High-fat, high-fructose, high-cholesterol feeding causes severe NASH and cecal microbiota dysbiosis in juvenile Ossabaw swine.

Pediatric obesity and nonalcoholic steatohepatitis (NASH) are on the rise in industrialized countries, yet our ability to mechanistically examine this relationship is limited by the lack of a suitable higher animal models. Here, we examined the effects of high-fat, high-fructose corn syrup, high-cholesterol Western-style diet (WD)-induced obesity on NASH and cecal microbiota dysbiosis in juvenile Ossabaw swine. Juvenile female Ossabaw swine (5 wk old) were fed WD (43.0% fat; 17.8% high-fructose corn syrup; 2% cholesterol) or low-fat diet (CON/lean; 10.5% fat) for 16 wk ( n = 6 each) or 36 wk ( n = 4 each). WD-fed pigs developed obesity, dyslipidemia, and systemic insulin resistance compared with CON pigs. In addition, obese WD-fed pigs developed severe NASH, with hepatic steatosis, hepatocyte ballooning, inflammatory cell infiltration, and fibrosis after 16 wk, with further exacerbation of histological inflammation and fibrosis after 36 wk of WD feeding. WD feeding also resulted in robust cecal microbiota changes including increased relative abundances of families and genera in Proteobacteria ( P < 0.05) (i.e., Enterobacteriaceae, Succinivibrionaceae, and Succinivibrio) and LPS-containing Desulfovibrionaceae and Desulfovibrio and a greater ( P < 0.05) predicted microbial metabolic function for LPS biosynthesis, LPS biosynthesis proteins, and peptidoglycan synthesis compared with CON-fed pigs. Overall, juvenile Ossabaw swine fed a high-fat, high-fructose, high-cholesterol diet develop obesity and severe microbiota dysbiosis with a proinflammatory signature and a NASH phenotype directly relevant to the pediatric/adolescent and young adult population.

Effects of Elderberry Juice from Different Genotypes on Oxidative and Inflammatory Responses in Microglial Cells.

Many species of berries are nutritious food and offer health benefits. However, among the different types of berries, information on health effects of American elderberries (Sambucus nigra subsp. canadensis) has been lacking and little is known about whether elderberry consumption can confer neuroprotective effects on the central nervous system. Microglial cells constitute a unique class of immune cells and exhibit characteristic properties to carry out multifunctional duties in the brain. Activation of microglial cells has been implicated in brain injury and in many types of neurodegenerative diseases. Our recent studies demonstrated the ability for endotoxin (lipopolysaccharide, LPS) and interferon gamma (IFNγ) to induce reactive oxygen species (ROS) and nitric oxide (NO) in murine microglial cells (BV-2) through activating NADPH oxidase and the MAPK pathways. In this study, BV-2 microglial cells were used to examine effects of elderberry juice obtained from different genotypes on oxidative and inflammatory responses induced by LPS and IFNγ. Results show that 'Wyldewood' extract demonstrated antioxidant properties by inhibiting IFNγ-induced ROS production and p-ERK1/2 expression. On the other hand, most juice extracts exerted small effects on LPS-induced NO production and some extracts showed an increase in NO production upon stimulation with IFNγ. The disparity of responses on ROS and NO production from different extracts suggests possible presence of unknown endogenous factor(s) in the extract in promoting the IFNγ-induced iNOS synthesis pathway.

Determination of Anthocyanins and Total Polyphenols in a Variety of Elderberry Juices by UPLC-MS/MS and Other Methods.

