Council of Medical Research, Ansari Nager, New Delhi). All subjects were native to the village and voluntary informed consent to participation in the study was obtained from each by means of a written consent form, translated into Telugu and also read aloud to each subject by a fluent Telugu speaker. Demographic data and self-reported data on

  Samples of buccal mucosal cells were taken from all subjects using wire cytology brushes (Medical Wire and Equipment Company, UK) (Franch et al., 2010). Genomic DNA was extracted from these samples and blindly genotyped for polymorphisms at positions -174 (CCTTTAGCAT[C-G]GCAAGAC, rs 1800795), -572 (CAACAGCC[C-G]CTCACAG, rs 1800796)) and -1480 (ACCGTCTCT[C-G]TGTTTAG) in the IL6 gene by real time PCR

  4 detection

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	value); ii) high or low levels (counts) of each bacterium (both categorical data) and iii) absolute
	counts of each bacterium (continuous data). Subgroup analyses were performed in subjects divided
	by their periodontal status (healthy/periodontitis). Multiple logistic regression analysis adjusting for
	confounders (age, smoking, gender and type of toothbrushing) was performed to investigate these O associations (categorical variables). Linear logistic regression adjusting for confounders (age, r a smoking, gender and type of toothbrushing) was used to detect differences in bacterial counts
	l (continuous variables) between genotypes and by clinical diagnosis .
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RESULTS

A total of 251 subjects took part in the study. Their demographic and clinical characteristics have been reported elsewhere

(Franch et al., 2010)

(table

1

). Forty-two per cent and 19% of subjects were diagnosed with periodontitis, according to EWP1 and EWP2 definitions respectively

(Tonetti and Claffey, 2005)

. Thirteen subjects resulted IL6 -174 C homozygous, 46 CG and 185 GG, while 7

Association between IL6 genetic factors and bacterial detection IL6

  -174 GG genotype subjects exhibited high (above median) levels of A.actinomycetemcomitans compared to CC and CG individuals, irrespective of clinical diagnosis (adjusted p=0.043, O.R. = 1.89, 95% C.I. = 1.02-3.50) (figure1). This association was confirmed among the subgroup of non-

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	l a O r periodontitis individuals (according to EWP2) (adjusted p=0.037, O.R. = 2.11, 95% C.I. = 1.0-4.2).	Deleted: Mann-Whitney test (continuous variables) (Socransky et al., Deleted: as previously described
			2000)
	Among other bacteria, IL6 -174 GG genotype subjects showed increased detection, and high (above D i median) levels of C.sputigena (adjusted p= 0.003, O.R. = 2.60, 95% C.I. = 1.38-4.90 and p=0.009,
	O.R. = 2.29, 95% C.I. = 1.23-4.26 respectively). No statistically significant differences in s e percentage of bacterial complexes
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  Haffajee and coworkers is particularly striking. It needs to be highlighted that only two of the deepest pockets per subject were sampled in the present

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	Deleted: subgingival
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Table 1 .

 1 . Further studies should analyze the possible effect of these and other genetic factors (in both periodontitis and healthy populations) on a wider range of components of the oral microbiota, on different serotypes and clones of A.actinomycetemcomitans, and should investigate possible functional mechanisms for the observed associations. Representation of number of subjects with A.actinomycetecomitans above or below median according to their IL-6 -174 genotypes. GG vs CG/CC p=0.043, O.R.= 1.89, 95% C.I.=1.02-3.50. Pie charts with distribution of bacterial complexes between IL6 -174 GG and CG/CC individuals. % DNA probe counts for each bacterium was determined at each site, and averaged per patient. The % of the DNA probe count for each species in each complex (according to[START_REF] Socransky | Microbial complexes in subgingival plaque[END_REF] was summed and the proportions of each complex were calculated. N.mucosa: p<0.01 for differences in detection, high (above median) counts and absolute counts between healthy and periodontitis. Demographic and clinical characteristics of the participants, as divided by their periodontal status.

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ACKNOWLEDGEMENTS

This work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme. This study was also supported by the Periodontal Research Fund of the Eastman Dental Institute. The help of Professor Gareth Griffiths, Professor Hubert Newman, Dr Bidinger and the Institute for Rural Health Studies, Hyderabad, Andhra Pradesh, are gratefully acknowledged.


Richeal Ni Riordain
email: richeal.niriordain@ucc.ie

Sarah Meaney

Christine Mccreary
email: c.mccreary@ucc.ie

Impact of chronic oral mucosal disease on daily life: Preliminary observations from a qualitative study

Keywords: Qualitative study, Chronic oral mucosal conditions, Quality of life, Oral Medicine Qualitative study, Chronic oral mucosal conditions, Quality of life, Oral Medicine

Impact of chronic oral mucosal disease on daily life:

preliminary observations from a qualitative study Richeal Ni Riordain, Sarah Meaney, Christine Mccreary

diseases of the soft tissue of the mouth. These conditions, like other diseases of the mouth, can result in considerable morbidity with physical, social and psychological consequences for patients [START_REF] Mcgrath | A review of the influences of oral health on the quality of life[END_REF]. They are commonly managed in an oral medicine clinical setting and are often long standing or recurrent, painful disorders. Some of these diseases can predispose to or lead to the development of life-threatening conditions (Hegarty et al., 2002a). Not only can these chronic oral mucosal conditions have an impact on the every day lives of patients but many of the treatment options available, and their side effects, can also influence their daily lives (Ni Riordain & McCreary, 2010).

The importance of evaluating the patients' own perceptions of the impact of their health on daily living has been long established [START_REF] Bowling | Measuring Disease: A review of Disease Specific Quality of Life Measurement Scales[END_REF]. The Expert Patient was a document produced by the Department of Health in the UK in 2001 [START_REF] Doh Doh | The Expert Patient -A New Approach to Chronic Disease Management for the 21st Century In: DoH[END_REF], which outlines the vital role of the patient in their own healthcare, and the insight available to healthcare professionals from patients with chronic conditions:

"An observation often made by doctors, nurses and other health professionals…is 'my patient understands their disease better than I do.'...today's patients with chronic diseases need not be mere recipients of care. They can become key decision-makers in the treatment process."

This document further emphasises the value of the patients' contribution in providing key information regarding living with chronic conditions.

