Lineage divergence at the first TCR-dependent checkpoint: preferential γδ and impaired αß T cell development in nonobese diabetic mice.

The first TCR-dependent checkpoint in the thymus determines αß versus γδ T lineage fate and sets the stage for later T cell differentiation decisions. We had previously shown that early T cells in NOD mice that are unable to rearrange a TCR exhibit a defect in checkpoint enforcement at this stage. To determine if T cell progenitors from wild-type NOD mice also exhibit cell-autonomous defects in development, we investigated their differentiation in the Notch-ligand-presenting OP9-DL1 coculture system, as well as by analysis of T cell development in vivo. Cultured CD4 and CD8 double-negative cells from NOD mice exhibited major defects in the generation of CD4 and CD8 double-positive αß T cells, whereas γδ T cell development from bipotent precursors was enhanced. Limiting dilution and single-cell experiments show that the divergent effects on αß and γδ T cell development did not spring from biased lineage choice but from increased proliferation of γδ T cells and impaired accumulation of αß T lineage double-positive cells. In vivo, NOD early T cell subsets in the thymus also show characteristics indicative of defective ß-selection, and peripheral αß T cells are poorly established in mixed bone marrow chimeras, contrasting with strong γδ T as well as B cell repopulation. Thus, NOD T cell precursors reveal divergent, lineage-specific differentiation abnormalities in vitro and in vivo from the first TCR-dependent developmental choice point, which may have consequences for subsequent lineage decisions and effector functions.

Long-term "in vitro" proliferating mouse hematopoietic progenitor cell lines.

Long-term proliferating hematopoietic progenitor cell lines have been established from mouse bone marrow in tissue cultures on the M-CSF-deficient stromal cell line OP9. In the presence of stem cell factor (SCF), thrombopoietin, IL-3 and IL-6 pluripotent hematopoietic stem cells (pHSC) initiate proliferation. For 2-3 weeks they maintain long-term reconstitution capacity, as tested in adoptive transfer experiments into sublethally irradiated hosts, but later loose this capacity. Transfection with HOXB4 stabilises the pluripotency and long-term reconstitution capacity of these pHSC-like cell lines. Transfer into media containing SCF and FLT3L, the ligand for flt3, develops cell lines with myelopoietic and lymphopoietic potencies, reconstituting hosts with a wave of short-term reconstitutions of these cell lineages. Subsequent transfer into cultures containing SCF, FLT3L and IL-7 generates lines with lymphoid reconstitution capacities, i.e. able to develop T-lineage, B-lineage and NK-lineage cells. Again, this multi-lymphoid lineage developmental capacity is lost within 2 weeks, so that the remaining, proliferating cells generate B-lineage cells only, when induced to differentiate. These cell lines become capable to proliferate in IL-7 alone and now resemble pre BI-Type cell lines, as those previously isolated from fetal liver. Hence such preBI cell lines can be generated by a stepwise alteration of the cytokine milieu in culture from pHSC but intermediate differentiation stages still need to be stabilized in attempts to establish long-term proliferating cell lines at different stages of hematopoietic development.

A clinically significant incidence of bleeding in critically ill children receiving therapeutic doses of unfractionated heparin: a prospective cohort study.

Unfractionated heparin (UFH) is frequently prescribed for children for the prevention and treatment of thrombosis; however, its safety and efficacy have not been assessed. The aim of this single center, prospective cohort study was to determine the incidence of major bleeding and recurrent thrombosis in children receiving UFH. Major bleeding was defined a priori as: central nervous system or retroperitoneal bleeding, bleeding resulting in UFH being stopped or overt bleeding causing a drop in hemoglobin >20 g/dL in less than 24 h. Major bleeding events occurred in 9/38 children (24%, 95% CI 11-40%) and 2/38 (5%, 95% CI 0-18%) developed thrombosis. In conclusion, there is clinically significant bleeding in children receiving UFH.

Predictive value of persistent versus transient antiphospholipid antibody subtypes for the risk of thrombotic events in pediatric patients with systemic lupus erythematosus.

