Dose-response effect of in vivo administration of endotoxin on polymorphonuclear leukocytes oxidative burst.

In vitro, endotoxin primes polymorphonuclear leukocytes (PMNs) to respond with a greater oxidative burst. The purpose of the present study was to investigate the in vivo effect of a wide range of single endotoxin bolus doses using a rat model. PMNs were subsequently challenged in vitro with phorbol ester to produce reactive oxygen intermediates (ROI). Flow cytometric determination of ROI production by large doses induced a decrease in ROI production by the few PMNs that remained in the circulation. By 6 h after injection, ROI production had returned to basal levels after a high dose, and was still increasing after a low dose. Neutropenia occurred immediately after endotoxin injection. After 6 h, PMN counts returned to almost normal levels with a high dose, but rebound neutrophilia occurred with a small dose. In contrast to in vitro studies, in vivo injection showed a response pattern that varied widely with dose and time of observation.

Comparison of Staphylococcus aureus and Escherichia coli infusion in conscious rats.

The purpose of this study was to compare the pathophysiology of bacteremia produced by intravenous infusion of either a Gram-positive (Staphylococcus aureus) or a Gram-negative (Escherichia coli) organism. Conscious, unrestrained, instrumented rats received S. aureus, E. coli, or sterile saline over 120 min, followed by a 240-min monitoring period. The infusates produced 90% (S. aureus,) 80% (E. coli), and 0% (saline) mortality at 24 hr. Neither bacterial group produced hypotension during the entire 360-min study period. E. coli produced early tachycardia and increased glucose, followed by decreased stroke volume and increased lactate and pO2. S. aureus caused early tachycardia followed by decreased pH, stroke volume, and cardiac output and increased lactate and systemic vascular resistance. Respiration rate and central venous pressure were not affected by either bacterial infusion. Compared to E. coli, S. aureus produced decreased pH, glucose, pO2, heart rate, and cardiac output and increased lactate, hematocrit, pCO2, and systemic vascular resistance. These data document quantitative differences in the acute response of the conscious rat to bacteremia caused by these isolates of E. coli and S. aureus.

Cellular F-actin levels as a marker for cellular transformation: relationship to cell division and differentiation.

Transformation is associated with profound structural and quantitative changes in the cytoskeleton. Herein we report studies using F-actin, a major cytoskeletal protein, as a quantitative marker for transformation cells, focusing on separating the effects of the cell cycle, cell differentiation, and transformation. The model system for these studies consisted of three lymphoblastic cell lines, one untransformed line (RPMI) and two transformed lines, one (HL-60) of which can be induced to differentiate and the other (Daudi) which cannot. The relation of F-actin levels to cell cycle was studied by flow cytometry with the use of fluorescein-phalloidin to label F-actin and propidium iodide to label DNA. F-actin levels in transformed Daudi and HL-60 lines were only two-thirds that of the untransformed RPMI cells. Histograms of the distribution of F-actin showed that the transformed lines consisted of two cell populations, one having an F-actin content near that of untransformed cells and the other having much less. Cell cycle analysis showed that F-actin in untransformed cells increased 10-15% as cells entered the S compartment, remaining approximately constant through G2 + M phases of the cell cycle, but in transformed cells the major increase in F-actin occurred during G2 + M phase. Double-label studies with rhodamine-phalloidin for F-actin and KI-67 monoclonal antibody for dividing cells (cells at late G1, S, G2, and M) measured with quantitative fluorescence image analysis showed that the mean F-actin content of dividing cells was twice that of nondividing cells. These results suggested that most of the cell division-related F-actin increase occurred during late G1 phase in untransformed cells. Differentiation of HL-60 cells with dimethyl sulfoxide or retinoic acid normalized the F-actin content of the nondividing cell population, but dimethyl sulfoxide and retinoic acid produced no detectable change in F-actin in the undifferentiable Daudi cells. A tumor promoter (12-O-tetradecanoylphorphol-13-acetate) inhibits differentiation of hematopoietic cells, resulted in a 32% decrease in the mean F-actin content of RPMI cells due to the appearance of a new subpopulation of low F-actin content. The 12-O-tetradecanoylphorbol-13-acetate-induced changes reversed slowly after removal of 12-O-tetradecanolyphorbol-13-acetate but more rapidly in the presence of retinoic acid. These results indicate that F-actin quantification can serve as a marker for cellular transformation and provides a tool for studying the mechanisms of cellular differentiation that may lead to a better understanding of the oncogenic process.