Materials and Methods

A purposive sample of participants with a chronic oral mucosal condition was recruited from the Oral Medicine Unit of Cork University Dental School and Hospital. The recruitment resulted in a sample of 24 participants with various chronic oral mucosal conditions including oral lichen planus, mucous membrane pemphigoid, pemphigus vulgaris, recurrent aphthous ulceration and orofacial granulomatosis. The purpose and methods of the study were explained to each participant in detail, both verbally and in written format.

Participants were also made aware that their participation in the study was voluntary and each participant was interviewed with written informed consent.

This study was approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals. The focus groups and interviews were digitally recorded and field notes were also taken. The recordings were transcribed and participants were anonymised with each participant being assigned a participant number.

Transcripts were imported into NVivo software and a thematic analysis was

undertaken. An interpretive methodology was utilised in order to identify key factors influencing participants' experiences of the various chronic oral mucosal conditions they were suffering from on a daily basis. The transcripts were analysed on a line by line basis independently by 2 researchers (RNR/SM). The transcripts were coded, this is a means of defining the data that is about to be analysed. Coding involves identifying passages of text that exemplify a descriptive idea and linking them together under a single idea or code. This process then allows the transcripts to be categorised and hence facilitates the identification of key themes.

Results

Analysis of the data indicated four key themes in relation to patients' experiences of their chronic oral mucosal condition. These themes included:

1. biopsychosocial issues 2. treatment limitations and side effects 3. unpredictability of the conditions and the potential for malignant transformation 4. issues for the healthcare professionals

In the following paragraphs, direct quotations from patient interviews are used to illustrate these key themes.

Theme 1: Biopsychosocial Issues

There was clear evidence that chronic mucosal conditions had a major impact not only on physical functioning but also psychologically and socially. Each participant remarked on the difficulties which arose from the physical symptoms associated with their condition such as difficulties with the textures and temperatures of food irritating their mouths. However the majority of participants outlined how these difficulties lead to high levels stress and in some cases depression. The social element of this theme includes the attitudes and perceptions of family and friends regarding these conditions.

"they don't understand and like that…you know I could have a bad day at work but I am not inclined to say it is because of my mouth is really in agony"

Participant 21 Due to the unpredictable nature of the disease participants stated that they would need to be prepared at all times for any unexpected flare-up. "Well that would be my thing, the fear of cancer." Participant 18

"'The Big C' that's always a big concern for everybody" Participant 13

However participants clearly outlined their satisfaction with the frequency of regular review. One participant described this as been comforting.

Theme 4: Issues for the healthcare professionals Participants expressed clear frustration with the delay in diagnosing their condition, with some reporting a delay of up to a year.

Discussion

The use of qualitative methods in health services research has become more widespread over the last number of years. Qualitative data are gathered via in-depth interviews and focus groups allow issues to not only be explored in detail but also to allow clarification to be sought in a comfortable environment.

Focus groups are types of group interviews that rely upon communication between the research participants in order to obtain data and are particularly useful in investigating people's experiences of procedures or conditions (Travess et al., 2004). As to be expected with chronic oral mucosal conditions the pain and discomfort experienced by the patients were foremost when discussing experiences of their daily lives. With these symptoms of pain one could also expect interference with oral functioning such as alteration of the diet and limitation of the types of food and drinks consumed. Oral pain and limitation of function could also lead to difficulty in social settings resulting in embarrassment for patients.

Patient reported outcome measures

The unpredictable nature of these chronic conditions leads to uncertainty about the future and frustration. Patients with oral lichen planus were

particularly concerned with the malignant potential of the condition and patients with other diseases were worried about diseases progression and spread. Frustration with the chronicity of the conditions and the lack of a cure was widespread.

One of the themes uncovered in this qualitative process has implications not only for the treatment of an individual patient or of a condition but has wider significance for healthcare professionals. Diagnostic delay experienced by patients raises issues for practitioners at a primary care level. Patients 14 reported significant delay in referring patients with both general medical and dental practitioners having prescribed multiple antibiotics and antifungals in the treatment of these conditions. An apparent lack of awareness of the clinical features of these chronic oral mucosal conditions is worrisome. A deficiency in the knowledge of primary care practitioners regarding the detection of oral malignancy has been well documented in the literature with educational models proposed for amelioration of this issue [START_REF] Macpherson | The role of primary healthcare professionals in oral cancer prevention and detection[END_REF], Ni Riordain & McCreary, 2009[START_REF] Carter | Oral cancer awareness of general medical and general dental practitioners[END_REF], however, this issue has not previously been highlighted with regard to chronic oral mucosal diseases.

Another possible cause for the delay in diagnosis is a lack of familiarity with the discipline of oral medicine and hence inappropriate referrals. Patients have frequently been seen previously by at least one other healthcare practitioner before being referred to an oral medicine clinic [START_REF] Miller | Changing oral care needs in the United States: the continuing need for oral medicine[END_REF]. In fact, [START_REF] Haberland | Referral patterns, lesion prevalence, and patient care parameters in a clinical oral pathology practice[END_REF] found that patients have, on average, previously been seen by 2.2 health practitioners prior to an oral medicine visit.

The promotion of knowledge of the oral cavity and of the diseases of the soft tissue could lead to more accurate and prompt referral.

  but only those who have the condition can report on the more subjective dimensions of the condition. This type of data can therefore add to commonly recorded medical or epidemiological data and hence enhance patient care. The aims of this study were to use qualitative research methods to explore the experiences of patients with chronic oral mucosal conditions and to identify factors that influence patients' daily lives.

  are being used to a limited extent in oral medicine (Ni Riordain & McCreary, 2010) with the oral health related quality of life and general quality of life of patients with oral lichen planus (Tabolli et al., 2009), pemphigus (Paradisi et al., 2009) and recurrent aphthous stomatitis (Mumcu et al., 2007) having been reported in the current literature. These studies regarding quality of life and chronic oral mucosal diseases use questionnaire based outcome measures to quantify the impact of theses conditions on the lives of patients. This is the first study in which qualitative research methods were used to report patient own perceptions of the impact of chronic oral mucosal conditions. The use of focus groups and interviews The study highlights a number of areas of concern for patients with chronic oral mucosal conditions that are of relevance to healthcare workers, and in particular oral medicine practitioners.

  can lead to increased waiting list times for those really in need of oral medicine services, increased cost both financially to the patient and from a time management perspective for funding. The educational measures and the promotion of awareness of the speciality of oral medicine are therefore paramount to improve the management of chronic oral mucosal conditions. Chronic oral mucosal conditions impact upon the experience of daily life of patients in a variety of areas from physical health and functioning, to concerns about their future. The role of the oral medicine practitioner in the treatment of patients with chronic oral mucosal diseases extends beyond active management and symptomatic relief to the management of all aspects of these conditions that impact upon their daily lives. Improvement of the knowledge base of general practitioner with regard to chronic oral mucosal diseases and the promotion of the specialty of oral medicine must also be addressed to help deal with the impact of these conditions on patients' lives.

