Johann D Ringe
email: ringe@klinikum-lev.de

John G Doherty

Med Klinik

Absolute risk reduction in osteoporosis: Assessing treatment efficacy by number needed to treat

Postmenopausal osteoporosis is a chronic condition due to decreased bone mass, leading to reduced bone strength and increased fracture risk. Currently available pharmacological treatments include antiresorptive agents (bisphosphonates and raloxifene) and bone-forming agents (strontium ranelate and two different parathyroid peptides). Comparison via reduction in relative risk of fracture may produce artificially high reductions in fracture risk for some agents. Responder analysis based on absolute risk reduction (ARR, the arithmetic difference between events rates with and without treatment over a fixed time) and a related parameter, number needed to treat (NNT, the number of patients needed to treat over a fixed time to prevent one event) may provide more reliable parameters. We reviewed placebo-controlled, randomized, double-blind, pivotal phase 3 trials employed as part of the regulatory process, in order to calculate ARRs and NNTs for vertebral and hip fracture over 3 years for antiosteoporotic agents currently available in Europe. The NNT values to prevent one vertebral fracture over 3 years range from 9 for the strontium ranelate to 21 for ibandronate. NNT values for hip fracture over 3 years range from 48 for strontium ranelate to 91 for three of the bisphosphonates. Our analysis indicates that the bone-forming agent strontium ranelate may have the lowest NNT for the prevention of both vertebral and hip fracture. Responder analysis may enable translation of clinical trial results into guidance for routine clinical practice by indicating the amount of effort needed to prevent the same event in comparable populations with different treatment options.

Introduction

Osteoporosis is a chronic condition characterized by a reduction in bone mass, usually as a consequence of aging, leading to a reduction in bone strength and an increase in the risk of fracture. Because women are particularly susceptible to bone loss after the menopause, by far the most common form is postmenopausal osteoporosis. Current estimates place the lifetime risk of wrist, vertebral, or hip fracture at 45% in Caucasian women aged over 50 [START_REF]Report of the Surge on General's Workshop on Osteoporosis and Bone Health[END_REF], though risk estimates such as this are set to escalate in coming years as the population ages. Indeed, postmenopausal osteoporosis already has a phenomenal impact on health care budgets, which are currently expected to double for osteoporosis by the year 2050 [START_REF] Inster | Osteoporosis: a still increasing prevalence[END_REF]. This dramatic increase in cost takes into consideration that older patients are more likely to suffer hip fracture, which is the most disabling and costly osteoporotic fracture [START_REF] Oden | Lifetime risk of hip fractures is underestimated[END_REF].

It is important to take these issues into account when making health care decisions, particularly the selection of effective treatment for chronic disease. Postmenopausal osteoporosis is managed partly by lifestyle modification to prevent falls, and dietary supplementation with calcium and vitamin D. However, these strategies have been shown to be of limited effectiveness in reducing actual fracture risk [START_REF] Van Schoor | Prevention of hip fractures by external h ip protectors: a randomized controlled t rial[END_REF][START_REF] Grant | Oral v itamin D3 and calciu m for sec ondary prevention of low-trau ma fractures in elderly people (Randomised Evaluation of Calciu m Or vitamin D, RECORD): a randomised placebo-controlled trial[END_REF][START_REF] Jackson | Calciu m plus vitamin D supplementation and the risk of fractures[END_REF], and should be accompanied by a pharmacological treatment. Antiosteoporotic agents with known antifracture efficacy are currently divided into two categories: agents that reduce bone resorption (e.g., bisphosphonates and raloxifene); and agents that increase bone formation (e.g., teriparatide and strontium ranelate) [START_REF] Kanis | Eu ropean guidance for the diagnosis and management of osteoporosis in postmenopausal wo men[END_REF]. All of these agents have proven efficacy against vertebral fracture, and some of them against hip fracture. Despite this picture of a chronic condition, with a whole range of effective treatments to choose from, osteoporosis is currently at the center of debate surrounding the presentation of results of clinical trials guiding the selection of treatment strate gies.

Antiosteoporotic treatments are generally compared using reduction in relative risk (RRR) of fracture, which generally describe the percentage reduction in event rates versus placebo. In a recent article focusing on osteopenia in the British Medical Journal, Alonso-Coello et al [START_REF] Lonso-Coello | Drugs for pre-osteoporosis: prevention or disease mongering?[END_REF] argued that such comparisons lead to artificially high reductions in fracture risk for some of antiosteoporotic treatments, and that assessments based on absolute risk would be more realistic. In this review, we attempt to clarify the arguments behind absolute and relative risk for antiosteoporotic treatments currently available in Europe.

Absolute versus relative risk

The most common parameter used in reporting clinical trials in osteoporosis is the RRR, which is defined as the percentage difference in event rates, in this case fracture, between two groups (see Box 1) [START_REF] Barratt | Tips for learners of ev idence-based medicine: 1. Relative risk reduction, absolute risk reduction and number needed to treat[END_REF][START_REF] Replogle | Interpretation of absolute measures of disease risk in comparative research[END_REF]. In other words, RRR represents the probability of fracture in the treatment group in proportion to the probability of fracture in the placebo group. The problem with the RRR is that it fails to discriminate between large absolute treatment effects and very small effects in terms of absolute numbers. In contrast, absolute risk reduction (ARR) is defined as the arithmetic difference between events rates over a fixed period of time (see Box 1) [START_REF] Barratt | Tips for learners of ev idence-based medicine: 1. Relative risk reduction, absolute risk reduction and number needed to treat[END_REF][START_REF] Replogle | Interpretation of absolute measures of disease risk in comparative research[END_REF]. In the case of an antiosteoporotic agent, this might be the numerical difference between the rate of vertebral fractures over 3 years with treatment and with placebo.

In an example cited by Alonso-Coello et al [START_REF] Lonso-Coello | Drugs for pre-osteoporosis: prevention or disease mongering?[END_REF] an RRR of 75% with raloxifene over 3 years in osteoporotic or osteopenic women can mask an ARR of 0.9% [START_REF] Kanis | Effect of ralo xifene on the risk of ne w vertebral fracture in postmenopausal women with osteopenia or osteoporosis: a reanalysis of the Multiple Outcomes of Ralo xifene Evaluation trial[END_REF]. Clearly, this example is taken out of context, but it demonstrates the dangers of making treatment decisions on the basis of relative risk.

A related-and perhaps more accessible-parameter is the number needed to treat (NNT). This is the number of patients needed to treat over a fixed period of time to prevent one event occurring. NNT, which is a point estimate, is easy to calculate since it is the reciprocal of the ARR (NNT=1/ARR) [START_REF] Replogle | Interpretation of absolute measures of disease risk in comparative research[END_REF][START_REF] Cook | The number needed to treat: a clinically useful measure of treat ment effect[END_REF]]. In the above example, the ARR of 0.9% leads to an NNT of 111, implying that we would need to treat 111 osteoporotic or osteopenic women for 3 years with raloxifene to prevent one vertebral fracture from occurring.

The smaller the NNT, the more effective the treatment. How low can we reasonably expect NNT values to go for antiosteoporotic treatment? NNT values of 2 or 3 (i.e., approaching 1) indicates that a treatment is very effective, and have been found, for example, for antibiotic therapy against Helicobacter pylori infection [START_REF] Mcquay | Using numerical results fro m systematic reviews in clinical practice[END_REF]. On the other hand, higher values, up to 20 to 40 are considered to indicate clinica l effectiveness in the prevention of mortality in cardiac patients [START_REF] Mcquay | Using numerical results fro m systematic reviews in clinical practice[END_REF]. From this, we see that NNT is driven by absolute risk at baseline, i.e. in the case of osteoporosis the baseline absolute fracture risk [START_REF] Seeman | 7: Treat ment of osteoporosis: why, who m, when and how to treat. The single most important consideration is the individual's absolute risk of fracture[END_REF].

The advantage of the NNT over RRR is that it expresses both the risk without treatment and the risk reduction with treatment in a single figure. In addition, it informs physicians how much effort they must make to prevent one event with respect to no treatment at all, and allows for better comparison with other treatments. This may well be especially important in the absence of head-to-head trials. European regulatory authorities currently recognize NNT as a relevant and easy to use method of extracting benefit-risk data from individual clinical trials [START_REF]Reflect ion paper on benefit -risk assessment methods in the context of the evaluation of marketing authorisation applications of med icinal products for human use[END_REF]. Indeed, responder analysis (ie, ARR and NNT) may enable the translation of clinical trial results into guidance for routine clinical practice, by giving a better indication of treatment effect than RRR [START_REF] Lonso-Coello | Drugs for pre-osteoporosis: prevention or disease mongering?[END_REF][START_REF] So X Hc | Straight talk about disease prevention[END_REF]. Provided the primary outcome and the treatment duration are the same, NNTs and ARRs can be compared for different treatment options in osteoporosis to give a better measure of the impact of treatment than RRRs.

ARR and NTT for antiosteoporotic treatments

Even though the antiosteoporotic class is relatively small, it can be immensely complicated to navigate around the plethora of studies with differing treatment durations in populations at differing baseline risk of fracture. Comparison of antifracture efficacy is fraught with pitfalls, and comparison of NNTs is of no exception. One admirable attempt at this, in a position statement from the American Society of Health-System Pharmacists [START_REF] Maclaughlin | ASHP therapeutic position statement on the prevention and treatment of osteoporosis in adults[END_REF] cited no fewer than 28 different NNT values for just 7 pharmacological therapies in osteoporosis, and many more for dietary supplements and treatments in development.

