Showing papers in "Blood Cells Molecules and Diseases in 2000"

Clustered CD20 Induced Apoptosis: Src-Family Kinase, the Proximal Regulator of Tyrosine Phosphorylation, Calcium Influx, and Caspase 3-Dependent Apoptosis☆☆☆★

Abstract: Anti-CD20 antibodies may reduce or eliminate non-Hodgkin's lymphoma B cells in patients, although the mechanism of action is not clear. To explore mechanism(s), we examined the induction of signal transduction events using anti-CD20 monoclonal antibodies (mAb) in the human non-Hodgkin's lymphoma Ramos B cell line. We found that while Rituximab (a human-mouse hybrid mAb) alone induced apoptotic cell death, other murine anti-CD20 mAbs induced apoptosis of Ramos B cells only upon clustering with a secondary antibody. CD20 clustering was accompanied by activation of tyrosine protein kinase activity, PLCgamma2 phosphorylation, influx of Ca(2+), and activation of caspase 3. All signaling events, as well as the subsequent apoptosis, were blocked by PP2, a selective inhibitor of Src-family kinases. Treatment of Ramos with EGTA and BAPTA to block changes in cytoplasmic Ca(2+) likewise prevented CD20-induced apoptosis. Our findings support a model in which CD20 clustering activates members of the Src family of protein tyrosine kinases, leading to phosphorylation of PLCgamma2 and increased cytoplasmic Ca(2+). These early signal transduction events activate caspase 3 to promote apoptotic cell death of NHL B cells.

Human Bone Marrow Stromal Cell: Coexpression of Markers Specific for Multiple Mesenchymal Cell Lineages

Abstract: The role of hematopoietic stem cells in blood cell development is reasonably understood, whereas the identity and the function of bone marrow stromal cells are much less clear. Using stromal cells in bone marrow cultures of the Dexter type, a favorite medium for the study of hematopoiesis, we show that stromal cells actually represent a unique cell type. Conventional wisdom has held that stromal cells in Dexter cultures comprise a mixture of macrophages, hematopoietic cells, adipocytes, osteoblasts, fibroblasts, muscle cells, and endothelial cells. Our findings demonstrate that Dexter cultures consist of three cell types: macrophages ( approximately 35%), hematopoietic cells ( approximately 5%), and nonhematopoietic cells ( approximately 60%). We have purified the nonhematopoietic cells free of macrophages and hematopoietic cells to produce compelling evidence that they in fact represent a single cell type (multidifferentiated mesenchymal progenitor cell, MPC) which coexpresses genes specific for various mesenchymal cell lineages including adipocytes, osteoblasts, fibroblasts, and muscle cells. We further show that these multi- or pluridifferentiated MPCs are capable of supporting hematopoiesis by demonstrating the expression of several hematopoietic growth factors and extracellular matrix receptors including G-CSF, SCF, VCAM-1, ICAM-1, and ALCAM. Since the MPCs can be easily purified to near homogeneity (95%), they can be of value in enhancing engraftment of hematopoietic stem cells. Also, this new understanding of bone marrow stromal cells as "one cell with many different faces" promises to advance our knowledge of regulatory cellular interactions within bone marrow.

Phenotype determination of a common Pro-Leu polymorphism in human glutathione peroxidase 1.

Abstract: Oxidative stress has been implicated in human illness such as cardiovascular and neurodegenerative disease. The genetic mechanisms involved are only poorly understood. Here we describe the determination of the allelic frequency and phenotype of a common polymorphism in Se-dependent glutathione peroxidase 1 (GPX1) in Finnish/Swedish populations. A proline/leucine variant occurs at position 197 close to the C-terminus of the protein. The more common allele encoding the Pro variant is present at 59% in a Finnish/Swedish population (n = 66) and at 73% in a Swedish population (n = 315). The genotypes encoding Pro/Pro, Pro/Leu, and Leu/Leu are distributed according to the Hardy–Weinberg relationship. The Swedish population consisted of 101 stroke cases and 214 controls. No significant association between allele frequency and risk to suffer from stroke was evident. Erythrocyte GPX activity was determined in the Finnish/Swedish population and no significant differences were obtained between the genotypes. It can be concluded that the Pro/Leu genetic variation does not appear to compromise the defense against oxidative stress in red blood cells nor to be associated with stroke.

Effects of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish.

Abstract: The zebrafish Danio rerio is a new model system for studying the genetics of hematopoiesis. To define naturally occurring viruses which could infect and replicate within hematopoietic precursors of the zebrafish, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) were studied. Infection of whole fish with viral supernatants demonstrated infectious replicants for both viruses, indicating that the virus host range includes the zebrafish. In other species, infection with these viruses leads to prominent hematopoietic necrosis of the head kidney, the major site of adult hematopoiesis. We detected a transient toxicity of the virus to hematopoietic precursors and terminally differentiated red cells after viral infections. The kinetics of hematopoietic defects between IHNV and IPNV infection differed; fish infected with either virus, however, recovered by 6 days postinfection. In contrast to other fish infected with the virus, hematocrit did not change appreciably during this time. These studies are the first to demonstrate IHNV and IPNV infection of the zebrafish and reveal the potential for use of such viruses for gene transfer experiments to infect zebrafish hematopoietic cells.

