Immunomodulatory intervention with interferon-γ in Escherichia coli pyelonephritis.

PURPOSE: We investigated the efficacy of recombinant human interferon-γ in experimental pyelonephritis due to Escherichia coli. MATERIALS AND METHODS: Pyelonephritis was induced by intrapelvic inoculation of bacteria after ureteral ligation in 38 rabbits assigned to 1 of 3 groups, including group 1-16 controls, group 2-14 rabbits treated with intravenous recombinant human interferon-γ and group 3-8 rabbits treated with intravenous recombinant human interferon-γ plus amikacin. Bacterial counts, cytokines and malondialdehyde were measured in blood. Peripheral blood mononuclear cells were isolated to measure TNFα transcripts, cytokine stimulation and apoptosis. Survival was recorded, and the tissue bacterial load and myeloperoxidase activity were measured after sacrifice. RESULTS: The mortality rate in groups 1, 2 and 3 was 66.7%, 25% and 12.5%, respectively. The circulating bacterial count and tissue bacterial load were less in group 2 than in group 1. Circulating malondialdehyde negatively correlated with the bacterial load of the spleen. Although the number of TNFα transcripts in circulating peripheral blood mononuclear cells did not differ, peripheral blood mononuclear cells isolated from group 2 at 48 hours produced much greater concentrations of tumor necrosis factor-α after stimulation with Pam3Cys. In parallel, the apoptosis rate of circulating monocytes was increased in group 2 at 48 hours. Lung myeloperoxidase activity at 24 hours, serving as indirect evidence of neutrophil infiltration, was decreased in group 2. CONCLUSIONS: Recombinant human interferon-γ administration prolonged survival in rabbits with experimental E. coli urosepsis. Its action was probably related to increased bacterial phagocytosis after modulation of oxidant status and reversal of monocyte immunoparalysis.

Efficacy and pharmacodynamics of linezolid, alone and in combination with rifampicin, in an experimental model of methicillin-resistant Staphylococcus aureus endocarditis.

OBJECTIVES: To evaluate the efficacy of oral linezolid, with or without rifampicin, on valve vegetations and secondary foci of infection compared with vancomycin, in the absence or presence of rifampicin, in experimental endocarditis caused by methicillin-resistant Staphylococcus aureus. METHODS: Treatment groups were controls (n = 16), linezolid (n = 15), vancomycin (n = 15), linezolid and rifampicin (n = 15), vancomycin and rifampicin (n = 13), linezolid relapse (n = 11) and vancomycin relapse (n = 9). Therapy lasted 5 days in all groups, with survival of animals in the linezolid relapse and vancomycin relapse groups being recorded for an additional 5 days. Blood was drawn to determine the linezolid concentration, and valve vegetations, and kidney, liver, lung and spleen segments were collected for culture. RESULTS: Survival in each individual group was higher than that in the control group; bacterial load in valve vegetations was reduced by all treatment regimens, with linezolid exhibiting bactericidal effects. Bactericidal activity of linezolid was noted in all secondary foci of infection except the lung, where only the combination of rifampicin with linezolid was bactericidal. CONCLUSIONS: Orally administered linezolid is effective in limiting bacterial growth in the secondary foci of endocarditis. Co-administration of rifampicin favoured the suppression of bacterial growth in the lung.

The impact of multidrug resistance on the pathogenicity of Escherichia coli: an experimental study.

Based on the controversial findings of clinical studies regarding the influence of multidrug resistance on mortality, 10 susceptible and 10 multidrug-resistant (MDR) and extended-spectrum beta-lactamase-producing isolates of Escherichia coli were applied to stimulate monocytes isolated from healthy donors. Immune mediators were estimated in supernatants. Four susceptible isolates (Group A) and four MDR isolates (Group B) were used to initiate acute pyelonephritis in 48 rabbits following inoculation of the pathogen into the right renal pelvis. Survival was recorded and blood monocytes were isolated and incubated to estimate the ex vivo release of tumour necrosis factor-alpha (TNFalpha). Release of TNFalpha, interleukin (IL)-6 and IL-8 was higher after 2 h and 4 h of stimulation by MDR isolates compared with susceptible isolates. The opposite occurred for the release of IL-12. Death occurred in 22 rabbits in Group A (91.7%) compared with 12 in Group B (50.0%) (P=0.003). Monocytes isolated at 24 h from Group A rabbits released significantly higher TNFalpha than monocytes from Group B. Tissue bacterial load after animal death was significantly higher in the kidneys of Group A rabbits. It is concluded that susceptible and MDR E. coli stimulate monocytes resulting in a different pattern of release of pro-inflammatory cytokines, which is accompanied by prolonged survival following experimental sepsis by MDR isolates.

