Coeliac disease (CD) is a common gluten-dependent enteropathy. It is considered to be a T-cell–mediated disease with an uncontrolled response to gluten peptides in genetically susceptible individuals (1). The permanent sensitivity to gluten requires a lifelong gluten-free diet (GFD). Patients with CD have an increased risk of autoimmune diseases. The cumulative risk of 8.1% is reported at the age of 15 years (2) with, for example, co-occurring type 1 diabetes mellitus, autoimmune thyroiditis, liver diseases, rheumatoid diseases, skin diseases, and serious haematological disorders. Recently the association with aplastic anaemia was reported for the first time in 3 adult patients (3).
We present the case of a girl with an unusual initial presentation of CD with severe immune hepatitis that resolved on a GFD followed by severe aplastic anaemia. Subsequent allogeneic haematopoietic stem cell transplantation (alloSCT) resulted in the cure of her CD.
An 11-year-old girl presented with a short history with jaundice and elevated liver enzymes (bilirubin 91 μmol/L, alanine aminotransferase 2290 U/L). She was known to have mild abdominal pains. Serologic markers for autoimmune hepatitis were negative. The CD serological screening was positive for aspartylglucosaminidase and tissue transglutaminase. Small-bowel histology demonstrated typical subtotal villous atrophy (Marsh 3C), and liver biopsies showed mild lobular and septal hepatitis with portal fibrosis (Metavir 2). On a GFD all of the symptoms resolved, including jaundice, liver tests, and later the coeliac serology. The family history was positive for liver pathology. Her father has primary sclerosing cholangitis and his sister is known to have primary biliary cirrhosis combined with autoimmune hepatitis. All first-degree relatives had serologic coeliac screening, resulting in a CD diagnosis in the patient's asymptomatic sister (Marsh 3A).
A few months after presentation, pancytopenia developed and evolved into severe aplastic anaemia. Within the next year 2 cycles of immunosuppressive therapy (antithymocyte globulin, prednisone, cyclosporin) were given, without result. Finally, after a conditioning regimen containing cyclophosphamide (200 mg/kg) and antithymocyte globulin (20 mg/kg), she successfully received an alloSCT from her father, who was human leukocyte antigen (HLA) identical except for a mismatch in HLA-DQ (DQxyz in father vs abc in patient). Haematological recovery was uncomplicated. Analysis by XY-fluorescence in situ hybridization probe showed complete donor chimerism. Recovery of peripheral blood lymphocytes showed absolute counts of natural killer cells and B cells within the normal range and a recovery of T-cell count, with normal CD4/CD8 and CD4/CD45RA/RO ratio.
During this entire period the girl stayed on a GFD. Six months after transplantation a normal gluten-containing diet was reintroduced. She remained symptom free. Coeliac serology (aspartylglucosaminidase and tissue transglutaminase) remained negative during the next 16 months.
After 7 months on GFD, multiple duodenal biopsies were taken proximal and distal to the duodenal papilla to analyse histology (standard procedures) and to isolate polyclonal T-cell lines. The small intestinal biopsies were treated with dithiothreitol and ethylenediaminetetraacetic acid to remove epithelial cells and subsequently brought into culture with either a trypsin/pepsin digest of gluten (gluten) or with a trypsin/pepsin digest of gluten that was treated with tissue transglutaminase (tTG-gluten) for 5 days. Subsequently, interleukin (IL)-2 and IL-15 were added. Part of the cultured cells were analysed on a fluorescence-activated cell sorting probe for the presence of CD4 and CD8 T cells. Restimulation was performed with irradiated allogeneic peripheral blood mononuclear cell, IL-2, and IL-15. Proliferation assays to test for gluten reactivity were performed in triplicate in 150 μL Iscove's modified Dulbecco's medium supplemented with 10% human serum in 96-well flat-bottomed plates using 104 T cells stimulated with 105 irradiated HLA-DQ2–matched or autologous peripheral blood mononuclear cells (3000 RAD) in the presence or absence of antigen (1–10 μg/mL). After 48 hours at 37°C, cultures were pulsed with 0.5 μCi of 3H-thymidine and harvested 18 hours thereafter. HLA typing was performed according to standard procedures.
All of the small intestinal biopsies had normal histology with crypt/villous ratios of 1:4. Only a few patchy areas with focal intraepithelial lymphocytosis (CD3+) were seen at the level of the duodenal bulb. The fluorescence-activated cell sorting analysis of the isolated intestinal lymphocytes revealed the presence of single-positive CD4 and single-positive CD8 cells, as well as CD4- and CD8-negative cells. Polyclonal T-cell lines generated from the small intestinal biopsies were HLA typed and found to be primarily of donor origin. No reactivity against gluten or tTG-gluten could be detected in these celllines.
AlloSCT has resulted in the improvement or stabilisation of various autoimmune diseases including juvenile arthritis, systemic lupus erythematosus, severe aplastic anaemia, and CD. In CD there are few data for haematological cell transplantation. In adults, the results of alloSCT for enteropathy-associated intestinal lymphoma are disappointing, and improvement but no cure has been suggested for refractory CD (4). Conversely, 2 patients (1 child) were reported to experience clinical manifestations of CD after allogeneic bone marrow transplantation for the treatment of acute myelogenous leukaemia (5). In these cases the disease was most likely caused by the transfer of gluten-reactive T cells from the donor to the patient, but no isolation of lymphocytes from duodenal biopsies was performed to demonstrate that they were indeed of donor origin.
Improvement of clinical manifestation and normalisation of coeliac serology has been reported in a child with CD treated with alloSCT for co-occurring acute myelogenous leukaemia (6). This communication did not report on changes of small intestinal villous architecture, the criterion standard for the presence or absence of CD. In a previous study, we reported the presence of CD4+ gluten-reactive intestinal T cells in untreated children with CD (1). Such cells respond to gluten peptides bound to the disease predisposing HLA-DQ2 or -DQ8 molecules and are uniquely found in biopsies from patients with CD and never in biopsies from patients without CD (1). Using identical procedures we have now generated T-cell lines from the patient's intestinal biopsies after autologous haematopoietic stem cell transplants. The isolated cells were HLA typed and found to be primarily of donor origin. However, a weak signal corresponding to DPB1*0301 was found. Because the recipient was DPB1*0301 positive and the donor DPB1*0301 negative, this indicates that in the small intestine biopsies a few cells from the recipient were still present. Nevertheless, no response to gluten could be demonstrated. Whilst this does not completely rule out the possibility that gluten-reactive T cells are still present in the child, it is unlikely that these are abundant because we are usually capable of isolating such cells from small intestinal biopsies of children with CD (1).
In conclusion, in our patient normal villous architecture, absence of gluten-specific T cells, and small bowel chimerism indicate that a successful alloSCT cured CD in this patient, although long-term follow-up is required to establish whether this condition is stable.
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