Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of ...cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death.
Both acamprosate and naltrexone have demonstrated clinical utility in reducing relapse to alcohol use in recovering alcoholics. The present experiments examined the effects of acamprosate and ...naltrexone, either alone or in combination, on basal ethanol consumption in a limited-access model with the use of outbred Wistar rats. Naltrexone, 0.1 mg/kg, significantly reduced ethanol consumption as previously reported. Acamprosate, 50 mg/kg, did not significantly reduce ethanol consumption when administered alone and provided no evidence of additive or synergistic effects when combined with naltrexone. Acamprosate, 200 mg/kg, produced a modest reduction in ethanol consumption when administered alone but no evidence of additive or synergistic effects when combined with naltrexone. From these findings, it is suggested that a combination approach of these drugs may not be any more effective than monotherapy.
The use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality ...compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals.
A set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed.
None of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR-145 correlated with nadir CD4+ T cell count.
No associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection.
It has frequently been reported that a high percentage of individuals, identified as either alcohol- or cocaine-dependent, concurrently abuse both drugs. The experiments reported here represent a ...continuing effort to develop an animal model to predict the effects of a potential pharmacotherapeutic agent on concurrently available oral ethanol and cocaine. These experiments utilized drinkometer circuitry to assess the effects of gamma-vinyl-GABA (GVG), a γ-aminobutyric acid (GABA) transaminase inhibitor, on the consumption and temporal pattern of responses for orally self-administered ethanol and cocaine. The results of these experiments showed that GVG, at doses of 100, 200 and 300 mg/kg, reduced both ethanol and cocaine consumption in a dose-related manner. When compared to vehicle, GVG at all doses significantly reduced ethanol consumption while consumption of cocaine was significantly reduced only at 300 mg/kg. This is consistent with data showing that GVG reduces consumption of these drugs when administered alone and data showing that GVG is more potent in reducing ethanol-induced compared to cocaine-induced extracellular dopamine in the nucleus accumbens. Analysis of the temporal pattern of drinking across the session suggests that GVG's effects are due to a disruption of the reinforcing properties of ethanol and cocaine rather than a more general reduction in motor behavior. These data suggest that GVG has potential for clinical use in populations that abuse either alcohol or cocaine alone or in combination.
Background:
Small studies have recently suggested that there are specific plasma metabolic signatures in chronic obstructive pulmonary disease (COPD), but there have been no large comprehensive study ...of metabolomic signatures in COPD that also integrate genetic variants.
Materials and Methods:
Fresh frozen plasma from 957 non-Hispanic white subjects in COPDGene was used to quantify 995 metabolites with Metabolon's global metabolomics platform. Metabolite associations with five COPD phenotypes (chronic bronchitis, exacerbation frequency, percent emphysema, post-bronchodilator forced expiratory volume at one second FEV
1
/forced vital capacity FVC, and FEV
1
percent predicted) were assessed. A metabolome-wide association study was performed to find genetic associations with metabolite levels. Significantly associated single-nucleotide polymorphisms were tested for replication with independent metabolomic platforms and independent cohorts. COPD phenotype-driven modules were identified in network analysis integrated with genetic associations to assess gene-metabolite-phenotype interactions.
Results:
Of metabolites tested, 147 (14.8%) were significantly associated with at least 1 COPD phenotype. Associations with airflow obstruction were enriched for diacylglycerols and branched chain amino acids. Genetic associations were observed with 109 (11%) metabolites, 72 (66%) of which replicated in an independent cohort. For 20 metabolites, more than 20% of variance was explained by genetics. A sparse network of COPD phenotype-driven modules was identified, often containing metabolites missed in previous testing. Of the 26 COPD phenotype-driven modules, 6 contained metabolites with significant met-QTLs, although little module variance was explained by genetics.
Conclusion:
A dysregulation of systemic metabolism was predominantly found in COPD phenotypes characterized by airflow obstruction, where we identified robust heritable effects on individual metabolite abundances. However, network analysis, which increased the statistical power to detect associations missed previously in classic regression analyses, revealed that the genetic influence on COPD phenotype-driven metabolomic modules was modest when compared with clinical and environmental factors.