Elderberry (Sambucus spp.) juice contains a variety of polyphenols, mostly anthocyanins. In order to understand the variation of polyphenol levels by genotype, various elderberry juice samples were analyzed for total phenolics (TP), total monomeric anthocyanins (TMA) and individual anthocyanin content. The Folin-Ciocalteu total phenolic method and pH differential method were used to measure the TP and TMA content, respectively. The TP and TMA concentrations of elderberry were found to vary greatly among different genotypes. TMA content varied from 2.1% for 'Sperandio' to 60.6% for the 'Bob Gordon' cultivar. In addition, ultra-performance liquid chromatography with triple quadrupole mass spectrometry was used to separate and detect individual anthocyanins from samples prepared by solid phase extraction. Multiple-reaction-monitoring was used to process data for the reduction of false positives, maximizing selectivity, and reliable quantification. The quantitative performance of the method was validated, and a detection limit of 0.3 ng·ml-1 for cyanidin 3-O-glucoside was determined. This newly developed method may serve to characterize and profile various anthocyanins in elderberry juices for quality control, assessment of dietary intake, and anthocyanin-based biomedical studies.

Sterculic oil, a natural inhibitor of SCD1, improves the metabolic state of obese OLETF rats.

OBJECTIVE: Abnormal lipid metabolism and excess accumulation of lipid in non-adipose tissues are defining characteristics of obesity and its comorbidities. Expression and/or activity of stearoyl-CoA desaturase-1 (SCD1), a major regulator of lipid metabolism, is increased with obesity and the reduction/ablation of this enzyme is associated with an improved metabolic profile. Sterculic oil (SO), obtained from the seeds of the Sterculia feotida tree, contains a high concentration of cyclopropenoic fatty acids which are known inhibitors of SCD1. The purpose of this study was to determine the effects of SO supplementation on the development of obesity and insulin resistance in hyperphagic, obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. DESIGN & METHODS: Rats received either an AIN-93G diet (control) or an AIN-93G diet containing 0.5% SO for 10 weeks. RESULTS: SO did not alter body weight or body composition. Importantly, the desaturase indices, a proxy for the activity of SCD1, were reduced in the liver and adipose tissue of SO supplemented animals. This reduction in SCD1 activity was associated with a reduction in fasting blood glucose concentrations and improved glucose tolerance. In addition, SO reduced intra-abdominal fat mass and adipocyte size and resulted in a ∼3-fold increase in GLUT1 gene expression in intra-abdominal fat. Liver triglyceride content and lipogenic gene expression were reduced by SO. Consistent with an improved metabolic phenotype, SO also improved plasma cholesterol, LDL-cholesterol, and triglyceride concentrations. CONCLUSION: Overall, our data demonstrate an improved metabolic phenotype with SO supplementation and suggest further studies are required to better understand the therapeutic potential of SO.

Comparative genomics of bacteria from the genus Collimonas: linking (dis)similarities in gene content to phenotypic variation and conservation.

Collimonas is a genus of soil bacteria comprising three recognized species: C. fungivorans, C. pratensis and C. arenae. Collimonads share the ability to degrade chitin (chitinolysis), feed on living fungal hyphae (mycophagy), and dissolve minerals (weathering), but vary in their inhibition of fungi (fungistasis). To better understand this phenotypic variability, we analysed the genomic content of four strains representing three Collimonas species (Ter14, Ter6, Ter91 and Ter10) by hybridization to a microarray based on reference strain C. fungivorans Ter331. The analysis revealed genes unique to strain Ter331 (e.g. those on the extrachromosomal element pTer331) and genes present in some but not all of the tested strains. Among the latter were several candidates that may contribute to fungistasis, including genes for the production and secretion of antifungals. We hypothesize that differential possession of these genes underlies the specialization of Collimonas strains towards different fungal hosts. We identified a set of 136 genes that were common in all tested Collimonas strains, but absent from the genomes of three other members of the family Oxalobacteraceae. Predicted products of these 'Collimonas core' genes include lytic, secreted enzymes such as chitinases, peptidases, nucleases and phosphatases with a putative role in mycophagy and weathering.

[The phantom and stump phenomena interview (PSPI)]. / Ein strukturiertes Interview zur Erfassung von Phantom- und Stumpfphänomenen nach Amputation.

