M L Miniati

R Conforti

M Casale

Matucci-Cerinic

Maddali Bongi

Del Rosso

Conforti Ml

Matucci Cerinic
email: cerinic@unifi.it

Marco Matucci Cerinic

The rehabilitation of facial involvement in systemic sclerosis: efficacy of the combination of connective tissue massage, Kabat's technique and kinesitherapy -a randomized controlled trial

Keywords: Systemic Sclerosis, rehabilitation, connective tissue massage, Kabat method, physiotherapy;

    The rehabilitation of facial involvement in systemic sclerosis: efficacy of the combination of connective tissue massage, Kabat's technique and kinesitherapy: a randomized controlled trial

Introduction

Systemic Sclerosis (SSc) is characterised by induration of the skin and internal organs, by joint modifications and muscle impairment [START_REF] Wigley | Systemic Sclerosis: Clinical features[END_REF][START_REF] Casale | Systemic sclerosis (scleroderma): an integrated challenge in rehabilitation[END_REF][START_REF] Ranque | Systemic sclerosis-associated myopathy[END_REF][START_REF] Allanore | New therapeutic strategies in the management of systemic sclerosis[END_REF][START_REF] Cerinic | Therapeutic challenges for systemic sclerosis: facts and future targets[END_REF]. Skin fibrosis leads to tissue retraction and atrophy, and consequently to disability and reduction of patients quality of life (QoL).

Though most authors agree that specific SSc rehabilitation program can prevent and reduce disabilities deriving from skin and joint involvement [START_REF] Casale | Systemic sclerosis (scleroderma): an integrated challenge in rehabilitation[END_REF], only few studies on this matter are available [START_REF] Pizzo | Effects of a non surgical exercise program on the decreased mouth opening in patients with systemic scleroderma[END_REF][START_REF] Bongi | Connective massage and Mc Mennel joint manipulation may improve the function of Scleroderma hand[END_REF][START_REF] Matucci-Cerinic | Association of Kabat's technique, connective massage and kinesitherapy in the rehabilitative treatment of systemic sclerosis face[END_REF][START_REF] Matucci-Cerinic | Manual lymph drainage for the treatment of edematous hand in systemic sclerosis[END_REF]. The involvement of face and oral tissues is a typical feature of SSc patients, that causes important aesthetic changes and impairment of the self-image [START_REF] Del Rosso | Health-related quality of life in systemic sclerosis as measured by the Short Form 36: relationship with clinical and biologic markers[END_REF]. The face becomes amimic, the cutaneous furrows disappear. Around the mouth, vertical wrinkling, due to skin retraction, develops and the nose becomes sharp.

Face changes also include thinning and reduction of mouth width (microcheilia) and opening (microstomia), also favoured by osteolysis of mandibular angles and by fibrosis of soft tissues.

These modifications interfere with eating, speaking, oral hygiene measures, and dental treatment, thus deteriorating the quality of life of SSc patients [START_REF] Pizzo | Effects of a non surgical exercise program on the decreased mouth opening in patients with systemic scleroderma[END_REF][START_REF] Nagy | Analysis of the oral manifestations of systemic sclerosis[END_REF][START_REF] Naylor | The nonsurgical treatment of microstomia in scleroderma: a pilot study[END_REF][START_REF] Cazal | Oral complaints in progressive systemic sclerosis: two cases report[END_REF][START_REF] Albilia | Small mouths ... Big problems? A review of scleroderma and its oral health implications[END_REF][START_REF] Shah | Often forgotten manifestations of systemic sclerosis[END_REF] and in most severe cases leading to surgical interventions, such as bilateral commissurotomies [START_REF] Pizzo | Effects of a non surgical exercise program on the decreased mouth opening in patients with systemic scleroderma[END_REF][START_REF] Nagy | Analysis of the oral manifestations of systemic sclerosis[END_REF][START_REF] Naylor | The nonsurgical treatment of microstomia in scleroderma: a pilot study[END_REF][START_REF] Cazal | Oral complaints in progressive systemic sclerosis: two cases report[END_REF][START_REF] Albilia | Small mouths ... Big problems? A review of scleroderma and its oral health implications[END_REF][START_REF] Shah | Often forgotten manifestations of systemic sclerosis[END_REF].

Rehabilitation management of microstomia, is mainly based on self administered home-based exercise program including mouth-stretching and oral augmentation exercises. This traditional approach has been shown to have some positive effect on mouth opening in SSc patients [START_REF] Pizzo | Effects of a non surgical exercise program on the decreased mouth opening in patients with systemic scleroderma[END_REF].

Preliminary results from our group suggested that the combination of connective tissue massage, Kabat's technique and a specific kinesitherapy program [START_REF] Matucci-Cerinic | Association of Kabat's technique, connective massage and kinesitherapy in the rehabilitative treatment of systemic sclerosis face[END_REF]

Materials and methods

Forty SSc patients, fulfilling ACR criteria [START_REF]Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma)[END_REF] (6 males and 34 females; age: 57.28 ± 11.33 years; disease duration 9.4 ± 4.3 years), were enrolled from the outpatient clinic of the Department of Biomedicine -Division of Rheumatology, of the University of Florence. After a written informed consent, all patients underwent a clinical examination and were assessed according to international guidelines [17]. Entry criterion was face involvement assessed by a Rodnan skin score > 1 [START_REF] Clements | Inter and intraobserver variability of total skin thickness score (modified Rodnan TSS) in systemic sclerosis[END_REF], no gender or age limits.

The presence of secondary Sjögren syndrome was evaluated by the determination of SSa/Ro and SSb/La autoantibodies, salivary scintigraphy, salivary minor glands biopsy and the ophthalmologic tests including Schirmer e Lissamine green test ().

Patients were assigned, using a random number sequence, to 2 groups and assessed at baseline (T0), after 9 weeks of treatment (T1) and after 9 weeks of follow up (T2).

The 20 patients of interventional group were treated for 9 weeks twice a week (each session lasting 1 hour) with a combined four steps procedure of connective tissue massage [START_REF] Goats | Connective tissue massage[END_REF][START_REF] Alpiner | Rehabilitation in joint and connective tissue diseases. 1. Systemic Diseases[END_REF][START_REF] Greenman | Mobilization with and without impulse technique[END_REF] during the first 10 minutes, Kabat's method [START_REF] Greenman | Mobilization with and without impulse technique[END_REF][START_REF] Kabat | The practical application of proprioceptive neuromuscular facilitation[END_REF] for about 15 minutes and kinesitherapy for the following 15 minutes. Due to the stressful work of this combination of physiotherapy, the remaining 20 minutes was employed to perform relaxing exercise. All the patients applied also a home program of mimic exercises [START_REF] Pizzo | Effects of a non surgical exercise program on the decreased mouth opening in patients with systemic scleroderma[END_REF][START_REF] Naylor | The nonsurgical treatment of microstomia in scleroderma: a pilot study[END_REF][START_REF] Kendall | Muscles: functions and test[END_REF]. Control group (20 patients) performed for 9 weeks a program of daily home exercises for the face only [START_REF] Pizzo | Effects of a non surgical exercise program on the decreased mouth opening in patients with systemic scleroderma[END_REF][START_REF] Naylor | The nonsurgical treatment of microstomia in scleroderma: a pilot study[END_REF][START_REF] Kendall | Muscles: functions and test[END_REF].

In both groups, the home program of mimic exercises was continued after the assessment at the end of the treatment (T1) for further 9 weeks till the end of follow-up (T2), for a total duration of 18 weeks.

None of the patients was edentulous and none presented mandibular osteolysis at X rays.

All patients continued their pharmacological treatments (alprostadil-α-cyclodextran, calcium channel blockers, topical glyceryl trinitrate, proton pump inhibitors, clebopride, steroids, cyclophosphamide, azathioprine, methotrexate) with no changes throughout the study.

Rehabilitation Techniques

Connective tissue massage is a manual technique used to treat altered connective tissues, in order to increase local blood flow and the release of involved tissue by connective tissue stretching [START_REF] Goats | Connective tissue massage[END_REF][START_REF] Alpiner | Rehabilitation in joint and connective tissue diseases. 1. Systemic Diseases[END_REF][START_REF] Greenman | Mobilization with and without impulse technique[END_REF]. For the facial involvement of SSc is strictly recommend to treat not only the face but also the Kinesitherapy consisted in a specific passive, active and assisted exercises for temporalmandibolar joint (exercises for improving mouth opening and jaw lateralizing) [START_REF] Pizzo | Effects of a non surgical exercise program on the decreased mouth opening in patients with systemic scleroderma[END_REF][START_REF] Naylor | The nonsurgical treatment of microstomia in scleroderma: a pilot study[END_REF]. [Fig. 2e,f].

Program of home daily exercises. All patients were instructed by the same operator (G.L.) to perform daily of mouth stretching and opening exercises [START_REF] Pizzo | Effects of a non surgical exercise program on the decreased mouth opening in patients with systemic scleroderma[END_REF][START_REF] Naylor | The nonsurgical treatment of microstomia in scleroderma: a pilot study[END_REF] and in grimacing exercises for mimic muscles [START_REF] Kendall | Muscles: functions and test[END_REF] [Fig. 3a-c].Home exercises were divided in three sequences. In the first sequence (to be executed at least 5 minutes, 3 times/day), the patient placed thumbs into the mouth in order to enlarge the oral angles. The movements were done bilaterally and simultaneously (modified from 6). In the second exercise sequence (to be performed once a day) the patient inserted a number (as high as possible) of tongue depressors between the premolars of one arch towards the molars of the controlateral one to properly open the mouth. The tongue depressors should be maintained for a minimum of 8 minutes and, after a stop, the exercise should be repeated, if possible, for additional 8 minutes, by increasing the number of tongue depressors (modified from 6 and 12). The third exercise sequence consisted in mimic exercises to be performed once a day, based on a series of grimaces to exercise (oro)facial muscles [START_REF] Kendall | Muscles: functions and test[END_REF].

Assessment

Patients were assessed by the same operator at baseline (T0), at the end of the 9 weeks period of treatment (T1) and after 9 weeks of follow-up (T2). The following outcome measures were chosen:

Assessment of global health condition

Health Assessment Questionnaire disability index (HAQ-DI) [START_REF] Johnson | The HAQ disability index in scleroderma trials[END_REF][START_REF] Montagna | HAQ-DI Italian version in systemic sclerosis[END_REF] was used to measure disability; the Physical Synthetic Index (PSI) and the Mental Synthetic Index (MSI)], of SF36 were used to assess QoL [START_REF] Del Rosso | Health-related quality of life in systemic sclerosis as measured by the Short Form 36: relationship with clinical and biologic markers[END_REF][START_REF] Ware | The MOS 36-Item Short Form Health Survey (SF-36): I. Conceptual framework and item selection[END_REF][START_REF] Apolone | The Italian SF-36 Health Survey: Translation, Validation and Norming[END_REF].