Differing antibiotic regimens can influence both survival and the inflammatory state in sepsis. We investigated whether the addition and/or type of antimicrobial agent could effect mortality in a ...murine model of Pseudomonas aeruginosa pneumonia-induced sepsis and if antibiotics altered systemic levels of cytokines. FVB/N mice were subjected to intratracheal injection of pathogenic bacteria and were given gentamicin, imipenem, or 0.9% NaCl 2 h after surgery, which continued every 12 h for a total of six doses. Survival at 7 days (n = 24 in each group) was 100% for mice given gentamicin, 88% for mice given imipenem, and 8% for sham mice treated with 0.9% NaCl (P < 0.0001). Systemic interleukin (IL) 6 levels were assayed 6 h postoperatively on all mice to see if they were predictive of outcome. Plasma IL-6 levels above 3,600 pg/mL were associated with a 100% mortality, levels under 1,200 pg/mL were associated with a 100% survival, and levels between 1,200 and 3,600 pg/mL had no utility in predicting mortality. In a separate experiment, mice were sacrificed at 3, 6, 12 or 24 h after instillation of P. aeruginosa and were assayed for levels of TNF-alpha, IL-6, IL-10, and IL-12. Significant alterations in the proinflammatory cytokines TNF-alpha and IL-6 were present at all time points except 3 h between mice treated with antibiotics and sham controls. In contrast, statistically significant differences in the anti-inflammatory cytokine IL-10 were present between the groups only at 6 h, and levels of IL-12 were similar at all time points. These results indicate that both gentamicin and imipenem increase survival at least 10-fold in a model of pneumonia-induced monomicrobial sepsis, and this is predominantly associated with a down-regulation of proinflammatory cytokines.
Forage is an application which uses two neural networks for detecting single nucleotide polymorphisms (SNPs). Potential SNP candidates are identified in multiple alignments. Each candidate is then ...represented by a vector of features, which is classified as SNP or monomorphic by the networks. A validated dataset of SNPs was constructed from experimentally verified SNP data and used for network training and method evalutation. Availability: The package is available at biobase.biotech.kth.se/forage/ Contact: fredrik@biotech.kth.se Supplementary information: Additional results and method description can be found at biobase.biotech.kth.se/forage/
Despite having dysregulated iron metabolism, critically ill patients may receive exogenous iron for the treatment of anemia. Iron is associated with increased tissue apoptosis and may facilitate ...bacterial growth. We hypothesized that exogenous iron administration given after the onset of sepsis would lead to increased mortality rate. To discriminate between elevated cell death and bacterial overgrowth as potential mediators of mortality, we examined gut epithelial and lymphocyte apoptosis and systemic bacterial counts in animals given iron supplementation after the onset of sepsis.
Prospective, randomized, controlled study.
Animal laboratory in a university medical center.
Male C57BL/6 mice, 6-10 wks old.
C57BL/6 mice were subjected to cecal ligation and puncture (CLP), a well-accepted model of intra-abdominal sepsis, followed by daily subcutaneous injections of either 1 mL of iron dextran (5 mg/mL) or 0.9% NaCl for a total of five doses. Animals (n = 78) were followed for survival for 8 days. Separate cohorts (n = 76) were killed 24 or 48 hrs after cecal ligation and puncture or sham laparotomy and were assayed for gut epithelial and splenic apoptosis as well as for quantitative blood cultures.
Eight-day survival was 7% in animals that received iron and 26% in mice that received 0.9% NaCl (p < .005). Iron supplementation after cecal ligation and puncture increased apoptosis by both active caspase 3 and hematoxylin and eosin staining in both the intestinal epithelium and spleen at 24 hrs (p < .05). Iron supplementation after sham laparotomy did not cause mortality or elevated apoptosis. Quantitative blood cultures revealed no detectable differences between septic animals that received iron and those that received 0.9% NaCl.
High-dose iron supplementation with iron dextran after the onset of sepsis significantly increases mortality rate in this animal model. Iron-induced mortality may be mediated by an increase in gut epithelial and splenic apoptosis, whereas severity of bacteremia does not appear to play a causative role.
We recently reported that 6-β naltrexol, the major metabolite of naltrexone in humans, reduced ethanol consumption in rats. Two new experiments were designed to compare 6-β naltrexol and naltrexone ...across three dose levels on an ethanol or sucrose baseline using a limited-access procedure in Wistar rats. The results of Experiment 1 showed that both 6-β naltrexol and naltrexone reduced ethanol consumption across a range of doses. An in vivo assay showed that naltrexone was approximately 25 times more potent than 6-β naltrexol at comparable ED
50 doses. In addition, there was no indication of systematic development of tolerance to the effect of either drug across the 4 days of drug administration. In Experiment 2, both 6-β naltrexol and naltrexone reduced the consumption of a sucrose solution using a limited-access procedure. The implications of these data for the development of pharmacotherapeutic agents capable of reducing drinking in recovering alcoholics are discussed.
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