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  Unpredictability of the conditions and the potential for malignant transformation Every participant highlighted the frustration with the unpredictable nature of the condition. They outlined one of the difficulties of living with the condition on a daily basis as being how irregular and erratic the occurrence of periods of flare with these diseases. Participants also expressed frustration at been unable to pinpoint the triggers that aggravate or exacerbate their condition.

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		"They kind of look at you and say 'What, what?' And … it's as if you have
		leprosy." "I would have concern about all medicines really because most if you look up
	O r a l Participant 22 l Theme 2: Treatment limitations and side effects there's some side effect with everything, the smallest little drug you're taking O r a there's a side effect." Participant 13
		D i s e Participants also indicated difficulties with the medications prescribed for the trial and error whereby on occasion medications resulted in the symptoms of e s conditions. Some outlined how when initially diagnosed there was a period of i D Theme 3:
		a s the condition been aggravated. A number of participants also raised concerns regarding the possible side effects of some medications used in the a s
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"The tears poured down my eyes, I was so bad with the mouthwash stinging me that I said to her ' I just can't do it!' You're after doing it thinking it would do you good and I think that it was after three days I rang her and I said 'I just can't' my tongue was red raw on fire." Participant 17

"I went to the chemist to get it [prescribed medication]… it was actually last

Thursday week that I took the first one I think, you know after getting it and you know I was really afraid going into bed that night. I though 'Jesus I will never wake-up again' and I was glad to see daylight again on Friday morning!." Participant 6 "The side effects are terrible from it…and I'm saying like, you know, is the cure going to be worse than the disease?" Participant 6 "Because if there was a pattern you could actually follow, but there isn't you know. You could be good for a couple of months even and then all of a sudden they just get bad and then they continue bad for another couple of months and then and there's nothing that you can see that is happening regularly that you can see to cause it." Participant 4 "It can heal up temporarily and you think you got rid of it. And the next thing back it comes, fresh as paint." Participant 22

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	Mumcu G, Hayran O, Ozalp DO, Inanc N, Yavuz S, Ergun T and Direskeneli Table1. Characteristics of participants 21 F 51 Oral Lichen Planus Mild 8
	H (2007). The assessment of oral health-related quality of life by factor Participant Sex Age Condition Severity i Time since 22 F 61 Oral Lichen Planus Mild 13
	O r a l O r a l analysis in patients with Behcet's disease and recurrent aphthous stomatitis. J Oral Pathol Med 36: 147-52. Ni Riordain R and McCreary C (2009). Oral cancer--current knowledge, O r a l diagnosis (in months) 1 F 75 Oral Lichen Planus Moderate 69 2 F 48 Recurrent Severe 72 23 F 37 Orofacial Moderate 8 O Granulomatosis r l Granulomatosis a 24 F 33 Orofacial Moderate 37
	D i s e a s e s D i s e a s e s practices and implications for training among an Irish general medical Ni Riordain R and McCreary C (2010). The use of quality of life measures in oral medicine: a review of the literature. Oral Diseases 9999. Paradisi A, Sampogna F, Di Pietro C, Cianchini G, Didona B, Ferri R, Abeni D and Tabolli S (2009). Quality-of-life assessment in patients with pemphigus using a minimum set of evaluation tools. Journal of the American Academy of s e a s e s 3 F 66 Oral Lichen Planus Mild 79 4 F 68 Mucous Membrane Pemphigoid Severe 5 F 62 6 M 50 Oral Lichen Planus Mild 7 F 71 Oral Lichen Planus Mild 74 s 49 e Oral Lichen Planus Moderate 33 s a 54 e s practitioner cohort. Oral Oncol 45: 958-62. D i Aphthous Stomatitis D i i Mild = Exacerbations
	-M a n u s c r i p t -M a n u s c r i p t --Oral Lichen Planus Moderate 12 Streiner DL and Norman GR (2006). Health Measurement Scales 8 M 64 Dermatology 60: 261-269. A practical Guide to their Devleopemtn and Use, Oxford University Press: Oxofrd. Tabolli S, Bergamo F, Alessandroni L, Di Pietro C, Sampogna F and Abeni D (2009). Quality of Life and Psychological Problems of Patients with Oral M a n u s c r 9 F 72 Pemphigus Vulgaris Severe 42 10 M 69 Oral Lichen Planus Mild M a 70 11 F 60 Oral Lichen Planus Severe 75 12 M 59 Oral Lichen Planus Moderate 33 13 M 58 Mucous Membrane Pemphigoid n u s Moderate 26 c r Mucosal Disease in Dermatological Practice. Dermatology. Travess HC, Newton JT, Sandy JR and Williams AC (2004). The development i p t 14 F 44 Oral Lichen Planus Mild 18 15 F 54 i p Oral Lichen Planus Moderate 14 t
	C o p y C o p y of a patient-centered measure of the process and outcome of combined C 16 F 65 Oral Lichen Planus Moderate 46 17 F 58 Oral Lichen Planus Mild 74 C orthodontic and orthognathic treatment. J Orthod 31: 220-34; discussion 201-2. o p y 18 M 68 Oral Lichen Planus Moderate 36 19 M 66 Pemphigus Vulgaris Severe 18 o p y
	20	M	76	Oral Lichen Planus Moderate 13
				Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy	17 18 19


Pia López-Jornet

Fabio Camacho-Alonso

Paz Andujar-Mateos

Pía López-Jornet

Keywords: Medicine, Genetics Burning mouth syndrome, primary, secondary, parafunctional habits, tongue, treatment Burning mouth syndrome, primary, secondary, parafunctional habits, tongue, treatment. Oral Diseases -Manuscript

Objective: was to apply a tongue protector with habit-modifying therapy through selfcontrol, in the patients with burning mouth syndrome (BMS).

Methods:

A prospective, randomized study was made of 65 consecutive patients with BMS. Fifty subjects were randomized to two groups: group A (informed) and group B (informed and the application of a tongue protector). The symptoms were evaluated by VAS, while the psychological profile was assessed using the HAD, with application of the quality of life questionnaires SF-36 and OHIP-49. The duration of treatment was two months.

