We describe two brothers who suffered from hyper-IgM syndrome (HIGM1) with similar clinical features: recurrent infections, especially cryptosporidium gastroenteritis with cholangitis. Their activated T cells did not express CD40L. Nucleotide sequencing revealed a mutation in both boys with respect to intron 4 and exon 5 boundaries of the CD40L gene in Xq26. They underwent successful bone marrow transplantation (BMT) from HLA-geno-identical siblings. The Cryptosporidium infection and cholangitis resolved thereafter. At 6 months after BMT, expression of CD40L on activated T lymphocytes was normal. After 1 year, both boys are well, and immune reconstitution has improved. Based on these two successful experiences, BMT with a genoidentical sibling seems a reasonable therapeutic approach for HIGM1, if Cryptosporidium infection occurs.
Hyper-IgM Syndrome (HIGM1) is a primary immunodeficiency associated with mutations in the gene encoding the CD40L, located at Xq26. Patients suffer recurrent sinopulmonary and gastrointestinal infections, opportunistic infections due to Pneumocystis carinii, Cryptosporidium parvum, Histoplasmosa capsulata and Leishmania. Dysimmunity and malignancy are classical complications.1,2
CD40L, a membrane glycoprotein, is transiently expressed on activated CD4+ and CD8+ T cells, mast cells, basophils, eosinophils and platelets. CD40, the CD40L receptor, is a member of the TNF receptor family found on B cells, monocytes, dendritic cells, epithelial cells, fibroblasts and some malignant cells. CD40 expression can also be induced on different cells by gamma interferon (IFNγ).3,4 Thus, a potential mechanism contributing to C. parvum immunity could be a direct triggering of apoptosis in CD40+ infected cells by CD40L expressed on activated T cells. More recently, Hayward et al4 have shown in a mouse model that CD40 expression on intestinal epithelial cells is not required for C. parvum clearance. Moreover, C. parvum infection can be cleared only when CD40 is present on dendritic and mononuclear phagocyte cells. Interleukin 12 production by these antigen-presenting cells leads activated T cells (via CD40-CD40L pathway) to produce TH1 cytokines such as IFNγ.
C. parvum, a coccidian parasite, infects the intestine and sometimes the respiratory tract. In immunocompromised patients, it may cause severe intestinal fluid loss, with dehydration, chronic diarrhea and malnutrition. Several antimicrobial treatments have been tried but none has proven effective. As previously reported by Hayward et al,5 patients with HIGM1 are at increased risk of cholangiopathy, especially with C. parvum infection.
Curative therapy of HIGM1 relies on bone marrow transplantation (BMT), from an HLA-matched sibling, as first reported by Thomas et al,6,7 or from matched unrelated donors.8 However, the presence of C. parvum infection, especially when complicated by sclerosing cholangitis, impairs the results of BMT. Khawaja et al9 reported three cases where patients suffering from severe C. parvum infection died soon after BMT.9
In this report, we describe two brothers with HIGM1 who were both suffering from severe C. parvum infection with gastroenteritis and sclerosing cholangitis. They underwent successful BMT from a genoidentical HLA sibling and recovered from the C. parvum infection.
Materials and methods
Two brothers, had a family history of immune deficiency: two cousins suffered from HIGM1, one of whom died of diffuse C. parvum infection despite an HLA-sibling allogeneic bone marrow transplant together with an orthotopic liver transplant.
Thanks to family screening, the two brothers were diagnosed with HIGM1, confirmed by the absence of CD40L expression on activated T lymphocytes. Sequencing of PCR-amplified CD40L genomic DNA revealed the same mutation for the two brothers: a 10-nucleotide deletion (gTGTTACAGT) and a dinucleotide insertion (aa) at the intron 4/exon5 boundary. This mutation predicts a premature termination of translation and generation of a truncated protein. The brothers presented similar clinical features. Treatment was initiated with intravenous (i.v.) immunoglobulin and P. carinii prophylaxis.
At 6 years of age, brother A had Cryptosporidium gastroenteritis with sclerosing cholangitis resulting in persistent diarrhea and severe weight loss requiring continuous enteral nutrition. The sclerosing cholangitis worsened and at MR-cholangiography, dilated extra and intrahepatic bile ducts were observed. Needle liver biopsy revealed sclerosing cholangitis with prominent peri-portal concentric fibrosis, eosinophilic infiltration but no biliary cirrhosis (Figure 1). Liver function tests were abnormal (Table 1). Alkaline phosphatase was elevated and serum aspartate and alanine amino transferase were six times above normal. There was also chronic, but not severe, neutropenia and eosinophilia.
Brother B had recurrent common sinus infections. At the age of 8 years, he also suffered from a Cryptosporidium infection with cholangiopathy, requiring enteral nutrition. The results of MR-cholangiography were similar to those of A (Figure 2). Needle liver biopsy revealed cholangitis with mild, nonconcentric, fibrotic enlargement of the portal tracts and eosinophilic infiltration. Liver function tests were also abnormal. Alkaline phosphatase was elevated and serum aspartate and alanine amino transferase levels were five times above normal. Neutropenia and eosinophilia were also present.
