R Rouveure

P Faure

M O Monod

A New Radar Sensor for Coastal and Riverbank Monitoring

For environmental management, remote sensing techniques are of major interest as they can provide global and local coverage of natural environments. Considering the visibility conditions that are often poor in outdoor environments (day/night cycles, clouds, shadows, fog, etc.), microwave system offers an alternative solution in order to overcome the difficulties of optical sensors. In order to deal with the need of high spatial resolution, close temporal acquisition, and low-cost data availability, we developed a small-sized microwave radar sensor called K2Pi. This radar is based on the principle of Frequency-Modulated Continuous Wave (FM-CW) technology. This radar can be easily positioned on different vehicles, including boats. An application in simultaneous localization and mapping using scan matching of radar images (R-SLAM algorithm) has been developed in order to build radar map of the environment. The potentialities of the sensor are presented through an application of riverbank mapping.

INTRODUCTION

Natural forces such as wind, waves, currents, rain, flood, etc. are constantly shaping the interface between the hydrosphere and the lithosphere. The monitoring of the evolutions of this interface, based on erosion and accretion processes, is of major interest in order to better understand the functioning of rivers and coastal regions [START_REF] Wilson | Managing flow for geomorphic form: erosion and accretion of river benches[END_REF], [START_REF] Saravanan | Lake. (a) Aerial image from Google Maps. Yellow dots show GPS trajectory. Mark A indicates a lack of GPS information (GPS signal unavailable). A part of the trajectory ([END_REF]. The development of numerical models to monitor or predict coastal and riverbank evolutions imply the availability of accurate and redundant data. But robust solutions for perception in extended outdoor environments are not so frequent, particularly with regard to the problem of resistance to the environmental conditions [START_REF] Hague | Ground based sensing systems for autonomous agricultural vehicles[END_REF], [START_REF] Hellström | Autonomous Navigation for Forest Machines[END_REF]. Laser and video based systems are common optical based sensors, particularly well adapted in indoor environments. However, these systems are affected by visibility conditions that are often poor in outdoor environments: day/night cycles change illumination, weather phenomena as fog, rain or dust may deteriorate the quality of information provided. In the microwave range, data can be acquired independently of atmospheric conditions and time of the day. So radar can provide an alternative solution to overcome the shortcomings of optical sensors. Moreover, backscattering signal has an unquestionable potential for environmental applications, as it can be shown in satellite remote sensing [5].

In response to new requirements related to the monitoring of environment at lower scales in comparison to airborne radar systems, we developed a small-sized radar sensor easy to embark on board of different vehicles and we show in this paper its potentialities through a preliminary application in riverbank mapping.

Because of its intrinsic characteristics, microwave radar is particularly well suited for outdoor applications and large scale mapping, and particularly in outdoor mobile robotics. Microwave radar has been successfully used in numerous domains such as terrain imaging [START_REF] Scheding | Terrain imaging and perception using millimetre wave radar[END_REF], [START_REF] Brooker | Using Millimetre Wave Radar in Underground Mines[END_REF], [START_REF] Brooker | A W-band airborne interrupted FMCW imaging radar[END_REF], target tracking [START_REF] Brooker | Millimetre Wave Radar for Tracking and Imaging applications[END_REF], three-dimensional map building [START_REF] Foessel | Three dimensional map building with mmw radar[END_REF], vehicle localization and vehicle navigation [START_REF] Clark | The design of a high performance mmw radar system for autonomous land vehicle navigation[END_REF]. Moreover in recent years different simultaneous localization and mapping algorithms based on microwave radar sensor have been developed [START_REF] Dissanayake | A solution to the simultaneous localisation and map building (SLAM) problem[END_REF], [START_REF] Jose | Millimetre Wave RADAR Spectra Simulation and Interpretation for Outdoor SLAM[END_REF], [START_REF] Foessel | Radar Sensor Model for Three-Dimensional Map Building[END_REF].

This paper is divided into the following sections. The principle and the characteristics of the K2Pi radar imager is presented in Section II. Section III develops and describes the R-SLAM algorithm which is based on a scan matching approach and the description of the environment with an occupancy grid. Results obtained on the Ayday Lake (France) are presented in Section IV.

II. PRESENTATION OF THE K2PI RADAR IMAGER

For short-range distance measurements i.e. few hundreds of meters, the classical pulse radar technology is not the best adapted solution. For example, a precision of 1 meter in distance measurement imposes a precision of 3.3 ns in time measurement, therefore fast and expensive electronics. We developed the K2Pi microwave radar (meaning 360° scanning in K band) using the frequency modulation continuous wave (FMCW) principle. This principle is known and used for several decades [START_REF] Monod | Frequency modulated radar: a new sensor for natural environment and mobile robotics[END_REF], [START_REF] Collette | Un altimètre radioélectrique à modulation de fréquence[END_REF]. When the range is some hundreds of meters, frequency modulation presents the following advantages: -low transmission power, as it is the mean power which determines the range, which is safer for the user, -transposition of the temporal variables into the frequency domain: a very short delay time ∆t is transformed into a broad variation of frequency ∆f, easier to measure.

The principle of FMCW radar consists to mix the emitted signal following a linear modulation waveform, with the received signal delayed after reflecting on the targets present within the view of the antenna [START_REF] Skolnik | Introduction to radar systems[END_REF]. Let us consider a sawtooth modulation at frequency F m over a range ∆F and a distribution of i stationary targets at respective distances R i in the antenna beam. When the echo signal is heterodyned with a portion of the transmitted signal, a beat signal S b will be produced [START_REF] Monod | Frequency modulated radar: a new sensor for natural environment and mobile robotics[END_REF], [START_REF] Rouveure | Multiple targets detection with a FMCW radar dedicated to mobile robotics[END_REF]:

cos 2 2 i b e ri m i i R S k V V F F t c π     = ∆ + Φ         ∑ . ( 1
)
V e is the amplitude of emitted signal, V ri and Φ i respectively the amplitude of received signal and a phase term depending on target i, c the light velocity and k a mixer coefficient. When more than one target is present within the view of the radar, the mixer output will contain a sum ΣF bi of frequency components, each of them corresponding to a particular target i:

2 bi m i F F F R c = ∆ . ( 2
)
The range R i to each target may be determined by measuring the individual frequency components by the mean of a Fourier transform. At each angle of sight a power spectrum is computed. Contrary to laser measurement which gives the nearest echo received in the beam, the aperture of the radar beam allows multi-targets detection in the same angleof-sight. An example of radar power spectrum is presented in Fig. 1. We will notice that moving targets which introduce a Doppler frequency shift are not considered in this paper.

A so-called "panoramic radar image" covering 360° in the horizontal plane is carried out using a PPI (Plan Position Indicator) representation, which displays range of targets versus angle. Considering the combined effect of antenna rotation and vehicle motion (translation and rotation), one would observe intra-scan distortions on the final 360° image. To achieve a vehicle motion correction, two proprioceptive sensors are used to estimate the displacement of the vehicle and to take into account these distortions: an odometer to measure the longitudinal displacement of the vehicle and a gyrometer to measure the rotation of the vehicle.

Once the vehicle motion has been corrected, an anti-speckle filter is added. The speckle effect is a random amplitude modulation which exists in any radar image construction. Speckle noise reduction can be achieved either by spatial filtering or multi-look filtering [START_REF] Lee | Speckle filtering of synthetic aperture radar images: A review[END_REF], [START_REF] Guoqing | Study on speckle reduction in multi-look polarimetric SAR image[END_REF]. We chose the latter approach: n independent images of the scene are incoherently combined (by space-domain averaging) to produce a single final image.

During the Aydat Lake experiment, no odometric sensor is available to estimate the displacement of the radar (odometric information are used during the Vehicle Motion Correction and the Anti-Speckle Filter steps). Data from a kinematic GPS system (with a centimetric precision) could be employed, but we try to avoid the use of such a system for different reasons: -we want to build a radar imager independent of any localization system, -the cost of a kinematic GPS system is very high, -if no differential GPS correction signal is available, it is necessary to install its own GPS ground station and the cost of the GPS system is doubled, -considering the GPS satellite constellation, the terrain topography, the presence of high vegetation or various obstacles, etc., GPS signals can be intermittently available or completely unavailable. So a direct estimation process of the vehicle displacement was developed: it allows estimating of the movement of the vehicle within initial radar images (before vehicle motion and anti-speckle corrections).

The radar is monostatic, i.e. a common antenna is used for both transmitting and receiving. The rotating antenna achieves a complete 360° scan around the vehicle in one second, and a radar acquisition is realized each degree. The sensor includes microwave components, electronic devices for emission and reception, data acquisition and signal processing units. A general view of the K2PI radar is shown in Fig. 2.

Data acquisition and signal processing units are based on a Pentium M-2 GHz PC/104 processor card. Computed data are transmitted for visualization and further treatment using an Ethernet link. Main characteristics of the radar are described in Table 1.

III. MAP CONSTRUCTION: THE R-SLAM ALGORITHM

In the robotics domain, the simultaneous localization and mapping (SLAM) problem asks if it is possible for a mobile robot to be placed at an unknown location in an unknown environment and for the robot to build a consistent map of this environment, while simultaneously determining its location within this map. This problem has been intensively treated, particularly for autonomous navigation problems. In our application, we have been interested by the SLAM methodology to build a representation of the environment, using a succession of panoramic radar images. One interest of this approach is that this construction is "sensor-based" and avoids the use of an absolute positioning system like GPS. The majority of SLAM formulations are based on the ability of landmarks locations in the environment, assuming that these locations are time invariant. Whatever the sensor used, the most difficult problem for this approach is beacon validation and data association [START_REF] Ho | Detecting Loop Closure with Scene Sequences[END_REF]. It means that a major difficulty is (i) to confirm that the detected beacon is a valid beacon, and (ii) to associate it with a known beacon in the computed map. The data association process is an essential problem in SLAM algorithm. It has been addressed by looking for stable beacons in the environment, or using a combination of sensors (multisensorial approach) [START_REF] Zhang | An Efficient Data Association Approach to Simultaneous Localization and Map Building[END_REF].

