Kinetics of Plasma Cell-Free DNA and Creatine Kinase in a Canine Model of Tissue Injury.

BACKGROUND: Cell-free DNA (cfDNA) comprises short, double-stranded circulating DNA sequences released from damaged cells. In people, cfDNA concentrations correlate well with disease severity and tissue damage. No reports are available regarding cfDNA kinetics in dogs. OBJECTIVES/HYPOTHESIS: Cell-free DNA will have a short biological half-life and would be able to stratify mild, moderate, and severe tissue injury. Our study aims were to determine the kinetics and biological half-life of cfDNA and to contrast them with those of creatine kinase (CK). ANIMALS: Three groups of 10 dogs undergoing open ovariohysterectomy, surgery for cranial cruciate ligament rupture (CCLR), or hemilaminectomy. METHODS: Plasma for cfDNA and CK analysis was collected at admission, at induction of anesthesia, postsurgery (time 0) and at 6, 12, 24, 36, 48, 60, and 72 hours after surgery. RESULTS: The biological half-life of plasma cfDNA and CK were 5.64 hours (95% confidence interval [CI 95], 4.36-7.98 hours) and 28.7 hours (CI95, 25.3-33.3 hours), respectively. In the hemilaminectomy group, cfDNA concentrations differed significantly from admission at 6-12 hours after surgery. Creatine kinase activity differed among the surgical groups and reached a peak 6 hours after surgery. In the ovariohysterectomy and CCLR groups, plasma CK activity 72 hours after surgery did not differ from admission activity of the ovariohysterectomy group. In contrast, in the hemilaminectomy group, plasma CK activity after 72 hours did not return to the ovariohysterectomy group admission activity. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma CK activity has a longer biological half-life than previously thought. In contrast to plasma CK activity, cfDNA has a short half-life and could be a useful marker for peracute severe tissue injury.

Health behaviours and outcomes associated with fly-in fly-out and shift workers in Western Australia.

AIMS: To examine the association of health behaviours and outcomes with employment type in the West Australian adult population. METHODS: Cross-sectional study of employed adults aged 16 years and over using self-reported information collected in the WA Health and Wellbeing Surveillance System between 2008 and 2010. A total of 380 fly-in fly-out (FIFO) workers, 913 shift workers and 10 613 workers of other employment types were identified. RESULTS: FIFO workers exhibited similar health behaviours to shift workers but had a different sociodemographic profile. Compared with other employment types, FIFO workers were significantly more likely to be current smokers, drink alcohol at risky levels, and be overweight or obese, after adjusting for age, sex and survey sampling strategies. They were less likely to report current mental health problems. CONCLUSIONS: Self-reported health behaviours of FIFO workers differ from other employment types. FIFO workers are expected to increase in number over the next decade, as the mining and resources sector expands in Australia. Our findings suggest that health interventions, whether in the workplace or clinical settings, need to be informed by the demographic mix of the cohort of workers on entry as they are not a homogenous group, and targeted towards specific employment patterns (length of shifts and type of employment) to improve their current and future well-being.

Post-traumatic paraureteral urinoma in a cat.

A retroperitoneal urinoma (uriniferous pseudocyst) was diagnosed in a domestic shorthair cat exhibiting a sublumbar swelling two weeks after a road traffic accident. Plain radiography revealed a soft tissue opacity in the left retroperitoneal space. Intravenous urography and fine-needle aspiration were diagnostic. Contrast was seen pooling in the dilated ipsilateral renal pelvis and proximal ureter. Fluid aspirated from the retroperitoneal space had a creatinine level five times that of serum. Surgical drainage followed by nephrectomy and omentalisation of the pseudocyst was curative.

A pilot study of N-acetyl glucosamine, a nutritional substrate for glycosaminoglycan synthesis, in paediatric chronic inflammatory bowel disease.

