Detection, pathogenesis, and prevention of damage to human granulocytes caused by interaction with nylon wool fiber. Implications for filtration leukapheresis.

Granulocytes collected by reversible adhesion to nylon wool fiber (NWF) function relatively well in standard in vitro tests; however, they have an abnormally shortened survival time in the circulation. Assuming that this rapid disappearance represents clearance and that recognition by phagocytes is important for such clearance, we used an autologous in vitro cell:cell recognition assay to determine whether phagocytes can detect cellular changes induced by exposure of normal granulocytes to NWF. Human granulocytes incubated with NWF 1 h at 37 degrees C, eluted with 20% acid citrate dextrose plasma, and washed stimulated the hexose monophosphate shunt activity of normal granulocytes an average of twofold (193+/-40% of controls), indicating a recognition response. NWF-induced granulocyte recognition was not dependent on plasma factors or activated complement components but was dependent on the time that the granulocyte was on the NWF and was maximal by 60 min of exposure. After elution from NWF, granulocytes demonstrated resting glucose oxidation rates only slightly higher than normal; however, during the first 20 min of exposure to NWF, granulocytes increased their rate of (14)CO(2) production from [1-(14)C]glucose three- to five-fold. Therefore, experiments were performed to determine whether toxic oxygen metabolites produced by NWF-adherent cells might contribute to recognition. The results showed that (a) normal granulocytes exposed to NWF in the presence of scavengers of superoxide anion (superoxide dismutase) or free radicals (ascorbate, mannitol, or benzoate) and washed before assay did not stimulate glucose oxidation of indicator granulocytes; and (b) NWF granulocytes prepared from cells unable to generate high levels of toxic oxygen metabolites, i.e. cells prepared anaerobically or from a patient with chronic granulomatous disease, also failed to stimulate indicator granulocytes. Human granulocytes placed in contact with NWF show an oxidative burst and become recognizable to other phagocytes. Free radical scavengers are effective in minimizing this recognition conferred on NWF-procured granulocytes.

Isolation and characterization of glycosphingolipids from human leukocytes. A unique glycosphingolipid pattern in a case of acute myelomonoblastic leukemia.

Neutral glycosphingolipids and gangliosides were isolated from the malignant cells of a patient with acute myelomonoblastic leukemia. Structural analyses were performed by gas-liquid chromatography and by high-performance liquid chromatography combined with enzymatic hydrolysis of glycosphingolipids using glycosidases. We found that, in contrast to normal leukocytes and chronic leukemia cells which have only a single tetraosylceramide species, these acute myelomonoblastic leukemia cells have approximately equal amounts of both globo- and neolactotetraosylceramide. This is the first population of human leukocytes in which we found two families of neutral glycosphingolipids to be present. The ganglioside fraction was composed of appreciable quantities of both NeuAc alpha 2 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer (GM3, hematoside) and NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer (sialoparagloboside). These cells did not have the 'leukocyte-specific' N-acetylneuraminosyllactotriaosylceramide found in normal human lymphocytes and neutrophils. These results are discussed in relation to normal leukocyte differentiation and acute leukemia. The present study also illustrates the usefulness of combining enzymatic degradation with high-performance liquid chromatography for glycosphingolipid structural determination.

Uptake and metabolism of daunorubicin by human leukemia cells.

Radiolabeled daunorubicin was used to study in vitro uptake of daunorubicin (DNR) by the human promyelocytic leukemia cell line HL-60 and by leukemic cells from five previously untreated patients with acute nonlymphocytic leukemia (ANLL). Uptake of the metabolite daunorubicinol (DOL) and the metabolism of DNR were examined using high-performance liquid chromatography (HPLC). Uptake of DNR and DOL by HL-60 and ANLL cells exhibited a similar kinetic pattern. The uptake of DOL was 35%-50% of the uptake of DNR at the same test concentration in both HL-60 and ANLL cells. Approximately 5%-10% of intracellular DNR was metabolized to DOL by HL-60 and ANLL cells after 24 h of drug exposure. Measurements of DNR or DOL derived from liquid scintillation spectrometry and HPLC permit a sensitive and accurate assessment of the pharmacokinetics of these drugs in human leukemia cells. In addition, the HL-60 cell line can be used as a model for studying in vitro pharmacokinetics of the anthracyclines.

Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates.

