Differential voluntary feed intake and whole transcriptome profiling in the hypothalamus of young sheep offered CP and phosphorus-deficient diets.

A reduction in voluntary feed intake is observed in ruminants consuming nutrient-deficient diets, such as those with a low CP or P content, and has been attributed to active metabolic regulation, rather than a physical constraint. The hypothalamus is the key integrator of feed intake regulation in mammals. The objectives of this experiment were to (1) establish a model of metabolic feed intake regulation in ruminants consuming diets of variable CP and P content, and (2) determine key biochemical pathways and influential points of regulation within the hypothalamus. Merino wethers [n = 40; 23.7 ± 1.4 kg liveweight (mean ± SD)] were fed one of five dietary treatments (n = 8/treatment) for 63 days in individual pens. The treatments included targeted combinations of high (H) and low (L) CP (110 and 55 g/kg DM) and high and low P (2.5 and 0.7 g/kg DM) with 9 MJ metabolisable energy (ME) per kg DM which were fed ad libitum (UMEI; unrestricted ME intake) resulting in four experimental diets (HCP-HP-UMEI, LCP-HP-UMEI, HCP-LP-UMEI and LCP-LP-UMEI). An additional nutritional treatment (HCP-HP-RMEI) restricted intake of the HCP-HP diet to an equivalent ME intake of wethers consuming the LCP-LP-UMEI treatment. Wethers offered the LCP-HP-UMEI, HCP-LP-UMEI and LCP-LP-UMEI treatments consumed 42, 32 and 49% less total DM (P ≤ 0.05), respectively than the HCP-HP-UMEI treatment, and this was not attributable to any physical limitation of the rumen. Plasma concentrations of urea nitrogen and inorganic phosphate indicated that these nutrient deficiencies were successfully established. To assess potential mechanisms, RNA-seq was conducted on samples from the arcuate nucleus (ARC), ventromedial hypothalamus and lateral hypothalamus of the wethers, yielding a total of 301, 8 and 148 differentially expressed genes across all pairwise comparisons, respectively. The expression of NPY, AGRP and CARTPT, known for their regulatory role in mammalian feed intake regulation, had a similar transcriptional response in the ARC of wethers consuming nutrient-deficient treatments and those consuming a ME-restricted treatment, despite these wethers expressing behaviours indicative of satiated and hungry states, respectively. In addition, genes involved with glycolysis (TPI1), the citric acid cycle (CS, OGDH, GLUD1, GOT1) and oxidative phosphorylation (COX5A, ATP5MC1, ATP5F1B, ATP5MC3) were downregulated in the ARC of wethers fed a nutrient deficient (LCP-LP-UMEI) relative to the non-deficient (HCP-HP-UMEI) treatment. In summary, a model for voluntary feed intake restriction was established to determine genome-wide molecular changes in the hypothalamus of young ruminants.

Comparing the responses of grain fed feedlot cattle under moderate heat load and during subsequent recovery with those of feed restricted thermoneutral counterparts: metabolic hormones.

We set out to determine the impact of moderate heat load on the plasma concentrations of a suite of hormones involved in regulating energy metabolism and feed intake. The responses of the thermally challenged (TC) feedlot steers were compared to those of feed restricted thermoneutral (FRTN) steers. Two sequential cohorts of twelve 518 ± 23 kg Black Angus steers on finisher grain ration were housed in climate-controlled rooms (CCR) for 18 days and returned to outdoor pens for 40 days. The TC group was subjected to a diurnal range of 28-35 °C for 7 days (Challenge) but held in thermoneutral conditions beforehand (PreChallenge), and in Recovery (after Challenge). The FRTN group was held in thermoneutral conditions and feed restricted throughout. Blood was collected over the three periods in CCR and two periods in outdoor pens for 40 days (PENS and Late PENS). Plasma concentrations of prolactin, thyroid stimulating hormone, insulin, leptin, adiponectin and thyroxine (T4) were determined during the five periods. Whilst the pituitary hormones were relatively stable, there were differences in plasma leptin, adiponectin and T4 between the two groups during Challenge and Recovery, and occasionally in PENS. The interaction of the plasma hormone concentrations and rumen temperature and DMI were also investigated. Whilst the positive relationship between DMI and leptin was confirmed, we found a strong negative relationship between adiponectin and rumen temperature, and a strong positive relationship between adiponectin and dry matter intake (DMI) in the TC steers only.

