Metabolic adaptations and changes of the mammary immune response during beta-hydroxybutyrate administration

Elevation of ketone bodies occurs frequently after parturition during negative energy balance in high yielding dairy cows. Previous studies illustrated that hyperketonemia interferes with metabolism and it is assumed that it impairs the immune response. However, a causative effect of ketone bodies could not be shown in vivo before, because spontaneous hyperketonemia comes usually along with high NEFA and low glucose concentrations. The objective was to study effects of beta-hydroxybutyrate (BHBA) infusion and an additional intramammary lipopolysaccharide (LPS) challenge on metabolism and immune response in dairy cows. Thirteen dairy cows received intravenously either a BHBA infusion (group BHBA, n=5) to induce hyperketonemia (1.7 mmol/L), or an infusion with a 0.9 % saline solution (Control, n=8) for 56 h. Infusions started at 0900 on day 1 and continue up to 1700 two days later. Two udder quarters were challenged with 200 μg Escherichia coli-LPS 48 h after the start of infusion. Blood samples were taken one week and 2 h before the start of infusions as reference samples and hourly during the infusion. Liver and mammary gland biopsies were taken one week before the start of the infusion, 48 h after the start of the infusion, and mammary tissues was additionally taken 8 h after LPS challenge (56 h after the start of infusions). Rectal temperature (RT) and somatic cell count (SCC) was measured before and 48 h after the start of infusions and hourly during LPS challenge. Blood samples were analyzed for plasma glucose, BHBA, NEFA, triglyceride, urea, insulin, glucagon, and cortisol concentration. The mRNA abundance of factors related to potential adaptations of metabolism and immune system was measured in liver and mammary tissue biopsies. Differences between blood constituents, RT, SCC, and mRNA abundance before and 48 h after the start of infusions, and differences between mRNA abundance before and after LPS challenges were tested for significance by GLM of SAS procedure with treatment as fixed effect. Area under the curve was calculated for blood variables during 48 h BHBA infusion and during the LPS challenge, and additionally for RT and SCC during the LPS challenge. Most surprisingly, both plasma glucose and glucagon concentration decreased during the 48 h of BHBA infusion (P<0.05). During the 48 h of BHBA infusion, serum amyloid A mRNA abundance in mammary gland was increased (P<0.01), and haptoglobin (Hp) mRNA abundance tended to increase in cows treated with BHBA compared to control group (P= 0.07). RT, SCC, and candidate genes related to immune response in the liver were not affected by BHBA infusion. However, during LPS challenge the expected increase of both plasma glucose and glucagon concentration was much less pronounced in the animals treated with BHBA (P<0.05) and also SCC increased much less pronounced in the animals infused with BHBA (P<0.05) than in the controls. An increased BHBA infusion rate to maintain plasma BHBA constant could not fully compensate for the decreased plasma BHBA during the LPS challenge which indicates that BHBA is used as an energy source during the immune response. In addition, BHBA infused animals showed a more pronounced increase of mRNA abundance of IL-8, IL-10, and citrate synthase in the mammary tissue of LPS challenged quarters (P<0.05) than control animals. Results demonstrate that infusion of BHBA affects metabolism through decreased plasma glucose concentration which is likely related to a decreased release of glucagon during hyperketonemia and during additional inflammation. It also affects the systemic and mammary immune response which may reflect the increased susceptibility for mastitis during spontaneous hyperketonemia. The obviously reduced gluconeogenesis in response to BHBA infusion may be a mechanism to stimulated the use of BHBA as an energy source instead of glucose, and/or to save oxaloacetate for the citric acid cycle instead of gluconeogenesis and as a consequence to reduce ketogenesis.

The beta-isoform of neuronal nitric oxide synthase (nNOS) lacking the PDZ domain is localized at the sarcolemma

In skeletal muscles, the expression of neuronal NO synthase (nNOS) isoforms is uncharacterized at the protein level. We therefore conducted epitope mapping with anti-peptide-antibodies. Antibodies specific for the nNOS N-terminus recognized the 160-kDa alpha-isoform. In contrast, antibodies against the middle portion or the C-terminus of nNOS bound additionally to the truncated 140-kDa beta-isoform which lacks the PDZ-domain present in the alpha-isoform. All nNOS immunohistochemical reactivity was confined to the sarcolemma. Consistently, immunoblotting disclosed both nNOS-isoforms to be co-enriched in the membrane-associated fractions. The beta-isoform was co-immunoprecipitated with alpha-isoform antibodies in muscle extracts indicating an association of both nNOS-isoforms to direct the beta-variant to the sarcolemma.