RESEARCH QUESTION: A structured German-language interview was developed for the assessment of painful and non-painful phantom and stump phenomena after amputation. The aim was a thorough assessment of the quality, quantity and time course of these phenomena, which is of scientific as well as therapeutic relevance. METHODS: Each phenomenon was assessed using visual analogue scales as well as qualitative descriptors adapted from the McGill Pain Inventory and from literature reports. The factor structure and psychometric properties of the interview were evaluated in a sample of 139 upper and lower limb amputees. Test-retest coefficients were obtained in a subset of 20 amputees. RESULTS: As expected, all pain-related scales showed a two-dimensional internal structure with the factors "affective pain" and "sensory pain". For the non-painful phantom sensations, three factors "general/kinesthetic phantom sensations", "phantom movements" and "paresthesias" were obtained, while for non-painful stump sensations only one general factor emerged. The internal consistency was high with respect to the pain-related scales and was still satisfying for the scales that cover nonpainful phantom and stump phenomena. All scales have sufficient validity. Test-retest coefficients suggest a satisfactory stability of all scales that assess present phenomena, while the stability of the retrospective scales is markedly lower and in some cases insufficient. CONCLUSIONS: The phantom and stump phenomena interview is a highly reliable and valid instrument to assess present perceptual phenomena after amputation. Only the included retrospective scales apparently show low stability scores over time. This raises the more general question of the validity of retrospective pain reports.

Consumption of eicosapentaenoic acid and docosahexaenoic acid impair murine interleukin-12 and interferon-gamma production in vivo.

In mice, individual dietary omega-3 polyunsaturated fatty acids n-3 (PUFA) were found to be sufficient to effect the changes in circulating interleukin (IL)-12 and interferon (IFN)-gamma levels that were previously seen in fish oil-fed mice. Weanling female C3H mice were fed one of five experimental diets. All five diets met all known nutritional requirements for mice and differed only in the fat source. After 4 weeks, mice were challenged with live Listeria monocytogenes or sterile PBS. Twenty-four hours after infection, n-3 PUFA-fed mice had significantly lower circulating IL-12 p70 and IFN-gamma than mice fed the control diet (P<.01). In addition, splenic cytokine mRNA for IL-12 p40, tumor necrosis factor-alpha, and IL-1beta were lower in infected mice fed n-3 PUFA-containing diets than in mice fed the olive oil ethyl esters control diet. The reduction of IL-12 and IFN-gamma production by n-3 PUFA may have important implications for host infectious disease resistance.

The individual and combined effects of fumonisin B1 and moniliformin on performance and selected immune parameters in turkey poults.

Effects of feeding diets containing fumonisin B1 (FB1) and moniliformin (M), singly or in combination, on performance and immune response were evaluated in poults. Day-old poults were randomly assigned to one of four dietary treatments with four replicates of four poults each. Dietary treatments were 1) control; 2) 200 mg FB1, 0 mg M/kg diet; 3) 0 mg FB1, 100 mg M/kg diet; and 4) 200 mg FB1, 100 mg M/kg diet. In Experiment 1, poults were injected with 0.25 mL Newcastle disease virus (NDV) vaccine on Weeks 2 and 3 of the experiment, and anti-NDV antibody titers were measured 7 d after each injection. Compared with controls, poults fed FB1 had significantly lower (P < 0.05) secondary antibody response. Poults fed M and the combination of FB1 and M had significantly lower (P < 0.05) primary and secondary antibody response. Lower relative thymus weights were observed in poults fed diets containing FB1 or M. Decreased relative bursa and spleen weights were observed in poults fed M. In Experiment 2, poults were placed on dietary treatments for 3 wk. On Day 21, 2 x 10(6) peripheral lymphocytes were incubated with mitogens. Poults fed diets containing FB1 had a significantly lower (P < 0.05) proliferative response to mitogens in comparison to controls. In Experiment 3, poults were placed on the diets for 3 wk and were injected with 4.4 x 10(7) E. coli/kg body weight on Day 21. Significantly higher (P < 0.05) numbers of E. coli colonies were observed in the blood and tissue homogenates of poults fed M. In all three experiments, feed intake and body weight gains were significantly lower (P < 0.05) in turkeys fed diets containing M. Data from the present study suggest that FB1 and M are immunosuppressive in poults and that M not only suppresses immune response but also performance. However, neither synergistic nor additive effects between FB1 and M were observed for any of the parameters measured.

