Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant- induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium antagonist lanthanum chloride prevented xanthine- xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis.     

Current treatment for alcohol withdrawal [editorial]

The online version of the original article can be found at     

Patterns of cell proliferation and cell migration in the Sezary syndrome

The patterns of cell proliferation and cell migration were studied in three patients with the Sezary syndrome using autoradiographic techniques. Cell labeling patterns following pulse labeling with tritiated thymidine in vivo indicated that Sezary cells proliferate actively in skin and in lymph nodes but that few if any Sezary cells proliferate in the peripheral blood. In two of the patients serial samples were obtained. Label dilution patterns in skin and blood over time suggested that circulating Sezary cells originated in extracutaneous sites where cells were proliferating more rapidly than in the skin. Cells labeled in extracutaneous sites of proliferation appear rapidly in the blood, and their transit time through the peripheral blood compartment is short. Circulating Sezary cells may then be deposited in the skin where they resume proliferation at a low rate. Thus, while Sezary cells proliferate in both cutaneous and extracutaneous sites, proliferation appears to be more rapid in extracutaneous sites such as lymph nodes. This suggests that trials of systemic therapeutic approaches should be undertaken.     

Expansion of the growth fraction in multiple myeloma with alkylating agents

Patients with IgG multiple myeloma underwent serial studies of tumor cell kinetics including (1) estimation of the total body myeloma cell number (TBMC), (2) measurement of the myeloma cell tritiated thymidine labeling index (LI), and (3) calculation of the total number of myeloma cells undergoing DNA synthesis. Intermittent courses of chemotherapy with cycle-non-specific agents such as melphalan resulted in a marked increase in the LI of myeloma cells in patients who had a 75% reduction in TBMC. The long "plateau" phase of partial remission of myeloma in these patients was associated with a continued high LI: this suggests that the plateau resulted from a balance between the cytoreductive effects of chemotherapy and expansion of the growth fraction (GF) of the tumor. Preliminary attempts to capitalize therapeutically on this expansion of the GF in several patients included administration of the cycle-active agents vincristine and cytosine arabinoside. Vincristine appeared to induce a further reduction in tumor in several patients, although cytosine arabinoside appeared to be ineffective despite clear evidence of its inhibition of DNA synthesis in myeloma cells in vivo. Further clinical studies of the effects of cycle-active drugs on myeloma appear to be warranted; however, successful exploitation of the dynamic change in myeloma cell kinetics with chemotherapy will require the use of cycle-active agents with marked selective toxicity for myeloma cells.     

Synergistic cytotoxic effect of azidothymidine and recombinant interferon alpha on normal human bone marrow progenitor cells

Azidothymidine (AZT) and interferon alpha (IFN-alpha) are among the drugs showing strong in vitro activity against the human immunodeficiency virus type-1 (HIV-1). Each drug, however, has significant toxicity against normal marrow progenitor cells that frequently proves dose-limiting in patients. In this study, AZT and recombinant IFN-alpha 2a (rIFN-alpha 2a) were tested as single agents and in combination against normal myeloid (CFU-GM) and erythroid (BFU- E) colony forming cells in a standard methylcellulose culture assay. The data were analyzed using a quantitative computerized analysis based on the median-effect principle and the isobologram equation as described by Chou and Talalay (Adv Enz Regul 22:27, 1984). The ED90 for BFU-E and CFU-GM inhibition was then compared with previously measured in vivo plasma levels of each drug and the ED90 for the anti-HIV-1 effect in vitro. We demonstrate that (a) the drugs are strongly synergistic in inhibiting marrow progenitor cell growth and that this synergism occurs at drug levels that are within the range of measured plasma levels in phase I clinical trials, (b) BFU-E are more sensitive than CFU-GM to the inhibiting effects of AZT, rIFN-alpha 2a or both drugs in combination, (c) the drug concentrations in combination that synergistically inhibit bone marrow progenitors are much higher than those required to inhibit HIV-1 replication in vitro, and (d) the anti- HIV-1 effect for the combination of AZT and rIFN-alpha 2a was clearly superior to the effect of AZT or rIFN-alpha 2a alone as indicated by the combination index and the dose-reduction index. These data suggest that substantially lower doses of AZT and rIFN-alpha than those currently being tested in clinical trials might not only maintain a strong synergistic anti-HIV-1 effect but might also avoid significant hematologic toxicity.     

Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series

Recombinant human (rh) interleukin-3 (IL-3) stimulated the proliferation and differentiation of erythroid, granulocyte, macrophage, eosinophil (Eo), and mixed colonies as well as megakaryocytes from human bone marrow cells. rh IL-3 was a weaker stimulus than rh granulocyte-macrophage colony-stimulating factor (GM- CSF) for day 14 myeloid cell colonies. At day 7 of incubation, rh IL-3 stimulated a few G, M, and Eo clusters but no colonies. This loss of responsiveness of myeloid cells to rh IL-3 was accentuated with further differentiation of the cells. rh IL-3 stimulated very few or no clones after five-day incubation with enriched promyelocytes and myelocytes, whereas rh GM-CSF was an efficient stimulus. Responsiveness to rh IL-3 was completely lost in postmitotic mature neutrophils. Incubation of these cells with rh IL-3 did not result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells or superoxide anion production after stimulation with formyl-methyl-leucyl-phenylalanine (FMLP), although they could be stimulated by rh GM-CSF. In addition, preincubation of neutrophils with different concentrations of rh IL-3 failed to increase or decrease their response to rh GM-CSF. In contrast to neutrophils, mature Eos could be stimulated by rh IL-3 to kill antibody-coated tumor cells. These results show that cells of the neutrophilic myeloid series lose their responsiveness to h IL-3 as they differentiate and suggest that although h IL-3 may be an important therapeutic agent to use for hematopoietic regeneration in vivo, the lack of stimulation of mature neutrophil function makes it an unlikely sole candidate as adjunct therapy for treatment of infectious diseases.     