Effects of insulin on IGF-1 receptors in equine lamellar tissue in vitro.

Although it is understood that equine endocrinopathic laminitis can be triggered by high concentrations of insulin, it is unclear whether this represents a direct action on lamellar tissue via insulin receptors (InsR), an interaction with IGF-1 receptors (IGF-1R), or some other, indirect action. This uncertainty is because of the reported scarcity of InsR in lamellar tissue and the low affinity of insulin for equine IGF-1R. In the present study, the effects of insulin and IGF-1 (as a positive control) were examined using lamellar explants isolated from the hooves of healthy horses and incubated in cell culture medium for between 2 min and 48 h. In this system, a low physiological concentration of IGF-1 (10 nM; 1.31 ng/mL) caused a marked increase in the appearance of phosphorylated IGF-1R after 5 min (P < 0.05), and this effect was blocked by a human anti-IGF-1R monoclonal antibody (mAb). However, a high concentration of insulin (10 nM; 1,430 µIU/mL) appeared to cause dephosphorylation of the IGF-1R after 5 min (P < 0.01), 15 min, and 30 min (P < 0.001). Using 3H-thymidine as a marker, it was also demonstrated that insulin and IGF-1-stimulated cell proliferation in lamellar explants over the same concentration range as each other (1-100 nM), implying that each peptide acts via its own receptor (P < 0.001). Conversely, the effect of both peptides could be blocked using a selective anti-IGF-1R mAb (P < 0.001), implying that insulin acts via IGF1-R (either directly or indirectly). Notwithstanding this conundrum, the results demonstrate that insulin acts directly on lamellar tissue and suggest that a therapeutic anti-IGF-1R mAb could be useful in treating or preventing endocrinopathic laminitis.

Identification of monoclonal antibodies suitable for blocking IGF-1 receptors in the horse.

Prolonged hyperinsulinemia is thought to be the cause of equine endocrinopathic laminitis, a common and crippling disease of the foot, for which there are no pharmacologic treatments other than pain relief. It has been suggested that insulin causes its effects on the lamellae by activating IGF-1 receptors (IGF-1R), as insulin receptors (InsR) are scarce in this tissue, whereas IGF-1R are abundant and become downregulated after prolonged insulin infusion. As a first step toward confirming this mechanism and beginning to develop a therapeutic anti-IGF-1R monoclonal antibody (mAb) for horses, it was necessary to identify available human IGF-1R mAbs that would recognize equine receptors. Four IGF-1R mAbs were tested using soluble equine IGF-1R, with ELISA and flow cytometry. Frozen equine lamellar and liver tissue was also used in radioligand binding assays. The results demonstrated that only one of the mAbs tested (mAb1) was able to compete effectively with IGF-1 for binding to its receptors in equine lamellar tissue, with an IC50 of 5 to 159 ng/mL. None of the 4 mAbs were able to bind to equine hepatic InsR. This study has generated valuable structure-activity information and has identified a prototype anti-IGF-1R mAb suitable for further development.

Bacteremia in Childhood Life-Threatening Infections in Urban Gambia: EUCLIDS in West Africa.

BACKGROUND: The limited availability of microbiology services in sub-Saharan Africa impedes accurate diagnosis of bacterial pathogens and understanding of trends in prevalence and antibiotic sensitivities. We aimed to characterize bacteremia among hospitalized children in The Gambia and to identify factors associated with bacteremia and mortality. METHODS: We prospectively studied children presenting with suspected severe infection to 2 urban hospitals in The Gambia, between January 2013 and September 2015. Demographic and anthropometric data, clinical features, management, and blood culture results were documented. Urine screens for antibiotic activity were performed in a subset of participants. RESULTS: Of 411 children enrolled (median age, 29 months; interquartile range, 11-82), 79.5% (325 of 409) reported prehospital antibiotic use. Antimicrobial activity by urinary screen for antibiotic activity was detected in 70.8% (n = 80 of 113). Sixty-six bacterial pathogens were identified in 65 (15.8%) participants and Staphylococcus aureus predominated. Gram-positive organisms were more commonly identified than Gram-negative (P < .01). Antibiotic resistance against first-line antimicrobials (ampicillin and gentamicin) was common among Gram-negative bacteria (39%; range, 25%-100%). Factors significantly associated with bacteremia included the following: gender, hydration status, musculoskeletal examination findings, admission to the Medical Research Council The Gambia at London School of Hygiene & Tropical Medicine hospital, and meeting sepsis criteria. Those associated with increased mortality were presence of a comorbidity, clinical pallor, tachypnea, and altered consciousness. Tachycardia was associated with reduced mortality. CONCLUSIONS: The bacteremia rate in children with suspected childhood life-threatening infectious diseases in The Gambia is high. The pattern of pathogen prevalence and antimicrobial resistance has changed over time compared with previous studies illustrating the importance of robust bacterial surveillance programs in resource-limited settings.

