Non-nutritive sweeteners: no class effect on the glycaemic or appetite responses to ingested glucose.

There is considerable interest in whether non-nutritive sweeteners are sensed in the gastrointestinal tract to modulate appetitive or absorptive responses to ingested carbohydrate. We determined the effect of a panel of non-nutritive sweeteners, aspartame, saccharin and acesulfame-K, delivered in doses that would be consumed in normal usage. Each was given in combination with glucose, assessing their effect on glycemic responses and appetite in 10 healthy human subjects. There was no additional effect of aspartame or saccharin on the blood glucose response to oral glucose at any time point, although acesulfame-K exerted a small effect. However, none had an effect on perceptions of hunger or fullness. We conclude that there is no consistent evidence that non-nutrient sweeteners, when acutely consumed with glucose in dietetically relevant doses, have a class effect in modulating blood glucose in healthy human subjects. However, acesulfame-K may require further exploration.

Pattern recognition receptors in equine endotoxaemia and sepsis.

Pattern recognition receptors (PRRs) on host cells detect pathogens to activate innate immunity which, in turn, initiates inflammatory and adaptive immune responses. Successful activation of PRRs is, therefore, critical to controlling infections and driving pathogen-specific adaptive immunity, but overactivity of PRRs causes systemic inflammation, which is detrimental to the host. Here we review the PRR literature as it relates to horses and speculate on the role PRRs may play in sepsis and endotoxaemia.

Salmonella enterica serovar Typhimurium mutants completely lacking the F(0)F(1) ATPase are novel live attenuated vaccine strains.

The F(0)F(1) ATPase plays a central role in both the generation of ATP and the utilisation of ATP for cellular processes such as rotation of bacterial flagella. We have deleted the entire operon encoding the F(0)F(1) ATPase, as well as genes encoding individual F(0) or F(1) subunits, in Salmonella enteric serovar Typhimurium. These mutants were attenuated for virulence, as assessed by bacterial counts in the livers and spleens of intravenously infected mice. The attenuated in vivo growth of the entire atp operon mutant was complemented by the insertion of the atp operon into the malXY pseudogene region. Following clearance of the attenuated mutants from the organs, mice were protected against challenge with the virulent wild type parent strain. We have shown that the F(0)F(1) ATPase is important for bacterial growth in vivo and that atp mutants are effective live attenuated vaccines against Salmonella infection.

Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

Effect of Escherichia coli infection of the bovine uterus from the whole animal to the cell.

Following parturition, contamination of the uterine lumen by bacteria is ubiquitous, and uterine health is impaired in cattle because infection persists in 10% to 15% of animals as endometritis. Endometritis causes infertility for the duration of infection, and subfertility persists even after apparent successful resolution of the disease. Escherichia coli is the pathogenic bacterium most frequently isolated from the post partum uterus, and is associated with increased concentrations of peripheral plasma acute phase proteins and fetid vaginal mucus. The presence of E. coli is also associated with slower growth of the first post partum dominant follicle and perturbed oestradiol secretion. Furthermore, in animals that ovulate the first dominant follicle, the corpus luteum is smaller and secretes less progesterone. The endotoxin lipopolysaccharide (LPS), which is released from E.coli, can pass from the uterine lumen to the peripheral circulation and LPS concentrations are increased in cows with uterine infection. Infusion of E. coli LPS into the uterine lumen suppresses the pre-ovulatory luteinising hormone surge and disrupts ovulation in heifers. In vitro, endometrial explants produce prostaglandins in response to LPS. Addition of LPS or E. coli to stromal or epithelial cells increases cyclooxygenase-2 mRNA expression, and stimulates the production of prostaglandin E2 and prostaglandin F2α . Furthermore, uterine and ovarian cells express mRNA of the molecules required for recognition of LPS, Toll-like receptor-4 and CD14. In summary, E. coli is a common cause of infertility involving the perturbation of the hypothalamus, pituitary and ovary in dairy cows.

The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo.

Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2alpha (PGF) or prostaglandin E(2) (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n=7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.

Genotyping of Toll-like receptor 4, myeloid differentiation factor 2 and CD-14 in the horse: an investigation into the influence of genetic polymorphisms on the LPS induced TNF-alpha response in equine whole blood.

