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Biomedical subjects

J T Prchal

Publications and source records attributed to J T Prchal.

At least 55 records · Page 3Linked to original sources
PubMed1995

Clonality in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) is a myeloproliferative disease in which morbidity and mortality are primarily caused by nonhematopoietic organ failure from myelomonocytic infiltration or by failure of the normal bone marrow. Morphologic evidence of maturation arrest, karyotypic abnormalities, and progression to blast crisis are infrequent events. Viral infections and other reactive processes can initially mimic the clinical course of JCML, creating diagnostic problems. Because of the rarity of JCML and technical limitations, formal clonality studies have not been reported previously. Nine female JCML patients were identified by clinical criteria, characteristic 'spontaneous' in vitro cell growth, and negative cultures and titers for various viral agents. Peripheral blood and bone marrow samples were obtained at the time of diagnosis for cell separation and RNA and DNA isolation. To assess clonality, X-chromosome inactivation patterns were evaluated using three different, recently developed polymerase chain reaction-based clonality assays. All nine female JCML patients showed evidence for monoclonal origin of mononuclear cells at the time of diagnosis. Cell separation studies further traced the monoclonal origin back to at least the most primitive myeloid progenitor cell. Reversion to a polyclonal state was demonstrated after bone marrow transplant and also in one patient following treatment with 13-cis retinoic acid. This demonstration of clonality in JCML delineates it from the reactive processes and provides a basis for molecular genetic strategies to identify causally associated mutations.

Adult↗
PubMed1995

A TaqI polymorphism in the human erythroid beta spectrin gene.

Human erythroid spectrin consists of an alpha beta heterodimer. Abnormalities of spectrin are a common cause of hereditary haemolytic anaemias such as hereditary elliptocytosis (HE) and hereditary spherocytosis (HS). To identify the spectrin gene mutation one needs initially to establish which of the spectrin subunits is defective. For this purpose, the beta spectrin restriction fragment length polymorphism (RFLP) we describe here will be useful in linkage analysis. The elucidation of an Ala-->Gly beta spectrin gene mutation in a family with HE, highlights the importance of this TaqI polymorphism in establishing linkage.

Alanine↗
PubMed1995

Primary polycythemias.

In this review, primary polycythemic states are discussed in the context of other polycythemic disorders. Primary polycythemias result from an acquired or inborn mutation affecting hematopoietic and erythroid cells. The best-known type of primary polycythemia is polycythemia vera, which is caused by an acquired somatic mutation of a hematopoietic stem cell with exaggerated myeloid proliferation; the molecular events leading to this disease are not understood. In contrast, primary familial and congenital polycythemias result from inborn mutation affecting hematopoietic and erythroid cells. The molecular mechanisms causing primary familial and congenital polycythemias may be different in different families; some have already been defined. Progress in defining the molecular defect of primary polycythemias and the better understanding of altered signal transduction leading to excessive erythrocyte production should provide important insights into the pathogenesis of primary polycythemias, other leukemic disorders, as well as normal hematopoiesis. Such information should contribute to better understanding of and more effective therapeutic interventions in myeloproliferative and leukemic disorders.

Erythropoiesis↗
PubMed1995

A novel clonality assay based on transcriptional polymorphism of X chromosome gene p55.

