Treosulfan is an immuno-suppressive and myeloablative alkylating agent that has been introduced as a conditioning agent in stem cell transplantation (SCT). Most studies have been performed in adult patients with malignancy where a low incidence of regimen-related toxicity has been reported. We report the use of treosulfan in 32 consecutive children undergoing SCT for non-malignant disease. Patients received a total treosulfan dose of 36 or 42 g/m(2)/patient given in three daily, divided doses. A range of other conditioning agents and serotherapy was administered to patients who underwent family donor SCT (n = 11), or unrelated donor SCT (n = 21). One patient (3%) died early. Transplant morbidity was limited and mucositis was only mild. Dermatological toxicity was frequent but mild. Twenty-eight patients (87.5%) established donor cell engraftment. In 25 patients (78%) there was adequate, stable donor engraftment. Four patients have required additional transplant procedures to maintain adequate donor-derived haemopoiesis. Twenty-seven patients (84%) survive with a median follow up of 417 d. There were four late deaths due to progression of the underlying disease, graft-versus-host disease or infection. Treosulfan-based conditioning regimens achieve excellent engraftment with reduced regimen-related toxicity in children with non-malignant disease at high risk for both regimen-related toxicity and graft failure.

Haptoglobin (Hp) is a plasma protein synthesized primarily by hepatocytes. It exerts a broad range of anti-inflammatory activities and acts indirectly as a bacteriostatic agent and an antioxidant by virtue of its ability to bind free hemoglobin (Hb) and to facilitate its immediate clearance by macrophages. We identified Hp as a novel specific granule protein of neutrophils by means of immunoelectron microscopy, subcellular fractionation, and exocytosis studies. Consistent with these findings, blood cells from a patient with specific granule deficiency (SGD) lacked neutrophil-derived Hp. Neutrophils contained a large amount of highly glycosylated Hp (beta-chain 45-65 kDa) synthesized in neutrophil precursors and stored in specific granules and a small amount of Hp (beta-chain 39 kDa) endocytosed from plasma and stored in secretory vesicles. Subsequent binding studies revealed that Hp from specific granules binds to Hb. Finally, the CCAAT enhancer binding protein-epsilon (C/EBPepsilon) induced Hp transcription in a myeloid cell line, suggesting that Hp expression in myeloid cells, as in hepatocytes, is at least partially regulated by members of the C/EBP transcription factor family. Collectively, these findings demonstrate that Hp is stored in specific granules and is released by neutrophils in response to activation. Hence, neutrophil-derived Hp might reduce tissue damage and bacterial growth at sites of infection or injury by propagating anti-inflammatory activities and Hb clearance.

Hepatic veno-occlusive disease (VOD) is a clinical and pathological entity seen predominantly—but not exclusively—in children undergoing hematopoietic stem cell transplantation (HSCT), where it represents an important cause of morbidity and mortality. This review will highlight recent advances in our understanding of the aetiology and pathogenesis of VOD. In addition, risk factors, clinical features and various prophylactic and therapeutic options will be discussed. We specifically address how recent understanding of the alterations in local haemostasis and fibrinolysis gave clearer model of the pathogenesis, and hence contributed to early diagnosis and intervention both to prevent its occurrence and to treat established conditions.

Purpose: Hurler syndrome is a debilitating genetic disease with a typical life span of 5 to 8 years. Early hematopoietic stem cell transplantation (HSCT) mitigates disease symptoms and improves survival. However, morbidity and mortality associated with HSCT can limit its success. We describe the initial experience with combined use of enzyme replacement therapy (ERT, laronidase) and HSCT in Hurler syndrome.Methods: Thirteen transplants were performed in 12 patients. ERT was given at a standard dose of 0.58 mg/kg per week. Transplant conditioning regimen and donor graft source were determined by institutional protocol.Results: The median age at initiation of ERT was 12 months (range, 8 to 18 months). The median duration of pre-HSCT ERT was 12 weeks (range, 4 to 28). All but 1 patient tested showed decrease in urinary GAG excretion during ERT. ERT infusion-related toxicity was limited to mild reactions. Development of antibodies to laronidase did not correlate with infusion reactions or responses in urinary GAG excretion. ERT was given for a median of 7 weeks (range, 3 to 20) after HSCT. After transplantation, eight patients demonstrated complete donor engraftment and four suffered graft failure. Two patients required ventilator support and three developed acute GVHD. Eleven of the 12 patients are surviving with a median follow-up of 3 months (range, 1 to 7 months).Conclusions: In children with Hurler syndrome, ERT with HSCT is feasible and well tolerated. Development of antibodies against exogenous enzyme does not appear to correlate with infusion reactions or response to ERT. A prospective study is needed to determine the effect of concomitant ERT on transplant outcomes.

