Early outcomes after allogeneic hematopoietic SCT in pediatric patients with hematologic malignancies following single fraction TBI.

Fractionated TBI (FTBI) followed by allogeneic hematopoietic SCT results in donor engraftment and improves survival in children with high-risk hematologic malignancies. However, acute toxicities (skin, lung and mucosa) are common after FTBI. Late complications include cataracts, endocrine dysfunction, sterility and impaired neurodevelopment. Instead of FTBI, we used low-dose single fraction TBI (550 cGy) with CY as transplant conditioning for pediatric hematologic malignancies. GVHD prophylaxis included CYA and short-course MTX; methylprednisolone was added for unrelated donor transplants. A total of 55 children in first (40%) or second remission and beyond (60%) underwent transplantation from BM (65%) or peripheral blood; 62% from unrelated donors; 22% were mismatched. Median follow-up was 18.5 months (1-68). Overall survival and disease-free survival at 1 year were 60 and 47%, respectively. Acute toxicities included grade 3-4 mucositis (18%), invasive infections (11%), multiorgan failure/shock (11%), hemolytic anemia (7%), veno-occlusive disease (4%) and renal failure (4%). TRM was 11% at 100 days. Non-relapse mortality was 6% thereafter. Graft rejection occurred in 2%. Three patients (5%) died of GVHD. The regimen was well tolerated even in heavily pretreated children and supported donor cell engraftment; long-term follow up is in progress.

Allogeneic hematopoietic cell transplantation (HCT) in Hurler's syndrome using a reduced intensity preparative regimen.

Allogeneic hematopoietic cell transplantation (HCT) in patients with Hurler's syndrome can improve survival and ameliorate many aspects of Hurler's syndrome including neurologic decline and cardiac compromise. Unfortunately, the toxicity of traditional preparative regimens to organs affected by the syndrome may have deleterious effects. Additionally, despite the intensity of these regimens, achieving stable donor chimerism can be difficult. We report transplant outcomes following a reduced intensity, highly immunosuppressive preparative regimen consisting of alemtuzumab, fludarabine and melphalan prior to HCT in seven patients with Hurler's syndrome treated at two centers. Six patients received grafts from unrelated donors and one received a sibling donor graft. The preparative regimen was well tolerated. All patients had initial donor engraftment at 100 days; one patient had delayed loss of donor chimerism. There was no severe acute GVHD (no GI GVHD of grade II or more, no grade IV skin GVHD). Six of the seven children are surviving at a median of 1014 (726-2222) days post transplant. This reduced intensity preparative regimen has the potential to support engraftment and improve survival and outcome in patients with Hurler's syndrome undergoing HCT.

A novel reduced-intensity stem cell transplant regimen for nonmalignant disorders.

Bone marrow transplantation (BMT) benefits nonmalignant diseases but is limited by regimen-related toxicity, graft-versus-host disease (GVHD), donor availability, and graft rejection (GR). To overcome some of these barriers, we developed a new conditioning strategy for these patients. In total, 16 patients received Campath-1H (33/48 mg; days -21 to -19), fludarabine (150 mg/m(2); days -8 to -4), melphalan (140/70 mg/m(2); day -3), and transplant using related/unrelated stem cells. GVHD prophylaxis included cyclosporine/methylprednisolone for cord cells. Other recipients also received methotrexate. Risk factors for GR included multiple transfusions (6), low stem cell numbers (1), and immunologic/metabolic disorders (3). Donor engraftment was present in 14/16 recipients. Neutrophils (ANC>0.5 x 10(9)/l) and platelets (>50 x 10(9)/l) engrafted at a median of 13 and 24 days. Two patients died of Pseudomonas sepsis prior to engraftment, one of CMV disease, and another of intracranial hemorrhage. With median follow-up of 281 days (78-907), 12/16 are stable/improved, or cured. Acute GVHD was absent (n=10) or mild and transient (grade1-2 skin) (n=4). There was no chronic GVHD. Toxicities were predominantly early infections within 100 days, and correlated with lymphopenia (CD4+ T and B cells). Stable engraftment and low incidence of significant GVHD, irrespective of age or stem cell source, make this reduced-intensity regimen attractive for nonmalignant disorders.

Peripheral blood mononuclear cell DNA 6-thioguanine metabolite levels correlate with decreased interferon-gamma production in patients with Crohn's disease on AZA therapy.

