Ambiguous phenotypes and genotypes in 16 children with acute leukemia as characterized by multiparameter analysis

Ambiguous phenotypes and genotypes were observed in 16 children with acute leukemia. Surface marker, cytogenetic, molecular genetic, and DNA flow cytometric analyses as well as standard morphologic and cytochemical studies were used to divide the patients into three groups. The first group comprised five children with acute leukemia whose blast cells were morphologically lymphoid, while immunophenotyping disclosed simultaneous expression of early pre-B cell and myeloid features. Molecular genetic studies showed evidence of heavy-chain immunoglobulin (Ig) gene rearrangements in all patients. Cytogenetic data, available in three of these children, revealed t(4;11). In five of the 16 patients, morphologic and surface marker analyses indicated the coexistence of two separate cell populations, one with myeloid and the other with early pre-B cell features. Further evidence of B cell commitment in these patients was provided by demonstration of Ig heavy-chain gene rearrangements in all five patients. Surprisingly, one of the five patients showed oligoclonal Ig heavy-chain as well as monoclonal gene rearrangement for the beta chain of the T cell receptor (beta-TCR). The last group consisted of four cases with otherwise typical acute lymphoblastic leukemia (ALL), early pre-B cell phenotype, and coexpression of myeloid or T cell-associated antigens, and two children with unequivocal acute myeloid leukemia (AML) and coexpression of T cell antigens. Gene rearrangement of Ig heavy-chain could be demonstrated in five of six patients, additional Ig light-chain gene rearrangement in two children with ALL, and bigenotypic features (Ig heavy-chain and beta-TCR gene rearrangement) in one patient. In none of the 16 patients did flow cytometry disclose clonal abnormalities of leukemic cell DNA content. Based on these findings, we suggest that malignant transformation in the first and second group of patients took place at a stage ontogenetically close to the pluripotent stem cell, whereas ambiguous phenotypes in the third group resulted from aberrant gene expression or insufficient reagent specificity.     

Impaired nicotinamide adenine dinucleotide synthesis in pyruvate kinase- deficient human erythrocytes: a mechanism for decreased total NAD content and a possible secondary cause of hemolysis

Erythrocytes from individuals with pyruvate kinase (PK) deficiency have approximately half the total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD) of normal erythrocytes. In order to elucidate the mechanism(s) for the decrease in total NAD, we examined NAD synthesis in intact erythrocytes. It is demonstrated that NAD synthesis is impaired in PK-deficient erythrocytes to a degree that is dependent on the PK activity and adenosine 5'-triphosphate (ATP) concentration of these cells. After incubation in the presence of fluoride, which simulates the characteristics of PK deficiency by inhibiting enolase, normal erythrocytes had impaired NAD synthesis and decreased ATP concentrations. Fluoride did not inhibit NAD synthesis in a hemolysate system that is not dependent on glycolysis for ATP generation. These data suggest that fluoride does not inhibit the enzymes of NAD synthesis and that impairment of NAD synthesis by fluoride is mediated by decreased ATP formation. Thus, it is concluded that impaired NAD synthesis in PK-deficient erythrocytes is caused by decreased ATP formation due to the PK deficiency. Since the rate of glycolysis is limited by the availability of NAD+, it is suggested that impaired NAD synthesis causes further ATP depletion and thereby may enhance hemolysis in PK-deficient erythrocytes.     

Analysis of p53 mutations in a large series of lymphoid hematologic malignancies of childhood

p53 mutations are found in a wide variety of cancers, including hematologic malignancies. These alterations apparently contribute to development of the malignant phenotype. We analyzed a large series of lymphoid (330 cases) and a smaller series of myeloid (29 cases) malignancies of childhood for p53 mutations by single-strand conformational polymorphism (SSCP) following polymerase chain reaction. Samples with abnormal SSCP were reamplified and analyzed by direct sequencing method. p53 mutations were detected within the known mutational hotspots (exons 5 to 8) in 8 of 330 lymphoid malignancies, and in none of 29 myeloid malignancies, showing that the frequency of p53 mutations in childhood lymphoid malignancies was very low (8 of 330 cases [2%]). Four of these patients had very aggressive, fatal acute lymphocytic leukemia (ALL). None of 13 infants and none of 48 patients with T-lineage leukemia had detectable p53 mutations in their ALL cells. Exceptionally, p53 mutations were comparatively frequent in a small sample of B-cell non-Hodgkin's lymphomas (2 of 8 cases). Mutations were detected in samples from two patients with ALL at relapse; these were not detected in samples at initial diagnosis from the same patients, suggesting that p53 mutations may be associated with progression to a more malignant phenotype. Seven of eight alterations of p53 were missense mutations, and seven of eight samples may be heterozygous for the mutant p53, indicating that p53 protein may act in a dominant negative fashion.     