But non-ambiguous target detection and identification based on radar signal interpretation are still open problems [START_REF] Chen | Recent advances in simultaneous localization and map-building using computer vision[END_REF]. This is particularly true in outdoor and natural environment, where landmarks which are detected by the radar cannot be modeled as simple geometric primitives. Power/range spectra contain a large amount of information mixed with various sources of noise, so detection and tracking of particular landmarks are not simple problems [START_REF] Jose | Millimetre Wave RADAR Spectra Simulation and Interpretation for Outdoor SLAM[END_REF]. The detection of landmarks in power/range spectra can be treated through constant false-alarm rate (CFAR) techniques that estimate the level of noise and the setting of a threshold [START_REF] Rohling | Radar CFAR thresholding in clutter and multiple target situations[END_REF]. But in very poor structured environment, the clutter, which is traditionally considered as noise in radar applications, must be taken into account because it can be the only available information (clutter traditionally refers to natural echoes such as ground, sea, precipitation -rain, snow or hail-, sand storms, animals -especially birds-or insects, etc). In such situations, a detection approach based on threshold selection will fail. It is for that reason we developed a particular R-SLAM algorithm which does not treat the landmarks individually, but considers the entire radar scan. A radar image is constituted by a scan over 360° around the vehicle and is considered as an occupancy grid (matrix of cells), which divides the area of interest in regular cells. Each cell is related to a small area (20 cm × 20 cm), the occupation level of which is given by the power of the reflected signal. The global grid is updated for each 360° scan, using a data merging process. The first scan is directed at 0° referring to the initial direction of the vehicle.

The absolute orientation in reference to magnetic north may be obtained by the addition of a compass.

The R-SLAM process includes two main steps: -map matching between the current radar scan and the constructed map, in order to estimate the inter-scan displacement, -global map updating to integrate the current radar scan through a data merging process.

A. Map Matching

Classical feature extraction and matching imply to recognize the same feature repeatedly during the map construction. Then it is necessary to detect persistent and reliable features in the scan. But because of limited angle and distance resolutions, radar scans can not offer the same resolution than optic images or laser scans. Another problem is that target's reflected power depends on the orientation of the target, so the reflected signal will be different according to the angle of observation: such a property is particularly disadvantageous in performing long-term tracking of individual landmarks.

Considering the particularities of radar data, the best adapted method here is scan-matching, which performs a 3D cross correlation (two translations and one rotation), between the current image and the constructed map. The maximum of the correlation matrix gives the estimate of the radar displacement and rotation between two scans.

B. Global Map Updating

Once the inter-scan displacement and rotation estimated, the observation from the current radar scan has to be integrated in the global radar map.

The radar data interpretation requires a representation tool that facilitates the study of sensor-scene interaction. In that sense occupancy grids are powerful and flexible representation tools that capture the relevant scene aspects for radar-data interpretation [START_REF] Martin | Robot Evidence Grids[END_REF]. Occupancy grid represents environment by fine-grained, metric grids of variables that reflect its occupancy. Each cell of the occupancy grid contains a number that represents accumulated evidence of occupancy or emptiness in the space. In the R-SLAM process, the number contained in the cell is proportional to the microwave power of the reflected signal. This reflected power is proportional to the radar cross section (RCS) of the target. For most common types of targets such as buildings, vegetation and terrain, the radar cross section does not necessarily bear a simple relationship to the physical area, except that the larger the target size, the larger the cross section is likely to be. As the radar moves, a data merging process is applied in order to estimate the value of the cell according to the successive values of the reflected radar signal.

This data merging process faces an ambivalent situation: -the necessity to take into account the new information from the current radar image, -the necessity to preserve the information previously stored in the global map. The principle retained is to consider that the probability for a target to appear in the map is proportional to the number of times it has been observed at the same place.

IV. RESULTS

A preliminary experimentation has been done on the Aydat Lake near Clermont-Ferrand, France (latitude 45°39'51" N, longitude 2°59'09" E, elevation 840 m). The radar is positioned at the front of an experimental boat from the Cemagref Institute (see Fig. 3). The radar antenna is 1.5 m above the water surface. Radar data are acquired and stored in real time during the experiment. The R-SLAM algorithm has been implemented in Matlab language on a Quadri-Core 2.5 GHz Pentium PC with an off-line process. It takes 3 seconds to compute a radar image and to update the radar map. Simultaneously with radar data acquisitions, GPS data are recorded in order to have a localization reference.

An aerial view of the lake (from Google Maps, date: April 24th, 2004) is presented in Fig. 4(a). One can observe that a part of the lake is hidden by clouds. The covered distance is about 4 km, with a mean velocity of 1.2 m/s. Yellow dots indicate the successive GPS positions. A total of 3176 panoramic radar images was stored and processed to work out the global map and trajectory (red dots) presented in Fig. 4(b). This trajectory is directly estimated with radar data, without using GPS sensor.

A detail of the experiment is also presented in Fig. 5.

V. CONCLUSION

For outdoor environment perception, microwave radar is a sensor which presents a considerable potential, overcoming the limitations of vision-based sensors. In order to construct consistent maps of the environment, the R-SLAM algorithm presented in this paper exploits all the richness of radar signal, avoiding the detection of particular landmarks. The algorithm builds simultaneously a map of the environment and a localization of the vehicle within the map, without using a global positioning system. The range of radar, combined with the R-SLAM algorithm performances, makes it possible to deal with extended and low structured environments.

Figure 1 .

 1 Figure 1. Example of radar power spectrum. The frequency peaks indicate the presence of two targets (A and B) in front of the antenna.

Figure 2 .

 2 Figure 2. General view of the K2Pi radar.


MD Markus Kraemer
email: markus.kraemer1977@gmx.de

MD Peter Berlit

Dr. med Markus Kraemer

MD ; P Berlit

Background and Purpose: Primary central nervous system vasculitis (PCNSV) represents a rare and poorly-understood form of vascular inflammatory disease restricted to the brain and spinal cord. This study was undertaken to better characterize demographic and clinical features and to evaluate treatments and outcome.

Methods

Results:

The mean age at onset of symptoms was 42.48 years. In 6 patients (28.6%) of patients PCNSV diagnosis was established based on positive CNS tissue histopathology. Additional angiography was only performed in 2 of these 6 patients and was negative in both cases. For 13 patients (61.9%), the diagnosis was confirmed by positive cerebral angiogram. The most frequent clinical presentations over the whole follow up period were hemiparesis (76.2%), cerebral ischemia (66.7%), headache (57.1%) and altered cognition (52.4%). Patients with MRtomographic evident microangiopathy showed significantly worse modified Rankin disability scores during the onset and at last visit compared to the other patients (p=0.047, p=0.039).

Conclusion: Clinical features and course of PCNSV of Europeans analyzed in this

German study are comparable to American results but some further reported characteristics of different subtypes could not be duplicated. Although this well defined analysis adds to our knowledge of PCNSV, it also raises important open questions. The enigma of PCNSV could only be solved by implementing large prospective multicenter databases and elucidating animal models. Primary central nervous system vasculitis (PCNSV) also known as primary or isolated angiitis of the central nervous system is an uncommon disease in which lesions are limited to the brain and spinal cord. [START_REF] Salvarani | Primary CNS vasculitis with spinal cord involvement[END_REF] It still represents one of the enigmas of clinical neurology [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF]. Although it is a rare disease its seriousness makes it important to consider in clinical differential diagnosis. The affliction remains poorly recognized. Since there are no uniform diagnostic criteria, the low specifty and sensifity of the different diagnostic approaches and the scarceness of the disease our existing knowledge is based on incompatible and small case series or single case reports. It is still a major diagnostic and therapeutic challenge and is magnified by numerous unanswered questions regarding epidemiology, predisposition, pathophysiology and outcome [START_REF] Calabrese | Primary central nervous system vasculitis: progress and questions[END_REF]. Recently, a working group from the Mayo Clinic, USA, published the retrospective analysis of the largest cohort of such patients ever reported. [11, 28, 35-37, 39, 41] Their analysis included 101 patients with PCNSV seen in Mayo Clinic over two decades from 1983 through 2003 [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. As we frequently are asked to re-evaluate patients with uncommon causes o f stroke thought to have inflammatory (PCNSV and secondary caused vasculitides) and non-inflammatory vasculopathies (Moyamoya disease or Sneddon´s syndrome), we had the opportunity to include 21 patients with definite PCNSV treated within the short period of 5 years.

The design of this study was orientated on the protocols of the most relevant American studies [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF] to enable a comparison.

Material and Methods

Patient Selection

We identified all patients diagnosed as "cerebral vasculitis" evaluated at the neurological department of the Alfried Krupp Hospital, Essen, Germany from 2003 through 2008 using our institutional diagnostic linkage system. Inclusion criteria were definite diagnosis of PCNSV. We excluded patients in which vasculitis of the CNS was not definite because other differential diagnoses were discussed. Furthermore we excluded patients who had vasculitis outside the CNS and patients with secondary vasculitis. For cases of uncertain diagnoses we re-evaluated the diagnosis based on the diagnostic criteria proposed by Calabrese and Mallek [START_REF] Calabrese | Primary angiitis of the central nervous system. Report of 8 new cases, review of the literature, and proposal for diagnostic criteria[END_REF] after reviewing the complete medical records again.