Characterization of insulin and IGF-1 receptor binding in equine liver and lamellar tissue: implications for endocrinopathic laminitis.

Although it is well established that equine laminitis can be triggered by extreme hyperinsulinemia, the mechanism of insulin action is not known. High concentrations of insulin lead to separation of the weight-bearing apparatus from the hoof wall and are associated with an increased cycle of cell death and proliferation in the lamellae. Gene expression and immunohistochemistry studies have indicated that the lamellae are sparsely populated with insulin receptors, whereas IGF-1 receptors (IGF-1R) are abundant, suggesting that the action of insulin may be mediated by insulin binding to the IGF-1R. To investigate this possibility, cell membrane fragments containing IGF-1R were extracted from the livers of 6 horses and the lamellae of >50 horses euthanized for nonresearch purposes at an abattoir. Radioligand-binding studies using 125I-IGF-1 and 125I-insulin confirmed an abundance of high-affinity IGF-1R in the liver (KD 0.11 nM, Bmax 223 fmol/mg protein) and lamellae (KD 0.16 nM, Bmax 243 fmol/mg protein). However, the affinity of insulin for binding to the lamellar IGF-1R (Ki 934 nM) was >5,800 fold less than that of IGF-1, suggesting that insulin is unlikely to bind to equine IGF-1R at physiological concentrations. Although insulin receptors could be detected in the liver (KD 0.48 nM, Bmax 123 fmol/mg protein), they were barely detectable in lamellae (estimated Bmax 14 fmol/mg protein). There was no evidence to support the presence of insulin/IGF-1 hybrid receptors in either tissue. These findings suggest that insulin does not act directly through IGF-1 receptors and that an alternative theory is required to explain the mechanism of insulin action in laminitis.

Low sensitivity of a test for anti-Mullerian hormone to assess presence of ovaries in prepubertal bitches.

BACKGROUND: Anti-Mullerian hormone (AMH) is currently used in several species as an indicator of the number of antral and pre-antral follicles within the ovaries. Currently, there is some uncertainty on the precision of a single AMH test for detecting the presence of ovarian tissue in prepubertal, pubertal and spayed bitches. The purpose of this study was to investigate the specificity of AMH levels determined using the Gen II AMH ELISA to detect the presence or absence of ovarian tissue in bitches of varying ages. METHODS: From a large cohort of dogs located at an animal shelter, 36 bitches were assigned to three age groups (< 6 months; 6-18 months and > 2 years of age) plus a group of six spayed bitches. RESULTS: AMH was below the detectable limit for each spayed bitch (< 0.010 ng/mL) and for 9/10 intact bitches aged less than 6 months. AMH levels were therefore significantly different for these two groups compared with older intact bitches (6-18 months, 0.302 ± 0.135 ng/mL; > 2 years, 0.237 ± 0.210 ng/mL). AMH was undetectable in two intact bitches aged > 2 years of age, which gave a sensitivity of 82% in that group. Overall, the sensitivity of the test was 90% for all bitches aged over 6 months, which highlights that a small percentage of intact females will be incorrectly diagnosed as having no ovarian tissue. CONCLUSION: AMH testing had very low sensitivity in bitches aged less than 6 months and thus it is advisable to delay testing in very young bitches.

Dosing obese cats based on body weight spuriously affects some measures of glucose tolerance.

