Somatic cell hybrid analyses of hematopoietic differentiation

A differentiated cell expresses an entire set of specialized features. Somatic cell hybridization provides a method to examine control of gene regulation. We studied the expression of tissue-specific features in hybrids between human promyelocytes (HL-60) and human Burkitt's lymphoma cells (P3HR-1). Two hybrid lines, HP-1 and HP-2, and 18 hybrid clones were established and confirmed by karyotype, isozyme, and surface antigen analyses. The hybrids extinguished the 10 myeloid (HL- 60) features that we examined including myeloid morphology, histochemistry, and functions that included response to colony- stimulating factor and ability to differentiate to granulocytes or macrophages. In contrast, the hybrids synthesized immunoglobulin and expressed Epstein-Barr nuclear, early, and viral capsid antigens similar to the P3HR-1 lymphoid parental line. Results are contrasted to the findings when P3HR-1 lymphocytes are fused to human erythroid- myeloid cells (K562). Taken together, our results suggest that phenotypic differences between human myeloid and lymphoid cells in the hematopoietic lineage involve mutually exclusive programs and may possibly be mediated by the activity of diffusible, transacting molecules.     

Mutation and protein expression of p53 in acquired immunodeficiency syndrome-related lymphomas

p53 mutations are found in a variety of neoplasia. B-immunoblastic lymphoma (BIBL) is a rapidly progressive, aggressive lymphoma. As patients with acquired immunodeficiency syndrome (AIDS) live longer, BIBL is becoming an increasing problem. We asked three questions in our study. What is the frequency of p53 mutations in BIBL? Is it more frequent in patients with AIDS? Can immunohistochemical staining of lymph nodes for expression of p53 substitute for mutational analysis of p53 to detect lymphomas with mutated p53? Exons 5, 6, 7, 8 of the p53 gene (hot-spots for mutations) were amplified and examined for mutations by single-strand conformation polymorphism (SSCP) analysis. Altered migration was observed in 7 of 52 BIBL samples. Of these, 4 of 25 were from individuals infected with human immunodeficiency virus (HIV) and 3 of 27 were not infected with HIV. Direct sequencing of amplified material confirmed the presence of mutations in exons 5, 7, 8 of p53. A total of 26 BIBL as well as other lymphoma/leukemia samples, stained strongly by immunohistochemistry with three antibodies directed against human p53. Five of 6 BIBL samples with p53 mutations stained strongly for p53, but 20 lymphoma samples with no detectable p53 mutations also stained strongly for p53. Of note, however, 10 hyperplastic, nonmalignant lymph nodes from individuals either infected or not infected with HIV had negligible staining for p53 protein. In conclusion, p53 mutations occur in about 14% BIBL samples; the frequency of p53 mutations in BIBL in individuals with and without AIDS was similar. Positive p53 immunohistochemistry did not correlate with detectable p53 mutations in the same tissue, but positive immunohistochemical staining for p53 was only found in neoplastic lymph nodes. This latter finding provides a strong warning that p53 immunochemistry with available reagents cannot be used to determine which tumors have mutations of p53.     

Late Results of Pulmonary Balloon Valvuloplasty in Adults Using Double Balloon Technique

Between July 1985 and July 1987, 18 consecutive adult patients with congenital pulmonary stenosis underwent pulmonary balloon valvuloplasty (PBV). There were 11 males and 7 females, aged 15–45 years (mean 25). A double balloon technique was used in 14 patients and a single balloon in four. The size of the balloon used was 0.9 to 1.4 times the size of the pulmonary annulus. Eleven patients were restudied by repeat cardiac catheterization 6 months later. Student's t-test was used for comparison of data. Right ventricular (R V) systolic pressure before dilatation ranged from 84 to 180 (mean 123 ± 28.3) mmHg and the right ventricular to pulmonary artery (PA) peak systolic gradient ranged from 60 to 165 (mean 105 ± 30.2) mmHg. Immediately after dilatation, the RVsystolic pressure dropped to 30–80 (mean 53 ± 14.2) mmHg (P < 0.001). RV to PA peak systolic gradient dropped to 10–57 (mean 32 ± 14.2) mmHg (P < 0.001). A restudy of 11 patients 6 months later showed a further drop of RV systolic pressure to 35–65 (mean 49 ± 11.3) mmHg (P < 0.05). RV to PA peak systolic gradient continued to drop to 10–48 (mean 26 ± 11.3) mmHg (P < 0.01). Cardiac index improved from 2.68 ± 0.73 to 3.03 ± 0.40 L/min/m², P < 0.05. No complication was noted apart from either sinus bradycardia or extrasystole in a few patients. It was noted that balloon to annulus ratios of 1.1 to 1.4 produced sustained relief of the pulmonary valve stenosis. We concluded that PBV is the treatment of choice for congenital pulmonary stenosis. It reduced the hospital stay to 2 days and avoids the ri±k of open heart surgery. (J Interven Cardiol 1988:1:1)     