Clinical Data Collection

In living patients diagnosed with PCNSV < 1 year before data collection, the asked features were obtained from recent clinical visits. In living patients diagnosed > 1 year before data collection we tried to obtain follow up information by telephone interview or from outside hospital charts. A standard data collection form was used for the definite cases. Assessment was focussed on demographic, clinical and outcome features. Analysis included demographic data like gender and age at first symptom presentation and the period from symptom onset to diagnosis, clinical presentation at onset of disease and during follow-up period, results from MRI, angiography, CNS biopsy and laboratory investigations. Furthermore treatment data were collected as well as physician's judgment about the response to therapy. Functional status at first visit and latest follow-up was classified by using the modified Rankin score (MRS). Angiograms were regarded as highly probable for vasculitis if signs of multiple stenoses or occlusions or areas of vessel-wall segmental thinning or dilatations were observed without evidence for arteriosclerosis or other better explaining conditions. Histopathological marks were considered positive if definite vasculitis was evident, unspecific features like thickened vessel-walls or features of ischemia were judged as vasculitis-negative.

Statistics and Data Analysis

Statistical comparisons were made using the Mann-Whitney U test (Wilcoxon test) for numeric data. Comparisons of categorical information were done using Chi-Quadrate-test or Fisher's exact test when samples were small. All p values were twosided. P < 0.05 was considered to be statistical significant. Statistical analysis was completed with SPSS version 17.0.

Results

Demographic data and diagnosis

We identified 21 patients treated at Alfried Krupp Hospital, Essen, Germany between 2003 and 2008 regarded as having definite PCNSV. In the preceding process of data collection we excluded 12 uncertain patients because PCNSV was not definite and possible differential diagnoses were considered. The most relevant differential diagnosis was arteriosclerotic vasculopathy. Furthermore we excluded 16 cases of secondary vasculitis.

Table 1 shows the tests used to establish the diagnosis of PCNSV. In 6 patients (28.6%) of patients PCNSV diagnosis was established based on positive CNS tissue histopathology. Additional angiography was only performed in 2 of these 6 patients and was negative in both cases. For 13 patients (61.9%), the diagnosis was confirmed by positive cerebral angiography. This group of patients diagnosed by angiography included 8 patients who did undergo biopsy. From the 5 angiographypositive patients who underwent biopsy no specimen was positive. In two patients we decided to include them in the analysis despite the angiogram being negative and biopsy was not performed or negative. According to diagnostic criteria of PSNSV formally these were not definite cases of PSNV. However after once again reviewing the medical records and clinical re-evaluation, we were convinced to establish this diagnosis in these two cases and so did not excluded them from data collection in contrast to the other doubtful 12 cases already mentioned.

The mean age of the 21 included PCNSV patients (13 female and 8 male patients) at time of symptom onset was 42.48 years (median, 43, range, 11-65 years). One patient was of Irish, one of Swiss, one of Turkish and 18 patients (85.7%) of German family origin. The majority of patients (71.4%) were older than 37 years at first presentation of the disease. Regarding the time from symptom onset and correct diagnosis, in the majority of patients (76.2%) this duration was within 5 months. The median duration from first presentation to correct diagnosis was 2 months with a mean of 18.29 month (range, 0 -184 months). The mean follow up period was 42.6 months (median, 24 months, range, 1 -226 months). There was no statistical difference regarding age on first symptom onset, duration to correct diagnosis and follow-up time dividing patients in angiography-positive or biopsy-positive patient and even in patients with microangiopathy or with gadolinium enhancement. In 14 of the PCNSV patients (66.6%) additional diseases were documented during follow-up.

Despite age-related and treatment-related diseases it is notable that in five patients other autoimmune diseases were documented (Hashimoto-thyreoditis in 2, psoriasis in 2 and autoimmune hepatitis in 2 patients). Cerebrovascular risk factors were noted in 16 of 21 patients (76.2%) which were smoking in 10 patients (47.6%) and arterial hypertension in 9 patients (42.9%). In these patients with cerebrovascular risk factors, this aspect was associated with a higher incidence of ischemic insult over the whole follow-up (p=0.025) and MR-tomographic evident microangiopathy (p=0.025). Furthermore cerebrovascular risk factors were found only in three of six biopsy-proven patients (p=0.045). Patients with cerebrovascular risk factors did not differ in other demographic, clinical, diagnostic or outcome features.

Clinical Symptoms

The most frequent clinical presentations over the whole follow up period were hemiparesis (76.2%), cerebral ischemia (66.7%), headache (57.1%) and altered cognition (52.4%). Clinical symptoms at onset of disease were similar to clinical features during follow-up. However headaches seemed to be more frequent at onset of the disease (42.9% versus 28.6%) as well as hemiparesis (61.9% versus 38.1).

The reverse was true for transient ischemic attacks (0% versus 23.8%) and seizures (9.5% versus 33.3). When patients were divided by diagnostic modality (angiographypositive, biopsy-positive, patients with microangiopathy, patients with gadolinium enhancement), most symptoms appeared with comparable frequency in the different groups (table 2). However, incidence of amnestic syndrome at first presentation was greater in patients with gadolinium enhanced lesions (p=0.042) and gait disorder at the onset seemed to be more frequent (p=0.056) in patients with MRI gadolinium enhancement. Comparing patients with and without microangiopathy those with MRtomographic small vessel involvement showed more often hemiparesis, cerebral ischemia over the whole follow up period (p=0.025, p=0.017, respectively) and more often cognitive decline and cerebral ischemia during follow up (p=0.024, p=0.032).

Intracranial hemorrhage was very infrequent, it repeatedly occurred in only one biopsy-proven patient. Furthermore none of the patients characterised headache as "thunderclap" headache.

Magnetic resonance findings

MRI was performed and revealed abnormal findings in all patients (100%). Ischemic infarctions with diffusion disturbances were seen as the most common feature in MRI in 76.2% of all patients (50% in biopsy-positive and 84.6% in angiography-positive patients). All patients showed bilateral MRI abnormalities. Signs of microangiopathy were also a regularly observed aspect in MRI (66.7% for all, 66.7% in biopsy-positive, 61.5% for angiography-positive patients, respectively). Patients with or without signs of microangiopathy significant differed regarding hemiparesis and ischemic stroke over the whole follow-up and cognitive decline and ischemic stroke in follow-up.

Furthermore they presented with a worse MRS. Gadolinium-enhanced lesions were detected in 8 patients (biopsy-positive patients, 66.7%; angiography-positive patients, 30.8, respectively). Patients with gadolinium enhancement were more often men (p=0.018) and had more frequent amnestic syndromes at disease onset (p=0.042) and a trend to a more frequent symptom onset with gait disturbances (p=0.056) compared with patients without gadolinium enhancing lesions. Patients with gadolinium enhancement were more often treated with cyclophosphamide compared to those without enhancing lesions (p=0.049). MR-angiography (MRA) was performed in 17 of 21 patients and was suggestive for vasculitis in 8 of 17 patients (47.1%). There was no statistical difference detectable between patients with or without positive MRA regarding demographical, clinical, diagnostic, treatment or outcome features.

Conventional cerebral angiography

Conventional cerebral angiography was carried out in 17 of 21 patients (81.0%) and was indicative for vasculitis in 13 of 17 patients (76.5%). Out of the 8 MRangiography-positive patients 6 also showed remarkable conventional angiography findings whereas in 6 angiography-positive patients MR-angiography was normal.

Conventional angiography was only performed in 2 of 6 biopsy-approved patients and was normal in both cases. Accordingly, conventional angiography and MRA revealed the same result in 9 of 16 patients (56.2%) who underwent both investigations. Angiographic-positive and -negative patients did not differ from each other according to demographic data, clinical or diagnostic and outcome features.

The association of positive color duplex sonography in angiography-proven patients failed to reach statistical significance (p=0.063).

Histological Features

CNS biopsy specimens were histological analysed in 12 of 21 patie nts (57.1%).

Histopathological examinations revealed definite diagnosis of PCNSV in 6 of 12 patients (50%). Out of these 6 patients, only in 2 an additional angiogram was performed and both were negative. Patients with positive CNS biopsy differed from the others by a significant lower quantity of abnormal color duplex sonography findings (p= 0.015). There were no other statistical differences detectable between biopsy-positive and biopsy-negative patients. Patients who underwent biopsy had a lower incidence of headaches in the whole follow up (p=0.024) and transient ischemic attacks in the follow up (p=0.08) but did not differ in other parameters.

Laboratory and Cerebrospinal Fluid Studies

In 19 of 21 patients (90.5%), CSF examinations were performed and disclosed abnormal findings (cell count > 5 cells /μl or total protein concentration > 45 mg/dl) in 13 of them (68.4%) (table 3). Samples were obtained in 12 of 13 angiography-proven (92.3%) and in 5 of 6 biopsy-proven patients (83.3%). In both diagnostic groups pathological results of cerebrospinal fluid (CSF) occurred in the similar incidence (60.0% for biopsy-based and 66.7% for angiography-based diagnoses, respectively).

The typical finding in CSF was a mild lymphocytic pleocytosis combined with an elevated protein level. Cell count was elevated in 68.4% and total protein was increased in 52.6% of patients. The mean cell count in PCNSV patients was 34.2/μl (median, 18/μl, range, 5-202/μl), the mean concentration of total protein was 88.4 mg/dl (median, 83.1 mg/dl, range 54-142 mg/dl). Oligoclonal banding was found occasionally. In 17 documented data sets with repeated lumbar taps oligoclonal banding was evident at least at one time in 4 patients (23.5%) with a similar distribution in both diagnostic groups (25.0% in biopsy-proven and 27.3% in angiography-proven cases). In all examinations, there were no hints for alternative diagnoses, particularly infections or neoplastic meningeal infiltrations. Patients with pathological CSF results did not differ from the others in almost all test criteria despite a higher frequency of gait disturbances at symptom onset (p=0.044).

