Selenium Speciation Analysis Reveals Improved Antioxidant Status in Finisher Pigs Fed L-Selenomethionine, Alone or Combined with Sodium Selenite, and Vitamin E.

Conditions associated with selenium (Se) and/or vitamin E (VitE) deficiency are still being reported in high-yielding pigs fed the recommended amounts. Here, the dietary effects of Se source (sodium selenite, NaSe, 0.40 or 0.65 mg Se/kg; L-selenomethionine, SeMet, 0.19 or 0.44 mg Se/kg; a NaSe-SeMet mixture, SeMix, 0.44-0.46 mg Se/kg) and VitE concentration (27, 50-53 or 101 mg/kg) on the antioxidant status of finisher pigs were compared with those in pigs fed non-Se-supplemented diets (0.08-0.09 mg Se/kg). Compared to NaSe-enriched diets, SeMet-supplemented diets resulted in significantly (p < 0.0018) higher plasma concentrations of total Se (14-27%) and selenospecies (GPx3, SelP, SeAlb; 7-83%), significantly increased the total Se accumulation in skeletal muscles, myocardium, liver and brain (10-650%), and enhanced the VitE levels in plasma (15-74%) and tissues (8-33%) by the end of the 80-day trial, proving better Se distribution and retention in pigs fed organic Se. Injecting lipopolysaccharide (LPS) intravenously half-way into the trial provoked a pyrogenic response in the pigs followed by a rapid increase of inorganic Se after 5-12 h, a drastic drop of SeMet levels between 12 and 24 h that recovered by 48 h, and a small increase of SeCys by 24-48 h, together with a gradual rise of GPx3, SelP and SeAlb in plasma up to 48 h. These changes in Se speciation in plasma were particularly significant (0.0024 > p > 0.00007) in pigs receiving SeMet- (0.44 mg Se/kg, above EU-legislated limits) or SeMix-supplemented (SeMet and NaSe both at 0.2 mg Se/kg, within EU-legislated limits) diets, which demonstrates Se metabolism upregulation to counteract the LPS-induced oxidative stress and a strengthened antioxidant capacity in these pigs. Overall, a Se source combination (without exceeding EU-legislated limits) and sufficient VitE supplementation (≥ 50 mg/kg) improved the pigs' antioxidant status, while doubling the allowed dietary organic Se increased the Se in tissues up to sixfold without compromising the animal's health due to toxicity. This study renders valuable results for revising the current dietary SeMet limits in swine rations.

Qualitative and Quantitative Changes in Total Lipid Concentration and Lipid Fractions in Liver Tissue of Periparturient German Holstein Dairy Cows of Two Age Groups.

Fatty liver syndrome (FLS) is a common disease in high-producing dairy cows. Studies in humans suggest that the different hepatic lipid fractions play a role in this context. In dairy cows, little is known about the composition of fat stored in the liver, its periparturient dynamics, and the effect of cows' age. Therefore, our goal was to generate primary data in healthy cows to serve as reference values for future studies. Eight healthy German Holstein cows (2nd lactation, n = 3; ≥3rd lactation, n = 5) were examined 14 d antepartum and 7, 28, and 42 d postpartum. The examinations included clinical assessment, liver biopsy, blood sampling, and recording of milk yield. Total lipids (TL) in liver tissue were measured gravimetrically. The TL were separated into lipid fractions (triacylglycerol, TAG; phospholipids, PL; non-esterified fatty acids, NEFA; and cholesterol esters) using thin-layer chromatography, followed by gas chromatography for fatty acid determination. Concentrations of NEFA, ß-hydroxybutyrate, and cholesterol were analyzed in blood. Concentrations of TL, TAG, NEFA, and cholesterol esters in liver tissue and NEFA in blood increased in the periparturient period. The older cows had higher hepatic TL, TAG, and PL concentrations, higher relative hepatic concentrations of TAG in TL, higher NEFA concentrations in blood, a greater decrease in body condition, and higher milk yields between d 9 and 40 than the younger cows. We proposed that due to higher milk yield, older cows mobilized and deposited more fat in the liver, and the increase in hepatic TAG concentration was longer-lasting than in younger cows. Higher levels of structural lipids (PL) in older cows could be explained by higher demand for storage of TAG and cholesterol esters in lipid droplets or for the export of TAG via very-low-density lipoproteins. Results show that hepatic fat storage is a reversible process and does not necessarily cause clinical disease. Nevertheless, older cows have a more sustained and greater increase in hepatic TAG concentration, which may explain their increased risk of FLS. The results are limited in their extrapolation due to the small sample size and thereby possible selection bias but present a valuable basis for future studies.

