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Biomedical subjects

N P Anagnou

Publications and source records attributed to N P Anagnou.

At least 19 recordsLinked to original sources
PubMed2005

Use of the hereditary persistence of fetal hemoglobin 2 enhancer to increase the expression of oncoretrovirus vectors for human gamma-globin.

The development of oncoretrovirus vectors for human gamma-globin has been hampered by problems of low expression and gene silencing. In order to address these problems, we investigated an enhancer element identified from individuals with deletional hereditary persistence of fetal hemoglobin 2 (HPFH2), a genetic condition characterized by elevated levels of gamma-globin in adults. Plasmid transfection studies in erythroid MEL (murine erythroleukemia) cells demonstrated the HPFH2 element could function synergistically with the beta-globin locus control region to enhance the expression of an Agamma-globin gene with a truncated -382 bp promoter. A series of oncoretrovirus vectors were subsequently generated that contain an expression cassette for Agamma-globin linked to various combinations of the HPFH2 enhancer, the alpha-globin HS40 enhancer, and several versions of the promoter from Agamma-globin or beta-globin. Expression analysis in transduced MEL cell clones revealed very high levels of promoter-autonomous silencing that was at least partially abrogated by the HPFH2 enhancer. The vector containing a combination of a -201 bp Agamma-globin gene promoter with the Greek HPFH -117 point mutation and both the HPFH2 and HS40 enhancers exhibited no signs of vector silencing and was expressed at 248+/-99% per copy of mouse alpha-globin (62% of total alpha-globin). This represents a significant improvement over previously reported oncoretrovirus vectors for Agamma-globin, and demonstrates the capacity of the HPFH2 enhancer to abrogate sequence-autonomous silencing of the Agamma-globin promoter in the context of a gene transfer vector.

Animals↗
PubMed2004

Acute myeloid/NK precursor cell leukemia with trisomy 4 and a novel point mutation in the extracellular domain of the G-CSF receptor in a patient with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) has been well recognized as a granulocytic disorder not associated with increased risk to malignant transformation. Four cases, however, of acute myeloid leukemia have been recently reported in patients with CIN. In the current paper, we report on a CIN patient who developed acute myeloid/natural killer (NK) precursor cell leukemia 11 years after diagnosis and 4 months after initiation of treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Leukemic cells had trisomy 4 as the sole cytogenetic abnormality and, also, a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSFR) leading to truncated protein with a loss of 36 amino acids. There was no evidence that this receptor transmitted signals even in the presence of high doses of rhG-CSF in the cultures. We consider that CIN may be a preleukemic condition, at least in a subset of patients, and that rhG-CSF administration is unlikely to be involved in the leukemic transformation in this patient, although such a possibility could not be completely ruled out.

Base Sequence↗
PubMed2004

Congenital bronchopulmonary sequestration presenting as a thoracic tumor: a case report.

The accurate diagnosis of fetal thoracic tumors still remains unclear despite the progress in imaging technology. The differential diagnosis between tumors and congenital anomalies of the fetus respiratory system, largely depends on the diagnostic approaches involved. We report a case of a 25-year-old woman, gravida 3 para 0, who was seen at the 23rd gestational week for routine obstetric examination. The ultrasound scan detected a lung mass, occupying the whole left hemithorax with a significant shifting of the mediastinum exhibiting features compatible with cystic adenomatoid malformation (CAM). No other congenital anomalies were noted. Color Doppler ultrasound failed to detect any blood supply to the mass. Amniocentesis disclosed a normal male karyotype. Pregnancy termination was performed according to the parents' request, with the use of misoprostol and a 500 g dead fetus was delivered. The autopsy followed by detailed histological examination, disclosed the diagnosis of pulmonary sequestration. It is important to emphasize that the initial impression concerning the sonographic appearance and the size of the mass is not always in accordance with the diagnosis of the lesion and the outcome of the pregnancy. These data suggest that in cases of fetal pulmonary tumors, a thorough and comprehensive combination of imaging approaches should be employed followed by a pathologic examination of the congenital anomaly in order to establish a definitive diagnosis.