Laboratory screening was unremarkable for complement factors C3 and C4, antinuclear antibodies, antibodies to extractable nuclear antigens, antineutrophil cytospasmic antibodies and HIV in all patients, rheumatic factors were mildly elevated in one patient only and special coagulation studies revealed an abnormal result in one patient (heterozygote factor V Leiden mutation).

Electroencephalopathy and Color Duplex Sonography

Electroencephalograms were obtained in 15 of 21 patients and showed abnormal findings with dysrhythmias in 4 of them (26.7%). All patients underwent extra -and transcranial color duplex sonography. Ten of them presented with intracranial vascular abnormalities, 10 with normal intracranial color duplex sonography and only one patient had mild extracranial signs for arte riosclerosis. Out of the 10 patients with pathological color duplex sonography studies, all 9 cases in which cerebral angiography was done were suggestive for vasculitis in angiogram. Positive MRA was associated with positive duplex color duplex sonography in 7 of 9 cases (77.7%).

Dividing patients in biopsy-approved and angiography-approved diagnostic groups an opposite proportion of color duplex sonography positive patients could be noted (biopsy-based diagnoses, 16.7%, angiography-approved, 69.2%). Biopsy-positive patients showed a significant lower fraction of pathological color duplex sonography results compared with the other patients (p=0.015), while higher incidence of positive color duplex sonography in angiography-positive patients failed to reach statistical significance (p= 0.063).

Treatments

Thrombocyte antiaggregation drugs (ASS or clopidrogrel) were prescribed in 12 of 21 patients (57.1%). An immunosuppressive therapy was documented in the grand majority of patients (85.7%). In detail this was intravenous glucocorticoid therapy in 50%, oral glucocorticoid therapy in 77.8%, intravenous and/or oral cyclophosphamide in 38.9%, azathioprine in 27.8%, methotrexate in 22.2% and mycophenolate mofetile in 22.2% of immunosuppressive treated patients, respectively. A positive response to therapy was noted in 13 of 18 sufficiently documented patients (72.2%). There was no statistical significant difference observable in biopsy-approved and angiographyapproved diagnostic groups. Patients with gadolinium enhanced lesions were more often treated with cyclophosphamide (p=0.049).

Outcome

Complications of treatments or diagnostic procedures were recognized over the course of the disease in 8 of 20 documented cases (20%). These were 2 complications after CNS biopsy with cerebral abscess or mild cerebral hemorrhage, respectively. In 6 patients mild complications of treatment like diarrhoea, cough, pneumonia, fever or mild polyneuropathy were observed. Three patients died within documented follow-up, two of them due to progressive cerebral disease, one patient died due to complications associated with the medical therapy. Modified Rankin disability scores were assessed at first presentation and at the last follow-up examination. Median MRS was 2 (range 1-4) at first presentation and 2 (range 1-6) at last visit information. MRS at both times and a functional outcome of 3 or worse was not significant related to different diagnostic groups (angiography-approved, biopsyapproved) or to performance of biopsy, appearance of gadolinium enhanced lesions, pathological CSF results or occurrence of cerebrovascular risk factors. Patients with MR-tomographic evident microangiopathy showed significantly worse MRS at onset and at last visit compared with the other patients (p=0.047, p=0.039). Functional outcome of 3 or worse failed to reach significant difference in patients with microangiopathy compared with the other patients (p=0.063).

Discussion

Primary angiitis of the central nervous system (PCNSV) is still one of the enigmas in clinical neurology [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF]. It seems to be very rare with an estima ted incidence from 2.4 :

1 000 000 to at less than 1 : 2 000 000 [29, [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Despite the growing knowledge on processes of CNS inflammation, this entity remains poorly understood. Its pathogenesis is still regarded as a mystery, its diagnosis and treatment is still a clinical challenge [START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Our existing understanding of the disease is based on a number of smaller case series. Since the first histological description in 1922 [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF] about 500 cases have been published worldwide [START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF]. One of the most relevant studies is the recent series of 101 patients from the Mayo Clinic [START_REF] Miller | Biopsy findings in primary angiitis of the central nervous system[END_REF][START_REF] Salvarani | Angiography-negative primary central nervous system vasculitis: a syndrome involving small cerebral vessels[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis with prominent leptomeningeal enhancement: a subset with a benign outcome[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis: comparison of patients with and without cerebral amyloid angiopathy[END_REF][START_REF] Salvarani | Primary CNS vasculitis with spinal cord involvement[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF][START_REF] Salvarani | Efficacy of tumor necrosis factor alpha blockade in primary central nervous system vasculitis resistant to immunosuppressive treatment[END_REF][START_REF] Salvarani | Prominent perivascular enhancement in primary central nervous system vasculitis[END_REF].

In our study we present data from a cohort of 21 European white patients with PCNSV with details about clinical manifestations, diagnostic and outcome features.

This study was intended to discover similarities or differences of our data compared to the previously published cases. Our analysis should enable a better characterization and understanding of this disease [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF].

As no reliable prospective data is available our view of the disease is based on retrospective analyses with all possible bias to date. Inclusion and exclusion criteria of the different studies may be a source of inaccuracy by falsely including patients with other differential diagnoses or by falsely excluding PCNSV patients which do not fulfill all diagnostic criteria. The previous ly suggested diagnostic criteria for PCNSV include symptoms of multifocal or diffuse CNS-disorder with remitting or progressive course, cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI) findings supporting the diagnosis of vasculitis, and finally either an angiography with a vasculitis pattern with multilocular narrowing and vessel occlusions or a leptomenigeal and parenchymatous biopsy proving vasculitis [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF][START_REF] Calabrese | Primary angiitis of the central nervous system. Report of 8 new cases, review of the literature, and proposal for diagnostic criteria[END_REF]29]. Diagnostic criteria, sensivity and specifity of angiography and biopsy have to be discussed later. However, we have to point out that our analysis not only includes 6 biopsy-approved and 13 angiography-approved patients but also 2 patients with negative or notperformed biopsy and negative angiogram. Inclusion of these 2 cases in contrast to 12 other doubtful cases was decided after detailed re-assessment and review because of clinical confidence of definite PCNSV. So considering of these two cases can be seen as an advantage in the effort to achieve a more representative characterization of the disease.

Nevertheless, in order to avoid misdiagnoses it is important to rule out relevant differential diagnoses based on a sophisticated diagnostic test battery and clinical judgment [START_REF] Ceccarelli | Primary angiitis of the central nervous system: a misinterpreted clinical onset of CNS vasculitis[END_REF]. Due to our clinical experience the spectrum of differential diagnoses in patients admitted to us for second opinion in evaluation of PCNSV consists of noninflammatory vasculopathy due to arteriosclerosis [START_REF] Kis | Severe supraaortal atherosclerotic disease resembling Takayasu's Arteritis[END_REF] or drugs [START_REF] Ringelstein | Case of inflammatory vasculopathy and encephalopathy caused by treatment with tacrolimus[END_REF], Moyamoya disease and syndrome [START_REF] Kraemer | Moyamoya disease in Europeans[END_REF] and Sneddon´s syndrome [START_REF] Kraemer | Miscarriage, peripheral thromboses and aortic aneurysm in antiphospholipid-antibody-negative Sneddon's syndrome[END_REF][START_REF] Kraemer | Cerebral haemorrhage as the presenting form of Sneddon's syndrome[END_REF][START_REF] Kraemer | Long-term course of antiphospholipid-antibody-negative Sneddon's syndrome[END_REF][START_REF] Kraemer | The spectrum of differential diagnosis in neurological patients with livedo reticularis and livedo racemosa. A literature review[END_REF] as well as atypical cases of multiple sclerosis [START_REF] Kis | Clinical characteristics of patients with late-onset multiple sclerosis[END_REF]. One other important differential diagnosis of PCNSV is bacterial endocarditis [START_REF] Berlit | Isolated angiitis of the CNS and bacterial endocarditis: similarities and differences[END_REF], because both conditions may present with similar clinical and auxiliary findings but effort a different treatment regimen [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF]. PCNSV should also be distinguished from vasoconstriction syndromes associated with drugs, migraine, hypertension, eclampsia or the post-partum-period [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. As in spasms after subarachnoid hemorrhage reversible vasoconstriction syndromes (the so-called Call-Fleming syndrome) often presented with thunderclap headache and are more frequent than PCNSV [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF][START_REF] Calabrese | Primary central nervous system vasculitis: progress and questions[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. As mentioned above we excluded 12 uncertain PCNSV patients in which important differential diagnoses were considered and 16 patients with secondary vasculitis [START_REF] Kraemer | Systemic, secondary and infectious causes for cerebral vasculitis: clinical experience with 16 new European cases[END_REF]. These cases were associated with systemic disorders like Behcet syndrome [START_REF] Berlit | Behcet's disease is associated with increased concentrations of antibodies against phosphatidylserine and ribosomal phosphoproteins[END_REF][START_REF] Kramer | Myalgia in Neuro-Behcet's disease. Medication side effects or expression of underlying disease[END_REF] (7 cases), Wegener granulomatosis, giant cell arteritis, Churg-Strauss-syndrome [START_REF] Berlit | Diagnosis and therapy of Churg-Strauss allergic granulomatosis[END_REF], sarcoidosis, connective tissue disorders or infectious diseases like borreliosis and neurolues. Unfortunately, we were not able to identify the exact number of patients frequently referred to us to re-evaluate suspicion of CNS vasculitis in which this diagnosis was clearly excludable. In the Mayo clinic's cohort this information is also missing [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF].

