Circulating PIG-A mutant T lymphocytes in healthy adults and patients with bone marrow failure syndromes.

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematological disorder with acquired PIG-A gene mutations and absent surface expression of proteins utilizing glycosylphosphatidylinositol (GPI) anchors. PNH often follows aplastic anemia, suggesting PIG-A mutant cells have relative dominance over normal hematopoietic cells. Somatic PIG-A mutations could arise after aplasia, or healthy persons could have rare PIG-A mutant cells that expand under selection pressure. METHODS: We developed an in vitro negative selection method to isolate GPI-deficient T lymphocytes using aerolysin, an Aeromonas toxin that binds GPI anchors and induces cell lysis. Peripheral blood mononuclear cells (PBMC) from normal adults and patients with PNH or other bone marrow failure syndromes were analyzed. RESULTS: From healthy adults, 166 T lymphocyte clones with deficient GPI-linked surface protein expression (CD55, CD59) were isolated. The mean mutant frequency (M(f)) of aerolysin-resistant clones was 17.8 +/- 13.8 per 10(6) PBMC, range 5.0-59.6 per 10(6) cells. Clones had a Class A complementation defect and distinct PIG-A mutations. Patients with PNH had elevated aerolysin-resistant M(f) values averaging 19 x 10(-2), a 10,000-fold difference. Two patients with Fanconi anemia and two others with mild aplastic anemia had M(f) values less than 15 x 10(-6), but two with recovering aplastic anemia had M(f) values of 20 x 10(-4), representing an intermediate value between normal persons and PNH patients. CONCLUSION: Identification of PIG-A mutant T lymphocytes in healthy adults suggests PNH could develop following intense negative selection of hematopoiesis, with clonal outgrowth of naturally occurring PIG-A mutant stem cells.

Neural cell adhesion molecule (CD56)-positive acute myelogenous leukemia and myelodysplastic and myeloproliferative syndromes.

The CD56 antigen is normally expressed on natural-killer cells but has additionally been shown to be present on a variety of hematologic malignancies, including a subset of acute myelogenous leukemia (AML). There is disagreement, however, about its prognostic significance and its association with specific cytogenetic abnormalities. All clinical samples from June 1994, through September 1995, with increased myeloblasts were analyzed by multiparameter flow cytometry for anomalous expression of CD56. Patients with CD56+ blast cells were selected, and morphologic review was performed. Clinical information was obtained, and cytogenetic data were reviewed. Southern blot analysis to detect rearrangement of the mixed lineage leukemia (MLL) gene was performed when possible. The samples from 23 of 114 patients studied demonstrated anomalous expression of CD56 on myeloblasts, including patients with AML, myelodysplastic syndromes (MDS), and chronic myelogenous leukemia in blast crisis. The samples from 10 of 15 patients with CD56+ AML demonstrated at least partial monocytic differentiation. Dysplastic features were displayed in the samples of 12 patients. Correlation with specific cytogenetic abnormalities was not found. The MLL gene was rearranged in five of 18 patients. Seventeen patients have died, with a median survival of 4.6 months for patients with AML. Three have sustained a complete remission. One has findings of high-grade myelodysplastic syndrome. Two were unavailable for follow-up. Expression of CD56 was found in 20% of patients with increased myeloblasts, including patients with high-grade MDS, chronic myelogenous leukemia in blast crisis, and AML. This phenotype was associated with dysplasia, monocytic differentiation, and rearrangement of the MLL gene.

Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family.

The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.

Astrocyte apoptosis induced by HIV-1 transactivation of the c-kit protooncogene.

HIV-1 infection of the central nervous system (CNS) frequently causes dementia and other neurological disorders. The mechanisms of CNS injury in HIV-1 infection are poorly understood. Apoptosis of neurons and astrocytes is induced by HIV-1 infection in vitro and in brain tissue from AIDS patients, but the apoptotic stimuli have not been identified. We report herein that HIV-1 infection of primary brain cultures induces the receptor tyrosine kinase protooncogene c-kit and that high levels of c-Kit expression are associated with astrocyte apoptosis. Overexpression of c-Kit in an astrocyte-derived cell line in the absence of HIV-1 induces rapid apoptotic death. The apoptotic mechanism requires the c-Kit tyrosine kinase domain. The mechanism of c-kit induction by HIV-1 involves transactivation of the c-kit promoter by the HIV-1 Nef protein. These studies demonstrate that c-Kit can induce astrocyte apoptosis and suggest that this mechanism may play a role in CNS injury caused by HIV-1 infection. We propose that c-Kit can serve dual functions as a growth factor receptor or apoptosis inducer.

