Dynamics of Salivary Adenosine Deaminase, Haptoglobin, and Cortisol in Lipopolysaccharide-Challenged Growing Pigs.

Infectious and inflammatory conditions are common especially in growing pigs. Lipopolysaccharide (LPS) is an important antigenic structure of Gram-negative bacteria and can be used to induce inflammation experimentally. As pigs are usually group-housed in commercial conditions, it is difficult to detect sick individuals, particularly at an early stage of illness. Acute phase proteins such as haptoglobin (Hp) are known indicators of an activated innate immune system whereas adenosine deaminase (ADA) is a relatively novel inflammatory biomarker in pigs. Both parameters can be measured in saliva and could be used as indicators of inflammation. Compared with blood sampling, saliva sampling is a less stressful procedure that is rapid, non-invasive and easy to perform both at group and at individual level. In this blinded randomized clinical trial, 32 female pigs at their post-weaning phase were allocated to one of four treatments comprising two injections of the following substance combinations: saline-saline (SS), ketoprofen-saline (KS), saline-LPS (SL), and ketoprofen-LPS (KL). First, ketoprofen or saline was administered intramuscularly on average 1 h before either LPS or saline was given through an ear vein catheter. In all groups, saliva was collected prior to injections (baseline) and at 4, 24, 48, and 72 h post-injection for determination of ADA, Hp, and cortisol concentrations. A multivariate model was applied to describe the dynamics of each biomarker. Pairwise relationships between ADA, Hp, and cortisol responses from baseline to 4 h post-injection within the SL group were studied with Spearman correlations. A significant increase in the SL group was seen in all biomarkers 4 h post-injection compared to baseline and other time points (pairwise comparisons, p < 0.01 for all) and ketoprofen alleviated the LPS effect. We found a significant positive correlation between ADA and Hp within the SL group (r = 0.86, p < 0.05). The primary and novel findings of the present study are the response of ADA to LPS, its time course and alleviation by ketoprofen. Our results support the evidence that ADA and Hp can be used as inflammatory biomarkers in pigs. We suggest further studies to be conducted in commercial settings with larger sample sizes.

A Proposed Role for Pro-Inflammatory Cytokines in Damaging Behavior in Pigs.

Sickness can change our mood for the worse, leaving us sad, lethargic, grumpy and less socially inclined. This mood change is part of a set of behavioral symptoms called sickness behavior and has features in common with core symptoms of depression. Therefore, the physiological changes induced by immune activation, for example following infection, are in the spotlight for explaining mechanisms behind mental health challenges such as depression. While humans may take a day off and isolate themselves until they feel better, farm animals housed in groups have only limited possibilities for social withdrawal. We suggest that immune activation could be a major factor influencing social interactions in pigs, with outbreaks of damaging behavior such as tail biting as a possible result. The hypothesis presented here is that the effects of several known risk factors for tail biting are mediated by pro-inflammatory cytokines, proteins produced by the immune system, and their effect on neurotransmitter systems. We describe the background for and implications of this hypothesis.

The Effect of LPS and Ketoprofen on Cytokines, Brain Monoamines, and Social Behavior in Group-Housed Pigs.

Poor health is a risk factor for damaging behaviors, but the mechanisms behind this link are unknown. Injection of pigs with lipopolysaccharide (LPS) can be used to model aspects of poor health. Recent studies have shown that LPS-injected pigs perform more tail- and ear-directed behavior compared to saline-injected pigs and suggest that pro-inflammatory cytokines may play a role in these behaviors. The aims of this study were to test the effect of LPS on the social behavior of pigs and the neurotransmitters and modulators in their brains and to test the effect of a nonsteroidal anti-inflammatory drug on the effects of LPS. Fifty-two female pigs (11-12 weeks) were allocated to four treatments comprising two injections: saline-saline (SS), saline-LPS (SL), ketoprofen-saline (KS), and ketoprofen-LPS (KL). Activity was scan-sampled every 5 min for 6 h after the last injection in the pen. Social behavior was observed continuously in 10 × 15-min bouts between 8 a.m. and 5 p.m. 1 day before (baseline) and 1 and 2 days after the injection. Saliva was analyzed for cortisol and plasma for tryptophan and kynurenine. The frontal cortex, hippocampus, hypothalamus, and brain stem were sampled 72 h after the injection and analyzed for cytokines and monoamines. LPS activated the HPA axis and decreased the activity within 6 h after the injection. Ketoprofen lowered the effect of LPS on cortisol release and attenuated the behavioral signs of sickness in challenged pigs. SL pigs manipulated the ears of their pen mates significantly longer than SS pigs 2 days after the injection. LPS had no observed effect on IFN-γ, TNF-α, and IL-18. At 72 h after the injection, plasma tryptophan was depleted in SL pigs, and tryptophan and kynurenine concentrations in the frontal cortex and brain stem of SL pigs were significantly lower compared to those in SS pigs. Dopamine concentrations in the hypothalamus of SL pigs were significantly lower compared to those in SS pigs. Serotonin concentrations in the hypothalamus and noradrenaline concentrations in the hippocampus of SL pigs were significantly lower compared to those in KL pigs. In conclusion, LPS influenced the different neurotransmitters and modulators in the brain that are hypothesized to play an important role in the regulation of mood and behavior.

Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay.