The Bovine Hepatic Cell Line BFH12 as a Possible Model for Hepatosteatosis in Dairy Cows.

Hepatosteatosis is a common metabolic disorder of dairy cows, especially during early lactation. Currently, there are a few models of bovine hepatic steatosis available, including primary hepatocytes, liver slices, and animal models. Studies that elucidate the influence of single fatty acids on lipid classes, fatty acid pattern, gene expression, and phenotypic changes are still limited. Hence, we investigated the suitability of the fetal bovine hepatocyte-derived cell line BFH12 as a model for hepatosteatosis. To create a steatotic environment, we treated BFH12 with stearic acid, palmitic acid, or oleic acid in non-toxic doses. Thin-layer chromatography and gas chromatography were used to analyze lipid classes and fatty acid pattern, and qPCR was used to quantify gene expression of relevant target genes. Lipid droplets were visualized with confocal laser scanning microscopy and evaluated for number and size. Treatment with oleic acid increased triglycerides, as well as lipid droplet count per cell and upregulated carnitine palmitoyl transferase 1, which correlates with findings of in vivo models. Oleic acid was largely incorporated into triglycerides, phospholipids, and non-esterified fatty acids. Stearic acid was found mainly in non-esterified fatty acids and triglycerides, whereas palmitic acid was mainly desaturated to palmitoleic acid. All three fatty acids downregulated stearyl-CoA-desaturase 1. In conclusion, BFH12 can acquire a steatotic phenotype by incorporating and accumulating fatty acids. Oleic acid is particularly suitable to produce hepatosteatosis. Therefore, BFH12 may be a useful in vitro model to study bovine hepatosteatosis and its underlying molecular mechanisms.

HOX Gene Expressions in Cultured Articular and Nasal Equine Chondrocytes.

Osteoarthritis the quality and span of life in horses. Previous studies focused on nasal cartilage as a possible source for autologous chondrocyte implantation (ACI) in cartilage defects in humans. "HOX gene-negative" nasal chondrocytes adapted articular HOX patterns after implantation into caprine joint defects and produced cartilage matrix proteins. We compared the HOX gene profile of equine chondrocytes of nasal septum, anterior and posterior fetlock to identify nasal cartilage as a potential source for ACI in horses. Cartilage was harvested from seven horses after death and derived chondrocytes were cultured in a monolayer to fourth subcultivation. HOX A3, D1, D8 and chondrocyte markers COL2 and SOX9 were analyzed with qPCR in chondrocytes of three different locations obtained during passage 0 and passage 2. HOX gene expression showed no significant differences between the locations but varied significantly between the horses. HOX genes and SOX9 remained stable during culturing. Cultured nasal chondrocytes may be a target for future research in cell-based regenerative therapies in equine osteoarthritis. The involvement of HOX genes in the high regenerative and adaptive potential of nasal chondrocytes observed in previous studies could not be confirmed.

Lipid classes in adipose tissues and liver differ between Shetland ponies and Warmblood horses.