Granulocyte-macrophage colony-stimulating factor: signals for its mRNA accumulation

Granulocyte-monocyte colony-stimulating factor (GM-CSF) is an important hematopoietic growth factor. Mesenchymal cells produce abundant GM-CSF in response to tumor necrosis factor alpha (TNF). We wished to determine (1) what cellular pathways enhanced levels of GM-CSF mRNA, and (2) if TNF used any of these pathways. Modulation in levels of GM- CSF mRNA in human fibroblasts (WI-38) was studied by using Northern blot analysis. Markedly increased levels of GM-CSF mRNA occurred in these cells after exposure to sodium fluoride (NaF) and the effect of NaF was slightly enhanced by aluminum chloride; these results suggest that accumulation of GM-CSF mRNA can occur by activating a G-binding protein. Stimulators of protein kinase C dramatically increased levels of GM-CSF mRNA; however, blockade of protein kinase C activity did not attenuate accumulation of GM-CSF mRNA stimulated by TNF and NaF. Exposure to ouabain increased levels of GM-CSF mRNA and this effect was prominently enhanced in the presence of low concentrations of extracellular K+ and was almost abolished in high concentrations of extracellular K+. A monovalent ionophore (monensin) also increased levels of GM-CSF mRNA. Both ouabain and monensin can increase intracellular Ca++ concentration (Cai++) through Na+-Ca++ exchange. A calcium channel blocker (diltiazem) blocked the increased levels of GM- CSF mRNA mediated by ouabain, but could not block the stimulation mediated by TNF alpha. Ca++ ionophores also increased levels of GM-CSF mRNA and rapidly increased levels of Cai++. TNF did not increase Cai++ and, moreover, was able to stimulate accumulation of GM-CSF mRNA in the absence of extracellular Ca++. Taken together, we have found that several different cellular pathways can lead to prominent accumulation of GM-CSF mRNA in mesenchymal cells including (1) activation of protein kinase C, (2) increase in Cai++, and (3) stimulation of G-binding protein. Our studies show that TNF appears to increase levels of GM-CSF mRNA independent of protein kinase C activity or levels of Cai++.     

Quantification of blood flow with dynamic MR imaging and presaturation bolus tracking

A technique is described for rapid imaging of blood flow and dynamic measurement of its velocity. The method is a combination of bolus tracking and low-flip-angle gradient-echo cine angiography. This method provides precise determination of velocity with high temporal resolution in a single measurement. Unlike what occurs in phase imaging techniques, flow is displayed directly, eliminating potential errors that result from non-flow-related sources of phase shifts. Manipulation of raw data sets is avoided. Results obtained from a flow phantom, healthy volunteers, and a patient with an aortic aneurysm demonstrate the capability of the technique to track flow at low and high velocities and to differentiate flowing blood from thrombus. Because of its conceptual simplicity, rapidity, and lack of susceptibility to extraneous phase shifts, this technique may prove ideal for in vivo flow measurement and evaluation of flow patterns.     

Tailored hormonal therapy in secretory adrenocortical cancer

Mitotane is often considered the front-line hormonal therapyof adrenocortical carcinoma (ACC). An illustrative case concerningthis issue and the rationale to ponder other alternatives isreported. A 69 year-old woman, diagnosed with ACC was admittedwith hypertensive crisis, supraventricular tachycardia, congestiveheart-failure, diarrhoea and rabdomyolisis. Two years earlier,she had undergone