Assessing experimental visceral pain in dairy cattle: A pilot, prospective, blinded, randomized, and controlled study focusing on spinal pain proteomics.

Few studies have verified the validity of behavioral and physiological methods of pain assessment in cattle. This prospective, blinded, randomized controlled experimental study aimed to validate different methods of pain assessment during acute and chronic (up to 21 d postintervention) conditions in dairy cattle, in response to 3 analgesic treatments for traumatic reticuloperitonitis. Cerebrospinal fluid (CSF) biomarkers and mechanical sensitization were measured as indicators of centralized pain. Proteomics in the CSF were examined to detect specific (to pain intensity) and sensitive (responsive to analgesia) markers. Recordings of spontaneous behavior with video analysis, telemetered motor activity, pain scales, electrodermal activity, and plasma cortisol concentration were quantified at regular intervals. Cows were assigned to group 1 (n=4, standard control receiving aspirin), group 2 (n=5, test group receiving preemptive tolfenamic acid), or group 3 (n=3, positive control receiving preemptive multimodal analgesia composed of epidural morphine, plus tolfenamic acid and butorphanol). Rescue analgesia was administered as needed. Generalized estimating equations tested group differences and the influence of rescue analgesia on the measurements. All 3 groups demonstrated a long-term decrease in a CSF protein identified as transthyretin. The decrease in transthyretin expression inversely correlated with the expected level of analgesia (group 1<2<3). Moreover, in group 1, CSF noradrenaline decreased long term, cows were hypersensitive to mechanical stimulation, and they demonstrated signs of discomfort with higher motor activity and "agitation while lying" recorded from video analysis. Decreased "feeding behavior," observer-reported pain scales, electrodermal activity, and plasma cortisol concentration were inconsistent to differentiate pain intensity between groups. In summary, changes in CSF biomarkers and mechanical sensitization reflected modulation of central pain in dairy cows. The spontaneous behavior "agitation while lying" was the only behavioral outcome validated for assessing acute and chronic pain in this visceral pain model.

Retrospective study to estimate the prevalence and describe the clinicopathological characteristics, treatments received, and outcomes of HER2-low breast cancer.

BACKGROUND: Approximately 80% of all breast cancers (BCs) are currently categorized as human epidermal growth factor receptor 2 (HER2)-negative [immunohistochemistry (IHC) 0, 1+, or 2+/in situ hybridization (ISH) negative]; approximately 60% of BCs traditionally categorized as HER2-negative express low levels of HER2. HER2-low (IHC 1+ or IHC 2+/ISH-) status became clinically actionable with approval of trastuzumab deruxtecan to treat unresectable/metastatic HER2-low BC. Greater understanding of patients with HER2-low disease is urgently needed. PATIENTS AND METHODS: This global, multicenter, retrospective study (NCT04807595) included tissue samples from patients with confirmed HER2-negative unresectable/metastatic BC [any hormone receptor (HR) status] diagnosed from 2014 to 2017. Pathologists rescored HER2 IHC-stained slides as HER2-low (IHC 1+ or IHC 2+/ISH-) or HER2 IHC 0 after training on low-end expression scoring using Ventana 4B5 and other assays at local laboratories (13 sites; 10 countries) blinded to historical scores. HER2-low prevalence and concordance between historical scores and rescores were assessed. Demographics, clinicopathological characteristics, treatments, and outcomes were examined. RESULTS: In rescored samples from 789 patients with HER2-negative unresectable/metastatic BC, the overall HER2-low prevalence was 67.2% (HR positive, 71.1%; HR negative, 52.8%). Concordance was moderate between historical and rescored HER2 statuses (81.3%; κ = 0.583); positive agreement was numerically higher for HER2-low (87.5%) than HER2 IHC 0 (69.9%). More than 30% of historical IHC 0 cases were rescored as HER2-low overall (all assays) and using Ventana 4B5. There were no notable differences between HER2-low and HER2 IHC 0 in patient characteristics, treatments received, or clinical outcomes. CONCLUSIONS: Approximately two-thirds of patients with historically HER2-negative unresectable/metastatic BC may benefit from HER2-low-directed treatments. Our data suggest that HER2 reassessment in patients with historical IHC 0 scores may be considered to help optimize selection of patients for treatment. Further, accurate identification of patients with HER2-low BC may be achieved with standardized pathologist training.