The clonal nature of cell populations in malignant and myeloproliferative disorders can be determined in female subjects by random-inactivation assays of the X chromosome. Assays utilizing either expression of the G6PD isozymes or DNA-methylation differences between the active and inactive X chromosomes have significant short-comings. We developed a test based on nucleotide #1311 exonic polymorphism of G6PD that allows detection of clonality by determining the transcriptional polymorphism of the active X chromosome using a reverse transcription-polymerase chain reaction-ligase detection reaction (rtPCR-LDR). Since only 18% of females in the United States are informative (heterozygous) for this chromosome with this assay, we searched for other exonic X chromosome polymorphisms. Concentrating on a discrepancy (G or T at cDNA #358) of published sequences of ubiquitously expressed gene, palmitoylated membrane protein for p55, we confirmed this conservative polymorphism at the cDNA level. To detect the genotype of this polymorphism, we established the intron/exon boundary of the first 5' exons and determined the whole sequence of the second intron. We found that the polymorphic site is at the third exon, nine nucleotides downstream from the 294-bp second intron. This close proximity to the intron necessitated the development of a separate PCR-LDR reaction with oligonucleotides for cDNA and genomic DNA. Furthermore, we determined that the p55 gene is subject to X chromosome inactivation. Based on these observations, we developed a novel p55 clonality assay that is reproducible, quantitative, and very sensitive. Screening of 37 randomly selected healthy females of Caucasian, African-American, and Asian origin revealed that 38% of females are informative when this assay is used. We demonstrate a multiple crossover between the G6PD and the p55 polymorphisms (separated by approximately 200 kb), suggesting that these two polymorphisms are in linkage disequilibrium; thus, approximately 50% of female subjects are informative for clonality studies using the two assays.

Base Sequence↗
PubMed1994

Accelerated healing of chronic sickle-cell leg ulcers treated with RGD peptide matrix. RGD Study Group.

Leg ulcers are a chronic manifestation of sickle-cell disease (SCD) and are often painful, disabling, and difficult to treat. RGD peptide matrix treatment is a novel therapy designed to provide a topical synthetic extracellular matrix that can act as a temporary substitute for the damaged natural matrix at the ulcer site. In this randomized, placebo-controlled, double-blind, prospective, multicenter investigation, SCD patients with full-thickness leg ulcers were treated with standard therapy plus RGD peptide matrix or saline placebo once weekly for up to 10 weeks. Healing in patients with chronic ulcers (2 months or greater in duration) was significantly accelerated (P = .0085) in RGD peptide matrix recipients compared with the placebo group. In these chronic ulcer cases, the average percent ulcer closure (decrease in ulcer surface area) in the RGD peptide matrix group (54.4% +/- 8.9%) exceeded that in the placebo group (19.0% +/- 24.3%) nearly threefold by study endpoint. Furthermore, RGD peptide matrix was equally effective in promoting healing of long persistent ulcers and ulcers of shorter duration. In contrast, standard therapy plus placebo was significantly less effective (P = .001) in promoting healing for ulcers of progressively greater duration. The results of this study provide preliminary evidence that RGD peptide matrix treatment may significantly accelerate healing of chronic sickle-cell leg ulcers.

Adolescent↗
PubMed1994

Anti-erythropoietin (EPO) receptor monoclonal antibodies distinguish EPO-dependent and EPO-independent erythroid progenitors in polycythemia vera.

Erythroid progenitor cells isolated from patients with polycythemia vera (PV) proliferate and differentiate in methylcellulose in the absence of exogenous erythropoietin (EPO). To investigate the potential role of the erythropoietin receptor (EPO-R) in the pathogenesis of PV, we cultured bone marrow-derived or peripheral blood-derived erythroid progenitors in the presence of neutralizing monoclonal antibodies (MoAbs) specific for EPO or EPO-R. Mononuclear cells were obtained from 9 healthy adults and 9 PV patients by Ficoll-Hypaque gradients and cultured with or without EPO in methylcellulose for 12 days under standard or serum-free conditions. Neutralizing anti-EPO and anti-EPO-R MoAbs, added to cultures at day 0, caused dose-dependent growth inhibition of all normal burst-forming units-erythroid (BFU-E) derived from health adult controls. The MoAbs had no effect on the growth of nonerythroid progenitor cells under the same culture conditions. In contrast, neutralizing antibodies distinguished two classes of BFU-E derived from PV patients. Class I BFU-E from PV patients were EPO-dependent. These progenitors, like those derived from healthy adults, had normal EPO dose-dependent growth characteristics and showed a normal period of EPO requirement in vitro that extended 6 days after the initiation of culture. These results indicate that EPO exerts its critical effect early during erythroid differentiation; the addition of neutralizing antibodies to normal progenitors after 6 days had no effect on the subsequent size or maturation of the colonies. Class II BFU-E from PV patients were EPO-independent. They proliferated and differentiated even in the presence of high concentrations of neutralizing anti-EPO or anti-EPO-R MoAbs. We conclude that the class II BFU-E from PV patients are independent of free EPO.