Neutrophil elastase produces 52-kD and 30-kD glucocorticoid receptor fragments in the cytosol of human leukemia cells

Characterization of glucocorticoid receptors in leukemia cells is important to understand mechanisms of glucocorticoid resistance but has been impeded by receptor fragmentation in cytosol extracts. We recently found that formation of 52- and 30-kilodalton (kD) glucocorticoid receptor fragments in cytosol of leukemia cells is due to proteolysis and is blocked by diisopropylfluorophosphate (DFP). In the present study, we identify a 28-kD serine protease in cytosol of leukemia cells that binds [3H]DFP and correlates with the formation of 52- and 30-kD receptor fragments. This protease is immunoprecipitated by antiserum to neutrophil elastase. Limited digestion of [3H]dexamethasone-21-mesylate- labeled receptors by purified neutrophil elastase produces 52- and 30- kD receptor fragments. Receptor fragmentation in the cytosol of leukemia cells in inhibited by methoxysuccinyl-alanyl-alanyl-prolyl- valyl-chloromethylketone, a highly specific inhibitor of neutrophil elastase. The addition of as few as 5% neutrophils to a lymphoid cell suspension provides sufficient elastase to produce receptor fragmentation. Our findings indicate that neutrophil elastase is responsible for receptor fragmentation in the cytosol of leukemia cells. The neutrophil elastase may be endogenous to the leukemia cells or may come from neutrophils that contaminate leukemia cell suspensions.     

Plasmic degradation of crosslinked fibrin. I. Structural analysis of the particulate clot and identification of new macromolecular-soluble complexes

Plasmic degradation of crosslinked fibrin has been studied to identify the proteolytic cleavages that convert the clot into a soluble lysate and also to identify the derivatives that are likely to circulate during clot dissolution. Initial polypeptide chain cleavages do not disrupt the solid clot matrix. With continued exposure to plasmin, high molecular weight derivatives are produced that remain attached to the clot by noncovalent forces. Further degradation then results in the liberation into solution of several large, noncovalently bound complexes. Progressive degradation of the largest, initially liberated complexes to the terminal derivatives, DD/E, DD, and E, occurs in solution after their release from the clot. As the fibrin clot is exposed to plasmin for longer intervals, progressive dissolution occurs, but the structure of the covalently bound insoluble fibrin core, the noncovalently attached derivatives, and the liberated complexes remains constant. Since much of the initially liberated protein is in complexes larger than DD/E, these derivatives probably represent the more prevalent plasmic degradation products of crosslinked fibrin in vivo.     

Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria

The hematologic disorder paroxysmal nocturnal hemoglobinuria (PNH) occurs following an acquired somatic mutation in the Piga gene within a bone marrow stem cell. The progeny of this mutated cell cannot synthesize glycosylphosphatidylinositol (GPI) anchors, with a resultant deficiency in surface expression of all GPI-linked proteins. The protean clinical manifestations of PNH presumably result from the deficiency of these GPI-linked surface proteins. To explain the observation that neutrophils are affected at a significantly higher percentage than circulating erythrocytes and to analyze the proliferative rates of erythroid production in PNH, we studied 25 patients using flow cytometry. The fluorescent dye thiazole orange was used to detect reticulocytes, and CD59 monoclonal antibody was used to identify GPI-deficient cells. In contrast to the mature circulating erythrocytes, the percentage of abnormal reticulocytes was similar to the percentage of affected neutrophils. However, the vast majority of reticulocytes was completely GPI-deficient, ie, were type III cells, even in patients with only modest numbers of circulating type III erythrocytes. In addition, greater than 5% type II reticulocytes were identified in only 3 patients, although greater than 5% type II mature erythrocytes were identified in 10 of 25 patients. The results show that the erythroid and neutrophil bone marrow precursors have an equivalent proliferative advantage in PNH. The data also have important implications for the origin of type-II erythrocytes in PNH.     

Studies on the Mechanism of the Lower Esophageal Sphincter Pressure Response to Alkali Ingestion in Humans

To clarify the lower esophageal sphincter (LES) pressure response to alkali ingestion, normal subjects and postantrectomy patients with either a gastroduodenostomy or gastrojejunostomy were studied in a double-blind controlled fashion. LES pressure and serum gastrin concentrations were measured after ingestion of a 100 ml bolus of either 0.4 M NaHCO3 or 0.4 M NaCl. In addition, the effect of a therapeutic dose (30 ml) of a commercial antacid preparation was studied in a double-blind fashion in 14 patients with gastroesophageal reflux disease. Peak increases in LES pressure above basal were significantly higher (p less than 0.05) after NaHCO3 than after NaCl in normal subjects and in patients with vagotomy and Billroth I antrectomy, but not in patients with vagotomy and Billroth II antrectomy. Serum gastrin concentrations were unaffected by alkali. Thirty milliliters of liquid antacid containing aluminum and magnesium hydroxide resulted in a small sustained rise in LES pressure over the first 50 min after ingestion, but this was not statistically different than the placebo response. It is suggested that: 1) neither the antrum nor intact vagi nor gastrin were required for NaHCO3 ingestion to increase LES pressure; 2) the increase in LES pressure with NaHCO3 ingestion appears to rely upon an intact duodenum and may relate to volume and osmolarity of the alkali load; and 3) therapeutic doses of a liquid commercial antacid does not significantly increase LES pressure in the presence of an intact stomach.