Influence of oxytetracycline on carprofen pharmacodynamics and pharmacokinetics in calves

A tissue cage model of inflammation in calves was used to determine the pharmacokinetic and pharmacodynamic properties of individual carprofen enantiomers, following the administration of the racemate. RS(±) carprofen was administered subcutaneously both alone and in combination with intramuscularly administered oxytetracycline in a four‐period crossover study. Oxytetracycline did not influence the pharmacokinetics of R(?) and S(+) carprofen enantiomers, except for a lower maximum concentration (C_max) of S(+) carprofen in serum after co‐administration with oxytetracycline. S(+) enantiomer means for area under the serum concentration–time curve (AUC_0–96h were 136.9 and 128.3 μg·h/mL and means for the terminal half‐life (T_½k₁₀) were = 12.9 and 17.3 h for carprofen alone and in combination with oxytetracycline, respectively. S(+) carprofen AUC_0–96h in both carprofen treatments and T_½k₁₀ for carprofen alone were lower (P < 0.05) than R(?) carprofen values, indicating a small degree of enantioselectivity in the disposition of the enantiomers. Carprofen inhibition of serum thromboxane B₂ ex vivo was small and significant only at a few sampling times, whereas in vivo exudate prostaglandin (PG)E₂ synthesis inhibition was greater and achieved overall significance between 36 and 72 h (P < 0.05). Inhibition of PGE₂ correlated with mean time to achieve maximum concentrations in exudate of 54 and 42 h for both carprofen treatments for R(?) and S(+) enantiomers, respectively. Carprofen reduction of zymosan‐induced intradermal swelling was not statistically significant. These data provide a basis for the rational use of carprofen with oxytetracycline in calves and indicate that no alteration to carprofen dosage is required when the drugs are co‐administered.     

Effects of flunixin meglumine and ketoprofen on mediator production in ex vivo and in vitro models of inflammation in healthy dairy cows

Donalisio, C., Barbero, R., Cuniberti, B., Vercelli, C., Casalone, M., Re, G. Effects of flunixin meglumine and ketoprofen on mediator production in ex vivo and in vitro models of inflammation in healthy dairy cows. J. vet. Pharmacol. Therap.  36 , 130–139. In this study, ex vivo assays were carried out in dairy cows to evaluate the anti‐inflammatory effects of two nonsteroidal anti‐inflammatory drugs: ketoprofen (KETO) and flunixin meglumine (FM). Twelve healthy Holstein dairy cattle were randomly allocated to two groups (n=6): group 1 received FM and group 2 received KETO at recommended therapeutic dosages. The anti‐inflammatory effects of both drugs were determined by measuring the production of coagulation‐induced thromboxane B₂ (TXB₂), lipopolysaccharides (LPS) (10 μg/mL)‐induced prostaglandin E₂ (PGE₂), and calcium ionophore (60 μm )‐induced leukotrien B₄ (LTB₄). Cytokine production was assessed by measuring tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ) and interleukin‐8 (CXCL8) concentrations after incubation in the presence of 10 μg/mL LPS. The IC₅₀ of FM and KETO was determined in vitro by determining the concentration of TXB₂ and PGE₂ in the presence of scalar drug concentrations (10^?9–10^?3 m ). Both FM and KETO inhibited the two COX isoforms in vitro, but showed a preference for COX‐1. FM and KETO showed similar anti‐inflammatory effects in the cow.     

Carprofen pharmacokinetics in plasma and in control and inflamed canine tissue fluid using in vivo ultrafiltration