Fatty acids, as key components of cellular membranes and complex lipids, may play a central role in endocrine signalling and the function of adipose tissue and liver. Thus, the lipid fatty acid composition may play a role in health status in the equine. This study aimed to investigate the fatty acid composition of different tissues and liver lipid classes by comparing Warmblood horses and Shetland ponies under defined conditions. We hypothesized that ponies show different lipid patterns than horses in adipose tissue, liver and plasma. Six Warmblood horses and six Shetland ponies were housed and fed under identical conditions. Tissue and blood sampling were performed following a standardized protocol. A one-step lipid extraction, methylation and trans-esterification method with subsequent gas chromatography was used to analyse the total lipid content and fatty acid profile of retroperitoneal, mesocolon and subcutaneous adipose tissue, liver and plasma. Fatty acids were grouped according to their degree of saturation and their conjugated double bond into the respective lipid classes. In the adipose tissues, saturated fatty acids (SFAs) and n-9 monounsaturated fatty acids (n-9 MUFAs) were most present in ponies and horses. N-6 polyunsaturated fatty acids (n-6 PUFAs), followed by SFAs, were most frequently found in liver tissue and plasma in all animals. Horses, in comparison to ponies, had significantly higher n-6 PUFA levels in all tissues and plasma. In liver tissue, horses had significantly lower hepatic iso-branched-chain fatty acids (iso-BCFAs) than ponies. The hepatic fatty acid composition of selected lipid classes was different between horses and ponies. In the polar PL fraction, horses had low n-9 MUFA and n-3 PUFA contents but higher n-6 PUFA contents than ponies. Furthermore, iso-BCFAs are absent in several hepatic lipid fractions of horses but not ponies. The differences in fatty acid lipid classes between horses and ponies provide key information on the species- and location-specific regulation of FA metabolism, thus affecting health status such as inflammatory responses.

Evaluation of the hepatocyte-derived cell line BFH12 as an in vitro model for bovine biotransformation.

The knowledge of drug metabolising enzymes (DMEs) in cattle is rather limited. The capability of the bovine foetal hepatocyte-derived cell line BFH12 to serve as model for biotransformation was evaluated. Gene expression analysis of DMEs was performed by reverse transcription PCR (RT-PCR). The presence of efflux transporters was visualised by immunocytochemistry, and functional induction of cytochrome P450 (CYP) 1A was assessed by the ethoxyresorufin-O-deethylase (EROD) assay. The production of bile acids was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RT-PCR revealed the expression of cytochromes 1A1, 1A2, 3A4 and phase II enzymes UGT1A1, UGT1A6 and GSTM1. Immunofluorescence demonstrated efflux transporters ABCG2 and ABCC1. The EROD assay revealed a dose-dependent CYP1A induction after treatment with benzo[a]pyrene (BP). LC-MS/MS analysis of cell culture supernatants showed the production of bile acids including taurocholic acid, tauro-chenodeoxycholic acid, taurodeoxycholic acid and taurolithocholic acid. The results strongly suggest the applicability of the cell line BFH12 for subsequent experiments in the emerging field of bovine biotransformation.

Lipid composition of membrane microdomains isolated detergent-free from PUFA supplemented RAW264.7 macrophages.

Profound alterations in the lipid profile of raft and non-raft plasma membrane microdomains were found when RAW264.7 macrophages were supplemented with polyunsaturated fatty acids (PUFAs) in physiologically relevant concentrations. For the first time lipids in the detergent-free isolated membrane domains of phagocytic immune cells were characterized by mass spectrometry. The extent of remodeling of the membrane lipids differed with different n3 and n6 PUFA supplements. The mildest effects were detected for α-linolenic acid (LNA) and linoleic acid (LA), the C18 precursors of the n3 and n6 families, respectively. When the effects of highly unsaturated PUFAs were compared, eicosapentaenoic acid (EPA) caused more extensive restructuring of membrane lipids than docosahexaenoic acid (DHA) or arachidonic acid (AA). The supplements altered the lipid species composition of both the raft and non-raft membrane fractions. The rafts containing elevated proportions of highly unsaturated lipid species may relocate sterically incompatible lipids and proteins originally belonging to this microdomain. Such effect was evident for sphingomyelin, which favored non-rafts instead of rafts after EPA supplementation. The current work suggests that the different functional consequences found previously when supplementing macrophages with either EPA or DHA have their origin in the different effects of these PUFAs on membrane architecture.

Differentiation and Functionality of Bone Marrow-Derived Mast Cells Depend on Varying Physiologic Oxygen Conditions.