Face Assessment

The functional involvement of face and mouth was assessed by the Mouth Handicap in Systemic Sclerosis (MHISS) scale [START_REF] Mouthon | Development and validation of a scale for mouth handicap in systemic sclerosis: the Mouth Handicap in Systemic Sclerosis scale[END_REF] [START_REF] Miniati | Matucci-Cerinic M. Mouth Handicap in Systemic Sclerosis scale (MHISS) Italian version[END_REF].

Mouth opening was assessed in centimeters measuring the distance between the tips of upper and lower right incisive teeth (mean of two consecutive measurements).

Skin involvement of the face was assessed by means of a modified Rodnan skin score [START_REF] Clements | Inter and intraobserver variability of total skin thickness score (modified Rodnan TSS) in systemic sclerosis[END_REF] to be applied to two areas of the face (right and left cheek).

Statistical Analysis

Data are presented as mean ± standard deviation. Student's T test or Mann Whitney tests, when necessary, were used to compare groups characteristics at T0. ANOVA for repeated measures was used for all outcome measures. Data analysis was performed using the SPSS statistical package 12.0 for Windows.

Clinical trial registration

The study was registered with Current Controlled Trials (www.controlled-trials.com) no ISRCTN43633950.

Results

The general features of the samples are reported in table 1. No patient dropped out from the study.

Assessment of global health condition

SF36 and HAQ. In both groups, neither the combined treatment nor the home exercise program modified the Physical and Mental Summary Indexes of SF36 and HAQ-DI, as well at the end of the study (T1) and at follow-up (T2) (table 2 and3). In control group, the home exercise program did not provide any significant change in MHISS throughout the study (table 2 and3).

Mouth opening. Patients of both groups benefited from the treatments for mouth opening. In interventional group, a significant increase of mouth opening was shown at T1 and still maintained at T2 (p<0.05 and p<0.001 versus T0, respectively).

In control group, the home exercise program improved mouth opening at T1 (p<0.001 versus T0), but the significance was lost at T2 (table 2 and3).

Skin score. In interventional group, the combined treatment decreased significantly skin score at T1 (p< 0.001 versus T0). The decrease of the skin score was still significant at T2 (p< 0.01 versus T0).

In controls the home exercise program did not modify skin score throughout the study (table 2 and 3).

Discussion

Our data show that, in SSc, a combined rehabilitation approach is significantly more effective than a home exercise program in reducing skin thickness of the face, in recovering mouth opening and in improving self-reported face and mouth related symptoms. It is however clear that the treatment of the face did not exert any effect on global function and QoL. SSc leads to fibrosis of facial soft tissues with reduction of mimics and disappearance of the cutaneous furrows. These features, together with diminished mouth opening and width, altered dentition, difficulties in dental intervention, concomitance of sicca syndrome and changes at temporo-mandibular joints often lead to impairment of QoL . In fact, the fibrotic involvement of hands and face becomes one of the SSc patients' greatest complaints [START_REF] Joachim | Life with a rare chronic disease: the scleroderma experience[END_REF][START_REF] Benrud-Larson | Body image dissatisfaction among women with scleroderma: extent and relationship to psychosocial function[END_REF], with concern about disfigurement [START_REF] Paquette | Cutaneous concerns of scleroderma patients[END_REF] and personal self-esteem [START_REF] Van Lankveld | Appearance selfesteem in systemic sclerosis-subjective experience of skin deformity and its relationship with physician-assessed skin involvement, disease status and psychological variables[END_REF]. It was also shown that, in SSc, skin deformities are a core stressor of the disease, only preceded by fatigue [START_REF] Van Lankveld | Appearance selfesteem in systemic sclerosis-subjective experience of skin deformity and its relationship with physician-assessed skin involvement, disease status and psychological variables[END_REF].

In order to deal with the facial involvement of SSc patients, we have chosen to use the combination of connective tissue massage (able to modify, by stretching, bloodstream and releasing the connective tissues) and Kabat's method (a neurorehabilitation technique, that reinforces neuromuscular recruitment) [START_REF] Ware | The MOS 36-Item Short Form Health Survey (SF-36): I. Conceptual framework and item selection[END_REF][START_REF] Apolone | The Italian SF-36 Health Survey: Translation, Validation and Norming[END_REF]. These techniques were added to the traditional kinesitherapy and home exercises for mouth and mimic muscles. At the best of our knowledge, this is the first study combining these techniques in a rehabilitation program specifically conceived for the face of SSc patients.

It is interesting to note that the improvement of mouth opening, the only effect of home exercises, was lost at follow up. Contrarily, the effects of combined protocol on mouth opening, skin score and self-perceived functional mouth and face handicap, were maintained after 9 weeks from the end of the study. This can be due to the efficacy of the different techniques acting sinergically and to the fact that patients continued, for a 9 week period after the end of the treatment, the program of mimic and stretching exercises in a home self management program. This evidence suggests that the continuity of care is mandatory in the rehabilitation of patients affected with chronic rheumatic diseases, such as SSc, in order to maintain its efficacy [START_REF] Dagfinrud | Physiotherapy interventions for ankylosing spondylitis[END_REF].

Home exercises for orofacial muscles were of some efficacy. In fact, in the short term period, they were able to increase mouth opening in the group of SSc patients that were assigned to the home program only.

In the chronic care model, patients are encouraged to become part of the health care team and the concept of self-care or self-management plays a central role [START_REF] Bodenheimer | Patient self management of chronic disease in primary care[END_REF]. Some evidence exists about the effectiveness of self-management in SSc patients. Recently, Mugii et al studied the efficacy of selfadministered stretching of each finger in SSc patients, showing that amelioration of range of motion was present in each finger after 1 month and maintained within 1 year [START_REF] Mugii | The efficacy of self-administered stretching for finger joint motion in Japanese patients with systemic sclerosis[END_REF].

The amelioration of MHISS scale suggests that, in SSc, the combined approach improved also the subjective perceptions and the self perceived disability due to face and mouth involvement. This result is important because, although problems related to mouth involvement seem to have less weight than hand related problems in total disability, mouth problems are felt as an important handicap by SSc patients. MHISS is a new scale with an excellent reliability and good construct validity, in the specific assessment of disability involving the mouth in SSc patients [START_REF] Kabat | The practical application of proprioceptive neuromuscular facilitation[END_REF]. In our study, MHISS was helpful in following-up SSc patients over time for evaluating the outcome due to rehabilitation.

From our data, it is evident that the treatment of the face had no effects on QoL and disability. This is probably due to the relatively short period of the treatment and to the fact that the patients were treated with techniques not aimed to the overall body, but specifically conceived for the involvement of the face.

Due to the complexity and the severity of SSc, the rehabilitation program proposed in the present work should be regarded as a support tool in SSc management. However this combined program should be integrated with global rehabilitation techniques on top of the pharmacological treatments for the disease [START_REF] Allanore | New therapeutic strategies in the management of systemic sclerosis[END_REF][START_REF] Cerinic | Therapeutic challenges for systemic sclerosis: facts and future targets[END_REF].

The limitations of our study may be considered the short period of treatment and follow-up that did not allow us to identify how long the effect of the combined treatment could last after the end of the study.

In conclusion, the combination of Kabat's technique, connective tissue massage and kinesitherapy with home based exercises is effective in the rehabilitative treatment of SSc face. This combination Table 1. Patients characteristics.

Table 2.

Mean and SD of the items assessed at baseline (T0), at the end of treatment (T1) and after 9 weeks of at follow-up (T2) in interventional group.

Table 3.

Mean and SD of the items assessed at baseline (T0), at the end of treatment (T1) and

  can be usefully used for the treatment of facial involvement in SSc. The aim of our work was to evaluate in SSc patients the efficacy of a rehabilitation program for the face based on the sequential combination of connective tissue massage, Kabat's technique, kinesitherapy and traditional home-based exercise program of mimic exercises versus home-based exercise program of mimic exercises alone.

  neck [Fig. 1a] and the clavear regions. [Fig. 1b] Kabat's method is a neurorehabilitation technique that uses spiral and diagonal movement patterns in conjunction with stretch, resistance and other proprioceptive facilitation techniques to reinforce neuromuscular recruitment [21, 22]. The Kabat's method concern the stimulation of the orbicularis oris [Fig. 1c], zygomaticus [Fig. 1d], levator labii [Fig. 1e], nasalis [Fig. 1f] buccinator [Fig. 2a], frontalis [Fig. 2b,c] and corrugator [Fig. 2d] muscles.

  In interventional group, the combined treatment significantly improved the scores of MHISS scale at T1 versus T0 (16.25± 5.64 vs 17.20± 5.15; p<0.001). At T2, MHISS scores (18.50±5.23) worsened both versus scores obtained at T1 and at T0. This indicates that the home based program did not maintain the effect obtained by the combined program during the previous 9 weeks.

LegendsFig. 1

 1 Fig. 1 Physioterapic intervention with connective tissue massage of the neck (a) and clavear region

Fig. 2

 2 Fig.2Physiotherapic intervention with Kabat's method for the involvement of buccinators (a)

Fig. 3

 3 Fig. 3 Program of home daily exercises: mouth stretching (a) and mimic exercices (b,c).

treatment may lead to benefits for mouth opening and skin score, maintained also after the end of the program and is more effective than a simple home exercise program. Further studies are needed to evaluate the long term effects of this combined program also in the two SSc subsets. after 9 weeks of at follow-up (T2) in control group.


Antonella Fioravanti
email: fioravanti7@virgilio.it

Luca Cantarini

Maria Romana Bacarelli

Arianna Lalla

Linda Ceccatelli

Patrizia Blardi

Fioravanti Antonella

Cantarini Luca

Romana

Lalla Arianna

Ceccatelli Linda

Blardi Patrizia

Effects of Spa therapy on serum leptin and adiponectin levels in patients with knee osteoarthritis

Keywords: osteoarthritis, leptin, adiponectin, spa therapy

INTRODUCTION

Leptin is a peptidic molecule synthesized almost exclusively by adipocytes, which regulates appetite and energy expenditure at the hypothalamic level [START_REF] Henry | Adipose tissue hormones and the regulation of food intake[END_REF]. In the past few years, additional actions have been attributed to this molecule, specifically, modulating the immune response and affecting the bone and cartilage metabolism [START_REF] Fantuzzi | Adipose tissue, adipokines, and inflammation[END_REF][START_REF] Loeser | Systemic and local regulation of articular cartilage metabolism: where does leptin fit in the puzzle?[END_REF].

