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Rheumatology 2001; 40: 1299-1307
© 2001 British Society for Rheumatology

Paediatric Rheumatology

Autologous stem cell transplantation for paediatric-onset polyarteritis nodosa: changes in autoimmune phenotype in the context of reduced diversity of the T- and B-cell repertoires, and evidence for reversion from the CD45RO+ to RA+ phenotype

Paediatric Rheumatology/Series Editor: P. Woo

L. R. Wedderburn, R. Jeffery, H. White1, A. Patel, H. Varsani, D. Linch2, K. Murray and P. Woo

Paediatric Rheumatology Unit, Institute of Child Health and Department of Molecular Pathology, University College London,
1 Molecular Immunology Unit, Institute of Child Health and
2 Department of Haematology, University College London Hospital, London, UK

Abstract

We have studied immune reconstitution in a patient with paediatric-onset polyarteritis nodosa treated with high-dose immunosuppressive agents followed by stem cell rescue. The patient developed several new autoimmune phenomena over the 18 months after immunosuppression and stem cell rescue. Flow cytometry, reverse transcription–polymerase chain reaction (RT-PCR) heteroduplex and isotype-specific RT-PCR analysis of immunoglobulin expression showed that the T- and B-cell repertoires were highly restricted in the first few months after treatment. The dominant T-cell clones seen after reconstitution were persistently expanded, were different from those which could be demonstrated before autologous stem cell transplantation, and were in the CD8+ population. Our data also show that 12 months after treatment these expanded T-cell clones were within the CD45RA+ population, suggesting that reversion from the CD45RO+ to the CD45RA+ phenotype had occurred in vivo.

KEY WORDS: T-cell receptor, Repertoire, CD45, Memory, Autologous stem cell transplantation, Vasculitis, Immune reconstitution, High-dose immunosuppression.

The treatment of systemic vasculitis has advanced considerably in the past 20 yr with the introduction of immunosuppressive therapies, including cyclophosphamide, intravenous (i.v.) immunoglobulin (Ig) and plasmapheresis [1]. However, a proportion of cases remain difficult to control, and these carry a poor long-term prognosis. Treatment of a range of autoimmune disorders, including rheumatoid arthritis (RA), systemic lupus erythematosus and scleroderma, using intense immunosuppression followed by reconstitution with a peripheral blood stem cell or bone marrow transplant has recently gained increasing attention [2–7]. In children, severe juvenile idiopathic arthritis and systemic sclerosis have been treated by this approach, with promising early results [8–10]. To date there have been few reports of the use of this approach as a treatment for vasculitis. In other studies of immune reconstitution, several groups have reported large T-cell expansions present after autologous stem cell transplantation (ASCT). However, whether these expansions arise from new naive cells derived from infused stem cells or from previously primed mature T cells is still unclear. In this study we report a patient who presented with polyarteritis nodosa at age 14 yr, whose disease proved increasingly unresponsive to extensive immunosuppressive therapy. The patient therefore underwent peripheral blood stem cell harvesting, intensive immunosuppression and rescue with autologous CD34+ stem cells.

Our study shows several novel features. During the 18 months after the procedure, the patient developed several new autoimmune conditions: we discuss the implications of this in the context of the treatment of autoimmune conditions by immunosuppression and stem cell rescue. By sequence-specific T-cell receptor (TCR) heteroduplex (HD) analysis of purified T-cell subsets, we show that clonal T-cell expansions, present within 2 months of stem cell rescue when the majority of the T cells express CD45RO+, are subsequently within the CD45RA+ T-cell subset at 1 yr after ASCT. These data suggest that T cells undergo reversion from CD45RO+ to RA+ in vivo. Therefore, the utility of the CD45 isoforms alone as markers of memory or naive cells is limited in patients who have undergone immune reconstitution.

