Effect of clarithromycin in experimental empyema by multidrug-resistant Pseudomonas aeruginosa.

Evidence from a recent randomized study of our group suggests that intravenous clarithromycin resulted in earlier resolution of ventilator-associated pneumonia. The need to understand the mechanism of action of clarithromycin guided to the study of a model of experimental empyema by multidrug-resistant Pseudomonas aeruginosa in 40 rabbits. Animals were randomized into controls (group A); treatment with clarithromycin (group B); treatment with piperacillin/tazobactam (group C); and treatment with both agents (group D). Pleural fluid was collected at regular time intervals for quantitative culture, estimation of cell apoptosis and of concentrations of tumour necrosis factor-alpha (TNFα). After 7 days, animals were euthanized for estimation of tissue growth. Bacterial growth in the pleural fluid of group D was significantly decreased compared with the other groups on day 5. Lung growth of group D was lower than group A. That was also the case of cytokine stimulation by pleural fluid samples on U937 monocytes. It is concluded that administration of clarithromycin enhanced the antimicrobial efficacy of piperacillin/tazobactam and decreased bacterial growth in the pleural fluid and in tissues. It also attenuated the pro-inflammatory phenomena induced by the ß-lactam.

Effect of clarithromycin in inflammatory markers of patients with ventilator-associated pneumonia and sepsis caused by Gram-negative bacteria: results from a randomized clinical study.

One recent, double-blind, randomized clinical trial with 200 patients showed that clarithromycin administered intravenously for 3 days in patients with ventilator-associated pneumonia (VAP) accelerated the resolution of pneumonia and decreased the risk of death from septic shock and multiple organ dysfunctions (MODS). The present study focused on the effect of clarithromycin on markers of inflammation in these patients. Blood was drawn immediately before the administration of the allocated treatment and on six consecutive days after the start of treatment. The concentrations of circulating markers were measured. Monocytes and neutrophils were isolated for immunophenotyping analysis and for cytokine stimulation. The ratio of serum interleukin-10 (IL-10) to serum tumor necrosis factor alpha (TNF-α) was decreased in the clarithromycin group compared with the results in the placebo group. Apoptosis of monocytes was significantly increased on day 4 in the clarithromycin group compared with the rate of apoptosis in the placebo group. On the same day, the expression of CD86 was increased and the ratio of soluble CD40 ligand (sCD40L) to CD86 in serum was unchanged. The release of TNF-α, IL-6, and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) by circulating monocytes after stimulation was greater in the clarithromycin group than in the placebo group. The expression of TREM-1 on monocytes was also increased in the former group. These effects were pronounced in patients with septic shock and MODS. These results suggest that the administration of clarithromycin restored the balance between proinflammatory versus anti-inflammatory mediators in patients with sepsis; this was accompanied by more efficient antigen presentation and increased apoptosis. These effects render new perspectives for the immunotherapy of sepsis.

An early circulating factor in severe sepsis modulates apoptosis of monocytes and lymphocytes.

We hypothesized that a factor may circulate in serum early during sepsis, modulating apoptosis of monocytes and lymphocytes. Serum was collected from 20 healthy volunteers and from 48 patients with severe sepsis/shock within 12 h from signs of the first failing organ. PBMCs were isolated from 20 healthy volunteers and incubated with collected sera. Apoptosis and expression of CD95 were determined by flow cytometry; experiments were run in the presence of caspase-8 and caspase-9 inhibitors and of CaCl(2). Activity of caspase-3 was determined in cell lysates by a chromogenic kinetic assay. Incubation with serum of patients induced apoptosis of CD4 lymphocytes and inhibited apoptosis of CD14 monocytes. This was attenuated after diluting serum or mixing with healthy serum. Activity of caspase-3 was consistent with these findings. Induced apoptosis of CD4 lymphocytes was greater among nonsurvivors, and it was inhibited in the presence of caspase inhibitors. Inhibitors did not modify the effect of patients' serum on apoptosis of CD14 monocytes. CaCl(2) reversed the inhibitory effect on apoptosis of CD14 moncytes. The above findings support the hypothesis for the existence of an early circulating factor in severe sepsis/shock, modulating apoptosis of CD4 lymphocytes and of CD14 monocytes by interaction with the two apoptotic pathways.

