Schistosoma mansoni: eicosanoid production by cercariae

Cercariae of Schistosoma mansoni are stimulated to penetrate skin by certain free fatty acids. The cercariae have an active arachidonate cascade, presumably using host skin essential fatty acids as cascade precursors. Exposing cercariae to 3.3 mM linoleate for 1, 10, and 60 min resulted in production of a wide variety of eicosanoids. Using high-performance liquid chromatography, eicosanoids coeluting with prostaglandin E2, D2, and A2, leukotriene B4, and 5-hydroxyeicosatetraenoic acid standards were identified, as well as unidentified peak positions. Radioimmunoassay confirmed the presence of immunoreactive prostaglandin E1, and E2, and 5- and 15-hydroxyeicosatetraenoic acids in cercarial extracts. No eicosanoid production occurred when cercariae were exposed to 3.3 mM oleate and 1 or 330 microM linoleate. Both high-performance liquid chromatography and radioimmunoassay data indicated that cercariae regulate the production of eicosanoids through time. It is postulated that arachidonate metabolism and subsequent eicosanoid production are required for successful cercarial penetration.     

ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20-59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats ("55" genotype) than in subjects with one deletion (d) (three or four repeats) and one common ("d5" genotype) allele or with two deletion ("dd") alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk.     

Influence of dietary fish on eicosanoid metabolism in man

Two groups of 40 volunteers were given a dietary supplement consisting of 135 g of mackerel or meat (control) paste per day for 6 weeks. Compliance was about 80% in both groups and the daily intake of 20:5(n-3) and 22:6(n-3) from the mackerel supplement was about 1.3 and 2.3 g, respectively. In collagen-activated platelet rich plasma, the potency of blood platelet to produce HHT from arachidonic acid (AA) clearly reduced in the mackerel group, whereas the formation of HHTE from timnodonic acid (TA) increased slightly. Changes in the formation of HHT and HHTE, measured by HPLC, correlated significantly with those of TxB2 and TxB3, respectively, measured by GC/MS. Changes in the formation of the lipoxygenase products HETE (ex AA) and HEPE (ex TA) were qualitatively similar to that seen for the cyclo-oxygenase products, but quantitatively the responses were smaller. Formation of ir TxB2 in clotting blood significantly reduced in the mackerel group. In collagen-activated, citrated whole blood, TxB2 formation tended to be reduced in the mackerel-supplemented volunteers. Mackerel consumption was associated with the formation of considerable amounts of PGI3, as judged from the appearance of 2,3-dinor-delta 17-6-keto-PGF1 alpha in urine. The amount of the major metabolite of PGI2, 2,3-dinor-6-keto-PGF1 alpha was not reduced, or even increased. The daily amount of tetranor prostaglandin metabolites in the urine did not change significantly, which indicates that mackerel supplementation did not alter the formation of prostaglandins E and F.     

Infuence of dietary fish on eicosanoid metabolism in man

Two groups of 40 volunteers were given a dietary supplement consisting of 135 g of mackerel or meat (control) paste per day for 6 weeks. Compliance was about 80% in both groups and the daily intake of 20:5(n−3) and 22:6(n−3) from the mackerel supplement was about 1.3 and 2.3 g, respectively. In collagen-activated platelet rich plasma, the potency of blood platelet to produced HHT from arachidonic acid (AA) clearly reduced in the mackerel group, whereas the formation of HHTE from timnodonic acid (TA) increased slightly. Changes in the formation of HHT and HHTE, measured by HPLC, correlated significantly with those of TxB₂ and TxB₃, respectively, measured by GC/MS. Changes in the formation of the lipoxygenase products HETE (ex AA) and HEPE (ex TA) were qualitatively similar to that seen for the cyco-oxygenase products, but quantitatively the responses were smaller. Formation of ir TxB₂ in clotting blood significantly reduced in the mackerel group. In collagen-activated, citrated whole blood, TxB₂ formation tended to be reduced in the mackerel-supplemented volunteers. Mackerel consumption was associated with the formation of considerable amounts of PGl₃, as judged from the appearance of 2,3-dinor-Δ 17-6-keto-PGF_1α in urine. The amount of the major metabolite of PGl₂, 2,3-dinor-6-keto-PGF_1α was not reduced, or even increased. The daily amount of tetranor prostaglandin metabolites in the urine did not change significantly, which indicates that mackerel supplementation did not alter the formation of prostaglandins E and F.     

