Treosulfan-containing regimens achieve high rates of engraftment associated with low transplant morbidity and mortality in children with non-malignant disease and significant co-morbidities.

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 is synthesized during granulocyte differentiation, stored in specific granules, and released by neutrophils in response to activation.

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.

The impact of monitoring Epstein-Barr virus PCR in paediatric bone marrow transplant patients: can it successfully predict outcome and guide intervention?

BACKGROUND: Epstein-Barr virus (EBV) associated lymphoproliferative disease is a complication of haemopoietic stem cell transplantation (HSCT). In certain groups (unrelated and mismatched donor transplants, T-cell depleted) the risk may be as high as 25% with significant morbidity and mortality. Strategies to predict the impending development of this disorder and allow early intervention have therefore assumed importance. We routinely screen the peripheral blood of all recipients of allogeneic HSCT to detect EBV DNA by quantitative polymerase chain reaction (PCR) technology and report here how this correlates with clinical disease and management. PROCEDURE: Data on 28 successive patients who underwent HSCT at our institution were reviewed. The relationship between EBV reactivation demonstrated by quantitative PCR and development of post transplant lymphoproliferative disease (PTLD) was determined. RESULTS: EBV reactivation occurred in 68% of patients, however only 7% developed clinical PTLD. Patients with high level reactivation (n = 9) had more frequent episodes of reactivation and all patients who progressed to overt PTLD were found in this group. In contrast none of those patients with low level reactivation (n = 10) or persistently negative results (n = 9) showed any signs of clinical disease. Anti-CD20 monoclonal antibody (Rituximab) therapy was instigated in both cases of proven PTLD and three cases of high level reactivation with successful outcomes. Response to treatment was associated with a prompt decline in viral copy number. CONCLUSIONS: Our results indicate that EBV reactivation is a common occurrence in the paediatric allogeneic transplant setting and that only a proportion of patients will progress to PTLD. Frequent monitoring may help to predict those at highest risk and guide intervention.

Highly glycosylated alpha1-acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils.

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.

Safety and efficacy of enzyme replacement therapy in combination with hematopoietic stem cell transplantation in Hurler syndrome.

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.