Characterization of the expression and inflammatory activity of NADPH oxidase after spinal cord injury.

Reactive oxygen species (ROS) and the NADPH oxidase (NOX) enzyme are both up-regulated after spinal cord injury (SCI) and play significant roles in promoting post-injury inflammation. However, the cellular and temporal expression profile of NOX isotypes, including NOX2, 3, and 4, after SCI is currently unclear. The purpose of this study was to resolve this expression profile and examine the effect of inhibition of NOX on inflammation after SCI. Briefly, adult male rats were subjected to moderate contusion SCI. Double immunofluorescence for NOX isotypes and CNS cellular types was performed at 24 h, 7 days, and 28 days post-injury. NOX isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on injury status. NOX2 and 4 were found in all cell types assessed, while NOX3 was positively identified in neurons only. NOX2 was the most responsive to injury, increasing in both microglia and astrocytes. The biggest increases in expression were observed at 7 days post-injury and increased expression was maintained through 28 days. NOX2 inhibition by systemic administration of gp91ds-tat at 15 min, 6 h or 7 days after injury reduced both pro-inflammatory cytokine expression and evidence of oxidative stress in the injured spinal cord. This study therefore illustrates the regional and temporal influence on NOX isotype expression and the importance of NOX activation in SCI. This information will be useful in future studies of understanding ROS production after injury and therapeutic potentials.

Measurement of PAF in blood by radioimmunoassay. Examination of interfering factors and problems involved in accurate quantification.

A recently developed radioimmunoassay for PAF was applied to blood extracts with the aims of defining and overcoming problems that lead to erroneous results and establishing optimum conditions for the accurate determination of PAF levels. The high lipid content of blood was found to interfere with the assay, and it appeared that phosphatidylcholine and/or sphingomyelin might be among the lipids responsible. Interference was eliminated by either dilution or preparative TLC of blood extract prior to RIA although dilution is unlikely to be generally useful due to the low amounts of PAF normally present in human blood. Lipid extraction of whole blood followed by preparative TLC proved to be necessary in the preparation of samples prior to performance of the RIA. The problems encountered in the measurement of PAF levels in blood by RIA highlight the importance of determining the correct method of sample preparation for any tissue/fluid prior to its inclusion in the assay.

Quantitation by radioimmunoassay of PAF in human saliva.

Approximately 75% of the PAF present in saliva is recovered on extraction of whole saliva (0.8 vol) with chloroform/methanol/water (2:2:1, v/v/v). PAF levels, determined by our recently developed radioimmunoassay, in saliva extracts ranged from 0.5-21 ng/mL with 59% between 2-6 ng/mL. These figures, for apparently healthy subjects, are higher than previously reported levels obtained by platelet assays. The validity of our radioimmunoassay results was checked by isolating and quantitating the PAF fraction from whole saliva. In addition, when we examined our saliva samples by platelet aggregation, low levels of PAF, comparable with the values found in the literature, were detected. Investigations revealed the presence of a substance(s) which inhibited PAF-induced platelet aggregation but which did not affect the radioimmunoassay.

Antibodies to platelet-activating factor (PAF) inhibit PAF-induced platelet aggregation.

Platelet-activating factor (PAF) is the most potent platelet agonist known. PAF-induced platelet aggregation was blocked by pre-incubation of PAF with the immunoglobulin fraction from sheep or rabbit anti-PAF anti-serum. Inhibition was specific for PAF and was dependent upon immunoglobulin and PAF concentrations. Antibody-mediated inhibition of PAF-activity may be a valuable method for studying the biological effects of PAF in some systems.

Stability of platelet activating factor (PAF) in human saliva. Quantitation by radioimmunoassay.

Platelet activating factor (PAF) is thought to mediate many inflammatory processes and its involvement in health and disease may be clarified by examining PAF levels in human secretions. The known presence of PAF, the ease of obtaining samples and the relative stability of PAF in saliva, makes this fluid a preferred source for examination of PAF in health and disease. The activity of PAF-acetylhydrolase (the PAF degrading enzyme) in saliva was 1,000-fold lower than that found in human plasma. Extraction of saliva with chloroform/methanol/water resulted in 70-90% recovery of PAF. Using the radioimmunoassay (RIA), PAF levels in the range 0.5-21 ng/ml were found in normal human salivas. These values were significantly higher than those reported from bioassay studies based on washed platelets. The validity of the RIA was checked by isolating and quantitating the PAF fraction from whole saliva extract, and by treatment of the extracts with the enzyme phospholipase A2. Direct comparison of salivary PAF levels, determined by both platelet aggregation (PA) and RIA confirmed our original finding that values obtained were lower using the bioassay method. Furthermore, these bioassay values compared favourably with those in the literature. Investigations revealed the presence of a substance(s) in saliva which inhibited PAF-induced platelet aggregation but which did not affect the radioimmunoassay.