Platelet storage: changes in cytosolic Ca2+ actin polymerization and shape

Platelets gradually lose their disc shape during storage. The authors studied simultaneous changes in platelet cytosolic Ca2+ (Cai) and the polymerization state of actin as related to the shape. Platelet concentrates were stored under blood bank conditions for up to 10 days. Aliquots were removed and analyzed as follows: platelet Cai and increments in Cai induced by adenosine diphosphate (ADP) were determined by fluorescence of fura-2-loaded cells; loss of disc shape was determined by differences in light scattering intensity induced by stirring; and the ratio of globular and total actin (G/T) of platelets in plasma was determined by a modification of the DNase inhibition assay. Globular actin was found to be 86 +/- 3% of total actin in freshly drawn platelets suspended in plasma. The following changes occurred during storage: G/T in platelet concentrates increased from 63 +/- 5 (day 0) to 74 +/- 2% in the first 24 hours then fell to 33 +/- 6% by day 10. The percent discoid platelets also increased from day 0 to day 1 then fell in the ensuing days. There was an initial drop in Cai from day 0 to day 1, after which Cai increased on days 3 and 6. Globular actin polymerization during storage closely correlated with the change in percent discs (r = 0.95). During 6 days of storage Cai was highly correlated with shape change (r = 0.97) and to a lesser extent (r = 0.87) with the ratio of globular actin. The authors conclude that actin polymerization, shape, and Ca2+ change in a related fashion during storage.     

Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae

Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms.Water is an essential molecule whose absence can lead to a variety of detrimental and often lethal effects on cells and organisms (1–3). Severe water removal, termed desiccation, has been proposed to lead a variety of detrimental stresses (3). Which of these stresses leads to lethality in desiccation-sensitive organisms is unclear. Organisms capable of surviving desiccation, commonly termed anhydrobiotes, are found among bacteria, fungi, plants, and invertebrates (1, 3). Anhydrobiotes harbor stress effectors that are known or postulated to mitigate the different stresses associated with desiccation (2, 4). These stress effectors include osmolytes, heat shock proteins, redox balancing enzymes, nonreducing disaccharides (trehalose, sucrose), and hydrophilins (short unstructured hydrophilic proteins—also known as LEAs) (1). A reasonable assumption might be that many if not all of these stress effectors are necessary for desiccation tolerance given the multitude of stresses imposed by desiccation. However, this assumption is challenged by the uncertainty in the number and degree of lethal stresses generated by desiccation and the versatility and coordination/cooperation of multiple stress effectors in ameliorating such lethal stresses. Thus, a critical question in the anhydrobiosis field is whether a single stress effector is sufficient to promote desiccation tolerance.One of the most studied desiccation-associated stress effectors is the simple nonreducing disaccharide, trehalose (α,α-1,1-glucoside) (5). It is found in extremely high concentrations in most anhydrobiotes, including in the model organism Saccharomyces cerevisiae (6, 7). In this yeast, exponentially dividing cells have very low levels of trehalose and are extremely desiccation sensitive (8). However, in saturated cultures, yeast cells accumulate high levels of a number of stress effectors, including extremely high levels of trehalose (up to 15% of dry cell mass) (6, 7). We recently showed that high levels of trehalose are necessary for yeast cells in saturated cultures to survive weeks to months of desiccation (long term), but not a few days (short term) (9). Trehalose dispensability during short-term desiccation is due in part to overlapping functions with the heat shock factor Hsp104. This overlap led us to discover that trehalose functions as a chemical chaperone capable of preventing the aggregation of both membrane and cytoplasmic proteins (9). Work in the nematode Caenorhabditis elegans demonstrated that worms unable to synthesize trehalose display hallmarks of membrane damage, consistent with trehalose playing a role in preserving membrane structure (10). Indeed, trehalose has been found to be lipidated in nematodes and these “maradolipids” are required for efficient desiccation tolerance (11). Due to the different and versatile mechanisms by which trehalose confers desiccation tolerance in anhydrobiotes, we hypothesize that trehalose, in the absence of other stress effectors, will be sufficient in conferring desiccation tolerance.A simple way to address this hypothesis is to increase the intracellular levels of trehalose in desiccation/dehydration-sensitive cells or organisms then assess whether they acquire desiccation tolerance. Two strategies for increasing intracellular trehalose have been previously used. These were engineering high level expression of trehalose biosynthetic enzymes or importing extracellular trehalose via fusion with lipid vesicles (12–16). Both methods only generated small increases in trehalose levels and minor increases in dehydration but not desiccation tolerance. This weak effect could reflect the need for additional stress effectors. Alternatively, trehalose alone could indeed be sufficient for desiccation tolerance but was missed for two reasons. First, high physiological levels of trehalose observed in desiccation-tolerant organisms were not reached so a potential critical threshold level of trehalose was not met. Second, the biosynthetic strategy not only increased trehalose but also trehalose-6-phosphate, a potent regulator of glucose metabolism that has deleterious effects on cell and organism fitness. Thus, it remains untested whether trehalose alone is sufficient for generating desiccation tolerance.The correlative evidence for trehalose being sufficient for desiccation tolerance was provided from our previous study comparing desiccation sensitivity of saturated and exponentially dividing cultures of yeast (8). Cells in a saturated culture rapidly lose desiccation tolerance when they divide upon dilution into fresh media. Shortly, after dilution, the levels of many stress factors, including trehalose, diminish. Trehalose levels drop as a consequence of activation of two intracellular trehalases, NTH1 and NTH2, and the inhibition of the trehalose biosynthetic enzyme Tps1 (6, 7). The diluted cells retained their desiccation tolerance significantly longer when trehalose depletion was slowed by inactivation of the trehalases (9). This result is consistent with the notion that sustaining high trehalose levels, while reducing the levels of other stress effectors, is sufficient to promote desiccation tolerance. Encouraged by this result, we decided to investigate further the potential sufficiency of trehalose for desiccation tolerance, exploiting the ability of the AGT1 sugar transporter to import extracellular trehalose (17). Here, we show that when AGT1 overexpressing cells are grown in the presence of trehalose, they acquire high levels of intracellular trehalose and desiccation tolerance similar to that of saturated cultures. We characterize this novel acquisition of desiccation tolerance and provide important insights into the roles of trehalose concentration and trehalose structure in both short- and long-term desiccation tolerance.     

