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BACKGROUND: Lethal B. anthracis infection produces high proinflammatory peptidoglycan (PGN) burdens in hosts. We investigated whether the lethality and inflammation anthrax PGN can produce are related. METHODS: At 6 h before and the start of 24 h anthrax PGN infusions, rats (n = 198) were treated with diluent (controls) or one of three IV-doses of either hydrocortisone (125, 12.5 or 1.25 mg/kg) or TNF-soluble receptor (TNFsr; 2000, 1000 or 333 µg/kg), non-selective and selective anti-inflammatory agents, respectively. RESULTS: Compared to controls, hydrocortisone 125 and 12.5 mg/kg each decreased 7-day lethality (p ≤ 0.004). Hydrocortisone 125 mg/kg decreased IL-1ß, IL-6, TNFα, MCP, MIP-1α, MIP-2, RANTES and nitric oxide (NO) blood levels at 4 and 24 h after starting PGN (except MCP at 24 h). Each decrease was significant at 4 h (except MIP-1α that was significant at 24 h) (p ≤ 0.05). Similarly, hydrocortisone 12.5 mg/kg decreased each measure at 4, 24 and 48 h (except TNFα at 24 h and MIP-1α at 24 and 48 h and NO at 48 h). Decreases were significant for IL-6 and NO at 4 h and RANTES at 48 h (p ≤ 0.05). Hydrocortisone 1.25 mg/kg had non-significant effects. Each TNFsr dose decreased lethality but non-significantly. However, when doses were analyzed together, TNFsr decreased lethality in a potential trend (p = 0.16) and IL-6 and NO significantly at 4 h (p = 0.05). CONCLUSIONS: Peptidoglycan-stimulated host inflammation may contribute to B. anthracis lethality.
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Although lethal toxin (LT) and edema toxin (ET) contribute to lethality during Bacillus anthracis infection, whether they increase vascular permeability and the extravascular fluid accumulation characterizing this infection is unclear. We employed an isolated perfused Sprague-Dawley rat lung model to investigate LT and ET effects on pulmonary vascular permeability. Lungs (n ≥ 6 per experimental group) were isolated, ventilated, suspended from a force transducer, and perfused. Lung weight and pulmonary artery (Ppa) and left atrial pressures were measured over 4 h, after which pulmonary capillary filtration coefficients (Kf.c) and lung wet-to-dry weight ratios (W/D) were determined. When compared with controls, LT increased Ppa over 4 h and Kf.c and W/D at 4 h (P < 0.0001). ET decreased Ppa in a significant trend (P = 0.09) but did not significantly alter Kf.c or W/D (P ≥ 0.29). Edema toxin actually blocked LT increases in Ppa but not LT increases in Kf.c and W/D. When Ppa was maintained at control levels, LT still increased Kf.c and W/D (P ≤ 0.004). Increasing the dose of each toxin five times significantly increased and a toxin-directed monoclonal antibody decreased the effects of each toxin (P ≤ 0.05). Two rho-kinase inhibitors (GSK269962 and Y27632) decreased LT increases in Ppa (P ≤ 0.02) but actually increased Kf.c and W/D in LT and control lungs (P ≤ 0.05). A vascular endothelial growth factor receptor inhibitor (ZM323881) had no significant effect (P ≥ 0.63) with LT. Thus, LT but not ET can increase pulmonary vascular permeability independent of increased Ppa and could contribute to pulmonary fluid accumulation during anthrax infection. However, pulmonary vascular dilation with ET could disrupt protective hypoxic vasoconstriction. NEW & NOTEWORTHY The most important findings from the present study are that Bacillus anthracis lethal toxin increases pulmonary artery pressure and pulmonary permeability independently in the isolated rat lung, whereas edema toxin decreases the former and does not increase permeability. Each effect could be a basis for organ dysfunction in patients with this lethal infection. These findings further support the need for adjunctive therapies that limit the effects of both toxins during infection.