Results: Fifty patients (46 females and 4 males) completed the study. The VAS scores in group B were 8.2 at baseline and 4.5 after two months. The respective scores in group A were 7.1 and 5.6 -the differences between the two groups being significant (p<0.001). In group B the OHIP-49 yielded lower scores for most of the scales, with significant differences versus group A. In group B the SF 36 yielded significant differences versus group A in physical role, bodily pain, general health and emotional role.

Conclusions: Parafunctional traumatism of the tongue should be taken into account in the pathogenesis of BMS with a view to exploring new therapeutic options.

INTRODUCTION

Burning mouth syndrome (BMS) is characterized by a subjective sensation of burning or itching of the oral mucosa, in the absence of clinical and laboratory test data capable of accounting for the symptoms, and with a duration of at least 4-6 months [START_REF] Grushka | Burning mouth syndrome[END_REF][START_REF] Klasser | Burning mouth syndrome: recognition, understanding, and management[END_REF][START_REF] Fedele | Burning mouth syndrome (stomatodynia)[END_REF]. The prevalence of BMS varies between 0.7-4.5% (Scala et al., 2203;[START_REF] Maltsman-Tseikhin | Burning mouth syndrome: will better understanding yield better management?[END_REF]. It is mainly seen in postmenopausal women (Baker et al., 2005). There is debate in the medical literature regarding the etiopathogenesis of the disorder, and several factors and concepts have been proposed [START_REF] Scala | Update on burning mouth syndrome: Overview and patient management[END_REF][START_REF] Lamey | Burning mouth syndrome: approach to successful management[END_REF][START_REF] Bergdahl | Cognitive therapy in the treatment of patients with resistant burning mouth syndrome: a controlled study[END_REF]Patton et al, 2007;[START_REF] Danhauer | Impact of criteria-based diagnosis of burning mouth syndrome on treatment outcome[END_REF][START_REF] Rojo | Prevalence of psychopathology in burning mouth syndrome[END_REF][START_REF] López -Jornet | Quality of life in patients with burning mouth syndrome[END_REF][START_REF] Sardella | Causative or precipitating aspects of burning mouth syndrome: a case-control study[END_REF]Zakrzewska et al, 2003). The postulated causal factors have been grouped into local factors (infections, irritants, etc.), systemic factors (diabetes mellitus, anemia through iron or folic acid deficiency, etc.), and psychogenic factors; but these causal factors contradict the definition of BMS and so constitute another disease condition separate from BMS Burning mouth syndrome is considered to be idiopathic/primary when the cause is impossible to determine [START_REF] Klasser | Burning mouth syndrome: recognition, understanding, and management[END_REF]) and secondary when it is possible to identify etiologic factors for the syndrome. Some investigators have suggested that the disorder may be a manifestation of somatization, while others have reported it to be more closely related to neuropathic pain than to somatoform chronic pain syndromes.

Parafunctional habits such as tongue thrusting or certain tics in the form of continuous rubbing of the teeth or dentures, lip, cheek or tongue nibbling, compulsive movements and hyperactivity of the tongue may contribute to induce and maintain the syndrome (Lopez-Jornet et al, 2009a). Pain in BMS is most often bilateral and symmetrical in the anterior two-thirds of the tongue (71-78%), followed by the dorsal and lateral surfaces of the tongue, the anterior portion of the hard palate, and the lip mucosa and gingiva.

The condition often manifests in several locations [START_REF] Scala | Update on burning mouth syndrome: Overview and patient management[END_REF].

Considering the chronic nature of BMS and its prevalence, optimum treatment must be established, since no effective therapies have been developed to date. Many treatments have been tested with a view to securing symptoms relief, including benzodiazepines, tricyclic antidepressants, gabapentin, serotonin reuptake inhibitors, lipoic acid and cognitive behavioral therapy -with variable results. It is therefore important to study and understand the physiopathological mechanisms of stomatodynia in order to select the best possible treatment [START_REF] Klasser | Burning mouth syndrome: recognition, understanding, and management[END_REF][START_REF] Maina | Personality disorders in patients with burning mouth syndrome[END_REF][START_REF] Serra | Pharmacological treatment of burning mouth syndrome: a review and update[END_REF][START_REF] Richard | Topical clonazepam in stomatodynia: a randomized placebo-controlled study[END_REF][START_REF] Femiano | Síndrome de boca ardiente. Estudio de la psicoterapia, medicación con ácido alfa-lipoico y combinación de terapias[END_REF][START_REF] Lopez-Jornet | Efficacy of alpha lipoic acid in burning mouth syndrome: a randomized, placebo-treatment study[END_REF] The proposed pharmacological protocols have not consistently proved to be predictable and effective in all BMS subjects, in this sense the aim of the present study was to apply a tongue protector with habit-modifying therapy through self-control, and to evaluate the symptoms and quality of life in the patients with burning mouth syndrome (BMS). Inclusion criteria for participating in the study were a clinical history of continuous symptoms of oral burning or pain on a daily or almost daily basis, during all or part of the day for more than 6 months, without paroxysms, and independent of the nervous pathway. Likewise, the included patients presented no clinical abnormalities that could account for the symptoms. Furthermore, the patients had to present normal blood test findings (complete blood count, blood glucose, serum iron and transferrin levels, serum vitamin B12 and folate), and were required to provide informed consent (Gremeau [START_REF] Richard | Topical clonazepam in stomatodynia: a randomized placebo-controlled study[END_REF]. Patients with pain attributable to other conditions (angiotensinconverting enzyme inhibitor use, candidiasis, lichenoid reactions, sores, tongue atrophy, etc.) were excluded, as were those presenting problems with dentures, biochemical anomalies and a history of hypersensitivity or allergy to the material used. Patients with known neurological disorders and those previously treated, even irregularly, with antidepressants, anticonvulsants, other psychotropic drugs, or psychological therapy were also excluded from the study. Patients occasionally using anxiolytics to induce sleep were accepted. Subjects with signs of lingual and labial parafunctional activity were also considered (tongue rubbing, lip or cheek nibbling) (Figure1).

