Clonal stability of blood cell lineages indicated by X-chromosomal transcriptional polymorphism.

The idea that stem cells oscillate between a state of activity and dormancy, thereby giving rise to differentiating progeny either randomly or in orderly clonal succession, has important implications for understanding normal hematopoiesis and blood cell dyscrasias. The degree of clonal stability in individuals also has practical implications for the evaluation of clonal lymphomyeloproliferative diseases. To evaluate the clonality pattern of the different types of blood cells as a function of time we have validated the applicability, sensitivity, and reproducibility of a thermostable ligase reaction to detect transcripts of the G6PD allele on the active X-chromosome in normal heterozygous females. While the ratio of the two X-chromosome-derived allelic transcripts varied widely among hemopoietic tissues in a given individual, this allelic ratio was virtually identical in all types of mature myeloid and lymphoid cells. Longitudinal studies indicated constancy of the G6PD allelic ratio in blood cells over a 912-d period of observation in healthy females. The individual variability observed in this allelic ratio suggests that the progeny of a relatively small number of original embryonic hemopoietic stem cells, approximately eight, contribute to the sustained production of all types of blood cells in healthy individuals.

Rapid determination of clonality by detection of two closely-linked X chromosome exonic polymorphisms using allele-specific PCR.

We reported two specific, reproducible, and quantitative clonality assays based on detection of exonic polymorphisms of the X chromosome genes p55 and G6PD using rtPCR-LDR. These assays are inconvenient for screening purposes. This study sought to develop a simple, reproducible assay, practical for screening genomic DNA samples for p55/G6PD genotypes, rapid clonality determination, and to determine the linkage relationship between these closely related loci. The salient feature of ASPCR is the performance of two PCR rounds. The first generates template; the second, using one aliquot of first-round products in two reaction tubes, each containing one allele-specific primer, detects each allele. ASPCR and rtPCR-LDR produced identical p55/G6PD results in 91 normal female genomic DNAs, and in 12 clonal hematopoietic disorder cDNAs, confirming assay validity. 209 female and 207 male genomic DNA samples were analyzed for p55/G6PD genotype by ASPCR; 60% of females were heterozygous at one or both loci. G6PD and p55 allelic frequencies were significantly different among African-American men and women, but were not significantly different among Caucasian men and women. These loci were in linkage equilibrium among African Americans, but not among Caucasians. ASPCR is a rapid, sensitive, and specific method for screening large numbers of genomic DNAs, and for rapid clonality determination.

Angiotensin II stimulates proliferation of normal early erythroid progenitors.

Angiotensin II exerts a mitogenic effect in several in vitro models, but a direct effect on erythroid progenitors has not been documented. Angiotensin-converting enzyme inhibitors and losartan, an angiotensin II type 1 receptor (AT1) antagonist, ameliorate posttransplant erythrocytosis, without altering serum erythropoietin levels. We studied erythroid differentiation and the effect of angiotensin II on proliferation of erythroid progenitors by culturing CD34+ hematopoietic progenitor cells in liquid serum-free medium favoring growth of erythroid precursors. Aliquots of cells were collected every third day, and were used for RNA preparation. AT1 mRNA was detected after 6 d. In these same samples, erythroid-specific mRNA (erythropoietin receptor) was also detected. AT1 protein was detected in 7-d-old burst-forming units-erythroid colonies by Western blotting. The CD34+ cell liquid cultures were used to incubate erythroid precursors with angiotensin II from days 6-9. After incubation, cells were transferred to semisolid medium and cultured with erythropoietin. Angiotensin II increased proliferation of early erythroid progenitors, defined as increased numbers of burst-forming units-erythroid colonies. Losartan completely abolished this stimulatory effect of angiotensin II. Moreover, we observed increased numbers of erythroid progenitors in the peripheral blood of posttransplant erythrocytosis patients. Thus, activation of AT1 with angiotensin II enhances erythropoietin-stimulated erythroid proliferation in vitro. A putative defect in the angiotensin II/AT1 pathway may contribute to the pathogenesis of posttransplant erythrocytosis.

