Antibody responses induced by Trichobilharzia regenti antigens in murine and human hosts exhibiting cercarial dermatitis.

Cercariae of bird schistosomes (genus Trichobilharzia) are able to penetrate the skin of mammals (noncompatible hosts), including humans, and cause a Th2-associated inflammatory cutaneous reaction termed cercarial dermatitis. The present study measured the antibody reactivity and antigen specificity of sera obtained after experimental infection of mice and natural infection of humans. Sera from mice re-infected with T. regenti showed a bias towards the development of antigen-specific IgM and IgG1 antibodies and elevated levels of total serum IgE, indicative of a Th2 polarized immune response. We also demonstrate that cercariae are a source of antigens triggering IL-4 release from basophils collected from healthy human volunteers. Analysis of sera from patients with a history of cercarial dermatitis revealed elevated levels of cercarial-specific IgG, particularly for samples collected from adults (> 14 years old) compared with children (8-14 years old), although elevated levels of antigen-specific IgE were not detected. In terms of antigen recognition, IgG and IgE antibodies in the sera of both mice and humans preferentially bound an antigen of 34 kDa. The 34 kDa molecule was present in both homogenate of cercariae, as well as cercarial excretory/secretory products, and we speculate it may represent a major immunogen initiating the Th2-immune response associated with cercarial dermatitis.

Membrane penetration of cytosolic phospholipase A2 is necessary for its interfacial catalysis and arachidonate specificity.

To determine the mechanism of calcium-dependent membrane binding of cytosolic phospholipase A2 (cPLA2), we measured the interactions of cPLA2 with phospholipid monolayers and polymerizable mixed liposomes containing various phospholipids. In the presence of calcium, cPLA2 showed much higher penetrating power than secretory human pancreatic PLA2 toward anionic and electrically neutral phospholipid monolayers. cPLA2 also showed ca. 30-fold higher binding affinity for nonpolymerized 2, 3-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-1-phosphoglycerol (D-BLPG) liposomes than for polymerized ones where the membrane penetration of protein is significantly restricted. Consistent with this difference in membrane binding affinity, cPLA2 showed 20-fold higher activity toward fluorogenic substrates, 1-O-(1-pyrenedecyl)-2-arachidonoyl-sn-glycero-3-phosphocholine, inserted in nonpolymerized D-BLPG liposomes than the same substrate in polymerized D-BLPG liposomes. Furthermore, cPLA2 showed much higher sn-2 acyl group specificity (arachidonate specificity) and headgroup specificity in nonpolymerized D-BLPG liposomes than in polymerized D-BLPG liposomes. Finally, diacylglycerols, such as 1, 2-dioleoyl-sn-glycerol, selectively enhanced the membrane penetration, hydrophobic membrane binding, and interfacial enzyme activity of cPLA2. Taken together, these results indicate the following: (1) calcium not only brings cPLA2 to the membrane surface but also induces its membrane penetration. (2) This unique calcium-dependent membrane penetration of cPLA2 is necessary for its interfacial binding and substrate specificity. (3) Diacylglycerols might work as a cellular activator of cPLA2 by enhancing its membrane penetration and hydrophobic membrane binding.

A phospholipase A2 kinetic and binding assay using phospholipid-coated hydrophobic beads.

A novel kinetic and membrane-binding assay for phospholipase A2 (PLA2) has been developed utilizing phospholipid-coated hydrophobic styrene-divinylbenzene beads (5.2 +/- 0.3 microm diameter). Phospholipids formed a stable monolayer film on styrene-divinylbenzene beads with average surface packing density of (1.3 +/- 0.2) x 10(-2) molecule/A2. Secretory PLA2 readily hydrolyzed 1-palmitoyl-2-[3H]-oleoyl-sn-glycero-3-phosphoglycerol coated on styrene-divinylbenzene beads which could be easily monitored by measuring the radioactivity of fatty acid released to solution in the presence of bovine serum albumin. For human cytosolic PLA2 with high specificity for sn-2 arachidonyl group, styrene-divinylbenzene beads coated with 1-stearoyl-2-[14C]-arachidonyl-sn-glycero-3-phosphocholine and dioleoylglycerol (7:3, mol/mol) were used as substrate. PLA2 activity was linearly proportional to the enzyme concentration in the range from 1 to 150 nM for human class II secretory PLA2 and from 1 to 20 nM for cytosolic PLA2; the specific activity was 1.6 and 1.7 micromol/min/mg, respectively. Finally, styrene-divinylbenzene beads coated with polymerized 1,2-bis[12-(lipoyloxy) dodecanoyl]-sn-glycero-3-phosphoglycerol were used to measure the membrane binding affinity of PLA2, which in conjunction with kinetic data provides important insights into how PLA2 interacts with membranes.