Effects of moniliformin on performance and immune function of broiler chicks.

Three trials were conducted to evaluate the effect of moniliformin (M) on performance and immune function in chicks. Day-old chicks were randomly assigned to four dietary treatments (0, 50, 75, or 100 mg M/kg diet). In Trial 1, chicks were placed on treatments for 3 wk and were injected intravenously with 4.6 x 10(6) Escherichia coli on Day 21. Blood samples were collected at 60, 120, and 180 min after inoculation, and liver, spleen, and lung were collected at 180 min postinjection. Compared with control chicks, chicks fed 75 and 100 mg M/ kg diet had higher (P < 0.05) numbers of E. coli colonies in the circulation, liver, and spleen. In Trial 2, chicks were placed on diets for 4 wk and were injected with 0.5 mL Newcastle disease virus (NDV) vaccine intramuscularly on Weeks 2 and 3 of the experiment. The primary and secondary anti-NDV antibody titers were measured 7 d after each injection. Chicks fed 100 mg M/kg diet had lower (P < 0.05) secondary antibody titers than did control chicks. In Trial 3, lymphocyte proliferation in chicks exposed to M in vivo and in vitro was determined. Results of the in vivo study showed that cell proliferation in response to mitogens from control- and M-fed chicks did not differ (P > 0.05). For the in vitro study, lymphocyte proliferation decreased linearly (P < 0.01) with increased concentrations of M. In all three trials, chicks fed 100 mg M/kg diet had lower (P < 0.05) feed intake and weight gain than did control chicks. Data from the current study suggested that M decreased performance and immune response in chicks at the level of 75 mg/kg diet.

Effects of fumonisin B1 on selected immune responses in broiler chicks.

Three experiments were conducted to evaluate immune responses in chicks fed fumonisin B1 (FB1). Day-old male chicks were randomly allotted to dietary treatments: 0, 50, 100, or 200 mg FB1/kg diet. In Experiment 1, chicks were fed diets for 3 wk and were injected intravenously with 4.6x10(6) Escherichia coli on Day 21. Blood samples were collected at 60, 120, and 180 min postinjection, and liver, spleen, and lung were collected after 180 min. Chicks fed 200 mg FB1/kg diet had significantly higher numbers of bacterial colonies in blood, spleen, and liver (P<0.05) than control chicks. In Experiment 2, chicks were placed on the diets for 4 wk and were injected with 0.5 mL inactivated Newcastle Disease virus vaccine on Weeks 2 and 3 of the experiment, and primary and secondary antibody titers were measured 7 d after each injection. The secondary antibody response in chicks fed 200 mg FB1/kg diet was significantly lower (P<0.05) than that of control chicks. In Experiment 3, lymphocyte proliferation in chicks exposed to FB1 in vivo or in vitro was determined. Results of the in vivo study showed that cell proliferation in response to mitogens was lower (P<0.05) in chicks fed 200 mg FB1/kg diet than in control chicks. For the in vitro study, cell proliferation was lower (P<0.05) when cells were exposed to > or = 2.5 microg FB1/mL. Data of the current study suggested that FB1 is immunosuppressive in chicks when present in the ration at 200 mg FB1/kg diet.

Effect of vitamin E and selenium on iron utilization in neonatal pigs.