Interparticle Forces of a Native and Encapsulated Metal-Organic Framework and Their Effects on Colloidal Dispersion.

The colloidal properties of suspended metal-organic frameworks (MOFs) are critical for device fabrication and application. Herein, van der Waals attractive, electric double layer repulsive, and steric repulsive forces of a native and encapsulated MOF are quantified for the first time. The van der Waals attractive forces were investigated by conducting environmental ellipsometric porosimetry (EEP) and spectroscopic ellipsometry (SE) on submicron, optical-quality nanoparticle films. The repulsive forces were determined from colloid and material characterization measurements. These data were used to predict suspension properties via extended Derjaguin, Landau, Verwey, and Overbeek theory. The state of dispersion was quantified for comparison with theoretical predictions for nine solvents. The MOF encapsulated with a surface-selective modification showed superior suspension in hydrophobic solvents. These findings should expedite the formulation of MOF colloidal suspensions for future works.

Large supra- and infra-tentorial occipital encephalocele encompassing posterior sagittal sinus and torcular Herophili.

Occipital encephaloceles are the most common type of encephalocele encountered in the Western Hemisphere. The occipital encephalocele is typically classified according to the relationship of the herniated tissue to the torcular. We report the unusual case of a premature infant with a rare, large occipital encephalocele which encompassed the posterior superior sagittal sinus and torcular. We discuss the variable venous anatomy with occipital encephaloceles and review the different options to employ in their surgical repair.

Evidence of species succession during chlorobenzene biodegradation.

We have previously reported the disappearance of a specific strain degrading chlorobenzene from a functionally stable bioreactor. In the present work, we investigated this species succession and isolated a new dominant strain, identified as Pandoraea pnomenusa sp. strain MCB032. A specific 16S rRNA-targeted oligonucleotide probe was designed and validated to identify strain MCB032 using fluorescence in situ hybridisation (FISH). The results confirmed the presence of strain MCB032 in samples collected over time, and showed that it was primarily located within the biofilm. Denaturing gradient gel electrophoresis (DGGE) provided evidence that the species succession occurred early in the operating period. The application of these biomolecular tools highlighted the remarkable stability of this new strain during the 15 months of reactor operation. The succession was attributed to the competitive kinetic behaviour of strain MCB032, which exhibited faster growth (micro(max) = 0.34 h(-1)) and higher substrate affinity (K(s) = 0.35 mg L(-1)) than strain JS150. Finally, this study contributed to the characterisation of the recently established Pandoraea genus, an emerging group in the biodegradation field.

Cholesterol and bile acid balance in Macaca fascicularis. Effects of alfalfa saponins.

We determine the effects of alfalfa top saponins on cholesterol and bile acid balance in eight cynomolgus macaques (Macaca fascicularis). The monkeys ate semipurified food containing cholesterol with or without added saponins. The saponins decreased cholesterolemia without changing the levels of high density lipoprotein-cholesterol; hence, they reduced the total cholesterol/high density lipoprotein-cholesterol ratio. Furthermore, they decreased intestinal absorption of cholesterol, increased fecal excretion of endogenous and exogenous neutral steroids and bile acids, and decreased the percent distribution of fecal deoxycholic and lithocholic acids. The fecal excretion of fat was also slightly increased, but steatorrhea did not occur. We saw no signs of toxicity in the monkeys after 6 or 8 wk of saponin ingestion. The data suggest that alfalfa top saponins may be of use in the treatment of patients with hypercholesterolemia, but long-term studies on possible toxicity are needed before this therapy can be recommended for humans.