The primary objective was to investigate whether dosing glucose by body weight results in spurious effects on measures of glucose tolerance in obese cats because volume of distribution does not increase linearly with body weight. Healthy research cats (n = 16; 6 castrated males, 10 spayed females) were used. A retrospective study was performed using glucose concentration data from glucose tolerance and insulin sensitivity tests before and after cats were fed ad libitum for 9 to 12 mo to promote weight gain. The higher dose of glucose (0.5 vs 0.3 g/kg body weight) in the glucose tolerance tests increased 2-min glucose concentrations (P < 0.001), and there was a positive correlation between 2-min and 2-h glucose (r = 0.65, P = 0.006). Two-min (P = 0.016 and 0.019, respectively), and 2-h (P = 0.057 and 0.003, respectively) glucose concentrations, and glucose half-life (T1/2; P = 0.034 and <0.001 respectively) were positively associated with body weight and body condition score. Glucose dose should be decreased by 0.05 g for every kg above ideal body weight. Alternatively, for every unit of body condition score above 5 on a 9-point scale, observed 2-h glucose concentration should be adjusted down by 0.1 mmol/L. Dosing glucose based on body weight spuriously increases glucose concentrations at 2 h in obese cats and could lead to cats being incorrectly classified as having impaired glucose tolerance. This has important implications for clinical studies assessing the effect of interventions on glucose tolerance when lean and obese cats are compared.

Diagnosis of prediabetes in cats: glucose concentration cut points for impaired fasting glucose and impaired glucose tolerance.

Diabetes is typically diagnosed in cats once clinical signs are evident. Diagnostic criteria for prediabetes in cats have not been defined. The objective of the study was to establish methodology and cut points for fasting and 2-h blood glucose concentrations in healthy client-owned senior cats (≥8 yr) using ear/paw samples and a portable glucose meter calibrated for feline blood. Of the 78 cats, 27 were ideal (body condition score [BCS] 4 or 5 of 9), 31 overweight (BCS 6 or 7), and 20 obese (BCS 8 or 9); 19 were Burmese and 59 non-Burmese. After an 18-24-h fast and an ear/paw blood glucose measurement using a portable glucose meter, glucose (0.5 g/kg bodyweight) was administered intravenous and blood glucose measured at 2 min and 2 h. Cut points for fasting and 2-h glucose concentrations were defined as the upper limits of 95% reference intervals using cats with BCS 4 or 5. The upper cut point for fasting glucose was 6.5 mmol/L. Of the overweight and obese cats, 1 (BCS 7) was above this cut point indicating evidence of impaired fasting glucose. The cut point for 2-h glucose was 9.8 mmol/L. A total of 7 cats (4 with BCS 8 or 9 including 1 Burmese; 3 with BCS 6 or 7, non-Burmese) were above this cut point and thus had evidence of impaired glucose tolerance. In conclusion, the methodology and cutpoints for diagnosis of prediabetes are defined for use in healthy cats 8 yr and older with a range of BCSs.

Plasma prolactin concentrations during lactation, pouch young development and the return to behavioural oestrus in captive koalas (Phascolarctos cinereus).

Plasma prolactin (PRL) concentrations in captive koalas during lactation were determined by serial blood sampling. PRL concentrations were low (1.3 ± 0.1 ng mL-1; n = 5) during early lactation until pouch young (PY) began to emerge from the pouch (around Day 130) before significantly (P < 0.05) increasing between Day 161 and Day 175 (5.3 ± 1.0 ng mL-1). A significant (P < 0.001) peak in PRL (7.7 ± 0.6 ng mL-1) coincided with maturing young between Day 189 and Day 231. All females failed to exhibit any signs of oestrous behaviour until Day 268.8 ± 8.5 (n = 4), some 102 ± 19 days before PY were weaned following achieving target weights of 2.5-2.7 kg. Throughout lactation, plasma LH concentrations were relatively high (range 4.9-8.7 ng mL-1) and LH responses to exogenous gonadotrophin-releasing hormone were observed in all koalas at all times during lactation.

Use of the gonadotrophin-releasing hormone antagonist azaline B to control the oestrous cycle in the koala (Phascolarctos cinereus).

