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Haemophagocytic lymphohistiocytosis (HLH) is a rare and rapidly progressive disease which, untreated, invariably leads to death. Gaucher disease is a rare lysosomal storage disorder. The acute neuronopathic variant; type II, is also rapidly progressive. We report an infant with Gaucher disease type II manifesting as HLH. Immunoblot revealed a deficiency of Munc 13-4, an intracellular protein responsible for docking of secretory lysosomes. This, and other possible pathogenetic mechanisms to explain the link are discussed.

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.

Evans syndrome is a rare cause of hemolysis in pediatric patients. The authors describe two severely affected patients who had previously been heavily treated, and who subsequently developed severe pulmonary hypertension. Both patients were successfully managed by a combination of immunosuppression and anti-pulmonary hypertension treatment. The first patient to present, case A, received an allogeneic bone marrow transplant with subsequent cure of both Evans syndrome and pulmonary hypertension and is now on a weaning dose of sildenafil. Case B is being worked up for allogeneic bone marrow transplantation. The authors speculate that the pulmonary hypertension was caused by the underlying immune dysregulation and hemolysis and that Evans syndrome joins the list of other hemolytic anemias that cause pulmonary hypertension, such as sickle cell disease, thalassemia, and paroxysmal nocturnal hemoglobinuria. However, they suggest a vasculitic process as the main cause.

We report a favorable outcome in a child who underwent cardiac transplantation for severe early post-anthracycline-induced cardiomyopathy, 9 months after completion of treatment for acute myeloid leukemia (AML). The child suffered a relapse of AML 2 months after cardiac transplantation and then underwent a successful bone marrow transplant. This case is unique in the literature. We believe it offers an alternative strategy for children with hematologic malignancies, where severe early post-anthracycline cardiotoxicity might preclude bone marrow transplantation.

The optimal preparation for stem cell transplantation (SCT) in children with congenital immunodeficiencies is currently unknown. In all, 81 children with immunodeficiency underwent 82 SCTs using reduced-intensity conditioning (RIC). The incidence of significant GVHD was low; viral reactivation was prominent with an unexpected increase in EBV reactivation; immune reconstitution was similar between different donor groups and comparable to conventional SCT. Overall, 68/81 (84%) survive with no significant difference between donor types or between severe combined immunodeficiency (SCID) and non-SCID diseases. Findings suggest a significant survival advantage in the unrelated donor setting for RIC compared to conventional SCT.

The broader application of stem cell transplantation (SCT) for paediatric diseases has been limited by a lack of human leucocyte antigen (HLA)-matched donors. Virtually all children, however have at least one haploidentical parent who could serve as a donor. Such a donor is immediately available and the considerable costs of additional HLA typing, registry and banking expenditures that are necessary to procure an unrelated donor, could be reduced. Recent technological advances appear to have overcome the historical problems of graft rejection and severe graft versus host disease in the haploidentical setting, and in the latest studies the overall survival for children undergoing haploidentical SCT for leukaemia is now comparable with that following unrelated donor bone marrow or cord blood transplantation. Post-transplant infectious complications and leukaemia relapse remain the most important barriers yet to overcome, and new directions in the use of adoptive cellular immunity appear to be promising in this respect. Haploidentical SCT is now a viable option for those children who do not have an HLA compatible sibling or fully matched unrelated donor. The relative merits of a haploidentical family donor versus mismatched unrelated bone marrow or cord blood donation needs to be assessed in prospective, randomized clinical trials.

Stem cell transplantation (SCT) remains the only cure for thalassaemia major. Recent advances in medical treatment make it even more important that accurate information is available regarding outcome of SCT in relevant patient populations in order to guide informed decisions regarding the most appropriate treatment for individual thalassaemia patients. We report the results of 55 consecutive first related allogeneic bone marrow transplants (BMT) for children with beta-thalassaemia major performed in two UK paediatric centres over 10 years. Between February 1991 and February 2001, 55 children underwent 57 allogeneic BMT. The median age at BMT was 6.4 years and the majority of patients (73%) originated from the Indian subcontinent. Using the Pesaro risk classification, 17 patients were class 1, 27 were class 2 and 11 were class 3. Actuarial overall survival and thalassaemia-free survival at 8 years were 94.5% (95% CI 85.1-98.1) and 81.8% (95% CI 69.7-89.8) respectively. Despite the majority of patients being in class 2 or 3, transplant-related mortality was low (5.4%). The principal complication was graft rejection accompanied by autologous reconstitution that occurred in 13.2% of transplants. Following modification of the conditioning regimen in 1993, the rejection rate fell to 4.6% and remained low. Acute graft-versus-host disease (GVHD) of grade II-IV occurred in 31% and chronic GVHD in 14.5%. These data compare favourably with survival with medical treatment for thalassaemia major and suggest that allogeneic BMT remains an important treatment option for children with beta-thalassaemia major, particularly when compliance with iron chelation is poor.