Language barriers and paediatric burns: does education make a difference?

A focused first aid education campaign was conducted in Sydney in 1996 for parents of children from Non-English speaking backgrounds (NESB) to improve their knowledge of correct burns first aid treatment (BFAT). This study sought to determine the correlation, if any, between the reported beneficial results of this education campaign and the subsequent severity of burns in children. A retrospective review of all patients admitted to the state Burns Unit at The Children's Hospital at Westmead (CHW) over a 7-year period from January 1995 to December 2001 was performed. Number of patients, language spoken at home (English, Chinese, Arabic, Vietnamese or Other) and the rate of burns requiring skin grafting was assessed. A total of 1,283 patients were admitted over 7 years. Skin grafting was required in 544 (42%). NESB children required grafting in up to 88% of cases prior to the first aid education campaign, reducing to a minimum of 37% at 1 year and 65% at 5 years following the campaign. This compared to 40 and 36%, respectively, for English speaking children. While the initial decrease in grafting requirements may reflect an effective education campaign, the subsequent rise suggests the need for reinforcement.

Human cord blood long-term engrafting cells are CD34+ CD38-.

There have been controversies about CD34 and CD38 expression by human cord blood (CB) stem cells. Using the newborn NOD/SCID/beta2-microglobulin-null mouse assay that we recently developed, we examined the in vivo engrafting capability of human CB cells. Almost all of the 4-5 months engrafting cells were found in CD34(+) population. The capability of secondary reconstitution was found only in the CD34(+) cells. When the CD34(+) CB cells were separated into CD38(-) and CD38(+) subpopulations and tested for engraftment, the majority of the engrafting cells were detected in the CD38(-) subpopulation. These findings are consistent with the results from studies of murine stem cells and strongly indicate that the phenotype of human CB stem cells is CD34(+) CD38(-).

Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells.

We used the human/sheep competitive engraft model to investigate the in vivo engraftment potential of human CD34-, Lin- cells. In three separate studies, transplantation of CD34+, Lin- and CD34-, Lin- cells from the same normal human donors into preimmune fetal sheep resulted in long-term engraftment and multilineage hematopoietic cell/progenitor expression. Human cell/progenitor activities of the CD34+ group were CD3, 0.52%; CD13, 0.36%; CD45, 1.02%; glyA, 1.78%; HLA-DR, 0.99%; colony-forming units (CFU)-Mix, 7.1%; and granulocyte-macrophage CFU (CFU-GM), 13.7%; those of the CD34- group were CD3, 0.24%; CD45, 2.49%; glyA, 0.75%; HLA-DR, 0.3%; CFU-Mix, 7.1%; and CFU-GM, 4.3%. Of special interest was the detection of highly significant numbers (1 x 10[9] cells) of human CD34+ cells in animals transplanted with CD34- cells. Secondary transplantation and limiting dilution studies confirmed the presence of cells with long-term engraftment potential in CD34- populations. The results presented here demonstrate that the CD34- fraction of normal human bone marrow contains cells capable of engraftment and differentiation into CD34+ progenitors and multiple lymphohematopoietic lineages in primary and secondary hosts.

Comparative effects of alfalfa saponins and alfalfa fiber on cholesterol absorption in rats.

Intestinal absorption of cholesterol was measured in control rats fed semipurified diets and in rats fed alfalfa meal, in which saponins had been previously extracted, or this extracted material plus alfalfa saponins. A dose of 2 mg radioactive cholesterol was administered intragastrically, and fecal excretion of labeled neutral steroids measured. Absorption of cholesterol was about 76% in control animals, and about 47% in alfalfa-red rats. Extraction of saponins from alfalfa eliminated the cholesterol absorption-lowering effect, while addition of 0.26% alfalfa saponins to the extracted alfalfa restored its activity. The results demonstrate that alfalfa saponins are responsible for the effect of alfalfa meal in reducing cholesterol absorption, and that alfalfa fiber is not involved in this activity.