Adiponectin is also synthesized by adipose tissue, and one of its main actions is to improve insulin sensitivity [START_REF] Henry | Adipose tissue hormones and the regulation of food intake[END_REF]. In skeletal joints, adiponectin may act as a pro-inflammatory agent and may be involved in matrix degradation [START_REF] Lago | A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and proinflammatory cytokines in chondrocytes[END_REF]. In patients with osteoarthritis (OA), leptin and adiponectin are detected in both synovial fluid and in plasma [START_REF] Presle | Differential distribution of adipokines between plasma and synovial fluid in patients with osteoarthritis. Contribution of joint tissues to their articular production[END_REF]. The adipokines exhibit different patterns of distribution in the joint and the circulating compartment: plasma levels of adiponectin exceed those in the paired synovial fluid, whereas leptin concentrations in synovial fluid are higher than their plasma counterparts [START_REF] Presle | Differential distribution of adipokines between plasma and synovial fluid in patients with osteoarthritis. Contribution of joint tissues to their articular production[END_REF].

Spa treatments have been applied for therapeutic purposes in rheumatic diseases since ancient times [START_REF] Sukenik | The role of spa therapy in various rheumatic diseases[END_REF].

The mechanism by which mud-packs and/or balneotherapy improves the symptoms of rheumatic diseases is still not fully understood [START_REF] Sukenik | The role of spa therapy in various rheumatic diseases[END_REF]. The effects of thermal baths are, in part, related to temperature [START_REF] Sramek | Human physiological responses to immersion into water of different temperatures[END_REF] , and balneotherapy also induces a neuroendocrine reaction which causes an increase in serum levels of corticosteroids and catecholamines [START_REF] Laatikainen | Response of plasma endorphins, prolactin and catecholamines in women to intense heat in a sauna[END_REF][START_REF] Cima | Neuroendocrin effects of a cycle of fangotherapy[END_REF] and a reduction in circulation levels of tumour necrosis factor α (TNF-α) and Interleukin-1β (IL-1 β) [START_REF] Cozzi | Anti-inflammatory effect of mud-bath application on adjuvant arthritis in rats[END_REF].

Since serum levels of leptin and adiponectin are influenced by temperature [START_REF] Zeyl | Interactions between temperature and human leptin physiology in vivo and in vitro[END_REF][START_REF] Imbeault | Cold exposure increases adiponectin levels in men[END_REF], catecholamines [START_REF] Trayhurn | Regulation of leptin production: a dominant role for the sympathetic nervous system?[END_REF], corticosteroids [START_REF] Miell | Dexamethasone induces an acute and sustained rise in circulating leptin levels in normal human subjects[END_REF] and TNF-α and IL-1 β [START_REF] Zumback | Tumour necrosis factor increases serum leptin levels in humans[END_REF][START_REF] Delaigle | Induction of adiponectin in skeletal muscle by inflammatory cytochines: in vivo and in vitro studies[END_REF], we evaluated whether Spa therapy modify plasma levels of leptin and adiponectin in patients with OA treated with a cycle of a combination of daily locally-applied mud packs and bicarbonate-sulphate mineral bath water.

MATERIALS AND METHODS

Patients

As a part of a recently-published randomized single-blind controlled clinical trial [START_REF] Fioravanti | Short and Long-Term Effect of Spa Therapy in Knee Osteoarthritis[END_REF] on the efficacy of spa therapy in knee OA, we included thirty patients of both sexes with knee OA fulfilling the ACR criteria [START_REF] Altman | Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association[END_REF] ] ]. . . Radiological staging was carried out using the Kellgren method [START_REF] Kellgren | Radiological assessment of osteo-arthrosis[END_REF]; patients with a radiological score of I-III were included in the study (Table I).

Exclusion criteria for the study were previously presented [START_REF] Fioravanti | Short and Long-Term Effect of Spa Therapy in Knee Osteoarthritis[END_REF]. In addition, for the purposes of this study, we excluded patients with history of diabetes mellitus, instable weight history, those treated with medications known to affect body weight and heavy smokers.

All selected patients resided in the area near the spa at Rapolano Terme (Siena, Italy) and continued to live at home and go about their normal daily routines.

Treatment

The patients were treated daily at the spa centre at Rapolano Terme with a combination of mud packs applied on both knees for 20 min at an initial temperature of 45°C and with bicarbonatesulphate mineral bath water at 38°C for 15 min, for a total of 12 applications carried out over a period of 2 weeks. [START_REF] Fioravanti | Short and Long-Term Effect of Spa Therapy in Knee Osteoarthritis[END_REF] .

It was recommended to patients that they should continue their established non-pharmacological treatments (physical exercise) and non steroidal anti-inflammatory drugs (NSAIDs) (150 mg Diclofenac tablets, 20 mg Piroxicam tablets, 750 mg Naproxen tablets, 200 mg Aceclofenac tablets) and/or analgesics (500 mg acetaminophen tablets), which were to be consumed liberally and noted daily in a diary. Furthermore, we advised patients not to utilize corticosteroids or hyaluronic acid infiltrations.

Leptin and Adiponectin measurements

Leptin and adiponectin plasma levels were assessed at baseline time and after 2 weeks, upon completation of the spa treatment period.

Baseline blood samples (6 ml) were drawn from an antecubital vein, in the supine position in the morning after an overnight fast, and again after two weeks under the same conditions. The blood was immediately centrifuged and serum was stored at -80°C.

Plasma leptin levels were determined by enzyme-linked immunosorbent assay method using ready kits of Quantikine® human Leptin Immunoassay (R&D Systems Europe, Abingdon, United Kingdom) [START_REF] Blardi | Rett syndrome and plasma leptin levels[END_REF].

Sensitivity of undiluted samples was 7•8 pg/ml. Inter-and intra-assay coefficients of variation were 5•4% and 3•3%, respectively.

Plasma adiponectin levels were detected by enzyme-linked immunosorbent assay method using ready kits of Quantikine® human Adiponectin Immunoassay (R&D Systems) [START_REF] Blardi | Rett syndrome and plasma leptin levels[END_REF]. Sensitivity of undiluted samples was 0•246 ng/ml. Inter-and intra-assay coefficients of variation were 6•9% and 4•7%, respectively.

Assessment parameters

All the patients were evaluated on a 0 to 100 mm visual analogue scale (VAS) with 0 for the absence of pain, as well as the Lequesne Index of severity of knee OA [START_REF] Lequesne | Indexes of severity for osteoarthritis of the hip and knee. Validation-value in comparison with other assessment tests[END_REF].

Acetaminophen consumption was calculated by the number of daily tablet intake. The NSAID consumption score was expressed as mg equivalence of diclofenac, according to a previously validated and published scale [START_REF] Dougados | Score d' équivalence des AINS[END_REF].

The clinical assessments were performed before and at the end of the period of spa therapy.

Statistical analysis

All the parameters of this study are expressed as mean and standard deviation (SD). For all tests, a p value less than 0.05 was considered as statistically significant.

The difference in clinical parameters (VAS, Lequesne Index, NSAID and analgesic consumption) and the plasma leptin and adiponectin levels before and after spa therapy were assessed using the Student's paired t-test.

Correlation analysis was performed using Pearson's correlation coefficient or Spearman's rank correlation coefficient For all analyses, SAS System v. 9.0 statistical software (Carry, USA Inc.) was used.

RESULTS

The baseline recorded variables in this series of 30 OA patients treated with spa therapy are shown in Table I.

At basal time, plasma leptin levels were significantly correlated with BMI (r=0.41, p<0.001) and gender (r=0.61, p<0.001). However, no significant correlation was found between serum leptin and patient age at the time of the study (r= 0.04, p= NS) , duration of the disease (r= 0.07, p= NS), radiographic severity of knee OA according to Kellgren and Lawrence's score (r=0.18, p=NS), VAS score (r=0.20, p=NS) or Lequesne index (r=0.15, p=NS).

There was no correlation between the plasma adiponectin level and BMI (r= -0. After spa treatment (two weeks), we observed a statistically significant reduction (p<0.001), in spontaneous pain and in the Lequesne index score for gonarthrosis (Table II). This symptomatic effect was confirmed by the significant reduction of NSAID consumption.

At the end of the cycle of mud-bath therapy, serum leptin levels showed a slight, but not significant increase, while we found a significant decrease (p<0.05) in serum adiponectin levels (Table II). However, leptin and adiponectin concentrations after the treatment were not correlated with other clinical parameters (VAS and Lequesne index score) described in Table II (data not shown).

Finally, it should be noted that no changes whatsoever were made in terms of diet or physical activity levels, and no modifications in body weight were observed.

DISCUSSION

This preliminary report was designed to investigate the possible effect of spa therapy on plasma levels of leptin and adiponectin in OA patients treated with mud-packs and balneotherapy. Our results demonstrated only a slight, but not significant, increase in plasma leptin after 2 weeks of spa therapy with no evident correlation with parameters of clinical efficacy.

Leptin induces the expression of growth factors such as IGF-1 and TGF-β1, stimulates proteoglycan and collagen synthesis, and increases the stimulatory effects of proinflammatory cytokines on nitric oxide (NO) production in chondrocytes [START_REF] Dumond | Evidence for a key role of leptin in osteoarthritis[END_REF][START_REF] Van Beuningen | Osteoarthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-beta injections[END_REF][START_REF] Otero | Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1[END_REF][START_REF] Simopoulou | Differential expression of leptin and leptin's receptor isoform (Ob-Rb) mRNA between advanced and minimally affected osteoarthritic cartilage;effect on cartilage metabolism[END_REF]. Leptin may also trigger cartilage destruction by increasing matrix metalloproteases (MMP)-9 and MMP-13 [START_REF] Simopoulou | Differential expression of leptin and leptin's receptor isoform (Ob-Rb) mRNA between advanced and minimally affected osteoarthritic cartilage;effect on cartilage metabolism[END_REF]. The present study shows for first time that there is no significant change in leptin concentration following a cycle of spa therapy. However, leptin concentration, measured after the treatment, did not correlate with the clinical parameters (VAS, Lequesne Index).

In our study, spa therapy produced a significant decrease (p<0.05) in serum adiponectin levels in patients with knee OA.

There is some evidence that adiponectin in skeletal joints may have proinflammatory effects and may be involved in cartilage degradation [START_REF] Lago | A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and proinflammatory cytokines in chondrocytes[END_REF][START_REF] Gomez | Adipokines in the skeleton: influence on cartilage function and joint degenerative diseases[END_REF].