Clarithromycin is an effective immunomodulator when administered late in experimental pyelonephritis by multidrug-resistant Pseudomonas aeruginosa.

BACKGROUND: To apply clarithromycin as an immunomodulatory treatment in experimental urosepsis by multidrug-resistant Pseudomonas aeruginosa. METHODS: Acute pyelonephritis was induced in 40 rabbits after inoculation of the test isolate in the renal pelvis. Therapy was administered upon signs of sepsis in four groups: A, controls; B, intravenous clarithromycin; C, amikacin; and D, both agents. Survival and vital signs were recorded; blood was sampled for culture and estimation of pro-inflammatory mediators; monocytes were isolated for determination of apoptotic rate and ex vivo TNFalpha secretion. Quantitative cultures and biopsies of organs were performed after death. RESULTS: Increased rectal temperature and oxygen saturation were found in groups B and D compared to A and C. Mean survival of groups A, B, C and D was 2.65, 7.15, 4.25 and 8.70 days respectively. No differences were noted between groups concerning bacterial load in blood and tissues and serum endotoxins. Serum MDA and total caspase-3 activity of monocytes of group D decreased following treatment compared to other groups. Negative correlation was detected between cytoplasmic caspase-3 and ex vivo secretion of TNFalpha of blood monocytes of group A; similar correlation was not found for any other group. Pathology scores of liver and lung of group B were lower than group A. CONCLUSION: Clarithromycin administered late in experimental urosepsis by multidrug-resistant P. aeruginosa prolonged survival and ameliorated clinical findings. Its effect is probably attributed to immunomodulatory intervention on blood monocytes.

The significance of oxidant/antioxidant balance for the pathogenesis of experimental sepsis by multidrug-resistant Pseudomonas aeruginosa.

OBJECTIVE: The significance of lipid peroxidation as an independent factor leading to sepsis by multidrug-resistant Pseudomonas aeruginosa. Design experimental study. METHODS: Twenty-six rabbits were applied. They were divided into two groups; A (n=6) comprising controls, and B (n=20) comprising animals infected by the injection of 1x10(8) cfu/kg inoculum of the test pathogen into the left inner jugular vein. Six rabbits of group B were followed-up to estimate survival; all of the remaining were sacrificed. Blood was sampled for the determination of serum malondialdehyde (MDA) by the thiobarbiturate assay, total antioxidant status (TAS) by a chromogenic assay, tumor necrosis factor alpha by a bioassay on fibrosarcoma L929 cell line, and endotoxins (LPS) by the QCL-1000 LAL assay. RESULTS: Mean survival of group B was 60.0+/-15.8 h. MDA was significantly higher in group B compared to group A at 30, 60, 120 and 150 min. TAS was statistically decreased in group B compared to group A at 30 and 60 min. Increases of MDA in group B were followed by reciprocal decreases of TAS (P of correlation <0.001). Hemodynamic instability was recorded in group B compared to group A 160 min after bacterial challenge. CONCLUSIONS: Early alterations of oxidant/antioxidant balance occur in experimental sepsis by multidrug-resistant P. aeruginosa followed by hemodynamic instability. Results highlight the perspective of the administration of antioxidants as immunomodulatory treatment of sepsis in animal studies.

Experimental sepsis using Pseudomonas aeruginosa: the significance of multi-drug resistance.

In order to clarify whether susceptible and multidrug-resistant Pseudomonas aeruginosa differ in the mechanism of induction of sepsis, three different isolates were used; one susceptible (isolate A) and two (isolates B and C) multidrug-resistant. Isolate B had moderately elevated MICs of antipseudomonal antimicrobials and isolate C highly elevated MICs. Each isolate was infused by a catheter inserted into the right jugular vein of six rabbits. Survival was recorded; blood was sampled at regular time intervals for estimation of bacterial blood counts, malondialdehyde (MDA) and tumour necrosis factor-alpha (TNFalpha). Quantitative cultures of various organs were performed after death or sacrifice. Mean survival after challenge by isolates A, B and C was 0.73, 2.58 and 11.00 days, respectively (P of comparisons A versus B, 0.0048; A versus C, 0.0012; B versus C, 0.0005). The number of viable organisms in the blood after challenge using isolates A and B was greater than the viable counts of C. Serum MDA was lower after challenge with B and C compared with A. Serum TNFalpha levels were higher after challenge by isolate A compared with isolate C. The bacterial loads of the liver, lower right lung lobe, spleen and mesenteric lymph nodes were greater after challenge by isolate A than the other isolates. It is concluded that infection by multidrug-resistant P. aeruginosa is accompanied by increased survival compared with infection by susceptible isolates; that finding might be explained by the different mechanisms leading to sepsis. Further studies must be done to clarify the significance of these observations for therapeutics.