Effect of alfalfa saponins on intestinal cholesterol absorption in rats.

Five to 20 mg of saponins obtained from alfalfa tops or roots were introduced intragastrically in rats also receiving oral and intravenous ring-labeled cholesterol. The saponins were tested before and after partial acid hydrolysis. Absorption of cholesterol was determined by estimation of fecal sterols and by a dual isotope technique involving assay of plasma radioactivity. Alfalfa top saponins (nonhydrolyzed) reduced absorption of cholesterol. Acid hydrolysis of alfalfa top or root saponins enhanced their ability to inhibit cholesterol absorption.

Central sensitization as a result of surgical pain: investigation of the pre-emptive value of pethidine for ovariohysterectomy in the rat.

The development of central hypersensitivity as a result of a routine surgical procedure, midline ovariohysterectomy, was investigated in rats using the paw pressure test (PPT) and tail-flick latency (TFL) tests of spinal reflex activity. In addition, the modulating effect of pre-emptive versus post-operative administration of pethidine (a short-acting pure mu-opioid agonist) on the development of central hypersensitivity was studied. Initial experiments indicated that pethidine (15 mg/kg, i.m.) gave sub-maximal increases in thresholds for 60 min, and also that the administration of an anaesthetic did not unduly prolong the action of pethidine. Subsequently, 24 female Wistar rats were allocated to 1 of 4 treatment protocols: (1) anaesthesia without analgesics; (2) anaesthesia and surgery (midline ovariohysterectomy) without analgesics; (3) anaesthesia and surgery with pre-operative administration of pethidine; (4) anaesthesia and surgery with post-operative administration of pethidine. Thirty-five minutes after the end of anaesthesia thermal and mechanical nociceptive thresholds were measured at stepwise increasing intervals for 480 min. Changes were expressed as percentage changes from baseline (PPT) or deviation from expected values (TFL). Area under the threshold versus time response curves (AUCs) were also calculated for the following time sectors: 30-90, 90-150, 150-270, 270-390 and 390-510 min post-anaesthetic. Results of the TFL testing did not indicate the development of any significant hyperalgesia in any groups, but the results of the PPT did. In the time sectors 150-270 and 270-390 min post-anaesthetic, the AUCs in rats subjected to anaesthesia and surgery with either post-operative administration of pethidine or surgery with no analgesic drug administration, were significantly lower than the AUCs in rats given analgesics pre-operatively or those subjected to general anaesthesia alone (P < 0.01), Mann-Whitney). In summary, it appears that pethidine, in this protocol, prevented the development of surgically induced hyperalgesia when it was given pre-emptively.

Alfalfa saponins and alfalfa seeds. Dietary effects in cholesterol-fed rabbits.

Since alfalfa meal prevents hypercholesterolemia and atherosclerosis in rabbits and alfalfa saponins prevent the expected rise in cholesterolemia induced by dietary cholesterol in monkeys, the experiments being reported here were performed to determine whether alfalfa saponins affect atherogenesis in rabbits. In addition, the effects of alfalfa seeds were studied. Cholesterol-feb rabbits were randomly assigned to 3 groups: (a) control animals (N = 18); (b) animals maintained on a diet containing 1.0 to 1.2% alfalfa saponins (N = 18); and (c) animals maintained on a diet containing 40% alfalfa seeds (N = 17). Results after a 4-month observation period demonstrated that alfalfa saponins and alfalfa seeds reduce hypercholesterolemia, aortic sudanophilia, and the concentration of cholesterol in aortic intima-plus-media and in the liver, but do not induce changes in the hematocrit.

Binding of [3H]clonidine to membranes from cow neurohypophysis and cerebral cortex of the rat.