Patients and methods

Patient
A 22-yr-old Caucasian female with an 8-yr history of juvenile-onset polyarteritis nodosa (PAN) was studied before and after ASCT. She presented aged 14 yr with fevers, headache, arthralgia, fatigue and erythema nodosa. She had a high acute-phase response, was negative for rheumatoid factor, antinuclear antibodies, anti-double-stranded DNA and antineutrophil cytoplasmic antibodies (ANCA), and had normal thyroid function. Hepatitis B and C serology was negative. Angiography was performed for diagnostic purposes, and demonstrated bilateral intrarenal aneurysms of medium-sized arteries. In view of the angiographic findings she was treated with cyclophosphamide and steroids. Over the following 8 yr she had multiple flares of disease (which were mirrored by rises in both erythrocyte sedimentation rate and C-reactive protein concentration on each occasion) with fevers, headaches, myalgia and severe abdominal pain. Over this time, her disease was difficult to control despite the use of oral and i.v. corticosteroids, cyclophosphamide (to a total dose of 51 g), oral colchicine, i.v. Ig and plasmapheresis. In the 18 months before ASCT she suffered increasingly frequent flares and repeat angiography showed new aneurysms in the hepatic arteries. It was felt that her disease was progressive and increasingly refractory to therapy, and she was therefore offered treatment with intensive immunosuppression and autologous stem cell rescue. Stem cells were harvested by leucopheresis after mobilization with cyclophosphamide (1.5 g/m2 on day -10) and granulocyte-colony stimulating factor (Lenograstim 10 µg/kg/day on days 10–8 before leucopheresis). CD34+ cells were purified by magnetic bead selection using the Clinimacs system (Miltenyi Biotec, Bisley, Surrey, UK) [11]. Depletion of T cells by a factor of more than 3 log was achieved; this is within recommended limits [12, 13]. Immunosuppressive conditioning was with 20 mg CAMPATH-1H (Schering Health Care, Burgess Hill, UK) (days -9 to -5), fludarabine 30 mg/m2 (days -8 to -4) and cyclophosphamide 1g/m2 on days -3 and -2. CD34+ cells (2x106 cells/kg) were returned on day 0. At the time of stem cell infusion, CAMPATH-1H would still have been active in vivo, resulting in further T-cell depletion of the graft.

Cell preparation, separation and flow cytometry
Venous blood samples were taken before and at various time-points after ASCT, with full informed consent. Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation. CD4+ and CD8+ PBMC populations were separated using magnetic beads (Miltenyi Biotec), and CD45RA+ and CD45RO+ purified T-cell populations by cell sorting on a Vantage cytometer (Becton Dickinson, San Jose, CA, USA), giving fractions which were routinely >=96% pure. For flow cytometry, PBMC were washed in phosphate-buffered saline/1% fetal calf serum (FCS)/0.1% sodium azide before standard three-colour analysis. We used monoclonal antibodies to CD3, CD4, CD8, CD28, CD45RO, CD45RA, HLA-DR and CD20 conjugated to fluorescein isothiocyanate, phycoerythrin or quantum red (Sigma, Poole, UK). 20 000–30 000 events were collected per condition on a FACStar cytometer (Becton Dickinson) and data were analysed using CellQuest software (Becton Dickinson).

Proliferative capacity of T cells
Proliferation of T cells was followed by labelling with the dye carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA). Briefly, PBMC were incubated in 2.5 µM CFSE for 5 min at 37°C, washed in RPMI/10% FCS, and then resuspended at 106 cells/ml in the presence of 1 µg/ml phytohaemagglutinin (Sigma) and 10 ng/ml interleukin IL-2 (Eurocetus, Harefield, UK) for 7 days. Cells were washed three times and stained for CD3, CD4 and CD8 before analysis. CFSE was detected in the FL-1 channel using standard flow cytometric methods.

RNA and cDNA synthesis
Total PBMC or purified cell populations (2x106 cells) were used to prepare total RNA using RNAzol (Biogenesis, Poole, UK) and 5 µg RNA was used for first-strand cDNA synthesis with oligo-dT using Superscript RT (Gibco, Paisley, UK) according to the manufacturer's instructions.

HD analysis and analysis of Ig repertoire
Reverse transcription–polymerase chain reaction (RT-PCR) HD analysis was performed as described [14]. One 60th of cDNA was used in each of 26 PCR reactions for TCR, using TCRBV (beta variable region) primers and an internal TCRBC (beta constant region) primer [14] in parallel with 26 HD carrier PCR reactions using an external TCRBC primer. Twenty microlitres of sample PCR product was mixed with 200 ng of carrier product; the mixture was heated to 95°C for 5 min and then annealed at 50°C for 1 h. HD products were analysed on a 12% non-denaturing polyacrylamide/0.5% Tris/borate/EDTA gel, blotted onto Hybond N+ membrane and probed using a carrier-specific TCRBC probe [15]. Isotype-specific RT-PCR analysis of Ig expression was performed as described [16]. Briefly, RT-PCR was performed on cDNA made from samples equalized for B-cell numbers. In the first round of PCR, primers specific for heavy chain variable gene 1 (VH1) (forward) and IgM, A or G1 (reverse) were used for 30 cycles [16]. A common heavy chain joining gene (JH) {gamma}33P dATP-labelled primer was used for nested run-off PCR (12 cycles), and the labelled products were resolved on a 5% denaturing sequencing gel.