BACKGROUND: The breakdown of glycosaminoglycans is an important consequence of inflammation at mucosal surfaces, and inhibition of metalloprotease activity may be effective in treating chronic inflammation. AIM: To report an alternative approach, using the nutriceutical agent N-acetyl glucosamine (GlcNAc), an amino-sugar directly incorporated into glycosaminoglycans and glycoproteins, as a substrate for tissue repair mechanisms. METHODS: GlcNAc (total daily dose 3-6 g) was administered orally as adjunct therapy to 12 children with severe treatment-resistant inflammatory bowel disease (10 Crohn's disease, 2 ulcerative colitis). Seven of these children suffered from symptomatic strictures. In addition, similar doses were administered rectally as sole therapy in nine children with distal ulcerative colitis or proctitis resistant to steroids and antibiotics. Where pre- and post-treatment biopsies were available (nine cases), histochemical assessment of epithelial and matrix glycosaminoglycans and GlcNAc residues was made. FINDINGS: Eight of the children given oral GlcNAc showed clear improvement, while four required resection. Of the children with symptomatic Crohn's stricture, only 3 of 7 have required surgery over a mean follow-up of > 2.5 years, and endoscopic or radiological improvement was detected in the others. Rectal administration induced remission in two cases, clear improvement in three and no effect in two. In all cases biopsied there was evidence of histological improvement, and a significant increase in epithelial and lamina propria glycosaminoglycans and intracellular GlcNAc. CONCLUSIONS: GlcNAc shows promise as an inexpensive and nontoxic treatment in chronic inflammatory bowel disease, with a mode of action which is distinct from conventional treatments. It may have the potential to be helpful in stricturing disease. However, controlled trials and an assessment of enteric-release preparations are required to confirm its efficacy and establish indications for use.

Topical tacrolimus may be effective in the treatment of oral and perineal Crohn's disease.

BACKGROUND: Crohn's disease of the mouth or perineum is more common in young people, and notably resistant to treatment. However, there is increasing evidence that topical therapy with tacrolimus (FK506) may be effective in skin diseases resistant to cyclosporin because of its high uptake in inflamed skin and subsequent reduction in keratinocyte chemokine production. PATIENTS AND METHODS: Tacrolimus ointment was made up inhouse from the intravenous or oral formulation and suspended in appropriate vehicles for perioral or perianal administration at an initial concentration of 0.5 mg/g. This was administered open label to eight children (aged 5-18 years) with treatment resistant oral (three patients) and/or ulcerating perineal (six patients) Crohn's disease. RESULTS: Marked improvement was seen in 7/8 patients within six weeks and healing within 1-6 months. One child with gross perineal and colonic disease showed little response. Two of the responders showed rebound worsening when tacrolimus was stopped or the dosage reduced rapidly, and one of these eventually required proctectomy. Slower weaning of drug concentration has been successful in 6/8 patients, with four receiving intermittent treatment and two on regular reduced dosage (0.1-0.3 mg/g) with follow up times of six months to 3.5 years. Serum concentrations of tacrolimus were undetectable in all patients. CONCLUSIONS: Topical tacrolimus at low concentrations (0.5 mg/g) shows promise in the management of childhood perineal and oral Crohn's disease, with no evidence of significant systemic absorption. However, rapid weaning or abrupt cessation of therapy may cause rebound worsening of disease. Further controlled studies are required to assess the efficacy and safety of this treatment.

International maternal mortality reduction: outcome of traditional birth attendant education and intervention in Angola.

BACKGROUND: As a result of war and periodic natural disasters, Angola has among the highest infant and maternal mortality rates in the world. In response to the acute health needs of the population, the International Medical Corps (IMC) developed a traditional birth attendant educational course designed to reduce the preventable causes of maternal and infant mortality. METHODS: From 1994 until 1998, Angolan traditional birth attendants (TBAs) participated in an intensive 38-hr training course on prenatal, delivery, and postnatal care. Following the birth of a child, the trained TBAs completed a registration form containing information regarding the health of the mother. Previous studies of Angolan maternal mortality served as historic comparisons. FINDINGS: Complete data including maternal mortality data were available for 19,666 deliveries (83% of total). Fifty five maternal deaths were recorded, which corresponds to a maternal mortality rate of 293 per 100,000 live births. The average historic maternal mortality rate for available comparison groups was 1241 per 100,000 live births. INTERPRETATION: The maternal mortality rate was reduced among women managed by IMC-trained TBAs when compared with historical control data.

Preparation of barbiturate optical isomers and their effects on GABA(A) receptors.