Comparison of human and Xenopus GATA-2 promoters.

GATA proteins comprise a family of transcription factors that are required for appropriate development of hematopoietic lineages. In order to understand the transcriptional regulation of GATA genes, we have cloned the human GATA-2 gene and identified and characterized its promoter. Comparison with the Xenopus GATA-2 promoter demonstrates highly conserved CCAAT box elements, which are essential for appropriate Xenopus expression. Unlike the Xenopus gene, the human GATA-2 gene lacks GATA binding motifs within the first 800 bp of 5' flanking sequence. In addition, the human GATA-2 promoter has two highly conserved ets sites that resemble the binding site for a recently described ets repressor factor, ERF. These conserved DNA sequence motifs provide strong candidate regions for the regulation of GATA-2.

Complex regulation of human c-kit transcription by promoter repressors, activators, and specific myb elements.

The c-kit proto-oncogene is expressed in several tissues during development. To understand the mechanisms controlling the expression of this gene, we characterized the human c-kit promoter. Expression is controlled transcriptionally. The 5'-flanking DNA was used to make promoter deletion-reporter constructs that were tested in cells that were either positive or negative for endogenous c-Kit. The results demonstrate that DNA, to at least position -4100, directs transcription well in both positive and negative cells. Addition of DNA from position -4100 to -5500 causes a reduction in expression to near-basal levels in c-Kit-negative cells but has little effect in c-Kit-positive cells. The DNA from -4100 to -5500 was tested for repressor function. It inhibits transcription from some heterologous promoters in c-Kit-negative cells. Likewise, this segment inhibits transcription from the homologous proximal promoter in a cell-specific manner, but the entire promoter is necessary for complete repression in c-Kit-negative cells. Two Myb binding motifs were also identified, and their role in regulating transcription was examined by mutation and functional testing. One, MYB1, acts as a partial repressor, whereas the other, MYB2, is a positive element that appears essential for expression. Binding proteins to both sites were characterized by several methods. MYB1 binds and responds functionally to c-Myb, but MYB2 does not. The results of these studies indicate that the regulation of c-kit transcription is complex, involving interactions among several activators and repressors.

The c-kit proto-oncogene receptor is expressed on a subset of human CD3-CD4-CD8- (triple-negative) thymocytes.

The c-kit receptor is a tyrosine-kinase transmembrane receptor first identified as an oncogene in the HZ4-feline leukemia virus and later found to be important in hematopoiesis in mice. The ligand for this receptor (Steel factor) can stimulate hematopoiesis both in vitro and in vivo. To study the pattern of c-kit receptor expression in normal human hematopoietic progenitor cells, we prepared a monoclonal antibody (9B9) against human c-kit receptor by using a synthetic peptide (amino acids 476-501) from the extracellular domain of c-kit receptor to immunize Balb/c mice. Monoclonal antibody 9B9 bound to recombinant c-kit protein, the erythroleukemic line HEL, the megakaryocytic line MEG-01, and the murine mast cell line P815. Monoclonal antibody 9B9 also bound to the surface of the CD7+CD3-CD4-CD8- T cell lymphoid cell lines DU.528 and HSB2T, and also to 1 to 4% of normal bone-marrow cells. The majority (67 +/- 6%) of CD34+ bone-marrow progenitor cells coexpressed c-kit receptor. Flow-cytometry analysis of immature CD3-CD4-CD8- (triple-negative) thymocytes indicated 30 +/- 9.5% expressed the c-kit receptor, and thymidine incorporation assay revealed that the receptor is functional. Indirect fluorescent microscopy of human thymic tissue, using a monoclonal antibody against Steel factor, revealed its presence on scattered mononuclear cells within the intralobular septae and the subcapsular cortex, which are regions where the triple-negative thymocytes are also localized. These data provide evidence that the c-kit receptor is present on human hematopoietic bone marrow and intrathymic T cell progenitor cells, and that it likely plays a role in early T cell lymphopoiesis.

Two new polymorphisms but no mutations of the KIT gene in patients with myelodysplasia at positions corresponding to human FMS and murine W locus mutational hot spots.