In order to simplify this situation and increase the accessibility of reliable data, in this article we review the data for antiosteoporotic agents from placebo-controlled, randomized, double-blind, pivotal phase 3 trials for registration by the European regulatory agencies, carried out in postmenopausal osteoporotic patients with one prevalent fracture at baseline. We restricted our analysis to agents that are currently available in Europe for the treatment of postmenopausal osteoporosis. We used the reported data to calculate ARRs and NNT for each trial for vertebral and hip fracture over 3 years (Tables 1 and2, Figure 1). We chose these two outcomes since they are the most common sites for osteoporotic fracture. We selected hip fracture, as opposed to -nonvertebral‖ fracture, which was reported in some of the studies, since these outcomes were composites of fractures at different sites (hip, wrist, and/or ankle) with differing definitions according to the clinical trial.

Antiresorptive agents

NNT to prevent vertebral fracture

Pivotal vertebral fracture data for the bisphosphonate alendronate were reported by the Fracture Intervention Trial (FIT) research group in 1996 [START_REF] Black | Randomised trial of effect of alendronate on risk of fracture in wo men with existing vertebral fractures. Fracture Intervention Trial Research Group[END_REF]. This first report from the FIT investigators included postmenopausal women aged between 55 and 81 years with at least one prevalent vertebral fracture and a femoral neck T-score of <-2.1. Later reports from the FIT trial did not meet our selection criteria. The rate of vertebral fracture in FIT was 15% in the placebo group versus 8% in the alendronate group. There was a 7% ARR for alendronate 10 mg/day for vertebral fracture over 3 years versus placebo, which leads to an NNT of 15 (Table 1).

More recently, the bisphosphonate ibandronate was assessed in the Oral Ibandronate

Osteoporosis Vertebral Fracture Trial in North American and Europe (BONE) [START_REF] Chesnut | Effects of oral ibandronate administered daily or intermittently on fracture risk in postmenopausal osteoporosis[END_REF]. in 2946 osteoporotic postmenopausal women. The BONE participants were aged between 55 and 80 years and had at least one prevalent vertebral fracture, and with an average lumbar spine T-score of -2.8. Over 3 years, the rate of vertebral fracture was 4.7% with oral ibandronate 2.5 mg/day versus 9.6% with placebo (P=0.0001), which translates into an NNT of 21 (Table 1).

The data for the other once-daily bisphosphonate in Table 1, risedronate, come from Vertebral Efficacy with Risedronate Therapy-North America (VERT-NA) [START_REF] Harris | Effects of risedronate treatment on vertebral and nonvertebral fractures in wo men with postmenopausal osteoporosis: a randomized controlled trial. Vertebral Efficacy W ith Risedronate Therapy (VERT) Study Group[END_REF], which was carried out in a similar population of patients aged <85 years with at least one vertebral fracture, and with an average lumbar spine T-score of -2.4. The parallel multinational study (VERT-MN) did not meet our selection criteria. The 3-year event rate for vertebral fracture was 16.3% in the placebo group versus 11.3% in the risedronate group (P=0.003). The ARR for vertebral fracture over 3 years with risedronate 5 mg/day was 5.0% compared with placebo, which gives an NNT of 20.

The bisphosphonates are currently being prescribed in longer-acting formulations, leading to once-weekly, once-monthly, or even once-yearly regimens. Despite this, there are few pivotal trials with these long-acting formulations, since most authorizations are based on bridging trials, which generally use surrogate end points as evidence of efficacy, as opposed to reduction in fracture (bone mineral density or biochemical markers of bone turnover) [START_REF] Cosman | Clinical evaluation of novel bisp hosphonate dosing regimens in osteoporosis: the role of co mparative studies and imp licat ions for future studies[END_REF][START_REF] Adami | Bisphosphonate antifracture efficacy[END_REF]. There is therefore only one pivotal trial of a long-acting formulation of zoledronate in the Health Outcomes and Reduced Incidence with Zoledronic Acid Once Yearly (HORIZON) trial [START_REF] Black | Once-yearly zoledronic acid for treat ment of postmenopausal osteoporosis[END_REF]. Osteoporotic women were included in HORIZON if they were aged between 65 and 89 years and had a femoral neck T-score of <-2.5 with one or no prevalent vertebral fracture, or <-1.5 if they had more than one prevalent vertebral fracture. The HORIZON participants received a single 15-minute infusion of 5 mg zoledronic acid every 12 months. There was a substantial difference in event rates between zoledronate (3.3%) and placebo (10.9%) over 3 years (Table 1). Responder analysis gives an ARR of 7.6% and an NNT of 14, which is compatible with the other bisphosphonates.

The antifracture efficacy of the selective estrogen receptor modulator (SERM) raloxifene was explored in the Multiple Outcomes of Raloxifene Evaluation (MORE) in postmenopausal osteoporotic women aged <80 years [START_REF] Ettinger | Reduction of vertebral fracture risk in postmenopausal wo men with osteoporosis treated with ralo xifene. Results fro m a 3-year rando mized clin ical trial[END_REF]. In MORE participants with at least one prevalent vertebral fracture, treatment with raloxifene 60 mg/day reduced the rate of fracture over 3 years versus placebo (14.7% versus 21.2%) ( Table 1). These data correspond to an ARR of 6.5% and an NNT for vertebral fracture of 16 for raloxifene at 60 mg/day.

NNT to prevent hip fracture

There are three pivotal studies of antiresorptive treatments that give NNT values, all of which are bisphosphonates (Table 2). In the FIT study, the rate of hip fracture was reported for postmenopausal osteoporotic women aged between 55 and 81 years with at least one prevalent vertebral fracture; their femoral neck T-score was <-2.1 [START_REF] Black | Randomised trial of effect of alendronate on risk of fracture in wo men with existing vertebral fractures. Fracture Intervention Trial Research Group[END_REF].

The rate of hip fracture over 3 years was 1.0% with alendronate 10 mg/day versus 2.1% with placebo. The substantial 52% RRR should be considered alongside a 1.1% ARR and an NNT for hip fracture of 91.

The same ARR (1.1%) and NNT (91) values were found for hip fracture over 3 years for risedronate and zoledronate in the Hip Intervention Program (HIP) [START_REF] Mcclung | Effect of risedronate on the risk of hip fracture in elderly wo men. Hip Intervention Program Study Group[END_REF] and HORIZON [START_REF] Black | Once-yearly zoledronic acid for treat ment of postmenopausal osteoporosis[END_REF] studies, respectively (Table 2). In HIP, administration of risedronate 2.5 or 5 mg/day to postmenopausal osteoporotic women (aged 70 to 79 years; femoral neck T-score <-4) led to a 2.8% rate of hip fracture versus 3.9% in the placebo group.

The event rates in HORIZON were slightly lower, at 1.4% with zolendronate acid versus 2.5% with placebo. The HORIZON population comprised postmenopausal osteoporotic women aged between 65 and 89 years with a femoral neck T-score of <-2.5 with one or no prevalent vertebral fracture, or <-1.5 if they had more than one prevalent vertebral fracture [START_REF] Black | Once-yearly zoledronic acid for treat ment of postmenopausal osteoporosis[END_REF].

Bone-forming agents

NNT to prevent vertebral fracture

There are only two bone-forming agents for which there are pivotal trials yielding data for responder analysis, strontium ranelate and teriparatide (Table 1). The antifracture efficacy of strontium ranelate was examined in the Spinal Osteoporosis Therapeutic Intervention (SOTI) trial for vertebral fracture [START_REF] Meunier | The effects of strontium ranelate on the risk of vertebral fracture in wo men with postmenopausal osteoporosis[END_REF]. The patients in this trial were aged >50 years; they had diagnosed postmenopausal osteoporo sis with at least one prevalent vertebral fracture and in average with a femoral neck T-score of -2.8 and lumbar spine T-score of -3.5. The event rate for vertebral fracture over 3 years in women receiving 2 g/day strontium ranelate was 20.9% versus 32.8% in the placebo group. These data translate into an 11.9% ARR and an NNT of 9 [START_REF] Meunier | The effects of strontium ranelate on the risk of vertebral fracture in wo men with postmenopausal osteoporosis[END_REF].

The randomized, double-blind, pivotal phase 3 trial for registration of teriparatide lasted for a median duration of 21 months [START_REF] Neer | Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis[END_REF]. This study was performed in a population aged >50 years with at least one prevalent vertebral fracture and a femoral neck or lumbar spine T-score of <-1. The event rates for vertebral fracture were 5.0% for teriparatide 20 μg/day over 21 months and 14% with placebo, leading to an ARR of 9% and an NNT of 12 [START_REF] Neer | Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis[END_REF].

NNT to prevent hip fracture

There is one pivotal trial in the prevention of peripheral fracture with strontium ranelate (Table 2). The Treatment of Peripheral Osteoporosis (TROPOS) trial [START_REF] Reginster | Strontiu m ranelate reduces the risk of nonvertebral fractures in postmenopausal women with osteoporosis: Treat ment of Peripheral Osteoporosis (TROPOS) study[END_REF] included an analysis of postmenopausal osteoporotic women aged 74 years or older with a femoral neck T-score -2.4. In this population, the event rate for hip fracture in patients receiving strontium ranelate 2 g/day was 4.3% versus 6.4% in the placebo group, leading to an ARR of 2.1% [START_REF] Reginster | Strontiu m ranelate reduces the risk of nonvertebral fractures in postmenopausal women with osteoporosis: Treat ment of Peripheral Osteoporosis (TROPOS) study[END_REF]. The NNT with strontium ranelate 2 g/day to prevent one hip fracture is therefore 48.