Results

Altered autoimmune phenotype after immune reconstitution
Haematological reconstitution occurred rapidly after ASCT, neutrophils reaching>0.5x109/l on day 11. The patient had no major infective complications. One month after ASCT the white blood cell count was 4.7x109/l (neutrophils 83%), although the T-cell count remained low for a further 8 months. One month after ASCT the patient developed a mild flare with fever and abdominal pain, which responded well to low-dose steroids and i.v. Ig. For the next 5 months she remained well and off immunosuppressive medication other than low-dose prednisolone (<10 mg/day). Over the ensuing year she developed several new autoimmune phenomena which were not present before ASCT. These included a new vasculitic rash on the lower extremities, which settled with oral steroids and monthly i.v. Ig for 3 months. Fourteen months after ASCT, she developed autoimmune hyperthyroidism, with tremor, resting tachycardia and proximal myopathy. Thyroid-stimulating hormone was<0.06 mU/l and thyroxine >77 pmol/l. The patient was positive for thyroglobulin antibodies (1:2560) and p-ANCA (anti-myeloperoxidase, 1:242), the latter probably due to cross-reactivity of anti-thyroperoxidase antibodies [17, 18]. Carbimazole and propranolol controlled these symptoms. At 18 months, the patient developed autoimmune thrombocytopenia, presenting with spontaneous bruising, petechiae and a platelet count of 14x109/l. A bone marrow aspirate showed a greatly increased number of megakaryocytes, and platelet-associated IgG and IgA were strongly positive, consistent with idiopathic thrombocytopenic purpura (ITP). The platelet count recovered with i.v. Ig and oral steroids. Eighteen months after ASCT, it is noteworthy that the patient has had no other flares of her previous PAN symptoms and, in particular, no abdominal symptoms, which had been predominant previously.

Immune reconstitution
Equal cell numbers of total PBMC or purified cell populations were used to prepare RNA and cDNA for molecular analysis of TCR expression by HD analysis, and Ig repertoire by RT-PCR spectratyping. A summary of the phenotype of T-cell populations over time is shown in Table 1Go. As previously reported in other patients undergoing stem cell reconstitution, the CD4:CD8 ratio remained inverted until 9 months after ASCT, with a more rapid expansion of CD8+ than of CD4+ T cells. Both 3 and 9 months after reconstitution, the T-cell response to a mitogenic stimulus in the presence of exogenous IL-2 showed an increased number of rounds of cell division compared with that of T cells before immunosuppression and ASCT, as indicated by a CFSE-labelled proliferation assay (Fig. 1Go). This phenomenon occurred with both CD8+ and CD4+ T cells (data not shown) and there was no intercurrent viral infection or febrile illness at the time of sampling. B cells recovered more rapidly than T cells, and in the first 6 months after stem cell rescue the majority of B cells were IgM+ (data not shown).


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  		TABLE 1. Phenotype of T-cell populations before and up to 16 months after stem cell rescue

 


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  		FIG. 1. Analysis by flow cytometry of proliferation in response to phytohaemagglutinin and IL-2 for 7 days by peripheral blood T cells before and 9 months after stem cell rescue. The histograms show CFSE-labelled cells; both plots are gated on CD3+ cells. Peaks to the left of the x-axis represent cells which have divided during the assay. One month before immunosuppression and stem cell rescue, 38% of the CD3+ cells were in the 6th peak of division (M2) and only 1.5% in the 7th peak (M1); 9 months after stem cell rescue, the largest peak, representing seven divisions (M1), contained 57% of the CD3+ cells.

