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Rheumatology 2001; 40: 1135-1140
© 2001 British Society for Rheumatology
Original Papers |
Clinical significance of anti-topoisomerase I antibody levels determined by ELISA in systemic sclerosis
Department of Dermatology, Kanazawa University School of Medicine, Kanazawa, Japan
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Abstract |
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Objective. To determine the clinical associations of the levels of anti-topoisomerase I (topo I) antibody in patients with systemic sclerosis (SSc).
Methods. Anti-topo I antibody levels were determined by enzyme-linked immunosorbent assay. In a longitudinal study, 125 sera from 21 patients were analysed during a follow-up period of 0.2–4.7 yr.
Results. Anti-topo I antibody levels were correlated positively with skin thickness score and renal vascular resistance, and inversely with percentage vital capacity. In the longitudinal study, five patients with a low anti-topo I antibody level at their first visit exhibited a stable antibody level or a small decrease in the level during the follow-up period, and their skin sclerosis was stable. Of 16 patients with a high anti-topo I antibody level at their first visit, seven showed a stable level, four had an increasing level and five had a decreasing level. The decreasing levels were accompanied mainly by atrophic skin change during the follow-up period, whereas the increasing levels were associated with new onset or worsening of organ involvement.
Conclusions. These results suggest the potential clinical significance of anti-topo I antibody levels in evaluating disease severity and the prognosis in SSc.
KEY WORDS: Systemic sclerosis, Anti-topoisomerase I antibody, Modified Rodnan total skin thickness score, Autoantibody level, Longitudinal study.
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Introduction |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Systemic sclerosis (SSc) is a multisystem disorder of connective tissue characterized by excessive fibrosis in the skin and various internal organs. The presence of antinuclear antibodies (ANAs) is a central feature of SSc as they are detected in more than 95% of patients [1, 2]. DNA topoisomerase I (topo I), centromere proteins, RNA polymerases I, II and III and some nucleolar components have been identified as intracellular targets of ANAs [1]. Although the precise mechanisms involved in the pathogenesis of SSc remain unknown, autoimmunity is considered to be involved, as the ANAs are detected exclusively in SSc and are closely associated with subsets of patients characterized by combinations of clinical manifestations [1].
The presence of anti-topo I antibodies has been clinically associated with a more severe form of SSc that exhibits diffuse cutaneous involvement, digital pitting, severe joint involvement and lung involvement [1, 3–5]. However, one study showed that 31% of SSc patients with anti-topo I antibodies had limited cutaneous involvement and that 53, 64 and 13% of patients did not have digital tip ischaemia, arthritis and pulmonary interstitial pneumonia respectively [4]. In addition, Steen et al. [3] reported that 18% of patients with limited cutaneous SSc (lSSc) were positive for anti-topo I antibody. Thus, the clinical manifestations of SSc in patients with anti-topo I antibodies are heterogeneous. However, no serological indicators of milder disease in SSc patients who are positive for anti-topo I antibody have been identified.
The clinical significance of changes in levels of SSc-specific autoantibodies in the long term has not been clarified fully. Such data are of special importance in view of the prognostic significance of these autoantibodies and of the possible association between autoantibody levels and disease manifestations. A long-term longitudinal study suggested that anticentromere antibody levels were relatively stable over time [6]. Similarly, two reports suggested that there were no major long-term changes in the level of anti-topo I antibody [7, 8]. However, these two reports showed conflicting data concerning the clinical associations of the anti-topo I antibody level, as Hildebrandt et al. [7] reported that an increase in anti-topo I antibody level was associated with the development of new involvement of major organs; however, this was not confirmed by Vazquez-Abad et al. [8]. Recently, Kuwana et al. [9] reported that 20% of anti-topo I antibody-positive SSc patients lost anti-topo I antibody during the disease course and had a favourable outcome, suggesting the clinical importance of the anti-topo I antibody level in patients with SSc.
In this study, we assessed the clinical associations of the anti-topo I antibody level by the use of the enzyme-linked immunosorbent assay (ELISA) in relatively early SSc patients. In addition, we performed a retrospective longitudinal study of the anti-topo I antibody level in some of these SSc patients to determine whether the anti-topo I antibody level is stable over time or whether there are fluctuations in the level that are associated with specific clinical manifestations.