To our knowledge, there is no pivotal study showing a reduction in rates of hip fracture with the other bone-forming agent teriparatide.

Discussion

The use of NNT to assess antifracture efficacy has been endorsed by international and national guidelines [START_REF]Reflect ion paper on benefit -risk assessment methods in the context of the evaluation of marketing authorisation applications of med icinal products for human use[END_REF][START_REF] Maclaughlin | ASHP therapeutic position statement on the prevention and treatment of osteoporosis in adults[END_REF][START_REF]Traitement de l'ostéoporose postménopausique[END_REF]. It translates the relatively complex results of clinical trials into a single accessible number, and allows comparison of the amount of effort needed to prevent the same event in comparable populations with other treatment options, which can aid in treatment decisions in daily clinical practice. It provides useful information about the efficacy of treatment, incorporating the effects of treatment and no treatment (placebo) into a single number. In limiting our metaanalytical approach to the selection of studies to pivotal phase 3 studies lasting 3 years on agents currently available in Europe for the treatment of postmenopausal osteoporosis, we hope to have simplified the comparison of antifracture efficacy within the class.

NNT values were also found over the longer-term, according to data available for 5 years' treatment in osteoporosis. To date, among all the antiosteoporotic agents, only strontium ranelate has a prespecified analysis of efficacy against vertebral and 01/ 12/ 2009 nonvertebral fracture over 5 years [START_REF] Reginster | Effects of long -term strontiu m ranelate treatment on the risk of non-vertebral and vertebral fractures in postmenopausal osteoporosis: results of a 5-year, rando mized, placebo-controlled trial[END_REF]. This long-term extension of the TROPOS trial for strontium ranelate 2 g/day reported an NNT of 21 for the prevention of one new osteoporotic fracture at any site (vertebral or nonvertebral) over 5 years in osteoporotic women with baseline lumbar and femoral neck T-scores <-2.4 [START_REF] Reginster | Effects of long -term strontiu m ranelate treatment on the risk of non-vertebral and vertebral fractures in postmenopausal osteoporosis: results of a 5-year, rando mized, placebo-controlled trial[END_REF]. This study demonstrated that strontium ranelate has antiosteoporotic efficacy in preventing vertebral and nonvertebral fractures (including hip) over 5-year follow-up. While these data are impressive and confirm the long-term anti-fracture efficacy of this agent, it is difficult to compare with other NNT values with other treatments for osteoporosis, due to the paucity of comparable data in terms of study design, duration, and population.

Another factor to take into consideration when considering anti-fracture efficacy in the clinical setting is adherence to treatment. Indeed, poor adherence to treatment can substantially increase the NNT to prevent an outcome. This issue was addressed in a recent report on the impact of low versus high adherence to bisphosphonate treatment on NNT in osteoporosis [START_REF] Curtis | The Benefit of Adherence with Bisphosphonates Depends on Age and Fracture Type: Results Fro m an Analysis of 101,038 New Bisphosphonate Users[END_REF]. The NNT values in this report (ranging from >100 to well into the thousands) are systematically higher than the values in our paper, because even 80% adherence can increase the NNT relative to a clinical trial setting. This underlines the importance of selection of agents with good acceptability to improve patient compliance with treatment.

Our analysis is not without its limitations. Confining the study to pivotal phase 3 trials used as part of the European regulatory processes may have eliminated those with better or worse NNT values from our analysis, as well as agents in development that

have not yet received approval for clinical use. Another limitation is possible differences in the baseline characteristics of the populations concerned. However, our aim was to clarify the comparison of absolute risk reduction in the management of osteoporosis in real clinical practice and simplify the selection of trials. The similarity of the values we found within the bisphosphonate class may support our analysis.

Our simplified approach shows that all of the currently available antiosteoporotic agents have acceptable NNT values for efficacy in preventing vertebral fracture, ranging from NNT=9 for strontium ranelate 2 g/day over 3 years to NNT=21 for ibandronate 2.5 mg/day. The NNT values for the prevention of hip fracture are higher, as would be expected by the increased risk of these patients, and range from NNT=48 for strontium ranelate 2 g/day to NNT=91 for bisphosphonates. On the basis of our analysis, it appears that bone-forming agents such as strontium ranelate have the lowest NNT for the prevention of both vertebral and hip fracture. With this analysis, we have demonstrated that pharmacological treatment of osteoporosis is worthwhile in the prevention of fracture, and that the antiosteoporotic class is not entirely homogeneous in terms of NNT. Box 1. Risk, absolute risk, and number needed to treat. Adapted and modified from Barratt et al [START_REF] Barratt | Tips for learners of ev idence-based medicine: 1. Relative risk reduction, absolute risk reduction and number needed to treat[END_REF].

Event rate: The number of patients experiencing an event (e.g., vertebral fracture) over a fixed period of time, as a proportion of the number of patients in the whole population.

Relative risk reduction (RRR):

The percentage difference in the event rates between two groups (e.g., percentage reduction in vertebral fracture rate in patients receiving treatment versus those receiving placebo), usually expressed as a proportion of the event rate in the untreated (placebo) group.

Absolute risk reduction (ARR):

The arithmetic difference between events rates in two groups (e.g., difference in vertebral fracture rates in patients receiving treatment and in those receiving placebo).

Number needed to treat (NNT):

The number of patients needed to treat over a fixed period of time to prevent one event (e.g., vertebral fracture) occurring, calculated as the reciprocal of the ARR (NNT=1/ARR).

01/ 12/ 2009

Figure 1 .

 1 Figure 1. Absolute risk reduction (ARR) with various antiosteoporotic therapies versus placebo for vertebral fracture (top) and hip fracture (bottom) over 3 years in postmenopausal osteoporotic women. *Assessment over 18 to 21 months. Hip fracture data fo r ibandronate, ralo xifene, and teriparatide not available.

Table 1

 1 Absolute risk reduction (ARR) and number needed to treat (NNT) for vertebral fracture over 3 years with various antiosteoporotic treatments calculated from the results of randomized, double-blind, pivotal phase 3 trials versus placebo.

		S tudy	Number of patients (placebo/active treatment)	Mean BMD (T-score or absolute value) Active Placebo treatment	ARR	NNT	P †
	Antiresorptive agents
	Alendronate	FIT [18]	965/981	FN BM D 0.56 g/cm 2	FN BM D 0.57 g/cm 2	7.0%	15	<0.001
				LS T-score	LS T-score
	Ibandronate	BONE [19]	975/977	-2.8 FN T-score	-2.8 FN T-score	4.9%	21	<0.0001
				-2.0	-2.0
				LS BM D	LS BM D
	Risedronate	VERT-NA [20]	820/821	0.83 g/cm 2 FN BM D	0.83 g/cm 2 FN BM D	5.0%	20	0.003
				0.60 g/cm 2	0.59 g/cm 2
				LS BM D	LS BM D
	Zoledronate	HORIZON [23]	3861/3875	0.79 g/cm 2 FN BM D 0.53 g/cm 2	0.79 g/cm 2 0.53 g/cm 2 FN BM D	7.6%	14	<0.001
				LS BM D	LS BM D
	Raloxifene	MORE [24]	770/769	0.75 g/cm 2 FN BM D	0.75 g/cm 2 FN BM D	6.5%	16	<0.001
				0.57 g/cm 2	0.57 g/cm 2
	Bone-forming agents
				LS BM D	LS BM D
	Strontium ranelate	SOTI [26]	719/723	0.72 g/cm 2 FN BM D	0.73 g/cm 2 FN BM D	11.9%	9	<0.001
				0.59 g/cm 2	0.59 g/cm 2
				LS BM D	LS BM D
	Teriparatide*	Neer et al [27]	448/444	0.82 g/cm 2 FN BM D	0.82 g/cm 2 FN BM D	9.0%*	12*	<0.001
				0.64 g/cm 2	0.64 g/cm 2
	* Assessment over 21 months. †Significance of d ifference in event rates (treatment versus plac ebo).
	BMD, bone mineral density; FN, femo ral neck; LS, lu mbar spine.

FIT, Fracture Intervention Trial; BONE, Oral Ibandronate Osteoporosis Vertebral Fracture Trial in North America and Europe; VERT-NA, Vertebral Efficacy with Risedronate Therapy-North America; HORIZON, Health Outco mes and Reduced Incidence with Zoledronic Acid Once Yearly; MORE, Multiple Outcomes of Ralo xifene Evaluation; SOTI, Spinal Osteoporosis Therapeutic intervention.

Table 2

 2 Absolute risk reduction (ARR) and number needed to treat (NNT) for hip fracture over 3 years with various antiosteoporotic treatments calculated from the results of randomized, double-blind, pivotal phase 3 trials versus placebo. Significance of difference in event rates (treatment versus placebo). Data for ibandronate, ralo xifene, and teriparatide not available. BMD, bone mineral density; FN, femoral neck; LS, lu mbar spine. HIP, Hip Intervention Program; HORIZON, Health Outcomes and Reduced Incidence with Zoledronic Acid Once Yearly; TROPOS, Treat ment of Peripheral Osteoporosis; FIT, Fracture Intervention Trial.

