Intramanual and intermanual transfer of the curvature aftereffect

The existence and transfer of a haptic curvature aftereffect was investigated to obtain a greater insight into neural representation of shape. The haptic curvature aftereffect is the phenomenon whereby a flat surface is judged concave if the preceding touched stimulus was convex and vice versa. Single fingers were used to touch the subsequently presented stimuli. A substantial aftereffect was found when the adaptation surface and the test surface were touched by the same finger. Furthermore, a partial, but significant transfer of the aftereffect was demonstrated between fingers of the same hand and between fingers of both the hands. These results provide evidence that curvature information is not only represented at a level that is directly connected to the mechanoreceptors of individual fingers but is also represented at a stage in the somatosensory cortex shared by the fingers of both the hands.
Introduction
The neural representation of haptic information can be investigated using different approaches. The representation of object shape perceived with the fingers has mainly been studied using neurophysiological tools. It has been found that especially slowly adapting type I (SAI) mechanoreceptors in the finger but also fast-adapting type I (FAI) receptors are sensitive to curvature (Goodwin et al. 1997; Jenmalm et al. 2003). In order to perceive curvature, a combination of responses from a population of receptors is required (Goodwin and Wheat 2004). This processing occurs along several stages up to at least the somatosensory cortex (SI) (Gardner and Kandel 2000). Taking a neurophysiological approach is useful to uncover the pathways underlying curvature processing, but is less appropriate to establish the levels at which perceived curvature is essentially represented.
A psychophysical approach that has been successful in providing greater insight into the neural representation of perceived properties is the study of the aftereffect, and especially, the transfer of the aftereffect. In vision, for example, the finding of partial, interocular transfer of the motion aftereffect has been explained by the involvement of both monocular and binocular cells in the processing of motion information from the stimulus (Moulden 1980; Wade et al. 1993; Tao et al. 2003). In a similar way, establishing the transfer characteristics of a haptic curvature aftereffect would provide insight into the representation of shape information. Finding aftereffect transfer between different fingers would indicate that curvature is represented at a level shared by these fingers, whereas no transfer would imply that each finger has a separate representation of curvature.
A curvature aftereffect is the phenomenon whereby a flat test surface feels concave following prolonged contact with a convex adaptation surface (see Fig. 1a). Curvature aftereffects have been found for different shapes and exploration modes. Gibson (1933) reported that a flat cardboard edge felt concave after the prolonged dynamic exploration of a convex cardboard edge. Vogels et al. (1996) demonstrated the existence of an aftereffect when the whole hand was placed on spherically curved shapes. They performed extensive experiments to examine the characteristics of this static curvature aftereffect. They found a linear relationship between the magnitude of the aftereffect and the curvature of the adaptation stimulus. Furthermore, they showed that the magnitude of the aftereffect increased with the adaptation time up to about 10 s. Finally, they found a decrease of the aftereffect with an increase of the interstimulus interval. In a follow-up study, they showed that the aftereffect also existed for alternative exploration modes, like touching a stimulus with only the five fingertips of the hand or performing small movements of the hand over the stimulus surface (Vogels et al. 1997). Given the strength and consistency of these findings, we supposed that curvature aftereffects should also occur for alternative ways of touching, such as the situation in which curved surfaces are statically being touched with only a single fingertip. However, this phenomenon has not yet been investigated, and consequently, any curvature aftereffect transfer between the fingers also remains unexplored.Fig. 1a Schematic overview of a haptic curvature aftereffect: when you first touch a convex (concave) surface for some time, say 10 s, and subsequently touch a flat surface, this latter surface feels concave (convex). b Schematic drawings of the cross-sections of a convex and a concave stimulus. The stimuli had a cylindrical shape with a spherical top (see illustration a). The distance from the bottom to the centre of the top (h) was consistently 30 mm. The diameter of the cylinders (d) was also 30 mm. c Examples of two psychometric curves. The circular data points and the fit through these points results from adaptation to the convex adaptation stimulus. The PSE is represented by PV. The square data points and the fit through these points result from adaptation to the concave adaptation stimulus. In this case, the PSE is represented by PC. The magnitude of the aftereffect (AE) is defined as the difference between PV and PC
The purpose of the present study was to obtain a better understanding of the representation of haptically perceived shape information, by probing the transfer of the curvature aftereffect. In the first experiment, we established the existence of an aftereffect when a curved surface is touched by a single finger and measured whether this aftereffect transferred to other fingers of the same hand. The second experiment was set up to determine whether the aftereffect depended on the finger used. Finally, in the third and fourth experiments, we investigated the transfer of the aftereffect between fingers of both hands.
Materials and methods
Subjects
A total number of 40 subjects participated [n = 8 for experiments 1, 2 and 4, n = 16 for experiment 3; 18 were male and 22 were female; the mean age was 22 years; 37 were right-handed, 3 were left-handed, according to a standard questionnaire (Coren 1993)]. Subjects in experiments 1 and 2 received course credit for their participation. Subjects in the third and fourth experiments received monetary compensation.
Stimuli
The stimuli comprised of a compound of polyurethane foam and artificial resin (Cibatool BM 5460). A computer-controlled milling machine was used to produce cylinders with a flat bottom and a spherically curved top. The top was either pointing outward (convex) or inward (concave). Both convex and concave adaptation stimuli were used, with curvature values of +36 and −36 m−1, respectively; the curvature of the nine test stimuli ranged from −16 to +16 m−1, in steps of 4 m−1. Illustrations of the stimuli and their cross-sections are given in Fig. 1a, b, respectively.
Procedure
A subject was seated behind a table. The preferred arm rested on a platform, which was 30 mm above the tabletop. In the third and fourth experiments, both arms rested on the platform. Only the fingertips projected over the platform. The experimenter placed the stimulus underneath a fingertip. A curtain prevented the subjects from seeing the stimulus. During a trial, the tip of one finger was placed on an adaptation stimulus for 10 s. Subsequently, the subject placed a finger on a test stimulus and had to judge whether this test stimulus felt convex or concave. Subjects were not allowed to move the finger over the stimulus surface, and the experimenter checked for this. No instructions were given on the force to contact the stimulus, nor was it measured. No feedback was provided on the response.
Three conditions were measured in the first experiment. In all conditions, the adaptation stimulus was touched with the index finger. In one condition, the test stimulus was also touched with the index finger. In the other two conditions, the test stimulus was touched with the middle finger or the little finger of the same hand, respectively. Each condition consisted of 10 repetitions of a group of 18 trials (two adaptation stimuli × nine test stimuli) with trials randomized within a group. One complete condition was measured in a single session of about one and a half hours. The separate sessions were spread over different days. The order in which the conditions were conducted was counterbalanced for the first six subjects and randomly chosen for the last two subjects.
In the second experiment, both the adaptation and the test stimuli were touched by the middle finger. In the third and fourth experiments, the adaptation stimulus was contacted by the index finger of the preferred hand; the test stimuli were touched with the index finger (third experiment) or middle finger (fourth experiment) of the non-preferred hand.