Selective inhibition of NF-kB activation and TNF-alpha production in macrophages by red blood cell-mediated delivery of dexamethasone.

Abstract: Glucocorticoids are a widely used class of anti-inflammatory and immunosuppressive drugs, but their therapeutic use is limited by endocrine and metabolic side effects that they produce when given systemically. Since cells of the monocyte/macrophage lineage play an important role in the pathogenesis of several autoimmune and inflammatory diseases, a drug-delivery system which targets phagocytic cells was studied. We had previously demonstrated that dexamethasone, a potent glucocorticoid analogue, can be encapsulated in erythrocytes and selectively delivered to macrophages. In addition, lipopolysaccharide (LPS) stimulation of dexamethasone-targeted macrophages results in the suppression of TNF-alpha secretion. In this paper we demonstrate that the administration of dexamethasone to macrophages by means of opsonized red blood cells allows efficient interference with NF-kB activation. This NF-kB repression was in part mediated by induction of IkBalpha gene transcription and, as a consequence, by an increased rate of IkBalpha protein synthesis. Furthermore, NF-kB inactivation correlated with downmodulation of TNF-alpha mRNA expression, demonstrating that suppression of TNF-alpha production in dexamethasone-targeted cells occurs at the transcriptional level.

Hereditary hemochromatosis: HFE mutation analysis in Greeks reveals genetic heterogeneity.

Abstract: ABSTRACT Hereditary hemochromatosis (HH) is common among Caucasians; reported disease frequencies vary from 0.3 to 0.8%. Identification of a candidate HFE gene in 1996 was soon followed by the description of two ancestral mutations, i.e., c.845G→A (C282Y) and c.187C→G (H63D). To these was recently added the mutation S65C, which may represent a simple polymorphism. The incidence of HH in Greece is unknown but clinical cases are rare. Also unknown is the carrier frequency of the two mutant alleles. A first estimate of the latter is given in the present report. It is based on data from the genetic analysis of 10 unrelated patients of Greek origin who were referred to our center for genotyping and 158 unselected male blood donors. The allele frequencies for the C282Y and H63D mutations were 0.003 and 0.145, respectively. The C282Y allele was detected in 50% of HH patients. This is considerably lower than the frequencies reported for HH patients in the U.S.A. (82%) and France (91%) and closer to that reported in Italy (64%). Five patients did not carry any known HFE mutation; three may represent cases of juvenile hemochromatosis, given their early onset with iron overload, hypogonadism, and heart disease. We suggest that genetic heterogeneity is more prominent in Southern Europe. It is also possible that the penetrance of the responsible genes is different across the Mediterranean.

Erythroid differentiation in vitro is blocked by cyclopamine, an inhibitor of hedgehog signaling.

Abstract: Adult hematopoietic differentiation is a developmental process that employs many of the same molecular mechanisms as embryogenesis. To explore the possibility that hedgehog signaling is involved in the control of hematopoietic differentiation, we screened a panel of human leukemia cell lines for the expression of Patched1 and Smoothened, the receptor and coreceptor for hedgehog ligands. Expression was found in multiple cell lines, and Patched1 expression was detected in normal marrow. Induction of myeloid differentiation in cell lines downregulated expression of both genes. When normal marrow mononuclear cells were grown in semisolid medium in the presence of 10 microM cyclopamine, development of colonies of granulocytic/monocytic lineage was unaffected in terms of both number and morphology. The number of erythroid colonies, however, was significantly reduced (P < 0.01). Furthermore, hemoglobinization was substantially delayed relative to controls in those erythroid colonies that did form. Incubation of hematopoietic progenitors with Shh-N and GM-CSF resulted in increased granulocyte/monocyte colonies (P < 0.01); the increase was blocked by cyclopamine. Incubation of hematopoietic progenitors with Shh-N and stem cell factor resulted in larger erythroid colonies. These results suggest that elements of the hedgehog signaling pathway are involved in the control of hematopoietic differentiation.

Comparative efficacy of dose regimens in enzyme replacement therapy of type I Gaucher disease.