				Mean BMD
			Number of	(T-score or absolute value)
		S tudy	patients (placebo/active			ARR NNT	P*
			treatment)	Placebo	Active treatment
	Antiresorptive agents
	Alendronate	FIT [18]	1005/1022	LS BM D 0.79 g/cm 2 FN BM D	LS BM D 0.79 g/cm 2 FN BM D	1.1%	91	0.047
				0.56 g/cm 2	0.57 g/cm 2
	Risedronate	HIP [25]	3134/6197	FN T-score -3.7	FN T-score -3.7	1.1%	91	0.02
				LS BM D	LS BM D
	Zoledronate	HORIZON [23]	2853/2822	0.79 g/cm 2 FN BM D 0.53 g/cm 2	0.79 g/cm 2 0.53 g/cm 2 FN BM D	1.1%	91	0.002
	Bone-forming agent
				LS T-score	LS T-score
	Strontium ranelate	TROPOS [28]	995/982	-3.2 FN T-score	-3.2 FN T-score	2.1%	48	0.046
				-3.6	-3.6

*

has received congress grants and honoraria from Sanofi Aventis; MSD, Novartis, P&G, Roche and Servier; J. G. Doherty has received educational, travel, and congress grants from MSD, Alliance for Better Bone Health, Lilly, and Servier


Jakub Swadzba

Teresa Iwaniec

Increased level of tumour necrosis factor-in patients with antiphospholipid syndrome marker not only of inflammation but also of the prothrombotic state

Keywords: TNF-, sIL-2R, antiphospholipid syndrome, inflammation, thrombosis

Connections between inflammation and thrombosis are intriguing, especially in a condition such as an antiphospholipid syndrome (APS), a disease characterized by immune mediated thrombosis. Tumour necrosis factor alpha (TNF-α) is a cytokine which shares proinflammatory and prothrombotic actions, while a soluble form of interlukin-2 receptor (sIL-2R) is considered a typical marker of (auto)immune inflammation with not known direct links to thrombosis. The differences in the pathogenesis of APS as compared to other autoimmune diseases might be connected with different serum levels of both mediators.

To answer this question, we studied 147 patients with systemic lupus erythematosus (SLE), 21 with SLE-like syndrome (SLE-LS), 20 with isolated APS (primary antiphospholipid syndrome -PAPS), and 32 healthy controls. Thirty six patients from the SLE group fulfilled the updated APS criteria (secondary APS -SAPS).

In comparison to healthy subjects, TNF-α concentration was increased in all patients, while sIL-2R rose significantly in the SLE group only. APS (both, SAPS and PAPS) was characterized by the highest levels of TNF-α. Moreover, patients with lupus anticoagulant or elevated levels of IgG anticardiolipin or IgG anti-β 2 -glycoprotein I antibodies had higher TNF-α levels than patients without the presence of any type of antiphospholipid antibodies (aPL).

In conclusion, the presence of aPL is associated with higher TNF-α level, whereas increased level of sIL-2R is rather connected with definite SLE where inflammatory processes prevail. It might be hypothesized that TNF-α plays a major role in pathogenesis of APS thrombotic phenomena.

Introduction

other systemic autoimmune diseases by its propensity to develop thrombosis. Systemic lupus erythematosus (SLE) is the prototypic example of an immune mediated, generalized inflammatory disease. Both diseases often occur together leading to the interplay between inflammatory and prothrombotic phenomena [START_REF] Hoffman | Links between the immune and coagulation systems: How do "antiphospholipid antibodies" cause thrombosis?[END_REF].

In general, systemic inflammation is a potent prothrombotic stimulus. Inflammatory mechanisms upregulate procoagulant factors, downregulate natural anticoagulants and inhibit fibrinolytic activity [START_REF] Esmon | Inflammation and thrombosis[END_REF]. Endotoxin, tumour necrosis factor alpha (TNF-α) and interleukin-1α induce tissue factor (TF) expression, primarily on endothelial cells and monocytes/macrophages, promoting blood coagulation [START_REF] Bevilaqua | Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: Characterization and comparison with the actions of interleukin 1[END_REF]. Activation of the complement C5b-C9 complex changes the cell surface to a more procoagulant phenotype by the shift of negatively charged phospholipids from the inner to the outer membrane [START_REF] Sims | Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity[END_REF]. Inflammatory reaction is also accompanied by the increase in fibrinogen and C-reactive protein (CRP) blood levels. CRP itself increases TF and decreases TF pathway inhibitor (TFPI) concentrations, what may be important in pathogenesis of arterial thrombosis and myocardial infarction [START_REF] Wu | C-reactive protein enchances tissue factor expression by vascular smooth muscle cells[END_REF].

Of the natural anticoagulants, protein C pathway appears to be the most strongly influenced by inflammation with thrombomodulin (TM) and the endothelial cell protein C receptor (EPCR) being both downregulated by TNF-α [START_REF] Conway | Tumor necrosis factor suppresses transcription of the trombomodulin gene in endothelial cells[END_REF].

On the other hand, thrombotic processes enhance inflammatory reactions, mainly through the action of TF and thrombin [START_REF] Chu | Tissue factor mediates inflammation[END_REF]. Activation of platelets leads to the release of CD40 ligand, which in turn induces TF expression and increases IL-6 levels [START_REF] Miller | CD40L-CD40 interaction regulate endothelial cell surface tissue factor and thrombomodulin expression[END_REF]. Thrombin also augments leukocyte adhesion and activation, stimulates endothelial cells to produce platelet activating factor and increases an expression of P-selectin [START_REF] Pierangeli | Thrombogenic effects of antiphospholipid antibodies are mediated by intracellular adhesion molecule-1, vascular cell adhesion molecule-1 and P-selectin[END_REF].

A common inhibitory pathway for thrombosis and inflammation also exists. Activated protein C (APC) acting directly as an anticoagulant, functions also as an anti-inflammatory and cytoprotective agent through specific receptors: endothelial cell protein C receptor (EPCR) and protease activated receptor-1 (PAR-1) [START_REF] Crawley | Cytoprotective effect of activated protein C: specificity of PAR-1 signaling[END_REF].

It is possible that TNF-α is a proinflammatory cytokine with the strongest prothrombotic action. TNF-α stimulates monocyte and neutrophil adhesion to endothelium, inhibits protein C system, impairs fibrinolysis and increases TF expression on the cell surface [START_REF] Esmon | Inflammation and thrombosis[END_REF]. Produced mainly by activated monocytes, macrophages, and T lymphocytes, this cytokine has been found to be elevated in patients suffering from both, SLE [START_REF] Studnicka-Benke | Tumour necrosis factor alpha and its soluble receptors parallel clinical disease and autoimmune activity in systemic lupus erythematosus[END_REF] and APS [START_REF] Forastiero | Circulating levels of tissue factor and proinflamatory cytokines in patients with primary antiphospholipid syndrome or leprosy related antiphospholipid antibodies[END_REF]. We hypothesized that TNF-α might be more elevated in APS patients, with thrombosis as its prominent feature, than in those with other autoimmune diseases where immune mediated inflammation prevails.

In contrast to TNF-α, a possible role of soluble interleukin-2 receptor (sIL-2R) in thrombosis and prothrombotic states is unknown. Soluble IL-2R is a key marker of lymphocyte T activation, crucial for the regulation of an autoimmune inflammatory response [START_REF] Rubin | The soluble interleukin-2 receptor: biology function and clinical application[END_REF]. Binding of interleukin-2 (IL-2) to its receptor on the surface of T-lymphocyte triggers a series of intracellular signaling events that result in the activation and proliferation of resting T cells. Upon activation of T cells, IL-2 receptor molecules are expressed on the surface and a soluble form is released. It has been found that autoimmune processes are associated with elevated levels of sIL-2R [START_REF] Wolf | Soluble interleukin-2 receptors in systemic lupus erythematosus[END_REF][START_REF] Neish | Elevated serum soluble interleukin-2 receptor levels in subacute cutaneus lupus erythematosus[END_REF]. We speculated that sIL-2R could be less elevated in an isolated form of APS, not associated with other autoimmune diseases, where inflammatory process is less (if at all) pronounced. If both, TNF-α and sIL-2R seem to be markers of autoimmune diseases, we asked whether their serum levels might correlate with the thrombotic state on one hand, and the intensity of autoimmune inflammation from the other. To answer this question, we have measured serum levels of both mediators in subjects with autoimmune diseases with or without presence of antiphospholipid antibodies (aPL) and examined carefully the differences which can be specific for patients fulfilling updated criteria for APS [START_REF] Miyakis | International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)[END_REF].

Patients :

We studied 188 patients (165 women and 23 men, aged 18-72; mean age 40,5 years) referred to the Outpatient Clinic for Autoimmune Diseases in Jagiellonian University Medical College. SLE was diagnosed in 147 patients based on the presence of at least 4 American College of Rheumatology (ACR) criteria [START_REF] Hochberg | Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus[END_REF]. The group of SLE-like syndrome (SLE-LS), defined by the presence of 2 or 3 of these criteria including antinuclear antibodies, consisted of 21 cases. Thirty six patients in the SLE group fulfilled recently revised APS classification criteria ( 16) (SAPS subgroup). There was no APS patient in the SLE-LS group. In 20 patients APS was not accompanied by any features of another autoimmune disease (PAPS). The groups were not statistically different in terms of sex and age. The control group consisted of 32 healthy subjects, matched by sex and age with the group studied.

A detailed history (a uniform set of questions) was taken from all the patients by a trained physician. All available medical records were also carefully analysed. Objective data confirming typical clinical involvement and laboratory test abnormalities of APS and SLE were required. All thrombotic episodes had to be confirmed by the imaging techniques (USG, CT and/or NMR). Pregnancy complications were defined according to recent updated APS classification criteria [START_REF] Miyakis | International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)[END_REF] and were confirmed by an experienced obstetrician based on available medical records.