Analysis
The data for each subject and each condition were analyzed separately for the convex and the concave adaptation stimuli. The percentage of “convex” responses was plotted against the curvature of the test stimulus. The point of subjective equality (PSE) was determined by fitting a psychometric function (cumulative Gaussian) to the data. The PSE represents the curvature value that in 50% of the test cases was judged “convex” and in 50% of the cases was judged “concave”. The magnitude of the aftereffect is defined as the difference between the PSE resulting from the adaptation to a convex surface and the PSE resulting from the adaptation to a concave surface. Examples of psychometric curves for a convex and a concave adaptation are given in Fig. 1c.
Results
The mean results for the aftereffect values are shown in Fig. 2. The error bars indicate the standard errors of the mean.Fig. 2Mean results of the aftereffect. The indicated error bars are the standard error in the mean for each condition. Experiment 1: eight subjects participated. Adaptation was performed by the index finger of the preferred hand. Testing was done using the index finger, middle finger, or little finger of the same hand. Experiment 2: eight subjects participated. Adaptation and testing was performed by the middle finger of the preferred hand. Experiment 3: sixteen subjects participated. Adaptation was performed by the index finger of the preferred hand; testing was done by the opposite index finger. Experiment 4: eight subjects participated. Adaptation was performed by the index finger of the preferred hand; testing was done by the middle finger of the non-preferred hand
Experiment 1
We tested the occurrence of an aftereffect in each condition by performing separate one-tailed t tests. A significant result was obtained in all conditions (t7 = 6.3, P < 0.001 for the index finger; t7 = 9.8, P < 0.001 for the middle finger; t7 = 3.4, P = 0.006 for the little finger). Subsequently, an ANOVA with a repeated measures design was performed to determine any differences between conditions. A significant main effect was found (F2,14 = 22.5, P < 0.001). Pairwise comparisons showed a significant difference between the index finger and the middle finger (P = 0.007) and between the index finger and the little finger (P = 0.004), but not between the middle finger and the little finger (P = 1.0). The P values were adjusted with a Bonferroni correction.
Experiment 2
A one-tailed t test showed that there was a significant aftereffect (t7 = 8.0, P < 0.001). Inspection of Fig. 2 shows that the aftereffect of the middle finger condition of the second experiment was comparable to the index finger condition of the first experiment and was much higher than the middle finger condition of the first experiment. Independent samples t test confirmed that there was no significant difference in the first case (t14 = 0.6, P = 0.6), but that there was a significant difference in the second case (t7.4 = 6.1, P < 0.001).
Experiment 3
A one-tailed t test highlighted a significant aftereffect (t15 = 2.7, P = 0.009). The magnitude of this aftereffect was much lower than for the index finger condition of the first experiment. This was confirmed by an independent sample t test (t22 = 5.0, P < 0.001).
Experiment 4
A significant aftereffect was obtained, as was confirmed by a one-tailed t test (t7 = 7.4, P < 0.001).
Discussion
The first novel observation of this paper is that the perception of surface curvature by a single fingertip is influenced by preceding contact of this finger with another curved surface. The magnitude of this curvature aftereffect did not depend on the finger employed, as shown by a comparison between the results of the first and the second experiment. Previously, Vogels et al. (1996, 1997) reported the existence of a static curvature aftereffect, when the whole hand was used. We suppose that our finding of a one-finger aftereffect falls in the same class of phenomena. A quantitative comparison between the results of Vogels et al. (1996) and our finding can be made by calculating the relative magnitude of the aftereffect, i.e. the aftereffect divided by the difference between the adaptation stimuli. This value equals 0.17 ± 0.02 for the results of Vogels et al., whereas it was 0.15 ± 0.07 for the index finger condition of the first experiment and 0.17 ± 0.06 for the middle finger condition of the second experiment, respectively. These values are in the same order of magnitude, irrespective of the differences in manner of touching and curvature range of the stimuli.
The second important finding of our study is that the aftereffect partially transfers between fingers of the same hand. This means that the sensation of shape with a certain finger influences the perception of a shape touched by another finger. This suggests that the sensations obtained by the different fingers share a common representation. However, the transfer is far from complete, indicating that curvature perception by each finger also yields a substantial, individual part in the representation. Interestingly, the aftereffect does not only transfer from the index finger to the neighboring middle finger, but also to the distant little finger. This result is unlike recently performed localization (Schweizer et al. 2000) and learning studies (Sathian and Zangaladze 1997; Harris et al. 2001), in which the reported transfer effects were obtained in the neighboring finger, but not in the distant fingers. This indicates that the processes involved in detecting the finger that is stimulated or increasing the skills to discriminate punctate pressure or roughness are quite different from those concerned in shape perception of an object.
The third interesting result of this study is that there was a small, but significant transfer of the aftereffect between fingers of both hands, irrespective of whether opposite fingers (experiment 3) or different fingers (experiment 4) were employed. This result is different from the result reported by Vogels et al. 1997, who did not find intermanual transfer. However, in their experiments, whole hands were involved, whereas only single fingertips were used in our experiment. Moreover, their conclusion was based on the performance of only 2 subjects, whereas 24 participants provided the data for our study. The results of the third and fourth experiments suggest that the representation of shape information obtained with one hand is not completely distinct from the representation of shape information received by the other hand, but shares a common, bilateral component.
How can our findings be interpreted in the context of neurophysiological literature? Firstly, our finding that the aftereffect only transfers partially between fingers of the same hand shows that a substantial part of the processing occurs at a stage where each finger is individually represented. On this stage, which spreads from the mechanoreceptors in the fingers up to area 3b in SI, no overlap occurs in signals from the slowly adapting receptors and the fast-adapting receptors (Gardner and Kandel 2000). Slowly adapting receptors respond with a sustained discharge when the finger is in contact with a surface, whereas fast-adapting receptors only respond at the onset and removal phase of the finger (Johansson and Vallbo 1983). Vogels et al. (1996) showed that the magnitude of their curvature aftereffect increased with an increase in adaptation time. These findings point to an important role for the slowly adapting receptors in the curvature aftereffect. Therefore, we suggest that the aftereffect at the stage related to an individual finger mainly originates from the processing of the slowly adapting receptors. Secondly, the fact that we found a transfer between the fingers of the same hand implies that a significant part of the processing of curvature information occurs at a level shared by the different fingers. In physiological terms, this indicates that at least area 1 or 2 of SI are involved, as receptive fields in these areas cover several fingers of a single hand (Gardner and Kandel 2000), but processing may also occur at an even higher stage. Thirdly, our finding of an intermanual transfer shows that the processing of curvature information also takes place on a higher, bilateral level. We can only speculate on the neural correlates of this bilateral processing. Possible candidates include area 2 of SI, areas 5 and 7 of the posterior parietal cortex, and the secondary somatosensory cortex (Iwamura 2000; Gardner and Kandel 2000).
