Ineffective erythropoiesis in beta-thalassemia major is due to apoptosis at the polychromatophilic normoblast stage.

Beta-thalassemia major is characterized by ineffective erythropoiesis, although it is difficult to define the dynamics of this process from the static information revealed by analysis of bone marrow (BM) aspirates. We aimed to study the kinetics of sequential erythroid differentiation in beta-thalassemia major. We isolated the progenitor cells (CD34(+) and CD34(+)CD38(-) cells) from BM of thalassemia major patients and studied in vitro erythropoiesis. This is the first report of an in vitro study in human beta-thalassemia major from purified BM CD34(+) progenitor cells, using erythroid culture conditions, which allow unilineage differentiation to mature enucleated red blood cells. In contrast to normal donors, a high proportion of BM CD34(+) and CD34(+)CD38(-) progenitors from beta-thalassemia major coexpressed the late erythroid lineage-specific protein glycophorin A and generated a higher proportion of erythroid colonies. However, despite the marked increase in erythroid clonogenicity of the progenitor population, erythroid cultures initiated from beta-thalassemia major BM CD34(+) cells expanded 10- to 20-fold less than from normal BM. There were less viable cells during differentiation, specifically after the polychromatophilic normoblast stage. There was a progressive increase in the apoptotic erythroid progeny with differentiation, and apoptosis occurred predominantly at the polychromatophilic normoblast stage. In thalassemia major, BM progenitor cells show increased erythroid clonogenicity, increased expression of late erythroid lineage-specific proteins, and accelerated erythroid differentiation. However, despite the apparent increased erythroid commitment, ineffective erythropoiesis occurs due to apoptosis at the polychromatophil stage. Identification of the differentiation stage at which apoptosis occurs will permit further studies of the underlying mechanisms and target therapeutic strategies to improve red cell production.

Cerebral vasculopathy in sickle cell anemia: diagnostic contribution of positron emission tomography.

Children with sickle cell anemia (SS) have an increased risk for cerebral vasculopathy with stroke (CVA) and cognitive impairment. The present study examines the extent to which adding positron emission tomography (PET) to magnetic resonance imaging (MRI) can improve the detection of cerebral vasculopathy. Whereas MRI has been the prime modality for showing anatomical lesions, PET excels at assessing the functional metabolic state through glucose utilization 2-deoxy-2 [18F] fluoro-D-glucose (FDG) and microvascular blood flow ([15O]H2O). Forty-nine SS children were studied. Among them, 19 had clinically overt CVA, 20 had life-threatening hypoxic episodes or soft neurologic signs, and 10 were normal based on neurological history and examination. For the entire sample of 49 subjects, 30 (61%) had abnormal MRI findings, 36 (73%) had abnormal PET findings, and 44 (90%) showed abnormalities on either the MRI or the PET or both. Of the 19 subjects with overt CVA, 17 had abnormal MRI (89%), 17 had abnormal PET (89%), and 19 (100%) had either abnormal MRI or PET or both. Among the 20 subjects with soft neurologic signs, 10 (50%) had abnormal MRI, 13 (65%) had abnormal PET, and 17 (85%) had abnormal MRI and/or PET. Six (60%) of the 10 neurologically normal subjects had abnormal PET. Among the 30 subjects with no overt CVA, 25 (83%) demonstrated imaging abnormalities based on either MRI or PET or both, thus, silent ischemia. Lower than average full-scale intelligence quotient (FSIQ) was associated with either overt CVA or silent ischemic lesions. Four subjects who received chronic red blood cell transfusion showed improved metabolic and perfusion status on repeat PET scans. In conclusion, (1) the addition of PET to MRI identified a much greater proportion of SS children with neuroimaging abnormalities, particularly in those who had no history of overt neurologic events. (2) PET lesions are more extensive, often bihemispheric, as compared with MRI abnormalities. (3) PET may be useful in management as a tool to evaluate metabolic improvement after therapeutic interventions, and (4) the correlation of PET abnormalities to subsequent stroke or progressive neurologic dysfunction requires further study.

An in vitro model of human red blood cell production from hematopoietic progenitor cells.

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.

Evidence of a dominant transcriptional pathway which regulates an undifferentiated and complete metastatic phenotype.