Mast cells (MCs) are long-living multifunctional innate immune cells that originate from hematopoietic precursors and specifically differentiate in the destination tissue, e.g., skin, respiratory mucosa, intestine, where they mediate immune cell recruitment and antimicrobial defense. In vivo these tissues have characteristic physiological oxygen levels that are considerably lower than the atmospheric oxygen conditions (159 mmHg, 21% O2; 5% CO2) traditionally used to differentiate MCs and to study their functionality in vitro. Only little is known about the impact of physiological oxygen conditions on the differentiation process of MCs. This study aimed to characterize the differentiation of immature murine bone marrow-derived MCs under physioxia in vitro (7% O2; 53 mmHg; 5% CO2). Bone marrow-derived suspension cells were differentiated in the presence of interleukin-3 with continuous, non-invasive determination of the oxygen level using a Fibox4-PSt3 measurement system without technique-caused oxygen consumption. Trypan blue staining confirmed cellular viability during the specified period. Interestingly, MCs cultivated at 7% O2 showed a significantly delayed differentiation rate defined by CD117-positive cells, analyzed by flow cytometry, and reached >95% CD117 positive population at day 32 after isolation. Importantly, MCs differentiated under physioxia displayed a decreased transcript expression level of hif-1α and selected target genes vegf, il-6, and tnf-α, but an increase of foxo3 and vhl expression compared to MCs cultivated under normoxia. Moreover, the production of reactive oxygen species as well as the amount of intracellular stored histamine was significantly lower in MCs differentiated under low oxygen levels, which might have consequences for their function such as immunomodulation of other immune cells. These results show for the first time that physioxia substantially affect maturation and the properties of MCs and highlight the need to study their function under physiologically relevant oxygen conditions.

Effects of transforming growth factor-ß and interleukin-1ß on inflammatory markers of osteoarthritis in cultured canine chondrocytes.

OBJECTIVE To determine effects of transforming growth factor (TGF)-ß and interleukin (IL)-1ß on inflammatory markers in cultured canine chondrocytes to clarify the role of these cytokines in osteoarthritis of dogs. SAMPLE Pooled chondrocytes isolated from the stifle joints of 4 adult dogs. PROCEDURES Chondrocytes were isolated, cultured, and frozen at -80°C. Pooled cells were incubated in medium with or without TGF-ß (1 or 10 ng/mL) and subsequently stimulated with IL-1ß (10 ng/mL). Concentrations of nitric oxide (NO) and prostaglandin (PG) E were measured in culture supernatants. Gene expression of matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-2, inducible NO synthase (iNOS), and cyclooxygenase (COX)-2 was quantified by use of real-time quantitative PCR assays. RESULTS Stimulation with IL-1ß increased gene expression of all inflammatory markers, except for TIMP-2. Incubation with TGF-ß resulted in a significant decrease in MMP-3 and TIMP-2 mRNA concentrations but had no effect on PGE and NO concentrations. For cells treated with TGF-ß followed by IL-1ß, concentrations of PGE and NO were lower, compared with concentrations for IL-1ß control cells. Furthermore, IL-1ß-induced gene expression of iNOS, MMP-3, and COX-2 was downregulated. However, the IL-1ß-induced downregulation of TIMP-2 gene expression was partially restored by pretreatment with TGF-ß. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that IL-1ß increased the expression of inflammatory genes and mediators, and TGF-ß largely attenuated the IL-1ß-mediated inflammatory response. Therefore, TGF-ß might be a novel target for use in the prevention and treatment of cartilage breakdown in dogs with osteoarthritis.

Hypoxia Modulates the Response of Mast Cells to Staphylococcus aureus Infection.