It is interesting to mention that the aftereffects in the intramanual transfer conditions (experiment 1) and the intermanual transfer conditions (experiment 3 and 4) are similar in magnitude. This suggests that no important curvature processing occurs at a level that is devoted to a single hand, but that all processing takes place at a higher stage. The similar results for experiments 3 and 4 provide further support that the hands and fingers are not somatotopically represented at this stage. From a previous study, it is known that subjects also performed similarly in intramanual and intermanual curvature discrimination tasks, but that higher performance was obtained when only a single finger was employed (Van der Horst and Kappers 2007). The analogy between that study and the current study is that curvature information is mainly represented at the level of the individual finger, but partly available at a higher, finger- and hand-independent level. We should be careful in ascribing a specific function to the involvement of the higher level areas in the processing of curvature information. The role of more cognitive aspects should not be excluded, since it is known that processes like tactile attention (Burton and Sinclair 2000; Spence and Gallace 2007), working memory (Burton and Sinclair 2000), and object recognition (Reed et al. 2004) also engage the somatosensory areas.
The aftereffect that we found in the present study is a similar phenomenon as the aftereffect that was previously reported by Vogels et al. (1996, 1997). However, this does not entail that the representation of curvature is identical for touching with a single finger or with the whole hand. Vogels et al. (1997) already showed that, although similar aftereffects were found when either the whole hand or only the five fingertips were used, only a small transfer between these exploration modes was obtained, which points to a limited overlap in representation. Similarly, we suppose that there is a difference in representation between curvature that is perceived by a single finger and curvature that is perceived by the whole hand. In the single finger case, the representation is mainly at the level of the individual finger, whereas in the whole hand case, the representation is spread over all fingers and the palm of the hand.
This study shows that establishing the intramanual and intermanual transfer of the aftereffect is a useful tool in obtaining more insight into the representation of object properties as perceived by the fingers. In general, studying aftereffect transfer is attractive, because it enables a connection between psychophysics and neurophysiology. The convergence of these approaches leads to a better understanding of human perception.

transfer curvature aftereffect touch mechanoreceptor somatosensory tactile


Physical fitness, fatigue, and quality of life after liver transplantation

Fatigue is often experienced after liver transplantation. The aims of this cross-sectional study were to assess physical fitness (cardiorespiratory fitness, neuromuscular fitness, body composition) in liver transplant recipients and to explore whether physical fitness is related to severity of fatigue. In addition, we explored the relationship between physical fitness and health-related quality of life. Included were 18 patients 1–5 years after transplantation (aged 48.0 ± 11.8 years) with varying severity of fatigue. Peak oxygen uptake during cycle ergometry, 6-min walk distance, isokinetic muscle strength of the knee extensors, body mass index, waist circumference, skinfold thickness, severity of fatigue, and health-related quality of life were measured. Cardiorespiratory fitness in the liver transplant recipients was on average 16–34% lower than normative values (P ≤ 0.05). Furthermore, the prevalence of obesity seemed to be higher than in the general population (17 vs. 10%). We found no deficit in neuromuscular fitness. Cardiorespiratory fitness was the only fitness component that was related with severity of fatigue (rs = −0.61 to rs = -0.50, P ≤ 0.05). Particularly cardiorespiratory fitness was related with several aspects of health-related quality of life (rs = 0.48 to rs = 0.70, P ≤ 0.05). Results of our study imply that cardiorespiratory fitness and body composition are impaired in liver transplant recipients and that fitness is related with severity of fatigue (only cardiorespiratory fitness) and quality of life (particularly cardiorespiratory fitness) in this group. These findings have implications for the development of rehabilitation programs for liver transplant recipients.
Introduction
Liver transplantation (LTx) is the only definitive treatment for end-stage liver disease. Since 1988, patient survival and liver function after LTx have improved markedly, due to improved technical expertise, better selection of patients, improved post-LTx management of complications, and improved immunosuppressive therapy (Adam et al. 2003). In Europe, LTx has achieved a 1-year survival rate of 82% (Burroughs et al. 2006).
Although studies have reported that quality of life improves after LTx (Gross et al. 1999; van der Plas et al. 2003), limitations in daily function still remain (Gross et al. 1999; van der Plas et al. 2003). Amongst these limitations, liver transplant recipients often experience fatigue (Aadahl et al. 2002; Belle et al. 1997; Leyendecker et al. 1993; van den Berg-Emons et al. 2006). Gross et al. (1999), and Belle et al. (1997) reported that, although the intensity of fatigue was reduced after LTx, fatigue remained the most distressing symptom 1 year after surgery. Leyendecker et al. (1993) found that 9 months after LTx, complaints of fatigue were more severe in the LTx group than in the general population. In a previous study, we found severe fatigue in 44% of patients up to 15 years after LTx, and these complaints did not decrease over time (van den Berg-Emons et al. 2006a).
Rehabilitation programs might be effective in reducing severity of fatigue after LTx, but, to develop appropriate programs, knowledge on the factors associated with fatigue after LTx is necessary. However, data on these factors are scarce. Aadahl et al. (2002) and van den Berg-Emons et al. (2006a) suggested that the fatigue experienced by liver transplant recipients is primarily physical, rather than psychological. Furthermore, van den Berg-Emons et al. (2006b) found that severe complaints of fatigue in liver transplant recipients are associated with low levels of everyday physical activity. A hypoactive lifestyle may lead to a negative spiral: hypoactivity leading to a reduction in physical fitness and deterioration of complaints of fatigue, leading to further hypoactivity.
Few studies have investigated the physical fitness of liver transplant recipients, but the limited data available suggest a reduced physical fitness after LTx (Beyer et al. 1999; Stephenson et al. 2001; Unnithan et al. 2001). Stephenson et al. (2001) found a 40–60% lower maximal oxygen uptake than predicted in liver transplant recipients. Beyer et al. (1999) reported that, although the cardiovascular and neuromuscular fitness in liver transplant recipients improved after a supervised exercise program during the post-operative year, maximal oxygen uptake and muscle strength remained 10–20% lower compared to healthy gender and age-matched individuals. Also in pediatric liver transplant recipients, deficits in cardiovascular fitness, and abdominal muscle strength have been reported (Unnithan et al. 2001).
It may be hypothesized that deficits in physical fitness in liver transplant recipients are associated with complaints of fatigue. Furthermore, deficits in physical fitness may lead to impaired health-related quality of life (HRQoL). However, to our knowledge, no studies are available on the relationships between these parameters in liver transplant recipients.
Because of the scarcity of studies on physical fitness and related parameters in liver transplant recipients, the present study assessed physical fitness (cardiorespiratory fitness, neuromuscular fitness, and body composition) in liver transplant recipients and explored whether physical fitness is related to severity of fatigue in this group. The relationship between physical fitness and HRQoL was also explored.