Supplying adequate iron (Fe) to neonatal pigs to support normal growth and hematological and antioxidant status, while preventing iron toxicity, is a challenge for producers. Three experiments were conducted to determine the effect of frequency and route of Fe administration with or without vitamin E (E) and selenium (Se) on growth, Fe, and antioxidant status of neonatal pigs. In Exp. 1, 12 pigs from dams with reduced E status were fed a semipurified diet without added Fe from d 3 to d 14 of age. At d 6 of age, pigs received the following i.m. injections: 1) FE, 1 mL containing 200 mg of Fe (iron dextran); 2) FEE, treatment FE plus 1 mL containing 300 IU of vitamin E (d-alpha tocopherol); or 3) FESEE, 1.03 mL containing 200 mg of Fe (iron dextran), .15 mg of Se (sodium selenite), and 15 IU of vitamin E (d-alpha tocopherol). Pigs were weighed daily and blood was collected at 3, 7, and 14 d of age. From d 8 to 14, growth was depressed (P < .05) in pigs injected with FESEE. At 14 d of age, pigs injected with FE or FEE had increased (P < .05) hemoglobin (Hb) concentration. Ceruloplasmin activity (CP) was greater (P < .05) at d 7 of age than at d 3 or 14 regardless of treatment. In Exp. 2, 3-d-old pigs (n = 94) received the following: 1) FE, 200 mg Fe (iron dextran) i.m.; (2) FEE, treatment FE plus 300 IU vitamin E i.m.; 3) EFE, 300 IU vitamin E i.m. followed by 200 mg Fe (iron dextran) i.m. 24 h later; or 4) OFE, 100 mg Fe and 10 mg Cu orally. On d 21 of age, one-half of the pigs in each treatment received a second dose of their respective treatment. Blood samples (n = 60) were obtained on d 3 and 21 of age. Pigs injected with FE, FEE, or EFE had greater (P < .05) Hb at d 21 than pigs given OFE. Copper/zinc superoxide dismutase (Cu/ZnSOD) activity was greater (P < .05) at d 21 with OFE than with the other treatments. At 65 d of age, ADG did not differ among treatments. In Exp. 3, pigs (n = 150, in three farrowing groups) were injected with 200 mg of Fe (iron dextran) on d 1 or d 1 and 14. Blood samples were obtained on d 7 and 21 of age. Hemoglobin concentration on d 21 was improved equally by both treatments. Catalase and Cu/ZnSOD activities were increased (P < .05) on d 21 of the experiment compared with d 7 regardless of treatment. Growth was not affected by injection frequency. Results from these experiments indicate that one Fe injection (200 mg) for pigs from sows fed adequate vitamin E will result in adequate growth and hemoglobin concentration with today's improved genetics.

Defluvibacter lusatiae gen. nov., sp. nov., a new chlorohenol-degrading member of the alpha-2 subgroup of proteobacteria.

The two Gram-negative bacterial strains S1 and S4 were isolated from activated sludge of an industrial waste water treatment plant and exhibited a stable capability to degrade 2,4-dichlorophenol, 4-chloro-2-methylphenol, 4-chlorophenol and phenol. The cells were short rods with a polar flagellum, being mesophilic, strictly aerobic, oxidase-positive, and chemoorganotrophic. They utilized a range of amino acids, but only a restricted number of carbohydrates. Reassociation experiments with DNA from strains S1 and S4 revealed high interstrain similarity, indicating, that both strains belong to the same species. The phylogenetic position was determined by comparison of the almost complete 16S rDNA sequence of strain S1 with sequences of related bacteria. Strain S1 clustered with members of the alpha-2 subgroup of the Proteobacteria by forming a separate lineage within the radiation of Mesorhizobium, Phyllobacterium and Sinorhizobium. Both strains can be differentiated from members of related taxa by a set of physiological and chemotaxonomic properties including the ability to grow with norvaline, L-tryptophan, putrescine, glutarate and malonate, and by the presence of spermidine as major polyamine and of 12:0 3OH as fatty acid. Strain S1 is described as type strain of a new species and assigned to a new genus with the proposed name Defluvibacter lusatiae.

Dietary omega-3 polyunsaturated fatty acids from fish oil reduce interleukin-12 and interferon-gamma production in mice.