Measurement of unbound drug concentrations at their sites of action is necessary for accurate PK/PD modeling. The objective of this study was to determine the unbound concentration of carprofen in canine interstitial fluid (ISF) using in vivo ultrafiltration and to compare pharmacokinetic parameters of free carprofen concentrations between inflamed and control tissue sites. We hypothesized that active concentrations of carprofen would exhibit different dispositions in ISF between inflamed vs. normal tissues. Bilateral ultrafiltration probes were placed subcutaneously in six healthy Beagle dogs 12 h prior to induction of inflammation. Two milliliters of either 2% carrageenan or saline control was injected subcutaneously at each probe site, 12 h prior to intravenous carprofen (4 mg/kg) administration. Plasma and ISF samples were collected at regular intervals for 72 h, and carprofen concentrations were determined using HPLC. Prostaglandin E2 (PGE₂) concentrations were quantified in ISF using ELISA. Unbound carprofen concentrations were higher in ISF compared with predicted unbound plasma drug concentrations. Concentrations were not significantly higher in inflamed ISF compared with control ISF. Compartmental modeling was used to generate pharmacokinetic parameter estimates, which were not significantly different between sites. Terminal half‐life (T½) was longer in the ISF compared with plasma. PGE₂ in ISF decreased following administration of carprofen. In vivo ultrafiltration is a reliable method to determine unbound carprofen in ISF, and that disposition of unbound drug into tissue is much higher than predicted from unbound drug concentration in plasma. However, concentrations and pharmacokinetic parameter estimates are not significantly different in inflamed vs. un‐inflamed tissues.     

Expression and regulation of cyclooxygenase-2 in normal and neoplastic canine keratinocytes

Squamous cell carcinoma (SCC) is one of the most common cancers in dogs, yet relatively little is known about the molecular events involved in its development. Increasing evidence implicates cyclooxygenase‐2 (COX‐2) in the pathogenesis of various cancers in humans and animals. COX‐2 overexpression has recently been demonstrated in canine SCCs. The objective of our study was to characterize the expression and regulation of COX‐2 in normal and neoplastic canine keratinocytes (CKs) to provide an in vitro system to investigate the implication of COX‐2 in SCC oncogenesis in dogs. Cell lines derived from normal CKs and neoplastic CKs (SCCs) were cultured in the absence or presence of agonists, and immunoblots, immunocytochemistry, radioimmunoassays and a cell proliferation assay were used to characterize COX‐2 expression and action. Results showed that neoplastic keratinocytes had a higher basal COX‐2 expression than normal keratinocytes. In both cell lines, stimulation with the tumour promoter phorbol 12‐myristate 13‐acetate induced a time‐dependent increase in COX‐2 protein, with COX‐2 induction being stronger in cancerous SCC than in normal CK cells. Moreover, SCC cells produced significantly more PGE₂ than CK cells, under both baseline and stimulated conditions (P < 0.05). NS‐398, a selective COX‐2 inhibitor, inhibited prostaglandin (PG)E₂ synthesis and decreased proliferation of CK and SCC cells (P < 0.05). Collectively, our results indicate that the canine neoplastic keratinocyte SCC cell line expresses more COX‐2 and produces more PGE₂ than the normal keratinocyte CK cell line, thus providing an in vitro system to study the molecular basis of elevated COX‐2 expression in SCCs in dogs.     

Detection of anti‐TNFα activity in canine hyperimmune serum using a TNFα inhibition assay

Background: Increased serum tumor necrosis factor‐α (TNFα) activity has been associated with onset of serious inflammatory diseases in dogs. Development of treatment with TNFα‐antagonists has been limited by the unavailability of suitable reagents and potency assays for TNFα. Objectives: The objectives of this study were to optimize a cell‐based assay to measure anti‐TNFα activity in serum and plasma from hyperimmune (vaccinated with an Escherichia coli J5 bacterin) and unvaccinated canine donors; to use the assay to determine whether hyperimmune serum inhibits TNFα activity in vivo; and to determine whether soluble TNF receptor‐1 (sTNFR1, a naturally occurring TNFα antagonist) contributes to anti‐TNFα activity. Methods: Commercial plasma and serum from hyperimmune‐frozen plasma (HFP) donors and unvaccinated fresh‐frozen plasma (FFP) donors were used in the study. An L929‐cell TNFα‐inhibition assay (LTIA) was optimized to measure anti‐TNFα activity. Using a rat subcutaneous pouch model of inflammation, the effects of HFP, FFP, a synthetic TNFα antagonist (Etanercept), and carprofen on TNFα activity were compared in vivo. Immunofluorescence was used to measure soluble sTNFR1 concentration. Results: Using the optimized LTIA, HFP serum but not FFP serum decreased canine TNFα activity (P<.01). HFP plasma and Etanercept (but not FFP plasma or carprofen) significantly decreased TNFα activity in pouch exudates (P<.05). A significantly higher concentration of sTNFR1 was found in HFP than FFP serum. Conclusions: Using the LTIA, anti‐TNFα activity is readily measured in canine serum and inflammatory exudates. sTNFR1 appears to contribute to anti‐TNFα activity in HFP serum. These results suggest HFP should be investigated further as a potential immunotherapeutic agent for controlling canine diseases in which TNFα is implicated.     