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Antígenos Bacterianos/toxicidad , Presión Arterial/efectos de los fármacos , Toxinas Bacterianas/toxicidad , Permeabilidad Capilar/efectos de los fármacos , Pulmón/irrigación sanguínea , Arteria Pulmonar/efectos de los fármacos , Edema Pulmonar/inducido químicamente , Animales , AMP Cíclico/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Masculino , Perfusión , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiopatología , Edema Pulmonar/metabolismo , Edema Pulmonar/fisiopatología , Ratas Endogámicas BN , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Ratas Wistar , Albúmina Sérica/metabolismoRESUMEN
BACKGROUND: Although anthrax immune globulin (AIG) improved survival in antibiotic-treated Bacillus anthracis-challenged animal models, whether it adds to the benefit of conventional hemodynamic support for B. anthracis toxin-associated shock is unknown. METHODS: We therefore tested AIG in sedated, mechanically ventilated canines challenged with 24-h B. anthracis lethal and edema toxin infusions and supported for 96 h with a previously demonstrated protective regimen of titrated normal saline and norepinephrine. RESULTS: Compared to controls, proportional survival (%) was increased with AIG treatment started 4 h before (33 vs. 100%, n = 6 each) or 2 h (17 vs. 86%, n = 6 and 7 respectively) or 5 h (0 vs. 67%, n = 3 each) after the start of toxin (p ≤ 0.05) and overall [3 survivors of 15 controls (20%) vs. 14 of 16 AIG animals (88%); p = 0.006]. Averaged across treatment times, AIG increased blood pressure at 48 h and decreased norepinephrine requirements at 72 h (p ≤ 0.02), increased left ventricular ejection fraction at 48 and 72 h (p ≤ 0.02), and increased urine output and decreased net fluid balance at 72 and 96 h (p ≤ 0.04). AIG also reduced acidosis and renal and hepatic injury markers between 24 and 96 h. CONCLUSIONS: These findings further support AIG's potential benefit for patients with B. anthracis infection and developing toxin-associated shock.
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Although edema toxin (ETx) and lethal toxin (LTx) contribute to Bacillus anthracis shock and lethality, the mechanisms underlying their cardiovascular effects are unclear. We have previously shown that ETx but not LTx inhibited phenylephrine-stimulated contraction of aortic rings prepared from healthy rats and that adefovir, a selective inhibitor of ETx cAMP production, blocked this effect. Here, we examined arterial function in rats that received 24-h ETx or LTx infusions. Compared with control rats, ETx reduced mean arterial pressure (MAP) and survival over 48 h (P ≤ 0.0003) and increased plasma cAMP at 4, 24, and 48 h (P < 0.0001) and nitric oxide (NO) at 24 and 48 h (P ≤ 0.01). Compared with control animals, at 24- and 48-h phenylephrine stimulation of aortic rings from ETx animals produced decreased maximal contractile force (MCF; P = 0.05 and 0.006) and in vivo phenylephrine infusion in ETx animals produced decreased proportional increases in MAP (P < 0.0001 and P = 0.05). In ETx-treated animals, compared with placebo-treated animals, adefovir treatment prevented all lethality (P = 0.01), increased MAP (P ≤ 0.0001), decreased plasma and aortic tissue cAMP at 24 and 48 h, respectively (P ≤ 0.03), and plasma NO at both times (P ≤ 0.004), and increased phenylephrine-stimulated increases in MCF in aortic rings and MAP in vivo at 48 h (P = 0.02). LTx decreased MAP and survival also, but it did not alter the response to phenylephrine of MCF in aortic rings prepared from LTx animals or of MAP in vivo. In conclusion, in rats, hypotension and lethality are associated with reduced arterial contractile function with ETx but not LTx and adefovir improves ETx-induced hypotension and lethality.NEW & NOTEWORTHY The most important aspects of the present study are the findings that 1) in vivo challenge with anthrax edema but not lethal toxin depresses arterial contractile function measured both ex vivo and in vivo and 2) adefovir inhibits the effects of edema toxin on arterial hypotension and improves survival with lethal dose of edema toxin challenge.
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Adenina/análogos & derivados , Antígenos Bacterianos/toxicidad , Arterias/efectos de los fármacos , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/toxicidad , Organofosfonatos/farmacología , Fenilefrina/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Choque/inducido químicamente , Choque/tratamiento farmacológico , Vasoconstrictores/farmacología , Adenina/farmacología , Animales , Aorta/efectos de los fármacos , Presión Arterial , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Choque/fisiopatologíaRESUMEN
Bacillus anthracis edema toxin (ET) consists of protective antigen (PA), necessary for host cell toxin uptake, and edema factor (EF), the toxic moiety which increases host cell cyclic AMP (cAMP). Since vasopressin stimulates renal water and sodium reabsorption via increased tubular cell cAMP levels, we hypothesized the ET would also do so. To test this hypothesis, we employed an isolated perfused rat kidney model. Kidneys were isolated and perfused with modified Krebs-Henseleit buffer. Perfusate and urine samples were obtained at baseline and every 10 min over 150 min following the addition of challenges with or without treatments to the perfusate. In kidneys perfused under constant flow or constant pressure, compared to PA challenge (n = 14 or 15 kidneys, respectively), ET (13 or 15 kidneys, respectively) progressively increased urine cAMP levels, water and sodium reabsorption, and urine osmolality and decreased urine output (P ≤ 0.04, except for sodium reabsorption under constant pressure [P = 0.17]). In ET-challenged kidneys, compared to placebo treatment, adefovir, an EF inhibitor, decreased urine cAMP levels, water and sodium reabsorption, and urine osmolality and increased urine output, while raxibacumab, a PA-directed monoclonal antibody (MAb), decreased urine cAMP levels, free water reabsorption, and urine osmolality and increased urine output (P ≤ 0.03 except for urine output with raxibacumab [P = 0.17]). Upon immunohistochemistry, aquaporin 2 was concentrated along the apical membrane of tubular cells with ET but not PA, and urine aquaporin 2 levels were higher with ET (5.52 ± 1.06 ng/ml versus 1.51 ± 0.44 ng/ml [means ± standard errors of the means {SEM}; P = 0.0001). Edema toxin has renal effects that could contribute to extravascular fluid collection characterizing anthrax infection clinically.