Abstract: Gaucher disease is caused by a deficiency of beta-glucocerebrosidase activity. The optimum dose and frequency of enzyme replacement therapy for Gaucher patients have not been determined. We set to compare the therapeutic effects of initiating treatment with macrophage-targeted glucocerebrosidase at a high dose followed by progressive dose reductions with that produced by initial treatment at a low dose in patients with type I Gaucher disease. The study included two parts: (i) Twelve patients received every 2 weeks enzyme replacement therapy at 60 IU/kg body wt for 24 months followed by sequential dose reduction every 6 months to 30 and then to 15 IU/kg body wt. (ii) Thirty-two patients received enzyme replacement therapy at 10 IU/kg every 2 weeks for 12 months. Hematologic parameters and liver and spleen volume were monitored in all patients. All patients had intact spleens. In patients who were started on high-dose enzyme replacement therapy, hemoglobin, acid phosphatase, and organ volume improved or remained unchanged at the end of each dose reduction. Platelet count decreased significantly when the dose of enzyme was reduced from 30 to 15 IU/kg body wt. Initiation of therapy at a low dose led to a significant improvement in all measured parameters at the end of 1 year. We conclude that the minimal effective dose for the nonskeletal manifestations of Gaucher disease can be achieved either by initiating enzyme replacement therapy with a high dose followed by a stepwise dose reduction or by starting treatment at the minimal dose. High dose provides a faster clinical response and should be considered for patients with more aggressive disease. The therapeutic threshold for macrophage-targeted glucocerebrosidase appears to be 10-15 IU/kg body wt every 2 weeks.

Resveratrol decreases early signaling events in washed platelets but has little effect on platelet in whole blood.

Abstract: Resveratrol, a polyphenolic compound found in red wines, is believed to be a contributor in decreasing the incidence of coronary heart disease Although its primary target is unknown, it blocks aggregation of washed platelets by an ill-defined mechanism We show that resveratrol, at 10-50 microM, blocked aggregation induced by collagen (5 microg/ml), thrombin (02 units/ml), and ADP (10 microM) This affect was not overcome by adding exogenous human fibrinogen to the assay, suggesting that an early (wave I) signaling step in the alpha(IIb)beta(3) activation cascade was impaired To explore this possibility we examined the effect of resveratrol on activation of MAP kinases In the platelet, MAP kinases become activated as a consequence of agonist binding and not of aggregation, which itself induces signaling events In fact, we find that collagen-induced activation of MAP kinases is superinduced in the presence of RGDS, an aggregation-blocking peptide Resveratrol, at concentrations of 10 microM and greater, inhibited MAP kinase activation induced by collagen (in the absence and presence of RGDS peptide), thrombin, and ADP These data indicate that resveratrol blocks receptor-mediated signaling events in washed platelets In comparison, resveratrol has poor antiplatelet activity in whole blood Under these conditions aggregation was not affected by 50-100 microM resveratrol Concentrations of 200 microM resveratrol were needed to cause a 30-60% decrease in platelet aggregation in whole blood Together these studies suggest that resveratrol is a potent inhibitor of platelet signaling responses, but its antiplatelet activity is weakened or masked in circulation Thus, although resveratrol may function as a protective agent of coronary heart disease, its affects are not solely attributed to its effects on platelets in circulation

Ribosomal Protein S19 Gene Mutations in Patients with Diamond-Blackfan Anemia and Identification of Ribosomal Protein S19 Pseudogenes

Abstract: Diamond-Blackfan anemia (DBA) is a rare congenital pure red cell hypoplasia characterized by a selective defect of erythropoiesis with a normochromic macrocytic anemia and reticulocytopenia often accompanied by various congenital anomalies. The critical region responsible for the pathogenesis of DBA has been mapped in some patients to chromosome 19q13.2 (P Gustavsson, E Garelli, N Draptchinskaia, et al. Am. J. Hum. Genet. 63:1388-1395, 1998) and the gene encoding ribosomal protein S19 (RPS19) is believed to be the candidate gene. Here we present molecular analysis of the RPS19 gene in DBA patients from the Czech National DBA Registry. We found that the RPS19 gene was mutated in 25% (5/20) of DBA patients (insertion, deletion, and point mutations, but no nonsense or splice site mutations). Point mutations were localized to hot spots defined by Willig (TN Willig, N Draptchinskaia, I Dianzani, et al. Blood 94:4294-4306, 1999). Moreover, we describe two processed RPS19 pseudogenes, which were not expressed. Possible models of the DBA pathogenesis in the view of RPS19 mutations are discussed.

Pulsed intravenous high-dose dexamethasone in adults with chronic idiopathic thrombocytopenic purpura.

Abstract: ABSTRACT The role of pulsed high-dose dexamethasone (DXM) in the treatment of patients with chronic idiopathic thrombocytopenic purpura (ITP) is still uncertain. Following an early report in which it was described as an effective and well-tolerated treatment with a sustained platelet response in 100% of cases, a number of subsequent studies have failed to confirm such favorable results. As all these studies were conducted on small numbers of patients, we investigated further the effectiveness and side effects of this therapeutic modality in a larger cohort. Thirty-two patients with chronic ITP were scheduled to receive six monthly courses of intravenous DXM at the dose of 40 mg/day for 4 consecutive days. All patients had ITP that had been resistant to between two and five different therapeutic regimens, including 9 patients who had already failed splenectomy. All patients had to be seen 2 weeks after each cycle to asses their response as well as secondary effects. Three patients failed to respond and clinically required other therapy. Thirteen patients (41%) had a partial (platelet count between 50 and 100 × 10 9 /liter) or complete (platelet count >100 × 10 9 /liter) response to treatment, responses being mostly transient. Responses were observed early during the course of treatment, usually right after the first cycle of DXM. There were no late responses. Side effects were mild and did not require discontinuation of treatment. No clinical or laboratory parameter was found to predict treatment outcome. We conclude that high-dose DXM has a limited effect in patients with chronic ITP. Novel approaches and controlled multicenter trials may help identify new therapeutic strategies for this disease.

