Effects of polymyxin-B on TNF-α production in equine whole blood stimulated with three different bacterial toxins.

Polymyxin-B is used to treat equine systemic inflammation. Bacterial toxins other than lipopolysaccharide (LPS) contribute to systemic inflammation but the effects of polymyxin-B on these are poorly defined. Whole blood aliquots from six healthy horses diluted 1:1 with RPMI were incubated for 21 hr with 1 µg/ml of LPS, lipoteichoic acid (LTA) or peptidoglycan (PGN) in the presence of increasing concentrations of polymyxin-B (10-3000 µg/ml). A murine L929 fibroblast bioassay was used to measure TNF-α activity. Polymyxin-B significantly inhibited the effects of all three bacterial toxins. Analysis of variance showed the IC50 value for polymyxin-B for TNF-α inhibition caused by LTA (11.19 ± 2.89 µg/ml polymyxin-B) was significantly lower (p = .009) than the values for LPS (46.48 ± 9.93 µg/ml) and PGN (54.44 ± 8.97 µg/ml). There was no significant difference in IC50 values between LPS and PGN (p > .05). Maximum inhibition of TNF-α was 77.4%, 73.0% and 82.7% for LPS, PGN and LTA, respectively and was not significantly different between toxins. At the two highest concentrations of polymyxin-B, TNF-α began to increase. These data suggest that polymyxin-B may inhibit the effects of bacterial toxins other than LPS and might be a more potent inhibitor of LTA than LPS or PGN.

Sequential plasma lactate concentrations as prognostic indicators in adult equine emergencies.

BACKGROUND: Sequential lactate concentration ([LAC]) measurements have prognostic value in that hospitalized humans and neonatal foals that have a delayed return to normolactatemia have greater morbidity and case fatality rate. HYPOTHESIS: Prognosis for survival is decreased in horses with a delayed return to normal [LAC]. ANIMALS: Two hundred and fifty adult horses presented for emergency evaluation excepting horses evaluated because of only ophthalmologic conditions, superficial wounds, and septic synovitis without systemic involvement. METHODS: Prospective observational study. [LAC] was measured at admission and then at 6, 12, 24, 48, and 72 hours after admission. The change in [LAC] over time ([LAC]deltaT) was calculated from changes in [LAC] between sampling points. RESULTS: Median [LAC] was significantly (P < .001) higher at admission in nonsurvivors (4.10 mmol/L [range, 0.60-18.20 mmol/L]) when compared with survivors (1.30 mmol/L [range, 0.30-13.90 mmol/L]) and this difference remained at all subsequent time points. The odds ratio for nonsurvival increased from 1.29 (95% confidence interval 1.17-1.43) at admission to 49.90 (6.47-384) at 72 hours after admission for every 1 mmol/L increase in [LAC]. [LAC]deltaT was initially positive in all horses but became negative and significantly lower in nonsurvivors for the time periods between 24-72 hours (- 0.47, P = .001) and 48-72 hours (- 0.07, P = .032) when compared with survivors (0.00 at both time periods) consistent with lactate accumulation in nonsurvivors. CONCLUSIONS AND CLINICAL IMPORTANCE: These results indicate that lactate metabolism is impaired in critically ill horses and [LAC]deltaT can be a useful prognostic indicator in horses.

KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel.

The M-current regulates the subthreshold electrical excitability of many neurons, determining their firing properties and responsiveness to synaptic input. To date, however, the genes that encode subunits of this important channel have not been identified. The biophysical properties, sensitivity to pharmacological blockade, and expression pattern of the KCNQ2 and KCNQ3 potassium channels were determined. It is concluded that both these subunits contribute to the native M-current.

Elapid snake envenomation in horses: 52 cases (2006-2016).

