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.

Plasma HMGB-1 and Nucleosome Concentrations in Horses with Colic and Healthy Horses.

BACKGROUND: Acute gastrointestinal disease occurs commonly in horses. Novel biomarkers might improve the understanding of SIRS and aid diagnosis and determination of prognosis. HYPOTHESES: Increased plasma concentrations of the biomarkers HMGB-1 and nucleosomes are associated with severity of gastrointestinal lesions in horses; concentrations of these biomarkers will be greater in horses with lesions more likely to cause SIRS; and will provide additional information compared with standard biomarkers fibrinogen and SAA. ANIMALS: Thirty horses with gastrointestinal disease, 22 healthy horses. METHODS: Prospective study. Plasma samples taken on admission were used for measurement of HMGB-1, nucleosomes, fibrinogen, and SAA. Values were compared between healthy horses and those with gastrointestinal disease, and between horses with gastrointestinal disease grouped by lesion type (inflammatory, strangulating, and nonstrangulating). Correlations between biomarkers were assessed. RESULTS: Plasma concentrations of all biomarkers were significantly higher in horses with gastrointestinal disease compared to healthy horses (P ≤ .001). HMGB-1 and nucleosomes were significantly higher in inflammatory and strangulating groups compared to healthy horses (3.5-fold and 5.4-fold increases, respectively, for HMGB-1 (P < .05) and 4.8-fold and 5.6-fold increases for nucleosomes (P < .05)), but concentrations in the group with nonstrangulating disease did not differ from healthy horses. There was significant correlation between HMGB-1 and nucleosomes (Spearman's r = 0.623; P < .001), and fibrinogen and SAA (Spearman's r = 0.801; P < .001) but not between other biomarkers. CONCLUSIONS AND CLINICAL IMPORTANCE: High mobility group box-1 and nucleosomes might have use as biomarkers for horses with gastrointestinal disease. Further studies are required to determine kinetics and prognostic value of serial measurements of these biomarkers in horses.

Plasma Peak and Trough Gentamicin Concentrations in Hospitalized Horses Receiving Intravenously Administered Gentamicin.

BACKGROUND: Gentamicin is an aminoglycoside antimicrobial commonly used in horses at 6.6 mg/kg IV once daily. Therapeutic drug monitoring (TDM) can confirm desired peak concentration is reached for common bacterial isolates, and detect toxicosis associated with high trough values. OBJECTIVES: Determine the relationship between gentamicin dose and plasma concentration in hospitalized horses, and identify a starting dose range to achieve peaks > 32 µg/mL. ANIMALS: Sixty-five horses (2002-2010) receiving once-daily gentamicin with TDM performed (N = 99 sets). METHODS: Retrospective study. Data from hospitalized horses including weight, dose, plasma peak, and trough gentamicin concentration, creatinine concentrations and presence of focal or systemic disease were collected from medical records. Peak concentrations measured 25-35 minutes after administration were included (N = 77). Data were divided into low (<7.7 mg/kg), medium (7.7-9.7 mg/kg) and high (>9.7 mg/kg) dose groups, and were grouped by the horse having focal or systemic disease. RESULTS: Peak concentrations resulting from doses ≥7.7 mg/kg were 5.74 µg/mL (SE 2.1 µg/mL) greater than peaks from doses <7.7 mg/kg (P = .007). Peak concentrations was 3.6 times more likely to be >32 µg/mL if dose was ≥7.7 mg/kg (P = .04). There were no significant effects of dose on trough or creatinine concentration. At a given dose, horses with focal disease had higher peaks than those with systemic disease (P = .039). CONCLUSIONS AND CLINICAL IMPORTANCE: These data suggest gentamicin dosage should be individually determined in horses using TDM, but support an initial once-daily dose of 7.7-9.7 mg/kg IV to achieve peaks >32 µg/mL and trough concentrations <2 µg/mL. Further studies evaluating the safety of doses >6.6 mg/kg are required.

Anti-inflammatory effects of four potential anti-endotoxaemic drugs assessed in vitro using equine whole blood assays.

Despite the severity and common occurrence of equine endotoxaemia, the available anti-endotoxic treatments do not effectively target key inflammatory mechanisms such as leucocyte activation and cytokine production. In this study, four compounds with potential anti-endotoxic effects, namely rolipram, azithromycin, ethyl pyruvate and metformin, were investigated in vitro using equine whole blood stimulated with bacterial lipopolysaccharide. TNF-α and IL-1ß production were measured in plasma. Rolipram was the most potent inhibitor of cytokine production (IC50 0.84 and 4.68 µm for TNF-α and IL-1ß, respectively) with almost complete inhibition of TNF-α, but inhibited IL-1ß by only 39.46%. Azithromycin produced almost complete inhibition of both cytokines, but tended to be less potent than rolipram (IC50 10.66 and 17.4 µm for TNF-α and IL-1ß, respectively). Metformin inhibited TNF-α production with similar potency to rolipram and azithromycin (IC50 3.35 µm) but showed significantly lower efficacy (45.93%; P < 0.05), and had no inhibitory effect on IL-1ß. Ethyl pyruvate was the least potent (IC50 68.35 µm and >10 mm for TNF-α and IL-1ß production, respectively). Further work is required to investigate whether these or related compounds may have potential use in the treatment of equine endotoxaemia in vivo.