Comparative study of the effects of acepromazine and structurally related compounds on the TNF-α release by monocytes stimulated by Chlamydia pneumoniae

Acepromazine (ACP), a member of the phenothiazine family, has antioxidant properties and interacts with reactive oxygen species produced by stimulated neutrophils ( Serteyn et al. 1999 ). We found that ACP reduced the differentiation of monocytes induced by an overnight incubation with a crude Chlamydia pneumoniae extract ( Serteyn et al. 2001 ). The same model was used to test the effects of phenothiazines on the TNF‐α release by activated monocytes. A crude Chlamydia pneumoniae extract was obtained by mechanical disruption and centrifugation (1 minute, 1500 r.p.m.) of 78 hours infected McCoy cells. Monocytes (THP1 cell line; 2 × 10⁶ cells by assay) were incubated overnight with 30 µL of Chlamydia pneumoniae crude extract (equivalent to an endotoxin charge of 3.5 pg) in the presence or absence of phenothiazines (from 10^?6 to 10^?4 M) ( Mouithys‐Mickalad et al. 2001 ). For estimation of TNF‐α release, the supernatants were collected, centrifuged (to eliminate the undifferentiated monocytes) and used for TNF‐α measurements (n = 6) (Quantikine HS human TNF‐α, R&D Systems, UK). Acepromazine was compared to other phenothiazines (chlorpromazine, trifluoperazine) or to structural analogues of phenothiazines (phenoxazine, thioxanthen‐9‐one and methylene blue). For each assay, cytotoxicity was evaluated by microscopic examination and blue trypan exclusion method. Mean values of TNF‐α were compared by a Student t‐test (p < 0.05). TNF‐α release by Chlamydia‐treated THP1 was significantly decreased by ACP in a dose‐dependent manner, 378 ± 30, 209 ± 38 and 189 ± 35 ng mL^?1 for 10^?6, 10^?5 and 10^?4 M compared to the control values 385 ± 9 ng mL^?1. Similar inhibitions of TNF‐α release were obtained with trifluoperazine (313 ± 25 and 265 ± 14 ng mL^?1 at 10^?6 and 10^?5 M) and chlorpromazine (323 ± 29 and 227 ± 13 ng mL^?1 at 10^?6 and 10^?5 M), but at 10^?4 M, these two drugs were cytotoxic. The other structurally parent compounds increased significantly the TNF‐α production: 630 ± 46 and 468 ± 60 ng mL^?1 for thioxanthen‐9‐one and 547 ± 17 and 331 ± 111 ng mL^?1 for methylene blue at 10^?5 and 10^?6 (M). At 10^?4 M, the two compounds were cytotoxic. Phenoxazine increased the TNF‐α production, slightly at 10^?6 and 10^?5 M (444 ± 39 and 424 ± 16 ng mL^?1, respectively) and significantly at 10^?4 M (959 ± 30 ng mL^?1). Further studies are needed to verify if the inhibition of TNF‐α release by some phenothiazines could be linked to a reduction of the signal transduction, especially the NF‐κB pathway. These results could be interesting for the anaesthesia or treatment of animals suffering from a systemic inflammatory reaction.     

Clinical and immunomodulating effects of ketamine in horses with experimental endotoxemia