METHODS

Primary outcome variable: oral symptoms were registered using a visual analog scale (VAS) consisting of a 10-cm vertical line marked from 0 (= no pain) to 10 (= most severe pain experienced) [START_REF] Maina | Personality disorders in patients with burning mouth syndrome[END_REF] The Hospital Anxiety and Depression scale (HAD) was used to evaluate the patient psychological profile. The HAD comprises two subscales relating to anxiety and depression. Each subscale contains 7 items pertaining to mood disorder. On analyzing the HAD scales, scores of over 10 indicated the probable presence of anxiety or depression, scores of 7 or less indicated no significant anxiety or depression, and scores of 8-10 were taken to be of borderline significance [START_REF] Bjelland | The validity of the Hospital Anxiety and Depression Scale. An updated literature review[END_REF].

The Oral Health Impact Profile (OHIP-49) is an instrument used to measure oral health.

With the OHIP-49, each item was scored as follows: 0 = "never", 1 = "hardly ever", 2 = "occasionally", 3 = "fairly often", and 4 = "very often". The OHIP-49 is divided into 7 different domains, and the possible score range for each domain is: "functional limitation" (9 items), from 0-36; "physical pain" (9 items), from 0-36; "psychological discomfort" (5 items), from 0-20; "physical disability" (9 items), from 0-36;

"psychological disability" (6 items), from 0-24; "social disability" (5 items), from 0-20;

"handicap" (6 items), from 0-24; and finally "overall OHIP score" (49 items), from 0-196. In this instrument, higher scores indicate a poorer state of health [START_REF] Lopez | Spanish version of the Oral Health Impact Profile (OHIP-Sp)[END_REF] .

The Short Form 36 (SF-36) Health Survey Questionnaire in turn has been designed to evaluate quality of life. The standard version of the SF-36 contains 8 areas (Alfonso et al., 1995): "physical functioning", "physical role limitations", "bodily pain", "general medical health", "vitality", "social functioning", "emotional role limitations", and "mental health". The scoring system is designed in such a way that higher scores indicate better health; thus, 0 is the worst state of health and 100 the ideal state of health.

Every new patient seen at the Oral Medicine Unit who met the eligibility criteria was asked to enter the trial. After being informed about the scope and methods of the study, the subjects who accepted signed the written informed consent form, and then were randomly allocated to one of the two arms of the study. The random allocation sequence was generated using software available online at http://graphpad.com/quickcalcs/randomize1. All the patients were instructed not to use any product for BMS during inclusion in the study.

All the patients in group A were informed in detail about their illness, and were instructed not to rub their tongue against their teeth and/or dentures. A self-control technique was used to this effect, the patients being given 10 printed habit-modifying reminder points to be placed in visible places. The same measures were adopted in group B, though in this case the patients were moreover programmed for tongue protector placement. The protector consisted of a transparent, low-density polyethylene sheath covering the tongue from the tip to the posterior third. These tongue protector s were single-use devices measuring 0.1 mm in thickness, with a standard size (67 mm in length and 66 mm wide), and were custom manufactured by our group (figure 2). Each patient received a kit with the protectors and the reminder points for treatment.

Instructions were provided on their use -the protector being worn during the daytime for a period of two months. We recommended use of the protector15 minutes / three times a day with the therapeutic aim of avoiding continuous rubbing against the teeth and/or dentures.

The patients in both groups were examined at the start of treatment and again after two months. One month after inclusion, the investigator called the patients in both groups by telephone as a reminder and to reinforce motivation. All patients completed the HAD questionnaire and the quality of life instruments OHIP 49 and SF-36 on each of the visits (after 0 and 2 months). On these visits the subjects were also questioned about tolerability and possible adverse effects.

The data were analyzed using the SPSS version 12.0 statistical package (SPSS ® Inc., Chicago, IL, USA). A descriptive study was made of each variable. The associations between the different qualitative variables were studied using Pearson's chi-squared test. We used the Student t-test for two independent samples in application to quantitative variables -in each case determining whether the variances were homogeneous. A probability of less than p ≤ 0.05 was accepted as significant.

RESULTS

Sixty-five patients referred to the Department of Oral Medicine for oral burning sensation were assessed for eligibility: 12 failed to meet the inclusion criteria and three refused to participate in the study. A total of 50 subjects were therefore finally included:

46 females and 4 males, aged 61.18±12.27 years (range 37-84). Table 1 shows the homogeneity of the two patient groups on the day of inclusion in the study. During the two-month duration of the study there were no dropouts in either group, and no adverse effects were observed.

The VAS scores in group B (tongue protector group) were 8.2 at baseline and 4.5 after two months. The respective scores in group A were 7.1 and 5.6) -the differences between the two groups being significant (p<0.001) (Table 2). Regarding the HAD, the scores showed no significant differences for either of the groups during the course of the study.

In group B the OHIP-49 (Table 3) yielded lower scores (i.e., better quality of life) in all domains, with significant differences between the two study time points (day 0 and after two months). Thus, the total OHIP-49 score in group B was 62.9 ±34.0 on day 0 and 44.5± 28.1 after two months. In contrast, group A showed only minimum changes, with a total score of 55.4±4 on day and 53.7±35.3 after two months. On comparing the two patient groups, significant differences were observed in the scales or domains corresponding to functional limitation, bodily pain, psychological discomfort, physical disability, psychological disability, and total score.

Lastly, as refers to the SF-36 (Table 4), the patients subjected to tongue protector treatment (group B) showed improvement in the scores between day 0 and the end of treatment (2 months) in practically all the subscales, while only minimal changes were observed in group A. On comparing the two groups, significant differences were recorded for physical functioning, physical role, bodily pain, general health and emotional role (Table 4).

DISCUSSION

Our results indicate clinical improvement among the patients for burning mouth syndrome (BMS). The VAS scores were modified in both groups during the treatment period (0-2 months), though significant differences were observed between them. On only considering overall improvement, the mean reduction in VAS score observed in group B was from 8.2 at baseline to 4.5 at the end of the study. The mean 1.4-point decrease in pain intensity observed in this study in group A may appear small, but obviously all patients did not respond in the same way to the active treatment.

Burning mouth syndrome is a diagnostic and therapeutic challenge because of its etiopathogenic variability (Patton et al, 2007;Kasser et al, 2008) (Patton et al, 2007;[START_REF] Serra | Pharmacological treatment of burning mouth syndrome: a review and update[END_REF]). The use of lipoic acid in BMS patients was presented as a promising alternative for management of the symptoms, with high levels of improvement -though lipoic acid is not effective in many cases (Lopez Jornet et al, 2009b[START_REF] Carbone | Lack of efficacy of alpha-lipoic acid in burning mouth syndrome: a double-blind, randomized, placebo-controlled study[END_REF].