Evaluation of naloxone therapy for Escherichia coli sepsis in the baboon.

This study evaluated the effects of naloxone hydrochloride in the treatment of Escherichia coli-induced shock in baboons. The baboons were studied for 12 hours and monitored for survival times. All baboons were intravenously infused for two hours with E coli and treated as follows: group 1, E coli (control); group 2, E coli plus naloxone hydrochloride, 0.5 mg/kg bolus plus 0.5 mg/kg/h for 9.5 hours; and group 3, E coli plus naloxone hydrochloride, 2.0 mg/kg bolus plus 2.0 mg/kg/h for 3.8 hours. Naloxone was administered after arterial pressure had reached the nadir (more than two hours following initiation of E coli infusion). Mean arterial pressure was supported by the lower dose of naloxone; however, sustained leukopenia and neutropenia were not reversed by its infusion. Naloxone prevented the increase in plasma beta-endorphin level and blunted the increase in plasma cortisol level. Despite these effects, naloxone did not prevent multiple-organ disease and did not decrease mortality.

Effects of prior administration of steroids upon recovery from lethal sepsis.

The effects upon survival of large doses of steroid administered to dogs prior to challenging them with lethal sepsis was evaluated in this study. Dogs were given 30 milligrams per kilogram of body weight per day of methylprednisolone sodium succinate for one, two or eight days and then were infused with 9.72 +/- 0.35 X 10(9) Escherichia coli per kilogram of body weight. All dogs in group 1 (n equals six) not given steroid died within 25 hours. Of the dogs in group 2 (n equals 12) given one or two doses of steroid previously, 42 per cent permanently survived (more than seven days). All dogs in group 3 (n equals five) given eight daily doses of steroid prior to infusion of Escherichia coli died within 17 hours. Dogs in group 4 (n equals six) were given eight daily doses of steroid prior to infusion of Escherichia coli and treated on the day of infusion of Escherichia coli with a regimen of methylprednisolone and gentamicin sulfate which results in a 100 per cent survival rate when given to dogs that have not received prior treatment with steroid. Thirty-three per cent of the dogs in group 4 permanently survived. One or two daily large doses of steroid did not detrimentally affect survival of the dogs. Eight days of steroid administration suppressed endogenous cortisol production. When the dogs were treated with six hours of steroid-antibiotic therapy, survival benefits were limited.

Tobramycin therapy for lethal sepsis in the dog.

Antimicrobial effectiveness and effect on survival of single-dose vs. multiple-dose aminoglycoside antibiotic therapy (with and without steroid) for lethal sepsis were evaluated. Adult dogs of either sex were anesthetized, divided into five groups, and infused iv for one hour with Escherichia coli. Group A was given no drug. Group B was given a 45-mg/kg, 10-min iv injection of tobramycin (TOB) at 65 min. Group C was given a 3-mg/kg, 10-min TOB injection at 65 min, followed by an 8.25-mg/kg iv infusion for 285 min, and three 11.25-mg/kg intramuscular injections at 6, 12, and 18 h (total 45 mg/kg). Group D was given the same TOB regimen as B, plus a 30-mg/kg iv injection and 30-mg/kg iv infusion of methylprednisolone sodium succinate (MPSS) from 15 to 360 min. Group E was given the same TOB regimen as C, plus the same MPSS regimen as D. Treated dogs also received 11.25 mg/kg of TOB daily for 4 days. The percent surviving more than 7 days was 0, 0, 17%, 83%, and 83%, for groups A through E, respectively. By 4 h, TOB-treated groups had significantly (p less than .05) lower E. coli blood levels than group A. Also E. coli levels in group B were significantly (p less than .05) lower than those in groups C, D, or E. High trough serum TOB concentrations were associated with death and very low levels with recovery. Serum urea nitrogen and creatinine concentrations increased in all groups, but returned to normal by 7 days in survivors.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of LD100 Escherichia coli septic shock with netilmicin and methylprednisolone in baboons.

Treatment efficacy with netilmicin sulphate/methylprednisolone sodium succinate in a severe septic shock baboon model, using an LD100 of live Escherichia coli, was evaluated. All the animals treated with both netilmicin and methylprednisolone were permanent (greater than or equal to 7 days) survivors, whereas none of the untreated baboons lived more than 24 hours. These results indicate that, in a baboon model, netilmicin is an effective alternative to gentamicin (with methylprednisolone) in the treatment of severe septic shock.