Abortion, Induced↗
PubMed2000

Increased serum IgA and decreased IgG3 strongly correlate with increased serum TGF-beta1 levels in patients with nonimmune chronic idiopathic neutropenia of adults.

OBJECTIVE: To study the changes in serum immunoglobulins and some closely related pro-inflammatory cytokines in patients with nonimmune chronic idiopathic neutropenia of adults (NI-CINA). METHODS: Serum levels of gamma-globulins, IgG, IgA, IgM, IgG subclasses, interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1) were evaluated in 83 NI-CINA patients and 65 normal controls using the respective conventional methods. RESULTS: We found that serum gamma-globulin, IgG and IgG1 levels were all significantly increased in the entire group of patients studied, compared to controls (p<0.001, p<0.01 and p<0.01, respectively), while the levels of IgG3 were significantly reduced (p<0.001). Serum IgA were increased in patients with severe neutropenia (p<0.001). No significant changes were noted in serum IgM, IgG2 and IgG4 levels. The infrequent occurrence of detectable amounts of IL-4 and IFN-gamma in the serum was similar in both, patients and control subjects. Serum levels of TGF-beta1 were increased in all groups of patients studied and they correlated inversely with the levels of IgG3 (p<0.001) and positively with the levels of IgA (p<0.001), suggesting the possible involvement of the cytokine in immunoglobulin class switching. CONCLUSION: Patients with NI-CINA have significant changes in serum immunoglobulins and some inflammation-related cytokines. These findings provide additional evidence for the existence of an unrecognized low-grade chronic inflammatory process in NI-CINA patients and coroborate our previously reported suggestion for the possible involvement of this inflammation in the pathogenesis of neutropenia in the affected subjects.

Adolescent↗
PubMed1997

Molecular cloning of the breakpoints of the hereditary persistence of fetal hemoglobin type-6 (HPFH-6) deletion and sequence analysis of the novel juxtaposed region from the 3' end of the beta-globin gene cluster.

Naturally occurring deletion mutations within the human beta-globin cluster lead to specific, phenotypically discrete syndromes (i.e., delta beta-thalassemias and hereditary persistence of fetal hemoglobin, HPFH), characterized by increased production of fetal hemoglobin in adult life. We have previously characterized an enhancer element, which is juxtaposed to the fetal G gamma-gene, by means of a deletion first described in a Thai family. To obtain further insights into the mechanisms involved in this deletion, we have now characterized several of its novel features. Following amplification by the polymerase chain reaction and sequencing of the 1.5-kb bridging fragment, we have shown that the 5' breakpoint of the deletion occurs 1260 bp 3' of the fetal G gamma-globin gene, whereas the 3' breakpoint lies 521 bp upstream of the EcoRI site of the enhancer element and 2845 bp upstream of the 3' breakpoint of the Chinese (A gamma delta beta) zero-thalassemia deletion. The total length of the deletion is 101 kb, which resembles that of HPFH-1 and HPFH-2 deletions and a set of two gamma delta beta-thalassemia deletions. Our data further support the hypothesis that these sets of large deletions with almost identical lengths are generated via the loss of a complete chromatin loop. To elucidate further the mechanisms leading to the deletion, we have sequenced the novel 0.5-kb region residing immediately 3' to the breakpoint and shown that it contains putative binding sites for several transcription factors, such as HNF-1, AP-1, and TFIID. Sequence comparison of the deletion breakpoints reveals no junctional homology, indicating an end-to-end joining of blunted ends; a pair of 7-nt complementary repeats adjacent to a set of a direct CCCT repeat flanks the breakpoints. This limited homology constitutes a frequent characteristic of a non-homologous recombination mechanism. All these features of the HPFH-6 deletion suggest that this mutation has resulted from a non-homologous recombination event.