Carbohydrate analysis has traditionally been viewed as a specialty science, performed only in a few well-established laboratories using conventional carbohydrate analysis technology (e.g. NMR, gas chromatography-mass spectroscopy, high-performance liquid chromatography, capillary electrophoresis) combined with the specialized technical training that has been essential for accurate interpretation of the data. This tradition of specialized laboratories is changing, due primarily to an increase in the number of scientists performing routine carbohydrate analysis. As a result, many scientists who are not trained in traditional carbohydrate analytical techniques now need to be able to perform accurate carbohydrate analysis in their own laboratories. This has created a need for technically simple and inexpensive methods of carbohydrate analysis. In this review, we present application vignettes of a technically simple, yet analytically powerful method called fluorophore-assisted carbohydrate electrophoresis (FACE). FACE can be used for performing routine oligosaccharide profiling, monosaccharide analysis, and sequencing of a variety of carbohydrates.

Direct measurement of unfractionated heparin using a biochemical assay.

A number of investigations have noted that functional biological assays for heparin are not always reliable and may not reflect the actual biochemical level of heparin in patients receiving anticoagulant therapy. This creates the possibility that patients receiving anticoagulant treatment may have an excess or deficiency of circulating levels of heparin. To address this problem, we have developed a direct biochemical measurement of heparin. The heparin assay uses fluorophore-assisted carbohydrate electrophoresis (FACE) to directly measure the predominate disaccharide of unfractionated heparin. In this study, unfractionated heparin was measured in vitro throughout a wide range of heparin concentrations in plasma. Seven in vivo pharmacokinetic studies in five normal subjects given 3,000 USP units of unfractionated heparin intravenously showed a three-phase elimination process with higher peak plasma levels and shorter elimination times than predicted from previous studies. At these doses, heparin is largely eliminated intact through urinary excretion. Body weight has a significant effect on heparin kinetics. When we compared the direct biochemical assay with two biological clotting assays, we found the latter can overestimate biochemical heparin concentrations. The FACE assay, due to its sensitivity, is also able to measure circulating levels of endogenous heparin in plasma and urine. Direct heparin measurement using the FACE technique is practical and useful for studies of the correlation of biochemical and biological activities.

Montagem de cariótipos de peixes assistida por computador / Computer assisted fish karyotyping

A obtenção de cariótipos de peixes desempenha um papel importante em estudos de citotaxonomia e evolução cromossômica das espécies. No entanto, poucos sistemas semi ou completamente automatizados para a obtenção de cariótipo de peixes estão disponíveis. Este trabalho propõe e avalia uma ferramenta baseada em imagens que auxilie a montagem de cariótipos de peixes. As espécies analisadas foram Hopliasmalabaricus (Bloch, 1794); Hypostomusancistroides (Ihering, 1911) e Parauchenipterusgaleatus (Linnaeus, 1766); popularmente conhecidas como traíra, cascudo e bagre-sapo, respectivamente.Um total de 100 metáfases foi analisado por dois métodos: 1 - geração semiautomática de cariótipo e 2 - geração automática de cariótipo. A avaliação do sistema foi feita por meio da correlação de Pearson, gráficos de diferenças e tabelas de contagens,utilizando-se como referência a média das contagens feitas por quatro usuários. No método 1, quatro usuários realizaram contagens e apresentaram correlação interobservador de r≥ 0.997. O número total de cromossomos identificados pelo método 1 foi 4348 e, para o método 2, foi 4135,o que resultou em uma identificação automática de aproximadamente 95,1% dos cromossomos, resultando em correlação entre os dois métodos de r= 0.93. Conclui-se que a ferramenta pode ser inserida no procedimento de cariotipagem de peixes para acelerar o processo com níveis aceitáveis de exatidão.(AU)

Fish karyotyping plays an important role in studies of cytotaxonomy and chromosomic evolution of species. However, few semi or completely automated fish karyotyping systems are available. This work proposes and evaluates an image-based tool to assist fish karyotyping. The analyzed species were Hopliasmalabaricus (Bloch, 1794), Hypostomusancistroides (Ihering, 1911), and Parauchenipterusgaleatus (Linnaeus, 1766). In Portuguese, these species are commonly referred to as traíra, cascudo, and bagresapo, respectively. A total of 100 metaphases were analyzed through two methods: (1) semi-automatic karyotype generation and (2) automatic karyotype generation. The results were analyzed using Pearson correlation, difference graphs and counting tables. The reference used for the evaluation of the system was the average of the counts made by four experts. In method 1, four users performed counts with interobserver correlation of r≥ 0.997. The total number of chromosomes identified by method 1 was 4348 and method 2 was 4135, excluding false positives, resulting in an automatic identification of approximately 95,1% of the chromosomes, resulting in a correlation between the methods of r= 0.93. The results indicate that the tool can be introduced for fish karyotyping procedures contributing for accelerating the process with acceptable accuracy.(AU)

Nonocclusive coronary disease after chronic exposure to nitrates: evidence for physiologic nitrate dependence.