The KIT proto-oncogene encodes a tyrosine kinase receptor which plays a critical role in haemopoiesis. We have screened genomic DNA from bone marrow mononuclear cells of 46 patients with myelodysplasia (MDS) for mutations/deletions of exons 6, 13, 17, and 21 of the KIT gene (stem cell factor receptor) using polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and autoradiography to detect single-stranded conformational polymorphisms (SSCP). These exons include positions analogous to those mutated in the FMS gene (colony-stimulating factor-1 receptor) in myelodysplastic syndrome (MDS) and mutated/deleted in the Dominant White Spotting mouse (W locus) which results in macrocytic anaemia. Two different gel running conditions were used for each exon. Polymorphisms were identified only at 4 degrees C in exon 17 (three out of 44 MDS samples and two of 21 DNA samples from normal subjects), and in the non-coding region of exon 21 (five out of 34 MDS samples and seven out of 19 normals). Direct sequencing identified a G to A base change at nucleotide 3169 within exon 21, and a C to T change at position 2415 in exon 17. No conformational changes suggestive of mutations or deletions have been found to date, although we cannot rule out low frequency clonal abnormalities undetectable by our method, which has a sensitivity in our hands of approximately 5%. Polymorphisms occur frequently in the KIT gene. Together with this study, a total of five have been described.

Cloning and structural analysis of the human c-kit gene.

The recent identification of the mouse White spotting and Steel loci as genes encoding the c-kit receptor and its ligand, respectively, has shed light on the importance of this ligand and receptor in embryogenesis, melanogenesis and hematopoiesis. In order to determine if the c-kit proto-oncogene is involved in human disease, we isolated seven overlapping lambda recombinants, using a fetal brain cDNA, and characterized the normal human gene (KIT). The longest mapped transcript is 5230 bp, is alternatively spliced and includes 21 exons that span more than 70 kb of DNA. From the exon-intron structure, we have localized an alternative splice site to the 3' end of exon 9. The overall c-kit gene structure closely resembles that found in the CSF-1R gene (c-fms). This similarity includes a large first intron, the same number of exons containing translated sequence and very similar exon-intron boundaries. Using pulsed-field gel electrophoresis, we have linked KIT to the platelet-derived growth factor receptor A gene, with both residing on a 700-kb BssHI fragment. These data will allow investigation into the control of KIT expression and the potential to identify mutations or altered expression of this gene in human disease.

Comparison of dipyridamole-echocardiography with dipyridamole-thallium scintigraphy for the diagnosis of myocardial ischemia.

After an intravenous infusion of dipyridamole (0.56 mg/kg), we performed both echocardiography and thallium scintigraphy in 63 patients who were referred for known or suspected coronary artery disease. Of those patients, 25 returned for coronary arteriography within 1 month after the tests, thus forming the study group for this report. Sensitivity for detection of coronary artery disease, when analyzed region-by-region, was 80% for thallium scintigraphy and 57% for echocardiography, whereas specificity was 85% and 98%, respectively. When evaluating individual patients for the presence or absence of ischemia, we found a sensitivity of 95% for scintigraphy and 58% for echocardiography; corresponding specificities were 50% and 100%. By using arteriography as the gold standard for comparison, it appears that thallium scintigraphy has a significantly higher sensitivity but lower specificity for the detection of coronary artery disease than does echocardiography. Echocardiography may, however, be a useful adjunct to thallium scintigraphy in the evaluation of patients with coronary artery disease.

Two-dimensional echocardiographic assessment of complications involving the Ionescu-Shiley pericardial valve in the mitral position.

The function of the Ionescu-Shiley bovine pericardial xenograft in the mitral position was investigated in 70 patients by two-dimensional echocardiography. Echocardiographic data from 21 patients with suspected bioprosthetic dysfunction and 49 patients with normal clinical findings were analyzed in a double-blind fashion. Confirmation of cardiovascular status was obtained by means of cardiac catheterization, surgery, autopsy or other techniques in 19 of the 21 symptomatic patients. Two-dimensional echocardiography correctly evaluated bioprosthetic function in 98% of the patients. Six of the seven patients with proven xenograft dysfunction demonstrated echocardiographic evidence of malfunction. There was one false negative evaluation but no false positive results. Valve dysfunction included endocarditic mass lesions, mitral regurgitation resulting from dehiscence of a leaflet or the sewing ring or mitral stenosis due to calcification or malposition of the valve. This experience indicates that two-dimensional echocardiography is useful for assessing the Ionescu-Shiley mitral bioprosthesis, particularly in those patients who develop nonspecific symptoms.