Reduction of the clinical severity of sickle cell/beta-thalassemia with hydroxyurea: the experience of a single center in Greece.

Abstract: The use of hydroxyurea for the prevention of sickle cell crises in patients with homozygous HbS disease is now well established. The beneficial effects of this compound stem from (a) selective enrichment of red cells containing an increased amount of fetal hemoglobin, which inhibits HbS polymerization, and (b) a decrease of leukocytes, platelets, and reticulocytes, which significantly limits their adherence to the vascular wall. We report the results of a clinical trial of hydroxyurea on 55 Greek-origin patients with sickle cell/beta-thalassemia and 14 patients with homozygous HbS disease who have been treated with hydroxyurea for several years. Such patients have a higher probability to benefit from hydroxyurea therapy, since in addition to its antisickling effect, the increase of gamma-chain synthesis is expected to diminish the deleterious effects of the unbound alpha-globin chains. Selection of patients and monitoring throughout the whole trial were done by the same clinicians. Quantitative expression of the clinical condition was done using a system scoring several outcome parameters. For a period of 52 months prior to starting treatment, the total score of severity for 59 evaluable patients was 1182 points (3068 patient-weeks), while for the 12,018 patient-weeks of the trial this parameter fell to only 82 points. Other observations of interest include the significant improvement of a group of patients with hepatic cholestasis, the development of leg ulcers possibly related to the treatment, and the dramatic increase of hemoglobin F, often in association with an increase of the total hemoglobin levels as a result of decreased hemolysis.

Dramatic decline in circulating intercellular adhesion molecule-1 concentration on quitting tobacco smoking.

Abstract: The concentration of soluble ICAM-1 (sICAM-1) is significantly elevated in smokers, but it is unclear if smoking is the direct cause of elevated sICAM-1 levels, if the relationship between smoking and sICAM-1 level is dose-dependent, and if smoking cessation may lead to a decline in sICAM-1. We sought to clarify the relationship between smoking and sICAM-1 in a group of smokers who quit smoking for 1 year (n = 30) and a control group who continued to smoke (n = 30). A dose-dependent relationship between plasma sICAM-1 concentration and daily cigarette consumption (P = 0.02), plasma cotinine level (P = 0.02), and expired CO level (P = 0.007) was observed at baseline (n = 60). The mean change in sICAM-1 concentration after 52 weeks was greater for quitters than for continuing smokers (mean difference = -71.1 ng/ml, P < 0.001). The influence of smoking on sICAM-1 needs to be carefully considered in clinical trials. Soluble ICAM-1 remains bioactive and may contribute to pathogenic processes; therefore, reduction in the concentration of circulating ICAM-1 molecules may directly contribute to the health benefits associated with smoking cessation.

Diamond-Blackfan anemia: report of seven further mutations in the RPS19 gene and evidence of mutation heterogeneity in the Italian population.

Abstract: ABSTRACT Diamond-Blackfan anemia (DBA) is a congenital disease characterized by defective erythroid progenitor maturation and physical malformations. Most cases are sporadic, but dominant or, more rarely, recessive inheritance is observed in 10% of patients. Mutations in the gene encoding ribosomal protein (RP) S19 have recently been found in 25% of patients with either the dominant or the sporadic form. DBA is the first human disease due to mutations in a ribosomal structural protein. Families unlinked to this locus have also been reported. In an investigation of 23 individuals, we identified eight different mutations in 9 patients. These include five missense, one frameshift, one splice site defect, and one 4-bp insertion in the regulatory sequence. Seven mutations are new; one has so far been found in 8 patients and is a relatively common de novo event. Two mutations are predicted to generate a truncated protein. We also report the prevalence of RPS 19 mutations in the Italian DBA population, as shown by an analysis of 56 patients. No genotype–phenotype correlation was found between patients with the same mutation. The main clinical applications for molecular analysis are clinical diagnosis of patients with an incomplete form of DBA and testing of siblings of a patient with a severe form so as to avoid using those who carry a mutation and a silent phenotype as allogeneic stem cell donors.

Oral isobutyramide therapy in patients with thalassemia intermedia: results of a phase II open study.