The inter- and intra-species differences in the response to lipopolysaccharides (LPS) are well recognised in mammalian species. It has been hypothesized that these differences can be attributed to genetic polymorphisms in the components involved in LPS signal transduction. These components include the cluster of differentiation factor 14 (CD-14), a membrane bound protein on the surface of mononuclear cells that recognises LPS and a receptor complex consisting of Toll-like receptor-4 (TLR-4) and myeloid differentiation factor-2 (MD-2). Sequencing of these three proteins in humans and mice revealed that all three are susceptible to polymorphic alterations, influencing the response to LPS. Previous experiments in the horse showed large inter-individual variations in the response to LPS. With the aim to assess this inter-individual variation, we performed a whole blood assay in 10 healthy horses as a functional assay to study the responsiveness to LPS. In 3 out of the 10 horses, LPS-induced TNF-alpha production was significantly lower compared to the overall mean. Subsequently the entire cDNA sequence encoding for the TLR-4, MD-2 and CD-14 protein was documented for each horse. Although mutations were observed in the sequence of TLR-4, these could not be related to an altered response to LPS in the concentration used in this study, as determined in the whole blood assay. Despite the various mutations found in the TLR-4 receptor protein, no alterations could be found in either the MD-2 or CD-14 gene, which are obviously more conserved structures.

Survey of pharmacists' awareness of veterinary medicines.

A survey of 186 pharmacies across Great Britain in 2004 showed that the pharmacists had little involvement in the supply of veterinary medicines. Most of them did not feel competent to be involved in the supply of prescription-only veterinary medicines, but 45 per cent of them expressed an interest in becoming competent, even at significant cost in time and money. The results suggested that the pharmacists had little awareness of the law relating to the supply of veterinary medicines or of their pharmacology.

Use of the cow as a large animal model of uterine infection and immunity.

For most of the reproductive cycle in both humans and animals, the uterus is clear of pathogenic bacteria. However, it is readily contaminated with pathogens, such as Escherichia and Tritichomonas species, during sexual intercourse and after parturition. Uterine infection is particularly common after parturition in cattle (Bos taurus), causing clinical disease and infertility. The endocrine and immune responses to uterine infection in cattle have been investigated in vivo and using tissue culture. Cattle are of sufficient size to permit monitoring of reproductive and immune function throughout uterine infections, and primary cell cultures are readily established. In the whole animal, uterine infections suppress GnRH and LH secretion, and inhibit the growth of ovarian follicles and their estradiol secretion. The immune response is characterized by an influx of neutrophils into the uterus and increased concentrations of acute phase proteins in peripheral plasma. In vitro, the endometrial and ovarian cell function is modified by challenge with bacteria, their products such as lipopolysaccharide or pro-inflammatory cytokines. However, it is interesting to note that the susceptibility to uterine infection and the immune response are partially regulated by the ovarian steroid hormone mileu. In conclusion, the ease of working with cattle, the availability of tissues and the similarity of uterine infection between mammals, make Bos taurus a good model for studying uterine infection and immunity.

Lipopolysaccharide and interferon gamma activate nuclear factor kappa B and induce cyclo-oxygenase-2 in equine vascular smooth muscle cells.

Equine endotoxaemia is an important cause of morbidity and mortality in horses caused by the interaction of bacterial lipopolysaccharide (LPS) with cells such as macrophages and vascular smooth muscle. In this study we isolated equine vascular smooth muscle from a variety of vessels and stimulated it with LPS and human interferon (hIFN)-gamma. Using reverse transcriptase polymerase chain reaction (rt-PCR) and Western blot analysis we show that cyclooxygenase-2 (COX-2) is readily expressed in equine vascular smooth muscle. Vascular smooth muscle cells produced prostaglandin E2 in response to LPS and hIFNgamma. Using similar approaches we saw very limited expression of inducible nitric oxide synthase (iNOS) in only one vascular smooth muscle preparation. LPS and IFNgamma caused translocation of the transcription factor nuclear factor kappa B (NfkappaB) to the nucleus in equine cells suggesting the limited iNOS production seen in our cells is not due to deficits in this signal transduction pathway. These data suggest that in equine vascular smooth muscle COX-2 and NfkappaB are likely to play important roles in the pathogenesis of equine endotoxaemia.

The annexin protein lipocortin 1 regulates the MAPK/ERK pathway.