Interferon production by human leukaemic leucocytes.

The Sendai virus-induced interferon (IF) production by partially purified human leucocyte suspensions of normal donors and of leukaemic patients have been investigated in vitro. (i) An increased production was observed with leucocytes taken from patients suffering from chronic myelogenous leukaemia (CML) during exacerbation, but the production was approximately normal with cells taken during remission. (ii) IF production was not influenced by large doses of cytostatics (DBM, 5-FU, FUDR, 5-HU, 6-MP, cyclophosphamide) irrespective of whether normal or CML leucocytes were used. (iii) In contrast, production was inhibited in both systems by inhibitors of RNA or protein synthesis (actinomycin D, puromycin, cycloheximide). (iv) CML leucocytes produced IF for a prolonged period of time as compared to normal leucocytes. (v) Leucocytes from children with acute blastic leukaemia and those from adults with chronic lymphoid or acute paramyeloblastic leukaemia produce, in contrast to normal leukocytes, approximately as much IF in the absence as in the presence of serum. It is concluded that the Sendai virus-induced IF synthesis in CML leucocytes requires de novo protein synthesis.     

Regulation of macrophage eicosanoid production by hydroperoxy-and hydroxy-eicosatetraenoic acids.

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the respective products of cyclo-oxygenase- and 5-lipoxygenase-catalysed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE and only relatively small amounts of PGE2. No LTC4 is formed under these conditions. In contrast, resident mouse peritoneal macrophages in cell culture for 4 days synthesized less PGE2 and LTC4 when exposed to zymosan. However, these macrophage populations continue to synthesize 12-HETE from exogenously added arachidonic acid. Zymosan induced the secretion of a lysosomal enzyme, N-acetyl-beta-glucosaminidase, equally in both 1- and 4-day cultures. Both 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs), the precursors of 12- and 15-HETE, were found to be irreversible inhibitors of the cyclo-oxygenase pathway and reversible inhibitors of the 5-lipoxygenase pathway in macrophages. 15-HETE were found to be reversible inhibitors of both pathways. Thus the oxidation of arachidonic oxidation of arachidonic acid to both prostaglandins and leukotrienes may be under intracellular regulation by products of 12- and 15-lipoxygenases.     

Preliminary tests on nisin and pediocin production using waste protein sources. Factorial and kinetic studies

Lactic acid bacteria, the object of current interest as bacteriocin producers, are microorganisms with complex requirements for peptidic sources, making them appropriate indicators for testing the suitability of formulations based on proteinaceous wastes for use as microbiological media. Different peptones obtained from visceral and fish muscle residues promoted growth of lactic acid bacteria when applied individually or in combination. Kinetic parameters and bacteriocin production were similar and, in some cases (pediocin), far superior (>500%) to those obtained with bactopeptones and commercial media specifically recommended for lactic acid bacteria growth. Visceral residues, especially when subjected to a brief process of autohydrolysis at 20 degrees C, were more efficient for bacterial growth than muscle, even when muscle was treated with pepsin.     

Effects of increasing dietary linoleic acid on phospholipid fatty acid composition and eicosanoid production in leucocytes and gill cells of Atlantic salmon (Salmo salar).

Diets containing linoleic acid at 10, 25 and 45% of total dietary fatty acids were fed to three groups of post-smolt Atlantic salmon (Salmo salar) for 18 weeks. Incorporation of linoleic acid into membrane phospholipids of leucocytes and gills increased in response to dietary intake. In general, there was an increase in arachidonic acid and a decrease in eicosapentaenoic acid in the individual phospholipids of both cell types in response to increasing dietary linoleic acid. These changes in eicosanoid precursors were reflected in significantly increased plasma concentrations of 6-keto-PGF1 alpha and TXB2 in salmon given the highest dietary linoleic acid. In whole blood stimulated with the calcium ionophore A23187, LTB4, 12-HETE and TXB2 were significantly increased and 12-HEPE significantly decreased in response to increasing dietary linoleic acid. In isolated gill cells stimulated with A23187, 12-HEPE, 12-HETE, 14-HDHE and TXB2 were all decreased in response to increasing dietary linoleic acid, although the ratio of 12-HEPE/12-HETE was also decreased.     