Abstract: ABSTRACT A pilot phase II open study on 12 patients with thalassemia intermedia (7 men, 5 women; age 31 ± 2.0 years SE) treated with oral isobutyramide, a derivative of butyric acid (150 mg/kg body wt/day), was performed in order to evaluate the effect of this compound in stimulating hemoglobin F (HbF) production. No patient underwent blood transfusion in the 1-year time frame prior to the study. Nine patients were splenectomized. Safety was monitored by clinical and laboratory tests. Efficacy was assessed in terms of the non-α/α globin chain biosynthetic ratio and the percentage increase of HbF. The study design consisted of a screening phase, a treatment phase of 28 days, and a posttreatment follow-up of 28 days. All patients completed the study. Compliance to treatment was 100%. No drug-related adverse event was recorded. We observed little or no increase in the non-α/α ratio in the majority of patients. Six patients showed a percentage increase of HbF at the end of treatment and in 5 of those 6 further increases at the end of the follow-up period were observed. The change in percentage of HbF over time was close to significance both in the treatment period (P = 0.06) and in the follow-up period (P = 0.08). These results indicate that butyrate derivatives can stimulate fetal hemoglobin in patients with intermediate thalassemia. Testing of the effects of different schedules of administration of isobutyramide will be required in order to determine the optimal use of this compound in the treatment of the β-thalassemia syndromes.

Immunophenotypic aberrations, DNA content, and cell cycle analysis of plasma cells in patients with myeloma and monoclonal gammopathies.

Abstract: We describe the immunophenotypic and gross DNA defects in 55 patients with myeloma and 50 patients with monoclonal gammopathy and review the literature on this subject (MedLine, 1994-2000). Our data confirmed previous reports indicating that in myeloma nearly all marrow plasma cells are abnormal (98.7 +/- 8.1%). In monoclonal gammopathy the fraction of abnormal plasma cells was 35.0 +/- 32.8%. In both myeloma and monoclonal gammopathy, the most frequent aberrant phenotypic features consisted of absence of expression of CD19, strong expression of CD56, and decreased intensity of expression of CD38; aberrant expression of CD10, CD20, CD22, or CD28 was observed in less than one-third of myeloma cases. The vast majority of cases had two or more phenotypic aberrations. In the DNA studies, 7% of myeloma cases were biclonal and 93% of cases were monoclonal. In those studies with only one plasma cell mitotic cycle, 37% had normal DNA content and 63% were aneuploid (hyperploid, 61%; hypoploid, 2%). The mean percentages of plasma cells in S- and G2M phases were 4.9 +/- 8.5 and 4.4 +/- 6.9%, respectively. Thirty-eight percent of cases had more than 3% of plasma cells in S phase. In monoclonal gammopathy, the DNA index of abnormal plasma cells ranged from 0.89 to 1.30 and the percentage of diploid (31%) and aneuploid (69%) cases was not different from the results found in myeloma. The differences in percentage of abnormal plasma cells in S- (7.4 +/- 8.6%) and G2M-phases (2.4 +/- 1.7%) in patients with monoclonal gammopathy were not statistically significant.

Anticoagulant proteins in childhood venous and arterial thrombosis: a review.

Abstract: Thrombotic disease is less frequent in children than in adults, but may result in severe morbidity and mortality. The coagulation system is balanced to provide rapid activation to stop bleeding and appropriate inhibition to prevent unwanted clot extension. It is regulated by fibrinolysis and by three major anticoagulant pathways: the protein C, antithrombin, and tissue factor pathway inhibitor systems. Acquired or inherited abnormalities of coagulation proteins or hemostatic regulatory mechanisms, particularly when combined with dehydration or the presence of indwelling catheters, may pose a high risk for thrombosis. Thrombosis in a child warrants investigation of potential underlying prothrombotic conditions. These include acquired antiphospholipid antibodies or the lupus anticoagulant as well as abnormalities of the inherited anticoagulant factors including protein C, protein S, antithrombin, and Factor V Leiden. Other abnormalities may result in heightened levels of otherwise normal coagulation proteins such as hyperprothrombinemia due to the prothrombin 20210 mutation. A large survey of children with thrombosis indicated that Factor V Leiden, protein C deficiency, and increased lipoprotein(a) were found most commonly. The most severe predisposition occurs with homozygous protein S or protein C deficiency with resultant purpura fulminans in the newborn. The risk of thrombosis in children with heterozygous deficiencies of anticoagulant proteins is not well defined, although it is clear that combined heterozygotes or a combination of an inherited and an acquired defect heightens the risk for thrombosis. Treatment of thrombosis primarily involves a rapidly acting anticoagulant such as heparin or low-molecular-weight heparin to prevent extension, and long-term anticoagulation with warfarin may be instituted to prevent recurrence. Fibrinolytic therapy is infrequently used because of the risk of serious bleeding complications and is reserved for selected cases of arterial thrombosis to initiate rapid reperfusion of ischemic tissue or used in those patients with a large venous thrombosis and pulmonary emboli causing hemodynamic compromise.

Clinical and Molecular Aspects of Juvenile Hemochromatosis in Saguenay–Lac-Saint-Jean (Quebec, Canada)

Abstract: ABSTRACT: We report the clinical, biochemical, and genetic characteristics of 13 hemochromatosis patients from Saguenay–Lac-Saint-Jean in whom the first symptoms appeared before age 30. Although the mean age at onset of the first symptoms was 21.5 years, their mean age at diagnosis was 23.8 years; the diagnosis was particularly delayed among women. Seventy-seven percent of the patients had hypogonadotrophic hypogonadism and 69% heart failure and/or cardiac arrhythmias. Genetic analysis of the HFE gene revealed heterozygosity for the C282Y mutation in 2 patients and for the S65C mutation in 2 others and homozygosity for the H63D mutation in 1 patient. The remaining 8 patients had no identified mutation in the HFE gene, although sequencing of all seven codons and intron–exon junctions was performed (5 patients). All 13 patients fulfill the clinical criteria of juvenile hemochromatosis and represent the largest cluster thus far reported.