Regarding demographic data and diagnostic test establishing the diagnosis, convincing similarities to Salvarini´s cohort are observable [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. Despite earlier reports of a higher frequency of PCNSV in males, in our cohort female to male ratio was 1.6 : 1 comparable to Salvarini´s 1.3 : 1. Diagnostic procedures to establish diagnosis of PCNSV were also utilized as frequent as in the cohort of 101 patients (angiography approved, 61.9% compared to 69.3%; biopsy-approved, 28.6% compared to 30.7%). Age distribution also was consistent to former reported ranges.

The time from first symptom to correct diagnosis was equally to the Mayo Clinic's data if regarding our median time but longer if regarding the mean time due to 4 patients in which time to diagnoses was much longer than one year [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF].

The strength of our demographic data is the accuracy in describing additional diseases and cerebrovascular risk factors. Both are missing in the American paper [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. In the analysis of 101 patients only one patient with chronic lymphoctic leukemia is noted. In contrast to this Salvarini´s working group identified 3 other medical diseases (hypothyreodism and Graves ophthalmopathy) in the analysis of 8 angiography-negative cases [START_REF] Salvarani | Angiography-negative primary central nervous system vasculitis: a syndrome involving small cerebral vessels[END_REF]. In the subset of 8 PCNSV patients with prominent leptomenigeal enhancement additional diseases were diabetes, hypothyreodism and Crohn's disease [START_REF] Salvarani | Prominent perivascular enhancement in primary central nervous system vasculitis[END_REF]. The frequency of co-occurring autoimmune diseases in our study (23.8%) points to the clinical problem in districting co-occurrence versus causative systemic disorders. After careful consideration, in individual cases cooccurring autoimmune disorders in patients and their family members should not be regarded as an argument against PCNSV but as a sign for their autoimmune predisposition. In difference to the American working group we assessed cerebrovascular risk factors. Despite confidence of diagnosis of definite PCNSV, in these patients with cerebrovascular risk factors this feature was associated with a higher incidence of ischemic lesions over the follow-up period (p=0.025) and MRtomographic evident microangiopathy (p=0.025) without differences in other clinical and outcome features. The occurrence of cerebrovascular risk factors with a great amount of smokers (47.6%) and patients with arterial hypertension (42.9%) seemed not to be surprising or unusual in persons over the age of 40 years. Although -agerelated-extracranial signs of arteriosclerosis with mild carotid plaques were recognized in one patient only we want to call attention to the diagnostic problem of differentiating the possibility of co-occurrence of PCNSV with arteriosclerosis from an isolated arteriosclerotic vasculopathy. To the best of our knowledge, this theme is neglected in the existing literature. Nevertheless, in clinical practice, patients with PCNSV should be motivated to avoid smoking and cerebrovascular risk factors should be corrected.

Clinical hallmarks of PCNSV are headaches, strokes, encephalopathy with cognitive disturbances and myelopathy [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF]. Headaches are known to occur in two-thirds of patients and are variable in quality and severity [START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. None of our 57.1% of patients with headache described it as "thunderclap" headache. Non-focal signs, such as neuropsychiatric findings or a decrease in memory [START_REF] Calabrese | Vasculitis in the central nervous system[END_REF] are typical for CNS vasculitis.

Cognitive alterations were found in similar frequency of patients in our and in the American series [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. In contrast to our and the Mayo Clinic´s statistics a former critical review of PCNSV denied stroke as the presenting symptom in any histological proven case [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. We have to discuss that stroke often reveals the disease but perhaps does not represent the retrospective evaluated initial manifestation. In our study only one biopsy-approved patient presented with intracranial hemorrhage, three recurrent hemorrhages appeared in this patient. Summarizing the data of our with the Mayo Clinic's cohort we disagree with former reflections that bleedings could be more prevalent than ischemic strokes in PCNSV [START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Pathophysiologically bleeding is thought to be caused from vessel wall weakness resulting from transmural inflammation [START_REF] Kristoferitsch | Cerebral granulomatous angiitis with atypical features[END_REF][START_REF] Negishi | Vasculitis presenting as primary leptomeningeal enhancement with minimal parenchymal findings[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Magnetic resonance imagings were abnormal in all of our patients and in over 96% in the Mayo Clinic's study [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. Normal MR-imaging almost excludes PCNSV. However, the abnormalities observed are absolutely nonspecific [START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF]. Schmidley pointed out that imaging is nonetheless important, as it might suggest other treatable etiologies for the patient's complaints [START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF]. Diffuse white matter lesions and microangiopathy were commonly observed. Despite pronunciation of disseminated T2 hypersignals in periventricular white matter in multiple sclerosis, MR-tomographic differentiation to PCNSV can be difficult [START_REF] Scolding | Can diffusion-weighted imaging improve the diagnosis of CNS vasculitis?[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Prominent gadolinium-enhancement of the leptomenix seemed to be less frequent than formerly supposed [START_REF] Chu | Diagnosis of intracranial vasculitis: a multi-disciplinary approach[END_REF][START_REF] Negishi | Vasculitis presenting as primary leptomeningeal enhancement with minimal parenchymal findings[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Gadolinium-enhanced lesions were observed in 38% of our patients and in about one third of Salvarini´s patients [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. Regarding the latest widespread usage of MRangiography for evaluation of suspected PCNSV, we want to stress that MRA was suggestive for vasculitis only in 59% of the American patients and 47.1% of our patients. Our rate of positive association of MR-and conventional angiography is worse than Salvarini´s data (56.2% versus 78%) [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. So we want to warn to overestimate the sensitivity of MRA which remains unquestionably lower than conventional angiography [START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Conventional cerebral angiography was indicative for vasculitis in 76.5% in our patients whereas 90% of the Mayo Clinic's cohort were positive [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. In contrast to the former opinion of a more benign course of PCNSV diagnosed on the basis of angiography alone our data does not indicate this comparable with the Mayo Clinic's results [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF][START_REF] Calabrese | Angiographically defined primary angiitis of the CNS: is it really benign?[END_REF][START_REF] Goldstein | Angiographically defined primary angiitis of the CNS: is it really benign?[END_REF]. Conventional angiography was only performed in 2 of 6 biopsy-approved patients and was not positive in both cases.

If the inflammatory findings are limited to arteries less than 500μm in diameter, angiography often is normal [START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Salvarini et al. observed 8 angiography-negative patients and concluded that this subtype of CNS vasculitis with small vessel involvement beyond resolution of conventional angiography has a more favourable outcome [START_REF] Salvarani | Angiography-negative primary central nervous system vasculitis: a syndrome involving small cerebral vessels[END_REF]. Furthermore, the American working group described some more characteristics like more common cognitive complaints, higher CSF cell and protein counts and more frequent gadolinium enhanci ng lesions [START_REF] Salvarani | Angiography-negative primary central nervous system vasculitis: a syndrome involving small cerebral vessels[END_REF]. In our series we only observed three patients with negative angiogram but could not duplicate the findings of Salvarini and colleagues [START_REF] Salvarani | Angiography-negative primary central nervous system vasculitis: a syndrome involving small cerebral vessels[END_REF]. The clinically relevant question is if the partly observed differences between the diagnostic groups in Salvarini´s and our cohort really represent heterogeneous subtypes of the disease. It is apparent, however, that angiography and biopsy do not provide the same information in all patients [START_REF] Calabrese | Angiographically defined primary angiitis of the CNS: is it really benign?[END_REF]. In Salvarini´s cohort 18 patients had positive angiograms but negative biopsy findings, in our cohort these were 5 patients [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. It could be suggested that affected vessels may be larger and were not reached by biopsy. In Yin´s Chinese study all cases diagnosed by angiography alone had large vessel involvement [START_REF] Yin | Primary angiitis of the central nervous system: report of eight cases from Southern China[END_REF]. The opposite conclusion can be reached from the fact that in our biopsy-approved patients positive color duplex sonography findings are significantly sparse which also could argue for small vessel involvement. Biopsy-approved PCNSV was found in 50% of our and 62% of Salvarini´s patients who underwent biopsy. Correct neurosurgical performance of biopsy is crucial for reaching positive results. The ideal diagnostic brain biopsy is a 1 cm 3 brain tissue with both gray and white matter as well as leptomeninges and preferably a cortical vessel [29,[START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. The risk of false-negative brain biopsy is not small and can be explained by the segmental involvement of vessels and a possible mismatch between radiological abnormalities and histological predominant lesions [START_REF] Oliveira | Cerebrospinal fluid and therapy of isolated angiitis of the central nervous system[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. It is consensus that open biopsy lessens chances of a false-negative result compared with stereotactic needle biopsy [START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. The morbidity rate of brain biopsy (0.03-2%) has to be balanced with the risk of unnecessary immunosuppressive treatments [START_REF] Alreshaid | Prognosis of patients with suspected primary CNS angiitis and negative brain biopsy[END_REF][START_REF] Goldstein | Primary central nervous system vasculitis: is biopsy appropriate?[END_REF][START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF]. Chu et al. found that morbidity associated with biopsy was less than with "blind" therapeutic immunosuppression [START_REF] Chu | Diagnosis of intracranial vasculitis: a multi-disciplinary approach[END_REF][START_REF] Goldstein | Primary central nervous system vasculitis: is biopsy appropriate?[END_REF]. In our cohort those patients which underwent biopsy differed from those without surgery by a lower percentage of headaches in the follow-up period and transient ischemic attacks. This difference obviously is not related to the biopsy procedure but is biased by selection of clinically untypical cases without headaches and transient ischemic attacks to biopsy. Patients with and without surgery do not differ regarding all other parameters. Despite the fact that angiography has become the preferred method of diagnosing PCNSV, we conclude that still biopsy should be regarded as the gold standard [START_REF] Goldstein | Primary central nervous system vasculitis: is biopsy appropriate?[END_REF]. It is important to realize that a negative biopsy certainly does not rule out the condition and it should be considered as an attempt to establish the diagnosis and exclude other conditions [START_REF] Goldstein | Primary central nervous system vasculitis: is biopsy appropriate?[END_REF]. However, it is still a source of controversy what should be done with patients suspected to have a PCNSV having a negative biopsy.