Myocardial dysfunction in endotoxin- and E. coli-induced shock: pathophysiological mechanisms.

Experimental studies have documented that myocardial dysfunction is precipitated between 3 and 6 hr after endotoxin or E. coli. This finding has now been confirmed in human septic shock. A "Hinshaw-modified" isolated working left ventricle preparation has been used to document and assess the degree of failure. It was found that the failure is often severe and reversible only temporarily by adrenergic agents but reversible by digoxin or insulin. The cause of the failure has not been identified, but evidence is presented against a myocardial depressant factor (MDF) being the causative factor. Hearts subjected to a 2-4 hr period of hypotension on the threshold of failure show no signs of failure when subjected to blood circulating from an animal in splanchnic arterial occlusion shock. Hearts from pancreatectomized animals subjected to endotoxin shock with their source of MDF removed demonstrate the typical failure in 4-6 hr. Other factors are suggested that contribute to myocardial dysfunction: hypotension or nonuniform perfusion of subendocardial regions of the heart, depressed responsiveness to inotropic and chronotropic stimuli, intracardiac ionic and fluid disturbances, and increases in heart chamber and muscle stiffness. Since steroid/antibiotic therapy increases the probability (p less than 0.05) that an animal will survive lethal sepsis, investigating the effect of this therapy on myocardial function may aid in determining whether or not this degree of heart failure contributes in the animal to irreversible shock and death.

Corticosteroid/antibiotic treatment of adrenalectomized dogs challenged with lethal E. coli.

Adrenalectomized animals are extremely sensitive to endotoxin and die quickly when given small doses. A six hour administration of the corticosteroid, methylprednisolone sodium succinate (MPSS), combined with the antibiotic, gentamicin sulfate (GS), promotes complete recovery of dogs with intact adrenals administered LD100 E. coli. The aim of the present study was to determine if this early administered treatment would protect adrenalectomized dogs from overwhelming lethal doses of E. coli. Dogs were infused with MPSS from fifteen minutes to six hours after the onset of E. coli administration and with GS after administration of all E. coli. Animals given only E. coli died in 2.6 (+/- 0.3) hours, while those given no E. coli, or E. coli plus steroid/antibiotic, survived longer than 100 hours. Arterial pressure, pH, pO2, hematocrit, lactate, and glucose concentrations were maintained near control values in animals receiving steroid/antibiotic infusions. Adrenalectomized dogs infused with corticosteroid/antibiotic recovered completely from shock even though the treatment period was limited to the first 6 hours after lethal E. coli infusion. Findings indicate that animals treated with MPSS/GS after E. coli ultimately succumbed to adrenal insufficiency rather than from the E. coli insult and thus recovery from shock itself was complete.

Evaluation of naloxone for therapy of Escherichia coli shock. Species differences.

Dogs and baboons were infused intravenously (IV) with Escherichia coli and treated with the opiate antagonist, naloxone hydrochloride, and the antibiotic, gentamicin sulfate, to determine the therapeutic efficacy of naloxone. Naloxone hydrochloride (2 mg/kg) was injected IV when one fourth of the E coli had been infused and then infused at 2 mg/kg/hr (six hours for dogs and 12 hours for baboons). Four of five naloxone-treated dogs survived permanently (greater than seven days), while all dogs that were given only E coli died. Arterial BP, blood glucose levels, PCO2, and PO2 were supported at higher levels and lesions of the gastrointestinal tract were prevented in naloxone-treated dogs. A steady decline in blood glucose levels after an initial hyperglycemia was observed in naloxone-treated baboons, indications of peripheral vasoconstriction were noted, and all baboons died within 42 hours.

A model for thrombin protection against endotoxin.

Infusion of dogs with thrombin (0.5 U/kg/min) for 90 minutes significantly increased percent survival following infusion of endotoxin (0.06 mg/kg/min) for 30 minutes. Nine of fourteen dogs infused with thrombin survived seven days (permanent survivors), whereas thirteen of fourteen dogs infused with saline died within 36 hours. Those dogs which survived responded immediately to endotoxin with enhanced anticoagulant and fibrinolytic activity as measured by the Xa one-stage and fibrin degradation product assays, respectively. Those dogs receiving saline instead of thrombin did not respond with anticoagulant or fibrinolytic activity until the end of the study. We concluded that thrombin in the correct amounts protected dogs from endotoxin and that this protection was associated with an early anticoagulant and fibrinolytic response to endotoxin infusion.

Effectiveness of modified steroid-antibiotic therapies for lethal sepsis in the dog.