Increasing fish production from wetlands at Lake Victoria, Uganda using organically manured seasonal wetland fish ponds

The processes driving primary productivity and its impacts on fish production were investigated in field trials in eight seasonal earthen wetland ponds ‘Fingerponds’ (192 m²) in Uganda between 2003 and 2005. The ponds were stocked by the seasonal flood with predominantly Oreochromis spp. at densities ranging from 0.1 to 0.5 fish m⁻². Chicken manure (521, 833 or 1,563 kg ha⁻¹) was applied fortnightly. Results showed that primary productivity was enhanced with maximum average net primary productivity (±Standard Error) of 11.7 (±2.5) g O₂ m⁻² day⁻¹ at the Gaba site and 8.3 (±1.5) g O₂ m⁻² day⁻¹ at the Walukuba site. Net fish yields were higher in manured ponds with up to 2,670 kg ha⁻¹ yield for a 310 day growth period compared to less than 700 kg ha⁻¹ in unmanured ponds. Fish production was limited mainly by high recruitment, falling water levels, light limitation from high suspended solids and turbidity, and low zooplankton biomass. It was concluded that Fingerponds have a high potential for sustainable fish production and can contribute to the alleviation of protein shortages amongst the riparian communities around Lake Victoria. Production can be enhanced further with improved stock management.     

Intraovarian cavity leucocytes of viviparous fish, Neoditrema ransonneti (Perciformes, Embiotocidae)

To obtain basic information on the properties of the intraovarian cavity leucocytes (IOCLs) of the viviparous teleost, Neoditrema ransonneti, morphological characteristics and numerical changes of IOCLs during the reproductive cycle were investigated. In the ovaries of newborn females, leucocytes exuded into the lumen were observed first in November, prior to insemination of semen. These cells were primarily macrophages, neutrophils and lymphocytes. Among them, macrophages were invariably the largest population throughout the reproductive cycle. They began to phagocytize spermatozoa in December, when spermatozoa were first detected in the ovary. The number of IOCLs gradually increased from November in the newborn female. However, this increase is not ascribed to the effect of copulation or the presence of semen, because the number of leucocytes also increased in non-mating fish. While developing embryos were discharged into the ovarian lumen at the latest in January, a number of spermatozoa and spermatozoa-phagocytizing macrophages were seen until March. Even after the extinction of sperm cells, numerous IOCLs remained in the lumen and coexisted with fetuses until their parturition. These results suggest that IOCLs play roles in successful pregnancy, besides elimination of remaining spermatozoa.     

Schistosoma mansoni: pH dependence of cercarial eicosanoid production, penetration, and transformation

The pH dependence of Schistosoma mansoni cercarial penetration and transformation was determined using a gelatin:agar matrix, containing 3mM linoleate, as a penetration substrate. Penetration was largely unaffected by pH, approaching 100% over the pH range of 5.4 to 8.2, while transformation had an optimum pH range between 6.2 and 7.4. Within this pH range, between 74 and 89% of cercariae lost their tails. Outside this range, transformation decreased to 0% above pH 7.8 and dropped to 57% at pH 5.4. Esculetin, a lipoxygenase inhibitor, also incorporated into the agar:gelatin plates at a concentration of 1 mM, had little effect on cercarial penetration, except between pH 6.5 and 6.65, where penetration rates fell to 50% at pH 6.63. Transformation, however, was inhibited by esculetin, except between pH 6.5 and 6.8, where transformation was statistically equivalent to controls (P = 0.064, two-tailed Student's t test). Cercarial eicosanoid production measured at pH 6.55 and 7.2 in the presence and absence of 1 mM esculetin has led to the tentative identification of a 5-lipoxygenase product associated with cercarial penetration: LTB4 or 6-trans LTB4, a breakdown product of LTB4. We discuss the importance of pH control in cercarial experiments as well as the possible modulatory role skin pH (surface, epidermal, and dermal) may have in regulating cercarial eicosanoid production.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Inhibition by adenosine of reactive oxygen metabolite production by human polymorphonuclear leucocytes.