Lago et al recently demonstrated that adiponectin induced the expression of type-2 nitric oxide synthase (NOS) and stimulated IL-6, MMP-3, MMP-9 and monocyte chemoattractant protein-1 (MCP-1) release [START_REF] Lago | A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and proinflammatory cytokines in chondrocytes[END_REF]. In view of these recent findings, the decrease of adiponectin after spa therapy demonstrated in our study may play a protective role in OA.

However, adiponectin presents metabolic properties it increases insulin sensitivity, improves glucose metabolism and decreases plasma triglycerides, and has antiatherogenetic actions [START_REF] Matsuzawa | Adiponectin: Identification, physiology and clinical relevance in metabolic and vascular disease[END_REF][START_REF] Ouchi | Cardioprotection by adiponectin[END_REF]. Furthermore, plasma levels of adiponectin are low in obese individuals and in those with cardiovascular disease [START_REF] Fantuzzi | Adipose tissue, adipokines, and inflammation[END_REF][START_REF] Matsuzawa | Adiponectin: Identification, physiology and clinical relevance in metabolic and vascular disease[END_REF][START_REF] Ouchi | Cardioprotection by adiponectin[END_REF]; obesity and cardiometabolic syndrome are frequently associated with OA [START_REF] Puenpatom | Increased prevalence of metabolic syndrome in individuals with osteoarthritis: an analysis of NHANES III data[END_REF]. Thus it is important to study the effects of spa therapy on certain cardiovascular risk factors.

Finally, it remains to be clarified which of the mechanisms of action of spa therapy [START_REF] Zeyl | Interactions between temperature and human leptin physiology in vivo and in vitro[END_REF][START_REF] Imbeault | Cold exposure increases adiponectin levels in men[END_REF][START_REF] Trayhurn | Regulation of leptin production: a dominant role for the sympathetic nervous system?[END_REF][START_REF] Miell | Dexamethasone induces an acute and sustained rise in circulating leptin levels in normal human subjects[END_REF][START_REF] Zumback | Tumour necrosis factor increases serum leptin levels in humans[END_REF][START_REF] Delaigle | Induction of adiponectin in skeletal muscle by inflammatory cytochines: in vivo and in vitro studies[END_REF] may have determined the changes in plasma levels of leptin and, above all, adiponectin that we observed. One possible hypothesis is that local application of heat, by causing an increase in the internal temperature of the knee, may determine a change in serum adipocytokine levels, probably due to localized production thereof [START_REF] Presle | Differential distribution of adipokines between plasma and synovial fluid in patients with osteoarthritis. Contribution of joint tissues to their articular production[END_REF]. Nevertheless, our study and other currently available data do not allow for specific identification of any specific mechanism of action.

In conclusion, our data show that spa therapy can modify plasma levels of the adipocytokines leptin and adiponectin, important mediators of cartilage metabolism.

Further studies on larger numbers of cases are needed to evaluate the effects of spa therapy on adipocytokines and the mechanisms with which said therapy may modify production thereof, as well as whether such modifications may have repercussions on cardiovascular risk factors.

Table I :

 I Baseline characteristics in 30 patients with knee OA treated

	with spa therapy
	Variable
	Age (years) (m±SD)		69.10±7.90
	Gender (M/F)		14/16
	BMI (Kg/m 2 ) (m±SD)		26.05±5.23
	Desease duration (years) (m±SD)	7.50±4.53
	Radiological score	I	8
		II	12
		III	10
	F = female; M =male; BMI = body mass index; OA = osteoarthritis
			14

Table

  II Assessment and biochemical parameters before and after spa therapy in our cohort of patients VAS= Visual Analogue Scale for spontaneous pain + NSAIDs = non-steroidal anti-inflammatory drugs (daily consumption as score);

	Measures	T0	T1
	VAS (mm) (m±SD)	41.18±15.6	29.42±14.36**
	Lequesne Index (m±SD)	10.13±2.95	7.68±3.14**
	Leptin (ng/ml) (m±SD)	14.338.±9.725	16.320±10.788
	Adiponectin (ng/ml) (m±SD)	13.715±4.566	11.215±4.068*
	Drugs consumption (m±SD)
	NSAIDs +	7.13±4.87	4.29±1.60**
	Acetaminophen ++	0	0
	++ Acetaminophen (daily consumption as tablets).
	*p<0.05; **p<0.001 vs basal time


Vrm Reddy

K S Satheesan

Bayliss Frcs

Outcome of Boyd-McLeod procedure for recalcitrant lateral epicondylitis of elbow Authors

Keywords: Tennis elbow, surgical release, Boyd McLeod procedure

Various surgical procedures including percutaneous and open release, arthroscopic procedures have been described to treat recalcitrant tennis elbow. We present the outcome of Boyd-McLeod surgical procedure for tennis elbow resistant to non-operative treatment in twenty-seven patients (twenty-nine limbs). Boyd McLeod procedure involves excision of the proximal portion of the annular ligament, release of the origin of the extensor muscles, excision of the bursa if present, and excision of the synovial fringe.

The average time interval from the onset of symptoms of tennis elbow until surgery was 28 months (range: 8 -72 months). 91% of the patients reported complete relief of symptoms with return to full normal activities including sports. Average post-op time for return to professional / recreational activity was five weeks. One case developed pain secondary to ectopic bone formation after surgery, which settled after excision and in another; there was no pain relief with Boyd McLeod procedure. Two patients had scar tenderness that did not affect the final outcome. We conclude that Boyd-McLeod procedure is an effective treatment option in patients with resistant lateral epicondylitis.

Introduction

Tennis elbow is a common orthopaedic problem that usually responds to non-operative treatment. Less than 5% are resistant or recur following conservative management.

Several theories have been proposed with regards to its aetiopathogenesis. Various surgical procedures have been described to treat recalcitrant tennis elbow. Systematic reviews have not established the superiority of one treatment over the other. Our study was undertaken to assess the clinical outcome of Boyd McLeod procedure in the management of resistant tennis elbow that has not responded to non-operative measures.

Material and Methods

Twenty-seven patients (Twenty nine limbs) with resistant lateral epicondylitis were reviewed after surgical management with Boyd-McLeod procedure. There were thirteen male, and fourteen female patients of which two had bilateral surgery. Average age of the patient was 47 years (range: 34-65). 93% were manual workers. 55.2% of the patients has bilateral tennis elbow. Dominant elbow involvement was present in 64% cases) odds ratio: 3.2). 25% of the patients waited for more than 12 months before presenting to their family doctor (range: 1-36 months, median: 6 months). All patients had an unsuccessful trial of conservative management including rest, splints, analgesia, physiotherapy and steroid injections. The mean number of local steroid injections given was 3( range: 1-10) resulting in an average pain free interval of 4.2 months (range: 0-18 months) before recurrence of symptoms. 89.7% used NSAIDS regularly for a defined period before surgery and 83% of the patients had physiotherapy prior to surgery. One patient had a failed open release of common extensor origin elsewhere.

The average time interval from the onset of symptoms of tennis elbow until surgery was 28 months (range: 8-72 months). Boyd McLeod procedure was done under general anaesthetic and tourniquet control as a day case procedure in all cases. Average tourniquet time for the surgical procedure was 32 minutes (range: 18-59 minutes). A 5 cm longitudinal incision is made 2 cm proximal to the lateral epicondyle extending distally. The common extensor origin is sharply dissected and reflected distally from the underlying bone and soft tissues. Adequate precautions are taken to protect the radial collateral ligament, and annular ligament. A small flap of the proximal portion of the annular ligament along with the synovial fold of the radio humeral joint is removed.

Debridement of the ECRB tendon is carried out.

The superior cortx of the lateral condyle is excised with a sharp osteotome and the bone decompressed with 2-3 drill holes. Finally, the common extensor tendon is sutured back to the lateral epicondyle.

Post-operatively, asling is worn for 4-48 hours and then the elbos is mobilised. Wound healing is checked at 10-14 days and physiotherapy is initiated if range of movement appears restricted.

The mean post-op follow up time was 16 months (range: 6-32 months) until discharge.

Surgical outcome was evaluated by a telephone questionnaire and a review of the case notes following the discharge of the patient.

Results

91% of the patients reported complete relief of symptoms with return to full normal activities including sports. Average post-op time for return to professional/recreational activity was five weeks. The number of steroid injections and its effectiveness did not have any bearing on the outcome of surgery.

Discussion

Tennis elbow is a common orthopaedic problem of the upper limb characterised by symptoms of increased lateral elbow pain with resisted wrist extension, particularly when the elbow is straight [START_REF] Cabot | Tennis elbow, a curable affliction[END_REF]. Contracture of the anterolateral elbow capsule explains the pain in extension.

The commonly accepted theory for aetiology of tennis elbow is due to repeated contraction of the wrist extensor muscles, especially ECRB, causing microscopic tendon tears that progress to degenerative tendinosis [START_REF] Bunata | Anatomic factors related to the cause of tennis elbow[END_REF][START_REF] Coonrad | Tennis elbow: Its course, natural history, conservative and surgical management[END_REF][START_REF] Nirschi | Elbow tendinopathy: Tennis elbow[END_REF][START_REF] Putnam | Painful conditions around the elbow[END_REF]. Bunata et al studied the bone structure and musculotendinous origin details in cadaveric elbows and concluded that the ECRB tendon has a unique anatomic location that makes its undersurface vulnerable to contact and abrasion against the lateral edge of capitellum during elbow motion [START_REF] Bunata | Anatomic factors related to the cause of tennis elbow[END_REF].

Pathoanatomy is non-inflammatory angiofibrobalstic tendinosis ECRB-RDC complex [START_REF] Nirschi | Elbow tendinopathy: Tennis elbow[END_REF].