The highly metastatic amelanotic C8161 human melanoma line was found to exhibit complete dominance of its undifferentiated and metastatic phenotype in multiple somatic cell hybridization studies designed to bypass the presence of potential tumor suppressor genes. In a three armed approach involving somatic cell fusions of C8161 with recipient lines of greater differentiation, different lineage, and different tumorigenicity status, the metastatic and undifferentiated phenotype of C8161 was promiscuously dominant. In somatic cell hybrids produced between the C8161 and a group of non-metastatic human melanoma lines which exhibited melanocyte differentiation markers including S100, HMB-45, NKI/C3, and melanin, the fusions were uniformly metastatic and undifferentiated. In somatic cell hybrids of C8161 and MCF-7 the fusions exhibited an estrogen independent and unresponsive, estrogen receptor (ER) negative, and highly metastatic phenotype. In fusions between C8161 and HMS-1, an immortalized 'benign' human myoepithelial line which produced an abundant extracellular matrix (ECM) and high levels of protease and angiogenic inhibitors including maspin, tissue inhibitor of metalloproteinase-1 (TIMP-1), alpha1-antitrypsin (alpha1-AT), protease nexin II (PN-II), thrombospondin-1 and soluble basic fibroblast growth factor (bFGF) receptors, the hybrids showed complete absence of matrix, absent maspin expression, markedly decreased protease inhibitor and angiogenic inhibitor production, high levels of proteases and angiogenic factors, and a highly metastatic phenotype. In our somatic cell fusions, the human-human hybrids represented true and complete fusions and not hybrid clones selected for by loss of dominant-acting growth suppressor genes. This finding was supported by detailed comparative genomic hybridization (CGH) studies, Q-banding karyotype analysis, and autofusions of representative clones. The purposeful creation of inherently unstable human-murine fusions between C8161 and B16-F1 where loss of putative suppressor loci would be expected, resulted in fusions exhibiting decreased growth and non-metastatic behavior with progressive chromosomal loss. Neither p53, nm23, DNA methyltransferase, activated ras, fibroblast growth factor-4 (FGF-4), or epidermal growth factor receptor (EGFR) mediated the acquisition of the metastatic or undifferentiated phenotype within the C8161-human fusions. These studies are the first studies ever to successfully transfer the complete metastatic phenotype by somatic cell fusion and support the presence of a new high level regulatory pathway(s) involving dominant trans-acting factors which act pleiotropically to regulate an undifferentiated and highly metastatic phenotype.

Nonspecific interstitial pneumonitis mimicking Pneumocystis carinii pneumonia.

Bronchoalveolar lavage (BAL) and transbronchial biopsies from 351 human immunodeficiency virus (HIV)-positive patients with presumed Pneumocystis pneumonia were analyzed to determine the spectrum and frequency of interstitial lung disease mimicking Pneumocystis pneumonia. Among 67 patients without Pneumocystis, nonspecific interstitial pneumonitis (NSIP) was the most common histologic diagnosis (n = 16). Tissue sections from patients with NSIP were tested by in situ hybridization for Epstein-Barr virus, cytomegalovirus (CMV), and HIV; sections were also tested with the polymerase chain reaction (PCR) for HIV env and gag protein DNA. In patients with NSIP, Epstein-Barr virus and CMV could not be detected by in situ hybridization; HIV nucleic acid was amplifiable with PCR in 10 of 15 formalin-fixed, paraffin-embedded tissue sections. Symptoms, physical findings, and blood gas values were similar in patients with NSIP and matched controls with Pneumocystis. Patients with NSIP presented earlier in the course of HIV, with higher weight, serum albumin levels, and CD4+ T-lymphocyte counts (492 +/- 828 cells/mm3 versus 57 +/- 60 cells/mm3), and more normal lactate dehydrogenase (LDH) levels (280 +/- 113 IU/L versus 432 +/- 141 IU/L; means +/- SD). Seven to 10 d later, improvement in blood gas values was of similar magnitude for the two groups. Only one other unequivocal, treatable infection was diagnosed only with transbronchial biopsy. These results indicate that NSIP may be the most common diagnosis mimicking Pneumocystis pneumonia in acquired immune deficiency syndrome (AIDS), and that NSIP may improve during empiric therapy.

Specific antibodies to Trypanosoma cruzi among blood donors in Los Angeles, California.