To study the antimicrobial function of immune cells ex vivo, cells are commonly cultivated under atmospheric oxygen concentrations (20-21%; normoxia), although the physiological oxygen conditions in vivo are significantly lower in most tissues. Especially during an acute infection, oxygen concentration locally decreases to hypoxic levels around or below 1%. The goal of this study was to investigate the effect of hypoxia on the activity of mast cells (MCs). MCs were cultivated for 3 or 24 h at 1% O2 in a hypoxia glove box and co-incubated with heat-inactivated Staphylococcus aureus. When incubating the cells for 24 h under hypoxia, the transcriptional regulator hypoxia-inducible factor 1α (HIF-1α) was stabilized and resulted in increased extracellular trap formation and decreased phagocytosis. Interestingly, while phagocytosis of fluorescent S. aureus bioparticles as well as the release of extracellular traps remained unaffected at 3 h hypoxia, the secretion of the prestored mediator histamine was increased under hypoxia alone. In contrast, the release of TNF-α was generally reduced at 3 h hypoxia. Microarray transcriptome analysis revealed 13 genes that were significantly downregulated in MCs comparing 3 h hypoxia versus normoxia. One interesting candidate is sec24, a member of the pre-budding complex of coat protein complex II (COPII), which is responsible for the anterograde transport of proteins from the ER to the Golgi apparatus. These data lead to the suggestion that de novo synthesized proteins including crucial factors, which are involved in the response to an acute infection like TNF-α, may eventually be retained in the ER under hypoxia. Importantly, the expression of HIF-1α was not altered at 3 h. Thus, our data exhibit a HIF-1α-independent reaction of MCs to short-term hypoxia. We hypothesize that MCs respond to short-term low oxygen levels in a HIF-1α-independent manner by downregulating the release of proinflammatory cytokines like TNF-α, thereby avoiding uncontrolled degranulation, which could lead to excessive inflammation and severe tissue damage.

Establishment and characterisation of a novel bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line.

Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 µM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.

LPS- or Pseudomonas aeruginosa-mediated activation of the macrophage TLR4 signaling cascade depends on membrane lipid composition.

It is well known that PUFA impede the LPS-mediated activation of the transcription factor NFkappaB. However, the underlying mode of action has not been clarified yet. To address this issue in a comprehensive approach, we used the monocyte/macrophage cell line RAW264.7 to investigate the consequences of a PUFA supplementation on the TLR4 pathway with a focus on (i) the gene expression of TLR4 itself as well as of its downstream mediators, (ii) the membrane microdomain localization of TLR4 and CD14, (iii) the stimulation-induced interaction of TLR4 and CD14. Our data indicate that the impairment of the TLR4-mediated cell activation by PUFA supplementation is not due to changes in gene expression of mediator proteins of the signaling cascade. Rather, our data provide evidence that the PUFA enrichment of macrophages affects the TLR4 pathway at the membrane level. PUFA incorporation into membrane lipids induces a reordering of membrane microdomains thereby affecting cellular signal transduction. It is important to note that this remodeling of macrophage rafts has no adverse effect on cell viability. Hence, microdomain disruption via macrophage PUFA supplementation has a potential as non-toxic strategy to attenuate inflammatory signaling.

Label free molecular sexing of monomorphic birds using infrared spectroscopic imaging.

The absence of sexual dimorphism in many birds often makes sex determination difficult. In particular immature birds and adults of monomorphic species show no external sex characteristics. Molecular techniques based on DNA hybridization or polymerase chain reaction (PCR) are standard methods for sex identification. However, these methods are expensive and time consuming procedures and require special sample preparation. Noninvasive methods for a rapid determination of bird's gender are of increasing importance for ornithologists, breeders as well as for successful captive-breeding programs. Fourier transform infrared (FT-IR) spectroscopy is one such technique that can provide gender specific information. In this study, using the example of domestic pigeons (Columba livia f. dom.) we demonstrate that only a small amount of the feather pulp is needed to determine the gender. FT-IR spectroscopic images of feather pulp suspensions were recorded in transmission mode. Principal component analysis (PCA) and linear discriminant analysis (LDA) were performed to identify the sex. The gender related information are described by 2nd and 4th principal component principle component (PC). The 2nd PC represents different amounts of proteins while the 4th PC shows variations within the amide I and amide II bands as well as in the region of phosphate vibrations of nucleic acids. Blood cells of male pigeons exhibit a significantly higher amount of proteins and nucleic acids than those of female pigeons. Feather pulp samples of male species were assigned with 100% accuracy. Seven from eight female samples were assigned correctly while one sample could not be classified. This study demonstrates that the sex of domestic pigeons can be accurately and and rapidly identified by infrared spectroscopic imaging.

[Investigations on the acute, carrageenan-induced inflammatory reaction and pharmacology of orally administered sodium salicylate in turkeys]. / Untersuchungen zur akuten, Karrageen-induzierten Entzündungsreaktion sowie der Pharmakologie oral applizierten Natriumsalicylats bei Puten.