Genomic structure of the human p47-phox (NCF1) gene.

Abstract: The cytosolic factor p47-phox, encoded by the NCF1 gene, is an essential component of the phagocyte NADPH-oxidase system. Upon activation of this multicomponent system, p47-phox translocates to the membrane and participates in the electron transfer from NADPH to molecular oxygen. A deficiency or absence of p47-phox is the most common autosomal form of chronic granulomatous disease (CGD). We have cloned and characterized the NCF1 gene from four bacteriophage clones, a P1 clone and genomic DNA from normal individuals. The gene is 15,236 base pairs long and includes 11 exons. It is 98.6% homologous in sequence to at least one pseudogene that maps to the same region of chromosome 7q11.23. Slightly more than half (50.37%) of the wild-type NCF1 gene consists of repetitive elements. In particular, the density of Alu sequences is high (1.4 Alu/kb); there are 21 Alu repeats interspersed through 10 introns. These findings are consistent with the observation that recombination events between the wild-type gene and its highly homologous pseudogenes account for the majority of potentially lethal mutations in p47-phox-deficient chronic granulomatous disease. Analysis of 1.96 kb of sequence 5' of the start of translation revealed a high homology (99.6%) between wild-type and pseudogene clones. Characterization of NCF1 establishes a foundation for detailed molecular analysis of p47-phox-deficient CGD patients as well as for the study of the regulation of the NCF1 gene and pseudogenes, both of which are present as full-length transcripts in normal individuals.

Proper Developmental Control of Human Globin Genes Reproduced by Transgenic Mice Containing a 160-kb BAC Carrying the Human β-Globin Locus☆

Abstract: ABSTRACT Four independent bacterial artificial chromosome (BAC) clones containing the human B-globin gene locus were obtained from a human genomic BAC library. A 160-kb clone (186D7) carrying the entire human B-globin locus including the B-globin gene family, locus control region (LCR), and 3′ regulatory elements was used to transform mice. Four transgenic lines were generated by microinjecting the purified BAC DNA into the fertilized eggs. RNase protection analysis showed that the expression of human B-globin genes is tissue- and developmental stage-specific and the expression level is similar among the three independent transgenic lines which carry the entire human B-globin locus; however, no B-globin gene expression was detected in the transgenic mice lacking the LCR region. The results suggest that the transgenic mouse model system that we have produced and that uses BAC to study the complex human B-globin gene cluster is stable and reproducible. Our results also indicate that some newly characterized HSs upstream from the LCR appear not to play an important role in globin gene expression and switching, while the traditional LCR can ensure correct human B-globin gene expression in transgenic mice. The BAC-mediated transgenic system can be used for further studies to determine which kinds of cis-acting elements are included in regulating the developmental timing and the level of human B-globin gene expression.

Distinct Phenotypic Expression Associated with a New Hyperunstable Alpha Globin Variant (Hb Heraklion, α1cd37(C2)Pro>0): Comparison to Other α-Thalassemic Hemoglobinopathies

Abstract: Clinical phenotypes associated with abnormal globin chain biosynthesis may result in thalassemia (deficient quantity) or hemolytic anemia (abnormal hemoglobins). However, the phenotypic expression of hyperunstable hemoglobin variants often includes features of thalassemia, along with variable peripheral hemolysis. Hemoglobinopathies caused by highly unstable beta-chain variants have a dominant thalassemia-like phenotype, in which carriers have a clinical expression of thalassemia intermedia, but highly unstable alpha-globin variants are usually only phenotypically apparent when they interact with other alpha-thalassemia mutations. In a child with clinical and hematological features consistent with beta-thalassemia intermedia, DNA analysis excluded any beta-globin gene mutations but characterized a novel deletion cd37(C2)Pro>0 (Hb Heraklion) in the alpha1 globin gene, in trans to a common Mediterranean nondeletion alpha-thalassemia mutation (alpha(Hph)alpha). The deletion of proline at alpha37(C2) is predicted to result in severe instability of the variant hemoglobin, which on interaction with a synthesis-deficient alpha-thalassemia mutation causes a relatively severe dyserythropoietic anemia, representing an alternative phenotype associated with highly unstable alpha-chain variants. Hb Heraklion is the fourth highly unstable alpha-globin variant that we have observed in patients from Greece and Albania. Two variants involve the alpha2-globin gene: Hb Agrinio (alpha29(B10)Leu>Pro) and Hb Adana (alpha59(E8)Gly>Asp), and two the alpha1-gene: Hb Aghia Sophia (alpha62(E11)Val>0) and (Hb Heraklion a37(C2)Pro>0). Each has been observed on interaction with a different alpha-thalassemia mutation and the phenotypes associated with these highly unstable alpha-variants are presented.