The stimulation of reactive oxygen metabolite production from human polymorphonuclear leucocytes by chemotactic peptide (fMet-Leu-Phe) was inhibited by adenosine with a K0.5 of 0.6 microM. Dipyridamole (0.1 microM), an inhibitor of adenosine uptake, did not prevent the effect of adenosine. Non-metabolizable analogues could substitute for adenosine in the potency order N-ethoxycarboxamideadenosine greater than 2-chloroadenosine greater than adenosine greater than L-N6-(phenylisopropyl)adenosine = D-N6-(phenylisopropyl)adenosine, which is characteristic of an A2 adenosine receptor. The effects of adenosine, 2-chloroadenosine and N-ethoxycarboxamideadenosine were reversed by 8-phenyltheophylline. When endocytosis was inhibited with cytochalasin B, cells were still susceptible to adenosine receptor agonists. 2-Chloroadenosine (10 microM) reduced the activation of respiration in response to chemotactic peptide from 3.3-fold to 1.4-fold. Activation of reactive oxygen metabolite production in response to latex beads was not reversed by adenosine or its analogues. It was concluded that adenosine acts at an A2 adenosine receptor to antagonize the activation of polymorphonuclear leucocytes by those stimuli, such as chemotactic peptide, which cause an increase in the intracellular free Ca2+ concentration.     

The effects of alcoholism and smoking on platelet eicosanoid production in vitro

Ethanol induces changes in eicosanoid synthesis in blood platelets and brain tissue. Cigarette smoking also causes alterations in eicosanoid formation. This preliminary report examined in vitro platelet sonicate eicosanoid production using 14C-arachidonic acid (14C-AA) and in separate experiments, 14C-PGH2, as substrates. Radiometric thin layer chromatography (TLC) was used to identify the products formed. Eicosanoid product formation in platelet sonicates collected from 28 abstinent male alcoholics were compared to those from 11 male control subjects. All but one of the alcoholics were chronic smokers and all control subjects were non-smokers. All smokers abstained from smoking for 12 h prior to the blood collection to control for any acute effects of cigarette smoke on eicosanoid production. Significant reductions in platelet sonicate production of PGD2 and PGE2 in vitro were observed in alcoholic smokers when 14C-PGH2, but not 14C-AA, was the substrate. These reductions were predicted equally well by two variables, smoking and alcoholism, using several statistical models. This is the first investigation that controlled for the acute effects of smoking and accounted for the potential effects of cigarette smoking on platelet eicosanoid production in alcoholics. Because cigarette smoking is prevalent among alcoholics, future studies on the role of eicosanoids in alcoholism should control for smoking.     

Schistosoma mansoni: cercarial eicosanoid production and penetration response inhibited by esculetin and ibuprofen

Cercariae of Schistosoma mansoni produce a wide variety of eicosanoids when stimulated by 3.3 mM linoleate. High-performance liquid chromatography indicated that 10(-5) M esculetin dramatically decreased eicosanoid production by cercariae. Ibuprofen (10(-4) M) also decreased eicosanoid production, but to a lesser extent. These results were confirmed by radioimmunoassay using time-dose curves for esculetin and time curves for ibuprofen. The results reported here indicated that, for cercariae, ibuprofen was neither a specific cyclo-oxygenase inhibitor, as has been reported for platelet and endothelial cells, nor was esculetin a specific inhibitor of lipoxygenase, as has been reported for platelets and mastocytoma cells. Rather, both drugs inhibited cyclo-oxygenase and lipoxygenase enzyme systems. Further, the data indicated that two forms of cyclo-oxygenase exist in cercariae (isozymes?), one initiating the conversion of gamma-dihomolinolenate into the 1-series prostaglandins and another acting on arachidonate forming the 2-series prostaglandins. The cyclo-oxygenase acting on arachidonate has a greater sensitivity to both ibuprofen and esculetin than the enzyme acting on gamma-dihomolinolenate. Cercarial lipoxygenases also varied greatly in their sensitivity to ibuprofen.     

Improved quantification of prostaglandins in biological samples by optimizing simultaneously the relationship eicosanoid/internal standard and using liquid chromatography tandem mass spectrometry

Although a wide variety of articles on quantification of eicosanoids by using internal standards are published every year, little has been done on how much internal standard should be added. This article demonstrates that the application of experimental design enables estimating the interaction eicosanoid/internal standard and to select confidently an optimal amount of internal standard and a response factor (RF) for the analysis of eicosanoids in a high number of samples, where the amount of sample is limited and the unknown levels of eicosanoids are spanned in a wide range of concentrations. The results revealed that the interaction eicosanoid/internal standard is an important factor that affects the validity of the RF and subsequently the accuracy of the analysis.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes.

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection. The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemia leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE2, PGD2, PGF2 alpha, TXB2 and 6-keto-PGF1 alpha. In all types studied PGE2 and TXB2 were the major products formed. The identification of PGE2 and TXB2 was confirmed by GC/MS with multiple ion monitoring. The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.