Alpha- versus beta-particle radiopeptide therapy in a human prostate cancer model (213Bi-DOTA-PESIN and 213Bi-AMBA versus 177Lu-DOTA-PESIN)

Recurrent prostate cancer presents a challenge to conventional treatment, particularly so to address micrometastatic and small-volume disease. Use of α-radionuclide therapy is considered as a highly effective treatment in such applications due to the shorter range and exquisite cytotoxicity of α-particles as compared with β-particles. (213)Bi is considered an α-emitter with high clinical potential, due to its short half-life (45.6 minutes) being well matched for use in peptide-receptor radionuclide α-therapy; however, there is limited knowledge available within this context of use. In this study, two novel (213)Bi-labeled peptides, DOTA-PEG(4)-bombesin (DOTA-PESIN) and DO3A-CH(2)CO-8-aminooctanoyl-Q-W-A-V-G-H-L-M-NH(2) (AMBA), were compared with (177)Lu (β-emitter)-labeled DOTA-PESIN in a human androgen-independent prostate carcinoma xenograft model (PC-3 tumor). Animals were injected with (177)Lu-DOTA-PESIN, (213)Bi-DOTA-PESIN, or (213)Bi-AMBA to determine the maximum tolerated dose (MTD), biodistribution, and dosimetry of each agent; controls were left untreated or were given nonradioactive (175)Lu-DOTA-PESIN. The MTD of (213)Bi-DOTA-PESIN and (213)Bi-AMBA was 25 MBq (0.68 mCi) whereas (177)Lu-DOTA-PESIN showed an MTD of 112 MBq (3 mCi). At these dose levels, (213)Bi-DOTA-PESIN and (213)Bi-AMBA were significantly more effective than (177)Lu-DOTA-PESIN. At the same time, (177)Lu-DOTA-PESIN showed minimal, (213)Bi-DOTA-PESIN slight, and (213)Bi-AMBA marked kidney damage 20 to 30 weeks posttreatment. These preclinical data indicate that α-therapy with (213)Bi-DOTA-PESIN or (213)Bi-AMBA is more efficacious than β-therapy. Furthermore, (213)Bi-DOTA-PESIN has a better safety profile than (213)Bi-AMBA, and represents a possible new approach for use in peptide-receptor radionuclide α-therapy treating recurrent prostate cancer.

Vitamin E reduces antidepressant-related beta-adrenoceptor down-regulation in cultured cells. Comparable effects on St. John's wort and tricyclic antidepressant treatment

The mode of action of antidepressants is still a matter of debate. Acute inhibition of neurotransmitter reuptake in central neuronal synapses, followed by a down-regulation of central postsynaptic beta-adrenoceptor (beta-AR) numbers were consistently observed in vivo, while a reduction in surface beta-AR density was found in cell cultures. Effects of the tricyclic antidepressant desipramine (DMI) were abolished by vitamin E (alpha-TOC) in vitro as well as in vivo. Alpha-TOC interfered with antidepressant-induced changes of cellular plasma membrane properties and with recycling of beta-AR. St. John's wort (SJW) extract reduced beta-AR numbers in cultured cells to a similar extent as DMI or the selective serotonin re-uptake inhibitor fluoxetine. We chronically co-exposed cell cultures to SJW extract and to alpha-TOC. Receptor down-regulation following exposure to the plant extract was inhibited in the presence of alpha-TOC suggesting a mode of action of SJW extract comparable to that of synthetic antidepressants. Inhibition of cell proliferation by the plant extract was also significantly reduced by alpha-TOC.

