Y.H. Thong

Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the hypaque-ficoll method

Data on the optimal conditions for the simultaneous purification of mononuclear (MN) and polymorphonuclear (PMN) leucocytes from human blood on Hypaque-Ficoll medium of one density is presented. The efficiency of separation of MN and PMN leucocytes by the Hypaque-Ficoll method was dependent on both the Ficoll concentration and the density of the medium. Best results were obtained with a medium consisting of a Ficoll concentration of 8.2% and a density of 1.114 g/ml. The separation procedure was much more efficient if carried out at room temperature than at 4 degrees C. Storing human blood markedly affected the efficiency of separation and best results were obtained using fresh blood. Data are also presented on the variability in the separation with respect to 40 adults and 80 children.

A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the hypaque-ficoll technique

A one-step procedure for purification of mononuclear and polymorphonuclear cells from human blood is described. It is a modification of the Hypaque-Ficoll method with density of 1.095 g/ml. Centrifugation at 200 X g for 20--30 min resulted in the separation of mononuclear and polymorphonuclear cells into 2 distinct bands at the interface. The mononuclear cell fraction contained 83.9 +/- 1.6% lymphocytes and 13.8 +/- 2.3% monocytes, while the other conssisted of highly purified neutrophils (96.4 +/- 1.0%). Leukocyte recovery by this method was always greater than 80% and viability exceeded 98%. Both cell fractions retained their immunological functions.

Separation of mononuclear and polymorphonuclear leucocytes from human blood by the one-step hypaque-ficoll method is dependent on blood column height

Further observations have been made in relation to separation of mononuclear (MN) and polymorphonuclear (PMN) leucocytes from human blood by the one-step procedure involving centrifugation of the blood on Hypaque-Ficoll medium. The distance separating the MN and PMN cell fractions was dependent on the height of the blood column but independent of the diameter of the tube, this distance being directly proportional to the height of blood. However, increasing the height of the blood column by diluting the blood in medium 199 did not increase the separation distance. Erythrocytes were shown to be important for separation of leucocytes into two distinct bands.

Immunoglobulin G subclass deficiency and predisposition to infection in Down's syndrome

Serum immunoglobulins and IgG subclasses were measured in 26 children with Downs syndrome using an enzyme-linked immunosorbent assay and monoclonal antibodies. Eighteen (69%) of the children had increased susceptibility to infection. None of the children had deficiencies of total IgG and IgM, and only one had an IgA deficiency. IgG4 deficiency was diagnosed in 14 (54%) children. One child had a deficiency of IgG2. There were no children with deficiencies of either IgG1 or IgG3. There was a significant correlation between IgG subclass deficiency and predisposition to infection (P < 0.05). Ninety percent of the patients with severe

Immunization with live amoebae, amoebic lysate and culture supernatant in experimental Naegleria meningoencephalitis.

Immunization with two doses of live Naegleria fowleri produced a survival of 34% of mice compared to 0% in unimmunized controls, whereas multiple doses of live N. fowleri resulted in loss of protective immunity. In contrast, multiple doses of N. fowleri lysate produced a survival of 30%, and multiple doses of N. fowleri culture supernatant produced a survival of 67 to 78%. Fractionation of the culture supernatant by column chromatography showed that all six fractions contained protective antigens, but the best protection occurred from immunization with the high molecular weight fraction (greater than 200,000 daltons).

Activation of complement by Naegleria

Neither Naegleria nor its culture supernatant was found to be directly chemotactic for human neutrophils. Interaction of Naegleria with human serum, however, resulted in the generation of a strong chemotactic stimulus. The reduction of serum activity by heat-inactivation indicated a dependence on serum complement for the interaction. The ability of Naegleria to activate the alternative complement pathway was demonstrated with the use of C2-deficient serum.

Simultaneous preparation of mononuclear and polymorphonuclear leucocytes from horse blood on ficoll-hypaque medium

Results presented show that highly purified populations of mononuclear (MN) and polymorphonuclear (PMN) leucocytes can be obtained from horse blood by a procedure similar to that previously described for the separation of these leucocytes from human blood. This involved centrifugation of horse blood on a Ficoll-Hypaque medium with a density of 1.095 g/ml. The procedure required approximately 1 h for completion and resulted in the simultaneous preparation of MN (greater than 98% purity) and PMN (greater than 96% purity) leucocytes. Cell viability exceeded 95% and cells retained immunological functions.

Site of expression of immunity to Naegleria fowled in immunized mice

Summary An experiment was performed which confirmed a previous finding that mice are protected against Naegleria fowleri infection by immunization with amoeba‐free supernatant from amoeba cultures. Histological observations suggested that this protection is expressed mainly at the nasal mucosa and possibly results from the combined effects of polymorphonuclear leucocyte‐mediated killing of the amoeba and mechanical elimination of the organisms by extensive shedding of necrotic epithelium.

Effect of mefloquine on the immune response in mice

The effect of mefloquine, a new antimalarial compound, on the immune response in mice was studied in vitro and in vivo. Slight inhibition of mitogen-induced lymphocyte proliferative responses was observed at a concentration of 1 microgram/ml, and marked cytotoxicity at 4 microgram/ml. In contrast, a higher proportion of human lymphocytes remained viable at the same concentration of mefloquine. Antibody responses to sheep erythrocytes were impaired in mice receiving a total dose of 60 mg/kg. At this dosage schedule, delayed-type hypersensitivity responses to the same antigen were not affected.

Modification of polymorphonuclear leucocyte function by imidazoles

The effect of five imidazole derivatives (metronidazole, tinidazole, clotrimazole, miconazole and ketoconazole) on human polymorphonuclear leucocytes (PMNL) was examined in vitro. Metronidazole and tinidazole had no apparent effect on either PMNL chemotactic response or PMNL fungal/bacterial killing. In contrast, clotrimazole, miconazole and ketoconazole inhibited PMNL chemotaxis. In addition, miconazole and ketoconazole were shown to depress the ability of PMNL to kill bacteria and fungi.