Prevention by tetrandrine of spontaneous development of diabetes mellitus in BB rats.

Treatment of BB rats with the plant alkaloid tetrandrine (20 mg.kg-1.day-1), a novel anti-inflammatory compound, from 35 to 120 days of age reduced the cumulative incidence of spontaneous diabetes from 75.5 to 10.9% (P less than 0.001). Dose-response studies with 0, 5, 10, and 20 mg.kg-1.day-1 of tetrandrine from 35 to 130 days resulted in spontaneous diabetes in 84.2, 63.1, 31.6, and 5.3% of the rats, respectively. When the start of treatment with 20 mg.kg-1.day-1 tetrandrine was delayed until 70 days of age, there was a significant reduction of the incidence of diabetes from 63.1 to 28.6% (P less than 0.01). Histological examination of the pancreases from tetrandrine-treated rats showed a lesser degree of insulitis than controls (P less than 0.01). Drug toxicity was not seen in the rats, as assessed by appearance, behavioral change, organ histology, and blood chemistry. These results provide some hope that tetrandrine may be of value in preventing diabetes and treating newly diagnosed diabetic subjects, either by itself or in combination with a more potent immunosuppressive agent.

Human monocyte adherence measured by the nylon fibre microcolumn technique.

A simple rapid method for the measurement of human monocyte adherence using nylon fibre microcolumns is described. The kinetics indicate the optimal contact time to be 30 min for monocytes, compared with 10 min for neutrophils. The optimal temperature is 37 degrees C; significantly low values were obtained for 4 degrees C and 45 degrees C, while intermediate values were obtained for 25 degrees C. Monocyte adherence was more sensitive to inhibition by fluoride than cyanide, suggesting that energy for adherence is mainly derived from the glycolytic pathway. The addition of phorbol myristate acetate enhances monocyte adherence. Significant decay in monocyte adherence occurred after isolation from whole blood for 24 h or longer.

Improved method for neutrophil hexose-monophosphate shunt assay using serum.

The addition of serum into the hexose-monophosphate shunt assay results in the reduction of neutrophil resting metabolic activity. This results in a higher stimulation index and consistent values.

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.

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.

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.

Development of a microassay technique for neutrophil adherence.

The development of a microassay for neutrophil adherence is described. The nylon fibre column consisted of 10 mg of fibre inserted into a micropipette tip. A vacuum harvester was constructed to aspirate the neutrophil-containing fluid in the columns into disposable test-tubes for quantitation of neutrophil concentrations. In this way, the neutrophil requirement was reduced from 10(7) cells to 10(5) cells. A complete assay can be performed efficiently and rapidly using less than 1 ml of blood.

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.

An immunoblot technique for identification of allergens of the Australian paralysis tick Ixodes holocyclus.

The blocking conditions appropriate for Western blot identification of allergens of the Australian paralysis tick, Ixodes holocyclus, have been investigated. The results suggest that human serum is a potent blocking agent which may have wide application in the immunodetection of antigens.

Uptake of 3H-deoxyglucose as a microassay of human neutrophil and monocyte activation.

The accumulation of 2-deoxy-D-[1-3H]glucose, a non-metabolised analogue of glucose provides a quantitative measurement of the state of activation of phagocytic cells. A microassay for 3H-deoxyglucose uptake by human neutrophils and monocytes is described. Optimal conditions for the assay include the use of 5 X 10(5) cells and 0.78 microCi/ml of deoxyglucose in a final volume of 0.2 ml per microtitre well, and an incubation time of 30 min at 37 degrees C. This simple, rapid and reproducible technique may find application in experimental immunology.