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Patients and methods |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Patients
We analysed serum samples from 30 Japanese SSc patients (25 females and five males) who were positive for anti-topo I antibody by the double immunodiffusion test (DID) at their first visit. All patients fulfilled the criteria for SSc proposed by the American College of Rheumatology (ACR) [10]. The patients were aged 2–73 yr (mean age 49) and the disease duration was 3.3±3.5 yr (range 0.08–10 yr). The duration of the disease was calculated from the time of onset of the first clinical event (other than Raynaud's phenomenon) that was a clear manifestation of SSc. Of 30 SSc patients with anti-topo I antibody, three were also positive for anticentromere antibody, one for anti-cardiolipin antibody, one for anti-SS-A antibody and one for anti-SS-A and SS-B antibodies. At their first visit, seven patients had been treated with low-dose steroids (prednisolone 5–20 mg/day) and 13 patients with D-penicillamine. None of the SSc patients had received immunosuppressive therapy. These patients were grouped according to the classification system proposed by LeRoy et al. [11]: six patients (all females) had lSSc and 24 (19 females and five males) patients had diffuse cutaneous SSc (dSSc).
In a retrospective longitudinal study, we analysed 125 serum samples from 21 of 30 SSc patients who had anti-topo I antibody at their first visit. These patients were aged 2–72 yr (mean age 46). The disease duration was 3.0±3.4 yr (range 0.2–10 yr). These patients had been followed up for 2.5±1.4 yr (0.2–4.7 yr), with 6±2.4 (2–10) time-points. Eighteen patients received low-dose steroids (prednisolone 5–20 mg/day) and eight received low-dose D-penicillamine (100–500 mg/day) throughout the follow-up period. At their first visit, five patients had been treated with low-dose steroids (prednisolone 5–20 mg/day) and two had been treated with low-dose D-penicillamine (100–200 mg/day). One patient received steroid pulse therapy, followed by 40 mg/day of oral prednisolone for subacute deterioration of interstitial pneumonitis during the follow-up period. In addition to anti-topo I antibody, one patient was also positive for anticentromere antibody, one for anti-cardiolipin antibody and one for anti-SS-A antibody. None of the SSc patients received immunosuppressive therapy during the follow-up period. Fresh venous blood samples were centrifuged shortly after clot formation. All samples were stored at -70°C before use.
Clinical assessment
At their first visit, all patients had a physical examination, laboratory tests were performed and a complete medical history was taken. There were limited evaluations during follow-up visits. Organ system involvement was defined as described previously [3, 12]: lung involvement was indicated by bibasilar fibrosis on chest radiography; oesophageal involvement by hypomotility shown on a barium radiograph; joint involvement by inflammatory polyarthralgias or arthritis; heart involvement by pericarditis, congestive heart failure or arrhythmias requiring treatment; and muscle involvement by proximal muscle weakness and an elevated serum creatine kinase concentration. Pulmonary function, including vital capacity (VC) and diffusion capacity for carbon monoxide, was also tested. Renal crisis was defined as malignant hypertension and rapidly progressive renal failure without any other explanation. The modified Rodnan total skin thickness score (TSS) was measured as described previously [13, 14]. Renal vascular damage was determined as a pulsatility index (PI) by colour-flow Doppler ultrasonography of both kidneys (SSD-2000 scanner; Aloka, Tokyo, Japan) [15, 16]. The PI, which represents vascular impedance, was calculated as A-B/mean, where A is the peak systolic frequency, B is the end diastolic frequency and the mean is the time-averaged frequency. The PI was calculated as the average value obtained for eight waveforms on the renal interlobar arteries of both kidneys. The PI was considered elevated when the value was higher than the mean+2 S.D. (1.4) for 16 normal persons [16]. At their first visit, all patients examined in this study were free of clinical symptoms of renal damage. In addition, urinalysis, 24-h protein excretion, plasma creatinine levels and endogenous creatinine clearance corrected for 1.73 m2 body surface area were within normal limits (data not shown).
The protocol for the study was approved by the Kanazawa University School of Medicine and Kanazawa University Hospital, and informed consent was obtained from all patients.