Antibodies, Monoclonal↗
PubMed1994

Evaluation of the methemoglobinemia associated with sulofenur.

A new class of antineoplastic agents, the diarylsulfonylureas entered clinical trials with the testing of Sulofenur (LY186641). Phase I trials and preclinical studies showed the dose limiting toxicity to be methemoglobinemia. We studied the incidence of methemoglobinemia, sulfhemoglobinemia and cytochrome b5 reductase deficiency in nine consecutive patients enrolled in a phase II trials using Sulofenur. The specific Malloy method as well as clinically standard co-oximeter measurements were used to determine methemoglobin levels and marked discrepancies were noted. One patient with symptomatic methemoglobinemia had enzyme levels and family history consistent with a heterozygous state for a cytochrome b5 reductase deficiency. We conclude that the clinical incidence of methemoglobinemia will be overestimated by co-oximeter measurements but that Sulofenur does produce clinically significant methemoglobinemia in cytochrome b5 reductase deficient patients.

Antineoplastic Agents↗
PubMed1994

Mutation in the negative regulatory element of the erythropoietin receptor gene in a case of sporadic primary polycythemia.

A 42-year-old Caucasian male with sporadic primary polycythemia has been followed by us for 13 years. During the time of observation, his hemoglobin had been stable, and he has never had an elevated white count or platelet count or any other stigmata of polycythemia vera (PV). Both of his parents, his three children, and all siblings have been hematologically normal. The in vitro culture of erythroid progenitors revealed an absence of autonomous erythropoietin (Epo)-independent erythroid colonies but demonstrated a marked increase in the sensitivity of erythroid progenitors to Epo. We have undertaken a study designed to determine whether a mutation in the Epo receptor (Epo-R) gene could cause the polycythemia phenotype seen in either dominant or recessive primary polycythemia described by us and others, or in polycythemia vera. We have sequenced the cytoplasmic positive and negative regulatory domains of the Epo-R genomic DNA, and a transversion of C to T in nucleotide 6148 was found in one of the patient's chromosomes. This mutation is located in the negative regulatory domain and results in a change from proline to serine (P488S). We have subsequently analyzed more than 40 chromosomes from unrelated normal subjects, as well as autosomal dominant, recessive, and sporadic primary polycythemia and polycythemia vera subjects. In no instance was the same or any other mutation in the Epo-R found. To determine if this Epo-R mutation is a cause of increased sensitivity of erythroid progenitors to erythropoietin, Ba/F3 cells (interleukin-3-dependent murine lymphoid line) were transfected with normal and mutated Epo-R cDNA, rendering the transfected cells viable and able to proliferate in Epo. Transfectants with wild-type and mutant Epo-R cDNA exhibited no difference in the presence of Epo. More recently, we were able to obtain DNA from the seven family members of the propositus and found that the nonpolycythemic mother and one of the siblings have the same Epo-R mutation. We conclude that this first described mutation of Epo-R encountered in humans does not appear on its own to explain the polycythemia phenotype; however, the possibility that it may interact with some other acquired or congenital abnormality in generating the polycythemia phenotype cannot be excluded.

Adult↗
PubMed1994

Clonal hematopoiesis and acquired thalassemia in common variable immunodeficiency.