Lipocortin 1 (annexin 1) is a calcium- and phospholipid-binding protein that modulates anti-inflammatory responses including those induced by lipopolysaccharide. To investigate the precise role of lipocortin 1 in regulating the lipopolysaccharide-induced signal transduction pathways, we generated stable RAW 264.7 macrophage cell lines expressing decreased and increased lipocortin 1 protein. Several RAW 264.7 clones with increased lipocortin 1 protein levels showed constitutive activation of the mitogen-activated protein kinase extracellular signal-regulated kinase, which was down-regulated following lipopolysaccharide treatment. Conversely, clones with decreased lipocortin 1 protein expression showed prolonged extracellular signal-regulated kinase activity, following lipopolysaccharide activation. Lipocortin 1 specifically regulates the components of the extracellular signal-regulated kinase pathway, since changes in lipocortin 1 protein expression had no affect on the related mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Lipocortin 1 modulated upstream components of the extracellular signal-regulated kinase pathway and associated with the adaptor protein growth factor binding protein. The downstream consequences of altered extracellular signal-regulated kinase activity were independent of the proinflammatory transcription factor nuclear factor kappa B. These data indicate that lipocortin 1 specifically regulates proximal signaling components of the extracellular signal-regulated kinase signal transduction pathway, resulting in the modulation of biochemical functions in RAW macrophages.

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.

Endotoxin induction of nitric oxide synthase and cyclooxygenase-2 in equine alveolar macrophages.

OBJECTIVE: To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. PROCEDURE: Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively. RESULTS: Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent. Conclusion-Lipopolysaccharide induces COX-2 and iNOS in equine macrophages. CLINICAL RELEVANCE: The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses.

Characterisation of the cardiovascular pharmacology of medetomidine in the horse and sheep.

Medetomidine was administered to sheep and horses at a dose rate of 5 microg kg(-1) (i.v.). Heart rate and blood pressure were recorded. Medetomidine induced bradycardia and a biphasic blood pressure response consisting of a transient hypertension followed by hypotension. Administration of prazosin (an alpha1 adrenoceptor antagonist; 100 microg kg(-1), i.v.) had no effect on the cardiovascular response to medetomidine (5 microg kg(-1), i.v.), but inhibited the cardiovascular response of methoxamine (an alpha1 adrenoceptor agonist; 75 microg kg(-1), i.v.). L-659,066 (an alpha2 adrenoceptor antagonist which does not cross the blood brain barrier; 264 microg kg(-1), i.v.) attenuated the medetomidine induced bradycardia, but had no effect on the cardiovascular response to methoxamine. L659,066 also reduced the medetomidine induced hypertension in sheep, but had less effect on the horse. It is concluded that both alpha1 and alpha2 adrenoceptors are important in the control of cardiovascular function in horses and sheep. Medetomidine appears to act on alpha2 adrenoceptors alone in the sheep. The cardiovascular effects of medetomidine in the horse are complex and may be influenced by central alpha2 adrenoceptor regulation or effects on other receptor subtypes as well as direct stimulation of peripheral alpha2 adrenoceptors.

Vascular endothelial growth factor upregulates constitutive cyclooxygenase 1 in primary bovine and human endothelial cells.

The effect of the angiogenic cytokine vascular endothelial growth factor (VEGF) on nitric oxide synthase (NOS) and cyclooxygenase (COX) expression was examined in human (HUVEC) and bovine (BAE) endothelial cells. VEGF (10 ng/ml) induced constitutive COX-1 expression in both HUVEC and BAE, but not the cytokine-inducible isoform, COX-2, inducible NOS or endothelial NOS. In HUVEC, VEGF (10 ng/ml) increased COX activity, but COX inhibitors had no effect on the proliferative response of endothelial cells to this cytokine. In conclusion the induction of COX-1 by VEGF is not involved in the mitogenic response of endothelial cells, but may be an important regulatory mechanism in the maintenance of vascular integrity.

Suppression by dexamethasone of inducible nitric oxide synthase protein expression in vivo: a possible role for lipocortin 1.

Western blot and densitometric analysis of organ homogenates from lipopolysaccharide (LPS)-treated rats (1-10 mg kg(-1), i.p.) exhibited a strong induction of inducible nitric oxide synthase (iNOS) expression seen at all the doses tested (1, 3, and 10 mg kg(-1), n = 3). In particular, 3 hr after challenge of rats with LPS, iNOS was detectable in the liver, kidney, aorta, spleen and lung. Dexamethasone (DEX) (0.1-1 mg kg(-1); -1 hr) dose-dependently reduced iNOS expression in lung homogenates after exposure to LPS (1 mg kg(-1); P < 0.05). A partial reversal of DEX-induced suppression of iNOS expression in lung homogenates 3 hr after challenge with LPS was observed in rats which received a specific anti-lipocortin 1 sheep serum (LCS3; 1 mL kg(-1) 24 hr prior to the steroid), with an inhibition of 35+/-8%, as compared to animals passively immunised with normal sheep serum where dexamethasone exhibited an inhibition of 60+/-7% (n = 4). Peritoneal macrophages collected from rats treated with LPS (1 mg kg(-1); 3 hr) and cultured for 16 hr, released significant amounts of nitrite (51+/-1 microM) into the cell supernatants; this was reduced (-70+/-6%) after pre-treatment with dexamethasone (0.3 mg kg(-1)) and this effect was neutralised if animals were passively immunised with LCS3 (P < 0.01; n = 4). Thus lipocortin 1 mediates, at least in part, the inhibitory action exerted by dexamethasone on both iNOS protein expression in lung and iNOS activity (as measured by nitrite release) in primary peritoneal cells of rats.