Base Sequence↗
PubMed1995

Sequences located 3' to the breakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhancer activity and can modify the developmental expression of the human fetal A gamma-globin gene in transgenic mice.

Expression of fetal gamma-globin genes in individuals with the deletion forms of hereditary persistence of fetal hemoglobin (HPFH) has been attributed either to enhancement by 3' regulatory elements juxtaposed to gamma-globin genes or to deletion of gamma-gene silencers normally residing within the beta-globin gene cluster. In the present study, we tested the hypothesis of imported enhancers downstream of beta-globin gene using the HPFH-3 deletion as a model. The abnormal bridging fragment of 13.6 kilobases (kb) containing the A gamma-gene with its flanking sequences and 6.2 kb of the juxtaposed region was microinjected into fertilized mouse eggs. Twelve transgenic mice positive for the fragment were generated. Samples from 11.5-day yolk sacs, 16-day fetal liver, and adult blood were analyzed for A gamma-mRNA using RNase protection assays. Three mice lacked A gamma expression in the yolk sac indicating non-optimal integration site. Four expressed A gamma-mRNA at the embryonic stage only, while two expressed A gamma-mRNA in both embryonic and fetal liver erythroid cells. Since the A gamma-gene with its normal flanking sequences and in the absence of the locus control region is expressed only in embryonic cells of transgenic mice, these data suggest that the juxtaposed sequences have altered the developmental specificity of the fetal gamma-globin gene. These sequences were further tested for the presence of an enhancer element, by their ability to activate a fusion reporter gene consisting of the CAT gene linked to the gamma-globin gene promoter, in erythroid (K562) and non-erythroid (HeLa) cells. A 0.7-kb region located immediately 3' to the breakpoint, enhanced chloramphenicol acetyltransferase activity by 3-fold in erythroid cells. The enhancer also activated the embryonic epsilon-globin gene promoter by 2-fold but not the adult beta- or delta-globin gene promoters. The enhancer represents a region of previously known complex tandem repeats; in this study we have completed the sequencing of the region encompassing the 0.7-kb enhancer element. Multiple areas of the enhancer region exhibit homology to the core element of the simian virus 40 enhancer and to the sequences of the human 3' A gamma- and the chicken 3' beta-globin enhancers. A consensus binding site for the erythroid specific GATA-1 transcription factor and seven consensus sites for the ubiquitous CP1 transcription factor are also included within the enhancer. These data suggest that these sequences located immediately 3' to the breakpoint of the HPFH-3 deletion, exhibit both the structure and the function of an enhancer, and can modify the developmental specificity of the fetal gamma-globin genes, resulting in their continued expression during adult life.

Animals↗
PubMed1994

Eastern European (delta beta) zero-thalassemia: molecular characterization of a novel 9.1-kb deletion resulting in high levels of fetal hemoglobin in the adult.

A novel deletion in the human beta-globin gene cluster associated with increased levels of fetal hemoglobin (HbF) in adult life was molecularly characterized in a member of a family of Eastern European descent. The phenotype of the deletion, documented in five members of the family, shows mild hypochromia and microcytosis (mean corpuscular Hb, 24 to 25.9 pg; mean corpuscular volume, 74 to 78.5 fL) but high production of HbF (13% to 24%) with heterocellular distribution (36% to 86% F cells). Extensive restriction enzyme mapping of the beta-globin cluster and sequencing of the region encompassing the breakpoints showed that the deletion starts 1,612 bp upstream of the cap site of the delta-globin gene, and terminates within the first intron of the beta-globin gene, deleting 9.1 kb of DNA. This length is definitely shorter than the average 12.0 kb of the previously characterized (delta beta) zero-thalassemias. The 5' breakpoint of the new deletion is close to that of the Yugoslavian delta beta-thalassemia deletion, whereas the 3' breakpoint is very close to those of the Turkish and the Greek beta zero-thalassemia deletions. The breakpoints of the deletion occur within a direct repeat containing a tetranucleotide exhibiting homology to a donor-splice site, and is symmetrically flanked by a set of 13- and 14-bp homologous complementary sequences, respectively. It is likely that the deletion may be the result of an "illegitimate" or "nonhomologous" recombination event to which these two short sequences may have contributed. It is of interest that the novel deletion (9.1 kb) is comparable to the Italian HPFH-5 deletion (12.9 kb), regarding both the size and the position of the breakpoints. However, the HPFH-5 deletion includes sequences flanking the breakpoints that are preserved in the new deletion. Considering the resulting two discrete phenotypes (ie, delta beta-thalassemia v HPFH), it can be hypothesized that the deleted sequences in the Italian HPFH-5 mutation may harbor regulatory elements that exert a negative control on the gamma-globin gene expression.