The objective of this study was to investigate the impact of feeding mice a diet rich in n-3 polyunsaturated fatty acids (PUFA) from fish oil on the interleukin-12 (IL-12) and interferon-gamma (IFNgamma) production during the early stage of an infectious challenge with Listeria monocytogenes. Weanling female C3H/HeN mice were fed AIN-93G experimental diets containing 20%, by weight one of three fat sources: lard (low PUFA), soybean oil (n-6 PUFA) or a mixture (9:1) of menhaden fish oil and corn oil (n-3 PUFA). After 4 weeks, mice were injected intraperitoneally with 10(5) Listeria monocytogenes and the concentration of IL-12(p70) and IFNgamma in serum was determined 24 h post-infection by ELISA. IL-12p35, IL-12p40 mRNA, and IFNgamma mRNA in the spleen were quantified by RNase protection assay. The number of IFNgamma-producing cells in the spleen was determined by flow cytometry using an intracellular staining procedure. We found that n-3 PUFA-fed mice had lower levels of circulating IL-12 at 24 h post-infection than n-6 PUFA- or low PUFA-fed mice (9.7+/-3.4 pg/ml vs. 61.6+/-10.6, and 44.4+/-12.5 pg/ml, respectively; P=0.002, n = 10/trt). The level of IL-12 p35 mRNA did not significantly differ among dietary treatment groups. However, IL-12p40 mRNA was significantly lower in n-3 PUFA- and n-6 PUFA-fed mice compared to low-PUFA-fed mice. Further, the n-3 PUFA group also had the lowest circulating IFNgamma (4.4+/-1.8 ng/ml vs. 9.1+/-1.0, and 9.7+/-2.1 ng/ml, respectively; P = 0.007. n = 8-10/trt). The n-3 PUFA-fed mice had significantly lower IFNgamma mRNA in their spleens compared to the mice fed the other fat sources. In agreement with having lower circulating IFNgamma and lower splenic IFNgamma mRNA, n-3 PUFA-fed mice had a significantly lower percentage of IFNgamma-producing cells in their spleens compared with the n-6 PUFA-fed group (2.1+/-0.6 vs. 4.2+/-0.7%; P = 0.037, n = 10/trt). In summary, feeding mice a diet rich in n-3 PUFA from fish oil significantly lowered the production of both IL-12 and IFNgamma during the early phase of a Listeria infection.

Dietary omega-3 polyunsaturated fatty acids reduce IFN-gamma receptor expression in mice.

Enrichment of immune cells in vivo or in vitro with omega-3 polyunsaturated fatty acid (n-3 PUFA) has been reported to diminish their response to interferon-y (IFN-gamma). We hypothesized that the n-3 PUFA-induced hyporesponsiveness to IFN-gamma is mediated, in part, by a reduction in the number of IFN-gamma receptors (IFNGR) expressed on the surface of these cells. To test this hypothesis, we fed mice experimental diets containing low or high amounts of n-3 PUFA. Thioglycollate-elicited peritoneal macrophages (PEC) were collected and tested for binding and internalization of [125I]-labeled recombinant murine IFN-gamma. High n-3 PUFA intake was associated with a significant (n = 2, p < 0.01) reduction in [125I]-IFN-gamma binding without affecting binding affinity (Kd). When studies were performed at 37 degrees C, high n-3 PUFA intake reduced internalization of [125I]-rmIFN-gamma by 20%-30% (n = 2,p < 0.001). Results from flow cytometric analysis of IFNGR-1 expression on the surface of murine splenocytes were in agreement with the binding studies. Further, total cellular IFNGR-1 from PEC and splenocytes was examined via immunoprecipitation and Western blotting. High n-3 PUFA diet was associated with a 50% decline (n = 3-6, p < 0.05) in total IFNGR-1 in both immune cell populations studied. These data suggest that reduced IFNGR expression may be responsible for immune cell hyporesponsiveness to IFN-gamma, which may, in part, explain some of the immunomodulatory and anti-inflammatory effects associated with the consumption of diets high in n-3 PUFA.