Aneurysm of the interatrial septum occurring as an isolated anomaly.

We report a case of aneurysm of the atrial septum occurring as an isolated abnormality in a patient with atypical chest pain. Apical two-dimensional echocardiography demonstrated phasic bulging of the fossa ovalis region of the atrial septum into the right atrium. Biplane cineangiography confirmed the presence of a large septal aneurysm in an otherwise normal heart. The incidence, pathogenesis, and complications of this unusual anomaly are briefly discussed.

Noninvasive assessment of the Ionescu-Shiley pericardial xenograft in the mitral position: preliminary experience.

To establish noninvasive criteria for assessment of the Ionescu-Shiley pericardial xenograft in the mitral position, 29 patients with a normally functioning bioprosthesis were studied with m-mode echocardiography and phonocardiography. Two-dimensional echocardiograms were also obtained in ten of the patients. Although two-dimensional echocardiography provided simultaneous visualization of a greater number of stents and leaflets than the m-mode technique, the superior resolution of m-mode ultrasound permitted more detailed analyses of the xenograft's motion patterns. The anterior leaflet, recorded in all patients, had an average excursion of 1.2 +/- 0.22 cm. Leaflet thickness measured 4 mm or less. Coarse diastolic vibration of the anterior leaflet was recorded in two patients in the absence of both aortic insufficiency and prosthesis dysfunction. Ultrasonic estimates of prosthesis height and orifice diameter did not correlate with micrometer measurements, possibly due to the limited resolution of pulsed ultrasound in the presence of a highly reflective substance. Opening and closing of the pericardial leaflets were associated with the production of high frequency vibrations on the phonocardiogram. The potential usefulness and limitations of echocardiography for evaluating the function of bioprosthetic valves are discussed.

Initial diastolic indentation of the mitral valve in aortic insufficiency.

An abnormal diastolic indentation of the mitral valve has been noted on short-axis two-dimensional echocardiograms in patients with aortic insufficiency. In order to assess the clinical usefulness of this finding, we reviewed the echocardiograms of 18 patients with this lesion who had undergone cineaortography. Results were compared with those of a control group of 100 patients. Initial diastolic indentation of the anterior mitral leaflet occurred in 8 of 12 patients with 3+ to 4+ aortic insufficiency. None of the patients with lesser regurgitation and no patients from the control group demonstrated this pattern. The presence of this abnormality appears to be a specific indicator of critical aortic insufficiency.

Absolute Determination of the Thermal Conductivity of Argon at Room Temperature and Pressures Up to 68 MPa.

The thermal conductivity of argon at room temperature and for pressures up to 68 MPa has been measured with a transient hot-wire technique in order to assess the accuracy of an instrument of this type. The data are presented for a nominal temperature of 300.65 K and comparison with other authors shows that our data is accurate to within ± 1 percent, and it is the most accurate set of data for pressures above 35 MPa. Experimental evidence of a thermal conductivity enhancement near the critical density for a temperature about twice the critical temperature is herein reported. The experimental data were compared with the values predicted by the hard sphere model and it has been found that the theory gives values that are about 4 percent lower than the experimental ones in the density range 0-400 kg/m3 and about 1 to 2 percent lower in the high density region 400-825 kg/m3.

Isolated mitral valve replacement with the Kay-Shiley prosthesis. Long-term follow-up and recommendations.

Eighty patients underwent isolated mitral valve replacement with the Kay-Shiley prosthesis during a 6 year period (September, 1966, in October, 1972) at Walter Reed Army Medical Center. One-hundred percent follow-up has been achieved with this group. Sixty-four percent of this group have experienced one or more thromboembolic episodes (TEEs), for a TEE rate of 28.7 episodes per 1,000 patient months at risk. The 5 year survival rate is 45%. Eleven of 43 (25%) late deaths resulted from TEEs, and 13 of 43 (30%) late deaths were secondary in the hemorrhagic complications of long-term anticoagulant therapy. The dismal TEE rate prompted as to initiate a policy of elective replacement of all Kay-Shiley mitral prostheses. Eighteen of the 26 survivors underwent replacement of the Kay-Shiley mitral prosthesis. The other eight patients either declined reoperation or were not considered suitable candidates. The reoperative mortality rate was 22% (4/18). Fifteen of 18 explained prostheses revealed a yellow disclored, grooved occluder disc with loosely adherent clot. We recommend elective replacement of Kay-Shiley mitral prostheses in all patients in whom the risk of operation is reasonable.