Adult↗
PubMed1991

Expression of embryonic globins by erythroid cells in juvenile chronic myelocytic leukemia.

Juvenile chronic myelocytic leukemia (JCML) is a rare hematopoietic neoplasia of early childhood with distinct hematologic and biochemical features. We studied the biologic properties and the globin synthetic profiles of JCML erythroid cells both in vivo and in vitro from a total of 24 patients. In these cases we observed the exuberant colony-forming unit-macrophage (CFU-M) colony growth, as reported previously. Furthermore, in contrast to previous reports, we found significant erythroid colony growth in most of our cases (average: 1,182 burst-forming unit-erythroid [BFUe] per 10(5) plated cells, range: 40 to 6,927). This growth was by and large erythropoietin-dependent and was not greatly influenced by other added cytokines. By several criteria all erythroid colony growth detected in vitro was derived from JCML progenitors. The globin synthetic profile of JCML erythroid cells showed high levels of fetal hemoglobin both in vivo and in vitro (gamma/gamma + beta: 53% to 94% in reticulocytes, 62% to 98% in BFUe-derived cells). In addition (in seven cases studied) we detected embryonic globins (epsilon and zeta) at the protein and messenger RNA level, a novel finding for primary leukemic cells. We speculate that the transformed erythroid cells in JCML harbor a trans environment supporting expression of developmentally earlier genes (fetal, embryonic). However, in contrast to other acute or subacute leukemias, JCML erythroid cells also have the ability to reach full maturation to the red cell level, thus allowing detection of this primitive program in vivo.

Bone Marrow↗
PubMed1991

Recurrent attacks of hemolytic uremic syndrome.

In 1955 Gasser and his co-workers were the first to describe the so-called hemolytic uremic syndrome (HUS); since then, the number of reports has steadily increased. Some authors consider HUS as a unique syndrome, while others suggest that HUS is both heterogenic and heterogeneous. It is generally emphasized that HUS never recurs. However, this view should be reconsidered due to the numerous reports on a recurrent form of HUS, which is beginning to be recognized as an important subset or variant of this syndrome. This report describes a case, where three similar recurrent episodes of hemolytic anemia, thrombocytopenia and uremia had occurred during the past eight years.

Adolescent↗
PubMed1990

Developmental and inducible patterns of human theta 1-globin gene expression in embryonic/fetal and adult erythroid cells.