Polycythemia vera. The in vitro response of normal and abnormal stem cell lines to erythropoietin.

Bone marrow cells from two glucose-6-phosphate dehydrogenase (G-6-PD) heterozygotes with polycythemia vera were cultured to determine whether progenitors which wre not of the polycythemia vera clone were present, and, if present, which cell lines contributed to the increase in erythroid colonies observed in response to added erythropoietin (ESF). To accomplish this, the G-6-PD isoenzyme activity of individual erythroid colonies was determined. All of the erythroid colonies analyzed in cultures without added ESF, contained the G-6-PD isoenzyme type characteristic of the abnormal clone. With higher ESF concentrations in the culture, however, there was an increase in the colonies that were not of the polycythemia vera clone. Analysis of the ratio of the various types of colonies indicated that normal and polycythemia vera cells are capable of responding to ESF in vitro. In selected patients, this technique permits analysis of the ratios of normal to abnormal cells during the course of the disease, in response to therapy and during late complications, such as myelofibrosis or leukemic transformation.

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.

Guinea pig serum erythropoietin (EPO) selectively stimulates guinea pig erythroid progenitors: human or mouse erythroid progenitors do not form erythroid burst-forming unit colonies in response to guinea pig serum EPO.

Erythropoietin (EPO) is the primary regulator of mammalian erythropoiesis, providing a proliferative and differentiative signal to the early EPO-responsive erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid, as well as to later EPO-responsive erythroid progenitors. EPO is secreted by the kidney in response to hypoxia and anemia. There is an extensive biological crossreactivity between human EPO and the EPOs of other mammals. Necas et al. have reported that this crossreactivity may not include the guinea pig (Cavia porcelllus). Because the specificity of the guinea pig's erythropoietic responses may be of biological significance, we compared guinea pig hypoxic serum with mouse (m) and human (h) recombinant (r) EPOs for their ability to induce erythroid progenitor proliferation and differentiation in semisolid cultures. Guinea pig bone marrow mononuclear cells (BMMCs) formed BFU-E colonies in response to guinea pig hypoxic serum, rhEPO, or rmEPO in a dose-dependent fashion. Neither human nor mouse BMMCs responded to guinea pig hypoxic serum; however, guinea pig hypoxic serum exerted no inhibitory effect on human or mouse in vitro erythroid differentiation in the presence of rhEPO or rmEPO. The intensity of the EPO band on Western blotting analysis of guinea pig hypoxic serum was significantly greater than in nonhypoxic serum. This suggests that guinea pig erythropoiesis is mediated by EPO and stimulated by hypoxia in a fashion similar to that observed in human and mouse erythropoiesis. Furthermore, guinea pig EPO did not stimulate human or mouse erythroid differentiation in vitro, whereas guinea pig erythroid progenitors could be stimulated by human or mouse EPO, suggesting structural differences in guinea pig EPO and EPO receptor (EPOR) compared with human or mouse EPO and EPOR. These differences probably evolved after the guinea pig's ancestors diverged from myomorph rodents. Further characterization of the guinea pig EPO and EPOR should facilitate our understanding of the interaction between EPO and EPOR.

Measurements of red blood cell methotrexate concentrations and lymphocyte subsets during therapy of rheumatoid arthritis.

Nine patients with rheumatoid arthritis were treated with low dose oral weekly methotrexate for 6 months. Successful therapy was not associated with changes in concentrations of total circulating lymphocytes nor with alterations of T lymphocytes in the helper-inducer, OKT4, or cytotoxic-suppressor, OKT8, subpopulations. Concentrations of methotrexate in circulating erythrocytes stabilized by 1 month of therapy and this measurement did not correlate with clinical efficacy or methotrexate toxicity in the long-term patient assessments.

B-lymphocyte colony formation in chronic lymphocytic leukemia.