One study showed that immunosuppressive treatment is not clearly superior in those patients compared to patients followed up with a wait and see strategy [START_REF] Abu-Shakra | Primary angiitis of the CNS diagnosed by angiography[END_REF][START_REF] Alreshaid | Prognosis of patients with suspected primary CNS angiitis and negative brain biopsy[END_REF][START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF]. In contrast to this, other authors conclude that immunosuppressive treatment remains appropriate for those with a consistent clinical and radiological presentation and a negative biopsy [START_REF] Goldstein | Primary central nervous system vasculitis: is biopsy appropriate?[END_REF].

Our laboratory results were mainly within normal ranges as in former studies [START_REF] Abu-Shakra | Primary angiitis of the CNS diagnosed by angiography[END_REF][START_REF] Calabrese | Primary angiitis of the central nervous system. Report of 8 new cases, review of the literature, and proposal for diagnostic criteria[END_REF]29]. Although a large-scale "laboratory screen" for autoimmune or infectious diseases is essential to exclude treatable differential diagnoses, one should be prepared for occasional "positive" results which are difficult to explain, and may at last be of minor clinical relevance [START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF]. The lower percentage of performance of lumbar tap in the American study (74.3% compared with 90.5%) is perplexing from the European point reports which are impossible to interpret [START_REF] Elkind | Cerebral vasculopathy does not equal primary central nervous system vasculitis[END_REF]. Combined aggressive therapy should be reserved for those patients with a deteriorating clinical status. Histological proven definite diagnosis should be mandatory in these patients but this once again raises the problem of false-negative biopsy. Alternative treatment regimes for example with intravenous gammaglobulines are not widely used [START_REF] Steinbrecher | Intravenous immunoglobulin treatment in vasculitis and connective tissue disorders[END_REF]. Furthermore it is often complex to estimate the success of treatments. Clinical assessment of therapy success is often influenced by impression of relapses and remissions. However, this also could be caused by normal recovery of cerebral ischemia [START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF]. To our experience, the activity of the disease under treatment is appreciable only by combined clinical, CSF and neuroradiological monitoring. Drop of CSF abnorma lities is reported corresponds with clinical improvement [START_REF] Oliveira | Cerebrospinal fluid and therapy of isolated angiitis of the central nervous system[END_REF]. The successful use of serial conventionally angiography has been reported [START_REF] Alhalabi | Serial angiography in isolated angiitis of the central nervous system[END_REF], but in clinical practise also MRI combined with color duplex sonography studies is increasingly used for follow-up [START_REF] Moritani | CNS vasculitis and vasculopathy: efficacy and usefulness of diffusion-weighted echoplanar MR imaging[END_REF][START_REF] Ritter | Follow-up examinations by transcranial Doppler ultrasound in primary angiitis of the central nervous system[END_REF]. A prolonged neurological supervision is absolutely necessary. Nevertheless, explicit guidelines for follow-up have not been established to date [START_REF] Calabrese | Primary central nervous system vasculitis: progress and questions[END_REF]. So our treatment and outcome data should be interpreted carefully. Clinically and pathophysiologically it makes sense that patients with microangiopathy have a worse outcome. Further interpretations about outcome concluded on the base of the different subgroups of Mayo Clinic's cohort should be regarded considering potential limitations and bias [START_REF] Miller | Biopsy findings in primary angiitis of the central nervous system[END_REF][START_REF] Salvarani | Angiography-negative primary central nervous system vasculitis: a syndrome involving small cerebral vessels[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis with prominent leptomeningeal enhancement: a subset with a benign outcome[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis: comparison of patients with and without cerebral amyloid angiopathy[END_REF][START_REF] Salvarani | Primary CNS vasculitis with spinal cord involvement[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF][START_REF] Salvarani | Efficacy of tumor necrosis factor alpha blockade in primary central nervous system vasculitis resistant to immunosuppressive treatment[END_REF][START_REF] Salvarani | Prominent perivascular enhancement in primary central nervous system vasculitis[END_REF].

The existing scientific literature on PCNSV is incomplete and difficult to evaluate as much of current understanding comes from smaller case series and single case reports [START_REF] Salvarani | Primary central nervous system vasculitis with prominent leptomeningeal enhancement: a subset with a benign outcome[END_REF].

Our investigation has a number of limitations. Most of the data was assessed retrospective, prospective follow-up was incomplete. It is possible that missing duplication of some characterizations of subgroups described by Salvarini and co-workers is caused by smaller study cohort [START_REF] Miller | Biopsy findings in primary angiitis of the central nervous system[END_REF][START_REF] Salvarani | Angiography-negative primary central nervous system vasculitis: a syndrome involving small cerebral vessels[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis with prominent leptomeningeal enhancement: a subset with a benign outcome[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis: comparison of patients with and without cerebral amyloid angiopathy[END_REF][START_REF] Salvarani | Primary CNS vasculitis with spinal cord involvement[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF][START_REF] Salvarani | Efficacy of tumor necrosis factor alpha blockade in primary central nervous system vasculitis resistant to immunosuppressive treatment[END_REF][START_REF] Salvarani | Prominent perivascular enhancement in primary central nervous system vasculitis[END_REF]. The possibility of referral bias has to be calculated. Sensivity and specifity of different diagnostic approves can influence interpretation of results. What should be done with patients presenting with false-negative biopsy and false-negative angiography but who are clinically considered as definite PCNSV?

Our investigation is elucidating and valuable because it represents one of the largest and most detailed descriptions of European patients with PCNSV.

The strength of our study is the large number of consecutive patients treated over the short period of 5 years. Beside the Mayo Clinic ´s cohort with 101 patients our cohort with 21 subjects is large compared with other case series in the existing literature [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. An important advantage of our short period of 5 years in which we were able to identify 21 patients with PCNSV isin contrast to the two decades in Mayo Clinic´s cohortthe greater change that quality of diagnostic measurements like MRI is equivalent [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. A further strong point in our analysis is the attempt to measure prospective data. Data collection was focussed on demographic, clinical, diagnostic and outcome data. Study design similar to the Mayo Clinic's cohort makes our investigation comparable [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. Furthermore this study clearly points out the remaining deficits in knowledge of the disease. To date it is still a challenge to diagnose, treat and follow-up patients thought to have PCNSV. Due to the missing clinical guidelines and the rarity of the disease diagnosis and treatment of PCNSV should be reserve to some clinical centers with great expertise in vasculitis. Furthermore the complex nature of the disease requires an intensive multiprofessional approach with good cooperation of neurological, rheumatologic, neurosurgical and neuroradiological experts in this field. In order to solve some aspects of its enigma, a prospective multicenter collaborative study collecting patients suspected to have cerebral vasculitis as recent demanded by Calabrese, Malloy and Singhal as well as by Berlit

:

  Our cohort included 21 white patients with PCNSV treated in a single German institution between 2003 and 2008. Clinical and diagnostic features were obtained by retrospective chart review; follow-up information and outcome were obtained prospectively. Clinical findings and outcomes were compared among patients categorized by different characteristics.

Acknowledgements:

The authors thank colleagues from the neurological, neurosurgical, neuroradiological and medical departments of Alfried-Krupp-Hospital and colleagues from outside hospitals for good cooperation in management of patients with CNS vasculitis.

Especially we want to thank M. W. Baumgaertel, MD (Muenster, Germany), R.R. Diehl, PhD (Essen, Germany), M. Herold, MD (Essen, Germany), A. Kilian (Essen, Germany) and C. Kraemer (Muenster, Germany).

Fundings:

The study was not supported by funding. of view. The major importance of CSF examinations also lies in excluding alterative diagnoses [START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF]. As usually in PCNSV, the typically -but unfortunately-nonspecific finding was mild lymphocytic pleocytosis and elevated protein [START_REF] Oliveira | Cerebrospinal fluid and therapy of isolated angiitis of the central nervous system[END_REF][START_REF] Schmidley | 10 questions on central nervous system vasculitis[END_REF]. In the recent published Chinese series of 8 cases the majority even of biopsy-approved patients presented with normal CSF results [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF][START_REF] Yin | Primary angiitis of the central nervous system: report of eight cases from Southern China[END_REF]. As mentioned above Salvarini et al. found a significant higher protein level and cell count in CSF in angiography-negative patients which could not be duplicated in our smaller cohort [START_REF] Salvarani | Angiography-negative primary central nervous system vasculitis: a syndrome involving small cerebral vessels[END_REF]. Unfortunately, in the American study information about oligoclonal banding is absent [START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. In our cohort oligoclonal banding was found occasionally (23.5%). Both electroencephalography and color duplex sonography are nonspecific. Our result with a significant smaller amount of abnormal intracranial color duplex sonography in biopsy-approved and a trend to more frequent abnormal intracranial duplex sonography in angiographyapproved patients may represent the different involvement of small or large vessels in both diagnostic groups.