BACKGROUND: Snake envenomation is a cause of morbidity and mortality in domestic animals worldwide. The clinical features of crotalid snake (pit viper) envenomation are widely reported and well described in horses but elapid snake envenomation is poorly characterised. OBJECTIVES: To describe the presentation, clinical and laboratory findings, treatment and outcome of horses with a diagnosis of elapid snake envenomation in Australia. STUDY DESIGN: Retrospective case series. METHODS: Medical records of horses with a diagnosis of elapid snake envenomation (2006-2016) at several university and private veterinary practices were reviewed. Inclusion criteria comprised one or more of the following: 1) observed snakebite, 2) positive snake venom detection kit (SVDK) result, 3) appropriate clinical response to treatment with antivenom or 4) supportive post-mortem findings. RESULTS: Fifty-two cases met the inclusion criteria. Most cases (94%) demonstrated clinical signs of neurotoxicity, characterised by generalised neuromuscular weakness. Associated neurologic signs included staggering gait, muscle fasciculations, recumbency, mydriasis, ptosis and tongue paresis. Concurrent clinically important conditions included rhabdomyolysis (50%) and haemolysis (19%). Of 18 urine samples evaluated with a SVDK, only three (17%) were positive. Overall survival was favourable (86%) among 49 horses who received antivenom. Eighteen surviving horses (43%) required more than one vial of antivenom. MAIN LIMITATIONS: Possible cases within the searchable database were not included if horses died acutely or responded to symptomatic treatment without receiving antivenom. CONCLUSIONS: Elapid snake envenomation is primarily a syndrome of neuromuscular weakness. Supportive anamnesis or an obvious bite site is rarely encountered. In endemic areas, this diagnosis should be considered for horses with generalised neuromuscular weakness, altered mentation, rhabdomyolysis and/or haemolysis; especially during spring and summer months. Diagnostic suspicion is best confirmed by response to treatment with antivenom.

Modulation and genetic identification of the M channel.

Potassium channels constitute a superfamily of the most diversified ion channels, acting in delicate and accurate ways to control or modify many physiological and pathological functions including membrane excitability, transmitter release, cell proliferation and cell degeneration. The M-type channel is a unique ligand-regulated and voltage-gated K(+) channel showing distinct physiological and pharmacological characteristics. This review will cover some important progress in the study of M channel modulation, particularly focusing on membrane transduction mechanisms. The K(+) channel genes corresponding to the M channel have been identified and will be reviewed in detail. It has been a long journey since the discovery of M current in 1980 to our present understanding of the mysterious mechanisms for M channel modulation; a journey which exemplifies tremendous achievements in ion channel research and exciting discoveries of elaborate modulatory systems linked to these channels. While substantial evidence has accumulated, challenging questions remain to be answered.

Changes in attitude toward methadone.

Staff and client attitudes toward heroin users and toward maintenance and abstient clients were assessed in 1970 and again in 1973. On each occasion there was striking agreement between the ratings made by staff and client groups. Abstinent clients were rated by all groups as significantly more effective and more responsible than either maintance clients or heroin users; maintenance clients were rated as more conservative, self-conscious, and self-effacing than were the other groups. While heroin users and abstinent clients were characterized similarly in the two rating periods, raters in the 1973 survey viewed methadone hydrochloride clients as more passive and less inclined to undertake adult responsibilities than was true of raters in the 1970 survey.

Controlling the abuse of illicit methadone in Washington, DC.

Methadone hydrochloride has been found to be medically safe when administered in the setting of a well-organized heroin addition treatment program. The abuse of illicit methadone, outside the therapeutic setting, has aroused considerable controversy, particularly with regard to the public health hazards of primary methadone addiction, overdose, abuse, and childhood poisoning. We attempted to document the nature and extent of these negative aspects of the diversion of methadone into the illicit drug market, using data collected between 1969 and 1974 in the District of Columbia. The data illustrate the severe problems created by the widespread availability of illicit methadone, and document that, with the appropriate controls, the large-scale use of methadone in addiction treatment is feasible with minimum risk of methadone addiction and overdose in the community.

Changes in mood, craving, and sleep during short-term abstinence reported by male cocaine addicts. A controlled, residential study.