A semisolid culture system for B-cell colony formation is described. The system includes pretreatment of B-cells by neuraminidase-galactose oxidase and help of mitomycin-treated T-cells. With this assay system, colony-forming B-cell precursors were detected in all eight patients we studied with B-cell chronic lymphocytic leukemia. These patients' own T-cell helper effect was less than that of normal T-cells.

Low serum vitamin B12 in Alzheimer-type dementia.

Serum vitamin B12 levels (as determined by radio-immunoassay) were measured in 20 subjects aged 65 years and over with Alzheimer-type dementia, 20 age-matched subjects with non-Alzheimer type dementia and 20 age-matched subjects with no dementia. Serum vitamin B12 levels were significantly lower and serum vitamin B12 deficiency was significantly more frequent in subjects with Alzheimer-type dementia and were independent of age, sex, haematological abnormality or serum folate.

Molecular basis for polycythemia.

This overview concentrates on familial and congenital polycythemias in the context of other polycythemic disorders, with emphasis on those with established molecular lesions. Recent advances in the regulation of erythropoiesis, as they may relate to polycythemic states, are discussed as a background for those well-defined polycythemic states wherein the molecular defect has not yet been elucidated. Primary familial congenital polycythemias and congenital and familial secondary polycythemias, including hemoglobin mutants, methemoglobinemias and congenital 2,3-bisphosphoglycerate deficiency, are discussed. The most common primary polycythemia, polycythemia vera, as well as the only likely endemic congenital secondary polycythemia, known as Chuvash polycythemia, are discussed.

Abnormalities of T-lymphocyte subsets in epileptic patients.

This study concerns the distribution of T-cell subsets as determined by specific monoclonal antibodies in 50 individuals with complex partial seizures (31) and primary generalized tonic-clonic seizures (19), and in 30 healthy controls. The epileptic group had significantly fewer circulating T4 "helper" lymphocytes and significantly greater number of T8 "suppressor" lymphocytes than the controls. The T4/T8 ratio was consistently significantly lower in the epileptic group. There was no relation between lymphocyte subsets or T4/T8 ratio and antiepileptic medication. The results suggest a derangement of cell-mediated immunity in individuals with epilepsy.

Persistent generalized lymphadenopathy in homosexual men: clinical, pathological and immunologic characteristics.

Eighteen homosexual men who had had lymphadenopathy in two or more extrainguinal sites for more than 5 months but had no evidence of other illnesses or infections were studied. All had extreme malaise, and 90% had additional symptoms (fever, night sweats, weight loss or gastrointestinal dysfunction). They were compared with 10 healthy homosexual and 10 healthy heterosexual male controls. The mean numbers of circulating T8 (suppressor) lymphocytes were increased equally in the two homosexual groups, but the mean number of T4 (helper) lymphocytes was decreased only in the group with lymphadenopathy. The response to testing for recall anergy was diminished in both homosexual groups but was significantly lower in the group with lymphadenopathy. The serum immunoglobulin and complement concentrations and the numbers of circulating B lymphocytes were normal in each group. Seven of nine lymph node biopsy specimens showed characteristic hyperplasia and confluence of follicles. Thus, idiopathic persistent, generalized lymphadenopathy in homosexual men without opportunistic infections or malignant diseases appears to be a distinct syndrome; it may also be related to the acquired immune deficiency syndrome.

Bone marrow cultures in dysmyelopoietic syndrome: diagnostic and prognostic evaluation.

Thirty-four patients with a probable clinical diagnosis of dysmyelopoietic syndrome (DMPS) were studied to assess the in vitro growth pattern of their hemopoietic progenitors, i.e. burst-forming unit erythroid (BFU-E) and colony-forming unit myeloid (CFU-C) progenitor cells. Twenty-one patients had DMPS confirmed by final diagnosis and were classified according to the French-American-British (FAB) recommendations. Our results indicate that the normal colony growth of hemopoietic progenitors in vitro excludes DMPS and other preleukemic conditions. In addition, within the DMPS group a low number of CFU-C (11 colonies or fewer) was a highly significant indicator for the development of acute leukemia. Analysis of the limited number of cytogenetic results in the DMPS patients did not reach statistical significance in relation to the development of acute leukemia.