No controlled therapeutic trials have been conducted in PCNSV to date [START_REF] Fountain | Control of primary angiitis of the CNS associated with cerebral amyloid angiopathy by cyclophosphamide alone[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. In an often cited review of 46 patients, 19 of the 20 non-treated patients rapidly progressed either to death or to persistence of severe sequelae [START_REF] Calabrese | Primary angiitis of the central nervous system. Report of 8 new cases, review of the literature, and proposal for diagnostic criteria[END_REF][START_REF] Zuber | Isolated angiitis of the central nervous system[END_REF]. Treatment regimes are often orientated on experiences in systemic vasculitides. In our cohort an immunosuppressive therapy was given in the grand majority of patients (85.7%). In detail this was intravenous glucocorticoid therapy in 50%, oral glucocorticoid therapy in 77.8%, intravenous and/or oral cyclophosphamide in 38.9%, azathioprine in 27.8%, methotrexate in 22.2% and mycophenolate mofetile in 22.2% of immunosuppressive treated patients, respectively. The Mayo Clinic's statement that "most patients showed a favourable response to glucocorticoids alone or in combination with cyclophosphamide" is very unprecise [START_REF] Elkind | Cerebral vasculopathy does not equal primary central nervous system vasculitis[END_REF][START_REF] Salvarani | Primary central nervous system vasculitis: analysis of 101 patients[END_REF]. The experiences on treatments are based on partly inadequately defined populations and on anecdotal is urgently needed [START_REF] Berlit | Primary angiitis of the CNS -an enigma that needs world-wide efforts to be solved[END_REF][START_REF] Calabrese | Primary central nervous system vasculitis: progress and questions[END_REF]. Furthermore the lack of an animal model represents a significant obstacle in achieving new insights in understanding pathophysiological mechanisms in PCNSV [START_REF] Calabrese | Primary central nervous system vasculitis: progress and questions[END_REF].

Finding

Table 3. Cerebrospinal Fluid Findings


MD Piotr Adrian Klimiuk

PhD, Assistant; Stanislaw Sierakowski

Professor and Chairman; Izabela Domyslawska

Assistant; Justyna Chwiecko

Serum chemokines in patients with rheumatoid arthritis treated with etanercept

Keywords: IL-8, RANTES, MCP-1, rheumatoid arthritis, etanercept

Objective. Chemokines promote leukocyte traffic into the synovium, leading to the initiation and progression of the rheumatoid arthritis (RA). The aim of the study was to determine the effects of etanercept, a soluble tumour necrosis factor receptor (sTNFr), on the serum chemokines levels in patients with active RA.

Methods. Patients were treated with 50mg of subcutaneous injection of etanercept per week and methotrexate (10-25mg/week). Serum levels of interleukin-8 (IL-8), RANTES (regulated upon activation, normal T cell expressed and secreted) and monocyte chemoattractant protein-1 (MCP-1) were assessed by ELISA at months 0, 3, 6, 9 and at month 12, prior to injection.

Results. Three months treatment with etanercept diminished serum concentrations of IL-8, RANTES and MCP-1 (p<0.05, p<0.01 and p < 0.001, respectively). Subsequent etanercept administrations prolonged decrease in serum chemokines levels and in the case of IL-8 even intensified the reduction of its concentration in serum. These changes were accompanied by significant decrease of disease activity score (DAS28) (in all cases p<0.001). Prior to the first etanercept administration serum concentrations of studied chemokines correlated with markers of RA activity such as the erythrocyte sedimentation rate (ESR) and DAS28. Following next drug injection such associations were less or not significant.

Conclusion.

Therapy with etanercept and MTX, not only caused a clinical improvement, but also diminished serum chemokines levels in RA patients. Further treatment with etanercept sustained chemokines suppression.

Key words: chemokines,

Introduction

Rheumatoid arthritis (RA) is characterized by infiltration of mononuclear cells into synovium.

Enhanced angiogenesis and the proliferation of the synovium-lining layer may also be noticed.

Macrophages, fibroblasts, lymphocytes and endothelial cells in rheumatoid synovium are supposed to contribute to the pathogenesis of the disease by several mechanisms including chemokines production [START_REF] Tarrant | Chemokines and leukocyte trafficking in rheumatoid arthritis[END_REF][START_REF] Vergunst | The role of chemokines in rheumatoid arthritis and osteoarthritis[END_REF]. Chemokines have an important role in the migration of mononuclear cells into synovium, leading to the initiation and progression of the disease.

These small proteins enhance the inflammation by recruiting and activating leukocyte subpopulations. Furthermore, chemokines like interleukin-8 (IL-8) or monocyte chemoattractant protein-1 (MCP-1) intensify infiltration of synovial tissue by inflammatory cells through the stimulation of neovascularisation of rheumatoid synovium [START_REF] Haringman | Chemokines in joint disease: the key to inflammation?[END_REF][START_REF] Szekanecz | Chemokines and angiogenesis in rheumatoid arthritis[END_REF]. Chemokine production is stimulated by inflammatory cytokines like interleukin-1 (IL-1) and tumour necrosis factor α (TNF-α) [START_REF] Koch | Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis[END_REF][START_REF] Pulsatelli | Chemokine production by human chondrocytes[END_REF]. In addition, chemokines promote synthesis of proinflammatory cytokines like IL-1, IL-6, TNF-α or matrix metalloproteinases engaged in the joint destruction in RA [START_REF] Haringman | Chemokines in joint disease: the key to inflammation?[END_REF][START_REF] Klimiuk | Serum matrix metalloproteinases and tissue inhibitors of metalloproteinases in different histological variants of rheumatoid synovitis[END_REF]. Abundant presence of such chemokines like IL-8, RANTES (regulated upon activation, normal T cell expressed and secreted) or MCP-1 in rheumatoid synovium even in early stage of the disease, support the role of chemokines in RA [START_REF] Katrib | Expression of chemokines and matrix metalloproteinases in early rheumatoid arthritis[END_REF][START_REF] Takahashi | The participation of IL-8 in the synovial lesions at an early stage of rheumatoid arthritis[END_REF][START_REF] Haringman | Chemokine and chemokine receptor expression in paired peripheral blond mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis[END_REF].

Several studies have shown that anti-TNF-α treatment with infliximab, a chimeric antitumour necrosis factor α antibody [START_REF] Lipsky | Infliximab and methotrexate in the treatment of rheumatoid arthritis[END_REF][START_REF] Maini | Infliximab (chimeric anti-tumour necrosis factor α monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial[END_REF] and etanercept, a soluble tumour necrosis factor receptor (sTNFr) [START_REF] Weinblatt | A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate[END_REF][START_REF] Van Der Heijde | Comparison of etanercept and methotrexate, alone and combined, in the treatment of rheumatoid arthritis: two-year clinical and radiographic results from the TEMPO study, a double-blind, randomized trial[END_REF][START_REF] Van Riel | Add Enbrel or Replace Methotrexate Study Investigators. Efficacy and safety of combination etanercept and methotrexate versus etanercept alone in patients with rheumatoid arthritis with an inadequate response to methotrexate: the ADORE study[END_REF] diminish clinical activity of RA. In our previous report, we demonstrated that infusions of infliximab reduce serum chemokines concentrations in RA patients [START_REF] Klimiuk | Regulation of serum chemokines following infliximab therapy in patients with rheumatoid arthritis[END_REF]. In the present study, we investigated the effect of the one-year therapy with etanercept on serum IL-8, RANTES and MCP-1 serum levels in patients with active rheumatoid arthritis.

Materials and methods

Patients and samples

Eligible patients (3 men and 14 women) met the American College of Rheumatology 1987 revised diagnostic criteria for RA [START_REF] Arnett | The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis[END_REF]. At the time of enrolment, patients had active RA, as manifested by at least 6 tender and 6 swollen joints, and two of the following: morning stiffness for more than 45 minutes, C-reactive protein (CRP) level of more than 20 mg/l, erythrocyte sedimentation rate (ESR) of more than 28 mm/h. None of the patients had previous history of tuberculosis or symptoms of infectious diseases in the previous 3 months. Chest x-rays performed prior to the first etanercept administration were normal in all patients.

Mean patients age was 41.6 years (range 19-71 years), and mean disease duration was 5.1±4.4 years. All subjects were receiving methotrexate (MTX) (median 17.5 mg/week, range 10-25 mg/week) in a stable dose for at least 2 months and nonsteroidal anti-inflammatory drugs (NSAIDs) in a stable dose for at least 4 weeks before enrolment into the study. Fourteen patients were receiving corticosteroids (median 7.5 mg/day of prednisone or equivalent, range 5-10mg/day) in a stable dose for at least 4 weeks before beginning of the study. Such treatment regimen with MTX, NSAIDs and corticosteroids was maintained throughout the study.

Patients were receiving etanercept, a soluble tumour necrosis factor receptor (sTNFr), 50mg subcutaneously weekly during 12 months. Clinical and laboratory assessment were conducted prior to injection at baseline and at months 3, 6, 9, and 12. Blood samples were clotted for 30 minutes and next centrifuged for 15 minutes at 1000 x g. Serum aliquots were stored at -80°C until assayed.

The local ethical committee approved the study protocol and all patients provided written informed consent.

Clinical and laboratory measures

The evaluation included the number of tender joints (of 28 joints assessed), the number of swollen joints (of 28 assessed), three variables disease activity score that include 28-joint counts (DAS28) [START_REF] Prevoo | Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis[END_REF], the duration of morning stiffness, blood cell counts, including differential white blood counts, erythrocyte sedimentation rate (ESR), rheumatoid factor level, transaminases activity and creatinine concentration in serum. Measurements were performed at baseline and at months 3, 6, 9 and 12 (prior to etanercept injection).

Enzyme-linked immunosorbent assays (ELISA)

The assessments of serum levels of studied chemokines (IL-8, RANTES, MCP-1) were determined by ELISA kits from R&D Systems, Wiesbaden-Nordenstadt, Germany, strictly according to the manufacturer's instructions. The sensitivity of the assays was 3.5 pg/ml (IL-8), 2 pg/ml (RANTES) and 5 pg/ml (MCP-1).