We examined changes over 28 days in mood states, craving for cocaine, and sleep during short-term abstinence reported by 12 male, predominantly intravenous-using, cocaine-addicted subjects residing in a research facility. For comparison, we examined 10 nonaddicted control subjects. There were no significant differences between cocaine addicts and controls regarding demographics and selected DSM-III-R diagnoses other than psychoactive substance use disorder and antisocial personality disorder. There were significantly higher scores of psychiatric symptoms reported by cocaine addicts 1 week before admission. Mood-distress and depression scores recorded at admission and during short-term abstinence were significantly greater than those reported by controls. Addicts' mood-distress scores and craving for cocaine were greatest at admission and decreased gradually and steadily during the 28-day study. There were no significant differences between groups regarding reports of sleep other than difficulty falling asleep and clearheadedness on arising. Although there were significant differences in resting heart rate at admission and over time, there were no significant differences in weight gain or blood pressure. Given the absence of a classic "withdrawal" pattern, "short-term abstinence" may be a more appropriate classification of psychological and physical phenomena experienced by cocaine addicts who initiate abstinence in a controlled environment.

An evaluation of drug treatments for adolescents in 4 US cities.

BACKGROUND: Little is known about outcomes of community-based treatment programs for adolescents with drug problems. METHODS: We studied 1167 adolescents (age range, 11-18 years; 368 females, 799 males) from 4 US cities (Pittsburgh, Pa; Minneapolis, Minn; Chicago, Ill; and Portland, Ore) using a naturalistic, nonexperimental evaluation design. These adolescents were consecutive admissions during the period from 1993 to 1995 at 23 community-based treatment programs in the Drug Abuse Treatment Outcome Studies for Adolescents. Included were 418 admissions to 8 residential programs, 292 admissions to 9 outpatient drug-free programs, and 457 admissions to 6 short-term inpatient programs. RESULTS: Adolescents in treatment typically had multiple problems (eg, 58.4% of them were involved in the legal system, and 63.0% met diagnostic criteria for a mental disorder). Nevertheless, less than half (43.8%) of all patients reported weekly marijuana use in the year following treatment (dropping from 80.4% in the year before admission). Similarly, there were decreases in heavy drinking (dropping from 33.8% to 20.3%), use of other illicit drugs (dropping from 48.0% to 42.2%), and criminal involvement (dropping from 75.6% to 52.8%). Additionally, patients reported better psychological adjustment and school performance after treatment. Longer stays in treatment were positively associated with several favorable outcomes, although length of time in treatment was generally short. CONCLUSIONS: Substance abuse treatment for adolescents is effective in achieving many important behavioral and psychological improvements. Strategies specific to adolescents to improve their treatment retention and completion are needed to maximize the therapeutic benefits of drug treatment.

The life of psychiatry.

Focusing on the definition, scope, and role of psychiatry today, the author discusses whether psychiatry is primarily medical, whether it is overstepping its boundaries in attempting to treat problems of living, and whether it is too involved with social questions. On the basis of an examination of the current scientific base of psychiatry, he predicts that psychiatry will continue to grow in size and diversity, that it will refocus substantially on biological issues, that it will become more humble about what it can do with regard to social problems, and that it will continue to yield new therapeutic measures and techniques.

The crisis in mental health research.

The author states that the mental health research program of the United States is facing a threat to its survival as a force for public health. He describes the antecedents of this crisis, which include the separation of the National Institute of Mental Health from the National Institutes of Health and other organizational changes, the public's disappointment that overly great expectations were not met, and monetary inflation coupled with budgetary cutbacks. He concludes that, because research cannot be separated from the goals of service and training, the success of the total mental health program in the United States depends on resolving the research support crisis.

Methadone maintenance: some client opinions.

The authors found similar attitudes toward methadone and methadone treatment programs in 75 detoxification and 115 methadone maintenance clients. Both groups expressed considerable ambivalence--although they viewed methadone as capable of helping them end their herioin addiction, they were concerned about possible methadone dependence and about side effects, both real and imagined. The authors stress the societal context of such concern and suggest that, althought they are not easily allayed, limiting the duration of methadone maintenace from the outset of treatment may be an ameliorative factor.

Breast cancer treatment: evolving approaches but stable results.