A 38-year-old man, exposed to notroglycerin in his work as an explosives expert, developed non-occlusive ischemic heart disease after withdrawal of exposure to organic nitrates. Despite the severity of his symptoms and the documented spasm of his right coronary artery, his electrocardiogram was at all times normal, as were results of a wide panel of laboratory tests. Sublingual nitroglycerin amerliorated the symptoms which have decreased with time.

Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils.

The lipid composition of immature myeloid cells from the bone marrow of normal persons and myeloblasts from patients with acute myeloblastic leukemia was studied and compared with the lipid composition of normal mature human neutrophils. Total cholesterol, phospholipid, and fatty acid composition was determined on each cell type. The leukemic cells showed decreased total cholesterol and cholesterol-to-phospholipid ratio, increase phosphatidylcholine and phosphatidylinositol, decreased phosphatidylethanolamine, and an increased percentage of unsaturated fatty acids when compared to normal mature neutrophils. A nearly identical pattern was seen in the normal immature myeloid precursors from normal bone marrow. We conclude that the altered lipid composition of acute myeloblastic leukemia cells is related to unexplained factors related to cell age and not to malignancy per se.

Isolation and chemical characterization of neutral glycosphingolipids of human neutrophils.

Six neutral glycosphingolipids were isolated from purified preparations of human neutrophils. The chemical structure of each compound was characterized by degradation with exoglycosidases, methylation analysis, and electron impact/desorption mass spectrometry. The following structures were assigned on the basis of these detailed analyses: Glc beta 1 leads to 1Cer Gal beta 1 leads to 1Cer Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Gal alpha 1 leads to 4Gal beta 1 leads to 1Cer GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer. Neutral glycosphingolipids containing N-acetylgalactosamine were not detected in human neutrophils. The major neutral glycosphingolipids were lactosylceramide and lactoneotetraosylceramide. Although lactoneotriaosylceramide accounts for only 10% of the neutral glycosphingolipid fraction, neutrophils are the most readily available source of this compound. We may conclude that human neutrophils, in contrast to human erythrocytes and platelets, contain as their major neutral glycosphingolipids lactoneo-type structures and smaller amounts of gala-type structures. These findings are discussed in terms of blood group antigens and glycosphingolipid changes due to malignancy.

Degranulation and abnormal bactericidal function of granulocytes procured by reversible adhesion to nylon wool.

Granylocyte bactericidal capacity, chemotaxis, hexose monophosphate shung activity (before and after phagocytic stimulus), and quantitative nitroblue tetrazolium reduction and enzyme content were examined in cells obtained by filtration leukaphresis (FL) and continuous-flow centrifugation (CFC). A decrease in the bactericidal efficiency of FL-produced cells compared to that of both normal and CFC-procured granulocytes was found; the decrease was 17% with a cell-to-bacteria ratio of 5:1, and 55% with a 1:1 ratio. Moreover, FL-acquired cells were often vacuolated and consistently contained less acid phosphatase and beta-glucuronidase than did normal granulocytes. When normal cells were incubated for 1-2 hr with nylon wool, 30% of the total acid phosphatase and beta-glucuronidase was released, with no evidence of cell death, thus suggesting degranulation. Similar results were obtained with glass, cotton, or polysulfone plastic fibers. Electron microscopic and peroxidase cytochemical studies of the adherence of normal granulocytes to nylon fibers were also carried out. After 30 min of incubation, cell-to-fiber attachment and cellular aggregation had occurred, although the cells per se appeared normal. After 60 and 120 min, other changes became apparent: (1) a decrease in the amount of cytoplasmic granules; (2) large, intracytoplasmic vaculoles; and (3) extracellular peroxidase on fiber surfaces. We conclude that granulocytes obtained by adherence to nylon fibers show both morphological and biochemical evidence of degranulation and diminished bactericidal capacity, and that these abnormalities may be causally related to decreased granulocyte survival in transfusion recipients.