Background: Ketamine has immunomodulating effects both in vitro and in vivo during experimental endotoxemia in humans, rodents, and dogs. Hypothesis: Subanesthetic doses of ketamine will attenuate the clinical and immunologic responses to experimental endotoxemia in horses. Animals: Nineteen healthy mares of various breeds. Methods: Experimental study. Horses were randomized into 2 groups: ketamine‐treated horses (KET; n = 9) and saline‐treated horses (SAL; n = 10). Both groups received 30 ng/kg of lipopolysaccharide (LPS, Escherichia coli, O55:B5) 1 hour after the start of a continuous rate infusion (CRI) of racemic ketamine (KET) or physiologic saline (SAL). Clinical and hematological responses were documented and plasma concentrations of tumor necrosis factor‐α (TNF‐α) and thromboxane B₂ (TXB₂) were quantified. Results: All horses safely completed the study. The KET group exhibited transient excitation during the ketamine loading infusion (P < .05) and 1 hour after discontinuation of administration (P < .05). Neutrophilic leukocytosis was greater in the KET group 8 and 24 hours after administration of LPS (P < .05). Minor perturbations of plasma biochemistry results were considered clinically insignificant. Plasma TNF‐α and TXB₂ production peaked 1.5 and 1 hours, respectively, after administration of LPS in both groups, but a significant difference between treatment groups was not demonstrated. Conclusions and Clinical Importance: A subanesthetic ketamine CRI is well tolerated by horses. A significant effect on the clinical or immunologic response to LPS administration, as assessed by clinical observation, hematological parameters, and TNF‐α and TXB₂ production, was not identified in healthy horses with the subanesthetic dose of racemic ketamine utilized in this study.     

Leukotrienes are Auto‐/Paracrine Factors in the Bovine Corpus Luteum: An In Vitro Study

The aim of this study was to determine leukotrienes (LTs) functions in the bovine corpus luteum (BCL) during the oestrous cycle. In steroidogenic CL cells we examined the effect of luteotropic [LH, prostaglandin E₂ (PGE₂)] and luteolytic (PGF_2α, cytokines) factors on: the levels of LTB₄ and C₄, the expression of 5‐lipoxygenase (LO), LT receptors type I (LTR‐I) and LTR‐II, and the effects of LTB₄ and C₄ stimulations on the levels of progesterone (P4), PGE₂, F_2α and nitric oxide (NO) metabolites. Both luteolytic and luteotropic factors stimulated 5‐LO expression on days 2–4 and 17–19 of the cycle. Leukotriene receptors type I expression increased after PGE₂ and tumour necrosis factor α with interferon γ (TNF/IFN) stimulation on days 2–4 of the cycle. Leukotriene receptor type II expression increased after PGE_2α and TNF/IFN stimulation on days 2–4 and 17–19 of the cycle, and LTR‐II expression on days 8–10 of the cycle was unchanged after cell stimulation with any factor. Leukotriene B₄ level increased after BSC incubation with luteotropic factors during all examined days of the cycle and after cytokine stimulation at early‐ and mid‐luteal stages, whereas luteolytic factors stimulated LTC₄ secretion over the entire cycle. Leukotriene B₄ stimulated P4 secretion at the mid‐luteal stage and stimulated NO secretion during all examined phases. Leukotriene B₄ stimulated PGE₂ secretion at the early‐ and mid‐luteal stage. Leukotriene C₄ inhibited P4 secretion at the mid‐ and regressing‐luteal stage, stimulated NO (entire cycle) and PGF_2α at mid‐ and regressing‐luteal phases. Leukotrienes modulate steroidogenic cells functions, depending on the stage of the cycle. Leukotriene B₄ plays a luteotropic role stimulating P4 and PGE₂ secretions; LTC₄ stimulates the secretion of luteolytic factors and enhances the luteolytic cascade within BCL.     

Regulation of uterine function by cytokines in cows: Possible actions of tumor necrosis factor-α, interleukin-1α and interferon-τ

When animals do not become pregnant, regression of the corpus luteum (CL) is essential for normal cyclicity because it allows the development of a new ovulatory follicle. Luteal regression is caused by a pulsatile release of prostaglandin (PG) F_2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF_2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF_2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF_2α secretion. There is increasing evidence that several cytokines mainly produced by immune cells modulate CL and uterine function in many species. Tumor necrosis factor‐α (TNF‐α) stimulates PGF_2α output from bovine endometrium not only at the follicular phase but also at the late luteal phase. Administration of TNF‐α at a high concentration prolongs luteal lifespan, whereas administration of a low concentration of TNF‐α accelerates luteal regression in cows. The data obtained from the authors’ previous in vitro and in vivo studies strongly suggest that TNF‐α is a crucial factor in regulating luteolysis in cows. The authors’ recent study has shown that interleukin‐1α mediates PG secretion from bovine endometrium as a local regulator. Furthermore, interferon‐τ (IFN‐τ) suppresses the action of TNF‐α on PGF_2α synthesis by the bovine endometrium in vitro, suggesting that IFN‐τ plays a luteoprotective role by inhibiting TNF‐α‐induced PGF_2α production in early pregnancy. The purpose of the present review is to summarize current understanding of the endocrine mechanisms that regulate uterine function by cytokines during the estrous cycle and early pregnancy in cows.     

COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

Pharmacokinetic/pharmacodynamic modelling of robenacoxib in a feline tissue cage model of inflammation

Pelligand, L., King, J. N., Toutain, P. L., Elliott, J., Lees, P. Pharmacokinetic/pharmacodynamic modelling of robenacoxib in a feline tissue cage model of inflammation. J. vet. Pharmacol. Therap.  35 , 19–32. Robenacoxib is a novel nonsteroidal anti‐inflammatory drug developed for use in cats. It is a highly selective COX‐2 inhibitor. Results from previous feline studies showed that, despite a short half‐life in blood, the effect of robenacoxib persisted for 24 h in clinical studies. A tissue cage model of acute inflammation was used to determine robenacoxib’s pharmacokinetics and its ex vivo and in vivo selectivity for COX‐1 and COX‐2 using serum TxB₂ and exudate PGE₂ as surrogate markers for enzyme activity, respectively. After intravenous, subcutaneous and oral administration (2 mg/kg), the clearance of robenacoxib from blood was rapid (0.54–0.71 L·h/kg). The mean residence time (MRT) in blood was short (0.4, 1.9 and 3.3 h after intravenous, subcutaneous and oral administration, respectively), but in exudate MRT was approximately 24 h regardless of the route of administration. Robenacoxib inhibition of COX‐1 in blood was transient, occurring only at high concentrations, but inhibition of COX‐2 in exudate persisted to 24 h. The potency ratio (IC₅₀ COX‐1: IC₅₀ COX‐2) was 171:1, and slopes of the concentration–effect relationship were 1.36 and 1.12 for COX‐1 and COX‐2, respectively. These data highlight the enzymatic selectivity and inflamed tissue selectivity of robenacoxib and support the current recommendation of once‐daily administration.     

Effects of tumour necrosis factor‐alpha and interleukin‐1 beta on in vitro development of bovine secondary follicles

The objective of this study was to determine the effects of TNF‐α and IL‐1β on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO₂ in air, for 18 days, in TCM‐199⁺ alone (cultured control) or supplemented with 10 ng/ml IL‐1β, 10 ng/ml TNF‐α or both TNF‐α and IL‐1β. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF‐9, c‐MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF‐α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL‐1β and a mixture of IL‐1β and TNF‐α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF‐α had the best‐preserved oocytes and granulosa cells. The presence of TNF‐α, IL‐1β or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL‐1β, TNF‐α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.     

High COX‐2 expression is associated with increased angiogenesis,proliferation and tumoural inflammatory infiltrate in canine malignant mammary tumours: a multivariate survival study

COX‐2 expression affects mammary tumourigenesis by promoting angiogenesis and cell proliferation, encouraging metastatic spread and tumour‐associated inflammation. Samples of canine mammary tumours (n = 109) were submitted to immunohistochemistry to detect COX‐2, CD31, VEGF, Ki‐67, CD3 and MAC387 expression. Concurrent high expression of COX‐2/CD31, COX‐2/VEGF, COX‐2/Ki‐67, COX‐2/CD3 and COX‐2/MAC was associated with elevated grade of malignancy, presence of intravascular emboli and presence of lymph node metastasis. Tumours with high COX‐2 (P < 0.001) and tumours with concurrent expression of high COX‐2 and high CD31 (P = 0.008); high VEGF (P < 0.001); high Ki‐67 (P < 0.001); high CD3+ T‐lymphocytes (P = 0.002) and elevated MAC387 macrophages (P = 0.024) were associated with shorter overall survival (OS) time. Interestingly the groups with high COX‐2/CD31 and high COX‐2/VEGF retained their significance after multivariate analysis arising as independent predictors of OS. Present data highlight the importance of COX‐2 in canine mammary tumourigenesis.     