BACKGROUND: Barbiturate anesthetics are optically active and usually exist in two mirror-image enantiomeric forms. Their stereoselective effects in mammals are well known, but remarkably few data are available concerning their effects on anesthetic targets in vitro. This is in part because of the lack of availability of pure barbiturate enantiomers. Such in vitro data could be used to test the relevance of putative molecular targets. METHODS: A high-performance liquid chromatography technique using a permethylated beta-cyclodextrin column was used to separate the optical isomers of three barbiturates in preparative quantities. The effects of the isomers on GABA-induced currents in stably transfected mouse fibroblast cells were investigated using the whole-cell patch-clamp technique. RESULTS: Highly purified optical isomers of hexobarbital, pentobarbital, and thiopental were prepared, and their effects were studied on a gamma-aminobutyric acid type A receptor of defined subunit composition. For each of the three barbiturates, both enantiomers potentiated gamma-aminobutyric acid-induced currents at pharmacologically relevant concentrations, with the S-enantiomer being more potent than the R-enantiomer by a factor of between 1.7 and 3.5. The degree of stereoselectivity did not vary greatly with anesthetic concentration. CONCLUSIONS: The rank order and degree of stereoselectivity that we have observed for the enantiomers of hexobarbital, pentobarbital, and thiopental acting on the gamma-aminobutyric acid type A receptor are entirely consistent with this receptor playing a central role in the anesthetic actions of barbiturates.

Stereoselective effects of etomidate optical isomers on gamma-aminobutyric acid type A receptors and animals.

BACKGROUND: The intravenous anesthetic etomidate is optically active and exists in two mirror-image enantiomeric forms. However, although the R(+) isomer is used as a clinical anesthetic, quantitative information on the relative potencies of the R(+) and S(-) isomers is lacking. These data could be used to test the relevance of putative molecular targets. METHODS: The anesthetic concentrations for a half-maximal effect (EC50) needed to induce a loss of righting reflex in tadpoles (Rana temporaria) were determined for both etomidate enantiomers. The effects of the isomers on gamma-aminobutyric acid (GABA)-induced currents in stably transfected mouse fibroblast cells was also investigated using the patch-clamp technique. In addition, the effects of the isomers on a lipid chain-melting phase transition were determined. RESULTS: The EC50 concentrations for general anesthesia for the R(+) and S(-) isomers were 3.4 +/- 0.1 microM and 57 +/- 1 microM, with slopes of n = 1.9 +/- 0.1 and n = 2.9 +/- 0.2, respectively. The R(+) isomer was also much more effective than the S(-) isomer at potentiating GABA-induced currents, although the degree of stereoselectivity varied with anesthetic concentration. R(+) etomidate potentiated the GABA-induced currents by increasing the apparent affinity of GABA for its receptor. Both isomers were equally effective at disrupting lipid bilayers. CONCLUSIONS: These data are consistent with the idea that the GABA(A) receptor plays a central role in the actions of etomidate. Etomidate exerts its effects on the receptor by binding directly to a specific site or sites on the protein and allosterically enhancing the apparent affinity of GABA for its receptor.

Stigma and incest survivors.

The impact of incestuous child sexual abuse continues to affect victims into their adult lives. Survey responses from college students indicate the predicted stigma surrounding this childhood event varies with gender as well as with length and type of relationship at the time of the disclosure. Women indicate differences in stigma when anticipating various types of relationships with male survivors. Particularly, women show dating as less comfortable than opposite sex friendship, and parenting as less comfortable than marriage. Men do not indicate these differences in stigma based on the type of relationship, but instead predict comfort with female survivors based on the length of relationship.

Adrenal vein and arterial levels of catecholamines and immunoreactive metenkephalin in canine endotoxin shock and their response to naloxone.

The alterations in plasma levels of immunoreactive metenkephalin (ir-metenkephalin) and catecholamines in adrenal vein and arterial blood in response to endotoxin, as well as the effects of subsequent naloxone administration, have been investigated in a canine model. Animals were anaesthetised with alpha chloralose and allowed to breathe spontaneously. The left lumbar adrenal vein was cannulated and an intermittent choke allowed retrograde sampling of the adrenal effluent. Severe shock was produced by the administration of a large bolus of E. coli endotoxin (5 mg/kg) followed by a continuous infusion (2 mg/kg per hour). One hour after induction of shock the circulating volume was expanded using a colloidal gelatin solution. Thirty minutes later one group of five animals received a bolus of naloxone (2 mg/kg) followed by a continuous infusion of (1.5 mg/kg per hour), while a control group of five animals was given an equivalent volume of isotonic saline. The production of endotoxin shock was associated with marked increases in adrenal vein and systemic levels of adrenaline and noradrenaline. Naloxone administration transiently limited the fall in adrenal vein levels of adrenaline and noradrenaline (P less than 0.05) following volume replacement and was associated with a sustained increase in systemic adrenaline levels (P less than 0.05). Changes in mean arterial pressure confirmed a significant haemodynamic response to naloxone (P less than 0.05). Alterations in ir-metenkephalin levels in the adrenal vein closely followed the changes in catecholamines, whereas arterial levels rose progressively and were unaffected by naloxone. We conclude that in canine endotoxin shock the opiate antagonist naloxone can transiently increase catecholamine levels in the adrenal effluent and produce a more sustained rise in systemic adrenaline levels. Moreover, the adrenal medulla is not the only source of circulating ir-metenkephalin.