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Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Riñón/efectos de los fármacos , Riñón/metabolismo , Sodio/metabolismo , Agua/metabolismo , Animales , Acuaporinas/análisis , AMP Cíclico/análisis , Inmunohistoquímica , Riñón/patología , Placebos/administración & dosificación , Ratas Sprague-DawleyRESUMEN
We showed previously that Bacillus anthracis edema toxin (ET), comprised of protective antigen (PA) and edema factor (EF), inhibits phenylephrine (PE)-induced contraction in rat aortic rings and these effects are diminished in endothelial-denuded rings. Therefore, employing rat aortic ring and in vivo models, we tested the hypothesis that nitric oxide (NO) contributes to ET's arterial effects. Compared with rings challenged with PA alone, ET (PA + EF) reduced PE-stimulated maximal contractile force (MCF) and increased the PE concentration producing 50% MCF (EC50) (P < 0.0001). Compared with placebo, l-nitro-arginine methyl-ester (l-NAME), an NO synthase (NOS) inhibitor, reduced ET's effects on MCF and EC50 in patterns that approached or were significant (P = 0.06 and 0.03, respectively). In animals challenged with 24-h ET infusions, l-NAME (0.5 or 1.0 mg·kg(-1)·h(-1)) coadministration increased survival to 17 of 28 animals (60.7%) compared with 4 of 27 (14.8%) given placebo (P = 0.01). Animals receiving l-NAME but no ET all survived. Compared with PBS challenge, ET increased NO levels at 24 h and l-NAME decreased these increases (P < 0.0001). ET infusion decreased mean arterial blood pressure (MAP) in placebo and l-NAME-treated animals (P < 0.0001) but l-NAME reduced decreases in MAP with ET from 9 to 24 h (P = 0.03 for the time interaction). S-methyl-l-thiocitrulline, a selective neuronal NOS inhibitor, had effects in rings and, at a high dose in vivo models, comparable to l-NAME, whereas N'-[3-(aminomethyl)benzyl]-acetimidamide, a selective inducible NOS inhibitor, did not. NO production contributes to ET's arterial relaxant, hypotensive, and lethal effects in the rat.
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Antígenos Bacterianos/farmacología , Aorta/efectos de los fármacos , Toxinas Bacterianas/farmacología , Hipotensión/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/metabolismo , Fenilefrina/farmacología , Vasoconstrictores/farmacología , Animales , Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Citrulina/análogos & derivados , Citrulina/farmacología , Inhibidores Enzimáticos/farmacología , Hipotensión/inducido químicamente , Hipotensión/mortalidad , Técnicas In Vitro , Masculino , Mortalidad , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Tasa de Supervivencia , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
BACKGROUND: Lethal and edema toxin contribute to shock and lethality with Bacillus anthracis. We showed previously in a 96-h sedated canine model that raxibacumab, a monoclonal antibody against protective antigen, augmented hemodynamic support (HS) and improved survival with lethal toxin challenge. Here we study raxibacumab further. Using this model, we have now studied raxibacumab with 24 h edema toxin challenges (Study 1), and lethal and edema toxin challenges together (Study 2). METHODS: Using our canine model, we have now studied raxibacumab with 24h edema toxin challenges (Study-1), and lethal and edema toxin challenges together (Study-2). RESULTS: In Study 1, compared to no treatment, HS (titrated fluid and norepinephrine) increased mean arterial blood pressure (MAP, p ≤ 0.05) but not survival [0 of 10 (0/10) animals survived in each group] or median survival time [43.8 h (range 16.8 to 80.3) vs. 45.2 h (21.0 to 57.1)]. Compared to HS, HS with raxibacumab treatment at or 6 h after the beginning of edema toxin increased MAP and survival rate (6/7 and 7/8, respectively) and time [96.0 h (39.5 to 96.0) and 96.0 h (89.5 to 96.0), respectively]; (p ≤ 0.05). HS with raxibacumab at 12 h increased MAP (p ≤ 0.05) but not survival [1/5; 55.3 h (12.6 to 96.0)]. In Study-2, survival rate and time increased with HS and raxibacumab at 0 h (4/4) or 6 h after (3/3) beginning lethal and edema toxin compared to HS [0/5; 71.5 h (65 to 93)] (p = 0.01 averaged over raxibacumab groups). CONCLUSIONS: Raxibacumab augments HS and improves survival during shock with lethal and edema toxin.