From the total group of patients, 66 subjects suffered from thrombosis (45-venous, 17arterial, 4-both) and 42 women experienced pregnancy complications. Among 20 patients with PAPS, there were 15 cases of thrombosis (9 -venous, 5 -arterial, 1 -both) and 9 cases of fetal loss (4 women had thrombosis and fetal loss). In the SAPS group 31 patients suffered from thrombosis (22-venous, 7-arterial, 2-both) and 13 women suffered from pregnancy morbidity. Remaining cases of thrombosis (n=20) and pregnancy complications (n=20) appeared in patients with SLE or SLE-like syndrome without laboratory signs of APS.

The study was approved by the local ethical committee. All of the patients provided informed consent.

Methods:

Lupus anticoagulant (LA) was detected in accordance with the three-step procedure recommended by the International Society on Thrombosis and Haemostasis (ISTH) [START_REF] Brandt | Criteria for the diagnosis of lupus anticoagulants: an update[END_REF]. We Serum levels of anticardiolipin (aCL) and anti-β 2 -glycoprotein I (aβ 2 GPI) antibodies (both of IgG and IgM classes) were measured using an in-house ELISA [START_REF] Swadźba | Anticardiolipin antibodies, anti-β2-glycoprotein I, antiprothrombin antibodies and lupus anticoagulant in patients with systemic lupus erythematosus with a history of thrombosis[END_REF]. Following the recent suggestions [START_REF] Ichikawa | A chimeric antibody with the human gamma1 constant region as putative standard for assays to detect IgG beta2-glycoprotein I-dependent anticardiolipin and anti-beta2-glycoprotein I antibodies[END_REF][START_REF] Wong | Consensus guidelines for anticardiolipin antibody testing[END_REF], monoclonal antibodies HCAL and EY2C9 (Sapporo Standards) against β 2 GPI, were used as calibrators for the construction of standard curves for IgG and IgM, respectively [START_REF] Swadźba | Valine/Leucine 247 polymorphism of β2GPI in patients with antiphospholipid syndrome: lack of association with anti -β2GPI antibodies[END_REF]. All the aPL measured values exceeding 99 percentile of a healthy control group were considered positive, if confirmed by a second determination at least 12 weeks apart.

Serum concentrations of TNF-and sIL-2R were determined by an immunoenzymatic method using commercially available kits on Immulite 1000 chemiluminescence automatic analyzer (Siemens, Germany). According to manufacturer, reference ranges for healthy population are lower than 8.1 pg/mL for TNF-and below 710 U/ml for sIL-2R.

assessed by means of Spearman's rank correlation coefficient.

Results

Among our patients LA was found in 38 subjects, aCL IgG in 79, aCL IgM in 57, aβ 2 GPI IgG in 36, and aβ 2 GPI IgM in 40 subjects. Altogether, at least one aPL at a level fulfilling the revised APS classification criteria (at least two measurements exceeding 99 percentile, at least 12 week apart) was found in 93 patients.

Serum concentration of TNF-was significantly elevated in all groups of patients (SLE, SLE-LS and PAPS) as compared to controls. The highest levels of TNF-were found in patients with PAPS, followed by these from the SAPS subgroup. In the SLE group, patients fulfilling APS classification criteria had significantly higher TNF-levels than the remaining patients suffering from SLE (Tab.1). Levels of sIL-2R were also higher in patients than in controls but the difference reached statistical significance for SLE group only. There were no differences in sIL-2R levels between studied groups of patients. Some trend toward higher levels of sIL-2R in SLE as compared to SLE-LS (p=0,06) and PAPS (p=0,07) patients could be seen. Interestingly, there was also a trend to the lower levels of sIL-2R in the PAPS group as compared to those with SAPS (p=0,06) (Tab. 1).

When aPL-positive patients (regardless of the concomitant presence of clinical APS symptoms) were compared to aPL-negative ones, higher TNF-values (but not sIL-2R) were seen in the former. Inside the SLE group these differences were evident for both markers (Tab.2).

Further analysis showed that the levels of TNF-α depended also on the type of aPL. TNF-α was higher in patients showing the presence of LA or ACL IgG or aβ 2 -GPI IgG antibodies as compared to those without any antibodies (Fig. 1). A weak, but significant correlation between aCL IgG and TNF-α levels (r=0.31; p=0.0001; by Spearman rank test) was found.

The presence of the APS clinical criteria (thrombosis and pregnancy complications)

were associated with a tendency toward the elevation of TNF-α. However, this difference did not reach statistical significance (data not shown).

The levels of cytokines were also analyzed in relation to historical presence of symptoms and signs of SLE (Tab. 3). Elevated levels of sIL-2R were associated with many SLE clinical symptoms, whereas TNF-α was significantly elevated in SLE patients with only renal involvement. As far as immunologic criteria of SLE is considered, patients with the presence of ds-DNA antibodies as well as those with decreased C4 concentrations showed higher values of both cytokines.

Discussion

In our study we have shown that: a) elevated levels of TNF-are present not only in SLE but also in iAPS patients; b) whilst the highest levels of TNF-were detected in patients with APS (PAPS and SAPS), sIL-2R levels were more elevated in SLE patients, especially within those showing symptoms and signs related to the generalized inflammatory reaction.

In SLE group of patients, elevation of both markers and their association with disease activity has already been shown [START_REF] Studnicka-Benke | Tumour necrosis factor alpha and its soluble receptors parallel clinical disease and autoimmune activity in systemic lupus erythematosus[END_REF][START_REF] Forastiero | Circulating levels of tissue factor and proinflamatory cytokines in patients with primary antiphospholipid syndrome or leprosy related antiphospholipid antibodies[END_REF][START_REF] Wolf | Soluble interleukin-2 receptors in systemic lupus erythematosus[END_REF][START_REF] Neish | Elevated serum soluble interleukin-2 receptor levels in subacute cutaneus lupus erythematosus[END_REF][START_REF] Davas | Serum IL-6, TNF , p55 srTNF , p75 srTNF , srIL-2 levels and disease activity in systemic lupus erythematosus[END_REF]. We support these findings by showing that patients with an active disease (decreased C4 levels, presence of ds-DNA autoantibodies and renal involvement) had significantly higher levels of TNF-and sIL-2R than the others.

Recently, it was published that in PAPS patient higher level of TNF-is connected with lower level of C3 and C4 and lupus anticoagulant activity [START_REF] Oku | Complement activation in patients with primary antiphospholipid syndrome[END_REF]. Patients showing other clinical symptoms (arthritis, serositis, skin involvement) show only elevated sIL-2R levels (Tab.3). It may be hypothesized that this is due to the fact that sIL-2R is a more sensitive marker of inflammation than TNF-. Levels of sIL-2R in SLE -LS and PAPS patients (negligible widespread inflammation) were not different from the controls (Tab.1). Possibly in these groups of patients lymphocyte T activation and inflammatory reactions are much less pronounced than in fully blown SLE. In this respect the PAPS group seems to be interestingly showing highest levels of TNF-α and a relatively low levels of sIL-2R. Our data may suggest that sIL-2R is a good marker of autoimmune activity but it does not reflect prothrombotic state, typical for APS.

In contrast, serum TNF-α was especially elevated in patients diagnosed with definite APS (Tab.1) and those with antiphospholipid antibodies (Tab. 2). Our results corroborate with those of Bertolaccini et al. [START_REF] Bertolaccini | Plasma tumor necrosis factor levels and the -238*A promoter polymorphism in patients with antiphospholipid syndrome[END_REF], who showed elevated TNF-levels in patients with APS. Also, in accordance with our findings, they were unable to show any differences between its primary and secondary form (Tab.1). It may indicate that elevated TNF-α levels are related rather to pathogenesis of thrombosis, common for both forms of APS, than to inflammationvery limited in PAPS. However, such a causal relation between TNF-α and thrombosis is not uniformly accepted. In the experimental setting it has been shown that TNF-α might exert anti-platelet and antithrombotic activity [START_REF] Cambien | Antithrombotic activity of TNF-α[END_REF].

TNF-α was found to be higher in patients positive for LA or aPL of the IgG class than in those with IgM antibodies. These LA or IgG-positive patients showed significantly higher levels of TNF-α as compared to aPL-negative group (Fig. 1). These findings indirectly strengthen the notion about the important pathogenic role of LA and IgG aPL (as opposed to IgM) in the development of APS-related thrombosis [START_REF] Swadzba | Revised classification criteria for antiphospholipid syndrome and the thrombotic risk in patients with autoimmune diseases[END_REF]. If eventually proven, it could also serve as yet another argument in the discussion about the negligible clinical significance of IgM aPL in APS [START_REF] Galli | Invitation to a debate on the serological criteria that define the antiphospholipid syndrome[END_REF][START_REF] Swadzba | More on: the debate on antiphospholipid syndrome classification criteria[END_REF].

It has been suggested that TNF-α plays an important role in inducing APS-related pregnancy loss [START_REF] Berman | TNF-alpha is a critical effector and a target for therapy in antiphospholipid antibody-induced pregnancy loss[END_REF], and thrombosis [START_REF] Forastiero | Circulating levels of tissue factor and proinflamatory cytokines in patients with primary antiphospholipid syndrome or leprosy related antiphospholipid antibodies[END_REF][START_REF] Tanne | Interaction of inflammation, thrombosis, aspirin and enoxaparin in CNS experimental antiphospholipid syndrome[END_REF]. However, in our study we were unable to find any significant differences in TNF-α levels between patients with and without APS clinical symptoms. This is not surprising as there were usually wide temporal gaps between TNF-α determinations and clinical complications which took place in the distant past.