Febrile neutrophilic dermatosis in acute myelogenous leukemia.

Three patients with acute myelogenous leukemia and adequate granulocyte reserves developed fever and painful indurated erythematous plaques on their extremities and faces. The plaques became studded with vesicles or bullae and occasionally became necrotic. Histologic examination revealed dermal edema, infiltration with granulocytes, and formation of intraepidermal vesicles. Efforts to relate the skin reaction to infiltration of leukemic cells, microorganisms, or allergic phenomena were unsuccessful. Empiric antibiotic therapy was without effect. The symptoms and signs responded dramatically to systemic administration of corticosteroids. These lesions, which may cause diagnostic confusion and needless therapy with potent antibiotics, may represent another previously uncharacterized nonspecific skin reaction in patients with acute myelogenous leukemia.

Coma, hyperthermia and bleeding associated with massive LSD overdose. A report of eight cases.

Eight patients were seen within 15 minutes of intranasal self-administration of large amounts of pure D-lysergic acid diethylamide (LSD) tartrate powder. Emesis and collapse occurred along with signs of sympathetic overactivity, hyperthermia, coma and respiratory arrest. Mild generalized bleeding occurred in several patients and evidence of platelet dysfunction was present in all. Serum and gastric concentrations of LSD tartrate ranged from 2.1 to 26 nanograms per ml and 1,000 to 7,000 mug per 100 ml, respectively. With supportive care, all patients recovered. Massive LSD overdose in man is life-threatening and produces striking and distinctive manifestations.

Neutral glycosphingolipids in hairy cell leukemia.

The neutral glycosphingolipids of hairy cells from a patient with hairy cell leukemia were chemically analyzed by thin-layer and gas-liquid chromatography, mass spectrometry, combined gas chromatography-mass spectrometry, and glycosidase treatment. These cells were found to have compounds containing one to four sugars with the following structures: Glc1 leads to 1Cer Gal beta 1 leads to 4Glc1 leads to 1Cer Gal alpha 1 leads to 4 Gal beta 1 leads to 4Glc1 leads to 1Cer GalNAc beta 1 leads to 3Gal alpha 1 leads to 4Gal beta 1 leads to 4Glc1 leads to 1Cer These compounds belong to the globo series of neutral glycosphingolipids and are similar to those found in human lymphocytes and chronic lymphocytic leukemia cells. They differ from the neutral glycosphingolipids found in human neutrophils and chronic myelogenous leukemia cells which are of the lactoneo and gala type. Neutral glycosphingolipids may be useful in classifying leukemias of uncertain origin.

Complex carbohydrates as differentiation markers in malignant blood cells: glycolipids in the human leukemias.

This article summarizes the data on chemical sequencing of human leukocyte cell surface glycolipids that we have obtained over the last 3 years. The purpose of the work was to determine if these cell surface glycoconjugates differ among different leukocyte populations and at different stages of development. In homogeneous form we purified large numbers of leukocytes from normal persons and persons with acute and chronic leukemias of myeloid and lymphoid types, using continuous flow centrifugation leukapheresis. We extracted the glycolipids from these cells and used thin-layer chromatography, gas-liquid chromatography, gas chromatography-mass spectrometry, direct probe mass spectrometry, and enzyme treatment to obtain complete structural information on these compounds. Twenty glycolipids were identified and sequenced, representing over 99% of the glycolipids of human leukocytes. The major findings are that human leukocytes possess glycolipid patterns that distinguish them from other blood cells. Additionally, leukocytes of "myeloid' and "lymphoid' types can be distinguished by their glycolipid type. Glycolipids in human leukemic cells show two main features: (1) even though the cells appear morphologically "undifferentiated', all leukemias can be classified on the basis of their glycolipids as "myeloid' or "lymphoid'; (2) the complexity of their cell surface glycolipids is correlated with their degree of morphologic "differentiation', the more differentiated leukemias having more complex structures. No glycolipids were found in leukemic leukocytes that were not found in the normal cells. Glycolipids would therefore appear to be useful as both cell- and possibly differentiation-specific markers in human blood cells. The antigenic differences seen in human leukemic cells may be due in part to an altered distribution of complex carbohydrates at the cell surface.