Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis of deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

Analysis of DNA methylation of potential age-related methylation sites in canine peripheral blood leukocytes.

Reliable methodology for predicting the age of mature dogs is currently unavailable. In this study, amplicon sequencing of 50 blood samples obtained from diseased dogs was used to measure methylation in seven DNA regions. Significant correlations between methylation level and age were identified in four of the seven regions. These four regions were then tested in samples from 31 healthy toy poodles, and correlations were detected in two regions. The age of another 11 dogs was predicted using data from the diseased dogs and the healthy poodles. The mean difference between the actual and calculated ages was 34.3 and 23.1 months, respectively. Further research is needed to identify additional sites of age-related methylation and allow accurate age prediction in dogs.

Gene analysis of signal-joint T cell receptor excision circles and their relationship to age in dogs.

The quantification of DNA excision circles produced during T cell receptor (TCR) rearrangement, termed signal joint TCR rearrangement excision circles (sjTRECs), has been employed as a measure of age and thymic function in humans and animals. δRec-ψJα sjTRECs are ring-shaped DNAs that are generated during TCRδ locus deletion that occurs at a late stage of T cell development. In this study, the nucleotide sequences of δRec-ψJα signal joints of canine δRec-ψJα sjTRECs were analyzed. The gene structure of canine δRec-ψJα signal joints was found to be similar to that of humans and mice. However, diversity of signal joints was detected and found to derive from N nucleotide insertions, recombination signal sequence combinational diversity and single-base substitutions at the recombination signal sequence. In addition, an adenine insertion or deletion was found approximately 280 bases from the ψJα signal end. Blood samples were collected from 46 dogs, ranging in age from 3 to 192 months, with a mean age of 96.4 and a SD of 51.5 months. Although δRec-ψJα sjTRECs were detectable in most of the dogs evaluated, the level did not significantly correlate with age. These results indicated that δRec-ψJα sjTREC levels were ineffective as a measure of age in dogs.

Refractory status epilepticus, circulatory collapse after cardiac arrest, and acute respiratory distress syndrome caused by severe isolated fluvoxamine poisoning: a case report.

Case: A 30-year-old female ingested 21.75 g fluvoxamine in a suicide attempt. She presented with grand mal seizures and vomiting on admission to our Emergency Center, with a fluvoxamine serum concentration of 4.58 µg/mL. The patient was diagnosed with status epilepticus, which could not be fully suppressed with the maximum dosage of benzodiazepines. The patient also developed circulatory collapse after resuscitation for sudden cardiac arrest and acute respiratory distress syndrome, believed to be secondary to aspiration. Outcome: With venoarterial extracorporeal membrane oxygenation, a massive infusion of propofol successfully suppressed status epilepticus, and both the circulatory collapse and acute respiratory distress syndrome gradually improved; venoarterial extracorporeal membrane oxygenation and propofol treatments were then terminated, and the patient was discharged without further disabilities. Conclusion: Compared to all other reported clinical cases of fluvoxamine poisoning, the patient in this study ingested the highest dose and developed the most severe symptoms, but was successfully treated without any disabilities.

Elevated S100B level in cerebrospinal fluid could predict poor outcome of carbon monoxide poisoning.

OBJECTIVE: S100B is a calcium-binding protein produced by astroglia in the brain and has been used as a marker of neuronal damage after brain trauma. We investigated the utility of S100B in cerebrospinal fluid (CSF) measured during the early phase of carbon monoxide (CO) poisoning in predicting the subsequent clinical course. METHODS: The study included 31 patients who were admitted to the hospital with loss of consciousness following CO poisoning. S100B levels were measured by enzyme-linked immunosorbent assay in CSF, and serum samples collected simultaneously within 24 hours and on the fourth day after CO exposure. All patients were followed for at least 3 months and divided into 3 groups based on the clinical course: persistent vegetative state (PVS), delayed encephalopathy (DE), and complete recovery with no complications (NC). RESULTS: During the 3-month period, 3 patients developed PVS, 5 developed DE, and 23 were classified as NC. The mean S100B levels in the CSF within 24 hours after CO exposure were higher in the PVS group (9.25 ng/mL) than in the DE (2.03 ng/mL) and NC groups (1.86 ng/mL). However, the mean serum S100B levels were not elevated in the 3 groups (0.21, 0.59, and 0.16 ng/mL, respectively). CONCLUSION: Early elevation of S100B in CSF after CO poisoning could be a suitable predictor of subsequent development of PVS.

Notable pink excreta and severe myocardial suppression in superwarfarin (difethialone) intoxication.

Patients rarely consult physicians before developing coagulopathy or bleeding in most reported cases of superwarfarin intoxication. A 57-year-old woman ingested red-dyed pellets of anticoagulant rodenticide containing difethialone and warfarin as well as tablets of nitrazepam. Although she presented to the hospital in a comatose state, notable pink-colored excreta hinted at the consumption of anticoagulant rodenticide, which led to the early diagnosis of superwarfarin intoxication. Supplementation of large doses of intravenous and oral vitamin K successfully prevented coagulopathy and bleeding. On the other hand, temporary and reversible myocardial suppression was extremely severe, and required the introduction of percutaneous cardiopulmonary support.

Aseptic meningitis in association with glyphosate-surfactant herbicide poisoning.