6-Mercaptopurine (6-MP) and its prodrug azathioprine (AZA) are well known for their lymphocytotoxic and bone marrow suppressive effects in the management of patients with leukemia. Although their immunosuppressive properties are mediated by the active AZA antimetabolite 6-thioguanine (6-TG), its mechanism of action is largely unknown. In IBD, a significant inverse correlation has been shown between erythrocyte 6-TG metabolite levels and disease activity, further supporting the proposed immunosuppressive role for 6-TG. Since leukocytes possess quantitatively different purine metabolic pathways compared to erythrocytes, this study aims to measure lymphocyte DNA 6-TG metabolites and correlate levels with the INF-gamma and IL-10 cytokine profile in patients with Crohn's disease (CD). Forty-six adult patients with CD, either naive (17) or on long-term (>4-month) AZA therapy (29), had erythrocyte and lymphocyte DNA 6-TG levels measured by reverse-phase HPLC under UV detection (6-TG, 340 nm). Lymphocyte DNA 6-TG was expressed as picomoles per milligram of DNA. Lymphocyte DNA 6-TG metabolite levels were correlated with INF-gamma and IL-10 cytokine profiles using the OptEIA kit (Pharmigen). Lymphocyte DNA 6-TG metabolite levels correlate with erythrocyte 6-TG levels (P < 0.03) but not total patient leukocyte levels. Erythrocyte 6-TG metabolite levels correlated (P < 0.01) inversely with INF-gamma but not IL-10 cytokine levels. This study suggests a preferential dampening of the TH1 response on exposure to 6-TG and a possible immunosuppressive mechanism of action for AZA. Future studies are needed to determine if cytokine profiles can be used to predict recalcitrant CD to AZA therapy.

Sialylation of the sialic acid binding lectin sialoadhesin regulates its ability to mediate cell adhesion.

The macrophage-specific cell surface receptor sialoadhesin, which is a member of the newly recognized family of sialic acid binding lectins called siglecs, binds glycoprotein and glycolipid ligands containing a2-3-linked sialic acid on the surface of several leukocyte subsets. Recently, the sialic acid binding activity of the siglec CD22 has been demonstrated to be regulated by sialylation of the CD22 receptor molecule. In the present work, we show that desialylation of in vivo macrophage sialylconjugates enhances sialoadhesin-mediated lectin activity. Herein, we show that receptor sialylation of soluble sialoadhesin inhibits its binding to Jurkat cell ligands, and that charge-dependent repulsion alone cannot explain this inhibition. Furthermore, we show that the inhibitory effect of sialic acid is partially dependent on the presence of an intact exocyclic side chain. These results, in conjunction with previous findings, suggest that sialylation of siglecs by specific glycosyltransferases may be a common mechanism by which siglec-mediated adhesion is regulated.

Temporal effects of ethanol on growth, thymidine uptake, protein and collagen production in human foetal lung fibroblasts.

Foetal Alcohol Syndrome is observed in 2/1000 live births worldwide and is characterized by decreased pre-natal and postnatal growth, physical anomalies and mental retardation. To determine the effects of ethanol (Et) on foetal cells cultured in the absence of hormones or growth factors, human foetal lung (HFL1) fibroblasts were exposed to Et-supplemented media (0.1-2% Et) for 6 hr to 7 days. Growth rates, thymidine incorporation into DNA, protein synthesis and degradation, and collagen production were assessed. For growth experiments, cells were seeded at 1 3 confluent density and incubated in Et-supplemented medium 24 hr later. Metabolic labelling was performed on confluent monolayers using [(3)H]thymidine (TdR), [(3)H]leucine or [(3)H]proline. Exposure to Et for 3 or 7 days decreased cell numbers but normal proliferation resumed when cells were re-plated in control medium. Exposure to 0.5% Et for 7 days resulted in a 3.5-fold increase in [(3)H]TdR uptake. Et suppressed protein production and enhanced degradation. The most significant decrease was seen at 6 hr, but was influenced by the amino acid used for labelling. Agarose-gel chromatography suggests that Et preferentially alters the lower-molecular-weight species. The percentage of the total protein secreted into the medium was not changed. Collagen production, as a percentage of total protein, decreased after a 48-hr label and a 7-day incubation with Et. The percentage of total collagen that was secreted into the medium was also not influenced by Et. The results indicate that, in the absence of endocrine or nutritional manipulation, acute exposure to Et in vitro inhibits cell growth and protein production; protein secretion, however, remains intact.

Changes in the agglutinability of red cells and the inhibition of specific agglutination by plasma from human blood taken into ACD and stored at 4 degrees C.

The effect of storage in ACD at 4 degrees C on red cell agglutinability and the inhibitory properties of the plasma to blood group reagents has been studied. It was found that the variability demonstrated was related to the origin of the reagent used, particularly between human and on-human sources. The significance of these findings with regard to biochemical and morphological changes in blood on storage is discussed.

The lecithin: cholesterol acyltransferase reaction, lysolecithin and red cell ageing in blood stored under normal transfusion service conditions.

The lecithin:cholesterol acyltransferase (LCAT) activity and lysolecithin content of human blood stored under standard blood transfusion service conditions at 4 degrees C for 6 weeks has been investigated. Cooling the blood to 4 degrees C rapidly inactivates the LCAT reaction, but the enzyme is not denatured during storage under these conditions. Citrate in the anticoagulant did not activate the LCAT reaction in freshly-taken whole blood. The total phospholipid and total lysolecithin content of whole blood decreased during storage at 4 degrees C for 6 weeks. The lysolecithin content of fresh red cells (2.0-3.0 mumol lysolecithin x 10(-11) per cell) showed no significant change during the storage period.