Human theta (theta 1)-globin gene represents a member of the alpha-like globin gene family residing on chromosome 16. theta 1-Specific transcripts have been detected so far only in erythroid tissues and in erythroleukemia K562 cells. To investigate systematically its inducible expression and developmental specificity, we analyzed at the RNA level five additional human erythroleukemia cell lines with diverse developmental globin programs, two somatic cell hybrids between K562 and mouse erythroleukemia (MEL) cells, a human fetal liver x MEL somatic cell hybrid, and reticulocytes and bone marrow cells from normal adults. theta 1-Globin gene was expressed in all cell types. Inducible expression (two- to sixfold) was documented both in HEL and K562 erythroleukemia cells after 5-azacytidine treatment. Like K562 cells, HEL cells also displayed hemin-inducible theta 1-globin gene expression. Following transfer of human chromosome 16 from embryonic/fetal K562 to the adult MEL cells, theta 1-globin gene remained active but lost its potential for inducibility, suggesting probably a trans regulation mechanism. Higher levels of theta 1 mRNA were found in fetal liver cells compared with trace amounts in reticulocytes and normal adult bone marrow cells. These data clearly show that in contrast to the embryonic and adult patterns of expression of zeta and alpha-globin genes, respectively, theta 1-globin gene displays a different profile, being active predominantly during the early stages of ontogeny, switching to lower levels of expression in adulthood.

Embryonic and Fetal Development↗
PubMed1990

Butyrate induces expression of transfected human fetal and endogenous mouse embryonic globin genes in GM 979 erythroleukemia cells.

We have analyzed the expression of endogenous murine genes and of transfected human fetal A gamma globin gene in GM 979, a mouse erythroleukemia line which produces adult as well as embryonic globins. Optimal induction of the endogenous murine adult globin genes was obtained with DMSO or HMBA while the epsilon y and beta h1 embryonic genes were preferentially induced by butyrate. Similarly, the transferred human A gamma-globin gene was preferentially induced by butyrate. These results as well as previous observations in vivo or in erythroid cell cultures suggest that butyrate preferentially induces the expression of fetal globin genes.

Acetamides↗
PubMed1990

Defective CSA-dependent granulopoiesis in patients with chronic drug-induced neutropenia.

Colony stimulating activity (CSA) and granulocyte-macrophage progenitor cells (GM-CFC) were assayed in the bone marrow and peripheral blood of 17 patients with drug-induced chronic neutropenia. Leukocyte-derived and monocyte/macrophage-derived CSA from the neutropenic patients was found to be significantly decreased compared to normal control. However, bone marrow and peripheral blood GM-CFC were within normal limits. These data suggest that in neutropenic patients monocyte/macrophages exhibit most likely a qualitative defect in CSA production, which may account at least in part, for the impaired granulopoiesis observed in drug-induced neutropenia.

Adolescent↗
PubMed1989

The human homolog of the Moloney leukemia virus integration 2 locus (MLV12) maps to band p14 of chromosome 5.

The Moloney leukemia virus integration 2 (MLV12) locus represents a common region for proviral integration and a putative oncogene involved in the induction of thymic lymphomas in rodents. The human homolog of the MLV12 locus has been cloned and studies have been initiated to determine its possible role in the induction and progression of human neoplasms. In this study we used a panel of human X rodent somatic cell hybrids and in situ hybridization to metaphase chromosomes to map MLV12 to the short arm of the human chromosome 5, band p14.

Animals↗
PubMed1988

Activation of developmentally mutated human globin genes by cell fusion.

Human fetal globin genes are not expressed in hybrid cells produced by the fusion of normal human lymphocytes with mouse erythroleukemia cells. In contrast, when lymphocytes from persons with globin gene developmental mutations (hereditary persistence of fetal hemoglobin) are used for these fusions, fetal globin is expressed in the hybrid cells. Thus, mutations of developmental origin can be reconstituted in vitro by fusing mutant lymphoid cells with differentiated cell lines of the proper lineage. This system can readily be used for analyses, such as globin gene methylation, that normally require large numbers of pure nucleated erythroid cells, which are difficult to obtain.

Animals↗
PubMed1988

A prospective randomized trial of HLA-matched versus mismatched single-donor platelet transfusions in cancer patients.