High level of TNF-α was rather connected with the present level of aPL than with the historical events of thrombosis. In future, hopefully, prospective studies can answer the question which groups of patients have the highest risk of getting thrombosis.

Several studies indicate that aPL [START_REF] Musiał | Thrombin generation measured ex vivo following microvascular injury is increased in SLE patients with antiphospholipid-protein antibodies[END_REF][START_REF] Pierangeli | Probing antiphospholipid mediated-thrombosis: the interplay between anticardiolipin antibodies and endothelial cells[END_REF] and TNF-α (34) can activate endothelium and induce prothrombotic phenotype of endothelial cells, leading to increased thrombin generation. Endothelial cell activation causes in vivo up-regulation of the tissue factor (TF) which has been proposed as a main potential mechanism of APS-related thrombosis [START_REF] Amengual | The role of the tissue factor pathway in the hypercoagulable state in patients with the antiphospholipid syndrome[END_REF][START_REF] Ch | Anthiphospholipid syndrome and tissue factor: a thrombotic couple[END_REF].

Once endothelial cells become activated, up-regulation of TF may be further augmented by a synergistic effect of TNF-α and factor Xa [START_REF] Hezi-Yamit | Synergistic induction of tissue factor by coagulation factor Xa and TNF: Evidence for involvement of negative regulatory signaling cascades[END_REF]. Other changes typical for this prothrombotic phenotype include increased expression of adhesion molecules (ICAM-1, VCAM-1, selectins E and P) [START_REF] Kaplanski | Increased soluble vascular cell adhesion molecule-1 concentrations in patients with primary or systemic lupus erythematosus-related antiphospholipid syndrome: correlations with the severity of thrombosis[END_REF][START_REF] Espinola | E-selectin mediates pathogenic effects of antiphospholipid antibodies[END_REF], and formation of endothelial microparticles [START_REF] Combes | In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant[END_REF]. Raschi et al. have shown that anti-beta 2 GPI antibodies induce translocation of NFκB in a manner similar to that elicited by LPS and TNF-α. Anti-beta 2 GPI antibodies bind to beta 2 GPI autoantigen, which acts as a toll-like receptor, and induce activation of endothelium via the MyD88 pathway [START_REF] Raschi | Role of the MyD88 transduction signaling pathway in endothelium activation by antiphospholipid antibodies[END_REF]. The other proposed mechanism for aPL-induced TF expression is the phosphorylation of p38 mitogen -activated protein kinase (MAPK) in endothelial cells and/or platelets [START_REF] Vega Ostertag | Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies[END_REF][START_REF] Bohgaki | The p38 mitogen-activated protein kinase (MAPK) pathway mediates induction of the tissue factor gene in monocytes stimulated with human monoclonal anti β2GPI antibodies[END_REF].

It is not clear whether aPL acts on endothelial cells directly or (as is the case with LPS) through TNF-α. The latter possibility finds support in the article of Dunoyer-Geindre et al. [START_REF] Dunoyer-Geindre | NFκB is an essential intermediate in the activation of endothelial cells by antibeta 2 -glycoprotein 1 antibodies[END_REF]. Independently from the mechanism, prothrombotic state, typical for APS, seems to be associated with both markedly elevated levels of aPL and increased concentrations of TNF-α.

  used partial thromboplastin time (PTT LA, Diagnostica Stago, France) and diluted Russell's viper venom time (DVV test, American Diagnostica, USA) for screening, and Staclot LA (Diagnostica Stago, France) and DVV Confirm (American Diagnostica, USA) for confirmation. Tests were run on Behring Coagulation Timer (BCT) and Fibrintimer analyzers (Siemens, Germany).

Figure 1 .

 1 Figure 1. Levels of TNF-α (A) and sIL2-R (B) depending on the presence and the type of aPL.

Table 1 .

 1 TNF-α and sIL2-R levels in the groups studied.

	Groups studied	Number of patients (n)	TNF-α [pg/mL]	sIL2-R [U/ml]
	SLE :	147	13,3 ± 7,2 a c	789 ± 591 a
	SAPS	36	15,1 ± 7,3 a c d	779 ± 396 a
	SLE-APS-	111	12,7 ± 7,1 a	791 ± 635 a
	SLE-LS	21	9,8 ± 4,2 a b	559 ± 224
	PAPS	20	16,6 ± 9,8 a	569 ± 190
	Controls	32	7,7 ± 2,3	427 ± 143
	Results are expressed as mean ± standard deviation (SD)

a -p<0,05 vs. controls b -p<0,05 vs. PAPS c -p<0,05 vs. SLE-LS d -p<0,05 vs. SLE-APS-

Table 2 .

 2 TNF-α and sIL2-R levels in patients with and without aPL.

	Group of patients	Number of patients (n)	TNFα [pg/mL]	sIL2-R [U/ml]
	All Patients	188
	aPL+	93	14,8 ±11,6 a	789 ± 660
	aPL-	95	11,7 ±5,8	692 ± 392
	SLE group	147
	aPL+	63	14,9 ±8,3 a	907 ± 778 a
	aPL-	84	12,0 ±6,0	709 ± 409
	Results are expressed as mean ± SD
	a -p<0,05 aPL+ vs. aPL-

Table 3 .

 3 TNF-α and sIL2-R levels in patients with and without SLE symptoms and signs.

	SLE symptoms and signs			TNF-α		IL-2R
			n		P		P
	Skin SLE criteria	+ -	107 81	13,66 ± 7,27 12,74 ± 11,4	NS	846,01 ± 657 598,08 ± 262	0,0008
	Arthritis	+ -	150 38	13,04 ± 7,28 14,15 ± 13,12	NS	783,5 ± 590 583,94 ± 238	0,01
	Serositis	+ -	29 159	13,94 ± 8,17 13,07 ± 9,56	NS	877,2 ± 506 697,25 ± 540	0,01
	Renal disorder	+ -	69 119	14,62 ± 8,05 12,48 ±9,85	0,02	864,37 ± 718 655,35 ± 353	0,01
	Neurologic disorder	+ -	24 164	14,07 ± 8,87 13,15 ± 9,34	NS	762,22 ± 408 733,92 ± 557	NS
	Haematologic disorder	+ -	128 60	13,48 ±10,03 12,79 ± 7,43	NS	705,09 ± 372 813,4 ± 793	NS
	Anti -Sm	+ -	9 179	16,17 ± 5,97 NS (0,06) 13,12 ± 9,39	1198 ± 756 716,25 ± 517	0,01
	Anti -RNP	+ -	24 164	15,1 ± 7,75 NS (0,07) 12,99 ± 9,46	1034,54 ± 1114 688,75 ± 347	NS
	Anti-Ro	+ -	84 104	12,95 ± 6,54 13,52 ± 11,01	NS	859,74 ± 716 634,13 ± 276	0,01
	Anti-La	+ -	60 128	12,71 ± 6,6 13,52 ± 10,29	NS	845,02 ± 770 686,77 ± 371	NS
	Anti-dsDNA	+ -	53 135	15,65 ± 7,8 12,33 ± 9,64	0,0004	855,11 ± 432 692,84 ± 567	0,001
	C4 decreased level	+ -	33 155	16,37 ± 10,09 12,6 ± 8,98	0,04	1063,36 ± 1041 675,12 ± 341	0,04

Our results suggest that while sIL-2R seems to be a good marker of a generalized (auto)immune inflammation, TNF-α might be crucial to the prothrombotic action of pathogenic aPL antibodies. If confirmed, it would offer an intriguing possibility of using TNF-alpha blockade as a therapeutic option in antiphospholipid syndrome.


Dr Andrea Emilio Salvi
email: andrea@orthopaedics.com

Giovanni Pietro Metelli

Elio Domeneghini

Antonio Cantalamessa

Andrea Emilio Salvi -Md

Pietro Giovanni

Metelli -Md

Elio Domeneghini - Md

Antonio Cantalamessa -Md

A clinical picture of chronic polyarticular tophaceous gout

Keywords: gout, urate, tophus, deposit, inflammation

with tophaceous deposits for 30 years. Descriptive clinical and radiographic images are furnished.

Brief Report

In gout, urate deposits in soft tissues (namely the so-called "tophi") are well recognized [START_REF] Jajić | Gout in the spine and sacro-iliac joints: radiological manifestations[END_REF] and since they frequently come out after several years of gout attacks, they represent the chronic phase of the disease process [START_REF] Ryu | Characteristic appearance of large subcutaneous gouty tophi in magnetic resonance imaging[END_REF]. Among the inflammatory arthritis group, gout is one of the most successfully treatable form but nevertheless, 5% of patients may not respond to medical management, and some of these may evolve to the tophaceous stage [START_REF] Sener | Surgical management of tophaceous gout in the hand[END_REF]. The development of multiple large tophi in patients with gout is rare [START_REF] Ryu | Characteristic appearance of large subcutaneous gouty tophi in magnetic resonance imaging[END_REF]. In this brief report it is our purpose to describe an unusual clinical presentation of polidistrectual gout disease. A 61-years-old patient has come to our attention for chronic tophaceus gout deposits, located at both the olecranons (especially on the right), at both the metatarsal-phalanx joints (mainly at the first), at the dorsum of the right foot and at both the second metacarpal-phalanx joint (Figure 1 and Figure 2). He has suffered from this pathology since 30 years, whose articular onset came out with heat and pain. The pathology started to appear at the first ray of the right foot, next appeared on the right elbow, then on the left elbow (since 5 years), subsequently on the left foot and so on. Since one year gout tophi appeared on both the hands. He had taken nimesulid when pain and allopurinol 1 tablet a day, hence half a tablet due to nosebleed. As epistaxis continued, one year ago he has given up allopurinol, that previously he has took discontinuously as he has sometimes forgotten to. Serum uric acid levels were equal to 9.9 mg/100 ml, C-reactive protein equal to 7.65 mg/l, erythrosedimentation speed equal to 26 mm and white blood count equal to 10.54. When gout is inadequately treated over a period of years, multiple shows voluminous polilobulated gouty tophus with superficial erosions of the olecranon. X-rays of both the foots shows calcifications in soft tissues and erosions at the base of the both the first phalanxes, and at both the first metatarsal heads and the 3 rd ,4th and 5th right metatarsal bases, with a similar, less marked features on the left foot. Radiographic findings are typical, since they include soft tissue tophi, punched out erosions with sclerotic margins, monoarticular or polyarticular joint involvement, and relative conservation of joint space [START_REF] Cornelius | Gouty arthritis in the adult[END_REF].

