Investigation of the Formal Pathogenesis of Green Liver Discoloration in Organically Reared Female Bronze Turkeys (Melleagris gallovapo).

Green liver discoloration (GL) in fattening turkeys is suspected to be a multifactorial disease complex with a compromised immune system as the key factor. This study aimed to identify the formal pathogenesis of GL and to investigate possible nutritional influences. A total of 360 Bronze turkey hens out of 10 flocks from 5 fattening farms were necropsied for detection of GL during 2 consecutive trials on 2 examination dates each (70th to 75th and 120th to 127th day of fattening, respectively). At each examination date, hematological and clinical chemistry analyses, as well as determination of vitamin E and selenium concentrations in the liver, were carried out in 6 hens with (if applicable) and 6 hens without GL, representing a total of 130 individuals. Raw nutrient, energy, amino acid, bulk and trace element, and vitamin E and D3 concentrations were analyzed in feed samples for each of the five feeding phases during each trial. The results of the hematological analyses, clinical chemistry analyses, and determination of vitamin E and selenium liver concentrations were statistically evaluated between: (i) individuals with and without GL, and (ii) individuals from flocks with and without turkeys with GL. At both fattening stages, the occurrence of GL was characterized by an inflammatory reaction. A subacute inflammatory reaction was detected in the early fattening stage, indicating a viral cause of the disease. In the late fattening stage, acute inflammation indicated a bacterial cause of the disease. The results of the feed sample analyses of the different flocks were generally quite homogeneous. However, the nutrient and energy content of the feed likely contribute to GL pathogenesis.

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane.

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.

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.

Chlamydia trachomatis inhibits apoptosis in infected cells by targeting the pro-apoptotic proteins Bax and Bak.

Apoptosis acts in defense against microbial infection, and many infectious agents have developed strategies to inhibit host cell apoptosis. The human pathogen Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that strongly inhibits mitochondrial apoptosis of its human host cell but there is no agreement how the bacteria achieve this. We here provide a molecular analysis of chlamydial apoptosis-inhibition in infected human cells and demonstrate that the block of apoptosis occurs during the activation of the effectors of mitochondrial apoptosis, Bak and Bax. We use small-molecule Bcl-2-family inhibitors and gene targeting to show that previous models cannot explain the anti-apoptotic effect of chlamydial infection. Although the anti-apoptotic Bcl-2-family protein Mcl-1 was strongly upregulated upon infection, Mcl-1-deficient cells and cells where Mcl-1 was pharmacologically inactivated were still protected. Ctr-infection could inhibit both Bax- and Bak-induced apoptosis. Apoptotic Bax-oligomerization and association with the outer mitochondrial membrane was reduced upon chlamydial infection. Infection further inhibited apoptosis induced conformational changes of Bak, as evidenced by changes to protease sensitivity, oligomerization and release from the mitochondrial porin VDAC2. Mitochondria isolated from Ctr-infected cells were protected against the pro-apoptotic Bcl-2-family proteins Bim and tBid but this protection was lost upon protease digestion. However, the protective effect of Ctr-infection was reduced in cells lacking the Bax/Bak-regulator VDAC2. We further found that OmpA, a porin of the outer membrane of Ctr, associated upon experimental expression with mitochondria and inhibited apoptosis, phenocopying the effect of the infection. These results identify a novel way of apoptosis inhibition, involving only the most downstream modulator of mitochondrial apoptosis and suggest that Chlamydia has a protein dedicated to the inhibition of apoptosis to secure its survival in human cells.

How Do Hexokinases Inhibit Receptor-Mediated Apoptosis?

The regulated cell death apoptosis enables redundant or compromised cells in ontogeny and homeostasis to remove themselves receptor-dependent after extrinsic signaling or after internal stress by BCL-2 proteins on the outer mitochondrial membrane (OMM). Mitochondrial BCL-2 proteins are also often needed for receptor-mediated signaling in apoptosis. Then, the truncated BH3-only protein BID (tBID) blocks retrotranslocation of the pro-apoptotic BCL-2 proteins BAX and BAK from the mitochondria into the cytosol. BAX and BAK in turn permeabilize the OMM. Although the BCL-2 proteins are controlled by a complex regulatory network, a specific mechanism for the inhibition of tBID remained unknown. Curiously, it was suggested that hexokinases, which channel glucose into the metabolism, have an intriguing function in the regulation of apoptosis. Recent analysis of transient hexokinase interactions with BAX revealed its participation in the inhibition of BAX and also BAK by retrotranslocation from mitochondria to the cytosol. In contrast to general apoptosis inhibition by anti-apoptotic BCL-2 proteins, hexokinase I and hexokinase 2 specifically inhibit tBID and thus the mitochondrial apoptosis pathway in response to death receptor signaling. Mitochondrial hexokinase localization and BH3 binding of cytosolic hexokinase domains are prerequisites for protection against receptor-mediated cell death, whereas glucose metabolism is not. This mechanism protects cells from apoptosis induced by cytotoxic T cells.

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.

DNA elements for constitutive androstane receptor- and pregnane X receptor-mediated regulation of bovine CYP3A28 gene.

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.

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 raft localization of TLR2 and its co-receptors is independent of membrane lipid composition.

BACKGROUND: Toll like receptors (TLRs) are an important and evolutionary conserved class of pattern recognition receptors associated with innate immunity. The recognition of Gram-positive cell wall constituents strongly depends on TLR2. In order to be functional, TLR2 predominantly forms a heterodimer with TLR1 or TLR6 within specialized membrane microdomains, the lipid rafts. The membrane lipid composition and the physicochemical properties of lipid rafts are subject to modification by exogenous fatty acids. Previous investigations of our group provide evidence that macrophage enrichment with polyunsaturated fatty acids (PUFA) induces a reordering of lipid rafts and non-rafts based on the incorporation of supplemented PUFA as well as their elongation and desaturation products. METHODS: In the present study we investigated potential constraining effects of membrane microdomain reorganization on the clustering of TLR2 with its co-receptors TLR1 and TLR6 within lipid rafts. To this end, RAW264.7 macrophages were supplemented with either docosahexaenoic acid (DHA) or arachidonic acid (AA) and analyzed for receptor expression and microdomain localization in context of TLR stimulation. RESULTS AND CONCLUSIONS: Our analyses showed that receptor levels and microdomain localization were unchanged by PUFA supplementation. The TLR2 pathway, in contrast to the TLR4 signaling cascade, is not affected by exogenous PUFA at the membrane level.

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.

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.

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.

The impact of membrane lipid composition on macrophage activation in the immune defense against Rhodococcus equi and Pseudomonas aeruginosa.

Nutritional fatty acids are known to have an impact on membrane lipid composition of body cells, including cells of the immune system, thus providing a link between dietary fatty acid uptake, inflammation and immunity. In this study we reveal the significance of macrophage membrane lipid composition on gene expression and cytokine synthesis thereby highlighting signal transduction processes, macrophage activation as well as macrophage defense mechanisms. Using RAW264.7 macrophages as a model system, we identified polyunsaturated fatty acids (PUFA) of both the n-3 and the n-6 family to down-regulate the synthesis of: (i) the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α; (ii) the co-stimulatory molecule CD86; as well as (iii) the antimicrobial polypeptide lysozyme. The action of the fatty acids partially depended on the activation status of the macrophages. It is particularly important to note that the anti-inflammatory action of the PUFA could also be seen in case of infection of RAW264.7 with viable microorganisms of the genera R. equi and P. aeruginosa. In summary, our data provide strong evidence that PUFA from both the n-3 and the n-6 family down-regulate inflammation processes in context of chronic infections caused by persistent pathogens.