After considering the poor prognosis of this disease, especially when it is complicated by Cryptosporidium infection, and given the family history, the parents gave informed consent for their two sons to undergo BMT. The donors were two different HLA-identical sisters.
The boys were conditioned with busulfan (4 mg/kg body weight per day for 4 days) followed by cyclophosphamide (50 mg/kg body weight per day for 4 days). Graft-versus-host disease (GVHD) prophylaxis consisted of short-term methotrexate (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, 11 after BMT), and cyclosporine A (3 mg/kg/day as a continuous i.v. infusion on days −1 to +30 and 6.25 mg/kg twice daily from days 31 to 180 post-BMT). Infection prophylaxis included isolation in a laminar air-flow unit, oral administration of nonabsorbable antibiotics and treatment with i.v. immunoglobulins for 1 year (200 mg/kg weekly for 3 months and then 400 mg/kg per month). The boys continued with enteral nutrition and anti-Cryptosporidium treatment (paromomycin 500 mg/day and azithromycin 500 mg/day).
On 27 July 2001, B received 1.6 × 108 nucleated marrow cells/kg and 3.36 × 106 CD34+ cells/kg, and A received 0.78 × 108 nucleated marrow cells/kg, and 5.8 × 106 CD34+ cells/kg on 3 August 2001.
Immunofluorescence detection Stools were prepared and processed using the monofluokit Cryptosporidium (Sanofi Pasteur, France), based on fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies directed against the oocyst wall. Cryptosporidium sp oocysts (5-7 μm) were detected by fluorescence microscopy.
PCR detection Stools were processed and DNA was extracted with QI Amp stool MiniKit (Quiagen). The reaction was performed using primers SB012F and SBR012R that amplified a 458 bp fragment specific for C. parvum.10
Clinical course after BMT
The clinical course for A was uneventful. At day 17, his absolute neutrophil count was over 500 per cubic millimeter and platelet counts were self-sustaining at levels above 50 000/μl at day 26. Neither GVHD nor veno-occlusive disease was observed. He had suffered from sinusitis (no organism identified) successfully treated by i.v. antibiotherapy. The Cryptosporidium infection disappeared soon after engraftment and was undetectable after day 60, despite immunological defects due to the allograft procedure. At 4 months after BMT, MR-cholangiography was significantly improved. After 19 months, stabilization of the cholangitis and decreased dilation of the extra and intrahepatic bile ducts was confirmed (Figure 2). Liver function tests normalized by day 60 post-BMT (Table 1). On day 200 post BMT, the quantitative and qualitative expression of CD40L on T cells was normal. DNA analysis revealed that 100% of lymphocytes and polynuclear cells were of donor origin. Immune functions have improved regularly. Today, the patient is well, requires no parenteral nutrition, has had no further infection, no GVHD, and is going to school.
At day 10, B had sinusitis that resolved with i.v. antibiotics. At day 13, he had grade 2 veno-occlusive disease that resolved with appropriate treatment. At day 49, he developed acute GVHD of the gut, successfully treated by corticotherapy. On day 15, an absolute neutrophil count higher than 500 per cubic millimeter was observed, and platelet counts were above 50 000/μl at day 20. Cryptosporidium was detectable until day 16 after BMT and then, as with his brother, the infection resolved. Results of MR-cholangiogram analysis and liver function tests were similar to those of A. On day 200 post BMT, quantitative and qualitative expression of CD40L on T cells was normal. DNA analysis revealed that 100% lymphocytes and polynuclear cells were of donor origin. Immune functions have improved regularly.
Today, the patient is well, requires no parenteral nutrition, has had no infections, no recurrence of GVHD, and is going to school.
At 1 month after cessation of i.v. immunoglobulin, normal production of IgM, IgG, IgG subclasses and specific antibodies to tetanus and diphteria toxoid occurred. The same time course of lymphocyte reconstitution was observed in the two brothers. The T-cell count, normal before BMT, improved gradually and was completely normal 19 months post BMT (1618 × 106/l for A and 1123 × 106/l for B). The T-cell subset, evidence of thymic function (CD4+ CD45RA+) simultaneously reached normal levels (412 × 106/l for A and 312 × 106/l for B). The absolute B-cell count, low before BMT, normalized within the first year after BMT. Moreover, memory B cells (CD19+CD27+), absent before BMT, were first observed 1 year post BMT.
Before BMT, in vitro proliferative responses to T-cell mitogens were normal but no proliferative response to tetanus toxoid antigen could be observed. At 1 year post BMT, response to mitogen completely normalized. The same normalization was observed after stimulation with tetanus toxoid antigen.
We describe two brothers with HIGM1. Both had a severe form of Cryptosporidium infection with sclerosing cholangitis requiring continuous enteral nutrition. Both underwent successful BMT without serious complications. Despite weak immune defences, the Cryptosporidium infection did not worsen but disappeared soon after BMT. After 1 year, immune reconstitution has gradually improved and both patients are currently well with a good quality of life.