Patients and methods
Patients
To obtain a representative sample of liver transplant recipients with respect to fatigue, we recruited liver transplant recipients with varying severity of fatigue, according to the distribution of severity of fatigue as found in our previous study in 96 liver transplant recipients (van den Berg-Emons et al. 2006a). Severity of fatigue in this previous study ranged from ‘no signs of fatigue’ to ‘most disabling fatigue’, and was assessed with the Fatigue Severity Scale of Krupp et al. (1989) (see below). Inclusion criteria for the present study were: LTx between 1 and 5 years ago, sufficient knowledge of the Dutch language, and age between 18 and 65 years. Exclusion criteria were: multiorgan transplant recipients, severe comorbidity, and contra-indication for a progressive maximal cycle ergometer test. Of the original sample of 96 patients in the previous study (van den Berg-Emons et al. 2006a), 4 patients had died, 1 patient had emigrated, 53 patients were transplanted more than 5 years previously, and 10 patients were not eligible because of contra-indications for a maximal cycle ergometer test. Of the 28 eligible patients, 18 patients (64%) agreed to participate. There were no significant differences in relevant characteristics between the patients who decided to participate and the non-participants. The study was approved by the Medical Ethics Committee of the Erasmus University Medical Center. Written informed consent was obtained from all subjects. Table 1 shows the characteristics of the study group.
Table 1Characteristics of the study group (n = 18)Age (years)48.0 ± 11.8Gender Male11 Female7Primary disease (n) a Chronic17 Acute1Time since transplantation (years)3.3 ± 1.1Immunosuppressive agentsb 116 21 31Results are presented as mean ± SD or numbersaChronic primary disease: cholestatic (n = 6), viral (n = 6), miscellaneous (n = 5); acute primary disease: intoxication (n = 1)bImmunosuppressive agents: 1 agent, cyclosporine or tacrolimus; 2 agents, prednisone with tacrolimus; 3 agents, prednisone with tacrolimus and azathioprine
Measurements
Physical fitness: cardiorespiratory
Cardiorespiratory fitness was measured with a progressive maximal aerobic test on a cycle ergometer (ER800, Jaeger Toennies, Breda, The Netherlands). The test was preceded by a 1-min warm-up period (20 W). The test started at 20 W, and resistance was increased every minute by 15 or 20 W, depending on the ability of the patients. Individual protocols were constructed such that the total exercise time ranged from 8 to 12 min. The pedal rate was 60 rpm and strong verbal encouragement was given during the test. The test was terminated when the subject voluntarily stopped due to exhaustion, or when the patient was unable to maintain the initial pedal rate. Gas exchange and heart rate (HR) were measured continuously using a breath-by-breath gas analysis system (K4b2, COSMED, Rome, Italy). Subjective strain was measured immediately after the final stage by the Borg Category Scale for Rating of Perceived Exertion (Borg 1982). Patients were asked to indicate how strenuous they had experienced the test by giving a number from 0 (no effort at all) to 10 (maximal effort). Cardiorespiratory fitness was defined as the mean oxygen uptake during the last 30 s of exercise [VO2peak, in ml kg−1 min−1 and in ml kg fat-free mass−1 min−1 (ml kgFFM−1 min−1)]. In addition, the ventilatory anaerobic threshold (VAT, expressed as percentage of predicted VO2peak) was estimated by the ventilatory equivalent method, when VE/VO2 and PetO2 increased while VE/VCO2 and PetCO2 remained stable (Reinhard et al. 1979; Wasserman et al. 1999).
Finally, patients performed the submaximal 6-min walk test (6MWT) (Guyatt et al. 1985). Patients were instructed to walk, not run, as far as they could along a 30-m marked tape in a hall during a 6-min period. Standardized encouragement was provided with the following phrases: “You are doing well” and “Keep up the good work”. Patients were allowed to stop and rest during the test, but were instructed to resume walking as soon as they felt able to do so. The 6-min walk distance (6MWD) was registered.
Physical fitness: neuromuscular
Isokinetic muscle strength of the knee extensors was assessed in both legs by a Biodex® dynamometer (Shirley, New York, USA), recording strength as torque in Nm. The patients were seated against a back-rest, firmly strapped at the hip and thigh. The rotational axis was aligned with the lateral femoral epicondyle. After five familiarization repetitions, isokinetic strength was measured at 60°/s with 5 maximal contractions and at 180°/s with 15 maximal contractions. Strong verbal encouragement was given during the test. Peak torque (PT) was defined as the maximum torque generated by the patients throughout one series of repetitions at each velocity.
Physical fitness: body composition
Height and body mass were measured without shoes. Body mass was measured using a Cormier Paribel® weighing chair (FH Balances Cormier, Romainville, France). Body mass index (BMI, kg m−2) was calculated from height and body mass. Waist circumference (cm) was measured mid-way between the lowest rib and the iliac crest while standing. Thickness of four skinfolds (biceps, triceps, subscapular, suprailiaca region) was measured twice at the right side of the body with a Harpenden Skin-Fold Caliper (Burgess Hill, UK). The mean of the two measurements was used as representative for each site. Percentage body fat (BF) was predicted from skinfold thickness according to the method of Durnin and Womersley (1974).
Severity of fatigue
Severity of fatigue was assessed by the Dutch version of the Fatigue Severity Scale (FSS) (Krupp et al. 1989). The FSS is a self-administered questionnaire with answers ranging from 1 (‘strongly disagree’) to 7 (‘strongly agree’). The mean score of the nine inquiries ranges from 1 (‘no signs of fatigue’) to 7 (‘most disabling fatigue’). Internal consistency, reliability, validity, and sensitivity of the FSS have been established in several patient groups (Krupp et al. 1989; Merkies et al. 1999).
In addition to the FSS, severity of fatigue was assessed with a horizontal visual analogue scale (VAS). Patients were asked to mark the 100-mm line according to how intense they had experienced fatigue during the last month (0 denotes ‘no fatigue experienced’ and 100 denotes ‘the most severe fatigue’) (ter Borg et al. 2004). Visual analogue scales have been found to yield reliable and valid data (Huskisson 1982; Scott and Huskisson 1979).
Health-related quality of life
HRQoL was assessed by the validated Dutch version (RAND-36) (van der Zee and Sanderman 1993) of the Medical Outcomes Study Short Form-36 (SF-36) (Ware and Sherbourne 1992). The SF-36 is a validated, self-administered questionnaire used internationally to measure health status with respect to different dimensions: physical functioning, social functioning, role limitations due to physical problems, role limitations due to emotional problems, pain, mental health, vitality, general health perception, and change in perceived health during the last 12 months. All raw scores were converted to a 0–100 scale, with higher scores indicating higher levels of functioning or well-being.
Procedure
On the day of the measurements, patients refrained from caffeine, nicotine, and heavy exercise. The order of the tests was standardized: patients started with the 6MWT, followed by the questionnaires (completed under supervision of the researcher), body composition measurements, strength test, and finally the progressive maximal aerobic test. Exercise tests were performed under supervision of a physician. There were sufficient rest periods between the tests.
Statistics
Statistical analysis was performed using SPSS 10.1 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as mean ± standard deviation (SD), range or numbers. Results on cardiorespiratory fitness were compared to normative values for sedentary persons and to normative values for people who exercise no more than 1–2 h a week (recreational) (Vos et al. 2001). Results on neuromuscular fitness were compared with the normative values of Akima et al. (2001) and 6MWD was compared with the normative values of Enright and Sherill in healthy adults aged 40–80 years (1998). When patients were younger than 40 years, the normative values of Gibbons et al. (2001) were used. Obesity was defined as a BMI ≥ 30 (World Health Organization 1989), a waist circumference ≥102 cm in men and ≥88 cm in women (Lean et al. 1995) or percentage BF ≥25% in men and ≥32% in women (Lohman 1992).