This report describes the outcome of 530 women with breast cancer diagnosed from 1968 through 1983 and represents a demographic population rather than a referred selected one. The data represents the results of evolving breast cancer treatment approaches during the past 2 decades and is particularly useful as a measure of the total population denominator, free of selection factors that confound reports detailing a surgical, radiation, or chemotherapy experience. During the time interval reviewed, the standard treatment approach of the primary changed from radical mastectomy to biopsy and radiation therapy. Chemotherapy policy evolved from single agent treatment for relapse to multiple drug programs as adjuvant or for relapse. The major findings were: The 5-, 10-, and 15-year survival rates for the intervals 1972-75, 1976-79, and 1980-83 were slightly better than the earliest interval 1968-71, but with no statistically significant improvement. The frequency of favorable disease (Stages Tis, 1) increased from 16 to 31 percent during the interval but the mean age remained the same suggesting that patient education programs, availability of health insurance, or mammography may have lead to identifying patients with more favorable disease. Mastectomy has been replaced by breast conserving surgery and radiation as the most common treatment of the primary. Patients treated by surgery and biopsy/radiation had identical survival outcomes. It was not possible to detect improved survival that could be ascribed to the adoption of multiple agent chemotherapy but the magnitude of the effect is calculated to be on the order of 2% of the total patient population diagnosed. Death due to breast cancer decreases with time after diagnosis but is still 4% per year, 10 years after treatment. The findings suggest that progress has been made in detection, breast conservation, and palliation of symptoms in many subpopulations, but the end results for the total breast cancer population have remained stable during an era when the treatment approach evolved markedly.

The Trypanosoma brucei mitochondrial ATP synthase is developmentally regulated at the level of transcript stability.

The mitochondrial ATP synthase is developmentally regulated throughout the life cycle of the Trypanosoma brucei. The alpha and beta subunits of the F(1) moiety, and subunit 9 of the F(0) moiety of the T. brucei ATP synthase have been previously cloned and characterized. Here we have determined the chromosomal localization and developmental regulation of these three key subunits of the complex. Southern blot analysis indicates that all three of these genes are present as single copies in the T. brucei genome. Pulsed field gel analysis demonstrates that these genes are encoded in different chromosomes, and are thus not part of the same gene cluster. A comparison between the protein and steady state transcript levels for these subunits suggests that regulation of expression occurs predominantly posttranscriptionally. Comparison of mRNA stability for procyclic and bloodstream forms shows that the half life of the three transcripts is much shorter in bloodstream forms. The differences in transcript stability in the procyclic form for subunit 9 is greater than that for alpha and beta subunits, while the differences at the protein levels are comparable. These results suggest that there may be further posttranscriptional regulation of subunit 9.

Cloning and characterization of the subunits comprising the catalytic core of the Trypanosoma brucei mitochondrial ATP synthase.

The Trypanosoma brucei mitochondrial F(1)-ATPase has been previously isolated and characterized. It is composed of five subunits of molecular weights 55000, 42000, 32000, 22000, and 17000 [1]. We have identified the alpha and beta subunits of the T. brucei F(1)-ATPase by N-terminal sequence determination together with analysis of cDNA and genomic clones. The genes for both subunits are homologous to the same subunits from other organisms. They contain the Walker A and B boxes of homology and a putative mitochondrial import sequence. The isolated T. brucei alpha subunit is unusually small at 42 kDa. The alpha cDNA clone encodes a protein of predicted size 59 kDa with a mitochondrial import presequence at the N-terminus. The predicted size was confirmed by expression of a 59 kDa protein from the cDNA clone in vitro. These results suggest that the alpha subunit may have an unusually large mitochondrial presequence of 159 amino acids. In contrast, the estimated size of the native beta subunit (55 kDa) correlates well with the size predicted from the cDNA clone, 57 kDa, from which a 21 amino acid presequence has been removed in vivo. The size of the beta subunit was confirmed by expression in an in vitro and an Escherichia coli expression system. The purified recombinant beta subunit, like the native F(1)-ATPase, can be labeled by the photoaffinity nucleotide analogue 8-azido ATP. Binding of the 8-azido ATP probe is best competed by the natural substrate ATP, and is significantly reduced by pretreatment with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazide as has been shown with beta subunits of other organisms. The differential binding of this photoaffinity analogue was used to resolve the identities of the alpha and beta subunits of the ATP synthase from T. brucei. These results are in contrast to results previously obtained for a related trypanosomatid Crithidia fasciculata.

