Nassef F. Hassan

Purification of human monocytes on gelatin-coated surfaces.

Human peripheral blood monocytes secrete a cell membrane-associated glycoprotein, cold insoluble globulin (fibronectin). Since fibronectin binds to gelatin-coated surface, we developed a simple technique for separation of human peripheral blood monocytes from whole mononuclear cell preparation. These preparations are characterized by a high monocyte purity (more than 90%), low platelet contamination and excellent viability.

Marrow transplantation in chronic granulomatous disease: An update, with 6-year follow-up*

enzymes of the coagulation cascade. Free iron, not plasma protein bound iron, is considered responsible for oxidative damage to cellular and organelle membranes? It may also inhibit function of protease enzymes of the coagulation cascade. Therapy for hemorrhage related to hepatotoxic coagulopathy involves coagulation factor replacement. If, however, there is functional impairment of coagulation factors because of peripheral inhibition by circulating free iron, it is unlikely the factor replacement or vitamin K would be beneficial. Thus therapy must include prompt removal of excess iron. Chelation therapy with deferoxamine is standard practice in severely iron-poisoned patients; unappreciated benefit may be the moderation of serious gastrointestinal hemorrhage. In many institutions rapid serum iron analysis is not available. This has led to the use of other factors (leukocytosis, hyperglycemia, vomiting, diarrhea, and radiographic demonstration of iron within the gastrointestinal tract) as indicators of thesever i ty of poisoning and the need for specific treatment. 6 If additional experience confirms the presence of an early coagulopathy in severe iron poisoning, the presence of early coagulation abnormalities could also suggest the need for deferoxamine therapy.

Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.

Microglia have been identified in the white matter of developing and adult mouse brain using different murine macrophage markers. While several techniques for the isolation of murine microglia have been described, the small cell yields and partial purification have limited the progress of these studies. We now describe the isolation of murine microglia using a modification of McCarthy and de Vellis method. Brain tissues from 1–2 day old newborn mice were mechanically and chemically dissociated and maintained in in vitro culture for 3 weeks. In primary dissociated brain cultures, microglia are observed after 10 days migrating from small colonies. After 16–20 days, brain‐derived microglia were isolated with high cell yields by continuous shaking of the cultures for 16 hr. In contrast to resident murine peritoneal macrophages, microglia express less Class II (la) antigen and a small percentage express L3T4 (CD4) antigen by flow cytometry.

Isolation and characterization of human fetal brain-derived microglia in in vitro culture

Human brain microglia may play a central role in immunopathogenesis of CNS diseases including HIV infection, multiple sclerosis and Alzheimers disease. In order to investigate the possible relationship between microglia and the mononuclear phagocyte system, human brain microglia were isolated from 14-18-week-old fetal brains, and maintained in in vitro culture. Enriched fetal brain microglia were stained for different monocyte/macrophage and glial cell markers. Fresh dissociated brain cells lacked macrophage surface markers. Isolated microglial cells stained positive for complement receptor C3bi, Class II [human leukocyte antigen-DR (HLA-DR)] antigen and with the lectin Ricinus communis. Microglia also share several functional properties with monocyte/macrophages, which include generation of superoxide anion and histochemically demonstrable intracellular acid phosphatase and non-specific esterase. Primary human dissociated brain cultures were maintained in culture for at least 28 weeks. Although microglia were not observed above the astrocyte cell layer after 5 weeks in culture, microglia-like cells appear below the astrocyte layer after 12 weeks in culture. These cells stained positive for non-specific esterase and displayed oxidative burst activity upon activation with phorbol myristate acetate. Thus, we have successfully isolated an enriched population of microglia from human fetal brain and have demonstrated that these cells possess markers and properties which are characteristics of mononuclear phagocytes.

Phorbol myristate acetate induced oxidation of 2',7'-dichlorofluorescin by neutrophils from patients with chronic granulomatous disease

The oxidative metabolic burst of stimulated human polymorphonuclear leukocytes (PMNs) has been evaluated by the measurement of oxygen consumption, chemiluminescence, and oxygen radicals (O2 ‐, H2O2,OH‐) derived from activation of the hexose monophosphate shunt (HMPS). PMNs from patients with chronic granulomatous disease (CGD) are shown to lack functional NADPH oxidase and undetectable oxygen radical generation. However, using single cell analysis by flow cytometry and 2′,7′‐dichlorofluorescin (DCFH) oxidation by H2O2, significant DCFH oxidation by the PMA stimulated CGD PMNs was observed. Furthermore, 1mM potassium cyanide enhanced DCFH oxidation by control and CGD PMNs. DCFH oxidation by cells from an obligate heterozygous mother of an X‐linked CGD patient was intermediate. These observations suggest that a PMA induced oxidase enzyme is present in CGD cells.

Stress-related neuroimmunomodulation of monocyte-macrophage functions in HIV-1 infection

Monocytes/macrophages play a central role in the afferent and efferent limbs of the immune system. Macrophages perform several immunological functions both in vivo and in vitro, including antigen presentation, tumor cell killing, phagocytosis, and bacterial and viral killing. Acquired immunodeficiency syndrome (AIDS), a disease characterized by a profound immunodeficiency, induces a wide range of neuropsychological abnormalities. The occurrence of severe psychological disturbances, including stress, depression, and anxiety increase psychological and physical indices of morbidity among patients. Stress influences several immunological responses in man and animals and is usually accompanied by altered blood levels of various CNS-related peptides or neurohormones. Monocytes/macrophages express surface receptors for different CNS-secreted molecules. In ARC and AIDS patients abnormal neuropeptide levels may be related to severe psychological disturbances. Neuropeptides and neurohormones may play a central role in stressed HIV-1-infected patients by affecting monocyte-macrophage functions, which may further trigger disease progression and immunologic deficiency. It is hypothesized that stress reactions lead to altered release of neurohormones and/or neuropeptides which affect monocyte-macrophage functions and favor progression of HIV-1-related disease.