The present study examined the effectiveness of the gonadotrophin-releasing hormone (GnRH) antagonist azaline B to suppress plasma LH and 17ß-oestradiol concentrations in koalas and its potential application for oestrous synchronisation. In Experiment 1, single subcutaneous injections of azaline B successfully blocked the LH response to exogenous mammalian (m) GnRH in a dose-dependent manner; specifically, 0 mg (n = 4) did not suppress the LH response, 1 mg azaline B (n = 6) suppressed the LH response for 24 h (P < 0.05), 3.3 mg azaline B (n = 8) suppressed the LH response significantly in all animals only for 3 h (P < 0.05), although in half the animals LH remained suppressed for up to 3 days, and 10 mg azaline B (n = 4) suppressed the LH response for 7 days (P < 0.05). In Experiment 2, daily 1 mg, s.c., injections of azaline B over a 10-day period during seasonal anoestrus (June-July; n = 6) suppressed (P < 0.01) the LH response to mGnRH consecutively over the 10-day treatment period and, 4 days after cessation of treatment, the LH response had not recovered. Experiment 3 was designed to test the efficacy of daily 1 mg, s.c., azaline B over 10 days to suppress plasma LH and 17ß-oestradiol concentrations and ultimately synchronise timed return to oestrus during the breeding season. Although azaline B treatment did not suppress basal LH or 17ß-oestradiol, oestrus was delayed in all treated females by 24.2 days, but with high variability (range 9-39 days). Overall, the present study demonstrates that the GnRH antagonist azaline B is able to inhibit the LH response in koalas to exogenous mGnRH and successfully delay the return to oestrus. However, although azaline B clearly disrupts folliculogenesis, it has not been able to effectively synchronise return to oestrus in the koala.

The use of a synthetic progesterone, levonorgestrel (LNG), to control the oestrous cycle in the koala.

This study investigated the efficacy of a synthetic progestogen, levonorgestrel (LNG), to control koala ovarian activity for the purposes of oestrous synchronisation. Captive koalas were administered either saline control or a 70-mg LNG implant on Day 2 of oestrus. Urogenital cytology, oestrous behaviour and plasma oestradiol-17ß and LH concentrations were monitored over a 6-week period. After LNG implant removal females were monitored to determine if the return to oestrus was synchronised. LNG-treated koalas immediately ceased displaying oestrous behaviour, showed no evidence of cornified epithelial cells in smears of urogenital cytology and exhibited low plasma oestradiol-17ß concentrations throughout the implantation period. In contrast, oestradiol-17ß levels in control koalas showed evidence of continued cyclic activity associated with behavioural oestrus and increased cornified epithelial cells in urogenital smears on Days 33 to 35 after saline injection. After implant removal, LNG-treated koalas exhibited oestrus at 13, 14, 17 and 30 days after implant removal. Plasma LH concentrations varied throughout the study period with no significant time (P = 0.49) or treatment (P = 0.13) effect. Overall results from this study suggest that LNG implants in koalas can inhibit oestrous behaviour and reduce circulating oestradiol-17ß levels before oestrus, most likely by preventing development of the pre-ovulatory follicle. However, there was no evidence of LH suppression by the LNG implants. Removal of LNG implants resulted in the synchronous return to oestrus in three of the four treated koalas. Further studies on a larger population are required to validate these findings.

Management of persistent purulent pericarditis using streptokinase for intrapericardial fibrinolysis.

Purulent pericarditis (PP) is a very serious condition with almost 100% mortality if untreated. Intrapericardial fibrinolysis is a preferred alternative to pericardectomy in the treatment of persistent PP, but there are no consensus guidelines on the standard protocol for this procedure in children. A 9-year-old boy was referred to the Medical Research Council Unit in The Gambia (MRC). He had been unwell for 18 days with a high continuous fever, cough, fast breathing, and dyspnoea on exertion. Prior to referral he had been treated for malaria and pneumonia with no improvement. At the MRC, he was diagnosed with purulent pericarditis caused by Staphylococcus aureus and after admission he was managed for 4 weeks with intravenous antibiotics, pericardial aspirations followed by saline lavage of the pericardium and intrapericardial antibiotic instillation. Despite these measures, massive re-accumulation of the purulent pericardial effusion continued. Once daily intrapericardial instillation of streptokinase at a dose of 18,000 i.u/kg diluted in 50 ml of normal saline, and saline washout of the pericardium after 2 hours was commenced on the 29th day of admission, in addition to the antibiotics. This technique of fibrinolysis employed for 2 days was effective in managing the persistent purulent pericarditis when pericardial aspiration and intravenous and intrapericardial antibiotics failed.

Maternal corticosterone exposure in the mouse has sex-specific effects on placental growth and mRNA expression.