Several other pathological processes can cause lateral elbow pain including periostitis, bursitis, synovial fringe of the radio humeral joint and, degenerative changes in the annular ligament [START_REF] Boyd | Tennis Elbow[END_REF]. Cyriax identified twenty-nine different pathological processes responsible for tenniselbow syndrome [START_REF] Cyriax | The pathology and treatment of tennis elbow[END_REF]. Ruch et al suggested synovial plicae of the elbow as a cause of the elbow as a cause of lateral elbow pain in patients with vague clinical symptoms simulating tennis elbow that does not respond to conservative management [START_REF] Ruch | The posterolateral plica: a cause of refractory lateral elbow pain[END_REF]. The site of maximal tenderness is posterior to the lateral epicondyle and centered at the posterior radiocapitellar joint. Mullett et al observed a collar like band radiocapitellar capsular complex impinging on the radial head and subluxating into the radiocapitellar joint with manipulation under direct vision [START_REF] Mullett | Arthroscopic treatment of lateral epicondylitis: clinical and cadaveric studies[END_REF]. Based on arthroscopic findings, they calssified this degenerative capsular fold relationship to the radial head into four types: Type I: radial head is completely exposed' Type II: partial coverage of the radial head by capsuloligamentous complex without interposition into the joint in any position, Type III: subluxation of the capsular edge into the joint and, Type IV: radial head completely obscured by capsuloligamentous complex throughout the range of motion. Bosworth suggested chronic inflammation of annular ligament due to repeated rotation of an eccentrically placed aymmetric radial head in certain position as a cause of tennis elbow and reported good results with partial excision of the annular ligament [START_REF] Bosworth | Surgical treatment of tennis elbow. A follow-up study[END_REF].

Non-operative management usually resolves all symptoms with restoration of normal elbow function. Calvert et al reported 92% success with complete relief of symptoms following 1 or 2 steroid injections and that repeated injections were not helpful [START_REF] Calvert | Simple lateral release in treatment of tennis elbow[END_REF]. Less than 5% cases are resistant or recur following non-operative management. Infrequently, surgery is required for patients suffering from recalcitrant tennis elbow that has recurred or failed to respond to conservative measures [START_REF] Cabot | Tennis elbow, a curable affliction[END_REF]. Bankes et al were of the opinion that day-case lateral release for resistant tennis elbow should be offered at an earlier stage in view of the simple and effective nature of the operation with little justification in pursuing with conservative treatment [START_REF] Bankes | Day-case simple extensor origin release for tennis elbow[END_REF].

Percutaneous extensor tenotomy for chronic tennis elbow preformed as an office procedure under local or general anaesthesis is a simple procedure resulting in good result, quick recovery and minimal complications [START_REF] Baumgard | Percutaneous release of the epicondylar muscles for humeral epicondylitis[END_REF][START_REF] Dunkow | A comparison of open and percutaneous techniques in the surgical treatment of tennis elbow[END_REF][START_REF] Yerger | Percutaneous extensor tenotomy for chronic tennis elbow: an office procedure[END_REF]. Dunkow et al reported better results and quicker recovery with precutaneous release in a radomised comparative trial

between percutaneous and open releases [START_REF] Dunkow | A comparison of open and percutaneous techniques in the surgical treatment of tennis elbow[END_REF]. Difficulty in visualisation and isolating ECRB tendon from common extensor origin is a drawback with percutaneous tenotomy [START_REF] Thomas | Patient satisfaction after open release of common extensor origin in treating resistant tennis elbow[END_REF]. Also, this procedure does not deal with soft tissues like capsule, synovial fringe that may be the cause of lateral elbow pain, or prominent lateral epicondyle process that may be responsible for ECRB abrasion.

Ruch et al successfully treated ten cases with arthroscopic synovial plica excision in which conservative management was unsuccessful [START_REF] Ruch | The posterolateral plica: a cause of refractory lateral elbow pain[END_REF]. Arthroscopic tennis elbow releaes has the advantages of minimal morbidity, allows intra-articular assessment and quicker rehabilitation [START_REF] Mullett | Arthroscopic treatment of lateral epicondylitis: clinical and cadaveric studies[END_REF][START_REF] Kalainov | Arthroscopic tennis elbow release[END_REF]. It allows resection of degenerative capsuloligamnetous complex of the radiocapitellar joint without extensive dissection. However, arthroscopic surgery is very expensive, technically demanding and therefore requires a long learning curve for beginner surgeons. Rubenthaler et al observed similar results with endoscopic technique as compared to the open technique for lateral epicondylitis [START_REF] Rubenthaler | Long-term follow-up of open and endoscopic Hohman procedures for lateral epicondylitis[END_REF].

Different techniques of open releases have been described in the literature with varying results. Carroll et al reported 50% success with ECRB tenotomy [START_REF] Carroll | Evaluation of the Garden Procedure for Lateral Epicondylitis[END_REF] where as other authors have reported 87-95% symptomatic relief with simple open release of the extensors [START_REF] Cabot | Tennis elbow, a curable affliction[END_REF][START_REF] Heyse-Moore | Resistant tennis elbow[END_REF][START_REF] Rayan | V-Y slide of the common extensor origin for lateral elbow tendonopathy[END_REF][START_REF] Verhaar | Lateral extensor release for tennis elbow. A prospective long-term follow-up study[END_REF][START_REF] Zingg | Debridement of extensors and drilling of the lateral epicondyle for tennis elbow: a retrospective follow-up study[END_REF]. Heyse-Moore observed similar results following lengthening of ECRB tendon and decompression of radial tunnel which could be due to the relief of tension on the lateral epicondyle and its adjacent structures following surgical division of the fibrous arch of the superficial leaf of the supinator independently of radial or posterior interosseous nerve decompression per se [START_REF] Heyse-Moore | Resistant tennis elbow[END_REF]. Wilhelm et al obtained 90% success by denervation of radial nerve branches supplying the lateral epicondylar region by careful exposure or blind deneravtion through disinsertion of certain muscles. Additional direct radial nerve release did not improve the results of denervation [START_REF] Wilhem | Tennis elbow: treatment of resistant cases by denervation[END_REF].

Boyd-McLeod procedure, a modification of Bosworth III procedure, was originally described in 1973 [START_REF] Boyd | Tennis Elbow[END_REF]. It is done as a day case procedure through a lateral approach of the elbow that involves excision of all the pathological tissues at the elbow that can produce symptoms attributable to tennis elbow including degenerative tissue from common extensor origin, decortication and decompression of lateral epicondyle, excision of synovium fringe and partial release of annular ligament and capsule. This procedure also enables the surgeon to evaluate radio humeral joint intra-operatively. There are few studies regarding the outcome of Boyd-McLeod procedure [START_REF] Boyd | Tennis Elbow[END_REF]. Our study revealed a high success rate and a low complication rate that were comparable wit hthe wide spectrum of surgical procedures described in the literature. Pain relief and restoration of elbow function can be reliably achieved with minimal morbidity and high rate of patient satisfaction. We conclude that Boyd-McLeod procedure is an effective treatment option in patients with resistant lateral epicondylitis that has recurred or not responded to nonoperative management.

  Two cases had poor results with no relief of pain post-operatively. One case developed pain secondary to ectopic bone formation after surgery, which settled after excision. Another patient, who had primary open release of tennis elbow elsewhere, did not have any pain relief with Boyd McLeod procedure. Two patients had scar tenderness, which did not affect elbow function. Intra-operative findings included early osteoarthritis of the radial head in one case who reported an excellent outcome after surgery. None of the patients suffered from significant complications or loss of limb function. The results in our study were comparable to other open releases published in the literature.


Dimitrios Yoo

M.D Andrew P Daoussis
email: jimdaoussis@hotmail.com

M.D Andonopoulos

Dr D Daoussis

Rituximab in the treatment of systemic sclerosis-associated interstitial lung disease. Comment on the article by

HAL is

Dear Sir

We read with interest the article by Yoo which reported a case of successful treatment of systemic sclerosis (SSc)-associated interstitial lung disease (ILD) with rituximab (RTX) in a patient resistant to steroid and cyclophosphamide (CYC) therapy. The author mentions that there is only one case report of improvement of SSc-associated ILD with RTX so far. This is incorrect. We have previously shown that RTX may improve lung function in patients with SSc-associated ILD, in a randomized, controlled, proof of principle study, of one year duration. [START_REF] Daoussis | Experience with rituximab in scleroderma: results from a 1year, proof-of-principle study[END_REF] This study was available online (Epub ahead of print) since May 2009, six months prior of the date this article was submitted. This is why we were surprised to notice that our study was not included in the references even though the author has performed an exhaustive literature search and has included all the articles regarding the use of rituximab in SSc, including two in abstract form.

Three studies so far have explored the potential clinical efficacy of RTX in SSc. [START_REF] Daoussis | Experience with rituximab in scleroderma: results from a 1year, proof-of-principle study[END_REF][START_REF] Lafyatis | B cell depletion with rituximab in patients with diffuse cutaneous systemic sclerosis[END_REF][START_REF] Smith | Rituximab in diffuse cutaneous systemic sclerosis: an open-label clinical and histopathological study[END_REF] Our study, however, was the only one specifically designed to test the effect of RTX on SSc-associated ILD, since the presence of ILD was an inclusion criterion and most patients had severe ILD. On the contrary the other two studies excluded patients with severe ILD and only a fraction of patients had evidence of mild ILD. We evaluated 14 patients with SSc. Eight patients were randomized to receive 2 courses of RTX at baseline and 24 weeks, whereas 6 patients (control group) received standard treatment alone. The median percentage of improvement of FVC in the RTX group was 10.25% whereas that of deterioration in the controls was 5.04% (p=0.002). The median percentage of improvement of DLco in the RTX group was 19.46% whereas that of deterioration in the control group was 7.5% (p=0.023). We also found evidence of skin remodelling with reduction of collagen deposition in most patients treated with RTX which correlated with skin B cell depletion. We have also published a case report describing dramatic improvement of lung function and skin fibrosis in a patient with severe, diffuse scleroderma following long term treatment with RTX. (4) Our data indicate a possible disease modifying role of RTX in SSc, particularly in SSc-associated lung disease. (5)

The case report by Yoo is in agreement with our data and suggests that RTX may have beneficial effects on SSc-associated ILD. It is worth mentioning that this particular patient showed a dramatic improvement in HRCT findings something that was not observed in our cohort, probably due to the longer disease duration of our patients which had more established fibrosis.

SSc is a multisystem disease with poor prognosis since no disease modifying drugs exist so far. Cyclophosphamide has shown modest results, however its toxicity restricts its long term use. Clinical, as well as basic research data indicate that targeting B cells with RTX may be a promising therapeutic approach in SSc. We propose that further exploration of the clinical efficacy of RTX in SSc-associated ILD is highly needed. A direct comparison of RTX versus cyclophosphamide, the gold standard of care so far, in a large, multicentre, randomized, double blind study, would seem a reasonable approach.

