S. De Boever

Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens

Real-time PCR has become a powerful tool for the detection of inflammatory parameters, including cytokines. Reference or housekeeping genes are used for the normalization of real-time RT-PCR results. In order to obtain reliable results, the stability of these housekeeping genes needs to be determined. In this study the stability of five genes, including beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phophoribosyl-transferase (HPRT), ubiquitin (UB) and glucose-6-phosphate dehydrogenase (G6PDH), was determined in a lipopolysaccharide inflammation model in chickens. beta-Actin appeared to be the most stable single gene in our model. Because the use of a single gene for normalization can lead to relatively large errors, the use of the geometric mean of multiple reference genes or normalization factor is preferred. The most stable combination for gene expression analysis in this lipopolysaccharide inflammation model in chickens is G6PDH and UB, since their correlation coefficients were 0.953 and 0.969, respectively (BestKeeper) and an M value of 0.34 and a low V(2/3) value of 0.155 (geNorm) were obtained. The use of HPRT and GAPDH should be avoided. The stable housekeeping genes, G6PDH and UB together, can be used to normalize the expression of pro-inflammatory cytokines in a lipopolysaccharide inflammation model in chickens.

Antipyretic effect of oral sodium salicylate after an intravenous e. coli LPS injection in broiler chickens

1. A study was set up to investigate the influence of sodium salicylate on fever and acute phase reaction after lipopolysaccharide (LPS) injection in broiler chickens. 2. An acute phase reaction was provoked through the intravenous injection of Escherichia coli LPS. Four oral doses of sodium salicylate were tested. Apart from body temperature, other inflammation indices, such as plasma corticosterone and ceruloplasmin, serum thromboxane B2 and zinc concentrations were monitored. 3. Intravenous LPS induced a fever of about 1°C. A dose-dependent attenuation of the fever response of the chickens in the salicylate treated groups was observed. LPS-injected chickens also showed elevated plasma corticosterone and ceruloplasmin, while serum thromboxane and zinc concentrations decreased. Except for thromboxane B2, no linear relationship with increasing salicylate dose could be shown for the other blood variables. 4. These data confirm that sodium salicylate is an effective antipyretic agent after injection of LPS in chickens, if used at an appropriate dosage. No dose-related change could be found for the other inflammation indices.

Species comparison of oral bioavailability, first-pass metabolism and pharmacokinetics of acetaminophen

Species differences in oral bioavailability, first-pass metabolism and pharmacokinetics of biopharmaceutics classification system (BCS) class I compound acetaminophen were studied. The absolute bioavailability was 42.2%, 39.0%, 44.5%, 75.5% and 91.0% in chickens, turkeys, dogs, pigs and horses, respectively. After hydrolysis of metabolites by beta-glucuronidase/sulfatase, apparent bioavailability increased significantly in all species (turkeys: 72.4%, dogs: 100.5%, pigs: 102.2%), except horses (91.6%). Mean metabolic ratios of [acetaminophen glucuronide]/[acetaminophen] between 0 and 1h were significantly higher after oral dosing in turkeys, dogs and pigs, revealing the role of first-pass metabolism in incomplete bioavailability. Evidence of species differences in acetaminophen metabolism is provided by differences in plasma clearance, which was inversely proportional to bioavailability. In conclusion, differences in BA appeared to originate predominantly from differences in first-pass metabolism, demonstrating that the BCS high permeability classification of acetaminophen is consistent across the mammalian species studied. In turkeys, however, incomplete absorption additionally seemed to contribute to the low BA.

The influence of age and repeated lipopolysaccharide administration on body temperature and the concentration of interleukin-6 and IgM antibodies against lipopolysaccharide in broiler chickens

Our objective was to create a standardized and reproducible inflammation model in chickens in order to study the pharmacodynamics of several anti-pyretic and anti-inflammatory drugs. We studied the influence of age and repeated lipopolysaccharide (LPS) administration on body temperature and the correlation of this with concentrations of interleukin-6 and IgM antibodies against LPS in plasma of chickens. Three-week-old and 5-week-old broilers were injected intravenously with LPS from Escherichia coli O127: B8 at a dose of 1 mg/kg. LPS administration was repeated after 2 or 7 days. After the first dose of LPS, the body temperature was initially decreased below normal and then later increased above normal. The second dose of LPS reduced the level of hypothermia and the duration of the febrile phase. Three-week-old birds responded to LPS with a higher maximum body temperature and a greater area under the body temperature versus time curve than 5-week-old chickens (P<0.05). Interleukin-6 reached its highest concentration 3 h after LPS administration and returned to baseline levels after 9 h. A second dose of LPS resulted in a significantly lower peak in interleukin-6. Significant higher levels of antibodies against LPS could be detected 7 days after LPS administration. However, there appeared to be no correlation between the reduced response to LPS and the presence of antibodies.

Species comparison of enantioselective oral bioavailability and pharmacokinetics of ketoprofen.

As a part of ongoing research to further elucidate frequent and species-specific causes of differences in oral bioavailability, a 3mg/kg dose of racemic ketoprofen, a high permeability/low solubility compound in the human biopharmaceutics classification system, was administered intravenously and orally to different species. Due to possible enantioselective disposition kinetics and inversion, enantiomers were quantitated separately using a stereospecific HPLC assay. The absolute bioavailability of R(-) and S(+) ketoprofen in chickens, turkeys, dogs and pigs was 31.5% and 52.6%, 42.6% and 32.5%, 33.6% and 89.1%, and 85.9% and 83.5% respectively. Incomplete bioavailability in poultry is probably due to incomplete absorption in addition to first-pass elimination. Low bioavailability of R(-) ketoprofen in dogs, strongly indicates first-pass metabolism. High bioavailability of S(+) ketoprofen in dogs and both enantiomers in pigs confirms that absorption of these substances is complete and controlled by gastric emptying rather than dissolution.