Human erythroid colony formation in vitro: evidence for clonal origin.

Human marrow cells, suspended in methylcellulose medium containing erythropoietin, give rise to discrete colonies of hemoglobin synthesizing cells. The presumption that such colonies originate from single progenitor cells has been tested directly in females with X-chromosome inactivation mosaicism using glucose-6-phosphate dehydrogenase (G-6-PD) as a marker. When individual colonies were grown from marrow cells obtained from two black females heterozygous for G-6-PD, only one or the other isoenzyme type was observed, but not both. These results are most consistent with the interpretation that human erythroid colonies arise from single cells.

Autosomal dominant polycythemia.

Two families with polycythemia inherited as an autosomal dominant trait are described. Serial hemoglobin determinations in multiple family members and RBC volume measurements in selected affected subjects documented their polycythemia. Measurements of arterial p02s, p50s, and blood oxygen affinity were normal in all affected individuals from each family who were tested. Erythropoietin (EPO) levels were low in affected individuals from family 1 and normal in affected members of family 2. Stimulation of in vitro CFU-E colony growth by low levels of EPO was significantly increased in subjects from family 1, but normal in those affected from family 2. We conclude that although the inheritance pattern for the polycythemia in both of these families appeared to be the same, the biologic defect leading to the disorder in each of these unique families was different. The precise mechanism of the increased EPO sensitivity noted in affected subjects from family 1 awaits elucidation.

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.

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.

Human hematopoietic progenitors express erythropoietin.

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Polycythemia vera: stem-cell and probable clonal origin of the disease.

Two women with polycythemia vera and heterozygosity (GdB/GdA) at the X-chromosome-linked locus for glucose-6-phosphate dehydrogenase were studied to determine the nature of the cellular origin of their polycythemia. In contrast to unaffected tissue, such as skin fibroblasts, which consisted of both B and A types, the glucose-6-phosphate dehydrogenase of the patients' erythrocytes, granulocytes and platelets was only of Type A. These results provide direct evidence for the stem-cell nature of polycythemia vera and strongly imply a clonal origin for this disease. The fact that no descendants of the presumed normal stem cells were found in circulation suggests that bone-marrow proliferation in this disorder is influenced by local (intramarrow) regulatory factors.

Congenital polycythemia in Chuvashia.

Familial and congenital polycythemia, not due to high oxygen affinity hemoglobin or reduced 2,3-diphosphoglycerate in erythrocytes, is common in the Chuvash population of the Russian Federation. Hundreds of individuals appear to be affected in an autosomal recessive pattern. We studied six polycythemic Chuvash patients <20 years of age from unrelated families and 12 first-degree family members. Hemoglobins were markedly elevated in the index subjects (mean +/- standard deviation [SD] of 22.6 +/- 1.4 g/dL), while platelet and white blood cell counts were normal. Although performed in only three of the index subjects, serum erythropoietin concentrations determined by both radioimmune and functional assays were significantly higher in polycythemic patients compared with first-degree family members with normal hemoglobin concentrations. Southern blot analysis of the Bgl 2 erythropoietin gene polymorphism showed that one polycythemic subject was a heterozygote, suggesting the absence of linkage of polycythemia with the erythropoietin gene, assuming autosomal recessive inheritance. Polymerase chain reaction (PCR) amplification of the GGAA and GA minisatellite polymorphic regions of the erythropoietin receptor gene showed no evidence of linkage of phenotype with this gene. We conclude that Chuvash polycythemia may represent a secondary form of familial and congenital polycythemia of as yet unknown etiology. This condition is the only endemic form of familial and congenital polycythemia described.