RESUMEN
Although direct myocardial depression has been implicated in the lethal effects of Bacillus anthracis lethal toxin (LT), in hearts isolated from healthy rats and perfused under constant pressure, neither LT or edema toxin (ET) in typically lethal concentrations depressed myocardial function. In the present study, we challenged rats with LT and ET and performed in vivo and ex vivo heart measures. Sprague-Dawley rats infused over 24 h with LT (n = 94), ET (n = 99), or diluent (controls; n = 50) were studied at 8, 24, or 48 h. Compared with control rats (all survived), survival rates with LT (56.1%) and ET (37.3%) were reduced (P < 0.0001) similarly (P = 0.66 for LT vs. ET). LT decreased mean arterial blood pressure from 12 to 20 h (P ≤ 0.05), whereas ET decreased it progressively throughout (P < 0.05). On echocardiography, LT decreased left ventricular (LV) ejection fraction at 8 and 48 h but increased it at 24 h and decreased cardiac output (P ≤ 0.05 for the time interaction or averaged over time). ET decreased systolic and diastolic volumes and increased LV ejection fraction at 24 h (P ≤ 0.05). In isolated hearts perfused for 120 min under constant pressure, LT did not significantly alter LV systolic or developed pressures at any time point, whereas ET decreased both of these at 24 h (P < 0.0001 initially). ET but not LT progressively increased plasma creatine phosphokinase and cardiac troponin levels (P < 0.05). In conclusion, despite echocardiographic changes, in vivo lethal LT challenge did not produce evidence of myocardial depression in isolated rat hearts. While lethal ET challenge did depress isolated heart function, this may have resulted from prior hypotension and ischemia.
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Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Cardiopatías/inducido químicamente , Corazón/efectos de los fármacos , Función Ventricular Izquierda/efectos de los fármacos , Animales , Biomarcadores/sangre , Presión Sanguínea/efectos de los fármacos , Creatina Quinasa/sangre , Corazón/fisiopatología , Cardiopatías/sangre , Cardiopatías/diagnóstico por imagen , Cardiopatías/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Perfusión , Ratas Sprague-Dawley , Volumen Sistólico/efectos de los fármacos , Factores de Tiempo , Troponina I/sangre , Ultrasonografía , Presión Ventricular/efectos de los fármacosRESUMEN
The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.
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Recolección de Muestras de Sangre/métodos , Arterias Carótidas , Cateterismo/métodos , Venas Yugulares , Animales , Cateterismo Venoso Central/métodos , Hemodinámica , RatasRESUMEN
INTRODUCTION: One proposed benefit of probiotic therapy is that probiotic bacterial cell-wall binding to intestinal cell pathogen-recognition receptors activates protective innate immunity. However, in critically ill patients, intestinal epithelium disruption by shock or other insults may compromise this compartmentalized response and cause systemic bacteria and cell-wall translocation. The effects of intravascular introduction of probiotic bacterial cell wall are unclear. METHODS: We investigated 24-hour infusions of purified cell wall from Lactobacillus gasseri ATC33323 (L. gasseri), a probiotic bacterium, in Sprague-Dawley rats (n = 49). RESULTS: Increasing cell-wall doses (0 (control), 10, 20, 40, 80, or 160 mg/kg over 24 hours) produced dose-ordered decreases in survival measured after 168 hours (11 survivors/11 total (100%), seven of seven (100%), seven of seven (100%), six of eight (75%), five of eight (63%), and one of nine (11%), respectively, P < 0.0001). The L. gasseri cell wall was equally or more lethal than Staphylococcus aureus cell wall, which was previously studied (100% to 88% survival with the same increasing doses). During challenge, compared with controls, L. gasseri cell wall produced increases in blood IL-1ß, IL-10, tumor necrosis factor-α, migratory inhibitory protein-1α, monocyte chemotactic protein-1, and nitric oxide, and decreases in neutrophils, lymphocytes, and platelets that were greater with higher versus lower doses (P ≤ 0.05). Medium-dose cell wall (40 and 80 mg/kg combined) progressively decreased blood pressure and increased heart rate, and all doses increased lactate, hepatic transaminases, and creatinine phosphokinase (P ≤ 0.05). CONCLUSION: Although L. gasseri, like other probiotic bacteria, is considered safe, its cell wall can stimulate the maladaptive inflammatory response associated with pathogenic bacteria. Such effects deserve study, especially regarding critically ill patients.