The binding of [3H]clonidine to membranes prepared from rat cerebral cortex and cow neurohypophysis was compared. Both tissues showed a high affinity site and a low affinity site when examined using a variety of techniques. The binding data, together with displacement studies using a variety of alpha 2-adrenoceptor agonists and antagonists, indicated that the binding activity of clonidine in the two tissues was very similar, however when the putative sigma opiate compound SKF 10,047 was examined it was found to have only a weak activity in displacing clonidine on the cortical membranes but a much stronger activity on the neurohypophysial membranes, other opiates showed little activity on binding of clonidine in either tissue. It is proposed that the binding of clonidine to neurohypophysial membranes may explain in part the various reports of suppression by clonidine of release of hormones from the neurohypophysis and that the effects of SKF 10,047 may be similarly associated with the reports of diuresis induced by phencyclidine.

Analgesic effects of intrathecally-applied alpha 2-adrenoceptor agonists in conscious, unrestrained sheep.

Intrathecal injections of small volumes of the alpha 2-adrenoceptor agonists, xylazine and clonidine, into the cervical region of the spinal cord of conscious unrestrained sheep produced a dose-dependent analgesia of the forelimbs as measured using a mechanical pressure device. Intravenous injection of the alpha 2-adrenoceptor antagonist, idazoxan completely abolished the analgesic effects of the intrathecally applied alpha 2-adrenoceptor agonists. Subsequent studies using [3H] clonidine injected at a similar dose and volume via the intrathecal catheters, indicated that the volume of drug used, 100 microliter, gave a localisation of the drug limited to about five vertebral segments around the catheter tip.

Endocardial pacing, cardioversion and defibrillation using a braided endocardial lead system.

The clinical efficacy and safety of a second-generation braided endocardial pacing, cardioversion and defibrillation lead system was evaluated in 25 patients with ventricular tachycardia (VT) or ventricular fibrillation (VF). The lead system consisted of two 8Fr active fixation endocardial leads each with pacing and defibrillation electrodes and a thoracic patch electrode. Monophasic and biphasic shocks were delivered using a triple-electrode configuration with a right ventricular common cathode and right atrial and thoracic patch anodes. VT and VF were electrically induced. Rapid VT (rate > or = 180 beats/min) and VF were initially terminated by 20 J (550 V) shocks and slow VT (rate < 180 beats/min) by 10 J (400 V) shocks. One hundred fourteen episodes (rapid VT/VF 73, slow VT 41) were treated with 128 shocks (monophasic 80, biphasic 48). Mean ventricular pacing threshold was 0.7 +/- 0.5 ms before and 0.9 +/- 0.5 ms after endocardial shock delivery (p > 0.2). Mean ventricular electrogram amplitude in sinus rhythm was 11.9 +/- 5.7 mV before and 11.4 +/- 5.1 mV after shock delivery (p > 0.2). Simultaneous monophasic endocardial shocks terminated 53% of VF episodes at < or = 20 J. Simultaneous biphasic shocks terminated 94% of all VF episodes at < or = 20 J (p < 0.03). Efficacy of > or = 10 J shocks for rapid VT/VF was greater for biphasic (92%) versus monophasic (74%) shocks (p < 0.05) at lower average shock energy (15 +/- 7 J vs 19 +/- 7 J, respectively, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Opiate-like analgesic activity in general anaesthetics.