Abstract

Several lines of evidence from basic research indicate that B cells may contribute to the pathogenesis of systemic sclerosis. Clinical studies as well, have provided preliminary data pointing to the direction that B cell depletion with rituximab might be a therapeutic option for patients with this debilitating disease. Key words: systemic sclerosis; scleroderma; rituximab; interstitial lung disease; B cells; fibrosis; skin thickening; rheumatic diseases


Luca Cantarini
email: cantariniluca@hotmail.com

Maria Orso

Rolando Lucherini

Maria Giuseppina Cimaz

Mauro Brizi

Galeazzi

Cantarini Luca

Lucherini Orso Maria

Cimaz Rolando

Brizi Maria Giuseppina

Galeazzi Mauro

Autoinflammatory disorders and patients with isolated serosal involvement

Keywords: recurrent ascites, recurrent pleuritis, recurrent idiopathic pericarditis, familial Mediterranean fever (FMF), tumor necrosis factor receptor-1 associated periodic syndrome (TRAPS)

We read with interest the article by Cakir et al [START_REF] Cakir | A rare cause of refractory ascites in a child: familial Mediterranean fever[END_REF] who describe a child who had febrile episodes of ascites, without any extraabdominal complaints at disease onset.

The patient did not fulfil the diagnostic criteria for familial Mediterranean fever (FMF; MIM no.#249100) [START_REF] Livneh | Criteria for the diagnosis of familial Mediterranean fever[END_REF] and genetic testing did not reveal any known mutation in the MEFV gene.

Nevertheless, the resolution of symptoms determined by colchicine within few days since its administration and the absence of disease flares at 12-month follow-up, both strengthened the presumptive diagnosis of FMF. The later onset of skin eruptions constituted a probable FMF diagnosis. Peritoneal effusion during abdominal attacks is commonly observed in FMF and is often related to serosal peritoneal inflammation. The effusion is usually mild and is not detectable by clinical evaluation, disappearing during symptom free intervals [START_REF] Sohar | Familial Mediterranean fever (1967) A survey of 470 cases and review of the literature[END_REF]. Only rarely it may develop into ascites with large amounts of peritoneal fluid [START_REF] Bellin | Encapsulating peritonitis in periodic disease. Apropos of a case studied by x-ray computed tomography[END_REF], and it is also uncommon that patients present ascites as the inaugural sign of FMF. To date, Cakir et al have described the first pediatric patient with this unusual presentation. Since an increasing interest is currently being devoted to serosal involvement in autoinflammatory disorders [START_REF] Cantarini | Innate versus acquired immune response in the pathogenesis of recurrent idiopathic pericarditis[END_REF], we would like to present some additional data on our personal experience with adult-onset autoinflammatory disorders and their presentation with isolated serosal involvement occurring with fever. Among monogenic periodic fever syndromes, the inflammatory involvement of the serosal membranes is reported both for FMF, caused by mutations in the MEFV gene, and for tumor necrosis factor receptor-associated periodic syndrome (TRAPS), caused by mutations in the TNFRSF1A gene [START_REF] Mcdermott | Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes[END_REF]. Both disorders are characterized by recurrent episodes of fever associated with abdominal and chest pain, arthralgia and/or arthritis, and cutaneous manifestations. FMF and TRAPS typically manifest in the pediatric population, with onset ranging from the first months to the first decade of life, while a limited number of patients experience disease onset during adulthood. Late-onset FMF is fairly rare, with 14-30% of FMF patients manifesting the disease in late adulthood [START_REF] Sohar | Familial Mediterranean fever. A survey of 470 cases and review of the literature[END_REF][START_REF] Sayarlioglu | Characteristics of patients with adult-onset familial Mediterranean fever in Turkey: analysis of 401 cases[END_REF] up to the age of 65, as we recently reported [START_REF] Cantarini | in press) Familial Mediterranean fever diagnosed in an elderly patient[END_REF]. Adult-onset FMF seems to be related to low-penetrance mutations and, although patients may experience milder disease, the clinical manifestations are usually similar to those of younger patients, except that arthritis and erysipela-like erythema occur at significantly lower frequencies in adults [START_REF] Sayarlioglu | Characteristics of patients with adult-onset familial Mediterranean fever in Turkey: analysis of 401 cases[END_REF]. Disease onset in adulthood has been described for TRAPS as well, up to the age of 63 [START_REF] Dodé | French Heraditary Recurrent Inflammatory Disorder Study Group (2002) The enlarging clinical, genetic, and population spectrum of tumor necrosis factor receptor-associated periodic syndrome[END_REF] and, as with FMF, is frequently related to low-penetrance mutations [START_REF] Dodé | French Heraditary Recurrent Inflammatory Disorder Study Group (2002) The enlarging clinical, genetic, and population spectrum of tumor necrosis factor receptor-associated periodic syndrome[END_REF][START_REF] Ravet | Clinical significance of P46L and R92Q substitutions in the tumour necrosis factor superfamily 1A gene[END_REF]. Patients with adult-onset TRAPS may be characterized by a phenotype that can mimic FMF in the duration of inflammatory attacks, which may last less than 1 week [START_REF] Dodé | A novel missense mutation (C30S) in the gene encoding tumor necrosis factor receptor 1 linked to autosomaldominant recurrent fever with localized myositis in a French family[END_REF]. In addition, patients with adult-onset TRAPS may present atypical clinical manifestations, such as recurrent serosal involvement of pericardium as the sole clinical manifestation [START_REF] Dodé | French Heraditary Recurrent Inflammatory Disorder Study Group (2002) The enlarging clinical, genetic, and population spectrum of tumor necrosis factor receptor-associated periodic syndrome[END_REF][START_REF] Cantarini | Idiopathic recurrent pericarditis refractory to colchicine treatment can reveal tumor-necrosis factor associated periodic syndrome[END_REF][START_REF] Cantarini | in press) Familial clustering of recurrent pericarditis may disclose tumor necrosis factor receptor-associated periodic syndrome[END_REF]. Among these subjects, we recently hypothesized the criteria for identifying patients for whom testing for genetic mutations of the TNFRSF1A gene should be carried out [START_REF] Cantarini | Idiopathic recurrent pericarditis refractory to colchicine treatment can reveal tumor-necrosis factor associated periodic syndrome[END_REF][START_REF] Cantarini | in press) Familial clustering of recurrent pericarditis may disclose tumor necrosis factor receptor-associated periodic syndrome[END_REF]. Atypical presentations of FMF have also been increasingly reported in the literature and, as with TRAPS, often manifest with isolated serosal involvement (pericardium, peritoneum and pleura) [START_REF] Cakir | A rare cause of refractory ascites in a child: familial Mediterranean fever[END_REF][START_REF] Tutar | Isolated recurrent pericarditis in a patient with familial Mediterranean fever[END_REF][START_REF] Lega | Isolated Recurrent Pleuritis Revealing Familial Mediterranean Fever in Adulthood[END_REF].

Recently, a diagnostic score for improving genetic diagnosis of recurrent periodic fever in children under the age of 10 has been proposed with the aim of increasing the probability of obtaining positive results [START_REF] Gattorno | A diagnostic score for molecular analysis of hereditary autoinflammatory syndromes with periodic fever in children[END_REF]. In addition, with the aim of improving genetic diagnosis in adults suspected with autoinflammatory disorders, we have recently identified some variables that appear to be strongly related to the probability of detecting gene mutations in MEFV and TNFRSF1A and we have also developed a diagnostic score for identifying patients at high risk to carry these mutations (unpublished data, manuscript submitted). Both scores might represent two evidence-based guidelines, thus helping in the diagnostic evaluation of patients (pediatric and adult), including those presenting with atypical and incomplete disease, thus helping clinicians to identify those who may be carriers of mutations in "autoinflammatory genes". Since all patients enrolled in both studies were of Italian origin, the multivariate analysis could not evaluate the ethnic origin among the possible discriminating variables. Nevertheless, the ethnic origin should obviously be taken into consideration when patients from populations with a high prevalence of mutations of a certain gene are examined [START_REF] Federici | A decision tree for genetic diagnosis of hereditary periodic fever in unselected patients[END_REF].

Cakir et al, as we did with recurrent pericarditis [START_REF] Cantarini | in press) Familial clustering of recurrent pericarditis may disclose tumor necrosis factor receptor-associated periodic syndrome[END_REF][START_REF] Tutar | Isolated recurrent pericarditis in a patient with familial Mediterranean fever[END_REF], suggested that clinicians should keep FMF in mind in the differential diagnosis of massive ascites especially in regions where hereditary inflammatory diseases are common.

Nevertheless, further evaluations are needed in order to establish criteria that can identify the few patients among those with isolated recurrent serositis that could be carriers of mutations in the genes of autoinflammatory diseases.


Sílvia Fraga
email: silfraga@med.up.pt

Milton Severo

; Diogo Costa

Carla Lopes

Elisabete Ramos

CLUSTERING BEHAVIOURS AMONG 13 YEAR-OLD PORTUGUESE ADOLESCENTS

Keywords: health behaviours, adolescents, clustering behaviours, schools

Aim To identify clustering of behaviours in young adolescents; b) to analyse differences in food intake according clusters.

Subject and Methods

A cross-sectional study was carried out on adolescents' behaviours comprising 2160 adolescents aged 13 years-old attending schools of Porto, Portugal. We used data of 875 girls and 737 boys with complete information for all variables considered in the cluster definition (tobacco and alcohol use, sport activities, fruit intake, daily sleep hours). The average method was used to find the natural structure of clusters.

Results

We identified three clusters: Cluster 1 characterized by never smokers and never drinkers represented those with higher intake of fruits and lower intake of soft drinks; cluster 2 comprises essentially tobacco and alcohol experimenters and was almost similar to cluster 1 regarding the other characteristics. Cluster 3 represented mostly regular smokers and drinkers, and those with the lowest daily sport activities, the lowest number of daily sleep hours, the lowest mean of fruits consumption and the highest intake of sweets and soft drinks.

Conclusion

Our findings show that even in early stages of adolescence there is a cluster of smoking, alcohol use and food intake, providing further evidence of the clustering of unhealthy behaviours.

INTRODUCTION

During the transition from childhood to adulthood, adolescents establish patterns of behaviour and make lifestyle choices that affect both their current and future health [START_REF] He | Defining and understanding healthy lifestyles choices for adolescents[END_REF]).

The Centers for Disease Control and Prevention [START_REF] Eaton | Youth risk behavior surveillance--United States, 2007[END_REF]) defined six categories of adolescents' unhealthy behaviours based on their contributing to the leading causes of morbidity and mortality among youth. Unhealthy dietary behaviour and physical inactivity are recognized as one of these six [START_REF] Eaton | Youth risk behavior surveillance--United States, 2007[END_REF].