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Pared Celular , Modelos Animales de Enfermedad , Mediadores de Inflamación/sangre , Lactobacillus , Probióticos/toxicidad , Síndrome de Respuesta Inflamatoria Sistémica/sangre , Síndrome de Respuesta Inflamatoria Sistémica/mortalidad , Animales , Inflamación/sangre , Inflamación/inducido químicamente , Inflamación/mortalidad , Lactobacillus/aislamiento & purificación , Probióticos/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Tasa de Supervivencia/tendencias , Síndrome de Respuesta Inflamatoria Sistémica/inducido químicamenteRESUMEN
BACKGROUND: Disseminated intravascular coagulation (DIC) appears to be important in the pathogenesis of Bacillus anthracis infection, but its causes are unclear. Although lethal toxin (LT) and edema toxin (ET) could contribute, B. anthracis cell wall peptidoglycan (PGN), not the toxins, stimulates inflammatory responses associated with DIC. METHODS AND RESULTS: To better understand the pathogenesis of DIC during anthrax, we compared the effects of 24-hour infusions of PGN, LT, ET, or diluent (control) on coagulation measures 6, 24, or 48 hours after infusion initiation in 135 rats. No control recipient died. Lethality rates (approximately 30%) did not differ among PGN, LT, and ET recipients (P = .78). Thirty-three of 35 deaths (94%) occurred between 6 and 24 hours after the start of challenge. Among challenge components, PGN most consistently altered coagulation measures. Compared with control at 6 hours, PGN decreased platelet and fibrinogen levels and increased prothrombin and activated partial thromboplastin times and tissue factor, tissue factor pathway inhibitor, protein C, plasminogen activator inhibitor (PAI), and thrombin-antithrombin complex levels, whereas LT and ET only decreased the fibrinogen level or increased the PAI level (P ≤ .05). Nearly all effects associated with PGN infusion significantly differed from changes associated with toxin infusion (P ≤ .05 for all comparisons except for PAI level). CONCLUSION: DIC during B. anthracis infection may be related more to components such as PGN than to LT or ET.
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Carbunco/sangre , Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Pared Celular/química , Coagulación Intravascular Diseminada/sangre , Peptidoglicano/toxicidad , Animales , Carbunco/patología , Antitrombina III , Bacillus anthracis , Coagulación Sanguínea , Coagulación Intravascular Diseminada/microbiología , Fibrinógeno/metabolismo , Óxido Nítrico/sangre , Tiempo de Tromboplastina Parcial , Péptido Hidrolasas/sangre , Inactivadores Plasminogénicos/sangre , Proteína C/metabolismo , Protrombina/metabolismo , Ratas , Ratas Sprague-Dawley , Tromboplastina/metabolismoRESUMEN
B. anthracis edema toxin (ET) and lethal toxin (LT) are each composed of protective antigen (PA), necessary for toxin uptake by host cells, and their respective toxic moieties, edema factor (EF) and lethal factor (LF). Although both toxins likely contribute to shock during infection, their mechanisms are unclear. To test whether ET and LT produce arterial relaxation, their effects on phenylephrine (PE)-stimulated contraction in a Sprague-Dawley rat aortic ring model were measured. Rings were prepared and connected to pressure transducers. Their viability was confirmed, and peak contraction with 60 mM KCl was determined. Compared with PA pretreatment (control, 60 min), ET pretreatment at concentrations similar to those noted in vivo decreased the mean (±SE) maximum contractile force (MCF; percent peak contraction) in rings generated during stimulation with increasing PE concentrations (96.2 ± 7.0 vs. 57.3 ± 9.1) and increased the estimated PE concentration producing half the MCF (EC50; 10(-7) M, 1.1 ± 0.3 vs. 3.7 ± 0.8, P ≤ 0.002). ET inhibition with PA-directed monoclonal antibodies, selective EF inhibition with adefovir, or removal of the ring endothelium inhibited the effects of ET on MCF and EC50 (P ≤ 0.02). Consistent with its adenyl cyclase activity, ET increased tissue cAMP in endothelium-intact but not endothelium-denuded rings (P < 0.0001 and 0.25, respectively). LT pretreatment, even in high concentrations, did not significantly decrease MCF or increase EC50 (all P > 0.05). In rings precontracted with PE compared with posttreatment with PA (90 min), ET posttreatment produced progressive reductions in contractile force and increases in relaxation in endothelium-intact rings (P < 0.0001) but not endothelium-denuded rings (P = 0.51). Thus, ET may contribute to shock by producing arterial relaxation.
