Lysis Of Sheep Red Cells Research Articles

Inhibition of human serum complement activity by diisopropylfluorophosphate and selected anticholinesterase insecticides

Activation of the human complement (C′) system, a major line of defense against infections, requires the participation of serine esterases. Since the widely used anticholinesterase insecticides inhibit serine esterases, the present study evaluated potencies of carbaryl, carbofuran, dichlorvos, and paraoxon to inhibit C′ activities of a panel of normal human sera. C′-mediated lysis of sheep red cells was measured with a modified assay (1) incorporating suboptimal concentrations of sensitizing antibody and (2) exhibiting increased sensitivity to serine esterase inhibitors. Test chemicals were added to diluted sera 2 hr prior to incorporation into C′ reaction mixtures. Potencies to inhibit C′ and serum cholinesterase (CHE) were compared to potencies of diisopropylfluorophosphate (DFP), a potent serine esterase inhibitor and a standard probe for C′ esterases. At 0.5 to 3.0 m m, carbaryl, carbofuran, dichlorvos, and DFP produced a dose-dependent inhibition of lysis, whereas paraoxon was not inhibitory. On a molar basis, carbaryl was three times more potent than DFP, and inhibited lysis 15–25 and 26–45% at 1.0 and 3.0 m m, respectively. Carbofuran, dichlorvos, and DFP were equipotent. Mean IC50's for inhibition of CHE (a marker for occupational exposure to organophosphates and carbamates) by DFP, paraoxon, dichlorvos, carbofuran, and carbaryl were 1.0 × 10 −8, 4.1 × 10 −8, 1.0 × 10 −7, 3.3 × 10 −6, and 1.8 × 10 −5 m, respectively. Potencies of the insecticides to inhibit CHE did not predict absolute or relative potencies to inhibit serum C′ activity.

Inhibition of Human Serum Complement Activity by Diisopropylfluorophosphate and Selected Anticholinesterase Insecticides

Inhibition of Human Serum Complement Activity by Diisopropylfluorophosphate and Selected Anticholinesterase Insecticides. CASALE, G. P., BAVARI, S., AND CONNOLLY, J. J. (1989). Fundam. Appl. Toxicol. 12, 460–468. Activation of the human complement (C') system, a major line of defense against infections, requires the participation of serine esterases. Since the widely used anticholinesterase insecticides inhibit serine esterases, the present study evaluated potencies of carbaryl, carbofuran, dichlorvos, and paraoxon to inhibit C' activities of a panel of normal human sera. C'-mediated lysis of sheep red cells was measured with a modified assay (1) incorporating suboptimal concentrations of sensitizing antibody and (2) exhibiting increased sensitivity to serine esterase inhibitors. Test chemicals were added to diluted sera 2 hr prior to incorporation into C' reaction mixtures. Potencies to inhibit C' and serum cholinesterase (CHE) were compared to potencies of diisopropylfluorophosphate (DFP), a potent serine esterase inhibitor and a standard probe for C' esterases. At 0.5 to 3.0 mM carbaryl, carbofuran, dichlorvos, and DFP produced a dose-dependent inhibition of lysis, whereas paraoxon was not inhibitory. On a molar basis, carbalyl was three times more potent than DFP, and inhibited lysis 15–25 and 26–45% at 1.0 and 3.0 mM respectively. Carbofuran, dichlorvos, and DFP were equipotent. Mean IC50's for inhibition of CHE (a marker for occupational exposure to organophosphates and carbamates)by DFP, paraoxon, dichlorvos, carbofuran, and carbaryl were 1.0×l0−8 4.1×l0−8, 1.0×l0−7 3.3×l0−6and 1.8×l0−5M, respectively. Potencies of the insecticides to inhibit CHE did not predict absolute or relative potencies to inhibit serum C' activity.

Distinction between fixation of C1 and the activation of complement by natural IgM anti-hapten antibody: Effect of cell surface hapten density

Naturally occurring human IgM anti-methotrexate and anti-folinic acid antibodies were used to study the mechanism of complement (C)-mediated lysis of sheep red cells (E) labelled with methotrexate (MTX) or folinic acid (FA). We found that IgM bound to these cells in three forms: one binds C1 and activates the lytic sequence, the second binds C1 with no subsequent lysis, and the third neither binds C1 nor activates C. The ratio of the three forms depended on the density of hapten on the cell surface. It was concluded that the binding of one antigen-reactive site in the IgM to a cell surface hapten is not sufficient to activate the lytic sequence but may be sufficient to bind C1.