The complex mechanisms of acute inflammation have been subject to veterinary investigations since a long time. However, knowledge on the role of specific inflammatory mediators, as well as pharmacokinetics (PK) and -dynamics (PD) of non-steroidal anti-inflammatory drugs (NSAID) in birds is limited. The objective of this work therefore was to establish a modified tissue cage-model to investigate the acute, carrageenan-mediated inflammatory response, as well as plasma and exudate-kinetics and the antiphlogistic effect of orally administered sodium salicylate on the elicited inflammatory reaction in turkeys. Within the class Aves, comparable studies have so far only been published in chicken. Following bilateral subcutaneous implantation of carrageenan-treated synthetic sponges in the lateral thoracic region, sodium salicylate was administered orally at a dose of 50 mg/kg body weight (BW; therapy group) twice daily on three consecutive days, while a control group received drinking water as a placebo (n = 24 per group). Combined PK and PD of sodium salicylate were evaluated on the basis of salicylate- and prostaglandin (PG) E2-plasma- and -exudate-concentrations, exudate volumes, as well as leukocyte exudate counts. Sodium salicylate was readily absorbed from the gastrointestinal tract and accumulated in the inflammatory exudate. At 4, 6, and 10 h after first application, sodium salicylate significantly reduced PG E2-concentrations in the inflammatory exudate when compared to the control group, whereas leukocyte exudate counts increased over time in both study groups, unaffected by sodium salicylate The described modified tissue cage-model can be beneficial for further research on the pathophysiology of avian inflammatory processes and the investigation of the combined pharmacodynamics and -kinetics of drugs in birds of adequate size.

The influence of polyunsaturated fatty acids on the phospholipase D isoforms trafficking and activity in mast cells.

The impact of polyunsaturated fatty acid (PUFA) supplementation on phospholipase D (PLD) trafficking and activity in mast cells was investigated. The enrichment of mast cells with different PUFA including α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) revealed a PUFA-mediated modulation of the mastoparan-stimulated PLD trafficking and activity. All PUFA examined, except AA, prevented the migration of the PLD1 to the plasma membrane. For PLD2 no PUFA effects on trafficking could be observed. Moreover, PUFA supplementation resulted in an increase of mastoparan-stimulated total PLD activity, which correlated with the number of double bonds of the supplemented fatty acids. To investigate, which PLD isoform was affected by PUFA, stimulated mast cells were supplemented with DHA or AA in the presence of specific PLD-isoform inhibitors. It was found that both DHA and AA diminished the inhibition of PLD activity in the presence of a PLD1 inhibitor. By contrast, only AA diminished the inhibition of PLD activity in the presence of a PLD2 inhibitor. Thus, PUFA modulate the trafficking and activity of PLD isoforms in mast cells differently. This may, in part, account for the immunomodulatory effect of unsaturated fatty acids and contributes to our understanding of the modulation of mast cell activity by PUFA.

Unsaturated fatty acids promote the phagocytosis of P. aeruginosa and R. equi by RAW264.7 macrophages.

In the present study, using the murine monocyte/macrophage cell line RAW264.7 as a model system, we analyzed the phagocytosis rate and the bactericidal capacity of polyunsaturated fatty acids (PUFA)-enriched macrophages against Pseudomonas aeruginosa and Rhodococcus equi. The P. aeruginosa strain ATCC 10145, the virulent R. equi strain ATCC 33701, and the non-virulent R. equi strain ATCC 6939 were examined. Flow cytometric detection of intracellular microorganisms in combination with viability assays were used to determine the impact of PUFA on the number of engulfed, surviving as well as replicating bacteria. Macrophage enrichment with PUFA resulted in an increase of the internalization rate of the microorganisms by the immune cells. Moreover, an impeding action of the unsaturated fatty acids on the intracellular survival rates of the virulent strains P. aeruginosa ATCC 10145 and R. equi ATCC 33701 could be observed. The n-3 fatty acid docosahexaenoic acid (DHA) as well as the n-6 fatty acid arachidonic acid (AA) showed the most pronounced effects. Taken together, our data support the idea of supplementing PUFA to immunocompromised individuals as well as to people suffering from chronic infections with P. aeruginosa or R. equi to improve macrophage phagocytic and microbicidal activity.