alpha-1-Acid glycoprotein (AGP) is an acute-phase protein produced by hepatocytes and secreted into plasma in response to infection/injury. We recently assessed the transcriptional program of terminal granulocytic differentiation by microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/ metamyelocytes (MYs), and BM neutrophils. These analyses demonstrated a transient, high mRNA expression of genuine secondary/tertiary granule proteins and AGP in MYs. In agreement with this, immunocytochemistry revealed the presence of AGP protein and the secondary granule protein lactoferrin in cells from the MY stage and throughout granulocytic differentiation. Immunoelectron microscopy demonstrated the colocalization of AGP and lactoferrin in secondary granules of neutrophils. This finding was substantiated by the failure to detect AGP and lactoferrin in blood cells from a patient with secondary/tertiary (specific) granule deficiency. In addition, Western blot analysis of subcellular fractions isolated from neutrophils revealed that neutrophil-derived AGP, localized in secondary granules, was abundant and highly glycosylated compared with endocytosed, plasma-derived AGP localized in secretory vesicles. Exocytosis studies further demonstrated a marked release of AGP and lactoferrin by activated neutrophils. Finally, induction of CCAAT/ enhancer-binding protein (C/EBP)-epsilon in a myeloid cell line was shown to increase AGP transcript levels, indicating that AGP expression in myeloid cells, like in hepatocytes, is partially regulated by members of the C/EBP family. Overall, these findings define AGP as a genuine secondary granule protein of neutrophils. Hence, neutrophils, which constitute the first line of defense, are likely to serve as the primary local source of AGP at sites of infection or injury.

Anti-CD20 monoclonal antibody treatment (Mabthera) has an increasing therapeutic role in the management of non-Hodgkin's lymphoma (NHL) (McLaughlin et al, 1998). This chimaeric monoclonal antibody binds specifically to CD20, a pan B-lymphocyte marker, and works to deplete B cells through complement and antibody-dependent cellular cytotoxicity. It is therefore not unreasonable to think of it as a salvage treatment for other immunoglobulin-mediated disorders, particularly those that do not respond satisfactorily to standard immunosuppressive regimens, such as cold haemagglutinin disease (CHAD). Complete remission of resistant CHAD secondary to low-grade non-Hodgkin's␣lymphoma has been reported in three cases to date following Mabthera (rituximab) (Lee & Kueck, 1998; Bauduer, 2001; Layios et al, 2001). We hereby report the first case of idiopathic CHAD to be treated by Mabthera, which induced a long-term remission (the longest to be reported to date among both secondary and idiopathic forms of CHAD). A 53-year-old man was referred in January 1997 for investigation of intermittent, frank, painless ‘haematuria’ for 2 months with acrocyanosis and erectile dysfunction. The former proved to be haemoglobinuria. He was unable to go outside his house without multiple layers of clothing and had taken to wearing electrically heated gloves. There was no history of chest infections and no lymphadenopathy was detected on a computerized tomography (CT) scan. His blood count revealed a mild normochromic anaemia, Hb 12·7 g/dl, with a reticulocytosis (118 × 109/l), but an otherwise normal total and differential white cell and platelet count. Serum biochemistry tests were within the normal ranges. He had normal immunoglobulin levels, and no detectable monoclonal paraprotein. His cold agglutinin titre was 1:524288 at 4°C demonstrating anti-I red-cell specificity. Other autoantibodies were not detected. Bone marrow aspirate and biopsy showed mild erythroid hyperplasia. Three reactive lymphoid follicles were seen on the trephine biopsy, but clonality was excluded by IgH gene rearrangement by polymerase chain reaction. The patient was started on cyclophosphamide 750 mg/week and was advised to avoid exposure to cold. Three weeks later the cold agglutinin titre had dropped from its previous level, but remained high at 1:16384. After 6 weeks of cyclophosphamide therapy the patient continued to be symptomatic despite a stable haemoglobin and cold agglutinin titre. Further treatments over the next 12 months were given: azathioprine 50 mg thrice daily (for 8 weeks), prednisolone 1 mg/kg (for 6 weeks), high-dose cyclophosphamide 3 g/m2 (with peripheral haematopoietic stem cell harvest) and, finally, cyclosporin A (for around 10 weeks). His symptoms, however, remained the same, as did the cold agglutinin titre. A repeat CT scan as well as other biochemistry revealed no underlying lymphoma. In early March 1998, four doses of Mabthera (Roche Products Limited, Welwyn Garden City, UK) were given (375 mg/m2 weekly). Cold agglutinin titres fell gradually. Prior to the third dose, the titre of anti-I was 1:512. The patient developed transient lymphopenia (0·28 × 109/l) but immunoglobulin levels remained normal. He started to feel better 2 months after the end of therapy. The cold agglutinin slowly resolved over a period of 9 months to become undetectable (Fig 1) and the patient remains symptom free at 3·5 years with a normal blood count and reticulocyte count. His erectile dysfunction did not improve with this treatment. Cold agglutinin titres following Mabthera therapy. Idiopathic CHAD is notoriously difficult to treat. Therapeutic measures that are often successful in warm autoimmune haemolytic anaemia such as steroids, intravenous immunoglobulins, and splenectomy are usually ineffective with CHAD (Rosse et al, 1997). Purine analogues treatment has also been largely disappointing (Berentsen et al, 2000). Lee & Kueck (1998) reported successful control of cold autoimmune-mediated haemolysis in one of their patients using Rituxan, steroids and cyclophosphamide. Their patient, however, had an underlying B-cell non-Hodgkin's lymphoma with an IgM monoclonal gammopathy. The duration of remission was not reported. Two more similar cases of CHAD secondary to low-grade NHL have been reported (Bauduer, 2001; Layios et al, 2001). Remission lasted for 7 months in the former, but was continuing at 9 months with the latter. Our case supports the role of anti-CD20 monoclonal antibody in the treatment of patients with refractory idiopathic CHAD, resulting in a continuing complete remission for more than 3 years. It represents the first idiopathic case of CHAD to be treated using Mabthera with the longest reported remission.