Differences in physical fitness between patients and normative values were tested with the non-parametric Mann-Whitney U Test. Relationships between physical fitness and severity of fatigue and between physical fitness and quality of life were explored using the non-parametric Spearman correlation coefficient (rs). A probability value ≤0.05 determined statistical significance. However, because of the relatively small study sample, also results on the α = 0.10 level are presented (indicating a trend).
Results
Physical fitness
Mean score on the Borg Scale was 6.3 ± 2.2, indicating that the patients experienced the maximal ergometer test on average as heavy to very heavy. The mean HRmax was 90 ± 10% of what was predicted (predicted HRmax = 220-age) (Fox et al. 1971).
The individual results of the patients are presented in Table 2. Table 3 shows the cardiorespiratory fitness and neuromuscular fitness of the patients compared with normative values. VO2peak in ml kg−1 min−1 was 15 ± 22% (P = 0.07) and 34 ± 15% (P = 0.00) lower than normative values for, respectively, sedentary persons and those who exercise recreationally. VO2peak in ml kgFFM−1 min−1 was 16 ± 19.2% (P = 0.02) and 33 ± 14.8% (P = 0.00) lower than normative values for, respectively, sedentary persons and those who exercise recreationally. 6MWD was 16 ± 14% (P = 0.01) lower than normative values. There was no significant deficit in neuromuscular fitness. Table 4 shows the body composition of the LTx group. According to the cut-off points for obesity based on BMI, waist circumference and percentage BF, respectively 17, 36, and 41% of the patients were classified as obese.
Table 2Individual results on physical fitness in the 18 liver transplant recipientsAge (year/Gender)BMI (kg m−2)Waist (cm)aBody fat (%)bVO2peak (ml kg−1 min−1)VO2peak (ml kgFFM−1 min−1)bVAT (% predicted VO2peak sed) VAT (% predicted VO2peak recr)6MWD (m)PT extension 60°/s (Nm)PT extension 180°/s (Nm)56/M28.3111.029.036.551.454.142.7652156.388.252/M29.032.716.724.849.138.9428101.873.142/F22.828.934.548.590.565.8530100.065.660/F38.4118.542.013.122.543.632.5495101.978.664/M29.7103.530.621.731.377.455.443099.962.624/F21.072.025.929.940.438.831.1660159.9111.546/M26.5100.025.828.238.048.638.7606151.688.563/M24.420.520.926.469.449.741074.444.842/M34.9132.022.830.925.8513145.9118.353/F23.977.530.123.133.0108.664.846540.035.753/M25.999.623.620.727.156.742.6510149.396.839/M23.787.017.431.237.851.543.5547169.0111.833/M24.088.019.835.143.767.959.9510155.9116.963/F33.0103.034.815.824.257.943.230367.239.445/F26.830.823.634.161.046.2514120.161.739/F18.775.022.730.238.156.246.8600115.967.932/M22.991.020.425.031.443.438.3576180.4124.658/M25.199.025.018.524.648.637.9495128.691.9Abbreviations: BMI (kg m−2), body mass index; Waist (cm), waist circumference; VO2peak (ml kg−1 min−1), peak oxygen uptake per kg; VO2peak (ml kgFFM−1 min−1), peak oxygen uptake per kg fat-free mass; VAT (% predicted VO2peak), ventilatory anaerobic threshold as percentage of predicted VO2peak; sed, sedentary normative values; recr, recreational normative values; 6MWD, 6-min walk distance; PT, peak torqueaWaist circumference: n = 14, because of thickness of the skin at the place of the cicatrice in four patientsbBody fat and VO2peak: n = 17, because the thickness of the subscapular skinfold could not be measured reliably in one patientTable 3Cardiorespiratory fitness and neuromuscular fitness in the liver transplant groupLTx groupNorm valuesaPCardiorespiratory fitness VO2peak (ml kg−1 min−1)24.8 ± 6.929.4 ± 7.4 (sed)0.07†37.5 ± 6.7 (recr)0.00* VO2peak (ml kgFFM−1 min−1)b34.0 ± 8.741.0 ± 7.8 (sed)0.02*50.9 ± 5.9(recr)0.00* 6MWD (m)513.6 ± 88.9609.6 ± 97.60.01*Neuromuscular fitness PT extension at 60°/s (Nm)123.2 ± 38.7138.5 ± 44.10.22 PT extension at 180°/s (Nm) 82.1 ± 27.986.1 ± 30.10.59Results are presented as mean ± SDAbbreviations: VO2peak (ml kg−1 min−1), peak oxygen uptake per kg; VO2peak (ml kgFFM−1 min−1), peak oxygen uptake per kg fat-free mass; VAT (% predicted VO2peak), ventilatory anaerobic threshold as percentage of predicted VO2peak; sed, sedentary normative values; recr, recreational normative values (people who exercise no more than 1–2 h a week); 6MWD 6-min walk distance; PT, peak torqueaVos et al. (2001), Enright and Sherill (1998), Gibbons et al. (2001), Akima et al. (2001)bn = 17, because the thickness of the subscapular skinfold could not be measured reliably in one patient* Significant (P ≤ 0.05) difference between patients and normative values† Difference between patients and normative values at the α = 0.10 level (trend)Table 4Body composition in the liver transplant groupBody compositionMean ± SDBody weight (kg)80.6 ± 18.3Height (m)1.74 ± .11Body mass index (kg m−2)26.6 ± 5.0Waist circumference (cm)a96.6 ± 16.8Body fat (%)b26.9 ± 6.4Results are presented as mean ± SDaWaist circumference: n = 14, because of thickness of the skin at the place of the cicatrice in four patientsbBody fat: n = 17, because the thickness of the subscapular skinfold could not be measured reliably in one patient
Relationships
Table 5 shows the correlation coefficients between the physical fitness parameters and severity of fatigue as assessed with the FSS and VAS. None of the parameters of neuromuscular fitness or body composition were significantly related with severity of fatigue, assessed with either the FSS or the VAS.