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Antígenos Bacterianos/farmacología , Aorta/efectos de los fármacos , Toxinas Bacterianas/farmacología , AMP Cíclico/metabolismo , Fenilefrina/farmacología , Sistemas de Mensajero Secundario/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Aorta/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Organofosfonatos/farmacología , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
BACKGROUND: Tumor necrosis factor (TNF) antagonists [e.g., TNF soluble receptor (TNFsr)] improved survival in preclinical but not clinical sepsis trials. However fluid support-itself beneficial-is standard clinically but rarely employed in preclinical sepsis models. We hypothesized that these therapies may not have additive benefit. METHODS AND RESULTS: Antibiotic-treated rats (n = 156) were randomized to intratracheal or intravenous Escherichia coli challenges (>LD50) and either placebo or TNFsr and 24 h fluid treatments alone or together. The survival effects of these therapies did not differ significantly comparing challenge routes. When averaged across route, while TNFsr or fluid alone decreased the hazard ratio of death significantly [ln ± standard error (SE): -0.65 ± 0.30 and -0.62 ± 0.30, respectively, p ≤ 0.05], together they did not (p = 0.16). Furthermore, the observed effect of TNFsr and fluid together on reducing the hazard ratio was significantly less than estimated (-0.37 ± 0.29 versus -1.27 ± 0.43, respectively, p = 0.027) based on TNFsr and fluid alone. While each treatment increased central venous pressure at 6 and 24 h, the observed effects of the combination were also less than estimated ones (p ≤ 0.0005). CONCLUSIONS: The individual survival benefits of TNFsr and fluids were not additive in this rat sepsis model. Investigating new sepsis therapies together with conventional ones during preclinical testing may be informative.
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Fluidoterapia , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Sepsis/mortalidad , Sepsis/terapia , Animales , Terapia Combinada , Masculino , Ratas , Ratas Sprague-Dawley , Tasa de SupervivenciaRESUMEN
INTRODUCTION: If thrombosis contributes to sepsis, heparin titrated using activated partial thromboplastin times may be efficacious. We investigated heparin in preclinical models. METHODS AND MAIN RESULTS: In unchallenged mice (n = 107), heparin at 100, 500, or 2500 units/kg produced activated partial thromboplastin time levels less than, within, or greater than a prespecified therapeutic range (1.5-2.5 times control), respectively. In animals (n = 142) administered intratracheal Escherichia coli challenge, compared to placebo treatment, heparin at 100, 500, or 2500 units/kg were associated with dose dependent increases in the hazard ratios of death (hazard ratio [95% confidence interval]: 1.08 [0.66, 1.76]; 1.34 [0.80, 2.24]; 3.02 [1.49, 6.10], respectively) (p = .001 for the dose effect). Compared to normal saline challenge, E. coli without heparin (i.e., with placebo) increased the activated partial thromboplastin time (p = .002) close to the therapeutic range. While heparin at 100 and 500 units/kg with E. coli further increased activated partial thromboplastin time (p < .0001 vs. placebo) within or above the therapeutic range, respectively, these did not decrease inflammatory cytokines or lung injury. In metaregression analysis of published preclinical studies, heparin improved survival with lipopolysaccharide (n = 23, p < .0001) or surgically induced infection (n = 14, p < .0001) but not monobacterial (n = 7, p = .29) challenges. CONCLUSION: Coagulopathy with sepsis or other variables, such as type of infectious source, may influence the efficacy of heparin therapy for sepsis.
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Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli , Heparina/administración & dosificación , Sepsis/tratamiento farmacológico , Sepsis/mortalidad , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Infecciones por Escherichia coli/mortalidad , Femenino , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Tiempo de Tromboplastina Parcial , Distribución Aleatoria , Valores de Referencia , Sepsis/microbiología , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
OBJECTIVE: W1-mAb is a chimpanzee-derived monoclonal antibody to protective antigen that improved survival when administered before anthrax lethal toxin challenge in rats. To better define W1-mAb's efficacy for anthrax, we administered it after initiation of 24-hr infusions of edema toxin and lethal toxin either alone or together in rats or following anthrax spore challenge in mice. INTERVENTIONS: W1-mAb or placebo treatment. METHODS AND MAIN RESULTS: In toxin-challenged rats treated with placebo, survival rates were lower with edema toxin (500 µg/kg) compared to lethal toxin either alone (175 µg/kg) or with edema toxin (175 µg/kg each) (8%, 33%, and 32%, respectively), but the median time to death was longer (36, 11, and 9 hrs, respectively) (p ≤ .01 for all comparisons). W1-mAb administered up to 12 hrs after edema toxin and 6 hrs after lethal toxin increased survival and reduced hypotension (p ≤ .01). However, only administration of W1-mAb at 0 hrs improved these variables with lethal toxin and edema toxin together (p ≤ .0002). In C57BL/6J mice challenged with anthrax spores subcutaneously, compared to placebo treatment (0 of 15 animals survived), W1-mAb administered beginning 24 hrs after challenge increased survival (13 of 15 survived) (p ≤ .0001). CONCLUSION: While rapidity of lethality may influence the effectiveness of delayed W1-mAb treatment, these rat and mouse studies provide a basis for further exploring this agent's usefulness for anthrax.