1 The interaction of naloxone with various anaesthetics was studied both in vivo and in vitro.2 Naloxone (10 mg/kg) did not significantly alter the anaesthetic duration of halothane, diethylether, ketamine, pentobarbitone or Althesin.3 Naloxone (10 mg/kg) reduced the analgesic activity of nitrous oxide, ketamine and morphine in the rat tail-flick test. With the exception of pentobarbitone and Althesin, the other anaesthetic agents also induced analgesia but were not antagonized by naloxone.4 Specific [(3)H]-dihydromorphine binding was displaced by the opiates naloxone (IC(50) = 7.6 nm), methionine-enkephalin (Met-enkephalin, IC(50) = 40 nm) and morphine (IC(50) = 54 nm). Similarly, displacement was observed with xylazine (IC(50) = 9 mum), ketamine (IC(50) = 130 mum) and Althesin (IC(50) = 150 mum); other anaesthetics agents tested were inactive in mm concentrations.5 Ketamine (IC(50) = 200 mum) and xylazine (IC(50) = 9.5 mum) were also capable of displacing specific [(3)H]-D-Ala(2)-enkephalin (D-Leu) binding, as were morphine (IC(50) = 95 nm) and Met-enkephalin (IC(50) = 40 nm).6 On the stimulated guinea-pig ileum, Met-enkephalin and morphine inhibited the contractions, the IC(50) values were 30 nm and 50 nm respectively. The anaesthetics ketamine (IC(50) = 10 mum) and Althesin (IC(50) = 8 mum) were active. Xylazine (IC(50) = 12 nm) exhibited considerable potency in inhibiting the contractions on this preparation. Naloxone 0.5 mum produced a 1000 fold shift in the opiate dose-response curve but the anaesthetic responses showed only slight sensitivity to antagonism by naloxone.7 The activity of Althesin was found to be due to the vehicle in which this anaesthetic is solubilised.8 Whilst several anaesthetic agents showed analgesic activity, specific dihydromorphine binding displacement or guinea pig ileum inhibiting activity, these effects showed variable sensitivity to naloxone.

The development of tolerance to ketamine in rats and the significance of hepatic metabolism.

1. A decrease in sleeping time in rats pretreated with ten daily doses of ketamine compared to controls is shown. 2. This decrease in sleeping time is associated with a more rapid decrease in circulating and brain levels of ketamine and its N-demethylated metabolite and higher levels of the subsequent oxidation metabolite in the pretreated animals. 3. Metabolism of ketamine to its N-demethylated metabolite by liver homogenates in vitro was more rapid when the livers were obtained from ketamine pretreated rats. 4. Microsomal preparations from rat liver were capable of metabolizing ketamine to its N-demethylated metabolite and this metabolite to the subsequent oxidation metabolite in vitro. The Vmax and Km for the first reaction calculated from loss of substrate were 433 mol mg-1 protein h-1 and 0.133 mM respectively and 199 nmol mg-1 protein h-1 and 0.121 mM for the second reaction. 5. The results indicate that tolerance to ketamine in rats is associated with increased hepatic metabolism which can also be demonstrated in vitro in liver homogenates.

Effects of clonidine and xylazine on body temperature in the rat.

Clonidine and xylazine injected intraperitoneally both produced a dose-dependent hypothermia in unanaesthetized, freely moving rats. The alpha 2-antagonist yohimbine produced an antagonism of the xylazine-induced hypothermia but a potentiation of the clonidine-induced hypothermia. The opioid antagonist naloxone had little effect on the xylazine hypothermia, but potentiated the clonidine hypothermia. It is concluded that whilst the two alpha 2-agonists produce a similar hypothermic effect in the rat, they are acting by different mechanisms.

The spinal antinociceptive activity of the alpha 2-adrenoceptor agonist, xylazine in sheep.

1. The intrathecal administration of xylazine (100 micrograms), via a chronic indwelling, cervical intrathecal catheter, produced a marked elevation of the mechanical nociceptive thresholds in the sheep. This antinociceptive effect was abolished by the prior intrathecal administration of the alpha 2-adrenoceptor antagonist, idazoxan. 2. The intrathecal administration of the selective alpha 2-antagonists, idazoxan (100 micrograms) and RX811059 (33 micrograms), significantly attenuated the antinociceptive activity of intravenous xylazine, with a 60-65% reduction in the area under the antinociceptive curve. The intrathecal administration of the antagonists alone had no significant effect on nociceptive thresholds. 3. Examination of the distribution of tritiated idazoxan (25 microCi in 100 microliters) indicated that the site of action of the drug was limited to the cervical spinal cord after intrathecal administration. 4. These studies demonstrate that a significant proportion of the antinociceptive effect of systemically administered xylazine is mediated by spinal alpha 2-adrenoceptors.