Statistical analysis

The normally distributed data were analyzed by paired Student t-test. Wilcoxon signed rank test was used to compare the differences between non-normally distributed data. Correlations between variables were assessed by Spearman rank order test. P values lower than 0.05 were considered statistically significant.

Results

Serum concentrations of chemokines

Etanercept decreased serum concentrations of IL-8 after three months of treatment as compared to baseline values (p<0.05) (Figure 1). Continuation of therapy even more significantly diminished serum IL-8 levels as assessed at months 6, 9 and 12 (in all cases p<0.001). Down regulation of IL-8 was accompanied by the fall of serum concentrations of RANTES as evaluated at months 3, 6, 9 and 12 (p<0.01, p<0.001, p<0.001 and p<0.01 respectively) (Figure 2). Three months treatment with etanercept suppressed also MCP-1 serum level (p<0.001) (Figure 3). Subsequent drug administrations prolonged MCP-1 reduction, although less significantly (p<0.01).

Correlations between serum levels of chemokines and clinical data Down regulation of circulating chemokines was accompanied by significant decrease of three variables disease activity score that include 28-joint counts (DAS28) as assessed at months 3, 6, 9 and 12 (in all cases p<0.001) (data not shown). At baseline we demonstrated significant correlations between IL-8 and RANTES serum concentrations, and such markers of RA activity as the erythrocyte sedimentation rate (ESR) or DAS28 (in all cases p<0.01) (Table 1).

Furthermore we observed significant correlations between MCP-1 serum levels and ESR or DAS28 (p<0.01 and p<0.05 respectively). During therapy with etanercept such associations were less or not significant. No associations between patient sex, age or disease duration with studied circulating chemokines were observed.

Discussion

Chemokines are proteins, which work as mediators of inflammation process by recruiting and activating particular leukocyte populations. Along with adhesion molecules, they control the migration of mononuclear cells into synovium, leading to the initiation and progression of the rheumatoid arthritis. Chemokine synthesis is regulated by inflammatory cytokines like interleukin-1 (IL-1) and tumour necrosis factor α (TNF-α) [START_REF] Koch | Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis[END_REF][START_REF] Pulsatelli | Chemokine production by human chondrocytes[END_REF]. Previously we revealed that another blocker of TNF-α, a chimeric anti-TNF-α monoclonal antibody (infliximab), not only suppress clinical activity of RA, but also down regulate serum levels of IL-8, RANTES and MCP-1 [START_REF] Klimiuk | Regulation of serum chemokines following infliximab therapy in patients with rheumatoid arthritis[END_REF]. In current study, we analysed serum concentrations of IL-8, RANTES and MCP-1 in patients with active RA treated with etanercept, a soluble tumour necrosis factor receptor (sTNFr). IL-8 is a member of CXC chemokine family and is produced by different cells like macrophages, fibroblasts, T cells, neutrophils, endothelial cells and chondrocytes [START_REF] Haringman | Chemokines in joint disease: the key to inflammation?[END_REF][START_REF] Pulsatelli | Chemokine production by human chondrocytes[END_REF].

Elevated IL-8 levels were found not only in serum [START_REF] Klimiuk | Histological patterns of synovitis and serum chemokines in patients with rheumatoid arthritis[END_REF][START_REF] Kaneko | Interleukin-6 and interleukin-8 levels in serum and synovial fluid of patients with osteoarthritis[END_REF] but also in synovial fluid [START_REF] Kaneko | Interleukin-6 and interleukin-8 levels in serum and synovial fluid of patients with osteoarthritis[END_REF] of RA patients compared to patients with OA or in healthy controls. In our present study, we demonstrated that treatment with etanercept decreased serum levels of IL-8 in RA patients.

Especially serum concentrations of that chemokine were reduced after 6 months of therapy and afterwards. Prior to etanercept administration serum IL-8 was correlated with clinical markers of disease activity like erythrocyte sedimentation rate (ESR) and disease activity score (DAS28). Circulating IL-8 was demonstrated by other investigators to correlate with serum MCP-1 [START_REF] Koch | Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis[END_REF]. We also observed the association between serum levels of IL-8 and MCP-1. Moreover, we noted the correlation between serum IL-8 and RANTES concentrations prior to the beginning of etanercept treatment. However, after subsequent drug injections such associations were less or not significant.

RANTES (regulated upon activation, normal T cell expressed and secreted) belongs to

CC chemokine family, produced by T cells and synovial fibroblasts [START_REF] Robinson | Chemokine expression in rheumatoid arthritis (RA): evidence of RANTES and macrophage inflammatory protein (MIP)-1 beta production by synovial T cells[END_REF][START_REF] Volin | RANTES expression and contribution to monocyte chemotaxis in arthritis[END_REF], and involved in monocyte and T lymphocyte migration [START_REF] Haringman | Chemokines in joint disease: the key to inflammation?[END_REF][START_REF] Ellingsen | In vitro migration of mononuclear cells towards synovial fluid and plasma from rheumatoid arthritis patients correlates to RANTES synovial fluid levels and to clinical pain parameters[END_REF]. Enhanced concentrations of RANTES were found in RA serum [START_REF] Klimiuk | Histological patterns of synovitis and serum chemokines in patients with rheumatoid arthritis[END_REF][START_REF] Boiardi | Relationship between serum RANTES levels and radiological progression in rheumatoid arthritis patients treated with methotrexate[END_REF] and synovial fluid [START_REF] Volin | RANTES expression and contribution to monocyte chemotaxis in arthritis[END_REF] compared to OA patients and healthy individuals. In rheumatoid arthritis this chemokine was demonstrated to be predictive of radiological erosions [START_REF] Boiardi | Relationship between serum RANTES levels and radiological progression in rheumatoid arthritis patients treated with methotrexate[END_REF]. In our study we showed that therapy with etanercept reduce serum levels of RANTES in RA patients. Further drug administrations prolonged circulating RANTES reduction in studied patients. Prior to the etanercept therapy serum RANTES concentrations were demonstrated by us to correlate with ESR and DAS28. Furthermore, serum RANTES was associated also with IL-8 and MCP-1 levels. As in the case of IL-8, after further etanercept administrations such correlations were less or not significant. MCP-1 (monocyte chemoattractant protein-1) is produced by leucocytes, fibroblasts, endothelial cells and chondrocytes and is member of CC chemokine family [START_REF] Koch | Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis[END_REF][START_REF] Pulsatelli | Chemokine production by human chondrocytes[END_REF]. It attracts not only monocytes, but also T cells, natural killer cells and basophils, and plays a role in T cell differentiation and angiogenesis [START_REF] Haringman | Chemokines in joint disease: the key to inflammation?[END_REF]. Elevated MCP-1 levels were revealed in serum and synovial fluid of RA patients [START_REF] Klimiuk | Histological patterns of synovitis and serum chemokines in patients with rheumatoid arthritis[END_REF][START_REF] Koch | Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis[END_REF][START_REF] Katrib | Expression of chemokines and matrix metalloproteinases in early rheumatoid arthritis[END_REF]. In the current study, serum MCP-1 levels were down regulated following 3 months therapy with etanercept. Further drug injections maintained MCP-1 suppression, however to a lesser extent. Also others demonstrated serum MCP-1 levels reduction in RA patients during 6 months treatment with etanercept [START_REF] Kageyama | Etanercept reduces the serum levels of macrophage chemotactic protein-1 in patients with rheumatoid arthritis[END_REF]. Other investigators correlated serum levels of MCP-1 with IL-8 concentrations [START_REF] Koch | Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis[END_REF], swollen joint count and Ritchie articular index [START_REF] Ellingsen | Plasma monocyte chemoattractant protein 1 is a marker for joint inflammation in rheumatoid arthritis[END_REF] in RA. In our analysis prior to etanercept administrations circulating MCP-1 was correlated with clinical markers of disease activity like ESR and disease activity score (DAS28). Furthermore, we also noted the associations between serum concentrations of MCP-1 and IL-8 or RANTES concentrations. Following later on etanercept administrations these associations were less or not significant.

We found no correlations between patient sex, age or disease duration with serum concentrations of studied chemokines. As in other studies [START_REF] Weinblatt | A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate[END_REF][START_REF] Van Der Heijde | Comparison of etanercept and methotrexate, alone and combined, in the treatment of rheumatoid arthritis: two-year clinical and radiographic results from the TEMPO study, a double-blind, randomized trial[END_REF][START_REF] Van Riel | Add Enbrel or Replace Methotrexate Study Investigators. Efficacy and safety of combination etanercept and methotrexate versus etanercept alone in patients with rheumatoid arthritis with an inadequate response to methotrexate: the ADORE study[END_REF]

Figure 1 .

 1 Figure 1. Serum concentrations of interleukin-8 (IL-8) in rheumatoid arthritis (RA) patients,

Figure 2 .

 2 Figure 2. Serum concentrations of RANTES (regulated upon activation normal T cell

Figure 3 .

 3 Figure 3. Serum concentrations of monocyte chemoattractant protein-1 (MCP-1) in RA

Table 1 .

 1 Correlations between serum concentrations of chemokines and DAS28 in RA and secreted; MCP-1, monocyte chemoattractant protein-1; ESR: erythrocyte sedimentation rate; DAS28: three variables disease activity score that include 28-joint count. Data expressed as r values (correlation coefficient) according to Spearman rank correlation: * p<0.05, ** p<0.01 DAS28 evaluations were performed, and blood samples were obtained on month 0, prior to first etanercept injection.

Acknowledgements

Study was support by the grant (3-60766L) from Medical University of Bialystok, Poland.

Conflict of interest statement: None