  Figures legend :

Figure 1 -

 1 Figure 1 -A -Tophus on the outer surface of the right olecranon. B -Tophus on the outer surface

Figure 2 -

 2 Figure 2 -X-ray of the right elbow shows swelling on the right olecranon. MRI of the right elbow


Donato Rigante
email: drigante@gmail.com

Piero Valentini

Daniela Rizzo

Andrea Leo

Gabriella Rosa

Roberta Onesimo

Alessia Nisco

Francesca Donatella

Adele Angelone

Angelica Bibiana Compagnone

Delogu

Donato Rigante

Gabriella De Rosa

Alessia De

Nisco • Donatella

Francesca Angelone

Adele Compagnone

Angelica Bibiana

Delogu D Rigante

D F Angelone

A Compagnone

G De

Rosa A De Nisco

A B Delogu

Responsiveness to intravenous immunoglobulins and occurrence of coronary artery abnormalities in a single-center cohort

Keywords: Kawasaki syndrome, intravenous immunoglobulins, coronary artery abnormalities

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

More than 40 years after the original description by the Japanese pediatrician Tomisaku Kawasaki about the clinical signs of Kawasaki syndrome (KS) we are still unable to define its etiology, understand the nature of endothelial damage, predict the responsiveness to treatment or the risk of its vascular complications. Although initially recognized as benign, this syndrome was subsequently acknowledged to have serious cardiovascular concerns, gaining the worldwide interest of pediatricians and pediatric cardiologists [START_REF] Kawasaki | Acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children[END_REF][START_REF] Burns | Commentary: translation of Dr. Tomisaku Kawasaki's original report of fifty patients in 1967[END_REF]: nowadays KS is the most common cause of acquired pediatric heart disease in the developed world [START_REF] Burns | Kawasaki syndrome[END_REF][START_REF] Holman | Kawasaki syndrome hospitalizations in the United States, 1997 and 2000[END_REF].

Diagnosis of KS requires that fever persisting for 5 days or more is accompanied by at least 4 of the following 5 clinical signs: polymorphous rash, bilateral conjunctival congestion, oral mucosal changes, non-purulent cervical lymphadenitis, abnormalities in the peripheral extremities and perineum [START_REF] Ozen | EULAR/PReS endorsed consensus criteria for the classification of childhood vasculitides[END_REF]. High-dose intravenous immunoglobulins (IVIG) are the most effective treatment to reduce KS acute symptoms and prevent the occurrence of coronary artery abnormalities (CAA), which develop in 15 to 25% of untreated children and even in 5-10% of children adequately treated [START_REF] Satou | Kawasaki disease: diagnosis, management and longterm implications[END_REF]. However, 5-15% of patients with KS do not respond to IVIG and this category shows the highest risk of developing CAA or even ischemic heart disease [START_REF] Dajani | Guidelines for long-term management of patients with Kawasaki disease: report from the Committee on rheumatic fever, endocarditis and Kawasaki disease, Council on cardiovascular disease in the young[END_REF].

Attempting to identify which patients should be unresponsive to treatment or predicting the ones who might display a significant risk of developing cardiologic sequelae are two issues of outstanding priority, still unresolved in the medical literature. Aim of our evaluation was to study retrospectively a cohort of Italian patients with diagnosis of KS, hospitalized in a single-centre during a period of 18 years, and analyze their general, clinical and laboratory data as possible predictors for resistance to IVIG and for CAA occurrence.

Cohort of patients, methods and statistical evaluation

A retrospective review of patients' medical charts was carried out for all children admitted to our Department between February 1990 and April 2009 and discharged from hospital with diagnosis of KS. Each chart was examined by both a pediatrician and a pediatric cardiologist. A written informed consent was obtained by each family. Exclusion criteria for our evaluation included prior treatment with IVIG or systemic steroids, presence of another disease known to mimic KS, previous diagnosis of KS and the presence of contraindications to IVIG or acetylsalicylic acid (aspirin).

Atypical forms of KS were excluded by our study.

Thirty-two Italian children were considered in our evaluation. For each patient the following data were collected: age, sex, clinical signs and all laboratory data at diagnosis, illness days before IVIG, IVIG dose, eventual IVIG additional dose, CAA at the time of diagnosis or over follow-up. Illness day 1 was defined as the first day of fever. Unresponsiveness to IVIG was defined as persistent fever (≥ 37.5°C) over 24 hours after the first IVIG infusion [START_REF] Kobayashi | Prediction of intravenous immunoglobulin unresponsiveness in patients with Kawasaki disease[END_REF]. For nonresponder patients one supplementary dose of IVIG was administered. Cardiologic evaluation and serial echocardiograms were performed at KS recognition after hospital admission, then with 3-day frequency during hospitalization and with weekly frequency over the first 6-8 weeks. After discharge periodic cardiologic evaluations were performed according to each patient's risk stratification at intervals of 6-12 months.

Normality of distribution was assessed through the use of the Kolmogorov-Smirnov test. Categorical data were compared using Fisher's exact test. Continues variables, normally distributed, were analysed using Student's t-test. Data that were not normally distributed were compared by using the Mann-Whitney U test. P-values 0.05 were considered statistically significant. All tests were two-tailed and the statistical analysis was performed with the Statistical Package for Social Sciences (SPSS) 11.0 program for Windows (Chicago, Illinois).

Results

Thirty-two patients included in our analysis (20 males and 12 females, male/female ratio: 1.7/1) had a mean age of 23.8 months and were of Italian ancestry.

Median age at diagnosis was 17,5 months, ranging from 2.3 to 80.2 months. All patients had fever (ranging from 38.2 to 40.1°C) and its median duration was 7 days (range 5-18 days). IVIG were administered to all patients: 10 children, treated before 1999, received 400 mg/kg/day for 5 days and 22 children, treated after 1999, received 2 g/kg in a single 12 hour-infusion. IVIG were given at a variable time since disease onset and were associated with oral high-dose aspirin (50-100 mg/kg/day) during the acute phase, followed by low-dose aspirin (3-5 mg/kg/day) for a variable period, depending on the echocardiographic results. None of patients who had recovered completely from the original episode had a recurrent disease.

Five patients (15.6%) out of the initial cohort were IVIG-resistant and the remaining 27 responded completely to the first IVIG infusion. Table 1 shows the comparison between the groups of IVIG-responders and nonresponders as for general and laboratory data. Table 2 lists the distribution of KS clinical signs between IVIGresponder and nonresponder groups. There was no statistically significant difference in age, sex, clinical signs and laboratory results between the two groups. Nonresponders were diagnosed and treated in a most precocious time when compared with responders: the median day of illness at which IVIG were infused in nonresponders was earlier than in responders (5 vs 7 days, p=0.08); in addition, the nonresponder group showed significantly higher C-reactive protein (CRP) in comparison with responders (p=0.04).

Five (15.6%) out of the 32 patients developed CAA, defined as coronary dilations more than 2 standard deviations (SD) higher than the expected mean, after body surface area adjustment. Ten patients with no apparent involvement of coronary arteries presented an endothelial brightness at a qualitative echocardiographic assessment. The proportion of CAA in IVIG-nonresponders was higher than in responders, though with no statistical significance (20% [1/5] vs 14.8% [4/27], p=1). In particular, 6 patients had an incomplete form of KS and 2 of them displayed CAA.

Ten children treated before 1999 received IVIG at the dose of 400 mg/kg/day for 5 consecutive days, according to the clinical practice of that period: we observed a higher percentage of IVIG-nonresponders in this group than in the 22 children treated with a single IVIG infusion of 2 g/kg after 1999, though with no statistical significance (20% [2/10] vs 13.6% [3/22], p=1). Although there was a trend for patients in the first group to have more CAA than those in the second one (respectively 30% [3/10] vs 9% [2/22]), this difference was not statistically significant (p=0.30), maybe due to the limited number of patients. In addition, patients with CAA had higher levels of white blood cell count at diagnosis in comparison with those without CAA (23.70 x 10 9 /l vs 16.05 x 10 9 /l, p=0.01). At a long-term follow-up all CAA resolved completely in all patients, even in the youngest patient, who had an incomplete clinical presentation.

Eleven patients (34.4%, 6 males and 5 females) out of the cohort were less than 1 year old: 2 (18.2%) of this 11 children were IVIG-nonresponders in comparison with 3/21 nonresponder aged more than 1 year (14.3%) with no statistical significance (p=1).