The absence of serious, lesions in the liver or other organs allowed us to perform a standard BMT with myeloablative conditioning. This was not the case for the cousin who also suffered from HIGM1, complicated by frequent pulmonary infections which caused bronchiectasis. He also had a C. parvum infection with liver lesions including sclerosing cholangitis. At the age of 12 years, he underwent orthotopic liver transplantation, followed by BMT. This was unsuccessful and he died of diffuse C. parvum infection with pulmonary and hepatic failure. This was probably due to the parasite, the advanced sclerosing cholangitis, as well as to the pulmonary insufficiency and patient's age at BMT. Owing to this case, the two brothers underwent BMT at a younger age, before serious lesions had occurred and in the absence of any other organ injury.
Khawaja et al9 reported two HIGM1 patients with severe liver disease and C. parvum infection, both of whom died soon after myeloablative BMT. One had chronic Cryptosporidium infection with diarrhea, sclerosing cholangitis, cirrhosis and pancreatitis, requiring total parenteral nutrition. After BMT, he died from fulminant liver failure associated with veno-occlusive disease, Cryptosporidium infestation, and GVHD. The other had a similar clinical pattern, and also extensive bilateral bronchiectasis. On the other hand, Amrolia et al8 reported nonmyeloablative stem cell transplants on eight patients with congenital immunodeficiency and severe organ dysfunction. Two, who suffered from HIGM1 with sclerosing cholangitis, were successfully treated. One of these had a Cryptosporidium infection before BMT that cleared after transplantation, suggesting significant recovery of functional immunity. However, Khawaja et al9 described an HIGM1 patient with a severe Cryptosporidium infection who died soon after nonmyeloablative BMT. He had cryptosporidial ascending cholangitis with portal inflammation, confirmed by liver histology. The Cryptosporidial enteritis worsened after BMT and this, together with a capillary leak syndrome, resulted in the patient's death. When severe liver failure is associated with HIGM1, orthotopic liver transplantation, together with nonmyeloablative BMT, could be an option. Hadzic et al11 reported the successful treatment of an 18-year-old HIGM1 patient with serious cirrhosis: they accomplished combined correction of the primary immune defect and secondary liver complications. Martinez Ibanez et al12 described three HIGM1 patients with sclerosing cholangitis who underwent only liver transplantation and suffered relapse of sclerosing cholangitis 10, 18 and 25 months later.12 Owing to the T-cell deficiency, HIGM1 patients are predisposed to autoimmune disease triggered by opportunistic infections. Thus, in HIGM1 patients, a liver transplant alone cannot prevent sclerosing cholangitis relapse and must be accompanied by a BMT to correct immune defect.12,13
When Cryptosporidium infection occurs during the wait for a transplant, antimicrobial treatment can be initiated. Many treatments have been studied in AIDS patients, including Paromomycin, Clarithromycin, Azythromycin and Sinefungine, but without any demonstrable benefit. In our two patients, Paromomycin did not clear the Cryptospridium infection. As previously mentioned, the CD40L-CD40 pathway is vital for C.parvum clearance.4 However, we have successfully used Nitazoxamide in a child with Cryptosporidium infection secondary to cord blood transplantation for acute myeloblastic leukemia (unpublished). This may be an interesting alternative antimicrobial treatment. Additional intravenous CD40L could be useful in temporary Cryptosporidium infection management, although Vestereng et al14 did not observe P. carinii clearance using recombinant CD40L in SCID mice.
Optimal HIGM1 management appears a challenge when severe hepatic lesions occur, especially in the presence of C. parvum.8,9,11,12,13 Myeloablative, or perhaps nonmyelo-ablative stem cell transplantation, should be performed as soon as C. parvum infection occurs. In cases of advanced hepatic damage, liver transplantation together with nonmyeloablative BMT should be considered. Nitazoxamide could decrease C. parvum infection before BMT, limiting conditioning regimen toxicity, and could be maintained during the profound post BMT immunodeficiency. Regular liver function checks and early C. parvum detection would be helpful for HIGM1 patients. PCR analysis together with immunofluorescence studies seem promising and sensitive tools for early parasite detection.10 PCR sensitivity could be improved by using competitive, quantitative or nested PCR. Early diagnosis could allow clinicians to consider BMT–especially when a genoidentical sibling is available–before any severe hepatic lesions occur.
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We thank Mrs D Binot Saintot and S Lapuyade for their excellent technical assistance.
The first two authors contributed equally to this work
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Cite this article
Dimicoli, S., Bensoussan, D., Latger-Cannard, V. et al. Complete recovery from Cryptosporidium parvum infection with gastroenteritis and sclerosing cholangitis after successful bone marrow transplantation in two brothers with X-linked hyper-IgM syndrome. Bone Marrow Transplant 32, 733–737 (2003). https://doi.org/10.1038/sj.bmt.1704211
- X-linked immunodeficiency with hyper-IgM
- CD40 Ligand
- cryptosporidum parvum gastroenteritis
- sclerosing cholangitis
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