Oral parafunctional habits have been widely implicated as factors that intervene in the development and perpetuation of other syndromes (Axel, 2008[START_REF] Dworkin | Research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specification, critique[END_REF] such as for example temporomandibular joint disorders. Parafunctional activity is defined as the potential lesions (depending on the tolerance of the individual in question) caused by a series of movements that occur in parallel to normal function, though lacking a functional purpose, and which generate traumatic forces characterized by an abnormal direction, excessive intensity, and a frequent and lasting character. Any type of hyperactivity -in this case of the tongue and/or lips -occurring without functional objectives or in a way that proves inadequate for the stomatognathic system, is regarded as parafunctional activity. In this context, the tongue protector used in our study offered protection, since it avoided direct friction or rubbing of the tongue mucosa against the teeth and/or dentures protect the tongue regarding the changes in temperature and taste, salivary flow (increased) though its use could also exert a placebo effect.

The management of BMS symptoms is the greatest challenge related with this syndrome. Several systemic and local therapies have been suggested to treat these patients, with frequently unsatisfactory results. Maybe this fact explains the high receptivity of BMS patients for new therapies and the good adherence to treatment observed in our study.

A possible confounding factor is represented by occult candidiasis. Some authors reported evidence for candida-induced burning mouth even in the absence of objective signs (Terai and Shimahara, 2007), thus suggesting the advisability of routine treatment with antifungal agents for at least one week before confirming the diagnosis of BMS.

As this issue was not taken into account in the present protocol, some patients may have had occult candidiasis, and therefore perhaps failed to respond to treatment.

Moreover, as the evaluation of symptoms could be altered by the interpersonal relationship between the patient and investigator, the same examiner followed-up on all of the patients in the study, in order to ensure that the results would be comparable.

It is important to consider quality of life in BMS patients, since the impact of the disease on the different domains or subscales is considerable. Accordingly, in our study use was made of two validated instruments, the OHIP-49 and SF-36. Our results show significant improvements in the OHIP-49 score in the group of patients treated with the tongue protector, in the domains relating to functional limitation, bodily pain, psychological discomfort, physical disability, psychological disability and in the total score.

As regards the SF-36, after two months of treatment group B showed improvement in all the domains, with significant differences versus group A as regards the subscales used to assess physical functioning, physical role, bodily pain, general health, and emotional role. Thus, the tongue protector could be an alternative for the management of patients with BMS. Self-inflicted damage secondary to parafunctional activity of the tongue should be taken into consideration in the pathogenesis of BMS, with a view to developing new management strategies.

Although our working hypothesis may be supported statistically, we included only a small patient series treated for a period of two months -a fact that makes it difficult to draw firm conclusions. Future studies are needed to reproduce our findings in larger series, over longer periods of time and involving an adequate sample of patients.

It -------------------------------------------------------------------------------------------------------P ---------------------------------------------------------------------------------------------------P

Group B (n=25): mean ± SD Group A (n=25): mean ± SD SF-36 ------------------------------------------------------------------------------------------------------P-value

A

  prospective, randomized study was made of 65 consecutive patients with BMS seen in the Department of Oral Medicine (University of Murcia, Spain). The study was carried out during the period between March 2007 and June 2009, and was authorized by the Bioethics Committee of the University of Murcia. A sample size was not calculated before starting the trial, as we were not able to predict the effect of the investigated treatment.

Table 1 .

 1 is essential to deepen our understanding of the physiopathological mechanisms of burning mouth in order to select the best treatment and develop new therapies which may use different mechanisms. Future research moreover should focus on the long-term effects of treatment and on the quality of life of patients with burning mouth syndrome, in order to establish the clinical impact of such interventions View of the tongue protector .Manufactured by our group with a standard size (67 mm in length and 66 mm wide) Homogeneity of the study groups on day 0 (baseline) in relation to age, gender and the VAS and HAD scores (Student t-test and Pearson χ 2 ).

	Oral Diseases -Manuscript Copy
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Table 2 .

 2 Evolution of the VAS and HAD scores during the two months of the study (Student t-test).

		Group B (n=25): mean ± SD	Group A (n=25): mean ± SD
	VAS and HAD	-

Table 3 .

 3 Evolution of the OHIP-49 score during the two months of the study (Student t-test).

		Group B (n=25): mean ± SD	Group A (n=25): mean ± SD
	OHIP-49	-

Table 4 .

 4 Evolution of the SF-36 score during the two months of the study (Student t-test).

	-value

Page 19 of 19 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy

  Social functioning (47.00 ± 39.73) -(59.50 ± 41.02) = -12.50 ± 25.51 (60.00 ± 32.87) -(62.50 ± 34.23) = -2.50 ± 10.20 0.075

		Evolution (day 0 -2 months)	Evolution (day 0 -2 months)
	Physical functioning	(57.40 ± 34.12) -(67.60 ± 38.04) = -10.20 ± 25.26 (54.00 ± 38.78) -(54.20 ± 39.01) = -0.20 ± 1.00	0.054
	Physical role	(26.50 ± 42.43) -(58.00 ± 49.23) = -35.50 ± 45.31 (44.00 ± 50.66) -(44.00 ± 50.66) = 0.00 ± 0.00	<0.001
	Bodily pain	(24.00 ± 31.38) -(39.50 ± 36.48) = -15.50 ± 26.65 (51.22 ± 28.61) -(54.22 ± 29.11) = -3.00 ± 7.63	0.029
	General health	(32.99 ± 29.38) -(44.16 ± 29.34) = -11.16 ± 23.31 (42.33 ± 27.94) -(42.99 ± 29.13) = -0.66 ± 3.33	0.031
	Vitality	(39.80 ± 25.55) -(52.00 ± 30.31) = -12.20 ± 23.80	(47.80 ± 27.99) -(50.80 ± 30.40) = -3.00 ± 8.03	0.073
	Emotional role	(34.66 ± 47.60) -(55.99 ± 48.80) = -21.33 ± 40.68	(42.66 ± 47.64) -(43.99 ± 46.86) = -1.33 ± 6.66	0.019
	Mental health	(36.16 ± 27.45) -(51.52 ± 29.08) = -15.36 ± 27.89	(47.04 ± 27.76) -(52.00 ± 30.19) = -4.96 ± 17.40 0.120
	Note: SD = standard deviation