The incidence of CAA was significantly higher in the group of less than 1-year-old children in comparison with the group of children over 1 year (36.4% [4/11] vs 4.8% [1/21], p =0.037). Table 3 shows the comparison of general and laboratory data with regard to the occurrence of CAA. Figure 1 shows the relationship between age at diagnosis and occurrence of CAA.

Discussion

Many clinicians have been making outstanding efforts to uncover the mysteries behind KS, though the definition of its outcome remains hazy, even for cases with echocardiographically normal coronary arteries. Even if KS remains a self-limited panvasculitis involving small and medium-sized muscular arteries in children of all ethnic backgrounds, the disease bears an intrinsic risk of severe cardiovascular sequelae: a continuous spectrum ranging from no apparent involvement in the majority of patients to the presence of multiple giant aneurysms is recognized [9]. The primary end-point of treatment in KS is the prevention of any vascular injuries and the risk of developing CAA is clearly decreased by IVIG administration during the acute phase [START_REF] Newburger | The treatment of Kawasaki syndrome with intravenous gamma globulin[END_REF]. In the last decade a single high-dose IVIG infusion (2 g/kg of body weight) has been considered the gold-standard treatment for KS [START_REF] Newburger | A single intravenous infusion of gamma globulin as compared with four infusions in the treatment of acute Kawasaki syndrome[END_REF].

It is widely known that more than 10% of KS patients fail to defervesce with initial IVIG treatment [START_REF] Wallace | Initial intravenous gammaglobulin treatment failure in Kawasaki disease[END_REF]. Egami et al showed that resistance to IVIG treatment can be predicted evaluating age, illness days, platelet count, serum transaminase level and CRP [START_REF] Egami | Prediction of resistance to intravenous immunoglobulin treatment in patients with Kawasaki disease[END_REF]. A multivariate analysis by Sano et al in 112 patients with KS, who received IVIG within 2 days, selected high CRP, total bilirubin and aspartate aminotransferase as independent predictors of low rate of response to initial IVIG treatment, finding that patients with at least two increased predictors of the three might result unresponsive to IVIG [START_REF] Sano | Prediction of non-responsiveness to standard high-dose gamma-globulin therapy in patients with acute Kawasaki disease before starting initial treatment[END_REF]. Failure of a single IVIG dose might occur in up to 11.6% of KS patients, especially if displaying low hemoglobin, high neutrophil count and low albumin [START_REF] Durongpisitkul | Immunoglobulin failure and retreatment in Kawasaki disease[END_REF]. In the most recent medical literature it has been accepted that an anticipated identification of IVIG-nonresponders might decrease the overall incidence of CAA through a "most intense" treatment and that the main predictor of CAA occurrence is the overall duration of fever longer than 8 days [START_REF] Kim | Predictive risk factors for coronary artery abnormalities in Kawasaki disease[END_REF].

In our evaluation 32 Italian patients with KS had a median duration of fever of 7 days: 15.6% of them were nonresponders to the initial IVIG infusion, receiving a second IVIG dose which led to disease remission, and the same percentage (15.6% of the cohort) developed CAA. There was no statistically significant difference for children treated with the two IVIG regimens both for the percentage of nonresponders and the occurrence of CAA. In addition, sex ratio in the nonresponder group showed that males were more represented than females (4 males to 1 female). IVIG-responders had a lower CRP (p=0.04) and showed a tendency to a longer duration of fever before IVIG infusion, when compared with the nonresponder group. The overall "amount of inflammation" resulted critical both for response to therapy and for development of CAA, as demonstrated by higher CRP levels in nonresponders and white blood cell count in patients with CAA. As observed in previous studies, we have found a higher incidence of CAA in IVIG-nonresponders compared to responders (20% vs 14.8%), though without statistical significance: this might be explained by the small sample studied, including incomplete forms and cases treated beyond 10 days of fever. However, children with less than 1 year have developed CAA in a higher statistically significant percentage if compared with children over 1 year of age (36.4% vs 4.8%, p=0.037). This observation has been confirmed by studies of surveillance in larger groups of patients with KS from Japan, Korea and Taiwan [START_REF] Nakamura | Epidemiologic features of Kawasaki disease in Japan: results from the nationwide survey in 2005-2006[END_REF][START_REF] Yeo | Incomplete Kawasaki disease in patients younger than 1 year of age: a possible inherent risk factor[END_REF][START_REF] Huang | Epidemiologic features of Kawasaki disease in Taiwan, 2003-2006[END_REF]. Possible genetic variants in the major histocompatibility complex region that are associated with KS might influence the responsiveness to IVIG and the development of CAA [START_REF] Kato | HLA antigens in Kawasaki disease[END_REF][START_REF] Lin | HLA-E gene polymorphism associated with susceptibility to Kawasaki disease and formation of coronary artery aneurysms[END_REF].

Limitations of the present report are common to most retrospective studies, though we want to underline the small number of patients, all deriving from a single-centre, and the administration of two different IVIG schemes.

It is known that at least transient CAA might develop in approximately 5-10% and giant aneurysms in 1% of children with KS despite prompt treatment with IVIG: these ones must be given within 7 to 10 days to be most effective in preventing the development of CAA. In addition, very young infants, especially those exhibiting an incomplete clinical KS presentation, have the highest rate of CAA and for this reason echocardiography should be performed on any infant younger than age 6 months with fever duration exceeding 7 days, increased CRP or no other explanation for the febrile illness [START_REF] Newburger | Diagnosis, treatment, and long-term management of Kawasaki disease: a statement for health professionals from the Committee on Rheumatic Fever, Endocarditis and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association[END_REF]. The relationship between the duration of illness before treatment and the frequency of occurrence of CAA was examined by Baer et al, who emphasized the relevance of the initial echocardiography as an adjunctive diagnostic test especially for incomplete forms of KS [23].

Coronary artery assessment by echocardiography needs to quantify internal coronary artery diameters: commonly used definitions have relied on the Japanese Ministry of Health criteria, which arbitrarily defined CAA as a maximum absolute internal diameter superior than 3 mm in children aged less than 5 years or superior than 4 mm in children aged more than 5 years [START_REF]Report of subcommittee on standardization of diagnostic criteria and reporting of coronary artery lesions in Kawasaki disease[END_REF][START_REF] Capannari | Sensitivity, specificity and predictive value of two-dimensional echocardiography in detecting coronary artery aneurysms in patients with Kawasaki disease[END_REF]. In 1998 de Zorzi et al reported that internal diameters of coronary arteries of KS patients adjusted for their body surface area, as z-scores calculated using a non-linear regression model, were significantly larger than normal non-febrile controls and that 27% of patients classified as having "normal" coronary arteries according to the Japanese Research Committee criteria would have had at least one coronary abnormality by their new criteria [START_REF] De Zorzi | Coronary artery dimensions may be misclassified as normal in Kawasaki disease[END_REF]. An increase in CAA recognition has been attributed to the widespread use of de Zorzi's criteria, which led to the discovery of larger body surface area-adjusted dimensions of coronary arteries in all phases of KS. Newer data are delineating vascular abnormalities as perivascular echogenicity or brightness, luminal irregularities and abnormal vessel tapering of the coronary arteries even in those KS patients in whom CAA were never detected and endothelial dysfunction is believed to be crucial to the pathogenesis of coronary vasoconstriction and myocardial ischemia [START_REF] Mccrindle | Coronary artery involvement in children with Kawasaki disease. Risk factors from analysis of serial normalized measurements[END_REF][START_REF] Furuyama | Altered myocardial flow reserve and endothelial function late after Kawasaki disease[END_REF]. During KS acute phase we observed a perivascular endothelial brightness of coronary arteries in 10 out of 32 patients, which did not evolve to any dilations: we used de Zorzi's criteria to measure at baseline left main and proximal right coronary artery dimensions in centimetres (normal values do not exist for distal segments of the circumflex artery), considering body surface area for each patient, and finding that 2/5 children having CAA did not meet the 1984 Japanese Ministry of Health criteria. In all our patients we observed the echocardiographic resolution of CAA, in accordance with a host of literature data related to large numbers of patients confirming that coronary ectasia or small coronary aneurysms have a good prognosis and a greater chance to undergo total regression.

In consideration of the overall risk of long-term cardiac sequelae, combined with the risk of a premature accelerated atherosclerosis and its consequences, we strongly believe that multicenter randomized clinical trials are needed to establish the best strategy to manage high-risk patients with KS, i.e. the youngest ones, by stricter monitoring or eventual administration of additional antinflammatory therapies, though the optimal surveillance modalities are still far to be unraveled especially for low-risk patients.

Figure 1 .

 1 Figure 1. Relationship between age (in months) at diagnosis of Kawasaki syndrome and occurrence of coronary artery abnormalities.

Table 1 .

 1 Comparison of general and laboratory data in the IVIG-responder and IVIGnonresponder groups of patients with Kawasaki syndrome.

	IVIG-	IVIG-	p
	responders	nonresponders
	(n = 27)	(n =5)

Table 2 .

 2 Distribution of clinical signs in the acute phase of Kawasaki syndrome between IVIG-responder and IVIG-nonresponder groups.

	IVIG-	IVIG-	p □
	responders	nonresponders
	(n = 27)	(n =5)

Table 3 .

 3 Comparison of general and laboratory data in patients with Kawasaki syndrome with regard to the occurrence of coronary artery abnormalities.

	Patients	Patients with	p
	without	coronary
	coronary	abnormalities
	abnormalities	(n =5)
	(n = 27)