Neuroendocrine and cardiovascular responses to high-dose corticosteroid therapy in canine endotoxin shock.

The neuroendocrine and cardiovascular responses to endotoxin administration and the effects of subsequent high-dose corticosteroid therapy have been investigated in dogs. Shock was induced in anaesthetised animals by a large bolus of E. coli endotoxin (5 mg/kg) followed by a continuous infusion (2 mg/kg per hour). One hour after induction of shock, the circulating volume was expanded using a colloidal gelatin solution. Fifteen minutes later, one group of five animals received a bolus of methylprednisolone sodium succinate 30 mg/kg, while a control group of five animals was given an equivalent volume of isotonic saline. The administration of endotoxin produced reductions in mean arterial pressure, cardiac index and left ventricular dp/dtmax, together with increases in systemic and pulmonary vascular resistances. These haemodynamic changes were associated with increases in arterial plasma levels of adrenaline, noradrenaline, cortisol, immunoreactive beta-endorphin and immunoreactive metenkephalin. Cardiovascular improvement followed volume replacement and was associated with reductions in circulating catecholamines. No significant haemodynamic or neuroendocrine changes were demonstrated in the 2 h following steroid therapy.

Alpha-adrenergic stimulation of corticotropin secretion by a specific central mechanism in man.

In a double-blind study in normal subjects, methoxamine, a highly selective agonist at alpha-1-adrenoceptors, significantly increased circulating ACTH and cortisol. The stimulant effect of methoxamine on cortisol secretion was dose dependent in the range 3.5-7 micrograms/kg/min, was abolished by concomitant administration of the strong alpha-1-adrenergic (and weak H1) antagonist thymoxamine but unaffected by the antihistamine, chlorpheniramine. In order to test whether the action of methoxamine on ACTH secretion was exerted centrally or peripherally, the effects of norepinephrine (NE), an alpha-1-agonist that does not cross the blood-brain barrier, were studied. Doses of NE (1-12 micrograms/min) that increased systolic blood pressure by amounts similar to the changes produced by methoxamine, did not result in any rise in plasma cortisol in normal subjects. The effect of methoxamine, which is more lipid soluble than NE, on plasma ACTH and cortisol, appears to be exerted on the CNS and not at the pituitary or via nonspecific peripheral mechanisms. In addition to its water solubility, NE differs from methoxamine in its beta-1-, beta-2- and alpha-2-agonist actions. However, prenalterol (2 mg) and salbutamol (250 micrograms), respectively beta-1- and beta-2-adrenergic agonist drugs, had no effect on the secretion of ACTH or cortisol and the alpha-2-antagonist yohimbine in an effective dose did not unmask a stimulant effect of intravenous NE on plasma cortisol. At high infusion rates, NE significantly inhibited cortisol secretion. Stimulation of central alpha-1-adrenergic mechanisms results in secretion of ACTH in man, presumably by increased release of a corticotropin-releasing factor.

Biochemical characterization of circulating Met-enkephalins in canine endotoxin shock.

The alterations in arterial, venous and adrenal vein levels of immunoreactive Met-enkephalins following endotoxin administration have been investigated in dogs by direct measurement and gel filtration chromatography. Animals were anaesthetized with alpha-chloralose and allowed to breath spontaneously. The left lumbar adrenal vein, limb vein and femoral artery were cannulated for blood sampling. Severe shock was produced by the administration of a large bolus of E. coli endotoxin followed by a continuous infusion. The production of endotoxin shock was associated with significant increases in adrenal vein and systemic venous plasma immunoreactive Met-enkephalin levels. Forty-five minutes after induction of endotoxin shock arterial immunoreactive Met-enkephalin levels were generally higher than baseline values. In resting anaesthetized animals a large 31,000 molecular weight form of Met-enkephalin, presumably proenkephalin, was found in plasma obtained from the adrenal vein, systemic and pulmonary circulations. Following endotoxin this enkephalin-containing peptide still predominated in arterial and venous plasma, whereas in the adrenal vein the proportion of Met-enkephalin immunoreactivity attributable to this large peptide fell. This was associated with the appearance of increasing amounts of smaller molecular forms (18,000, 8000, 3-5000 molecular weight and the pentapeptide itself). In this model enkephalin-containing peptides were not biochemically modified by their passage through the lungs.