BACKGROUND: Trypanosoma cruzi, the cause of Chagas' disease, is often transmitted by transfusion in Latin America. Previous studies showed that at least 1 in 1000 eligible blood donors at the Los Angeles County+University of Southern California (LAC+USC) Medical Center Blood Bank had specific antibodies to T. cruzi. In June 1993, serologic screening of prospective allogeneic donors at epidemiologic risk for T. cruzi infection was begun voluntarily. STUDY DESIGN AND METHODS: The risk of T. cruzi infection in all eligible donors was assessed by questionnaire. At-risk donors were screened serologically for antibodies to T. cruzi with an enzyme immunoassay, and confirmatory testing was done with a radioimmunoprecipitation assay. RESULTS: During the 29-month study period 1311 (39.5%) of 3320 donors were judged to be at risk for T. cruzi infection. Seven donors (1/475) were reactive by an enzyme immunoassay, and six of these seven (1/ 553) were positive in a radioimmunoprecipitation assay. All radioimmunoprecipitation assay-positive donors had been born in countries in which Chagas' disease is endemic. One person in this group had received a transfusion in his homeland. CONCLUSION: These results demonstrate that a substantive proportion of eligible blood donors at our institution have antibodies specific for T. cruzi and that a commercially available assay can be used to detect these antibodies. Our data suggest that the risk of transmission of T. cruzi by transfusion could be eliminated by serologic testing limited to persons born in or transfused in countries in which Chagas' disease is endemic.

Beta-globin haplotypes from blood spots for follow-up of newborn hemoglobinopathy screening.

The inheritance of sickle-cell anemia upon the background of the major beta-globin gene cluster haplotypes has been associated with differing risks for major organ failure, and more recently with response to hydroxyurea treatment. Early identification of beta-globin haplotypes in individuals with sickle-cell anemia may be a clinically useful prognostic factor for severity of disease expression. This report describes the use of whole-blood spots on filter papers from newborn hemoglobinopathy screening for beta-globin gene cluster haplotyping by the polymerase chain reaction.

Beta-S gene cluster haplotypes modulate hematologic and hemorheologic expression in sickle cell anemia. Use in predicting clinical severity.

PURPOSE: The rate of progression of major organ failure in sickle cell anemia is genetically controlled. It is the direct consequence of the sickle cell-evoked vasculopathy. PATIENTS AND METHODS: Presence of the beta S gene cluster haplotypes and alpha gene deletions as genetic markers indicate the expected frequency of illness and the risk of end-stage major organ failure. The risk of irreversible soft tissue organ failure is greatest in patients with a Central African Republic (CAR) chromosome, whereas morbidity is consistently lowest in patients with a Senegalese chromosome. Presence of alpha-thalassemia-2 decreases the risk of soft tissue organ failure in all haplotype combinations. RESULTS: Other laboratory abnormalities, when combined with haplotype and alpha gene status, also predict the risk of clinical morbidity. The mean hemoglobin level (or red blood cell count) is lowest in patients with the most severe clinical manifestations. On the other hand, the platelet count and leukocyte count as well as the plasma fibrinogen level are elevated in the sickest patients. A threshold level of hemoglobin F at 1.2 g/dl (approximately 20% hemoglobin F) decreases the risk of major organ failure and is attained most frequently in those with a Senegalese chromosome. Hemorheologic findings observed during the most stable state of patients with sickle cell anemia indicate two trends: (a) the mean percentage of dense red cells is nearly twice as high in the maximal severity patients as compared with the minimal severity patients; and (b) mean red cell rigidity is greatest in the maximal severity group and least in the minimal severity group. These findings suggest that a greater percentage of dense, poorly deformable red cells are present in sickle cell patients in the genotypic category of maximal severity. CONCLUSIONS: The combination of the beta S gene cluster haplotype and alpha-gene status correlates with both phenotypic laboratory findings (hematologic profile) and morbidity. These associations increase our ability to predict clinical severity and the future risk of major organ failure.

Sickle cell anemia. Beta s gene cluster haplotypes as genetic markers for severe disease expression.

Identification of the beta s gene cluster haplotype and alpha-gene status provides a useful tool for the detection of high-risk patients with sickle cell anemia. Analysis of the relationship of the long-term clinical course to the above parameters has revealed that those with the haplotype designated Senegal have decreased severity, those with the Benin haplotype have intermediate severity, and those with the Central African Republic (CAR) haplotype have the most severe clinical expression. Further modulation of the clinical course occurs with the coinheritance of alpha-thalassemia-2. In both Africa and the United States, the CAR beta s haplotype increased the risk (relative risk, 2.25; 95% confidence interval, 1.41 to 3.87) of developing a complication and death at an early age. Detection of the CAR haplotype identifies the child with sickle cell anemia at risk for a rapid rate of progression of sickle-induced microvasculopathy, ultimately leading to irreversible organ damage during the first three decades of life. In patients with the CAR haplotype, potential curative therapy, such as bone marrow transplantation or gene insertion, should be seriously considered during childhood, before organ failure is clinically evident.