Oral Diseases -Manuscript CopyOral Diseases -Manuscript Copy


Paola ; Solinas

University

PhD Michela Isola
email: misola@unica.it

E Proto

M Cossu

M.S Lantini

Reduced Statherin Reactivity of Human Submandibular Gland in Diabetes Running head: Statherin in Diabetic Submandibular Gland

Keywords: Cell, Pathology Oral Diseases - Statherin, Diabetes, Immunogold, Human Oral Diseases -Manuscript

BACKGROUND AND OBJECTIVE: Statherin is a salivary protein involved in the formation of enamel pellicle and in regulation of calcium homeostasis. Diabetes and other pathologies affect both salivary flow and protein secretion by salivary glands, causing increased susceptibility to mucosal infections, tooth demineralization, and caries. The purpose of this study was to compare the statherin expression in submandibular glands of healthy and diabetic subjects. MATERIALS AND METHODS. Fragments of submandibular glands obtained from diabetic and non diabetic patients were fixed, dehydrated, embedded in Epon Resin and processed for the immunogold histochemistry. The results were statistically evaluated.

RESULTS. Specific statherin labeling was demonstrated in secretory granules of acinar cells in both diabetic and normal samples. The staining was much more intense in the latter compared to those of diabetics. The labeling density was quantified by evaluating the number and spatial distribution of gold particles within the granules. The number of gold particles was significantly lower in glands from diabetics than in control glands.

CONCLUSIONS. The results obtained suggest that a reduced statherin secretion by salivary glands might be partly responsible for a less effective protection of the oral tissues, resulting in an higher incidence of caries and oral infections associated with diabetes.

Introduction

Statherin is a small salivary protein (43 aa) rich in negatively charged aminoacids, whose highly polar N-terminus contains two phosphorylated serine residues which confer to the molecule a high affinity for hydroxyapatite [START_REF] Schlesinger | Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva[END_REF]. Due to this distinctive property, it binds tooth enamel and form, with proline-rich proteins, the acquired enamel pellicle, an acellular layer which covers the entire surface of the dental crown [START_REF] Raj | Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization[END_REF][START_REF] Yao | Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches[END_REF]. Adsorbed on enamel, it plays multiple activities related to the maintenance of the dental health. The inhibitory activity of statherin on spontaneous precipitation of calcium phosphate has fundamental importance in promoting salivary supersaturation that allows optimal regulation of tooth demineralization and remineralization [START_REF] Raj | Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization[END_REF][START_REF] García-Godoy | Maintaining the integrity of the enamel surface: the role of dental biofilm, saliva and preventive agents in enamel demineralization and remineralization[END_REF].

Moreover, binding on enamel causes changes in the spatial conformation of statherin, so that the C-terminus becames available to bind microorganisms forming the dental plaque [START_REF] Gibbons | Human salivary acidic proline-rich proteins and statherin promote the attachment of Actinomyces viscosus LY7 to apatitic surfaces[END_REF]. Therefore, statherin is generally included among the numerous salivary proteins that participate in the protection of the mouth by means of antimicrobial, anti-inflammatory or other regulatory activities, such as lysozyme, lactoferrin, immunoglobulins, histatin and defensins [START_REF] Humphrey | A review of saliva: normal composition, flow and function[END_REF]Edgerton et al., 2002;Van Nieuw Amerongen and Veerman, 2002;[START_REF] Dodds | Health benefits of saliva: a review[END_REF].

Recently we investigated the ultrastructural localization of this peptide in human normal salivary glands by a post-embedding immunogold staining technique [START_REF] Isola | Electron microscopic detection of statherin in secretory granules of human major salivary glands[END_REF]. A strong statherin reactivity was demonstrated over the serous granules of parotid and submandibular glands, confirming that these glands are an important source of statherin in human saliva.

Considering the high incidence of caries, tooth loss, periodontal disease and candidiasis in patients affected by diabetes [START_REF] Lin | Dental caries in older adults with diabetes mellitus[END_REF][START_REF] Javed | Comparison of periodontal and socioeconomic status between subjects with type 2 diabetes mellitus and non-diabetic controls[END_REF][START_REF] Patiño Marín | Caries, periodontal disease and tooth loss in patients with diabetes mellitus types 1 and 2[END_REF][START_REF] Siudikiene | Dental caries increments and related factors in children with type 1 diabetes mellitus[END_REF] and given that no literature data document statherin concentration in diabetics, we undertook the present study in order to verify, using submandibular glands, if salivary glands of diabetic subjects have abnormal statherin expression, which could be in part responsible for oral complications.

Materials and Methods

Transmission Electron Microscopy Tissue Preparation.

Fragments of submandibular glands were obtained from 8 consenting adult patients, aged 40-60 years, 4 affected by type 2 diabetes and 4 non diabetic, undergoing surgery for the removal of tumors from the oral region at the Otorhinolaryngology Clinic, University of Cagliari. All samples investigated were taken avoiding the tissues adjacent to the tumors, and appeared normal by the histological analysis. The samples from non diabetics were the same examined in our previous paper [START_REF] Isola | Electron microscopic detection of statherin in secretory granules of human major salivary glands[END_REF]. All procedures were approved by the local Institutional Committee for human experimentation at the ASL 8 (Azienda Sanitaria Locale 8), Cagliari. Each sample was cut into small pieces and fixed for 2 hr in a mixture of 3% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2). Then, they were rinsed in cacodylate buffer added with 3.5% sucrose, dehydrated, and embedded in Epon Resin (Glycide Ether 100, Merk, Darmstadt). Ultrathin sections were collected on nickel grids and processed for the immunohistochemical analysis.

Immunohistochemical analysis.

The grids were treated with 1% bovine serum albumin (BSA) and 5% normal rabbit serum (NRS) in phosphate-buffered saline (PBS) solution to block non-specific binding. Then, they were incubated overnight at 4ºC with a goat polyclonal antibody specific for statherin (Santa Cruz Biotechnology), diluted 1:50 in 1% BSA and 5% NRS. After rinsing, the grids were incubated for 1 hr at room temperature with the secondary antiserum, a rabbit anti-goat IgG conjugated to 10 nm gold particles (Sigma) diluted 1:50 in BSA-PBS. Controls were incubated with a non immune goat serum or omitting the primary antibody. After washing with PBS and distilled water, they were stained with uranyl acetate and bismuth subnitrate, and finally observed and photographed in a JEOL 100S transmission electron microscope. Two grids from each patient were examined, and secretory cells rich in granules were randomly selected and photographed at low magnification.