Examination for platelet-activating factor production by preimplantation mouse embryos using a specific radioimmunoassay.

A specific and highly sensitive radioimmunoassay was used to measure platelet-activating factor (PAF) production by preimplantation mouse embryos in vitro. Levels of PAF greater than 1 pg per embryo were not observed in 24-h culture medium from 2-cell embryos, compacted morulae or blastocysts, or in extracts from these embryos. Synthetic PAF added to embryos at the start of culture could be almost totally recovered after the incubation period, indicating negligible degradation of PAF during culture. PAF was also not detected in embryo samples using a washed rabbit-platelet aggregation assay. It can be concluded that mouse embryos do not produce substantial levels of PAF, or any of the biologically active analogues of PAF detected by the assay.

Combining site specificities of rabbit antibodies to platelet-activating factor (PAF).

Anti-PAF sera from six different rabbits, immunized with C12- or C6-PAF as immunogen, were examined in hapten inhibition experiments in an attempt to define the fine structural recognition specificities of the antibody combining sites. Using a selection of naturally occurring lipids and PAF analogues, no significant cross-reactivity was observed with the lipids or with the inactive metabolite, lyso-PAF. Comparison of the structural specificity requirements of the antibodies from each rabbit showed some heterogeneity, with one antiserum demonstrating a different recognition specificity at position 1 on the glycerol backbone of the PAF molecule. A second rabbit antiserum showed a large degree of tolerance for analogues with increasing acyl chain length at position 2. In general, an ether group at position 1 and an acetyl at position 2 were required for inhibitory activity and a degree of tolerance was demonstrated at position 3, where the main structural requirement was for one or more methyl groups on the nitrogen atom of the phosphocholine moiety.

Inhibitory effects of (S)-3-chlorolactaldehyde on the metabolic activity of boar spermatozoa in vitro.

(S)-alpha-Chlorohydrin and 3-chloro-1-hydroxyacetone inhibited the oxidative metabolism of fructose by boar spermatozoa only after a period of incubation in which they presumably underwent conversion to (S)-3-chlorolactaldehyde, an inhibitor of sperm glyceraldehyde 3-phosphate dehydrogenase. With glycerol as substrate, 3-chloro-1-hydroxyacetone had a similar effect, (S)-alpha-chlorohydrin was ineffective while (R,S)-3-chlorolactaldehyde was immediately effective with either substrate. All three compounds caused the accumulation of fructose 1,6-bisphosphate and dihydroxyacetone phosphate from fructose but not from glycerol which led to the conclusion that inhibition of triosephosphate isomerase was also associated with the anti-glycolytic action of (S)-3-chlorolactaldehyde. (S)-3-Chlorolactaldehyde caused the depletion of ATP in incubates of boar spermatozoa metabolizing fructose but not glycerol. This suggests that futile substrate cycling may play an important function in the anti-glycolytic action of (S)-3-chlorolactaldehyde and/or that boar spermatozoa can synthesize ATP from glycerol by a mechanism not involving the glycolytic pathway.

Inhibition of triosephosphate isomerase in boar spermatozoa by (S)-3-chlorolactaldehyde.

Mature epididymal boar spermatozoa converted fructose and glycerol to carbon dioxide but in the presence of 3-chloro-1-hydroxyacetone these oxidations were inhibited. When the substrate was fructose, there was an increase in the amounts of fructose 1,6-bisphosphate and dihydroxyacetone phosphate but these glycolytic intermediates did not accumulate when glycerol was the substrate. Examination of enzyme activities in mature boar spermatozoa incubated with 3-chloro-1-hydroxyacetone, which is metabolised in vitro to (S)-3-chlorolactaldehyde, confirmed that glyceraldehyde 3-phosphate dehydrogenase and triosephosphate isomerase were both inhibited to equivalent degrees by this metabolite.