Platelet antibodies in thrombocytopenic patients.

Platelet antibodies either bound to the surface of platelets or free in the serum were sought in patients who had low platelet counts for a variety of reasons. They were detected by finding excess IgG on the surface of washed platelets either directly or after incubation of the serum with normal platelets. The technique used was a modification of that described recently (Dixon et al, 1975) in which the greater the amount of anti-IgG consumed by the reaction with platelets the less the subsequent lysis of sheep red cells coated with IgG. This test could be calibrated by adding known quantities of IgG to the antisera and thus the amount of bound IgG could be measured. Platelets from normal donors and those with thrombocytopenia due to non-immunological causes such as aplastic anaemia or acute leukaemia were found to have 15-70 ng IgG/10(7) platelets (mean 53 ng). 37 out of 38 thrombocytopenic patients in whom immune destruction of platelets was suspected were found to have excess IgG on their platelets ranging from 70 to 720 ng/10(7) (mean 297 ng, P less than 0.001) and there was a significant inverse correlation between this amount and the platelet count (r = 0.85, P less than 0.001). Antibody in the serum was found in 14 of 22 patients with 'idiopathic' thrombocytopenic purpura (ITP), three of four patients with underlying lymphoma and in all five cases of systemic lupus erythematosus (SLE). Four non-thrombocytopenic patients with autoimmune haemolytic anaemia (AIHA) due to IgG on the red cells were also studied and were shown to have no increase in platelet-bound IgG. Our results confirm the work of Dixon et al (1975) that platelet antibody as excess IgG can be readily detected on the surface of platelets in patients with immune thrombocytopenia. The clinical implications of these findings are discussed.

Effects of cortisol on immune cytolysis.

High concentrations of cortisol effectivelyabolish the lysis of sheep red cells induced by cytotoxic antibody and complement <i>in vitro. </i>This effect lies in the ability of the steroid molecules to pack closely among the aliphalic chains of the phospholipid monolayers present in cell membranes, thereby preventing the net uptake of ions by damaged cells and the consequent water influx causing enlargement of the complement holes and escape of hemoglobin.

Immune Hemolysis. II. Studies on the Mechanism of Enhanced Lysis of Sheep Red Cells in a High K+ Medium

Abstract In “Immune Hemolysis. II. Studies on the Mechanism of Enhanced Lysis of Sheep Red Cells in a High K+ Medium„ by John P. Leddy, Patricia A. Thiem, Pierre F. Leblond,Robert I. Weed and Peter K. Lauf in the February 1972 issue of the Journal of Immunology,Volume 108, Number 2, page 476, second column, under Effect of Ouabain, the first sentence should read: “A 10−3 M solution of ouabain (Sigma Chemical Co., St. Louis,Mo.) in Tris-NaCl was prepared at 56° and kept at 37°C prior to use.„

TWO LYTIC FACTORS INVOLVED IN INDUCED SYNERGISTIC HEMOLYSIS IN NORMALLY NONHEMOLYTIC STAPHYLOCOCCI.

Smith, Doyle C. (Kansas State University, Manhattan), V. D. Foltz, and T. H. Lord. Two lytic factors involved in induced synergistic hemolysis in normally nonhemolytic staphylococci. J. Bacteriol. 88:1700-1704. 1964.-Synergistic hemolysis of "normally nonhemolytic" staphylococci results from the combination of two factors: Z, produced by hemolytic staphylococci that elaborate a secondary hemolytic zone on blood-agar, and T, produced by nonhemolytic staphylococci. Factors Z and T are produced in supernatant fluids of broth cultures and are detected by the synergistic hemolysis (SH) test. Complete lysis of sheep red cells is observed in the SH test only when both factors Z and T are present. Production of Z and T is greatly enhanced by the presence of 10 or 20% CO(2) during incubation. Both Z and T are heat-stable, withstanding 100 C for 15 min. Neither factor is precipitated by saturated Na(2)SO(4) or (NH(4))(2)SO(4). Hydrogen ion concentrations between pH 5.0 and 8.5 do not exert any effect on the two factors. Factor Z is produced in greater quantity than factor T. It is suggested that factors Z and T are not proteinaceous, enzymatic, or lipid in nature.