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Stimulation-specific contribution of p38 and JNK to IFN-beta gene expression in human macrophages

Induction of interferon-beta (IFN-beta) gene expression is a tightly regulated process, and a plethora of studies identified the signal transduction pathway TANK-binding kinase-1 (TBK-1)/IFN regulatory factor-3 (IRF-3) as essential to the induction of IFN-beta gene expression. Data regarding the role of p38 and JNK are rare, however. We investigated the contribution of these kinases to IFN-beta expression in human macrophages treated with poly(I:C), lipopolysaccharide (LPS), Sendai virus, or vesicular stomatitis virus (VSV). We found that all the stimuli induced IFN-beta mRNA, albeit to a different extent. Whereas LPS and VSV induced the phosphorylation of p38 and JNK, neither poly(I:C) nor Sendai virus led to the detection of phosphospecific signals. When inhibiting p38, a VSV-triggered IFN-beta mRNA response was inhibited, whereas inhibiting JNK suppressed an LPS-triggered response, but only when macrophages were primed with IFN-gamma. Neither poly(I:C)-induced nor Sendai virus-induced IFN-beta mRNA expression was affected when p38 and JNK were inhibited. Collectively, the data show that the contribution of p38 and JNK to the expression of IFN-beta occurs in a stimulation-specific manner in human macrophages.

SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

Loss-of-function mutation of the SCN3B-encoded sodium channel {beta}3 subunit associated with a case of idiopathic ventricular fibrillation.

AIMS Loss-of-function mutations in the SCN5A-encoded sodium channel SCN5A or Nav1.5 have been identified in idiopathic ventricular fibrillation (IVF) in the absence of Brugada syndrome phenotype. Nav1.5 is regulated by four sodium channel auxiliary beta subunits. Here, we report a case with IVF and a novel mutation in the SCN3B-encoded sodium channel beta subunit Navbeta3 that causes a loss of function of Nav1.5 channels in vitro. METHODS AND RESULTS Comprehensive open reading frame mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, GPD1L, four sodium channel beta subunit genes (SCN1-4B), and targeted scan of RYR2 was performed. A novel missense mutation, Navbeta3-V54G, was identified in a 20-year-old male following witnessed collapse and defibrillation from VF. The ECG exhibited epsilon waves, and imaging studies demonstrated a structurally normal heart. The mutated residue was highly conserved across species, localized to the Navbeta3 extracellular domain, and absent in 800 reference alleles. We found that HEK-293 cells had endogenous Navbeta3, but COS cells did not. Co-expression of Nav1.5 with Navbeta3-V54G (with or without co-expression of the Navbeta1 subunit) in both HEK-293 cells and COS cells revealed a significant decrease in peak sodium current and a positive shift of inactivation compared with WT. Co-immunoprecipitation experiments showed association of Navbeta3 with Nav1.5, and immunocytochemistry demonstrated a dramatic decrease in trafficking to the plasma membrane when co-expressed with mutant Navbeta3-V54G. CONCLUSION This study provides molecular and cellular evidence implicating mutations in Navbeta3 as a cause of IVF.

Exposure assessment of extended-spectrum beta-lactamases/AmpC beta-lactamases-producing Escherichia coli in meat in Denmark

INTRODUCTION Extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases (AmpC) are of concern for veterinary and public health because of their ability to cause treatment failure due to antimicrobial resistance in Enterobacteriaceae. The main objective was to assess the relative contribution (RC) of different types of meat to the exposure of consumers to ESBL/AmpC and their potential importance for human infections in Denmark. MATERIAL AND METHODS The prevalence of each genotype of ESBL/AmpC-producing E. coli in imported and nationally produced broiler meat, pork and beef was weighted by the meat consumption patterns. Data originated from the Danish surveillance program for antibiotic use and antibiotic resistance (DANMAP) from 2009 to 2011. DANMAP also provided data about human ESBL/AmpC cases in 2011, which were used to assess a possible genotype overlap. Uncertainty about the occurrence of ESBL/AmpC-producing E. coli in meat was assessed by inspecting beta distributions given the available data of the genotypes in each type of meat. RESULTS AND DISCUSSION Broiler meat represented the largest part (83.8%) of the estimated ESBL/AmpC-contaminated pool of meat compared to pork (12.5%) and beef (3.7%). CMY-2 was the genotype with the highest RC to human exposure (58.3%). However, this genotype is rarely found in human infections in Denmark. CONCLUSION The overlap between ESBL/AmpC genotypes in meat and human E. coli infections was limited. This suggests that meat might constitute a less important source of ESBL/AmpC exposure to humans in Denmark than previously thought - maybe because the use of cephalosporins is restricted in cattle and banned in poultry and pigs. Nonetheless, more detailed surveillance data are required to determine the contribution of meat compared to other sources, such as travelling, pets, water resources, community and hospitals in the pursuit of a full source attribution model.