Study objectives were to determine, in children with systemic lupus erythematosus (SLE), (1) the association of antiphosholipid antibody (APLA) subtypes with thrombotic events (TEs) and (2) the predictive value of persistent versus transient antibodies for TEs. This is a cohort study of 58 SLE children in whom lupus anticoagulants (LAs), anticardiolipin antibodies (ACLAs), anti-beta2-glycoprotein-I (anti-beta2-GPI), and antiprothrombin (anti-PT) were assessed on at least 2 occasions (more than 3 months apart). Antibodies were classified as persistent (positive on at least 2 occasions) or transient (positive once). Outcomes were symptomatic TEs confirmed by objective radiographic tests identified retrospectively and prospectively. Seven of the 58 patients (12%) had 10 TEs; 5 patients had TEs during prospective follow-up. Persistent LAs showed the strongest association with TEs (P < .001). Persistent ACLAs (P = .003) and anti-beta2-GPI (P = .002) were significantly associated with TEs; anti-PT (P = .063) showed a trend. Persistent or transient LAs and anti-beta2-GPI showed similar strength of association, while ACLAs and anti-PT were no longer associated with TEs. Positivity for multiple APLA subtypes showed stronger associations with TEs than for individual APLA subtypes because of improved specificity. Lupus anticoagulant is the strongest predictor of the risk of TEs; other APLA subtypes provide no additional diagnostic value. Anticardiolipin antibodies and anti-PT require serial testing because only persistent antibodies are associated with TEs.

Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism.

Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Deltaasct/Deltaasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of beta-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of (13)C-enriched and (14)C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei.

Fibrinolytic response to venous occlusion is decreased in patients after Kawasaki disease.

Impaired fibrinolysis is considered a sensitive marker of endothelial dysfunction. Persistent endothelial dysfunction occurs in some patients following Kawasaki disease. The aim of the present study was to assess whether impaired fibrinolysis is present in long-term survivors of Kawasaki disease. The study included 42 children with a documented history of Kawasaki disease presenting with or without coronary lesions, and 26 healthy controls. Blood samples were collected from patients and controls prior to and following venous occlusion stress testing. Significantly decreased fibrinolytic response to venous occlusion was detected in patients compared with controls due to decreased tissue plasminogen activator. In addition, patients had significantly increased plasma concentrations of plasminogen and fibrinogen, which were related to similar increases of alpha2 -macroglobulin. Decreased fibrinolytic response was found in patients with coronary aneurysms but also in those without coronary lesions. In summary, a decreased fibrinolytic response to venous occlusion may reflect persistent endothelial damage following acute Kawasaki disease, potentially predisposing these patients to accelerated atherosclerosis and cardiovascular disease in early adult life.

Fibrinolytic system in adolescents: response to venous occlusion stress tests.

Recent studies indicate that the incidence of thromboembolic events is increasing as a secondary complication in children with serious underlying diseases. The mechanism to eliminate these thrombi via the thrombolytic system in children is unknown. The baseline fibrinolytic system is age dependent, with significant variation between children and adults. Adult studies would suggest that the fibrinolytic response to venous occlusion has more clinical relevance than the baseline fibrinolytic system. The aim of this study was to determine whether the fibrinolytic response to venous occlusion stress testing in healthy adolescents differs from the response in healthy adults. Healthy adolescents (13-18 y) from a school population and normal adults were recruited. Pre- and postvenous occlusion blood samples were collected using standard techniques. Plasma tissue plasminogen activator, plasminogen activator inhibitor-1, plasminogen, alpha(2)-antiplasmin, alpha(2)-macroglubulin, D-dimers, euglobulin lysis time, and fibrinogen were measured on each sample. Adolescents had significantly decreased tissue plasminogen activator antigen levels and increased plasminogen activator inhibitor-1 activity levels after venous occlusion, resulting in significantly prolonged euglobulin lysis times. The results of our study confirm developmental differences in the fibrinolytic response to venous occlusion stress testing. The age-related differences in fibrinolytic response to venous occlusion of younger children and the significance of these differences on the pathophysiology of thromboembolic events in children require further studies.