Achieving the WHO/UNAIDS antiretroviral treatment 3 by 5 goal: what will it cost?

The "3 by 5" goal to have 3 million people in low and middle income countries on antiretroviral therapy (ART) by the end of 2005 is ambitious. Estimates of the necessary resources are needed to facilitate resource mobilisation and rapid channelling of funds to where they are required. We estimated the financial costs needed to implement treatment protocols, by use of country-specific estimates for 34 countries that account for 90% of the need for ART in resource-poor settings. We first estimated the number of people needing ART and supporting programmes for each country. We then estimated the cost per patient for each programme by country to derive total costs. We estimate that between US5.1 billion dollars and US5.9 billion dollars will be needed by the end of 2005 to provide ART, support programmes, and cover country-level administrative and logistic costs for 3 by 5.     

Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a single- chain Fv fusion immunotoxin specifically targeting the CD3 epsilon moiety of the T-cell receptor

In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995). Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity. A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145-2C11 to a truncated form of the diphtheria toxin (DT390) gene. DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain. The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter. The protein was expressed in Escherichia coli and then purified from inclusion bodies. The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells. Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies. GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity. Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD. Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate. In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD. These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.     

Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain sequences

Human T-cell receptor (TCR) delta-chain diversity mainly originates from high junctional variability, since only a limited number of germline elements is available. This extraordinary diversity at the V.J junction, due to the use of two D delta elements and extensive incorporation of N nucleotides, constitutes a specific clonal marker for cell populations exhibiting rearranged TCR delta genes. To this end we amplified in vitro by polymerase chain reaction (PCR) the TCR delta junctional region of five acute lymphoblastic leukemias (ALL), isolated respective DNA fragments, and used them directly as clonospecific probes. The combination of PCR technology and hybridization to clonospecific probes permitted the detection of leukemia DNA at dilution of 1:100,000 in all five cases. Moreover, we were able to investigate one of the ALL patients 11 months after achieving continuous complete remission. Conventional Southern blot analysis failed to detect rearranged TCR genes at this stage. However, residual leukemic cells could readily be detected by PCR technique. We conclude that the strategy proposed here is a very sensitive tool to detect minimal residual disease in a significant proportion of human lymphoid neoplasias.     

Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.