A beta-2-adrenergic receptor activates adenylate cyclase in human erythrocyte membranes at physiological calcium plasma concentrations.

Abstract: More information is needed about the subtype of the beta-adrenergic receptor coupled to the G-protein-adenylate cyclase (AC) system in human erythrocytes and about the optimal experimental conditions to study this system. In this study we describe the characteristics of spontaneous and beta-agonist-activated AC in human erythrocytes. Human erythrocyte membranes were isolated and AC activity was utilized to assess the quantity of cAMP. Our data show that the subtype beta-2 is the functional beta-adrenergic receptor involved in such activation; this modifies the beta-adrenergic-stimulated activity of AC in human erythrocytes. Isoproterenol in a medium with calcium (1-10 mM, range that includes physiological plasma concentrations) enhances the activation of AC; this effect was blocked by propranolol, but not by atenolol. We conclude that in human erythrocytes subtype beta-2 is the functional beta-adrenergic receptor and that such a response depends to a large extent on Ca(2+) concentrations.

Cardiolipin enhances protein C pathway anticoagulant activity.

Abstract: The anticoagulant activity of activated protein C (APC) was studied using factor Xa-1-stage assays of both the procoagulant and anticoagulant activities of phospholipid vesicles containing phosphatidylserine or cardiolipin as active phospholipids. In the absence of APC, phosphatidylserine vesicles showed higher procoagulant activity than cardiolipin vesicles whereas cardiolipin vesicles supported APC-dependent anticoagulant activity better than phosphatidylserine vesicles. Enhancement of APC anticoagulant activity in plasma by cardiolipin was markedly stimulated by the APC cofactor protein S. In purified reaction mixtures, cardiolipin in phospholipid vesicles dose-dependently enhanced APC anticoagulant activity. This effect of cardiolipin was partially dependent on protein S, and immunoblotting studies showed that cardiolipin enhanced the APC-mediated cleavage of the factor Va heavy chain at Arg506 and Arg306. In solid-phase binding assays, increasing amounts of cardiolipin in multicomponent phospholipid vesicles increased the affinity for protein S and to a lesser extent APC. These data are consistent with the hypothesis that cardiolipin stimulates the anticoagulant protein C pathway by increasing the affinity of phospholipid surfaces for protein S:APC and by enhancing inactivation of factor Va by APC due to cleavages at Arg506 and Arg306 in factor Va. Based on this, it is further hypothesized that anti-cardiolipin or anti-oxidized cardiolipin antibodies may be thrombogenic because they inhibit phospholipid-dependent expression of the anticoagulant protein C pathway.

In Vivo Silencing of the Human γ-Globin Gene in Murine Erythroid Cells Following Retroviral Transduction

Abstract: ABSTRACT Increased expression of fetal hemoglobin can ameliorate the clinical severity of sickle cell disease. Whereas temporary induction of fetal hemoglobin can be achieved by pharmacologic therapy, gene transfer resulting in high-level expression of the fetal γ-globin gene may provide a permanent cure for sickle cell disease. We had previously developed a high-titer, genetically stable retroviral vector in which the human γ-globin gene was linked to HS-40, the major regulatory element of the human α-globin gene cluster. Based on experience in transgenic mice, the truncated promoter of the γ-globin gene of this vector should be active in adult erythroid cells. Our earlier studies demonstrated that this retroviral vector can give rise to high-level expression of the human γ-globin gene in murine erythroleukemia (MEL) cells. We have now utilized this vector to transduce murine bone marrow cells that were transplanted into W/W v recipient mice. Analysis of transduction of murine BFU-e's in vitro and peripheral blood cells from transplanted mice in vivo demonstrated efficient transfer of the human γ-globin gene. However, in contrast to the high level of expression of the human γ-globin gene of this vector in MEL cells, the gene was completely silent in vivo in all transplanted mice. These observations confirm that all the necessary regulatory elements responsible for the developmental stage-specific expression of the human γ-globin gene reside in its proximal sequences. They also emphasize the differences between gene regulation in MEL cells, transgenic mice, and retroviral gene transfer vectors. For this form of globin gene therapy to succeed, the proximal regulatory elements of the human γ-globin gene may have to be replaced with different regulatory elements that allow the expression of the γ-globin coding sequences in adult red cells in vivo.

Should whole-body red cell mass be measured or calculated?