 

Complexity of the expressed immune repertoires
The degree of diversity of the antigen-specific TCR was assayed by the HD technique [15]. This technique, which is based on TCR CDR3 sequence differences, can detect T-cell clones at a frequency of 1 cell in 50 000 [19]. Before intensive immunosuppression and stem cell rescue, our patient had a highly diverse repertoire by HD analysis, with no distinct expanded clones expressed in the 24 TCRBV families when analysed in total T-cell populations (data not shown). In contrast, HD analysis of the TCR expressed after stem cell rescue showed highly oligoclonal TCR expression with clear clonal bands in 20 out of 24 families. Representative HD analysis of TCRBV1–8, carried out on cDNA from total PBMC taken 3 months after ASCT, is shown in Fig. 2AGo. Many of these clonotypes were still clearly demonstrable 1 yr after stem cell rescue.



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  		FIG. 2. T- and B-cell repertoires after ASCT were highly restricted. (A) Heteroduplex analysis of TCR expression by CD3+ cells taken 3 months after ASCT. Representative TCR families (TCRBV1–8) are shown. Polyclonal T-cell populations produce a smear of DNA, while expanded clonal populations produce distinct HD bands, which are visible above the ‘carrier’ DNA bands (arrowed). (B) RT-PCR spectratyping of Ig expression from B cells taken before and after ASCT was performed using primers specific for IgM, IgG1 or IgA at the time-points shown. A polyclonal B-cell repertoire produce a smooth ladder of PCR products, each separated by three nucleotides, due to their CDR3 lengths. In contrast, a distorted repertoire (for example IgA and IgG1 at time +2 months) produce several dark bands due to clonally expanded B cells.

 
We analysed the diversity of the Ig repertoire by RT-PCR Ig spectratyping [16] (Fig. 2BGo). The IgA and IgG1 B-cell spectratypes expressed 2 months after ASCT showed a marked lack of diversity, evidenced by strong bands indicating the presence of large clonal expansions, compared with B cells before ASCT. The expressed Ig repertoire at 9 months was more diverse (Fig. 2BGo). These data suggest that the rapid increase in B-cell numbers has occurred through the expansion of relatively few clones, possibly with little T-cell help, given the low CD4+ numbers and constriction of the class-switched IgG1 and IgA repertoires. Whether the lymphocyte clones that expand rapidly during reconstitution after stem cell rescue do so in response to antigen or in an antigen-independent manner is unclear. To address this, we proceeded to identify the subpopulation of T cells containing clonal expansions.

CD45RO and RA+ T-cell phenotype: evidence for reversion of clonotypes
The expression of the CD45 isoforms was analysed on T cells from blood over the first 16 months after ASCT (Table 1Go). Immediately after ASCT, the majority of T cells were CD45RO+ and the majority of both CD4+ and CD8+ T cells were HLA-DR+. In the 12 months after ASCT the expression of CD45RA increased, and this was more rapid in the CD8+ than in the CD4+ cells (Table 1Go). The distinction between naive and memory cells based on CD45 expression is unclear, in particular for CD8+ cells, as many studies suggest that CD45RO+ CD8+ cells may revert to the CD45RA+ phenotype [20–22]. We compared the clonal TCR diversity of CD45RA+ and RO+ purified T cells from 1 yr after ASCT. Dominant HD clonotypic bands demonstrable at 1 yr were clearly visible in the CD45RA+ but not the CD45RO+ T cells (Fig. 3AGo). Separation of CD45RO+ from CD45RA+ cells was technically not possible 1 month after ASCT because of very low T-cell numbers. Parallel HD analysis of PBMC just after ASCT, CD8+ from 6 months and CD45RO+ or CD45RA+ purified T cells from 12 months after ASCT showed identical banding patterns (Fig. 3BGo). These data indicate that T-cell expansions, which were detectable as early as 1 month after reconstitution, subsequently had a CD45RA+ phenotype 1 yr after treatment.



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  		FIG. 3. Clonotypes of expanded T cells 1 yr after stem cell rescue were within the CD45RA+ population. (A) HD analysis (TCRBV6 and 8) was performed on CD45RA+ and RO+ purified T cells and run in parallel with PBMC reactions, all from 1 yr after treatment. (B) Parallel HD analysis (TCRBV6) of PBMC, CD8+ and CD45RA+ and RO+ purified T cells at the time-points shown.