Polysaccharide encapsulated bacterial infection in sickle cell anemia: a thirty year epidemiologic experience.

Annual age-specific incidence rates of Streptococcus pneumoniae or Haemophilus influenzae bacterial septicemia in sickle cell anemia (SS) were determined for the years of 1957 through 1989. Forty-nine patients had 64 episodes of septicemia among a population of 786 SS patients observed for 8,138 person-years. Peak frequency of infection occurred between 1968-1971 and 1975-1981 with a conspicuous absence of episodes in 1972, 1973, 1982-1984, and 1986-1987, thus demonstrating cycles of high and low attack rates. The annual age-specific incidence rate of septicemia varied from 64.5 (1965) to 421.1 (1980) per 1,000 person-years for those under 2 years of age and never exceeded 10.2 per 1,000 in those over 4 years of age. Following the introduction of pneumococcal polyvalent vaccine in 1978, incidence of infection decreased in SS children greater than 2 years of age. No modification of the risk of infection was observed in immunized children less than 2 years of age. During these three decades, there has been a ten-fold increase in the number of SS adults over 20 years of age. The relative risk of chronic sickle complications comparing the survivors of septicemia to the non-infected patients was: subsequent death 1.76, retinopathy 4.06, avascular necrosis 1.95, symptomatic cholelithiasis 1.33, stroke 1.30, and priapism 1.26. These data suggest that prognosis for lifetime severe SS is initially manifested as an increased risk of septicemia during childhood.

Chronic renal failure in sickle cell disease: risk factors, clinical course, and mortality.

OBJECTIVE: To determine the incidence, clinical course, and risk factors associated with the onset of chronic renal failure in sickle cell anemia and sickle C disease. DESIGN: A prospective, 25-year longitudinal demographic and clinical cohort study. A matched case-control study was conducted to determine risk factors. PATIENTS: A total of 725 patients with sickle cell anemia and 209 patients with sickle C disease who received medical care from the hematology service in a large municipal hospital. Most were observed from birth or early childhood. MEASUREMENTS: Thirty-six patients developed sickle renal failure: 4.2% of patients with sickle cell anemia and 2.4% of patients with sickle C disease. The median age of disease onset for these patients was 23.1 and 49.9 years, respectively. Survival time for patients with sickle cell anemia after the diagnosis of sickle renal failure, despite dialysis, was 4 years, and the median age at the time of death was 27 years. Relative risk for mortality was 1.42 (95% Cl, 1.12 to 1.81; P = 0.02) compared with patients who did not develop renal insufficiency. MAIN RESULTS: Histopathologic studies showed characteristic lesions of glomerular "drop out" and glomerulosclerosis. Case-control analysis showed that ineffective erythropoiesis with increasingly severe anemia, hypertension, proteinuria, the nephrotic syndrome, and microscopic hematuria were significant pre-azotemic predictors of chronic renal failure. The risk for sickle renal failure was increased in patients who had inherited the Central African Republic beta s-gene cluster haplotype. CONCLUSIONS: The pre-azotemic manifestations of hypertension, proteinuria, and increasingly severe anemia predict end-stage renal failure in patients with sickle cell disease. The rate of progression of renal insufficiency is genetically determined. Treatment of the uremic phase has been dismal, underscoring the need for the development of useful pre-azotemic therapeutic modalities.

Allelic losses of chromosomes 9, 11, and 17 in human bladder cancer.

Twenty-five human bladder tumors were examined for loss of heterozygosity of markers on chromosomes 6p, 9q, 11p, 14q, and 17p. These studies show that all of the markers were reduced to homozygosity in at least some of the tumors. They also confirmed earlier studies by Fearon et al. [Nature (Lond.), 318: 377-380, 1985] that approximately 40% of bladder tumors were reduced to homozygosity for markers on chromosome 11p. However, the greatest frequency of allelic loss was seen for chromosomes 9q (67% of informative cases) and 17p (63% of informative cases) with both chromosomes being lost concordantly in 10 out of 20 informative tumors. Allelic loss of chromosome 9q has not been previously observed with other human cancers; however, deletions of 17p have been reported in breast, lung, and colorectal carcinomas. The data raise the interesting possibility that allelic losses of specific chromosomes might be a feature of cancer in a particular differentiated cell type whereas loss of other chromosomes harboring more generally acting tumor suppressor genes might be a common feature of human cancers.