Allogeneic haematopoietic stem cell transplantation (HSCT) can be a highly successful treatment option for individuals with congenital immunodeficiency states. The strategy for HSCT is varied but in cases where there is preservation of residual T cell function, conditioning regimes have been used and have been based around a combination of busulphan and cyclophosphamide with or without serotherapy. In patients with coexisting organ damage this has resulted in significant morbidity and mortality. We have therefore used a low-intensity conditioning regime for transplantation in this group of immunodeficiency patients. Twenty-one patients with a variety of different immunodeficiencies were treated using the following conditioning regimes: (1) fludarabine/melphalan/ATG or Campath 1H (n=16), (2) fludarabine/cyclophosphamide/Campath 1H (n=1), (3) TBI/CyA/MMF (n=1), (4) fludarabine/melphalan/busulphan/ATG (n=3). In 13 cases matched (n=9) and 1 Ag mismatched (n=4) unrelated donors were used and in eight cases transplants from matched siblings (n=4), 1 Ag mismatched sibling (n=1), matched parent (n=1) and haploidentical parents (n=3) were performed. At a median follow-up of 13 months, 19 of 21 (90%) patients were still alive following the transplant procedure. Despite a T cell replete graft and the use of unrelated donor grafts in the majority of patients studied there was no evidence of significant organ disease. Immune reconstitution in terms of CD3+ and CD4+ T cell recovery and function was equivalent in comparison with a historical cohort. We believe that this low-intensity approach has significant implications for transplantation of individuals with immunodeficiency states with established organ disease.

Beta-thalassaemia major and sickle-cell disease (SCD) reduce lifespan and quality of life for >300000 children and young adults worldwide. The only cure for both disorders is allogeneic stem cell transplantation (SCT). The decision-making processes in recommending SCT for patients with thalassaemia and SCD are different. For thalassaemia, where transfusion-related iron overload is universal, SCT should be offered to all patients <17 years because long-term survival and thalassaemia-free survival are about 80 and 70% respectively. For thalassaemics unable to comply with medical treatment, SCT offers a significant survival advantage; however, for patients with optimal medical care, short-term survival after SCT is inferior to medical treatment, and SCT instead offers a life free from transfusions and iron chelation. The clinical heterogeneity of SCD means that SCT is recommended only for selected patients with severe disease, particularly sickle-related neurological problems, for whom long-term survival and SCD-free survival after SCT approach 92 and 86% respectively. We here review the evidence available to help physicians evaluate the role of SCT for individual patients with thalassaemia major or SCD.

We report the case of a 10-year-old boy with molecular relapse of CML following unrelated donor BMT who developed fatal grade 4 acute GVHD of the gut and liver following one antigen-mismatched donor lymphocyte infusion. Previous experience of donor lymphocyte infusion in the HLA-mismatched setting is reviewed and the role of adoptive immunotherapy in this situation is discussed.

We have prospectively assessed the relative contribution of host and donor to haemopoiesis following stem cell transplantation (SCT) in children with beta-thalassaemia major (n = 35), using karyotype analysis or Southern blot/polymerase chain reaction analysis of variable number tandem repeats on genomic DNA from peripheral blood. Early haemopoiesis was fully donor in origin in 24 out of 35 cases and remained so throughout the post-transplant course in all but one patient, who evolved to stable mixed chimaerism. The remaining 11 cases (31%) initially showed mixed chimaerism: four of these rejected, one eventually eradicated host haemopoiesis to become fully donor haemopoietic, and the remaining six had persistent mixed chimaerism, with 5--38% host haemopoiesis. The risk of graft rejection was high when > 15% host haemopoiesis was present at 3 months post transplant: four out of six such patients rejected their grafts; conversely, zero out of 29 patients with < 15% host haemopoiesis at 3 months rejected (P < 0.0001). There was a higher incidence of significant acute and chronic graft-versus-host disease in patients with full donor chimaerism. These studies confirm that the mixed chimaeric state is common following SCT for thalassaemia, often persists (with up to 4 years follow-up) and is compatible with long-term cure. Analysis of chimaerism in patients undergoing SCT for beta-thalassaemia enables monitoring of engraftment in the early post-transplant period, provides insight into the biology of engraftment and may be useful in identifying patients at high risk of rejection.