 

Recovery of CD8+ T-cell numbers in the context of a low-diversity TCR repertoire
Because the initial recovery of the CD8+ T-cell population was considerably more rapid than that of the CD4+ cells, we performed HD analysis to compare TCR expressed by separated CD4+ and CD8+ T cells. All of the dominant oligoclonal bands seen in the initial HD analysis segregated with the CD8+ T cells (Fig. 4AGo). Parallel analysis of CD8+ cells from before and after ASCT showed that, in some TCR families, clonal bands could be detected in the CD8+ cells before ASCT (Fig. 4BGo). This TCR repertoire clonality before treatment may have been related to previous intense immunosuppression or to the disease itself. However, it may also have been part of the normal spectrum, as CD8+ oligoclonality is a common finding in normal donors [23–25]. Even in these TCRBV families, the bands seen after ASCT were unique and different from the pre-existing clonotypes, indicating that they did not simply reflect the hierarchy of the T-cell clones present before ASCT (Fig. 4BGo).



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  		FIG. 4. Persistent clonal expansions after ASCT were in the CD8+ population, and differed from clonotypes seen before ASCT. (A) HD analysis (TCRBV families 6 and 13.2) performed on purified CD4 and CD8 T cells taken 4 months after treatment. TCR from CD4+ cells produced a smear, indicating a diverse set of TCR molecules; in contrast, CD8+ cells showed several clonal bands. (B) HD analysis for TCRBV6, 8, 13.2 and 20 performed on CD8+ cells taken before (Pre) and 3 months after (Post) ASCT treatment. Where specific clones were demonstrable in the initial CD8+ T cells (TCRBV20), these have fingerprints different from those of the expansions seen after ASCT, indicating different CDR3 region sequences.

 

Discussion

In this study of a patient with juvenile-onset PAN treated with intensive immunosuppression and stem cell rescue, we present several novel findings, both in the clinical features, and the analysis of antigen receptor expression in defined lymphocyte subsets. The clinical course after immune reconstitution was complicated by disorders which are considered to be autoimmune in origin, including a vasculitic rash, thyrotoxicosis with high titres of organ-specific antibodies, and autoimmune thrombocytopenia. The causal factors for new autoimmunity in our patient are unclear. However, it is interesting that in another recent study, of patients treated with the CAMPATH-1H (anti-CD52) antibody for multiple sclerosis, 30% of patients developed autoimmune thyroid disease after CAMPATH-1H treatment [26]. CAMPATH-1H has also been used as immunotherapy for other autoimmune conditions, including RA [27, 28]. Although these reports do not comment upon thyroid disease after CAMPATH-1H treatment, one other group has observed this complication in two of 53 RA patients treated with the antibody (J. Isaacs, personal communication). There is a report of Grave’s hyperthyroidism in a patient treated with anti-lymphocyte globulin for aplastic anaemia [29, 30], and thyroid disease has been reported after conventional bone marrow transplantation or ASCT, but this is generally transient and self-limiting [31–33]. Cases of ITP after peripheral blood stem cell transplantation have been reported previously, for example after treatment for acute myeloid leukaemia [34].

Given our experience and these reports, we suggest that all patients undergoing intense immunosuppression and immune reconstitution, in particular those who receive CAMPATH-1H, should be monitored for new autoimmune phenomena. It is possible that the breakdown of tolerance seen after ASCT in our patient mirrors that seen in animal models, in which naive T cells expand rapidly in a lymphocyte-depleted animal [35, 36]. This autoimmunity can be prevented by simultaneous infusion of regulatory T cells (Treg) whose phenotype is CD4+CD38+CD25+ [37]. At present, the lack of a specific marker for human Treg makes these cells difficult to identify.

In our patient we wished to compare the immune repertoires of both B cells and defined T-cell subsets before and after treatment with ASCT. Previous studies of TCR expression using spectratyping or sequence analysis after bone marrow reconstitution have seen marked oligoclonality in the TCR repertoire for several months after reconstitution [38–40]. We believe that this study is the first clonal TCR analysis of the CD45RA+ and CD45RO+ subsets analysed in parallel with CD4 and CD8 cell TCR expression, and the first application of the Ig spectratyping technique to this group of patients. As predicted, our patient had highly restricted T- and B-cell repertoires after ASCT. The TCR HD analysis demonstrated that large expansions of T cells were present immediately after ACST in the CD8+ T cells and that these persisted for at least 1 yr. It is likely that both the autoimmune disease itself and the drugs given to treat it, in particular the long-term use of cyclophosphamide, will have affected thymic function and peripheral immune repertoires before ASCT [41, 42]. We therefore compared clones seen after reconstitution with any detectable in CD4 and CD8 separated cells before ASCT. This showed that the T-cell clones seen after reconstitution were not simply a reflection of the dominant CD8+ clones present before ASCT. In addition we showed that, 12 months after ASCT, these expanded T cells were in the CD45RA+ population. Parallel analysis of serial samples showed that the pattern of these clonotypes was identical to that of the CD8+ clones present immediately after stem cell rescue (when the majority of T cells were CD45RO+), indicating that they represent the same set of TCR (since HD is sequence-specific).