Dogs permanently recover (survive at least seven days) from lethal doses of Escherichia coli when treated early with intravenous (IV) intermittent infusions of methylprednisolone sodium succinate and gentamicin sulfate. We evaluated the therapeutic effectiveness of constant or bolus IV infusion of methylprednisolone combined with gentamicin or netilmicin sulfate. Four groups of anesthetized dogs were infused for one hour with E coli and treated as follows (% survival indicated): no treatment (0%); constant infusion of methylprednisolone and gentamicin (100%); bolus infusion of methylprednisolone and gentamicin (57%); and constant infusion of methylprednisolone and netilmicin (83%). Constant or bolus infusion of methylprednisolone was begun 15 minutes after E coli infusion was started. Gentamicin or netilmicin administration was begun when all organisms had been infused. The probability of recovery from shock was significantly increased when dogs were treated with constant infusion of methylprednisolone and intermittent infusions of gentamicin or netilmicin, but was only moderately increased when treated with intermittent bolus infusions of methylprednisolone and intermittent infusions of gentamicin.

Detection of the 'hyperdynamic state' of sepsis in the baboon during lethal E. coli infusion.

This study was designed to document the early responses of the baboon to LD100 E. coli infusion with an emphasis on cardiovascular, respiratory, and metabolic alterations. Adult baboons were anesthetized, catheterized aseptically to monitor cardiopulmonary and systemic parameters, and infused with LD100 E. coli intravenously for 2 hours. Cardiac output was determined with an Edwards Laboratory Thermodilution Cardiac Output Computer. Oxygen contents of systemic and pulmonary artery blood were determined with a Van Slyke Manometric Apparatus. Results demonstrate early significant increases in cardiac output, oxygen uptake, heart rate, rectal temperature, respiratory rate and minute volume, and decreases in mean systemic arterial pressure and total peripheral resistance. Pulmonary vascular resistance remained relatively constant. In conclusion, an early 'hyperdynamic state' has been detected in the baboon during lethal E. coli infusion (similar to that documented in humans with sepsis) characterized by compensatory alterations in cardiovascular hemodynamics, respiration, and metabolism.

Prevention or amelioration of morphologic lesions in LD100 E coli-shocked baboons with steroid/antibiotic therapy.

We have documented the effectiveness of methylprednisolone sodium succinate (MPSS) and gentamicin sulfate (GS) therapy for LD100 E coli-induced shock in the baboon. We sequentially delayed initiation of MPSS infusion from 30 to 120 min and then to 240 min after onset of a 2-h E coli infusion. Treatment resulted in 100%, 85%, and 65% survival respectively. In this study we evaluated tissue taken at autopsy in the three MPSS/GS treatment studies including untreated baboons and those treated with GS only. When animals died (3-49 h) or were sacrificed (7-71 days), tissues were removed, coded, and prepared for histopathologic evaluation by light microscopy. On the basis of morphologic changes animals split into two groups: baboons with little or no tissue alterations (survivors), and those with multiple organ damage (nonsurvivors). Combinations of mild to massive congestion, edema, hemorrhage, fibrin thrombi, increased numbers of polymorphonuclear leukocytes (PMNs), and necrosis of the adrenal glands, liver, kidneys, lungs, and spleen of nonsurvivors were prevented or ameliorated in the MPSS/GS-treated surviving baboons. Data demonstrate the MPSS/GS therapy prevents or reverses the multiple organ damage and increases survival in lethal septic shock.

Preventing versus delaying death in shock therapy studies: evaluating "survival".

When results from a short-term experimental study of septic shock indicate that a therapy increases survival time (i.e., delays death), should the reader conclude that the therapy ultimately will increase the percentage of permanent survivors (i.e., prevent death)? If not, how long must animals be observed after lethal sepsis to reasonably validate that a therapy can be used to effect complete recovery? To help answer these questions, we reevaluated survival data from the symposium that entailed the largest number of separate shock therapy studies ever performed on a homogeneous group of dogs in one place at one time with a single lot of endotoxin. Twenty-eight scientists studied the responses of 115 treated and 38 untreated dogs given LD80 endotoxin by bolus injection to compare 15 therapies (most previously reported to have "increased survival"): the numbers of dogs alive were recorded at 0, 0.5, 1, 2, 4, and 7 days. Chi-square analysis indicated significantly more treated than untreated dogs alive only at 24 hr and significantly fewer treated dogs alive on day 7 than on day 2. Individual comparison of the proportion of animals alive in each of the 15 treated groups with the untreated group showed more dogs alive (P less than 0.05) in 3 treated groups at day 1 and in 1 treated group at day 2 and 4; so, four therapies appeared to "increase survival." However, by day 7 no therapy group had statistically more survivors than the untreated group. This evaluation indicates that in order to make a clear distinction between increasing survival time and increasing the percentage of permanent survivors, both treated and untreated dogs should be observed and compared after septic challenge until the percentage of survivors in both groups attains a steady state.