INTRODUCTION: The mechanisms underlying early central nervous system (CNS) signs and symptoms of glyphosate-surfactant herbicide (GlySH) poisoning are unclear. CASE PRESENTATION: A 58-year-old woman ingested approximately 150 mL of GlySH containing 41% glyphosate and 15% polyoxyethyleneamine. Two days later, she was admitted in the Emergency Center in a semicomatose state. Acute respiratory distress syndrome, circulatory collapse, acute renal failure, and disseminated intravascular coagulopathy were diagnosed. Meningitis was also suspected as she demonstrated Kernig's sign and significant neck stiffness with rigidity of the extremities as well as consciousness disturbance and fever (38.4°C). Investigations of cerebrospinal fluid (CSF) revealed the presence of glyphosate (122.5 µg/mL), significant elevation of IL-6 (394 µg/mL), and pleocytosis (32 cells/µL) with monocyte dominance. All bacteriological and virological tests were later found to be negative. She recovered completely after responding to aggressive supportive care in the intensive care unit. All signs and symptoms suggesting meningitis resolved as the concentration of glyphosate in CSF decreased. She was discharged on day 39 of hospitalization. DISCUSSION: These findings suggest that the present case involved aseptic meningitis in association with GlySH poisoning. CONCLUSION: CNS signs and symptoms induced by aseptic meningitis should be considered in cases of glyphosate-surfactant herbicide poisoning.

Pharmacokinetics of the active metabolites of ethyl loflazepate in elderly patients who died of asphyxia associated with benzodiazepine-related toxicity.

We determined the pharmacokinetics of ethyl loflazepate (Lof) in elderly patients who died of benzodiazepine-related toxicity. Three elderly patients with body mass indexes of less than 17 kg/m2 died of asphyxia after having taken maintenance doses of Lof for 2 to 3 weeks. We measured serum concentrations of the active metabolites of Lof using gas chromatography-mass spectrometry and a benzodiazepine receptor assay to determine the pharmacokinetics of each. On admission, the serum concentrations of the active metabolites of Lof ([Lofl) were 256 ng/mL, 425 ng/mL, and 177 ng/mL in cases 1, 2, and 3, respectively. Serum benzodiazepine-receptor binding activities, expressed as diazepam equivalent concentrations ([Bz]), were 1800 ng/mL, 2200 ng/mL, and 1500 ng/mL. The T1/2(beta) of [Lof] were 124 and 121 h in cases 1 and 2 and the T1/2(beta) of [Bz] were 75 and 87 h. The distribution volume in the elderly was reduced due to a small lipid compartment, and total drug clearance was decreased due to the decline in liver and kidney function. These changes did not prolong T1/2(beta) but did increase plasma concentrations of active metabolites, especially in case 2, and a slight decrease in protein binding increased the amount of free active metabolites greatly.

Effect of milk on intestinal fluid accumulation and renal injury following mercuric chloride ingestion in rats.

To investigate the effect of milk on intestinal fluid accumulation and renal injury following mercuric chloride (HgCl(2)) ingestion, 10 ml kg(-1) of saline or 10 mg kg(-1) of HgCl(2) dissolved in 10 ml kg(-1) of water or raw milk was administered enterally to rats and the mercury content in biological samples was determined by cold vapour atomic absorption spectrometry. Three hours after administration, the intestinal water content in rats that received HgCl(2) in water (group S2) was significantly higher than in rats that received saline (group S1) (P < 0.01) or HgCl(2) in milk (group S3) (P < 0.01). The amount of mercury detected per gram dry weight of small intestine was higher in group S2 than in group S3 (P < 0.05). Seventy-two hours after administration, both the serum urea nitrogen and creatinine concentrations in rats that received HgCl(2 )in milk (group L3) were significantly higher than in rats that received saline (group L1) (P < 0.05) or HgCl(2) in water (group L2) (P < 0.05). Mercury levels in many of the biological samples in group L3 were higher than in group L2 (P < 0.05). Milk may reduce the intestinal cytotoxicity of mercury but it promotes its absorption, which may lessen intestinal fluid accumulation but worsen renal injury.

Blockade of bradykinin B(2) receptor suppresses acute pancreatitis induced by obstruction of the pancreaticobiliary duct in rats.

1. The involvement of bradykinin (BK) B(2) receptor in acute pancreatitis induced by pancreaticobiliary duct ligation was investigated in rats. 2. The activities of amylase and lipase in the serum, the water content of the pancreas, and vacuolization of the acinar cells were significantly increased 2 h after obstruction of the duct in Sprague-Dawley rats. 3. Elevated serum amylase activity, increased pancreatic oedema, and damage of the pancreatic tissue were significantly less marked in plasma kininogen-deficient, B/N-Katholiek rats than in the normal strain, B/N-Kitasato rats 2 h after the ligation. 4. Obstruction of the pancreaticobiliary duct augmented the level of (1-5)-BK (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)), a stable BK metabolite, in the blood from 73.0+/-21.7 pg ml(-1) at 0 h to 149.8+/-38.0 pg ml(-1) at 2 h after the induction of pancreatitis in SD rats. 5. Administration of a BK B(2) receptor antagonist, FR173657 (100 mg kg(-1), p.o.) or Hoe140 (100 nmol kg(-1), s.c.), reduced the elevation of amylase and lipase activities in the serum and of pancreatic water content in a dose-dependent manner. The effective attenuation of oedema formation and vacuolization by the antagonists was also confirmed light-microscopically. In contrast, treatment with gabexate mesilate or indomethacin did not cause significant suppression of the pancreatitis. 6. These findings suggest a possible involvement of kinin B(2) receptor in the present pancreatitis model. Furthermore, they point to the potential usefulness of the B(2) receptor in clinical acute pancreatitis.