Maternal exposure to increased synthetic glucocorticoids (GC) during pregnancy is known to disturb fetal development and increase the risk of long-term disease. Maternal exposure to elevated levels of natural GC is likely to be common yet is relatively understudied. The placenta plays an important role in regulating fetal exposure to maternal GC but is itself vulnerable to maternal insults. This study uses a mouse model of maternal corticosterone (Cort) exposure to investigate its effects on the developing placenta. Mice were treated with Cort (33 µg/kg·h) for 60 h starting at embryonic d 12.5 (E12.5) before collection of placentas at E14.5 and E17.5. Although Cort exposure did not affect fetal size, placentas of male fetuses were larger at E17.5 in association with changes in placental Igf2. This increase in size was associated with an increase in placental thickness and an increase in placental junctional zone volume. Placentas from female fetuses were of normal size and had no changes in growth factor mRNA levels. The expression of the protective enzyme 11ß-hydroxysteroid dehydrogenase type 2 was increased at E14.5 but was decreased in males at E17.5. In contrast, the expression of Nr3c1 (which encodes the GC receptor) was increased during the Cort exposure and remained elevated at E17.5 in the placentas of male fetuses. Our study has shown that maternal Cort exposure infers a sex-specific alteration to normal placental growth and growth factor expression, thus further adding to our understanding of the mechanisms of male dominance of programmed disease.

Equine amnionitis and fetal loss: mare abortion following experimental exposure to Processionary caterpillars (Ochrogaster lunifer).

REASONS FOR PERFORMING STUDY: In Australia, there have been recent reports of unusual abortions in mid- to late-gestation mares. These were clinically distinct from other recognised causes of pregnancy loss and the term 'equine amnionitis and fetal loss' (EAFL) was adopted to describe this syndrome. Initial investigations concluded that possible causal factors included the presence on affected stud farms of Processionary caterpillars (Ochrogaster lunifer). OBJECTIVES: To determine if exposure of pregnant mares to Processionary caterpillars or their shed exoskeletons can induce EAFL. METHODS: Processionary caterpillars and their shed exoskeletons were collected and stored frozen. Mid-gestation mares were dosed with a slurry of caterpillars or shed exoskeleton by nasogastric intubation. Their clinical responses and times to abortion were recorded. All aborted fetuses were autopsied and samples taken for bacteriological and virological culture and histopathology. RESULTS: Intubating mares in mid-pregnancy with preparations of either whole Processionary caterpillars or shed caterpillar exoskeletons induced abortion with few impending clinical signs. The gross pathological and bacteriological findings of the aborted fetuses were similar to those observed in field cases of EAFL. POTENTIAL RELEVANCE: Possible exposure to Processionary caterpillars should be considered when examining cases of fetal loss in the mare. The present results provide a starting point to further explore the aetiology and pathogenesis of EAFL.

Association between Gc genotype and susceptibility to TB is dependent on vitamin D status.

Group-specific component (Gc) variants of vitamin D binding protein differ in their affinity for vitamin D metabolites that modulate antimycobacterial immunity. We conducted studies to determine whether Gc genotype associates with susceptibility to tuberculosis (TB). The following subjects were recruited into case-control studies: in the UK, 123 adult TB patients and 140 controls, all of Gujarati Asian ethnic origin; in Brazil, 130 adult TB patients and 78 controls; and in South Africa, 281 children with TB and 182 controls. Gc genotypes were determined and their frequency was compared between cases versus controls. Serum 25-hydroxyvitamin D (25(OH)D) concentrations were obtained retrospectively for 139 Gujarati Asians, and case-control analysis was stratified by vitamin D status. Interferon (IFN)-gamma release assays were also performed on 36 Gujarati Asian TB contacts. The Gc2/2 genotype was strongly associated with susceptibility to active TB in Gujarati Asians, compared with Gc1/1 genotype (OR 2.81, 95% CI 1.19-6.66; p = 0.009). This association was preserved if serum 25(OH)D was <20 nmol.L(-1) (p = 0.01) but not if serum 25(OH)D was > or =20 nmol.L(-1) (p = 0.36). Carriage of the Gc2 allele was associated with increased PPD of tuberculin-stimulated IFN-gamma release in Gujarati Asian TB contacts (p = 0.02). No association between Gc genotype and susceptibility to TB was observed in other ethnic groups studied.

Interferon-gamma release assays do not identify more children with active tuberculosis than the tuberculin skin test.

