A rapid centrifugation method for the isolation of polymorphonuclear leucocytes from human blood.

A rapid one-step gradient centrifugation method to prepare PMN leucocytes has been worked out by which a 98--99% pure, washed granulocyte suspension of 98% viability can be obtained in 30--40 minutes. The cells prepared by this method displayed higher NBT reduction upon ingesting the same soluble DNA-anti DNA complexes than those prepared by the dextran sedimentation method.

Serum histamine level in exercise-induced bronchospasm.

The changes in serum histamine levels were studied in 12 asthmatic patients: 6 patients gave positive responses and 6 gave negative responses to physical exercise. The serum histamine was determined fluorometrically, and the specific airway conductance was used as the spirometric criteria. The serum histamine levels were elevated in the patients who gave positive responses to exercise. In the negative responders to exercise, the serum histamine levels did not alter significantly. The changes in specific airway conductance and serum histamine levels were not in parallel during the entire reaction, suggesting the intervention of other immediate type hypersensitivity mediators in exercise-induced bronchospasm. The authors proved the role of histamine in the pathophysiology of exercise induced bronchospasm.

Measurement of lymphocyte activation by a chromatin topooptical reaction. II. Application for detecting drug allergy. A clinical and experimental study.

Lymphocyte chromatin activation was measured in 18 clinical cases of suspected or proven drug allergy as well as in 4 other persons with neither history nor signs of hypersensitivity. The different drugs were tested in the reaction in such dilution that the final concentrations ranged between 50 and 10.000 nanomoles. Within this range between 100 and 1250 nanomoles a bellshaped dose response curve was found in all drug-allergic subjects, measuring the neutral-red chromatin topooptical reaction. At the peak of this curve signs of cytotoxicity could be demonstrated with appreciable chromatin "desactivation", an increase of protein-like substance in the cells' supernatant and a morphological cellular damage. The exact drug concentration at which the lymphocyte autocytotoxicity occurred was inversely proportional with the extent of drug allergy in a given patient. The average lymphocyte chromatin birefringence measured at different non-cytotoxic drug concentrations was directly proportional with the extent of drug allergy. The ratio of the above characteristics gave a score (with the dimension of cm2/microM) which corresponded to the clinical picture. The score was low (55,7 +/- 5,8 cm2/microM) in the control subjects as well as in the drug allergic ones tested with other, i.e. nonsensitizing drugs. The score was high, (309,8 +/- 54,4 cm2/microM) however, when drug allergic patients' lymphocytes were challenged by the proper drug(s). There was neither false negativity in the positive group nor false positivity in the negative group of patients. Scores above 75 cm2/microM are considered as positive, those above 80 cm2/microM are undoubtedly positive. The relation of this rapid test to the lymphocyte transformation test as well as its advantages over the latter are discussed.

Measurement of lymphocyte activation by a chromatin topo-optical reaction. Mechanism and specificity of the test.

A rapid inexpensive method is presented for detecting peripheral blood lymphocyte chromatin activation by the neutral red "topo-optical" reaction, which causes strong and easily measurable birefringence in the lymphocyte nuclei. This reaction can be enhanced by fixing the cells with 150 mM/l NaCl in 70% ethanol and/or by treating the unfixed cellular suspensions with 0.2 M/l HCl to remove histones. In histone-removed preparations, 30 min DNase I treatment almost completely abolished the birefringent reaction, whereas RNase treatment resulted in only 18% loss. Chromatin activation induced by enzyme inhibition increased chromatin birefringence significantly. The same phenomenon could be induced in sensitive subjects' lymphocytes by specific antigens or haptens much more rapidly. The monocytes were not activated to a significant extent. In non-sensitive subjects different kinetics of antigen or hapten-dependent activation and no cytotoxic effects have been observed. Depletion of T-lymphocytes in vivo in SLE patients or by in vitro treatment with 0.5 mM/l KCN as well as with 0.02% trypsin has caused a significant drop in the mean chromatin birefringence. The effect of trypsin was reversible.

A new method for obtaining viable cells from dermal infiltrates. A study on 2,4 dinitrochlorobenzene induced contact dermatitis.

Experimental contact dermatitis has been induced in 2,4 dinitrochlorobenzene (DNCB) sensitized guinea pigs. The developing dermal infiltrate was excised and the infiltrating cells were obtained by mechanical extraction alone as well as by the combination with collagenase and elastase treatment. The most viable cells appeared in the elastase and mechanically extracted samples and the least in those subjected to mechanical treatment alone. The most cells in the enzyme-treated samples were present 24 h after re-exposure of the sensitized animals to DNCB consisting mainly of lymphocytes and of polymorphonuclear granulocytes. The optimum conditions for the action of enzymes including optimum duration of the treatment, buffer milieu, aspecific proteolytic effect on foreign substrate and action on T and B cell receptors have been elaborated. It was concluded that 80 min of collagenase treatment with gentle mechanical extraction under specified conditions does not affect any measurable immunologic properties of the liberated cells resulting in the second best yield. A comparison of these data with earlier reports and their significance is being discussed.

Immune-complex phagocytosis by human polymorphonuclear granulocytes.

A new two-step centrifugation technique is described for the rapid preparation of viable pure polymorphonuclear granulocytes from 9.5 ml of venous blood. The phagocytosis of these cells was stimulated by soluble DNA-anti-DNA complexes, produced in vitro by incubating high mol. wt. DNA with sera obtained from patients with severe SLE. The measure of phagocytosis was the amount of nitroblue-tetrazolium (NBT), reduced by the active microphages. The reduced NBT was estimated by counting the formazane positive cells or by extracting and measuring it spectrophotometrically. The immune-complex phagocytosis of control human granulocytes showed a linear relationship with the cell count per reaction mixture and displayed a bell-shaped dose-response curve as function of the immune-complex. The immune-complex phagocytosis by SLE granulocytes was decreased by about 40 per cent as compared to normal granulocytes.