Asunto(s)
Carbunco/tratamiento farmacológico , Anticuerpos Monoclonales/administración & dosificación , Antígenos Bacterianos/administración & dosificación , Bacillus anthracis/inmunología , Toxinas Bacterianas/administración & dosificación , Factores Inmunológicos/administración & dosificación , Animales , Carbunco/etiología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Ratones , Ratones Endogámicos C57BL , Pan troglodytes , Ratas , Ratas Sprague-Dawley , Esporas BacterianasRESUMEN
While anthrax edema toxin produces pronounced tachycardia and lethal toxin depresses left ventricular (LV) ejection fraction in in vivo models, whether these changes reflect direct cardiac effects as opposed to indirect ones related to preload or afterload alterations is unclear. In the present study, the effects of edema toxin and lethal toxin were investigated in a constant pressure isolated perfused rat heart model. Compared with control hearts, edema toxin at doses comparable to or less than a dose that produced an 80% lethality rate (LD(80)) in vivo in rats (200, 100, and 50 ng/ml) produced rapid increases in heart rate (HR), coronary flow (CF), LV developed pressure (LVDP), dP/dt(max), and rate-pressure product (RPP) that were most pronounced and persisted with the lowest dose (P ≤ 0.003). Edema toxin (50 ng/ml) increased effluent and myocardial cAMP levels (P ≤ 0.002). Compared with dobutamine, edema toxin produced similar myocardial changes, but these occurred more slowly and persisted longer. Increases in HR, CF, and cAMP with edema toxin were inhibited by a monoclonal antibody blocking toxin uptake and by adefovir, which inhibits the toxin's intracellular adenyl cyclase activity (P ≤ 0.05). Lethal toxin at an LD(80) dose (50 ng/ml) had no significant effect on heart function but a much higher dose (500 ng/ml) reduced all parameters (P ≤ 0.05). In conclusion, edema toxin produced cAMP-mediated myocardial chronotropic, inotropic, and vasodilatory effects. Vasodilation systemically with edema toxin could contribute to shock during anthrax while masking potential inotropic effects. Although lethal toxin produced myocardial depression, this only occurred at high doses, and its relevance to in vivo findings is unclear.
Asunto(s)
Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , AMP Cíclico/metabolismo , Corazón/efectos de los fármacos , Miocardio/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Animales , Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Dobutamina/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Organofosfonatos/farmacología , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND: Supporting its therapeutic application in sepsis, p38 mitogen-activated protein kinase (MAPK) inhibition decreases cardiopulmonary injury and lethality with lipopolysaccharide challenge. However, only one preclinical study has reported the survival effects of a p38 inhibitor (SB203580, 100 mg/kg) during infection. We therefore tested SB203580 in mice (n = 763) challenged with intratracheal Escherichia coli and treated with antibiotics and fluids. METHODS AND RESULTS: Compared with placebo, high dose SB203580 (100 mg/kg) pretreatment increased the hazards ratio of death (95% confidence interval) (3.6 [2.1, 6.1], p < 0.0001). Decreasing doses (10, 1, or 0.1 mg/kg) went from being harmful to having no significant effect (p < 0.0001 for the effect of decreasing dose). At 48 hours, but not 24 hours after E. coli, high and low dose SB203580 pretreatment decreased cardiac phosphorylated p38 MAPK levels and improved cardiac output either (p Asunto(s)
Inhibidores Enzimáticos/farmacología
, Infecciones por Escherichia coli/tratamiento farmacológico
, Imidazoles/farmacología
, Neumonía/tratamiento farmacológico
, Piridinas/farmacología
, Análisis de Varianza
, Animales
, Antibacterianos/farmacología
, Gasto Cardíaco/efectos de los fármacos
, Ecocardiografía
, Inhibidores Enzimáticos/administración & dosificación
, Ensayo de Inmunoadsorción Enzimática
, Infecciones por Escherichia coli/fisiopatología
, Fluidoterapia
, Imidazoles/administración & dosificación
, Lipopolisacáridos/farmacología
, Lesión Pulmonar/fisiopatología
, Ratones
, Placebos
, Neumonía/fisiopatología
, Modelos de Riesgos Proporcionales
, Piridinas/administración & dosificación
, Tasa de Supervivencia
RESUMEN