The optimal approach for stem cell transplantation in children with immunodeficiency has yet to be determined. Conditioning therapy is necessary for reliable engraftment and full immune reconstitution; however, the beneficial effect of cytoreductive conditioning is counterbalanced by increased short- and long-term treatment-related toxicity. Whether bone marrow transplantation with a nonmyeloablative preparative regimen was sufficient for the establishment of donor immune reconstitution, with the resultant correction of disease phenotype, was investigated. Eight patients with severe immunodeficiency states underwent T-cell replete bone marrow transplantation from a human leukocyte antigen-matched unrelated (n = 6) or sibling (n = 2) donor with nonmyeloablative conditioning using a fludarabine-melphalan-anti-lymphocyte globulin-based regimen. All patients had severe organ dysfunction that precluded transplantation with conventional conditioning. All patients were engrafted with predominantly donor hematopoiesis, and the duration of neutropenia was brief. Significant acute graft-versus-host disease (GVHD) did not develop, but one patient had limited chronic GVHD. One patient died of disease recurrence, and 3 have stable, mixed chimerism. At a median follow-up of 1 year, all patients have had good recovery of CD3(+) T-cell numbers, and 6 of 7 evaluable patients have normal phytohemagglutinin stimulation indices. The rate of immune reconstitution is comparable with that of historical controls undergoing standard myeloablative protocols. Two patients with CD40 ligand deficiency now show significant expression, and a patient with adenosine deaminase deficiency has improved deoxy adenosine triphosphate metabolites. In summary, it has been demonstrated that nonmyeloablative stem cell transplantation permits rapid engraftment from both sibling and unrelated donors with minimal toxicity even in the presence of severe organ dysfunction. If long-term immune reconstitution of patients treated with this protocol is demonstrated, it is believed this approach might offer significant advantages compared with standard protocols by combining adequate immune reconstitution with reduced short- and long-term toxicity. (Blood. 2000;96:1239-1246)

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human beta-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous beta- or alpha-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Expression of the genes of the human beta-globin cluster is subject to stringent developmental regulation. An integral component of this control is competition between the gamma- and beta-promoters for the upstream regulatory sequences of the Locus Control Region (LCR). We have defined two stage selector elements (SSE and SSE-2), located in the proximal gamma-promoter and gamma-genes 5'UTR which mediate the preferential interaction of the gamma-gene with the LCR in the foetal developmental stage. The activity of both elements is dependent upon the binding of foetal and erythroid-specific proteins. We have purified the protein binding the proximal SSE and characterised it as a complex between the ubiquitous transcription factor CP2 and a 40 hD partner protein. Comparative studies between human CP2 and the chicken stage selector protein, NF-E4, demonstrate homology between the protein complexes. These findings demonstrate that competitive silencing of globin genes mediated by developmentally-specific protein complexes is conserved in evolution.

Thiophenol and 4-aminothiophenol were used to study levels of toxicity in human red blood cells. Thiophenols caused conversion of oxyhemoglobin to methemoglobin. Reduction of corresponding disulfides by intracellular glutathione caused cyclic reduction/oxidation reactions, resulting in increased oxidative flux. Three levels of oxidative stress were observed in these experiments: the lowest level resulted from incubation with 0.25 mM thiophenol; the intermediate level with 0.50 mM thiophenol or 0.25 mM 4-aminothiophenol; the highest levels with 0.50 mM 4-aminothiophenol. Methemoglobin formation increased with increasing level of oxidative stress. Glycolysis and the hexose monophosphate shunt were inhibited at the intermediate and highest levels of stress, respectively. Above the highest level of stress non-intact hemoglobin was formed and cell lysis occurred. These metabolic responses were reflected in cellular levels of NADH, NADPH and reduced glutathione. At the lowest level of oxidative stress, both glycolysis and hexose monophosphate shunt were increased such that near-normal levels of NADH, NADPH and reduced glutathione were maintained and methemoglobin formation was kept to a minimum. The response of red cells to 0.25 mM thiophenol appears to represent a level of oxidative stress to which the cell is capable of adaptive metabolic response. Glycolysis contributes approximately one-quarter of the total reducing equivalents from glucose metabolism in response to the oxidative challenge by thiophenol. The results suggest that the metabolic response to autoxidation of endogenous thiols is thiol exchange with glutathione and reduction of resulting glutathione disulfide by the hexose monophosphate shunt.