Prenatal stress programs lipid metabolism enhancing cardiovascular risk in the female F1, F2, and F3 generation in the primate model common marmoset (Callithrix jacchus).

BACKGROUND: Many human diseases are modulated by intrauterine environment, which is called prenatal programming. This study investigated effects of prenatal glucocorticoids on the lipid metabolism of three filial generations of common marmosets. METHODS: Pregnant primates were treated with dexamethasone during pregnancy. Body weight and blood lipid parameters of adult female offspring (F1: n = 5, F2: n = 6, F3: n = 3) were compared with age-related female controls (n = 12). RESULTS: F1, F2, and F3 offspring showed significantly lower percentage of plasma n3 fatty acids than controls. F2 and F3 presented higher cholesterol levels, with significantly more LDL cholesterol, significantly less HDL triglycerides and an enhanced cholesterol/HDL cholesterol ratio. Body weight was not significantly affected. CONCLUSIONS: Prenatal dexamethasone led to higher amounts of cardiovascular risk factors and less protective parameters in female F1-F3 offspring. The intergenerational consequences suggest prenatal programming through epigenetic effects.

Unsaturated fatty acids as modulators of macrophage respiratory burst in the immune response against Rhodococcus equi and Pseudomonas aeruginosa.

In this paper, using the monocyte/macrophage cell line RAW264.7, we systematically investigate the impact of macrophage enrichment with unsaturated fatty acids on cellular radical synthesis. We found that the intracellular production of reactive nitrogen and oxygen intermediates depends on the activation status of the macrophages. For unstimulated macrophages PUFA enrichment resulted in an increase in cellular radical synthesis. For stimulated macrophages, instead, an impeding action of unsaturated fatty acids on the respiratory burst could be seen. Of particular importance, the impact of unsaturated fatty acids on the macrophage respiratory burst was also observed in RAW264.7 cells cocultivated with viable bacteria of the species Rhodococcus equi or Pseudomonas aeruginosa. PUFA supplementation of macrophages in the presence of R. equi or P. aeruginosa reduced the pathogen-stimulated synthesis of reactive nitrogen and oxygen intermediates. Furthermore, the unsaturated fatty acids were found to impede the expression of the myeloperoxidase gene and to reduce the activity of the enzyme. Hence, our data provide indications of a possible value of PUFA application to people suffering from chronic infections with R. equi and P. aeruginosa as a concomitant treatment to attenuate an excessive respiratory burst.

Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition.

In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.

LC-MS method for screening unknown microbial carotenoids and isoprenoid quinones.

The structure of secondary metabolites from microorganisms provides a useful tool for microbial characterization and chemotaxonomic classification. Microbial isoprenoid quinones, for example, are well described and used to distinguish among photosynthetic microorganism groups. In addition, isoprenoid quinones can also be found, together with carotenoids, in non-photosynthetic microorganisms. The aim of the present study was to develop a LC-MS/MS method which can analyze and identify these microbial isoprenoids. Positive atmospheric pressure chemical ionization (APCI) together with collisionally induced dissociation was applied for generation of informative fragment spectra by mass spectrometry. Enhanced product ion (EPI) scan in a linear ion trap with information dependent data acquisition (IDA) enabled generation of MS fragment data even from minor isoprenoids. The developed liquid chromatography method enabled separation of isoprenoid patterns from their ester derivatives. Discovery and structural characterization of isoprenoid quinones and carotenoids were carried out by comparing characteristics of fragment spectra from unknown compounds with fragment spectra of a range of isoprenoid standard compounds and using published data. Throughout the study 17 microorganisms (e.g., Acremonium butyri, Arthrobacter spp., Brevibacterium linens, Bullera variabilis, Exophiala dermatitidis, Lecythophora hoffmannii, Panthoea agglomerans, Rhodotorula spp., Xanthophyllomyces dendrorhous) were screened and probable structures of isoprenoid quinones and carotenoids were suggested. The method lays some foundations on the analysis of yet unknown isoprenoids in microorganisms by using LCMS/MS techniques.