OBJECTIVES: The in vivo inflammatory effects of the Bacillus anthracis cell wall are unknown. We therefore investigated these effects in rats and, for comparison, those of known inflammatory stimulants, Staphylococcus aureus cell wall or lipopolysaccharide (LPS). METHOD AND RESULTS: Sprague-Dawley rats (n = 103) were challenged with increasing B. anthracis cell wall doses (10, 20, 40, 80, or 160 mg/kg) or diluent (control) as a bolus or 24-h infusion. The three highest bolus doses were lethal (20-64% lethality rates) as were the two highest infused doses (13% with each). Comparisons among lethal or nonlethal doses on other measured parameters were not significantly different, and these were combined for analysis. Over the 24 h after challenge initiation with lethal bolus or infusion, compared to controls, ten inflammatory cytokines and NO levels were increased and circulating neutrophils and platelets decreased (P Asunto(s)
Bacillus anthracis/metabolismo
, Bacillus anthracis/patogenicidad
, Infecciones Bacterianas/metabolismo
, Infecciones Bacterianas/patología
, Toxinas Bacterianas/antagonistas & inhibidores
, Toxinas Bacterianas/toxicidad
, Animales
, Plaquetas/metabolismo
, Plaquetas/microbiología
, Pared Celular/microbiología
, Modelos Animales de Enfermedad
, Estimación de Kaplan-Meier
, Linfocitos/metabolismo
, Linfocitos/microbiología
, Neutrófilos/metabolismo
, Neutrófilos/microbiología
, Distribución Aleatoria
, Ratas
, Ratas Sprague-Dawley
, Staphylococcus aureus/metabolismo
, Staphylococcus aureus/patogenicidad
, Factores de Tiempo
RESUMEN
OBJECTIVES: The response of anthrax lethal toxin (LeTx) induced shock and lethality to conventional therapies has received little study. Previously, fluids worsened outcome in LeTx-challenged rats in contrast to its benefit with lipopolysaccharide (LPS) or Escherichia coli. The current study investigated norepinephrine treatment. MEASUREMENTS AND MAIN RESULTS: Sprague-Dawley rats (n = 232) weighing between 230 and 250 g were challenged with similar lethal (80%) 24-hour infusions of either LPS or LeTx, or with diluent only. Toxin-challenged animals were also randomized to receive 24-hour infusions with one of three doses of norepinephrine (0.03, 0.3, or 3.0 microg/kg/min) or placebo started 1 hour after initiation of challenge. All toxin animals received similar volumes of fluid over the 24 hours (equivalent to 4.0-4.3 mL/kg/hr). Although the intermediate norepinephrine dose (0.3 microg/kg/min for 24 hours) improved survival with LPS (p = 0.04) and increased blood pressure before the onset of lethality with LeTx (p < 0.0001), it did not improve survival with the latter (p = ns). Furthermore, neither increasing nor decreasing norepinephrine doses improved survival with LeTx. CONCLUSION: Hypotension with LeTx may not be a primary cause of lethality in this model. Rather, LeTx may cause direct cellular injury insensitive to vasopressors. These findings suggest that during anthrax infection and shock, along with hemodynamic support, toxin-directed treatments may be necessary as well.
Asunto(s)
Carbunco/tratamiento farmacológico , Carbunco/mortalidad , Presión Sanguínea/efectos de los fármacos , Norepinefrina/uso terapéutico , Choque Séptico/tratamiento farmacológico , Vasoconstrictores/uso terapéutico , Animales , Antígenos Bacterianos/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Tasa de SupervivenciaRESUMEN
BACKGROUND: Ethyl pyruvate (EP) treatment inhibits nuclear factor (NF)-kappaB-mediated inflammation and has been considered for sepsis. However, NF-kappaB is also protective, and its inhibition may have adverse effects. METHODS: We studied EP in lipopolysaccharide-challenged mice and systematically analyzed its efficacy in published sepsis models. RESULTS: After lipopolysaccharide, compared with placebo (n = 68), each of six doses of EP (0.01-100 mg/kg, n = 204) increased the hazards ratio of death. Although these increases were individually not significant (p = .13 to .37), when combined, they were (log mean +/- SEM, 0.26 +/- 0.13; p = .01). At 3 and 9 hrs after challenge, lipopolysaccharide increased lung NF-kappaB and 12 serum cytokines (p < or = .05 vs. phosphate-buffered saline challenge, except for interleukin-4 at 9 hrs). With lipopolysaccharide, although EP (100 mg/kg) decreased NF-kappaB and 11 of 12 cytokines at 3 hrs, it increased NF-kappaB and 11 of 12 cytokines at 9 hrs in patterns that differed (p < or = .05) across time points. In 14 published comparisons, EP's effects on the odds ratio of death varied (I2 = 85% [95% confidence interval, 74-91%], p < .0001), decreasing it significantly in five of the studies but not the other nine. In three of the latter, it increased time to death. CONCLUSION: Although EP has had promising effects in some preclinical sepsis models, it has not in others, and in the present model, it worsened outcome. Based on the complex role NF-kappaB has regulating both maladaptive and protective host responses, further defining factors that influence EP's effects is important if this agent is considered for patients with or at risk of sepsis.