Table 5Spearman correlation coefficients for the relationships between fitness parameters and severity of fatigue as measured with the Fatigue Severity Scale (FSS) and the Visual Analogue Scale (VAS) in 18 liver transplant recipientsPhysical fitnessFatigueFSSVASRsPRsPCardiorespiratory fitness VO2peak (ml kg−1 min−1)−0.400.10−0.520.03* % of sedentary norm−0.170.50−0.42.0.08† % of recreational norm−0.320.20−0.530.03*VO2peak (ml kgFFM−1 min−1)a−0.430.08†−0.510.04* % of sedentary norma−0.350.17−0.500.04* % of recreational norma−0.450.07†−0.610.01* VAT (% predicted VO2peak sed)0.220.380.030.92 VAT (% predicted VO2peak recr)0.170.49−0.060.82 6MWD (m)−0.440.07†−0.530.03* % of norm−0.250.32−0.520.03*Neuromuscular fitness PT extension at 60°/s (Nm)−0.390.11−0.310.22 % of norm−0.150.56−0.280.27 PT extension at 180°/s (Nm)−0.300.22−0.120.64 % of norm0.020.930.040.87Body composition Body mass index (kg m−2)0.030.910.060.82 Waist circumference (cm)b−0.070.82−0.060.83Body fat (%)a0.320.210.150.58Abbreviations: VO2peak (ml kg−1 min−1), peak oxygen uptake per kg; VO2peak (ml kgFFM−1 min−1), peak oxygen uptake per kg fat-free mass; VAT (% predicted VO2peak), ventilatory anaerobic threshold as percentage of predicted VO2peak; sed, sedentary normative values; recr, recreational normative values (people who exercise no more than 1–2 h a week); 6MWD, 6-min walk distance; PT, peak torquean = 17, because the thickness of the subscapular skinfold could not be measured reliably in one patientbn = 14, because of thickness of the skin at the place of the cicatrice in four patients* Significant (P ≤ 0.05) correlation† Correlation at the α = 0.10 level (trend)
VO2peak in ml kgFFM−1 min−1 (absolute and expressed as percentage of recreational normative values) and 6MWD (absolute) were related with severity of fatigue as assessed with the FSS at the α = 0.10 level (trend). With respect to the VAS, VO2peak in ml kg−1 min−1 (absolute and expressed as percentage of recreational normative values), VO2peak in ml kgFFM−1 min−1 (absolute and expressed as percentage of both sedentary and recreational normative values) and 6MWD (absolute and expressed as percentage of normative values) were significantly (P ≤ 0.05) correlated with severity of fatigue. VO2peak in ml kg−1 min−1 as percentage of sedentary normative values was related with severity of fatigue as assessed with the VAS at the α = 0.10 level (trend). The VAT was not correlated with severity of fatigue, assessed with the FSS or the VAS.
Table 6 shows the correlation coefficients between physical fitness parameters and HRQoL. There were several significant correlations between physical fitness and HRQoL and particularly between cardiorespiratory fitness on the one hand and ‘Physical functioning’, ‘Social functioning’ and ‘Vitality’ on the other hand.
Table 6Spearman correlations coefficients for the relationships between physical fitness and health-related quality of life as assessed with the RAND−36 in 18 liver transplant recipientsPhysical fitnessRAND-36 domainPFSFRlpRleMHVTBPGHCHCardiorespiratory fitness VO2peak (ml kg−1 min−1)0.55–––––––– % of sedentary norm–0.42–––0.50––– % of recreational norm0.48––––0.51––– VO2peak (ml kgFFM−1 min−1)a0.570.510.41–––––– % of sedentary norma–0.56–––0.59–0.48– % of recreational norma0.460.58–––0.58––– VAT (% predicted VO2peak sed)––––––––– VAT (% predicted VO2peak recr)––––––––– 6MWD (m)0.670.57––0.53–0.54–– % of norm–0.70–––0.51––0.44Neuromuscular fitness PT extension at 60°/s (Nm)0.44–––.41–––– % of norm–0.68––––0.56–– PT extension at 180°/s (Nm)––––––0.44–– % of norm–0.51–0.54––0.50––Body composition Body mass index (kg m−2)––––––––– Waist circumference (cm)b–––0.49–0.41––––– Body fat (%)a–––––––To enhance the clarity of the table, only significant correlation coefficients (P ≤ 0.05, bold) or trends (P < 0.10, not bold) are presentedAbbreviations: VO2peak (ml kg−1 min−1), peak oxygen uptake per kg; VO2peak (ml kgFFM−1 min−1), peak oxygen uptake per kg fat-free mass; VAT (% predicted VO2peak) ventilatory anaerobic threshold as percentage of predicted VO2peak; sed, sedentary normative values; recr, recreational normative values (people who exercise no more than 1–2 h a week); 6MWD, 6-min walk distance; PT, peak torque; PF, physical functioning; SF, social functioning; Rlp, Role limitations physical; Rle, Role limitations emotional; MH, mental health; VT, vitality; BP, bodily pain; GH, general health perception; CH, changes in healthan = 17, because the thickness of the subscapular skinfold could not be measured reliably in one patientbn = 14, because of thickness of the skin at the place of the cicatrice in four patients
Discussion
This is the first study in which relationships between several aspects of physical fitness and severity of fatigue are explored after LTx. The study is an initial step in identifying factors that are associated with fatigue in liver transplant recipients. A limitation of this study may be that the sample was relatively small. However, we believe that this sample is representative for patients after LTx and that the study provides important information for the development of rehabilitation programs for liver transplant recipients.
Physical fitness
We found average deficits in VO2peak of 16–34%, when we compared the results in the liver transplant recipients with normative values for, respectively, sedentary persons and those who exercise no more than 1–2 h a week. We believe that a value in between the sedentary and recreational normative values is representative for healthy Dutch persons of the same age as the liver transplant recipients that participated in our study. 6MWD was 16% lower than normative values. This subnormal level of cardiorespiratory fitness (both VO2peak and 6MWD) is in agreement with findings of previous studies (Beyer et al. 1999; Stephenson et al. 2001; Unnithan et al. 2001). Furthermore, the prevalence of obesity was higher in this study than in the general Dutch population; 17% of the patients had a BMI of more than 30 compared to 10% in the general Dutch population (Gezondheidsraad 2003).
Impaired cardiorespiratory fitness in liver transplant recipients may be due to the use of immunosuppressive medication, e.g. glucocorticoids and calcineurin inhibitors, which may influence both the cardiovascular system and skeletal muscles (Hokanson et al. 1995; Mercier et al. 1996). Immunosuppressive medication can also induce appetite stimulation and diabetes mellitus; this in association with emotional conditions can cause a change in eating habits (Correia et al. 2003; Hussaini et al. 1998). Furthermore, patients with primary biliary cirrhosis and some patients with other chronic liver disease are hypermetabolic. LTx in these patients may lead to a reduction in resting metabolic rate and can also cause an increase in BF (Hussaini et al. 1998; Richardson et al. 2001). Besides immunosuppressive medication and changes in appetite and metabolism, also deconditioning (both before and after transplantation) may contribute to the impaired cardiorespiratory fitness and body composition (Epstein et al. 1998; Grose et al. 1995). In a previous study we found that severe complaints of fatigue were associated with low levels of everyday physical activity in liver transplant recipients (van den Berg-Emons et al. 2006b).
Because of the immunosuppressive medication and deconditioning, we also expected a deficit in neuromuscular fitness. However, this was not demonstrated in the present study, in contrast to Beyer et al. (1999), who found a 10–20% lower muscle strength in liver transplant recipients compared to age- and sex-matched sedentary individuals. They contributed this deficit to muscle weakness as a side-effect of glucocorticoids.
The discrepancy between the findings of our study and those of Beyer et al. (1999) may be explained by the use of Tacrolimus instead of cyclosporine in the majority of our subjects. Tacrolimus is known to have less side effects than cyclosporine (Maes and Vanrenterghem 2004).