Abstract: The whole-body volume of red blood cells must be known for correct diagnosis of polycythemia vera. Since the venous hematocrit may not correctly reflect the absolute amount of red blood cells, the red cell mass (RCM) is usually determined by radioisotope labeling of red blood cells according to recommendations of the International Committee for Standardization in Haematology. We examined whether the radioisotope labeling procedure can be replaced by a simple calculation of RCM from the values of venous blood hematocrit and plasma volume, using an empirical factor (Ratio f) equal to the mean ratio between whole-body and venous hematocrit. A retrospective study was performed of 264 cases in which the RCM was assayed using (51)Cr or (99m)Tc for red cell labeling, and (125)I-labeled albumin was used for estimation of plasma volume. The results showed wide scattering of Ratio f (mean value 0.911; range from 0.76 to 1.15) that was responsible for substantial differences between the measured and calculated values of RCM. We also tested whether the calculated RCM may be used as an appropriate marker for polycythemia vera according to recommendations of the International Council for Standardization in Haematology. Our data showed that 146 patients had measured RCM values more than 25% above the mean normal predicted value. Using the calculated RCM, 17 of these patients would be lost from the polycythemia vera group, whereas 29 subjects with measured RCM levels equal to or lower than 125% of predicted values would incorrectly meet the RCM criterion for polycythemia vera. Thus, a total of 46 patients would be misclassified by using the calculated RCM. We conclude that the radioisotope labeling of red blood cells remains mandatory for correct determination of the whole-body red cell volume.

Characterization of zebrafish full-length prothrombin cDNA and linkage group mapping.

Abstract: In this paper, we report the complete cDNA sequence of zebrafish prothrombin. The cDNA sequence predicts that zebrafish prothrombin is synthesized as a pre-proprotein consisting of a Gla domain, two kringle domains, and a two-chain protease domain. Zebrafish prothrombin is structurally very similar to human and other vertebrate prothrombins. Zebrafish and human prothrombin share 53% amino acid identity whereas zebrafish and hagfish prothrombin share 51% identity. Amino acid alignments of various prothrombins identified conservation of many of the functional/structural motifs suggesting that the vertebrate prothrombins may have similar functions. The three-dimensional structure of prothrombin predicted by homology modeling also revealed that the prothrombin fragment 1 and the catalytic domain structures are well conserved except for the insertion of an extra 7-amino-acid loop in the connecting region (CR) between the Gla and kringle I domain of fragment 1. Linkage analysis revealed that the prothrombin gene locus on linkage group 7 in zebrafish is syntenic to the human chromosome 11-prothrombin region suggesting its preservation through evolution. The availability of this cDNA sequence in zebrafish adds to our knowledge of the zebrafish hemostatic system and provides support for the view that similarities between zebrafish and mammalian coagulation exist, thus underscoring the relevance of the zebrafish model for studying human hemostasis.

The ETS family member Tel antagonizes the Fli-1 phenotype in hematopoietic cells.

Abstract: The ETS family member Tel is rearranged in human leukemia of both myeloid and lymphoid origin while the ETS member Fli-1 is insertionally activated in Friend erythroleukemia in mice and is translocated to the EWS locus in Ewing's sarcoma. In previous studies we demonstrated that Tel binds to Fli-1 and blocks transactivation of megakaryocytic promoters by Fli-1. In this study we demonstrate that expression of Fli-1 in the leukemia cell line K562 induces a megakaryocytic phenotype and the expression of the platelet markers GPIX, GP1balpha, and GPIIb. Introduction of Tel blocked the megakaryocytic phenotype induced by Fli-1, suggesting a biological correlation to the biochemical interaction of Tel and Fli-1 reported previously.

SH2-Containing Protein Tyrosine Phosphatase-1 (SHP-1) Association with Jak2 in UT-7/Epo Cells☆

Abstract: ABSTRACT: We have investigated the interaction of the SH2-containing protein tyrosine phosphatase-1 (SHP-1) and Jak2 in an erythropoietin (Epo)-dependent human leukemia cell line, UT-7/Epo, using reciprocal immunoprecipitation and immunoblotting. The Epo-induced kinetics and dose response on phosphorylated Jak2 in anti-SHP-1 precipitates of UT-7/Epo cell lysates were similar to those in direct anti-Jak2 precipitates, suggesting that Jak2 coprecipitated with SHP-1. Furthermore, immunoblotting with anti-Jak2 and anti-SHP-1 antibodies indicated that SHP-1 appeared to be constitutively associated with non-tyrosine-phosphorylated Jak2 in UT-7/Epo cells in the absence of Epo and without phosphorylation of the Epo receptor (EpoR). Competition studies with C-terminal SHP-1 and Jak2 peptides decreased the amounts of SHP-1 and Jak2 detected in immunoprecipitates supporting the specific coprecipitation of SHP-1 and Jak2. In the presence of a recombinant GST-fusion protein containing both the N-terminal and C-terminal SH2 domains of SHP-1, anti-GST precipitated the fusion protein but not cellular Jak2. These studies suggest that SHP-1 and Jak2 are constitutively associated in UT-7/EPO cells. The association is not dependent upon Epo and is not mediated via SHP-1 SH2 binding. Sequential double immunoprecipitation demonstrated that only a small portion of intracellular Jak2 and SHP-1 molecules are constitutively associated. This partial association pattern may allow a more flexible and diverse regulation of Jak2 and SHP-1 activities. Whether Jak2 and SHP-1 are directly associated with each other or are part of a larger complex needs further investigation.