Oral Quantitative analysis of randomly chosen micrographs was done with Image Pro Plus (Media Cybernetics, Silver Spring, MD). The gold particles were counted inside 150 serous granules randomly selected from 40 electron micrographs (10 from each diabetic patient) and the labelling density was expressed as number of gold particles /µm 2 . The data values were expressed as mean ± standard deviation (SD). To determine if the differences were significant, statistical analysis was done using the Student's t-test. The values p < 0.05 were considered significant. The proportions of granules with no, weak and intense labelling were also calculated and expressed as percent of the 150 granules.

All the results were then compared with those obtained in micrographs of normal samples following the same procedure. In addition, the statherin labelling in diabetics was estimated as % of statherin labelling in controls.

Results

Statherin reactivity was found in all the submandibular gland samples from both normal and diabetic subjects. The labeling was confined to the serous cells, whose secretory granules appeared the specific site of gold deposition (Figg. 1, 2)

Within the positive granules, gold particles decorated uniformly the dense component in both diabetic and normal samples. However, a weaker staining was easily appreciated in diabetic samples with respect to the controls (Figg. 1, 2).

Statistical analysis showed significant reduction of statherin expression in diabetes (p<0.001), since immunoreactivity was decreased approximately 75% in the diabetic (mean 6,67 /µm 2 ± SD) versus normal glands (mean 24,25 /µm 2 ± SD) (Fig. 3). The relative amounts of positive and negative secretory granules also changed: statherin labelling was absent in 3% in normal samples, versus 22% in diabetics. Moreover, granules with intense labelling (10-49 gold particles /µm2),

were not found in diabetic cells, while represented the majority in normal samples (Fig. 4).

Discussion

This study first demonstrates that the statherin reactivity of submandibular glands is significantly decreased in diabetic status. This reduction is clearly demonstrated by the statistical analysis, but it is easily appreciable by the simple observation of the electron micrographs . On the other hand, [START_REF] Baumann | Sialochemical and morphology studies of the submandibular gland of the diabetic patient-a contribution to diabetic sialadenosis[END_REF][START_REF] Carda | Structural differences between alcoholic and diabetic parotid sialosis[END_REF], probably reflecting the absence of xerostomia and other oral complications in the patients.

The reduced statherin immunoreactivity provides new evidence that salivary glands are affected by diabetes and is thus a further example of altered parameters in diabetic subjects [START_REF] Hirsch | Diabetes and periodontitis[END_REF][START_REF] Venza | Salivary histamine level as a predictor of periodontal disease in type 2 diabetic and non-diabetic subjects[END_REF][START_REF] Rao | Proteomic identification of salivary biomarkers of type-2 diabetes[END_REF][START_REF] Rudney | Potential biomarkers of human salivary function: a modified proteomic approach[END_REF]. The dry mouth symptoms are undoubtedly effects of a reduction of the salivary flow [START_REF] Dodds | Health benefits of saliva: a review[END_REF][START_REF] Sabino-Silva | Na+-Glucose cotransporter SGLT1 protein in salivary glands: potential involvement in the diabetes-induced decrease in salivary flow[END_REF]. Great attention has been devoted to the protein composition of diabetic saliva, since protein alterations are generally believed to allow bacteria, bacterial plaque and caries to progress rapidly, significantly hampering the quality of life. Although literature data are often conflicting, the total protein content seems to remain unvaried, while the relative amounts of the different proteins appear significantly changed [START_REF] Dodds | Salivary alterations in type 2 (non-insulindependent) diabetes mellitus and hypertension[END_REF][START_REF] Rao | Proteomic identification of salivary biomarkers of type-2 diabetes[END_REF]. For example, lactoferrin and sIgA were increased, while a decrease is noticed for ghrelin [START_REF] Aydin | A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics[END_REF], salivatin [START_REF] Kimura | Reduction of incretin-like salivatin in saliva from patients with type 2 diabetes and in parotid glands of streptozotocin diabetic BALB/c mice[END_REF], EGF and prolin-rich proteins [START_REF] Kasayama | Epidermal growth factor deficiency associated with diabetes mellitus[END_REF][START_REF] Szczepanski | Expression and distribution of parotid secretory proteins in experimental diabetes[END_REF]. Salivary amylase may also be affected [START_REF] Baumann | Sialochemical and morphology studies of the submandibular gland of the diabetic patient-a contribution to diabetic sialadenosis[END_REF][START_REF] Aydin | A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics[END_REF][START_REF] Mednieks | Protein expression in salivary glands of rats with streptozotocin diabetes[END_REF]. The decreased statherin reactivity shown here suggests that diabetes causes reduced statherin synthesis or changes in its intracellular processing and storage in submandibular glands. Unfortunately, at present the biochemical measurement of salivary statherin in diabetics has not been performed to support this assumption. When confirmed, statherin decrease will represent a further evidence that vulnerability of oral tissues may be partly dependent on a defective enamel pellicle, which poorly protect the teeth, and by an unbalanced calcium metabolism. Moreover, although a direct bactericidal activity of statherin has been still not demonstrated, its decrease could be partly responsible for the increased susceptibility to oral infections, since its ability to bind some bacterial species also would be reduced. Besides, the importance of this peptide in the maintenance of tooth integrity was clearly signified by the strong correlation between its high amounts and absence of dental caries [START_REF] Vitorino | The role of salivary peptides in dental caries[END_REF].

Therefore, the present results could serve as a starting point for researches that will explain which metabolic signals determine salivary changes in the diabetic status and the relationship between the systemic disorders and the reduced salivary statherin.

Fig 1 .Fig 2 .Fig 3 .

 123 Fig 1. Serous cells of submandibular gland of a diabetic patient, low (A) and high (B) magnification. Gold particles appear exclusively confined within secretory granules. The majority of them express a weak reactivity for statherin. Moreover, a large number of granules are completely negative. 158x199mm (600 x 600 DPI)

Fig 4 .

 4 Fig 4. Proportions of submandibular secretory granules with no labelling (group 0), with 1-11 (group 1-11) , and with 12-46 (group 12-46) gold particles. 92x108mm (600 x 600 DPI)

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Acknowledgments

We wish to thank Mrs Silvana Bernardini and Mr Alessandro Cadau for technical assistance. The investigation was supported by Fondazione Banco di Sardegna (FBS) and PRIN 2008.