ELISA
Anti-topo I antibody levels were measured with specific ELISAs (Medical & Biological Laboratories, Nagoya, Japan). The human topo I protein (amino acids 163–765) was expressed as a ß-D-galactosidase fusion protein using the pEX expression vector system. Recovery and purification of the topo I protein from induced cell cultures were performed by the Zwittergent detergent method [17]. Ninety-six-well plates were coated with 5 µg/ml of recombinant human topo I. The serum samples were diluted 1:101 and incubated for 1 h at 20°C. After washing, the bound antibodies were detected with peroxidase-conjugated polyclonal anti-human IgG+IgM+IgA antibody. Colour was developed with 3,3',5,5'-tetramethylbenzidine and H2O2 and the plates were read at 450 nm. In this assay system, the cut off value was set at 16 U/ml, calculated as the mean+3 S.D. for 267 normal persons (data not shown). To determine the reliability of this ELISA system, we examined 118 serum samples from SSc patients diagnosed according to the ACR criteria. Twenty-eight serum samples were positive for anti-topo I antibody by both DID and ELISA. Of the remaining 90 serum samples, 89 were negative for anti-topo I antibody by both DID and ELISA and one serum sample was negative for anti-topo I antibody by DID but positive by ELISA. Therefore, this ELISA system had a sensitivity of 100% and a specificity of 98.9%.
Statistical analysis
The Mann–Whitney U-test was used to compare anti-topo I antibody levels. Spearman's rank correlation coefficient was used to examine the relationship between two continuous variables. A P value less than 0.05 was considered statistically significant. All data are shown as mean±S.D.
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Results |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Clinical associations of anti-topo I antibody levels
To evaluate the clinical associations of anti-topo I antibody levels, physical and laboratory findings were correlated with anti-topo I antibody level as determined by ELISA in 30 SSc patients with anti-topo I antibody. These patients were relatively active as the disease duration in 14 of the 22 patients was less than 5 yr (mean 3.3 yr). Only two patients showed the atrophic change of skin sclerosis. As shown in Fig. 1
, the anti-topo I antibody level correlated positively with the modified Rodnan TSS (r=0.529, P<0.01) and correlated inversely with percentage VC (%VC) (r=-0.486, P<0.01). There was a positive association between anti-topo I antibody level and renal vascular resistance, which was determined as the PI value in the renal interlobar arteries by colour-flow Doppler scans (r=0.486, P<0.05). Furthermore, the anti-topo I antibody level was significantly elevated in SSc patients with pitting scar/ulcer (P<0.02), short sublingual frenulum (P<0.02), arthritis/arthralgia (P<0.02) or oesophageal involvement (P<0.005) compared with SSc patients without these clinical features (Fig. 2). The anti-topo I antibody level was not related to the use and dose of oral steroids or D-penicillamine. Thus, the anti-topo I antibody level was correlated with disease severity in SSc patients with anti-topo I antibody.
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Longitudinal study of anti-topo I antibody level
To determine whether the anti-topo I antibody level is stable over time or whether the changes in the levels are correlated with new onset or worsening of major organ involvement, we analysed 125 serum samples from 21 of 30 SSc patients who had anti-topo I antibody at their first visit. At the first visit, these patients were relatively active as the disease duration in 17 of the 21 patients was less than 5 yr (mean 3.0 yr). None of these patients showed the atrophic change of skin sclerosis at their first visit. During the follow-up period, four patients entered the atrophic stage of skin sclerosis accompanied by improved skin sclerosis. Fourteen patients with SSc showed stable or slightly improved skin sclerosis in response to low-dose steroids during the follow-up period, whereas two patients exhibited worsening skin sclerosis regardless of treatment with low-dose steroids. The remaining one patient showed significant improvement of skin sclerosis. When the anti-topo I antibody level showed more than a 30% increase or more than a 30% decrease at one or more time-points during the follow-up period compared with the antibody level at the first visit, the change was defined as an ‘increase’ and a ‘decrease’ respectively. Otherwise, the antibody level was defined as ‘unchanged’. According to these criteria, 21 patients with SSc were classified as follows: nine (43%) patients had an unchanged level of anti-topo I antibody; eight (38%) had a decreased level; and four (19%) had an increased level (Figs 3 and 4).