BACKGROUND: Common variable immunodeficiency (CVID) is defined by hypogammaglobulinemia and increased susceptibility to infections. The gene defect responsible for CVID remains unknown. METHODS: During the course of their CVID disease, a female and three male patients developed microcytic anemia. The investigation of this anemia forms the basis for this report. RESULTS: Reticulocyte globin chain synthesis studies revealed the abnormal alpha/beta ratios that are pathognomonic of thalassemia. Through transcriptional analysis of the glucose-6-phosphate-dehydrogenase (G6PD) locus of the active X-chromosome in blood cells, we determined that the female patient has clonal reticulocytes, platelets, granulocytes, and B and T lymphocytes. CONCLUSIONS: The simultaneous presence of globin synthesis abnormalities and panhypogammaglobulinemia suggests that a common insult at the stem cell level could contribute to the development of CVID and acquired thalassemia.

Adolescent↗
PubMed1993

Biogenesis of erythrocyte membrane skeleton in health and disease.

To study the biogenesis of red cell membrane skeleton at various stages of erythroid differentiation, we have chosen the following model systems: a) Rauscher erythroleukemia cell line representing the early stages of differentiation, b) Friend erythroleukemia cells, and c) in vitro cultured human erythroblasts. The latter two systems represent terminally differentiated erythroblasts. Using these model systems, we have shown asynchronous synthesis of membrane proteins during erythroid differentiation. At the early stages of erythroid development, the synthesis of spectrin, ankyrin and band 4.1 proteins is initiated before that of the band 3 protein. Following erythroid induction with erythropoietin and dimethylsulfoxide (DMSO), there is a dramatic increase in the synthesis of the band 3 protein without noticeable changes in the synthesis of other membrane proteins. This increase in band 3 synthesis is accompanied by increased stability and recruitment of the skeletal proteins into the membrane skeleton, leading to increased steady state levels. The progressive increase in band 3 synthesis continues during terminal maturation of erythroblasts. This is accompanied by increased stability and assembly of spectrin and ankyrin on the membrane, despite their reduced synthesis. These results point to a key role for the band 3 protein in anchoring and stabilizing these proteins into the permanent skeletal network. Finally, to detect defects of skeletal biosynthesis, we have extended these studies to a patient with severe hereditary spherocytosis characterized by a combined deficiency of spectrin and ankyrin. We have shown that this combined deficiency is a consequence of reduced ankyrin synthesis and mRNA content representing a thalassemia-like membrane protein mutation.

Animals↗
PubMed1993

A novel clonality assay based on transcriptional analysis of the active X chromosome.

The clonal origin of malignancy and hematopoiesis is a principal tenet of modern biology and medicine. This paper describes a highly specific and sensitive assay for the detection of clonality in cells and cell lineages suitable for studies in a large proportion of females. The specific ligase chain and/or ligase detection reactions (LCR/LDR) are utilized at a polymorphic glucose-6-phosphate dehydrogenase (G-6-PD) locus for discrimination of the mRNA transcripts of the active X chromosome. This combination approach circumvents problems encountered with other currently used assays of clonality based either on peptide G-6-PD polymorphism or on DNA methylation differences between the active and inactive X chromosomes. The veracity of this assay was verified by analysis of 19 random healthy females as well as by the study of hemopoietic and nonhemopoietic tissues from a patient with clonal hemopoiesis/polycythemia vera. Furthermore, we demonstrate that the G-6-PD locus used in our clonal assay does not display marked differences in methylation between the active and the inactive X chromosomes.

Alleles↗
PubMed1993

Case report: alpha G-Philadelphia, beta O-Arab, and beta C globins present in a single patient.

The case of a 7-month-old Nigerian child who presented with anemia and microcytosis is described. Hemoglobin electrophoresis studies revealed a band with pronounced cathodic mobility. This represented a heterohybrid hemoglobin tetramer composed of an alpha-globin mutant, G-Philadelphia (alpha GPhil), and two variant beta-globin chains, beta C and beta O-Arab. The absolute amounts of alpha GPhil found in the propositus were less than expected for an alpha 2-globin gene product. It has not been established whether alpha G-Philadelphia interacting with beta O-Arab and beta C globin chains is the cause of the microcytosis.

Anemia↗