The use of histocompatability antigen (HLA)-matched platelets has been advocated for the support of thrombocytopenic cancer patients. We randomized 78 newly diagnosed cancer patients prospectively (before thrombocytopenia) to receive either HLA-matched or mismatched single-donor platelet transfusions. Three hundred forty-one platelet transfusions were given for 80 separate episodes of therapy-induced thrombocytopenia in 33 patients. Forty-five patients receiving intensive chemotherapy did not develop significant (less than 20,000 platelets/mm3) thrombocytopenia and did not receive a platelet transfusion. No marked difference was observed between the matched and mismatched groups in regard to number of total platelet transfusions per patient (median, 3 vs. 5, respectively; P = 0.076), number of platelet transfusions per episode (median, 3.0 vs. 3.5, respectively; P = 0.28), or days between transfusions (median, 2 vs. 2, respectively, P greater than 0.4). Bleeding episodes, although rare, tended to be of increased severity in the mismatched group. Febrile patients receiving mismatched platelets tended to have a lower posttransfusion increment increase than their nonfebrile counterparts (P = 0.068), although a similar trend could not be demonstrated between febrile and nonfebrile patients who received matched platelets (P = 0.22). Patients treated as outpatients had significantly higher posttransfusion increments than when transfused as inpatients when they were given mismatched platelets (P less than 0.0005). Development of antiplatelet antibody did not appear to affect response to platelet transfusions. Only one patient developed sustained high-level antibody titers. In patients where thrombocytopenia was significant, the transfusion of HLA-matched platelets did not appear to offer a significant advantage. However, HLA-matched platelet transfusions tended to be associated with higher posttransfusion increments in febrile patients and a trend toward fewer severe bleeding episodes. A multi-institution trial containing a large number of patients is needed to evaluate trends observed in this study.

Adolescent↗
PubMed1988

Molecular characterization of a novel form of (A gamma delta beta)zero-thalassemia deletion with a 3' breakpoint close to those of HPFH-3 and HPFH-4: insights for a common regulatory mechanism.

We have molecularly characterized a novel (A gamma delta beta)zero-thalassemia associated with increased synthesis of HbF in three members of a German family. The levels of HbF in the peripheral blood red cells of the heterozygotes ranged between 9.9% and 12.5% with a heterocellular distribution in the red cells, as detected by immunofluorescence. The mutation resulted from a deletion starting about 1.5 to 1.9 kb from the 3' end of the G gamma-gene and ending 27 +/- 0.5 kb 3' to the beta-globin gene. Thus, the total deletion is 52 +/- 0.5 kb. Its 5' breakpoint is similar to that of the previously described (A gamma delta beta)zero-thalassemias, while the location of the 3' breakpoint is placed very close to the 3' breakpoints of HPFH-4 and HPFH-3 deletions. The proximity of the 3' breakpoint of the German (A gamma delta beta)zero-thalassemia to those of HPFH-3 and HPFH-4 deletions raises the possibility that a common mechanism, such as the juxtaposition of an enhancer, might underlie the activation of the gamma-globin genes in these three mutants.

Chromosome Deletion↗
PubMed1988

Myelodysplastic syndrome with acquired hemoglobin H disease. Evolution through megakaryoblastic transformation into myelofibrosis.

A 64-year-old man was found to have a hemolytic anemia with a hematocrit of 0.28 (28%) during a routine evaluation. One month before this his hematocrit had been normal. Further studies revealed a myelodysplastic syndrome and acquired hemoglobin H disease. Eighteen months later this transformed into acute megakaryoblastic leukemia with disappearance of hemoglobin H, and shortly thereafter he had myelofibrosis develop. Acquired hemoglobin H disease, which is an alpha-thalassemia-like syndrome, results in the formation of an unstable hemoglobin composed of beta chain tetramers. This condition has been associated with preleukemia, sickle cell anemia, and hematologic malignancies. Although idiopathic myelofibrosis also has been described as a setting in which this thalassemic syndrome occurs, the present case is unusual in that the myelofibrosis was preceded by refractory anemia with leukemic transformation.

Cell Transformation, Neoplastic↗