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Seven of nine patients with an unchanged anti-topo I antibody level had a relatively high level (over 150 U/ml) of anti-topo I antibody at their first visit, while the remaining two patients had a low level (below 100 U/ml) of the antibody (Fig. 3A
). Six of seven patients with a high level of anti-topo I antibody had stable or slightly improved skin sclerosis (TSS changed from 21±8 at the first visit to 15±7 at the final visit), whereas the remaining one patient showed slightly worsened skin sclerosis during the follow-up (TSS changed from 18 to 24). Although the follow-up period was relatively short, two patients with a low level of anti-topo I antibody had stable skin sclerosis (TSS changed from 15 to 14 and from 4 to 4 in the two patients respectively). All but one of the nine patients with a stable level of anti-topo I antibody over time received low-dose steroids, suggesting that the low-dose steroid treatment did not affect the anti-topo I antibody level. None of the patients in this ‘unchanged’ group entered the atrophic stage of skin sclerosis during the follow-up period. Thus, regardless of the anti-topo I antibody level at the first visit, the antibody level remained stable in 43% of SSc patients, the majority of whom had stable skin sclerosis.
Of the eight patients in the ‘decreased’ group, four patients with a high level of anti-topo I antibody (over 150 U/ml) at the first visit showed a decrease in antibody level that paralleled their transition to the atrophic stage of skin sclerosis during the follow-up period (TSS changed from 25±10 to 12±8; Fig. 3B). These four patients received low-dose steroids. In three patients of the ‘decreased’ group who had a low level of anti-topo I antibody (below 100 U/ml) at their first visit, the antibody level decreased, possibly in response to treatment with low-dose steroids. However, the skin sclerosis in these patients was stable during the follow-up period (TSS changed from 12±8 to 11±8). The remaining one patient in the ‘decreased’ group exhibited a drastic decrease in anti-topo I antibody level parallel with significant improvement of skin sclerosis, as shown in Fig. 4. Eight months before her consultation, this patient presented with swollen hands. At her first visit, active skin sclerosis involving the chest, Raynaud's phenomenon, arthralgia and pitting scars was noted. After 20 mg/ml of prednisolone had been started, the high level of anti-topo I antibody decreased in parallel with the improvement of TSS. No major organ involvement was detected during the follow-up period. Thus, the decrease in anti-topo I antibody level was associated with atrophic change or the improvement of skin sclerosis in SSc patients with high levels of this antibody at their first visit.
The anti-topo I antibody level increased during the observation period in four patients who had a high level of anti-topo I antibody at their first visit (Fig. 3C). There were no patients with a low level of the antibody at first visit in this ‘increased’ group. One patient exhibited subacute deterioration of interstitial pneumonitis at the time-point with the highest level of anti-topo I antibody, leading to steroid pulse treatment followed by treatment with 40 mg/day of oral prednisolone. One patient who had not been treated with steroids developed new major organ involvement (scleroderma renal crisis with cardiac tamponade), which resulted in death at the time-point with the highest level of anti-topo I antibody. This patient showed very high renal vascular resistance at the first visit (PI value 1.68), and the value remained high during the follow-up period. These two patients had stable skin sclerosis during the follow-up period (TSS changed from 16 to 16 and from 13 to 13 in the two patients). One patient exhibited worsening skin sclerosis after childbirth (TSS changed from 13 to 24), which slightly improved after treatment with 20 mg/day of prednisolone. The remaining one patient who received low-dose steroids did not develop any new organ involvement and showed stable skin sclerosis (TSS changed from 19 to 20). Thus, an increasing level of anti-topo I antibody was correlated mainly with worsening or the development of major organ involvement.