High COX‐2 expression in canine mast cell tumours is associated with proliferation,angiogenesis and decreased overall survival

COX‐2 overexpression is associated with several hallmarks of carcinogenesis such as proliferation, angiogenesis, invasion and metastasis. Fifty cases of canine mast cell tumours (MCT) were retrospectively evaluated and submitted to immunohistochemistry for COX‐2, CD31, Ki‐67, MAC‐387 and CD3. Furthermore its relationship with clinicopathological variables and overall survival (OS) was analysed. COX‐2 intensity (P = 0.016), but not COX‐2 extension nor score was associated with decreased OS and higher grades of malignancy according to Patnaik (P = 0.002) and Kiupel (P < 0.001) grading systems. Cox‐2 intensity was also associated with higher Ki‐67 scores (P = 0.009), higher mitotic index (P = 0.022) and higher microvascularization density (P = 0.045). No association was observed for COX‐2 intensity and CD3‐T lymphocyte (P = 0.377) and macrophage infiltration (P = 0.261) by MAC‐387 immunollabelling, suggesting an active role of COX‐2 in MCT oncogenesis mainly through proliferation and angiogenesis stimulation making it a potentially clinical relevant prognosis marker and therapeutic target.     

Breed-specific pro-inflammatory cytokine production as a predisposing factor for susceptibility to sepsis in the dog

Objective: To determine whether 2 dog breeds with a high risk for parvoviral enteritis, a disease associated with sepsis, produce stronger pro‐inflammatory cytokine responses to a stimulus than dogs with a lower risk. Design: Blinded comparison. Setting: University outpatient clinic. Animals: Healthy, unrelated, purebred Doberman Pinschers (n=10) and Rottweilers (n=9) with age‐matched mixed‐breed dogs (n=7). Interventions: Heparinized, whole‐blood samples were collected from each dog and incubated for 6 hours with lipopolysaccharide. Plasma was collected, and bioassays were used to determine the concentrations of TNF‐α and IL‐6. The mean values obtained from the high‐risk breeds were compared with the mean obtained from the mixed‐breeds. Measurements and main results: The mean TNF‐α production from dogs with a high risk for parvoviral enteritis (1321±161 pg/mL; Doberman Pinscher and Rottweiler) was greater (P<0.05) than that from lower risk, mixed‐breed dogs (674±186 pg/mL). There were no differences in TNF‐α levels between Doberman (1128±247 pg/mL) and Rottweiler (1563±pg/mL) breeds or between any breeds with regard to IL‐6 production. Conclusions: The magnitude of TNF‐α production by peripheral blood monocytes was the greatest in the dogs with breed‐related risk for parvoviral enteritis. However, additional studies are needed to prove a causal relationship between high TNF and predilection for parvoviral enteritis. Regardless, breed appears to be a predisposing factor for variations in cytokine production that could impact the host response to infection and other inflammatory insults.     

Prostaglandin Endoperoxide Synthase 2 (PTGS2) and Prostaglandins F2α and E2 Synthases (PGFS and PGES) Expression and Prostaglandin F2α and E2 Secretion Following Oestrogen and/or Progesterone Stimulation of the Feline Endometrium

Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG‐endoperoxide synthase (PTGS2), PGF_2α synthase (PGFS) and PGE₂ synthase (PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E₂ and/or P₄ on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up‐regulated at the mid phase of pseudopregnancy. The effects of E₂ and/or P₄ treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up‐regulated by E₂ plus P₄ at oestrus and the mid phase of pseudopregnancy and was also up‐regulated by a single treatment with P₄ at late pseudopregnancy (p < 0.05). Simultaneous incubation with E₂ and P₄ up‐regulated PTGS2 gene expression at oestrus and mid‐luteal phase (p < 0.05). Progesterone plus E₂ significantly increased PGE₂ secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E₂ and/or P₄ affected neither PGF_2α secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up‐regulated at the mid phase of pseudopregnancy. An increase in PGE₂ secretion and up‐regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E₂ and P₄ at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE₂ are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats.     