Isolation and characterization of a calendic acid producing (8,11)-linoleoyl desaturase.

For the biosynthesis of calendic acid a (8,11)-linoleoyl desaturase activity has been proposed. To isolate this desaturase, PCR-based cloning was used. The open reading frame of the isolated full-length cDNA is a 1131 bp sequence encoding a protein of 377 amino acids. For functional identification the cDNA was expressed in Saccharomyces cerevisiae, and formation of calendic acid was analyzed by RP-HPLC. The expression of the heterologous enzyme resulted in a significant amount of calendic acid presumably esterified within phospholipids. The results presented here identify a gene encoding a new type of (1,4)-acyl lipid desaturase.

Fish oil source differentially affects rat immune cell alpha-tocopherol concentration.

We have previously reported that both the source of dietary fish oil and the chemical form of vitamin E supplied in the diet affect the vitamin E status of immune cells in rats. The purpose of this study was to investigate further the effect of fish oil source on immune cell vitamin E status using free alpha-tocopherol (alpha-T) at the AIN recommended level as the sole source of vitamin E. Sixty weanling female rats were fed semipurified, high fat (20 g/100 g) diets containing either tocopherol-stripped lard (LRD), menhaden fish oil (MFO), sardine fish oil (SRD) or cod liver oil (CLO) as the primary lipid source. Endogenous alpha-T concentration was measured and equalized to 150 mg/kg oil by addition of free RRR-alpha-T to each lipid source, allowing for a final concentration of alpha-T in the mixed diet of 30 mg/kg. An additional group of rats was fed LRD without supplemental vitamin E (LRD-) as a negative control. After feeding experimental diets for 5 or 10 wk, tissues were collected for alpha-T analysis by HPLC. After 5 wk, plasma and liver alpha-T (micromol alpha-T/g lipid) were significantly lower in SRD- and CLO-fed rats compared with LRD-fed rats. At 10 wk, only plasma alpha-T in CLO-fed rats remained significantly depressed. Plasma and liver alpha-T concentrations (micromol alpha-T/g lipid) were not significantly lower in MFO-fed rats than LRD-fed rats at either time point. Compared with LRD, feeding MFO to rats for 5 or 10 wk resulted in significantly greater alpha-T content of immune cells. In similar fashion, SRD-fed rats, compared with LRD-fed rats, also had significantly greater alpha-T content in splenocytes at both time points and greater thymocyte alpha-T at 10 wk. In all instances, the alpha-T status of rats fed CLO was indistinguishable from that of rats fed the vitamin E-free diet (LRD-). These data further demonstrate the complexity of the relationship between vitamin E status and dietary (n-3) polyunsaturated fatty acids (PUFA).

Dietary fish oil enhances circulating interferon-gamma in mice during listeriosis without altering in vitro production of this cytokine.

The objective of this study was to investigate the impact of feeding mice a diet rich in n-3 polyunsaturated fatty acids (PUFA) from fish oil on the interferon-gamma (IFN-gamma) response during an active infection with Listeria monocytogenes. Weanling female C3H/Hen mice were fed experimental diets containing 20% by weight one of the following fats: soybean oil, lard, or a mixture of menhaden fish oil and corn oil (17:3, w/w). After 4 weeks, mice were injected with 10(5) live L. monocytogenes, and the concentration of IFN-gamma in serum and spleen was determined 0, 2, 4, and 7 days postinfection by enzyme-linked immunosorbent assay (ELISA). Fish oil-fed mice showed significantly higher IFN-gamma in their blood at 2 and 4 days postchallenge compared with mice fed the soybean oil-containing or lard-containing diets (p < 0.001). A higher concentration of IFN-gamma was also found in the spleen homogenate of fish oil-fed mice on day 4 postchallenge (p < 0.005). To examine in vitro IFN-gamma production, splenocytes were isolated from fish oil-fed and soybean oil-fed mice on day 4 postchallenge and cultured with concanavalin A (1 microgram/ml and 10 micrograms/ml) for 24 and 48 h. There were no significant differences in the IFN-gamma concentration in cell culture supernatants between these diet treatments. This study demonstrated that the elevation in the concentration of IFN-gamma in blood and spleen during murine listeriosis is accentuated and prolonged by dietary n-3 PUFA, and these effects may not be due to changes in IFN-gamma production.