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Discussion |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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In this study, the anti-topo I antibody level was correlated positively with the modified Rodnan TSS and renal vascular resistance (Fig. 1
). A recent study indicated that modified Rodnan TSS correlates with outcome in SSc, as a high TSS at first visit predicted mortality and scleroderma renal crisis over the subsequent 4 yr [18]. We reported previously that SSc patients with elevated renal vascular resistance more frequently had digital pitting scars, a short sublingual frenulum, contracture of phalanges, pulmonary fibrosis, decreased %VC, heart involvement and elevated C-reactive protein compared with those with normal renal vascular resistance [16]. In addition, the anti-topo I antibody level was inversely correlated with %VC (Fig. 1), suggesting that the antibody level may be related to the severity of pulmonary fibrosis. Furthermore, the anti-topo I antibody level was significantly elevated in SSc patients with pitting scar/ulcer, a short sublingual frenulum, arthritis/arthralgia or oesophageal involvement (Fig. 2). In this study, we examined patients with relatively early SSc, as longer disease duration often results in atrophic changes of the sclerotic skin that may reduce the modified Rodnan TSS. Seven out of 30 SSc patients received low-dose steroids. However, none of the patients received immunosuppressive therapy or high-dose steroids, which have been reported to decrease the level of anti-topo I antibody [9]. Thus, our data suggest that the anti-topo I antibody level is correlated with the severity of SSc.
In the longitudinal study, patients with a low level of anti-topo I antibody at their first visit did not show any increase in antibody level during the follow-up period, but rather exhibited a stable level or a small decrease in the level, with relatively stable skin sclerosis (Fig. 3A and B). Therefore, a low level of anti-topo I antibody at first visit may identify a group of patients with milder disease among SSc patients with anti-topo I antibody. In patients with a high level of anti-topo I antibody at their first visit, this high level of antibody was associated predominantly with the development of new organ involvement or worsening skin sclerosis (Fig. 3C). Therefore, a high level of anti-topo I antibody at the first visit may identify a group of patients with more severe SSc. A decreasing level of anti-topo I antibody was accompanied mainly by atrophic skin change (Fig. 3B). However, it is noted that the anti-topo I antibody level may not reflect the severity of disease in patients who are in the atrophic stage, as pulmonary involvement still exists in these patients despite the decreased level of anti-topo I antibody. Thus, our data suggest that the anti-topo I antibody level at the first visit has prognostic significance.
In our study, we observed a decrease in the anti-topo I antibody level in some patients with SSc (Fig. 3B). Furthermore, a drastic decrease in anti-topo I antibody level in parallel with significant improvement in skin sclerosis was observed in one patient (Fig. 4). Kuwana et al. [9] reported a group of SSc patients in whom anti-topo I antibody disappeared during the course of the disease. These patients had a lower frequency of dSSc, their pulmonary fibrosis was less progressive and they had a better survival rate. The authors suggested that the loss of anti-topo I antibody in this patient group was due to loss of antigenic stimulation and not to the elimination of topo I-reactive T cells or B cells, as topo I-reactive T and B cells, after the loss of anti-topo I antibody, still have the capacity to produce anti-topo I antibody when they receive antigenic stimulation. Although anti-topo I antibody did not disappear in any of our patients, it is possible that some of our patients might lose anti-topo I antibody after a longer period of observation. The precise mechanisms of disappearance or reduction in the level of anti-topo I antibody remain unknown. However, the decreases in anti-topo I antibody level in our cases may also reflect diminished autoantigen stimulation.
Conflicting results have been reported concerning the association of increasing anti-topo I antibody level with clinical manifestations. Hildebrandt et al. [7] described two patients who developed myositis and worsening cardiac dysfunction with arrhythmia accompanied by a rise in the anti-topo I antibody level. In contrast, another study reported that changes in the anti-topo I antibody level were not associated with any particular clinical manifestations [8]. Similarly, Kuwana et al. [9] reported that none of the major increases in the anti-topo I antibody level was accompanied by the onset of serious manifestations of SSc. In the present study, three patients exhibited onset or worsening of major organ involvement parallel with an increase in antibody level (Fig. 3C). The reasons for the discrepancies among these studies are unknown, but may involve differences in the patient populations studied, the duration of the follow-up period and the assay systems used to detect anti-topo I antibody.
The results of this study suggest that the serum anti-topo I antibody level is clinically useful for evaluating the disease severity of SSc and predicting the prognosis. However, studies with larger numbers of SSc patients will be needed to confirm our findings.
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Notes |
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Correspondence to: S. Sato, Department of Dermatology, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan.
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