A stable luciferase reporter gene assay was developed based on the thyroid hormone responsive rat pituitary tumor GH3 cell line that constitutively expresses both thyroid hormone receptor isoforms. Stable transfection of the pGL4CP-SV40-2xtaDR4 construct into the GH3 cells resulted in a highly sensitive cell line (GH3.TRE-Luc), which was further optimized into an assay that allowed the detection of Triiodothyronine (T(3)) and Thyroxine (T(4)) concentrations in the picomolar range after only 24 h of exposure. The greater than 20-fold induction of T(3) relative to the solvent control is illustrative of the high responsiveness of the system. The assay was validated by the quantification of the agonistic effect of the natural hormones (T(3) and T(4)), the acetic acid derivatives of T(3) (triiodothyroaceticacid, or Triac) and T(4) (tetraiodothyroacetic acid, or Tetrac), hydroxy polybrominated diphenylethers (OH-PBDEs), hydroxy polychlorinated biphenyls (OH-PCBs) and the antagonistic action of sodium arsenite (NaAsO(2)). The putative antagonist Amiodarone, Bisphenol A (BPA) and its halogenated derivatives (TCBPA and TBBPA) for which effects reported in the literature are not consistent, showed comparable dose-response curves with a slight agonistic effect (5% of T(3)-max) followed by a slight antagonistic effect. The magnitude and reproducibility of the responses to various compounds confirms this assay as a promising tool for the identification and quantification of specific thyroid hormone receptor disrupting potency of compounds.

Sibling and self ovum donation for sisters with an intermediate FMR1 mutation: what's a program to do?

OBJECTIVE: To increase awareness of the unique clinical and ethical considerations invoked by the request of a patient with premature ovarian failure (POF) and her nulliparous sister, both with intermediate-size mutations in fragile X mental retardation 1 (FMR1), to pursue sibling ovum donation. DESIGN: Case report. SETTING: Academic medical center. PATIENT(S): A 32-year-old woman with POF and her 26-year-old sister with occult diminished ovarian reserve, both of whom are carriers of an intermediate-size mutation in FMR1. INTERVENTION(S): Prospective donor ovarian function testing, genetic testing and consultation, and psychological evaluation; institutional assisted reproduction ethics committee consultation, and controlled ovarian hyperstimulation-IVF with cryopreservation of embryos for potential future self-use. MAIN OUTCOME MEASURE(S): Successful cryopreservation of embryos for autologous use by the prospective donor (younger sister) before ovum donation. RESULTS(S): Three blastocysts were frozen. CONCLUSION(S): Requests for sibling ovum donation, while understandable and ethically sanctioned under typical circumstances, prove particularly challenging in some patients with POF given uncertainties regarding the prognosis of the currently asymptomatic sister, risks of genetic transmission of POF, and fiduciary responsibilities to address the reproductive interests of the prospective donor. A multidisciplinary approach was highly beneficial in this case.

An avian bioassay for environmental estrogens: the growth response of zebra finch (Taeniopygia guttata) chick oviduct to oral estrogens.

The rat uterotrophic assay is a recommended tier 1 screening assay for environmental estrogens, but no comparable assay exists for altricial birds. We orally dosed zebra finch chicks daily during their linear growth phase (days 5-11) with estradiol benzoate (EB), genistein, methoxychlor, or octylphenol, all dissolved or suspended in canola oil, or canola oil alone, as a vehicle control. On day 12, oviducts were removed, weighed and examined histologically. All doses of EB (0.1-1,000 nmol/g body wt), genistein at 100 nmol/g. and methoxychlor and octylphenol at 1,000 nmol/g, markedly increased oviduct weight, with the highest dose of EB inducing a 60-fold increase over controls. Oviducts were differentiated in a dose-depedent manner to the point of having tubular glands and a pseudostratified, ciliated epithelium at the higher doses of EB. Our earlier results show that EB at 100 and 1,000 nmol/g impairs reproductive performance of zebra finches. Thus, the zebra finch oviduct bioassay measures estrogenicity over a wide dose range and, for EB exposure, can predict impairment in adult reproductive performance. The responsiveness of chick oviducts to estrogen stimulation may serve as a useful marker of estrogen exposure in wild populations of songbirds.

Oral estrogen masculinizes female zebra finch song system.

It is well established that parenteral treatment of female zebra finch chicks with estradiol masculinizes their song control nuclei and that as adults they are capable of song. Concern over the widespread use of putative environmental estrogens caused us to ask whether oral exposure to estrogens (a natural route of exposure) could produce similar effects. We dosed chicks orally with estradiol benzoate (EB; 1, 10, 100, and 1000 nmol/g of body mass per day, days 5-11 posthatch), the non-ionic surfactant octylphenol (100 and 1000 nmol/g), or the pesticides methoxychlor (100 and 1000 nmol/g) and dicofol (100 nmol/g) and measured their song control nuclei as adults. EB treatment produced increases in song nuclei comparable to that induced by parenteral administration of estrogens. This is the first study of which we are aware to use an oral route of administration, which simulates the natural process of parent birds feeding their nestlings. We conclude that oral exposure to estradiol alters song control nuclei and we report in a related paper (Millam et al., 2001) that such exposure severely disrupts reproductive performance. Although we detected no influence of xenobiotics on induction of song control nuclei the possibility remains that oral exposure to xenoestrogens in high enough doses could affect development.