Tumour Necrosis factor-α Receptors are Present in the Corpus Luteum Throughout the Oestrous Cycle and During the Early Gestation Period in Pigs

To determine the physiological significance of tumour necrosis factor‐α (TNFα) in the regulation of luteal functions in pig, this study was conducted to identify the presence of functional TNFα receptors in porcine corpora lutea (CL) throughout the oestrous cycle and the early gestation. The CL were isolated from pigs on days 4, 6, 8, 12 or 15 of the oestrous cycle (n=3; day 0 = oestrus) and days 15, 20 or 25 of gestation (n=3; day 0 = mating). A Scatchard analysis revealed the presence of a high‐affinity binding site for TNFα in all samples (dissociation constant; 2.7 ± 0.51 to 5.8 ± 0.50 nM ). The concentration of TNFα receptors was higher on day 15 of the oestrous cycle than on days 4 and 8 of the oestrous cycle (p < 0.05). Furthermore, TNFα receptor concentrations in the CL on days 15, 20 and 25 of gestation were significantly lower than on day 15 of the oestrous cycle (p < 0.05). On day 9 of the oestrous cycle, exposure of cultured luteal cells to 0.06–60 nM TNFα stimulated prostaglandin (PG) F_2α and PGE₂ secretion in a dose‐dependent manner (p < 0.05). These results indicate that functional TNFα receptors are present in the porcine CL throughout the oestrous cycle and early gestation, and suggest that TNFα plays one or more physiological roles in regulating CL function throughout the oestrous cycle and the early gestation period. In addition, TNFα receptor concentration in the CL of the late luteal stage (day 15) of the oestrous cycle was higher than on the respective day in the early pregnant pig, suggesting that TNFα plays a role in accomplishing luteolysis in the porcine CL.     

Polysaccharides from Acanthopanax senticosus enhances intestinal integrity through inhibiting TLR4/NF‐κB signaling pathways in lipopolysaccharide‐challenged mice

To investigate the role of polysaccharide from Acanthopanax senticosus (ASPS) on lipopolysaccharide (LPS)‐induced intestinal injury, mice in three treatments were administrated orally with or without ASPS (300 mg/kg body weight) for 14 days, followed by challenge with LPS or saline. At 4 h post‐injection, blood and intestinal samples of six mice / treatment were collected. The results showed ASPS ameliorated LPS‐induced intestinal morphological deterioration, proven by improved villus height (P < 0.05) and villus height : crypt depth ratio (P < 0.05). ASPS also elevated the mucosal barrier of LPS‐challenged mice, supported by reduced plasma diamine oxidase (DAO) activity (P < 0.05) and L‐lactate (P < 0.05), increased mucosal DAO activity (P < 0.05) as well as enhanced intestinal tight junction proteins expression involving occludin‐1 (P < 0.05) and zonula occludens‐1 (P < 0.05). In addition, ASPS decreased LPS‐induced secretion of inflammatory mediators, including tumor necrosis factor (TNF)‐α (P < 0.05) and prostaglandin E₂ (P < 0.05). Also, ASPS down‐regulated messenger RNA expression of toll‐like receptor 4 (TLR4) and its downstream signals, including myeloid differentiation factor 88 (P < 0.05), TNF‐α receptor‐associated factor 6 (P < 0.05), as well as nuclear factor (NF)‐κB p65 (P < 0.05) and its protein expression. These findings suggest that ASPS improves intestinal integrity under inflammation conditions connected with inhibiting TLR4/NF‐κB signaling pathways.     

Potency and selectivity of carprofen enantiomers for inhibition of bovine cyclooxygenase in whole blood assays

Whole blood in vitro assays were used to determine the potency and selectivity of carprofen enantiomers for inhibition of the isoforms of cyclooxygenase (COX), COX-1 and COX-2, in the calf. S(+)-carprofen possessed preferential activity for COX-2 inhibition but, because the slopes of inhibition curves differed, the COX-1:COX-2 inhibition ratio decreased from 9.04:1 for inhibitory concentration (IC)₁₀ to 1.84:1 for IC₉₅. R(−) carprofen inhibited COX-2 preferentially only for low inhibition of the COX isoforms (IC₁₀ COX-1:COX-2 = 6.63:1), whereas inhibition was preferential for COX-1 for a high level of inhibition (IC₉₅ COX-1:COX-2 = 0.20:1). S(+) carprofen was the more potent inhibitor of COX isoforms; potency ratios S(+):R(−) carprofen were 11.6:1 for IC₁₀ and 218:1 for IC₉₀. Based on serum concentrations of carprofen enantiomers obtained after administration of a therapeutic dose of 1.4 mg/kg to calves subcutaneously, S(+)-carprofen concentrations exceeded the in vitro IC₈₀ COX-2 value for 32 h and the IC₂₀ for COX-1 for 33 h. The findings are discussed in relation to efficacy and safety of carprofen in calves.