The theory of critical behaviour proposed by Jessor [START_REF] Jessor | Adolescent development and behavioral health. Behavioural Health: A Handbook of Health Enhancement and Disease Prevention[END_REF] explains that problems of behaviour and health do not occur in isolation, but within a more comprehensive set of behaviours, highlighting an intersection area between these behaviours. This supports that engaging in an unhealthy behaviour may increase the likelihood of engaging in other unhealthy behaviours [START_REF] Jessor | Adolescent development and behavioral health. Behavioural Health: A Handbook of Health Enhancement and Disease Prevention[END_REF][START_REF] Breiman | Clustering of health-related behaviors among 18-year-old Australians[END_REF][START_REF] Talashek | Building teen power for sexual health[END_REF]). In the case of unrelated behaviours, programs targeting specific behaviours may be effective, but if adolescents' behaviours are related (to other sets of behaviours), prevention programs focusing on common determinants of unhealthy behaviours may turn more effective and should privilege approaches that take into account different behaviour-problems since they have similar patterns of development, not neglecting their specificity [START_REF] Guilamo-Ramos | Prevention programs for reducing adolescent problem behaviors: Implications of the co-ocurrence of problem behaviors in adolescence[END_REF][START_REF] Bartlett | Problem behaviours in adolescents[END_REF]). Thus, to know how health behaviours are interrelated is important in designing programs aiming to modify risk behaviours. Among adults, it has been shown that clustering of adverse health behaviours is common and increases the morbidity and mortality (Boden-Albala and Sacco 2000; [START_REF] Cardi | Health behavior risk factors across age as predictors of cardiovascular disease diagnosis[END_REF]. It was also demonstrated that unhealthy behaviours tend to aggregate since early ages [START_REF] Jessor | Adolescent development and behavioral health. Behavioural Health: A Handbook of Health Enhancement and Disease Prevention[END_REF][START_REF] Talashek | Building teen power for sexual health[END_REF][START_REF] Gorely | Patterns of sedentary behaviour and physical activity among adolescents in the United Kingdom: Project STIL[END_REF][START_REF] Velde | Patterns in sedentary and exercise behaviors and associations with overweight in 9-14-year-old boys and girls -a cross-sectional study[END_REF]), but it is still unknown if dietary habits cluster together with other known unhealthy behaviours in early adolescence, since it may be expected that at this age dietary choices are still under a relative high parental control [START_REF] Birch | Development of Eating Behaviors Among Children and Adolescents[END_REF][START_REF] Savage | Parental Influence on Eating Behavior: Conception to Adolescence[END_REF].

Health behaviours of schoolchildren are a challenge for worldwide policy-makers, and understanding the relationship between health behaviours in early ages may lead to the development of more effective strategies for promoting health [START_REF] Nelson | Adolescent physical activity and sedentary behavior: Patterning and long-term maintenance[END_REF][START_REF] Gorely | Patterns of sedentary behaviour and physical activity among adolescents in the United Kingdom: Project STIL[END_REF].

Although health promotion programs focusing on tobacco, alcohol and drugs make use of a combined approach, few of them take into account healthy dietary choices as part of an integrated approach.

Thus, this study aims a) to identify clustering of behaviours in young adolescents; b) and to analyse differences in food intake according to the identified clusters.

METHODS

The Epidemiological Health Investigation of Teenagers in Porto (EPITeen) cohort intends to follow adolescents from initial adolescence to adulthood stage, in order to study behaviours and chronic diseases like cardiovascular diseases in a life course approach. During the 2003/2004 school year, participants were selected as part of the assembling procedure of the EPITeen cohort [START_REF] Ramos | Family and school determinants of overweight in 13-year-old Portuguese adolescents[END_REF], which intends to follow adolescents born in 1990 and registered at every public and private schools of Porto, a large urban centre in the north-west of Portugal. To identify eligible students, every appropriated school in Porto (26 public and 24 private) were contacted. All public and 19 private schools agreed to participate, allowing the contact with parents and adolescents. In those schools we identified 2787 students (2126 public and 661 private). Forty-four (1.6%) adolescents could not be reached (missing classes frequently) and 2160 (77.5%) agreed to participate. A similar participation proportion in public (77.7%) and private (76.7%) schools (p=0.603) was observed.

The Ethical Committee of the University Hospital of São João, Porto, approved the study. Parents and children received written information explaining the purpose and the design of the study.

Additionally, the study steps were described in each school during special meetings arranged according to parents' convenience. Written informed consent was obtained both from parents and children.

The baseline evaluation required extensive data collection, comprising two self-administered questionnaires (one completed at home, another at school).

The home questionnaire was completed by adolescents with their parents' help and comprised information on demographic, social, and behavioural characteristics. The home questionnaire also evaluated dietary intake using a semi-quantitative food frequency questionnaire (FFQ) validated for the Portuguese adult population (Lopes 2000 (in Portuguese); [START_REF] Lopes | Intake and adipose tissue composition of fatty acids and risk of myocardial infarction in a male Portuguese community sample[END_REF], and adapted for adolescents adding some food items more frequently used at this age. Respondents were asked to report how often they usually eat each particular food over the prior 12-month period as the number of times per day, week, or month. FFQ has 92 food items and for this specific work we used the information of the following groups: sweets and pastry (other biscuits beyond simple, croissants, pastry, donut, cakes, chocolates, chocolate snacks, dairy desserts, ice creams, marmalade, compote, jelly, honey, sugar and candy), soft drinks (soda and carbonated juices), vegetables (cabbages, spinach, broccoli, onion, carrot, lettuce, peppers, tomato, cucumbers), fruits (all fresh fruits, including tropical fruit) and soup (vegetable soup). In addition to FFQ the specific question "how many fruit portions do you eat a week?" was asked as part of the global questionnaire. Information from this question assessing the number of fruit portions was used in the cluster definition since it had fewer missing information than the FFQ. For those with both information (fruits of FFQ and fruits in the single question) the correlation between them was 0.518.

Wakening-up and bedtime hours were recorded to compute daily sleeping hours.

We asked separately about time spent per week watching TV, playing computer and reading per week. Then we computed the total time spent on sedentary activities.

The school questionnaire was answered at school only by the adolescent. It collected information about sports practice, tobacco and alcohol use.

To evaluate the frequency of sport activities we considered only those activities taking at least 20 minutes a week and not included in school curriculum. To assess this information we used an fiveoption question (never, ≤ 1 time per month, 1 time per month to 1 time per week, 2 to 6 times per week, every day). Adolescents were classified regarding their use of alcohol and tobacco as never users, if they had never drunk alcoholic drinks and if they have never smoked; experimenters, if they reported only experimented those substances and used them less than one time per month; and drinkers if they drunk one drink at least one time per month or smokers if they smoke either occasionally or regularly.

To define clusters we used 1612 participants (875 girls and 737 boys) with complete information for all variables that were considered in the cluster definition (tobacco and alcohol use, sport activities, fruit intake per week and daily sleep hours). Compared to the excluded participants, the 1612 enrolled had a higher proportion of females, adolescents with higher parental educational level, and those reporting more frequency of sports activity (p-value < 0.001). No statistically significant differences were found according to tobacco and alcohol use (table 1). After to perform the analysis of clusters' definition, we have to consider one participant as outlier because he did not match with the characteristics of any of the clusters defined.

(Please, insert table 1 here)

Statistical Analysis

Quantitative continuous variables were presented as median (1 st quartile-3 rd quartile) and they were compared using Kruskal-Wallis test. Nominal and ordinal variables were described using proportions and compared by Chi-squared test.

Hierarchical cluster methods were used to find the natural structure of data. We compared Ward's, complete and average methods, and since the average method showed the highest prediction it was considered the final solution. Considering that the variables used to define clusters were a mixture of nominal, ordinal and interval variables, the dissimilarity between individuals was measured with the Gower coefficient [START_REF] Kaufman | Finding groups in data: An introduction to cluster analysis[END_REF] . Information on tobacco and alcohol use, sport activities, fruit intake per week and daily sleep hours were used to predict the classification of each method and define the clusters. The Rousseeuw's Silhouette internal cluster quality index was used to estimate the number of clusters [START_REF] Kaufman | Finding groups in data: An introduction to cluster analysis[END_REF]. Classification trees, using Gini index [START_REF] Breiman | Clustering of health-related behaviors among 18-year-old Australians[END_REF], were used to determine which variables were the most important to define the clusters.

Generalized linear models with log link function were used to estimate the frequency of consumption of sweets and pastry, vegetables, soft drinks and soup by cluster, adjusted for total energy intake and parents' education by cluster. To perform this analysis log transformation was applied to the dependent variable.

The software "R 2.8.0 version" was used to analyse data (R Development Core Team 2008).

RESULTS

Regarding tobacco and alcohol use, sport activities, fruit intake and daily sleep hours, tobacco and alcohol were the most important in the cluster definition as observable in the classification tree (figure 1).

(Please, insert here figure 1)

We should note that of 86 adolescents in the drinker category, 20 reported alcohol use at least one time per week. And regarding the 44 smokers, 25 adolescents reported daily tobacco smoking.

Our cluster analysis allowed us to identify 3 clusters: cluster 1 (n=735) was characterized by a high proportion of never smokers (89.0%) and never drinkers (100.0%). Cluster 2 (n=832) presented the experimenters of tobacco (27.9%) and alcohol (90.5%). Cluster 3 (n=44) represents all smokers (100%) and some drinkers (15.9%).

Results showed that cluster 1 also presented those adolescents with other healthiest behaviours, such as a high median of daily sleep hours (median= 9.0 hours (1 st Q -3 rd Q=8.5-9.5)) and lower median of time spend at sedentary activities (median= 10.0 hours (1 st Q -3 rd Q=9.6-10.5)). These results are similar in the cluster 2. Moreover, cluster 3 represented those with a lower median (1 st Q -3 rd Q) of fruits consumption (5 fruits (2-10)) and lower number of daily sleeping hours (8.8 hours (8.3-9.5)) (table 2). However no significant differences were found according frequency of sports practice.

Regarding sedentary activities according to the three clusters there is a direct dose-effect relation (P-value for trend=0.045), and cluster 3 showed a high median (1 st Q -3 rd Q) of sedentary activities (11.2 hours (9.4-12.9)) (table 2). Also significant differences in gender, type of school and parents' education were found between clusters (table 2). Cluster 1 had a higher proportion of males and cluster 3 had a higher proportion of females. Cluster 2 had the highest proportion of adolescents from private schools and a higher proportion (table 2).

(Please, insert table 2 here)

Concerning data from the FFQ, after adjustment for total energy intake and parents' education, cluster 3 showed a high mean of daily intake of soft drinks. Although without statistically significance, this cluster also presented the highest intake of sweets and pastry (table 3).

(Please, insert table 3 here)

DISCUSSION

Few studies have focused in the way unhealthy behaviours cluster together on early adolescence.