Effect of the inducible nitric oxide synthase inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine on zymosan-induced plasma extravasation in rat skin.

The effect of nitric oxide synthase (NOS) inhibitors on plasma extravasation in a rat model of zymosan-induced inflammation has been investigated. Plasma extravasation was determined in response to intradermal test agents over 0 to 45 min or 0 to 4 h by the accumulation of i.v. injected 125I-labeled human serum albumin. Zymosan (1-100 microg/site) produced a dose- and time-dependent plasma extravasation. N(G)-nitro-L-arginine methyl ester (30-300 nmol/site), but not aminoguanidine (AG; 10-300 nmol/site) or L-N6-(1-iminoethyl)lysine (L-NIL; 10-300 nmol/site), significantly (p < 0.01) inhibited zymosan-induced (10 microg/site) plasma extravasation over 0 to 45 min. However, both AG and L-NIL produced significant (p < 0.05) inhibition over 0 to 4 h. The inhibition produced by AG was reversed by i.v. L-arginine or by coinjection of the vasodilator, calcitonin gene-related peptide. Zymosan (10-100 microg/site) induced an increase in dermal blood flow (laser-Doppler flowmetry) and this was inhibited by AG. Neutrophils were depleted selectively with antiserum, but this did not affect plasma extravasation except at the highest dose of zymosan (100 microg/site). Furthermore, zymosan-induced edema was not modified at either time point by pretreatment with the cyclooxygenase inhibitor indomethacin (30 micromol/kg, s.c., -30 min). In conclusion, in this model of dermal inflammation, it is suggested that inducible NOS inhibitors selectively remove an inducible NOS component that, at least in part, acts to increase microvascular blood flow and thus the edema formation observed during 0 to 4 h. There is no evidence of a contributory role for neutrophils or cyclooxygenase products in this model.

Nitric oxide production by equine articular cells in vitro.

Recent research in several species has suggested nitric oxide (NO) as a mediator of articular cartilage damage and an inhibitor of cartilage matrix neosynthesis. This study investigated NO production by cultured equine articular chondrocytes in response to 2 arthritogenic molecules, namely lipopolysaccharide (LPS) and interleukin-1 beta (IL-1 beta), and compared NO production by cultured equine synoviocytes stimulated with LPS. Synoviocytes exhibited a low basal level of NO synthesis (measured as nitrite, a NO metabolite) that was neither significantly increased nor decreased by exposure to LPS. Basal NO synthesis by synoviocytes was not significantly reduced by competitive inhibitors of nitric oxide synthase (NOS). In contrast, chondrocytes treated with LPS or IL-1 beta synthesised nitrite in a dose-related manner. Inhibitors of NOS suppressed nitrite production to below the basal levels of release of unstimulated cells. Dexamethasone, an inhibitor of induction of the inducible isoform of NOS (iNOS), reduced nitrite synthesis by LPS-stimulated chondrocytes. Western blot analysis revealed expression, in response to LPS, of protein in the same molecular weight range as iNOS identified in other species. This work demonstrates that equine chondrocytes have the capacity to synthesise NO, although its exact roles in cartilage metabolism have yet to be determined.

Cardiopulmonary effects of medetomidine in sheep and in ponies.

Medetomidine was administered intravenously to six sheep at 5, 10 and 20 micrograms kg-1 and to one horse and four ponies at 5 and 10 micrograms kg-1. In both species medetomidine resulted in significant decreases in heart rate and cardiac output and, initially, in an increase in arterial blood pressure. In the ponies this increase in blood pressure was followed by a significant and prolonged decrease, but in the sheep the secondary decrease in blood pressure was not statistically significant. In the sheep, the three doses of medetomidine resulted in profound and significant decreases in arterial oxygen tensions, which were significantly dose related, but in the ponies the arterial blood oxygen tensions were not significantly decreased. In both species medetomidine caused a small but significant increase in arterial blood carbon dioxide tensions.