Clarithromycin is an effective immunomodulator in experimental pyelonephritis caused by pan-resistant Klebsiella pneumoniae.

OBJECTIVES: To apply clarithromycin as an immunomodulatory treatment in experimental infection caused by pan-resistant Klebsiella pneumoniae. METHODS: Acute pyelonephritis was induced in 80 rabbits after inoculation of the test isolate in the renal pelvis. Rabbits were divided into eight groups, with 10 animals in each group. In groups A-D, therapy was administered simultaneously with bacterial challenge as follows: A, controls; B, intravenous clarithromycin; C, amikacin; and D, both agents. In groups E-H, therapy was administered 24 h after bacterial challenge as follows: E, controls; F, intravenous clarithromycin; G, amikacin; and H, both agents. Blood was sampled for estimation of tumour necrosis factor-alpha (TNF-alpha) and malondialdehyde (MDA); monocytes were isolated for determination of intracellular activity of caspase-3 and ex vivo TNF-alpha secretion. Four days after bacterial challenge, animals were sacrificed for quantitative cultures and biopsies of organs. RESULTS: Serum TNF-alpha at 48 h was lower in groups B, C and D compared with group A. Activity of caspase-3 of monocytes was lower at 48 h in group D compared with group A. Bacterial loads of liver and spleen were decreased in group D compared with those of group A. The numbers of inflammatory cells of spleen of group B were lower compared with those of group A; those of kidney and mesenteric lymph nodes of group D were lower than those of group A. Serum MDA of group H was lower than that of group E and serum TNF-alpha of group F was lower compared with that of group E. TNF-alpha of monocyte supernatants and activity of caspase-3 of monocytes of group F were lower than those of group E. Bacterial tissue loads did not differ among groups E, F, G and H. The numbers of inflammatory cells of liver of groups F and H were lower compared with those of group E; those of kidney of groups F, G and H were lower compared with those of group E. CONCLUSIONS: Clarithromycin administered intravenously in experimental infection caused by pan-resistant K. pneumoniae attenuated systemic inflammatory response and local tissue damage. This effect is probably attributed to immunomodulatory intervention on blood monocytes.

Clarithromycin: immunomodulatory therapy of experimental sepsis and acute pyelonephritis by Escherichia coli.

The potency of clarithromycin as immunomodulator was assessed in an experimental model of sepsis based on acute pyelonephritis by susceptible Escherichia coli. 55 rabbits were utilized; 5 for preliminary pharmacokinetic study and 50 for treatment. The latter were divided into 5 groups of treatment, A: controls; B: clarithromycin pretreatment; C: amikacin pretreatment; D: clarithromycin treatment on presentation of pulmonary oedema; and E; amikacin treatment on presentation of pulmonary oedema. Survival was recorded; tumour necrosis factor-alpha (TNFalpha), and malondialdehyde (MDA) were estimated in serum; activities of caspase-3 in monocyte cytosolic extracts were studied; and bacterial counts made in various organs. Median survival of animals of groups A, B, C, D and E was 1.0, 21.0, 12.5, 2.0 and 5.0 d, respectively. TNFalpha and MDA and monocyte caspase-3 activity of group A increased over time; no increases were detected in groups B and C. Concentrations of MDA and activities of monocytic caspase-3 were decreased after administration of clarithromycin in group D, an effect not occurring in group E. Bacterial load was decreased in renal tissue of group D compared to group A. It is concluded that intravenous clarithromycin might constitute a promising immunomodulator in sepsis even in the advent of pulmonary oedema.

n-6 polyunsaturated fatty acids enhance the activities of ceftazidime and amikacin in experimental sepsis caused by multidrug-resistant Pseudomonas aeruginosa.