O-Phenylenediamine oxidation by phorbol myristate acetate-stimulated human polymorphonuclear leukocytes: Characterization of two distinct oxidative mechanisms

O-Phenylenediamine (OPD) oxidation has been extensively utilized for the measurement of peroxidase-mediated catabolism of hydrogen peroxide. However, until now this system has not been evaluated for the measurement of hydrogen peroxide produced upon activation of the hexose monophosphate shunt (HMPS) in polymorphonuclear leukocytes (PMNs). OPD oxidation by phorbol myristate acetate (PMA)-stimulated PMNs was mediated by both hydrogen peroxide and superoxide produced by the activation of the HMPS. Furthermore, OPD oxidation by an oxidative mechanism independent of the HMPS was observed by the PMA stimulation of PMNs obtained from patients with chronic granulomatous disease (CGD). This HMPS-independent OPD oxidation was inhibited by superoxide dismutase or 1 mM potassium cyanide (KCN). Superoxide dismutase, catalase, or 1 mM potassium cyanide inhibited 50% OPD oxidation obtained with PMA-stimulated normal PMNs. PMA treatment of purified human myeloperoxidase (MPO) produced OPD oxidation which is inhibited by superoxide dismutase or 1 mM KCN. These data indicate that OPD oxidation observed with CGD PMNs is mediated by a PMA-induced oxidase activity of myeloperoxidase. OPD oxidation in the presence of 1 mM KCN is a method comparable in sensitivity with ferricytochrome c reduction for the evaluation of HMPS activity. Furthermore, the OPD assay can measure myeloperoxidase oxidase activity in PMA-stimulated PMNs.

Use of colloidal silica (Sepracell-MN) for enrichment of dendritic cells from human peripheral blood: comparison with other methods.

Three methods are described for enrichment of dendritic cells from human peripheral blood. In method 1, mononuclear cells were incubated in plastic tissue culture flasks for two h. Nonadherent cells were removed. Adherent cells were washed to remove floating cells and incubated for 14 h at 37°C in 5% CO2. Carbonyl iron was added, and the flasks were incubated for another 2 h. Nonadherent cells were subjected to centrifugation over metrizamide gradient Phagocytic cells containing ingested carbonyl iron, small lymphocytes, and free carbonyl iron particles passed through the metrizamide, while the interface cell population was enriched for dendritic cells. The purity and yield of enriched dendritic cells were 52.8% and 0.05%, respectively. In method 2, adherent mononuclear cells were cultured overnight, and the released cells (released adherent cells) were centrifuged over metrizamide to separate low‐density cells. Monocytes from this low‐density cell population were removed by panning over human gamma globulin‐coated plastic Petri dishes. In this method the average purity and yield of DC were 63.8% and 0.1%, respectively. In method 3, released adherent cells were treated with anti‐CD5 and anti‐CD14 monoclonal antibodies plus baby rabbit complement for 15 min, washed, and centrifuged with colloidal silica (Sepracell‐MN). Centrifugation with Sepracell‐MN was repeated three times. Low‐density cells were panned twice over human gamma globulin‐coated plastic Petri dishes. Nonadherent cells were highly enriched for DC. Contamination of T cells, B cells, and NK cells was undetectable by flow cytofluorometry. Contamination of monocytes was < 2%. This method provided > 85.0% purity and 0.4% yield. This method (method 3) combines adherence, complement‐dependent lysis, centrifugation with colloidal silica, and panning and provides the best yield and purity; it is therefore recommended for optimal purification of DC.

Flow Cytometric Analysis of Oxidase Activity of Neutrophils from Chronic Granulomatous Disease Patients

Phagocyte cells, neutrophils and macrophages constitute the main defense mechanisms of the human body against invading microorganisms. The bactericidal activity of polymorphonuclear leukocytes (PMNs) involves the activation of a cell membrane associated NADPH oxidase enzyme“. Following activation of the NADPH oxidase enzyme, a cascade of oxidation/reduction reactions occurs, with the generation of several oxygen radicals. Superoxide (0 2 − ) hydrogen peroxide (H202), hydroxyl radical (HO) singlet oxygen (0) are the main oxygen by-products of the respiratory burst of PMNs2 and have an important role in killing of bacteria. The enzyme NADPH oxidase has a major function in the generation of O 2 − radical while other enzymes including superoxide dismutase3, myeloperoxidase4, catalase and glutathione peroxidase5 serve a regulatory activity in the respiratory burst. NADPH oxidase differs from other cellular oxidase enzymes (i.e., mitochondrial), since it is a cyanide insensitive enzyme6. The activation of the NADPH oxidase enzyme with different soluble stimulants as phorbol esters7, formyl-methionyl-leucyl-phenylalanine8 and γ-hexachlorocyclohexane9 results in the generation of active oxygen radicals and mimics the biochemical events which occur during phagocytosis in vivo.