Stephenson et al. (2001) reported an early VAT in liver transplant recipients (<45–50% of the predicted VO2max) compared to 50–60% of the VO2max in healthy persons found by Davis et al. (1997). They contributed this early anaerobic threshold in liver transplant recipients to the cyclosporine-induced decrease in mitochondrial oxygen consumption (2001). However, the European Respiratory Society (ERS Task Force 1997) reported that there is a wide range of normal predicted values (35–70%). Therefore, it is difficult to indicate whether the VAT in our patients was reduced compared to normal (58.6 ± 18.9 and 44.7 ± 11.1% of predicted VO2peak for, respectively, sedentary persons and those who exercise recreationally).
Physical fitness and severity of fatigue
Although relationships between severity of fatigue (as measured with the FSS and VAS) and physical fitness were not univocal, this study demonstrates a relationship between cardiorespiratory fitness and severity of fatigue after LTx. Patients with more severe complaints of fatigue had larger deficits in cardiorespiratory fitness than patients with less severe complaints of fatigue. Although this cross-sectional study does not allow us to conclude that a reduced cardiorespiratory fitness results in fatigue (or vice versa), there may be an interaction between parameters: complaints of fatigue leading to decreased physical activity (van den Berg-Emons et al. 2006b) and decreased physical fitness, leading to further deterioration of complaints of fatigue. It may then be hypothesized that rehabilitation programs, aimed at enhancing cardiorespiratory fitness, can be effective in breaking through this negative spiral and (partly) reduce complaints of fatigue in this population. However, this hypothesis has to be confirmed in future randomized trials.
In contrast to our expectations, the present study indicates that other aspects of physical fitness, neuromuscular fitness and body composition, do not seem to be related with severity of fatigue after LTx. However, it should be realized that our study sample was relatively small and some of the studied relationships may have failed to show statistical significance.
Physical fitness and HRQoL
Previous studies on healthy persons indicate that physical activity, fitness, and body fatness are associated with HRQoL and mood (Han et al. 1998; Stewart et al. 2003). There are also indications that physical activity is related to HRQoL after LTx (Painter et al. 2001; van den Berg-Emons et al. 2006b). Therefore, we expected to find a relationship between physical fitness and HRQoL in our study group. In agreement with the study of Stewart et al. (2003) in healthy elderly subjects, we found several significant correlations between physical fitness (particularly cardiorespiratory fitness) and HRQoL in our liver transplant recipients. Patients with large deficits in physical fitness experienced worse HRQoL than patients with small deficits in physical fitness. However, in contrast with studies in healthy persons (Han et al. 1998; Stewart et al. 2003), we found only few relationships between body composition and HRQoL.
Our results on the relationships between physical fitness and HRQoL imply that rehabilitation programs aimed at improving physical fitness (particularly cardiorespiratory fitness) may consequently result in improved HRQoL, particularly in improved physical and social functioning, vitality, and bodily pain. However, these implications have to be confirmed in future randomized trials on the effects of such rehabilitation programs in liver transplant recipients.
Conclusion
Cardiorespiratory fitness in the liver transplant recipients was distinctly impaired and the prevalence of obesity was higher than in the general population; there were no indications that neuromuscular fitness is impaired after LTx. Based on the relationships we found between cardiorespiratory fitness and severity of fatigue, a rehabilitation program aimed at enhancing cardiorespiratory fitness may help in reducing complaints of fatigue after LTx. Such rehabilitation programs may also result in improved HRQoL.

fatigue
liver transplantation
body composition
peak oxygen uptake
isokinetic muscle strength


Anti-CCP2 Antibodies: An Overview and Perspective of the Diagnostic Abilities of this Serological Marker for Early Rheumatoid Arthritis

The literature of the last 4 years confirms that the anti-CCP2 test is a very useful marker for the early and specific diagnosis of rheumatoid arthritis (RA). The anti-CCP2 test is very specific for RA (95–99%) and has sensitivity comparable to that of the rheumatoid factor (70–75%). The antibodies can be detected very early in the disease and can be used as an indicator for the progression and prognosis of RA. In this review, these interesting properties and some future possibilities of this diagnostic test are discussed.
The presence of autoantibodies in the serum of patients is a very typical phenomenon for autoimmune diseases. Most of these autoantibodies, however, can also be detected in patients with other conditions and are therefore not specific. A typical example is the rheumatoid factor (RF), which is present in most inflammatory conditions. However, in some cases, autoantibodies can give the clinician a more precise indication of the type of underlying disease because they occur specifically in a certain disease. For example, anti-Sm antibodies are linked almost exclusively to systemic lupus erythematosus (SLE); whereas anti-DNA topoisomerase-I antibodies are typically present in scleroderma patients. Among the most disease-specific autoantibodies described are the so-called ACPA (anti-citrullinated protein/peptide antibodies). These antibodies occur specifically in RA and can be measured most conveniently via the anti-CCP (anti-cyclic citrullinated peptide) antibody test.
The first generation CCP test (CCP1) used in early diagnostic studies (2000–2001) contained a single cyclic citrullinated peptide derived from filaggrin as the substrate [1]. It could detect ACPA in 68% of patients with established RA with a very high specificity (98%). Because filaggrin is not expressed in the synovium, it is most likely not the natural citrullinated antigen for ACPA. Other peptides, not related to filaggrin, could therefore potentially provide better epitopes for detection of ACPA. Via screening of a number of peptide libraries, novel citrullinated peptides were obtained and incorporated into a second generation CCP test (CCP2). This test is commercially available, and as all companies use the same type of CCP2 peptides, standardization is achieved quite easily. The diagnostic properties of this test will be discussed below.
The CCP2 Test is Sensitive and Highly Specific for RA
Since its appearance on the market in the second half of 2002, the diagnostic properties of the CCP2 test have been studied by many laboratories. This resulted in more than 120 publications dealing with this subject. The accumulated data, including only papers that appeared in PubMed till December 2006, are given in Table 1. It is clear that the accumulated data confirm the earliest reports on specificity and sensitivity of the CCP2 test. The anti-CCP2 test demonstrates an RF-like sensitivity with a very high specificity for RA (see also recent reviews: [2, 3]). It is also commonly recognized that anti-CCP2 antibody may be present in up to 40% of RF-negative RA sera [4, 5].
Table 1Cumulative anti-CCP2 diagnostic data published between 2002 and 2006Patient groupNumberCCP2+Sensitivity (%)Specificity (%)RA total14,18310,13571.5 Early3,8762,36561.0 Established10,3077,77075.4Controls15,1566834.595.6 Non-RA11,5026475.694.4 Healthy3,654361.099.0In total, 122 independent studies were included. The selection for early RA has been adopted from the original reports.
The anti-CCP2 test enables clinicians to distinguish RA patients from other arthritic diseases, especially in cases where the RF test is not discriminative. This is, for example, the case with chronic hepatitis C virus (HCV) infection, a disease that can easily be misdiagnosed as it often reveals RA-like arthropathies and, in many cases, is accompanied by a positive RF. Several examples of such studies are given in recent reviews on this subject [6].