Recent in vitro and ex vivo studies disclosed an enhancement of the activity of antimicrobials on multidrug-resistant Pseudomonas aeruginosa by n-6 polyunsaturated fatty acids (PUFAS); therefore their effect was evaluated in experimental sepsis in 60 rabbits. Solutions of gamma-linolenic acid (GLA) and arachidonic acid (AA) were administered intravenously with ceftazidime and amikacin in rabbits with sepsis caused by one multidrug-resistant isolate. Therapy was started after bacterial challenge in five groups comprising 12 animals in each group: A, normal saline; B, antimicrobials; C, 99% ethanol and antimicrobials; D, GLA and antimicrobials; and E, AA and antimicrobials. Blood was sampled for the estimation of levels of endotoxins in serum (lipopolysaccharide), leukocytes, tumor necrosis factor alpha (TNF-alpha) and antimicrobials. Animals were sacrificed 210 min after bacterial challenge for tissue cultures. All animals had considerable endotoxemia and evolved leukopenia. The number of viable cells in blood, lung, and mesenteric lymph nodes was significantly reduced in groups D and E compared to that in other groups. Levels of antimicrobials in serum were inadequate to achieve bacterial killing due to the level of resistance. n-6 PUFAs did not influence TNF-alpha. It is concluded that intravenous coadministration of n-6 PUFAs and antimicrobials enhanced antimicrobial bacterial killing in experimental sepsis caused by multidrug-resistant P. aeruginosa.

Early cutaneous alterations in experimental sepsis by Pseudomonas aeruginosa.

BACKGROUND: To evaluate whether histopathologic findings of skin in sepsis by Pseudomonas aeruginosa correlate with the clinical course. METHODS: Histological alterations after bacterial challenge by one susceptible (A) and two multidrug-resistant isolates (B and C) of P. aeruginosa were studied in 18 rabbits. Sepsis was induced by the intravenous infusion of 1 x 10(8) CFU by a catheter in the right jugular vein; blood was sampled for the estimation of tumor necrosis factor alpha (TNF-alpha) and malondialdehyde (MDA). Skin biopsies were collected along with a subcutaneous fat specimen for culture. RESULTS: The mean survival was 0.85, 1.75 and 11.00 days after challenge by isolates A, B and C, respectively. The main histologic findings of skin were: inflammation and swelling of the dermis; thickening of the endothelium and infiltration of vessel wall and lumen by polymorphonuclear leukocytes; extravasation of red blood cells, and necrobiotic changes of the hair follicles. Serum TNF-alpha was elevated in animals challenged by isolate A compared to challenge by isolates B and C. Concentrations of MDA were similar for all isolates. Mean log(10) of viable cells isolated from subcutaneous fat were 5.74, 2.74 and 1.40 after challenge by isolates A, B and C, respectively. CONCLUSIONS: Prolongation of survival was accompanied by lower serum TNF-alpha, decreased viable cells from subcutaneous fat and intensified inflammatory response in the dermis and subcutaneous tissue. These findings might be of importance for immunomodulatory intervention.

Immunomodulatory clarithromycin treatment of experimental sepsis and acute pyelonephritis caused by multidrug-resistant Pseudomonas aeruginosa.

Clarithromycin was administered intravenously to 55 rabbits to evaluate its effect on experimental sepsis caused by multidrug-resistant Pseudomonas aeruginosa. Acute pyelonephritis was induced after ligation of the right ureter and injection of 10(8) CFU of the test isolate per kg of body weight into the renal pelvis. The animals were divided into six groups: group A, controls; group B, rabbits that received one intravenous dose of 80 mg of clarithromycin per kg concomitantly with bacterial challenge; group C, rabbits that received two doses of clarithromycin, the second one of which was given 2 h after the first one; group D, rabbits that received 15 mg of amikacin per kg; group E, rabbits that received one dose of clarithromycin and amikacin; and group F, rabbits that received two doses of clarithromycin and amikacin. Serum endotoxin levels were estimated by the QCL-1000 Limulus amoebocyte lysate assay, tumor necrosis factor alpha (TNF-alpha) levels were measured by a bioassay, and malondialdehyde (MDA) levels were measured by the thiobarbiturate assay. Viable bacterial counts in various tissue samples were also assessed. The mean survival times of the animals in groups A, B, C, D, E, and F were 4.50, 7.69, 4.07, 4.55, 11.55, and 11.60 days, respectively (P = 0.033 for group D versus group F, P = 0.006 for group D versus group E, P = not significant for group B versus group E, P = 0.042 for group C versus group F). Serum endotoxin levels were similar between groups at all sampling times; TNF-alpha and MDA levels in groups B, C, E, and F decreased significantly over follow-up. The numbers of viable bacterial cells in the infected kidney were similar among the groups; those in the liver, spleen, lungs, and mesenteral lymph nodes were significantly decreased in groups B, E, and F compared to those in groups A and D. It is concluded that a prolongation of survival in animals with experimental sepsis caused by multidrug-resistant P. aeruginosa was achieved after coadministration of clarithromycin and amikacin and that the increased survival was probably attributable to the immunomodulatory properties of clarithromycin.