Subunit 9 of the mitochondrial ATP synthase of Trypanosoma brucei is nuclearly encoded and developmentally regulated.

We have previously shown that the mitochondrial ATP synthase is developmentally regulated through the life cycle of Trypanosoma brucei. The mechanism of this regulation is as yet unknown. We are currently examining regulation of expression of several key subunits of the ATP synthase to investigate this mechanism. In the work presented here, we have cloned, sequenced, and confirmed the identity of the ATPase subunit 9 homologue from T. brucei. The ATPase subunit 9 gene that we have identified from T. brucei has between 40 and 600% identity with subunit 9 from a variety of organisms. This gene possesses a putative mitochondrial import sequence at the N terminus of the encoded protein sequence. The protein expressed from this gene by in vitro transcription/translation comigrates with native protein isolated from inner mitochondrial membrane vesicles from T. brucei. We have shown that the cDNA identifies a copy of this gene in the nuclear genome, but does not identify a similar gene in kinetoplast DNA (kDNA) prepared from T. brucei. This gene does not show homology to any published sequence data from maxicircle DNA or edited maxicircle derived sequences. Steady state transcripts of a single size have been identified by Northern analysis and demonstrate significant developmental regulation through the T. brucei life cycle. Northern analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results show that the transcript is 10-14-fold higher in procyclic form than in early and late bloodstream forms.

Inhibition of M-current in cultured rat superior cervical ganglia by linopirdine: mechanism of action studies.

The mechanism involved in the blockade of M-current by linopirdine was studied in cultured rat superior cervical sympathetic ganglia using whole-cell patch clamp recording. The effects of modulators of intracellular signal transduction pathways on muscarine- or linopirdine-induced inhibition of M-current were compared. Intracellular addition of GDP-beta-S (500 microM) attenuated M-current block by muscarine (1 microM) but not that of linopirdine (10 microM). Intracellular dialysis of GTP-gamma)-S (100 microM) enhanced and prolonged muscarine-induced inhibition of M-current but had no effect on the activity of linopirdine. Intracellular BAPTA (20 mM) also inhibited the effects of muscarine without affecting those of linopirdine. Intracellular application of linopirdine had no effect on either basal M-current amplitude or the ability of linopirdine to block M-current when administered extracellularly. These results indicate that M-current inhibition by linopirdine is unlikely to be either G-protein-mediated or calcium-mediated or to involve an intracellular site of action and are, therefore, consistent with a direct block of the M-channel from its extracellular side.

Synthesis and antiarrhythmic and parasympatholytic properties of substituted phenols. 2. Amides.

Thirty amides patterned after the antiarrhythmic drug changrolin were synthesized and their antiarrhythmic and parasympatholytic activities were assessed. There was no correlation between antiarrhythmic and parasympatholytic activities. Several of the amides were found to be potent antiarrhythmic agents that possessed low parasympatholytic activity. All of the compounds appear to act by a class I mechanism.

Synthesis and antiarrhythmic and parasympatholytic properties of substituted phenols. 3. Modifications to the linkage region (region 3).

As part of a continuing program of systematically modifying the structure of the class I antiarrhythmic drug changrolin, we synthesized 15 analogues in which the linkage between the two aromatic regions was altered. High antiarrhythmic activity and low parasympatholytic activity was found when the linkage region, designated region 3, contained a carbonyl moiety, including ketones, amides, and ureas. Secondary amides were superior to tertiary amides, while amide reversal resulted in no change in activities. One compound in this series, 7, 2,6-bis(1-pyrrolidinyl-methyl)-4-benzamidophenol (ACC-9358), is undergoing preclinical evaluations.

Synthesis and antiarrhythmic and parasympatholytic properties of substituted phenols. 1. Heteroarylamine derivatives.

Twenty-four structural derivatives of the antiarrhythmic drug changrolin were synthesized and tested for antiarrhythmic and parasympatholytic activities. It was found that while the bis(pyrrolidinylmethyl)phenol pattern of changrolin appeared to be optimal in this series, a wide latitude existed for the heteroaryl substituent for maintaining good antiarrhythmic activity. Further, the antiarrhythmic and parasympatholytic activities tended to exhibit parallel changes.