Recently, there is also an interest to compare the diagnostic potential of anti-CCP2 with novel tests based on the use of a citrullinated antigen (for example, MCV = anti-mutated citrullinated vimentin [7], CPA = citrullinated protein antibodies, VCP = anti-viral citrullinated protein [8], antihuman fibrinogen alpha [9, 10], and CCP3 [11]). For a reliable comparison of these tests, it is essential that their performance is assessed under the same conditions, e.g., the sensitivities of the tests should be determined at the same level of specificity. Good examples of such stratified studies have been carried out recently by van der Cruijssen et al. [10] and Dejaco et al. [7]. For example, Dejaco et al. [7] showed, in a large cohort of patients (>600), that at a specificity of 98.7%, being the specificity of the anti-CCP2 test, the sensitivity of the anti-MCV test is 53.7% as opposed to 70.1% for the anti-CCP test. Coenen et al. [11] compared several commercial tests, including a very recent CCP3 test from Inova. At the cut-offs recommended by the various manufacturers, the positive predictive value of the three commercial CCP2 tests is about 90% with a specificity of around 96%. The specificity of the other tests (CCP3 = 88%, MCV = 90%, CPA = 94%) is lower as are their positive predictive values [11]. These numbers may improve a little bit when the cut-off values are adjusted to more realistic data; nevertheless, the data allow the statement that, in absolute percentages, none of the tests performs better than the anti-CCP2 test. They also seem to indicate that some tests detect RA patient groups that are negative in the anti-CCP test, illustrating again that the autoantibody repertoire of RA patients is very heterogeneous.
Another risk for the specificity of a test that is based on a citrullinated antigen is the possibility that antibodies are not directed exclusively to the citrulline-containing epitope but also to other possibly overlapping epitopes present in the substrate antigen. This is particularly important when citrullinated versions of proteins like vimentin or fibrinogen are used. For example, it is known that antibodies to vimentin are present in several diseases different from RA [12, 13]. This particular problem has been addressed for CCP2 by Vannini et al. [14]. They used ELISA plates containing the control CCP2 antigens (Arg instead of Cit in the same peptide context), produced and made available by Euro-Diagnostica, Arnhem, The Netherlands, in parallel to the normal CCP2 test. The results of these comparative studies showed that in RA and most non-RA rheumatic disease sera, anti-CCP reactivity indeed is citrulline-dependent. However, in some patients, particularly autoimmune hepatitis patients, citrulline-independent reactivity with the antigen may occur. A positive CCP test in a rheumatic disease (almost always citrulline-specific) may thus suggest the future development of RA as has been suggested by several studies [15, 16]. A positive test in a nonrheumatic disease (very often not citrulline-specific), for example, liver disease, should be interpreted with care [14].
Anti-CCP2 Antibodies are Present Early in Disease and have Predictive Potential
Because RA patients at first presentation often do not fulfill the criteria for the diagnosis/classification of RA, an early, highly predictive marker would greatly assist the clinician in reaching an early diagnosis. There are several studies indicating that the anti-CCP2 test provides this help (reviewed by [2]).
In the recently published EULAR recommendations for the management of early arthritis [16], a list of factors has been proposed that predict persistent and erosive disease. These factors include: number of swollen and tender joints, ESR or CRP, level of RF and anti-CCP antibodies, and radiographic erosions. Most of these factors were also mentioned as being important in the prediction of early erosive RA (Visser et al. [17]). Subsequent studies by the same group gave an indication of the relative importance of these factors. When expressed as odds ratios (OR), the data was as follows: arthritis of three or more joints, 5.0; radiographic erosions, 8.7; positive IgM-RF, 1.7; and positive anti-CCP2, 38.6 [18]. These and other data (see also [19]) clearly show that the presence of anti-CCP antibodies is an important and independent prognostic factor for radiographic progression in not only early arthritis but also in early rheumatoid arthritis [16, 20].
Recently, it has also been shown that IgM-CCP is present in early samples from both patients with undifferentiated arthritis (UA) and patients with RA as well as in follow-up samples from patients with RA. These data indicate the development of the anti-CCP isotype repertoire into full usage early in the course of arthritis and a continuous (re)activation of the RA-specific anti-CCP response during the further development of the disease [21].
It is, however, evident that besides the clinical and laboratory parameters mentioned above, some genetic factors are important as well. The effect of the HLA shared-epitope alleles on the development of ACPA has been firmly established. Citrullination is typically a process that occurs in apoptotic cells. Because such dying cells are generally removed from the environment via clearance by phagocytes, a process that is regulated by many genes, the immune system will normally not encounter citrullinated proteins. However, it has been shown that during inflammation, citrullinated proteins are detectable in the inflamed tissue, both in RA and non-RA patients [22]. This is probably caused by inefficient clearance of the massive numbers of dying cells, a process already described to occur in SLE [23]. This inefficient clearing could, in principal, also be the consequence of altered genes, i.e., the genetic background. The mere presence of citrullinated antigens in inflamed synovial tissue does not necessarily result in the occurrence of anti-CCP antibodies in serum or synovial fluid, neither in humans nor in mouse models of arthritis [24, 22]. Hill et al. [25] showed that the generation of anticitrulline antibody in mice actually is linked to the expression of the RA shared epitope (SE), and a similar link was also found in patients because the combination of SE, and anti-CCP has a very high predictive value for the future development of RA [26]. The specific structure of HLA molecules obviously plays an important role in the induction of autoimmunity to citrullinated proteins. These studies were extended by Huizinga et al. [27] who found that HLA-DRB1 alleles encoding the SE were only associated with anti-CCP positive RA and not with anti-CCP negative RA. In contrast, anti-CCP negative RA appeared to be associated with HLA-DR3 [28]. Therefore, the presence of citrullinated antigens, together with the appropriate genetic background (the SE and probably other types of HLA), appears to be the minimum requirement for an immune response to citrullinated polypeptides to be generated [29]. Next to that, other sensitizing genetic settings resulting from polymorphisms (e.g., PTPN22 [30]) might, given their association, aid in triggering of ACPA.
Conclusions and Future Perspectives
The present literature describes and confirms that the anti-CCP2 test is a very useful marker for the early and accurate diagnosis of RA. Anti-CCP antibody is very specific for RA and has a sensitivity comparable to that of RF. These antibodies can be detected very early in disease and may be used as an indicator for the progression and prognosis of RA. Initially, the test was available principally as a manual ELISA method through Euro-Diagnostica and Axis-Shield and their partners. More recently, fully automated anti-CCP assays have also been made available from Phadia (UniCap Elia CCP) and Abbott Diagnostics (AxSYM anti-CCP). A very novel format is the CCPoint, a Point-of-Care test for the detection of these antibodies in whole blood in 10 min [31]. A small drop of whole blood is applied onto the sample well followed by the addition of four drops of running buffer. After 10 min, the result is read by visual inspection of the detection zone for staining of the antigen line. As no special equipment is required to perform the assay, this test will bring the detection of anti-CCP antibodies into the office of the family doctor, facilitating a very fast referral to the rheumatologist when the test gives a positive answer. Such new applications will undoubtedly further enhance the utility of the anti-CCP autoantibody system in clinical practice.

ccp2 test
rheumatoid factor
autoantibodies
anti-cyclic citrullinated peptide