Relationship of serum gentamicin levels and methylprednisolone sodium succinate treatment in baboons challenged with Escherichia coli LD100.

Serum gentamicin bioassay determinations were performed on 15 adult baboons that had been challenged with an infusion of live Escherichia coli sufficient to kill all of the individuals (LD100) and then treated with either methylprednisolone sodium succinate (MPSS) (administered early in some animals and late in others) and gentamicin sulfate (GS) or with GS only. Initial peak gentamicin levels in all three groups were similar; however, subsequent valley levels revealed that when MPSS treatment was delayed or omitted, gentamicin concentrations remained elevated.

Current management of the septic shock patient: experimental basis for treatment.

Experimental research has shown that following the intravenous infusion of animals with bacteria or endotoxin a myriad of adverse vascular events occur resulting in deficient organ perfusion and cell death. The primary goals of therapy for sepsis and septic shock, therefore, should include elimination of the source of infection and/or infectious agents and prevention or reversal of adverse vascular events. The following review describes the evolution of an effective therapy for shock consisting of steroid in combination with antibiotic and discusses its relevance and application for humans in severe sepsis or septic shock.

Hematologic disturbances during sepsis: platelets and leukocytes.

This brief review summarizes recent observations about leukocyte and platelet involvements during sepsis and septic shock. Endotoxins are known to exert significant effects on leukocytes and platelets as well as monocytes, macrophages, endothelial, and mast cells. The presence of endotoxin itself is reported to enhance the phagocytic and killing capacity of neutrophils. Transfusion of additional white blood cells has been shown to increase the probability of recovery from sepsis. Defects in neutrophil function, impaired opsonization, sequestration of neutrophils in pulmonary capillaries, and depressed metabolic states adversely affecting neutrophils may significantly contribute to the lethal outcome of septic shock. Platelet responses in septic shock are reported to include aggregation accompanied by release of several agents, including vasoactive amines, ADP, and platelet factor 3. In summary, leukocytes and platelets are known to perform significant roles in sepsis and septic shock although the precise mechanisms of their involvement remain to be clearly defined.

Survival characteristics during septic shock in 39 baboons.

In septic shock nonsurvival is characterized by failure of multiple organ systems. The design of therapeutic measures to increase survival would be enhanced if critical responses could be identified early. Escherichia coli LD100 was given to 39 baboons by IV infusion over two hours followed by different therapy regimens in 31 [2--4]. There were 18 permanent survivors (seven days or more), all receiving antibiotic/steroid combination therapy. Responses of survivors and nonsurvivors were measured and compared during the first 12 hours from onset of infusion. Changes in blood pressure and acid-base parameters were not significantly different between groups. Five responses indicative of permanent survival were lower heart rates, less elevation of blood urea nitrogen, normal blood glucose at eight hours, hyperglycemia with normal insulin at 12 hours, and lower plasma lactate concentrations beginning at four hours.

Does LD100 E coli shock cause myocardial failure?

We have documented that myocardial dysfunction occurs in canine endotoxin shock and have designed this study to determine the effect of lethal live E coli-induced shock on the myocardium. Small adult heart "donor" dogs (wt range 6-9 kg) were infused with LD100 E coli (N = 12) or saline (N = 16) for 30 minutes. Two hours later, heart transfer surgery was initiated and once completed the isolated working left ventricle was allowed to equilibrate in the extracorporeal circuit of a "support" dog (wt range 22-32 kg). Myocardial performance was then evaluated by changing mean aortic pressure while controlling cardiac output. Three to five hours after E coli infusion, marked myocardial dysfunction occurred in 75% of the hearts as evidenced by increased left ventricular and diastolic pressures and depressed peak positive and negative dP/dt at every mean aortic pressure tested compared with control hearts. Myocardial efficiency and power were depressed, oxygen uptake was elevated, and coronary blood flow was unchanged in E coli-treated compared with control hearts. Data support the presence of heart dysfunction in gram-negative septic shock.