RIA/chromatographic evidence for novel opioid peptide(s) in Squilla mantis ganglia.

Proteins extracted from suboesophageal ganglia of Squilla mantis, an arthropod shown to be sensitive in vivo to opiates and to contain native opioid like peptide(s), were fractionated by gel filtration into three pools according to their molecular weight: A (Mr greater than 65,000), B (10,000 less than Mr less than 65,000) and C (Mr less than 10,000). None of these pools showed any immunoreactivity when radioimmunoassayed using antisera raised against Met-enkephalin either before or after sequential trypsin/carboxypeptidase B proteolysis. Further purification of pool C by HPLC followed by RIA using antibodies directed to Met-O-enkephalin,Leu-enkephalin,Dynorphin 1-13 and human beta-endorphin, showed only a trace amount of Met-enkephalin cross-reactivity (about 10 fmoles/mg of protein extract). No detectable amount of Leu- or Met-enkephalin was found after HPLC fractionation of proteolyzed pool B. Radioreceptor assay of HPLC fractions derived from trypsin/carboxypeptidase B treated pools B and C showed major areas of activity common to both pools, but nevertheless with differing retention times compared to the standard opioid peptides used.

The role of endorphins in septicaemic shock: a pilot study in burned patients.

There is recent evidence that circulating opioid peptides, or 'endorphins', act as chemical messengers responsible for the induction of the complex cardiovascular changes leading to hypotension in septicaemic shock. The pilot study of an investigation of opioid peptides in septicaemia in burned patients is presented. Serial measurements of plasma beta-endorphin and metenkephalin were performed throughout the recovery of six patients with large burns (20-70 per cent BSA). Our preliminary findings concur with previous evidence that opioid peptides may play a role in the hypotension of septicaemic shock.

Nitrous oxide inhalation does not influence plasma concentrations of beta-endorphin or Met-enkephalin-like immunoreactivity.

The possibility that nitrous oxide releases endogenous opioid peptides into the circulation has been tested in 10 pain-free, unstressed volunteers breathing 30% nitrous oxide in oxygen. Despite achieving plateau concentrations in venous blood, accompanied by subjective effects, there were no significant changes in plasma concentrations of immunoreactive beta-endorphin, methionine-enkephalin or ACTH. These results indicate that, in the absence of nociceptive input, the effects of the inhalation of nitrous oxide are unrelated to alterations in peripheral concentrations of these endogenous opioid peptides.

Neuroendocrine and cardiovascular changes in septic shock and after cardiac surgery: effect of high-dose corticosteroid therapy.

Plasma levels of beta-endorphin-like immunoreactivity (BLI) were similarly elevated in patients with septic shock (group A) and in normotensive subjects recovering from cardiac surgery (group B) (1231 +/- 483 pg ml-1 and 1,240 +/- 355 pg ml-1, respectively). In neither group was cardiac output reduced, but total peripheral resistance index (TPRI) was low in group A and low or normal in group B. Intravenous methylprednisolone (MP) 30 mg kg-1 variably suppressed BLI by a mean of only 30% in group A, while in group B, BLI usually rose and then fell following MP. In group A percentage changes in BLI were positively correlated with percentage changes in cardiac index (CI) and mean arterial pressure (MAP) (r = .83, P less than .01, r = .59, P less than .05 respectively). No such correlations were found in group B. These findings suggest that increases in circulating beta-endorphin are unlikely to be responsible for myocardial depression or hypotension in septic shock. ACTH levels were in general normal in group A but were consistently elevated in group B, although plasma cortisol was similarly elevated in both groups. Furthermore there was a good correlation between percentage changes in ACTH and BLI following MP in group B (r = .87, P less than .01) but not in group A. Possible explanations for these findings are discussed.