Characterization of an intravenous lipopolysaccharide inflammation model in broiler chickens

Intravenous administration of lipopolysaccharide (LPS) from Escherichia coli O127:B8 at a dose of 1,500,000 u/kg body weight evoked a hypothermic response followed by a fever phase in 5-week-old broiler chickens. The hypothermic phase coincided with a severe decrease in blood pressure. We assume that this decrease in blood pressure is, at least partly, responsible for the hypothermic phase of the body temperature curve. LPS administration also caused a decrease in circulating white blood cells. The heterophils were predominantly sequestered in the lungs. In LPS-treated chickens, far more apoptotic leukocytes were present in the circulation, compared with control chickens. The molecular players responsible for the LPS-induced inflammatory response could be TL1A, IL-1β and IL-6, since a slight increase in their mRNA levels in white blood cells was already seen 1 h after LPS administration. In accordance with these observations, the levels of secreted IL-6 were maximal 3 h after LPS administration. These parameters characterize this LPS-induced inflammation model in broiler chickens.

Flow cytometric differentiation of avian leukocytes and analysis of their intracellular cytokine expression

A flow cytometric method for the identification of chicken blood leukocyte subpopulations and thrombocytes was developed. An anti-chicken CD45 phycoerythrin-labelled antibody was used to separate leukocytes from red blood cell nuclei. Leukocytes and thrombocytes were identified using a combination of their CD45-positivity and their typical side scatter properties. The identity of the CD45-positive cells was confirmed by sorting the subpopulations and subsequent light microscopic evaluation. In these differentiated cell populations, intracellular expression analysis of the proinflammatory cytokines interleukin-1β and interleukin-6 was subsequently optimized on whole blood after in vitro stimulation with lipopolysaccharide from Escherichia coli strain O127:B8.

Pharmacodynamics of tepoxalin, sodium-salicylate and ketoprofen in an intravenous lipopolysaccharide inflammation model in broiler chickens.

The pharmacodynamic properties of tepoxalin, Na-salicylate and ketoprofen were determined in an intravenous lipopolysaccharide (LPS) inflammation model in broiler chickens. The drugs were administered orally at a dose of 30, 50 and 3 mg/kg, respectively. LPS administration induces an increase in the intracellular expression of interleukin (IL)-1β and IL-6 and the secreted IL-6 plasma concentration. Furthermore, an elevation in body temperature is noted. Despite pretreatment with a single dose of the drugs and LPS administration on the T(max) of the drug after a second dose, no decrease was seen in systemic IL-6 levels. The intracellular expression of IL-1β in the heterophils was slightly decreased if LPS was administered in combination with each of the three drugs. Tepoxalin and Na-salicylate administration had no significant effect on the LPS-induced increase in prostaglandin E(2) plasma concentration, in contrast to ketoprofen. None of the three drugs were able to influence the elevation in body temperature after LPS administration. The pharmacokinetic properties of Na-salicylate and ketoprofen were not altered in combination with LPS administration. However, LPS significantly decreased the AUC(0→6 h) of the active metabolite of tepoxalin, RWJ-20142, indicating a perfusion-limited elimination for this molecule.

Transsplenic portal catheterization combined with a jugular double-lumen catheter for pharmacokinetic and presystemic metabolization studies in pigs.

The reliability of a silicone double-lumen catheter implanted into the external jugular vein and tunnelled towards the neck region was investigated in eight pigs. Surgery was uneventful without interference with the normal homoeostasis during 8 days. After injection of amoxicillin/clavulanic acid through the distal port of the catheter, analysis of drug components in the simultaneous blood samples obtained by the proximal port and a Venoject system were comparable in one pig. Histological control of the catheterized jugular veins pointed to an acceptable tissue reaction while bacteriological examination of the tip of the catheters was negative in only three animals. A moulding of the intestinal veins was made in a pig cadaver to determine the optimal length of insertion of a silicone portal catheter from the splenic vein towards the portal vein. Surgery was straightforward in four pigs whereby the catheter was exteriorized towards the back region. No complications were encountered during and after surgery for 9 days. The technique of a double-lumen catheter placed into the jugular vein and a transsplenic portal catheter is a useful tool for the study of the pharmacokinetics and also the first-pass effect of drugs in experimental pigs.

Sodium salicylate attenuates lipopolysaccharide (LPS)-induced adipsia, but not hypophagia, in broiler chickens

1. A study was conducted to determine the influence of sodium salicylate on the behaviour and the food and water consumption of broiler chickens after lipopolysaccharide (LPS) injection. 2. An oral dose of 100 mg/kg sodium salicylate was given and an acute phase reaction in broiler chickens was provoked through the intravenous injection of Escherichia coli LPS. 3. Water intake was higher in the LPS and salicylate-treated group than in the positive control group. The salicylate treatment, however, did not restore the food intake, or influence the behaviour of the chickens. 4. These data show that sodium salicylate has a positive effect on the water intake after intravenous injection of LPS in chickens and suggests that there is a difference in mechanism of action of food and water consumption after LPS injection in chickens.