Our results show tobacco and alcohol as the most powerful in clustering unhealthy behaviours even in these young adolescents, which is in line with other studies that described tobacco and alcohol use as the "gateway" to other risk behaviours common during adolescence [START_REF] Breiman | Clustering of health-related behaviors among 18-year-old Australians[END_REF][START_REF] Ma | Clustering of lifestyle behaviors: the relationship cigarette smoking, alcohol consumtpion, and dietary intake[END_REF].

Our study shows that not only tobacco and alcohol are related, but also adolescents who smoke and drink (cluster 3) were characterized by longer periods spent in sedentary activities, lower consumption of fruit and a higher consumption of soft drinks. This results are particularly important since it is known that parents have a crucial role in the dietary behaviour of their children [START_REF] Savage | Parental Influence on Eating Behavior: Conception to Adolescence[END_REF], however in early adolescence, peer pressure begins to usurp parental authority and unhealthy diets may be initiated [START_REF] Gidding | Dietary recommendations for children and adolescents: a guide for practitioners[END_REF], reinforcing the need for early interventions.

The majority of studies show that males are more prone to engage in unhealthy behaviours [START_REF] Breiman | Clustering of health-related behaviors among 18-year-old Australians[END_REF][START_REF] Ma | Clustering of lifestyle behaviors: the relationship cigarette smoking, alcohol consumtpion, and dietary intake[END_REF][START_REF] Wu | Gender differences in health risk behaviors and physical activity among middle school students[END_REF]). However, recent research showed no differences among genders in health behaviours [START_REF] Lawlor | Socioeconomic Position, Cognitive Function, and Clustering of Cardiovascular Risk Factors in Adolescence: Findings From the Mater University Study of Pregnancy and Its Outcomes[END_REF] or even that the situation begins to reverse regarding some unhealthy behaviours [START_REF] Miller | Binge Drinking and Associated Health Risk Behaviours Among High School Students[END_REF]). Thus it is interesting to note that in cluster 3, which represents the unhealthiest pattern, the proportion of females is higher. This information reinforces the need for a constant updating of knowledge about the changes in the population that is targeted for intervention.

It is also interesting to observe that the healthier cluster (Cluster 1) does not have the highest proportion of adolescents from an upper social class (highest parental education) which supports that, indeed an increased knowledge (of parents) does not mean, by itself, proneness to the adoption of healthy behaviours. Nevertheless, our results on food intake were adjusted to parents' education and differences according clusters remained. However in our study, the prevalence of tobacco and alcohol use was very low, but the cluster analysis allowed discrimination of these features as a different group. And this cluster showed differences also for the other behaviours analysed, which reinforces the evidence that in this age, even a lower use of tobacco and alcohol represents an important sign to potential behavioural risk. Food intake is difficult to measure, particularly in young ages. However, it has already been described that adolescents of 13 years old are able to answer alone to the FFQ [START_REF] Rockett | Validation of a Youth/Adolescent Food Frequency Questionnaire[END_REF][START_REF] Papadopoulou | Validation of a questionnaire assessing food frequency and nutritional intake in Greek adolescents[END_REF]). To improve the quality of answers, we decided to get this information from both adolescents and parents. This option may have influenced some responses. However we expected that if any effect was presented it tend to minimize differences between clusters because it is expected that those with healthier intake report their actual intake but those with unhealthier intake tend to underestimate unhealthy consumption and to overestimate healthier food.

Despite the significant results obtained, we recognize that one important limitation of our study lies in the exclusion of 548 individuals who had no complete information for our analysis. Differences were observed in gender, type of school and sport activities. Girls and participants with a higher socioeconomic level tend to better fill questionnaires which may explain these differences. Since students of private schools belong to a higher social class and reported more frequently to involved in extra-curricular sport activities, this could explain why we have a large proportion of physical active adolescents. But it is important to note that no statistically significant differences were found according to tobacco and alcohol use, which are the most powerful predictors. However the number of girls and adolescents whose parents have higher education is overestimated in our sample. Nevertheless, we do not expect a different clustering pattern of behaviours for those participants lost or excluded from analysis. Although reducing analytic power, interpretation of results would most likely be similar.

Despite these limitations, the study has the advantage of having a homogeneous group of adolescents regarding their age (all with 13-year-old). Other studies tend to aggregate adolescents with different ages in the same category. In this stage of the life cycle, marked by transition and constant change, some behaviour vary within short periods of time, particularly the types of behaviours under analysis. Thus, the age homogeneity of our sample could make this crosssectional picture more accurate. Another important advantage lies in the use of school as a sampling source, since school attendance is compulsory at this age in Portugal.

CONCLUSION

Our findings show that even in early stages of adolescence there is a cluster of smoking, alcohol use and food intake, providing further evidence of the clustering of unhealthy behaviours. Our results reinforce the need for prevention programs to begin earlier in life. Prevention programs may benefit in effectiveness if these characteristics are considered. * p<0.001 † Adjusted for total energy intake (the log transformation was applied to the dependent variable) ‡ Adjusted for total energy intake and parents' education.

Figure 1 -

 1 Figure 1 -Classification tree for clusters.

Table 1 -

 1 Comparison of the distribution of complete available information to clusters' definition by demographic and behavioural characteristics of adolescents (The EPITeen cohort of Porto, Portugal, 2003/2004).

	Considered in analysis
	n (%)
	No	Yes
	548	1612	p-value

Table 2 -

 2 Social and behavioural characteristics according to different clusters (The EPITeen cohort ofPorto, Portugal, 2003[START_REF] He | Defining and understanding healthy lifestyles choices for adolescents[END_REF]).

		Cluster 1	Cluster 2	Cluster 3	p-value
	BEHAVIOURAL CHARACTERISTICS, n (%)
	Tobacco use
	Never	654 (89.0)	600 (72.1)	0 (0.0)
	Experimenter	81 (11.0)	232 (27.9)	0 (0.0)
	Smoker	0 (0.0)	0 (0.0)	44 (100.0)	<0.001
	Alcohol use
	Never	735 (100)	0 (0.0)	11 (25.0)
	Experimenter	0 (0.0)	753 (90.5)	26 (59.1)
	Drinker	0 (0.0)	79 (9.5)	7 (15.9)	<0.001
	Sport Activities
	Never	160 (21.8)	150 (18.0)	9 (20.5)
	≤ 1 time/month	86 (11.7)	84 (10.1)	7 (15.9)
	1 time/month-1 time/week	86 (11.7)	94 (11.3)	5 (11.4)
	2-6 times/week	288 (39.2)	392 (47.1)	16 (36.4)
	Every day	115 (15.6)	112 (13.5)	7 (15.9)	0.140
			Median (1 st Q-3 rd Q)
	Fruits/week	7 (4-10)	7 (4-12)	5 (2-10)	0.078
	Daily sleep hours	9.0 (8.5-9.5)	9.0 (8.5-9.5)	8.8 (8.3-9.5)	0.002
	Sedentary activities (hours) *	10.0 (9.6-10.5)	10.6 (10.2-11.0)	11.2 (9.4-12.9)	0.045 †

SOCIAL AND DEMOGRAPHIC CHARACTERISTICS, n (%) Gender

  Total hours watching TV, playing computer and reading † p-trend=0.045 .

	≥13	151 (21.4)	252 (31.6)	13 (31.7)	<0.001
	Female	383 (52.1)	459 (55.2)	33 (75.0)
	Male	352 (47.9)	373 (44.8)	11 (25.0)	<0.001
	Type of school
	Public	594 (80.8)	570 (68.5)	34 (77.3)
	Private	141 (19.2)	262 (31.5)	10 (22.7)	<0.001
	Parents' Education (yrs)
	0-4	123 (17.3)	90 (11.4)	5 (12.3)
	5-6	103 (14.5)	71 (8.9)	4 (9.7)
	7-9	148 (20.9)	157 (19.7)	8 (19.5)
	9-12	184 (25.9)	226 (28.4)	11 (26.8)

*

Table 3 -

 3 Frequency of food intake measured by FFQ according to different clusters (The EPITeen cohort ofPorto, Portugal, 2003[START_REF] He | Defining and understanding healthy lifestyles choices for adolescents[END_REF]).

		Cluster 1	Cluster 2	Cluster 3
	Frequency of daily intake	Mean (95% CI)	Mean (95% CI)	Mean (95% CI)
			CRUDE
	Sweets and pastry	2.08 (1.94-2.22)	1.98 (1.84-2.12)	2.08 (1.53-2.63)
	Soft drinks	1.07 (0.97-1.17)	1.20 (1.10-1.30)	1.37 (0.96-1.77)
	Fruits	2.56 (2.41-2.70)	2.56 (2.42-2.70)	2.18 (1.59-2.76)
	Vegetables	2.11 (1.95-2.27)	2.32 (2.16-2.48)	2.05 (1.40-2.70)
	Soup	0.82 (0.77-0.87)	0.90 (0.85-0.95)*	0.85 (0.64-1.06)
			ADJUSTED †
	Sweets and pastry	1.96 (1.86-2.06)	1.87 (1.77-1.97)	2.08 (1.64-2.51)
	Soft drinks	0.95 (0.87-1.03)	1.16 (1.08-1.23)*	1.31 (0.96-1.66)*
	Fruits	2.52 (2.38-2.65)	2.52 (2.39-2.65)	2.23 (1.69-2.77)
	Vegetables	2.06 (1.91-2.21)	2.25 (2.11-2.39)	2.09 (1.48-2.70)
	Soup	0.81 (0.76-0.86)	0.90 (0.85-0.95)*	0.86 (0.66-1.06)
			ADJUSTED ‡
	Sweets and pastry	1.95 (1.84-2.06)	1.91 (1.86-2.01)	2.10 (1.66-2.53)
	Soft drinks	0.92 (0.84-1.01)	1.18 (1.09-1.26) *	1.29 (0.95-1.63) *
	Fruits	2.53 (2.40-2.67)	2.47 (2.33-2.59)	2.21 (1.69-2.74)
	Vegetables	2.06 (1.91-2.22)	2.18 (2.03-2.33)	2.06 (1.47-2.65)
	Soup	0.81 (0.75-0.85)	0.85 (0.80-0.90)	0.86 (0.62-0.99)

ACKNOWLEDGEMENTS

We gratefully acknowledge grants from Fundação para a Ciência e Tecnologia (POCTI/SAU-ESP/62399/2004). Sílvia Fraga (SFRH/BD/44408/2008) and Diogo Costa (SFRH/BD/66388/2009) have a PhD grant from Fundação para a Ciência e Tecnologia.

The authors declare that they have no conflict of interest.