In the first 6 months after ASCT, the level of CD45RA+ expression increased more rapidly on CD8+ than CD4+ T cells, as observed previously [39]. Our data suggest that the clonal bands detected represent TCR from expanded T cells which have reverted from the CD45RO+ to the CD45RA+ phenotype over the year analysed. There is now powerful evidence that CD45RA+ is not a reliable marker of naive T cells for the CD8+ subset, because of the capacity of antigen-experienced CD8+ cells to revert to CD45RA+ expression [20–22]. In addition, the high level of HLA-DR expression by T cells immediately after ASCT suggests that the majority of these cells had an activated phenotype.

As in previous studies, our patient had a very high level of expression of CD45RO+ on peripheral T cells in the first 3 months after immune reconstitution [38, 43]. Several mechanisms may explain the origin of the CD45RO+ T cells after stem cell infusion. A small number of previously primed memory T cells may survive, whether in the patient or in the CD34+-selected graft, despite adequate T-cell depletion and the presence of CAMPATH-1H in vivo. If so, these may have an initial proliferative advantage over newly generated naive thymic emigrants. Alternatively, the expression of CD45RO+ may not indicate previous antigen priming, as newly generated clones may rapidly acquire a CD45RO+ phenotype while undergoing proliferative expansion. A recent mouse study has shown that T cells that are entirely naive with respect to their cognate antigen may become phenotypically identical to memory cells through rapid proliferation in a T-cell-depleted animal [44]. The measurement of TREC (T-cell receptor excision circles) DNA, a marker of recent thymic emigrants, in these cells will be valuable in determining the origin of these clones [45, 46]. An alternative would be to measure the expression of CD27, a marker of true naive status on CD45RA T cells [47]. Whatever the source of these CD45RO+ cells, we suggest that some of them undergo reversion from CD45RO+ to RA+ in vivo as the rate of expansion begins to slow. If so, then the quantitation of CD45RA+ T cells will be an unreliable measure of newly generated T cells after immune reconstitution, in particular for CD8+ T cells.

In conclusion, our study has provided novel insights into the restoration of T- and B-cell repertoires in a rapidly changing field, that of the application of stem cell transplantation to autoimmune disease. In particular, we propose that data which rely upon CD45 isoform expression to indicate that T cells are either primed/memory or new thymic emigrant naive cells should be interpreted with caution. In addition we suggest that, in patients who may have a genetic background which predisposes them to autoimmunity, rapid immune reconstitution can be associated with new autoimmune phenomena. A fuller understanding of the source and behaviour of the newly generated lymphocytes after stem cell transplantation is needed, to allow regimens for immunosuppression (or ‘conditioning’) to be optimized for patients with severe autoimmune diseases who are treated by this approach.

Acknowledgments

We are very grateful to the patient for the many samples of blood contributed to assist this research. We thank the staff of UCLH and Dr M. Watts for data on CD34+ purification. This work was supported by grants from The Wellcome Trust (LRW, AP) the UCLH Special Trustees (HV), the Arthritis Research Campaign (RJ), the Primary Immunodeficiency Association (HW) and the Medical Research Council (PW). This work was undertaken at UCLH and Great Ormond Street Hospital for Children NHS Trusts, which received a proportion of their funding from the NHS Executive. The views expressed in this publication are those of the authors and are not necessarily those of the NHS Executive.

Notes

Corresponding author: L. R. Wedderburn, Rheumatology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. Back

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Submitted 26 January 2001; Accepted 3 July 2001


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 Arch. Dis. Child.Home page
L R Wedderburn, M Abinun, P Palmer, and H E Foster
Autologous haematopoietic stem cell transplantation in juvenile idiopathic arthritis
Arch. Dis. Child., March 1, 2003; 88(3): 201 - 205.
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