Data are lacking on the performance of interferon-gamma release assays (IGRAs) in children. Although IGRAs are recommended for screening for latent tuberculosis infection (LTBI), many clinicians wish to employ them as a diagnostic test for active tuberculosis (TB). The objective of the present study was to compare the performance of the two commercially available IGRAs and the tuberculin skin test (TST) side-by-side in children with active TB and LTBI. In a prospective study, 209 children were investigated for active (n = 91) or latent TB (n = 118). TST, QuantiFERON-TB Gold In-tube (QFG-IT; Cellestis, Carnegie, Australia) and T-SPOT.TB (Oxford Immunotec, Abingdon, UK) assays were simultaneously used. For culture-confirmed active TB, the sensitivity of the TST was 83%, compared with 80% for QFG-IT and 58% for T-SPOT.TB. IGRAs did not perform significantly better than TST, although QFG-IT was significantly better than T-SPOT.TB. The agreement between QFG-IT and T-SPOT.TB in culture-confirmed TB was poor at 66.7%. In LTBI, the agreement between QFG-IT and T-SPOT.TB was very good (92%) with moderate agreement between TST and T-SPOT.TB (75%) and QFG-IT and TST (77%). A negative interferon-gamma release assay should not dissuade paediatricians from diagnosing and treating presumed active tuberculosis. If used for diagnosis of latent tuberculosis infection, interferon-gamma release assays could significantly reduce the numbers of children receiving chemoprophylaxis. Very good concordance between both tests was found.

Expression of pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R) in the ewe hypothalamus: distribution and colocalization with tyrosine hydroxylase-immunoreactive neurones.

We have examined the distribution of the pituitary adenylate cyclase activating polypeptide type I receptor (PAC1R) in the ewe hypothalamus by reverse transcription-polymerase chain reaction, in situ hybridization and immunohistochemistry. PAC1R mRNA was highly expressed in the mediobasal hypothalamus of the ewe, particularly in the arcuate nucleus and ventromedial hypothalamus, compared to other hypothalamic regions. Similar results were obtained from immunohistochemistry using a specific PAC1R antibody. Intense immunolabelling was observed in the arcuate nucleus, external zone of the median eminence and ventromedial hypothalamus. Only relatively weak immunolabelling was observed in other hypothalamic regions, including the paraventricular nucleus and supraoptic nucleus. In the ewe, PACAP acts via the arcuate nucleus to suppress prolactin secretion. Therefore we examined whether PAC1R was present on the tuberoinfundibular dopamine (TIDA) neurones in this nucleus. Dual immunofluorescence labelling for PAC1R and tyrosine hydroxylase revealed that 21.2 +/- 1.7% of dopaminergic neurones in the arcuate nucleus (A12 cell group) also stained for PAC1R. By contrast, other hypothalamic dopaminergic cell groups (A11, A13, A14 and A15) exhibited little (< 3%) or no colocalization. Overall, our results indicate that, in the ewe hypothalamus, PAC1R is most concentrated in the arcuate nucleus, where it is localized on a substantial proportion of dopaminergic neurones. These observations, together with previous in vivo studies, suggest that PACAP could act directly on TIDA neurones via PAC1R to increase dopamine release and consequently inhibit prolactin secretion in the sheep.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation.

Real-time Taqman RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P<0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P<0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the codon reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin secretion.

A prostaglandin f(2alpha) analog induces suppressors of cytokine signaling-3 expression in the corpus luteum of the pregnant rat: a potential new mechanism in luteolysis.

PRL and placental lactogen (PL) play key roles in maintaining the rodent corpus luteum through pregnancy. Suppressors of cytokine signaling (SOCS) have been shown to decrease cell sensitivity to cytokines, including PRL, and so here we have addressed the issue of whether luteolysis induced by prostaglandin F(2alpha) (PGF(2alpha)) might up-regulate SOCS proteins to inhibit PRL signaling. In d 19 pregnant rats, cloprostenol, a PGF(2alpha) analog, rapidly induced transcripts for SOCS-3 and, to a lesser extent, SOCS-1. We also found increased SOCS-3 protein in the ovary by immunoblot and in the corpus luteum by immunohistochemistry. Increased SOCS-3 expression was preceded by an increase in STAT3 tyrosine phosphorylation 10 min after cloprostenol injection and was maintained for 4 h, as determined by gel shift and immunohistochemistry. Induction of SOCS-3 was accompanied by a sharp decrease in active STAT5, as determined by gel-shift assay and by loss of nuclear localized STAT5. Four hours after cloprostenol administration, the corpus luteum was refractory to stimulation of STAT5 by PRL administration, and this was not due to down-regulation of PRL receptor. Therefore, induction of SOCS-3 by PGF(2alpha) may be an important element in the initiation of luteolysis via rapid suppression of luteotropic support from PL.