Effects of thermal environment on response to acute peripheral lipopolysaccharide challenge exposure in neonatal pigs.

OBJECTIVE: To evaluate effects of thermal environment on response to acute peripheral lipopolysaccharide (LPS) challenge exposure in neonatal pigs. ANIMALS: 26 neonatal pigs. PROCEDURE: Pigs were assigned to the following treatment groups: 1 warm environment/LPS; 2 warm environment/saline solution; 3 cool environment/LPS; and 4 cool environment/saline solution. For each pig given LPS, 1 littermate of the same sex was given saline solution. Sows with baby pigs were housed in a warm (32 C) or cool (21 C) thermal environment. At 28 days of age, pigs were given 150 micrograms/kg of body weight of Escherichia coli LPS or saline solution intraperitonealy as a control. Rectal temperature and signs of sickness were monitored for 3 hours after LPS administration, when pigs were euthanatized and blood samples were collected to determine serum concentrations of tumor necrosis factor (TNF) alpha and cortisol. To determine in vitro production of TNF alpha, alveolar macrophages were collected by tracheal lavage and incubated for 24 hours at 37 or 41 C, with or without LPS (10 micrograms/ml). RESULTS: Thermal environment had a significant (P = 0.0004) effect on rectal temperature; LPS administration induced a febrile response (P = 0.0007) only in pigs in the warm environment. All LPS-injected pigs developed signs of endotoxemia; serum TNF alpha and cortisol concentrations were significantly increased (TNF alpha, P = 0.003; cortisol, P = 0.0001); there was no significant in vivo thermal effect on serum TNF alpha and cortisol concentrations. LPS-stimulated alveolar macrophages produced significantly less (P = 0.0086) TNF alpha when incubated at 41 C. CONCLUSIONS: Thermal environment can have a significant impact on the response of neonatal pigs exposed to bacterial endotoxins.

An esterification protocol for cis-parinaric acid-determined lipid peroxidation in immune cells.

Loss of fluorescence from cis-parinaric acid (cPnA) is a sensitive indicator of lipid peroxidation. The purpose of this study was to utilize cPnA to determine, at the level of the intact immune cell, whether enrichment of membranes with polyunsaturated fatty acids (PUFA) increased lipid peroxidation. P388D1 macrophages were labeled by addition of cPnA as an ethanolic solution. Within two minutes of addition, in the absence-of serum, cPnA rapidly intercalated into the plasma membrane. Lipid peroxidation was initiated by addition of Fe(2+)-EDTA resulting in a dose-dependent decrease in fluorescence with increased oxidant concentration. Cells previously enriched with PUFA and labeled by intercalation showed no differences in spontaneous or Fe(2+)-induced lipid peroxidation. In separate experiments, 20 microM cPnA in ethanolic solution was injected into cell culture media containing 0.1% essentially fatty acid free bovine serum albumin (BSA). Cells were resuspended and incubated for 90 min at 37 degrees C. After washing with BSA to remove cPnA which had not incorporated, 0.5% (0.1 microM) of the added cPnA was found esterified within cellular lipids. This level of cPnA provided a 100-fold increase over basal autofluorescence levels. Cells labeled in this manner also lost fluorescence in a dose-dependent manner as levels of oxidant stress increased. Cells enriched with PUFA and labeled by esterification had significantly increased rates and total amounts of lipid peroxidation. Co-incubation with alpha-tocopherol and PUFA resulted in a decrease in lipid peroxidation which was not significantly different from control cells. In conclusion, esterification of cPnA into membrane phospholipids can sensitively detect changes in lipid peroxidation induced by alteration of membrane PUFA and/or vitamin E content.