Plasminogen activator regulation by transforming growth factor-beta in normal and neoplastic human urothelium.

Plasminogen activators (PA) are thought to participate in the invasive and metastatic processes of malignancies and are known to be modulated by certain growth factors (Danø, K; Andreasen, P.A.; Grondahl-Hansen, J.; Kristensen, P.; Nielsen, L.S.; Skriver, L. Adv. Cancer Res. 44:139-266; 1985 and Laiho, M.; Keski-Oja, J. Cancer Res. 49:2533-2553; 1989). This report describes the effect of TGF-beta on the regulation of secreted PA activity produced by human fetal urothelium and neoplastic urothelial cell lines. Epidermal growth factor was previously shown to induce substantially different effects on PA production by normal versus neoplastic urothelial (Dubeau, L.; Jones, P.A.; Rideout, W.M.; Laug, W.E. Cancer Res. 48:5552-5556; 1988). This report demonstrates that log phase normal urothelium, but not transformed cells, responded to TGF-beta (1-10 ng/mL) by diminishing the total secreted PA activity. Northern and western analyses showed that the reduction in protease activity resulted from an increased level of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. Additionally, northern analysis of total mRNA levels at varying cell densities demonstrated modulation of tPA, PAI-1, and TGF-beta transcripts in normal urothelial cells as a function of growth in vitro, suggesting the presence of an intact regulatory pathway to control extracellular proteolysis.

Expression of the MyoD1 muscle determination gene defines differentiation capability but not tumorigenicity of human rhabdomyosarcomas.

Several human rhabdomyosarcoma cell lines, cultured primary tumor explants, and biopsies of tumor and normal skeletal muscle tissue expressed a 2.0-kilobase transcript that hybridized to the mouse muscle determination gene MyoD1. This transcript was found in tumor cell lines and primary explants that developed multinucleated myotubes but was absent in Wilms' tumors or cell lines and primary explants that developed multinucleated myotubes but was absent in Wilms' tumors or cell lines derived from other mesenchymal tumor cell types. Expression of the human homolog of MyoD1 therefore can define a tumor as a rhabdomyosarcoma. Transfection of the mouse MyoD1 gene into the human rhabdomyosarcoma cell line RD increased the ability of the tumor cells to differentiate into multinucleated myotubes and enhanced myosin heavy-chain gene expression but did not decrease tumorigenicity in nude mice.

Complete nucleotide sequence of the neuraminidase gene of human influenza virus A/WSN/33.

The complete nucleotide sequence of the neuraminidase (NA) gene of WSN/33 (H1N1) virus was determined. The entire sequence was derived from the insert of cDNA clones, except the last 20 nucleotides, which were determined by primer extension. The WSN NA gene contained 1,409 nucleotides beginning at the 5' end (sense strand), with an untranslated region of 19 nucleotides followed by 1,359 nucleotides coding for 453 amino acids and finally ending with a 31-nucleotide sequence of untranslated region at the 3' termini. The amino acid sequence of WSN NA, as deduced from the DNA sequence, showed the presence of a stretch of 29 amino acids (7 to 35) enriched in hydrophobic amino acids, which may anchor the protein into the viral or cellular membrane. When compared with the PR8 NA sequence, WSN NA appeared to possess a similar structure, including the identical location of all cysteine and proline residues. However, WSN NA contained only three of the five potential glycosylation sites present in PR8 NA. Additionally, WSN NA contained a substitution of a five-amino acid sequence for a six-amino acid sequence in PR8 NA. The possible significance of these sequence changes in the primary structure of WSN NA in the unique role of WSN NA as a virulence factor in mouse brain and MDBK cells is discussed.

Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli.

Antigenic determinants of influenza virus hemagglutinin were expressed in Escherichia coli. DNA coding for presequences of hemagglutinin were removed and an ATG codon was placed before DNA coding for mature hemagglutinin. A number of expression plasmids were constructed in which various segments of this reconstructed hemagglutinin DNA were fused to DNA coding for bacterial beta-galactosidase. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.