Decrease of CD4-lymphocytes and apoptosis of CD14-monocytes are characteristic alterations in sepsis caused by ventilator-associated pneumonia: results from an observational study.

INTRODUCTION: The present study aimed to investigate changes of the immune response between sepsis due to ventilator-associated pneumonia (VAP) and sepsis due to other types of infections. METHODS: Peripheral venous blood was sampled from 68 patients with sepsis within 24 hours of diagnosis; 36 suffered from VAP; 32 from other nosocomial infections, all well-matched for severity, age and sex. Blood monocytes were isolated and cultured with/without purified endotoxin (lipopolysaccharide (LPS)). Estimation of tumour necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) in cultures' supernatants was done by an enzyme immunoassay. Flow cytometry was used to determine subpopulations of mononuclear cells and apoptosis. To mimic pathogenesis of VAP, mononuclear cells of healthy volunteers were progressively stimulated with increased inocula of pathogens; apoptosis was determined. RESULTS: In patients with VAP, the absolute number of CD3(+)/CD4(+) lymphocytes was significantly lower (P = 0.034) and apoptosis of isolated monocytes was increased (P = 0.007) compared to other infections. TNFalpha and IL-6 production from LPS-stimulated monocytes was lower in patients with VAP-related sepsis than with sepsis due to other infections. Apoptosis of monocytes was induced after in vitro stimulation of mononuclear cells by a mechanism mimicking VAP. CONCLUSIONS: Decrease of CD4-lymphocytes and immunoparalysis of monocytes are characteristic alterations of sepsis arising in the field of VAP.

Monocytes in systematic inflammatory response syndrome: differences between sepsis and acute pancreatitis.

AIM: To unravel the differences between systematic inflammatory response syndrome (SIRS) of acute pancreatitis compared to the same syndrome in sepsis. METHODS: Twenty-five patients were enrolled, 12 with sepsis and 13 acute pancreatitis. After diagnosis 20 mL blood was sampled. Half were assayed for isolation of monocytes and 10 mL was centrifuged for serum test of tumor necrosis factor alpha (TNFalpha and interleukin-6 (IL-6). Half of monocytes were incubated in the presence of patients' serum and supernatants were collected. The other half was treated for estimation of optical photometry under caspase-3 inhibition. TNFalpha and IL-6 were estimated by an enzyme immunoassay. RESULTS: median+/-SE of serum IL-6 in septic patients and acute pancreatitis patients was 192.30+/-35.40 ng/L and 21.00+/-16.05 ng/L, respectively (P<0.01). Respective values of caspase-3 were 0.94+/-0.17 pmol/min 10(4) cells and 0.34+/-0.09 pmol/min 10(4) cells (P<0.05). IL-6 of monocyte supernatants of patients with sepsis was significantly increased after addition of patients' serum, while that of patients with acute pancreatitis did not show significant difference. CONCLUSION: The data have shown that monocyte activity is different between acute pancreatitis and sepsis. This phenomenon might be explained as a different pathway to the pro-inflammatory cytokines release or could be a novel anti-inflammatory response in acute pancreatitis.

Kinetics of progenitor hemopoetic stem cells in sepsis: correlation with patients survival?

BACKGROUND: Current theories underline the crucial role of pro-inflammatory mediators produced by monocytes for the pathogenesis of sepsis. Since monocytes derive from progenitor hemopoetic cells, the kinetics of stem cells was studied in peripheral blood of patients with sepsis. METHODS: Blood was sampled from 44 patients with septic syndrome due to ventilator-associated pneumonia on days 1, 3, 5 and 7 upon initiation of symptoms. Concentrations of tumour necrosis factor-alpha (TNFalpha), interleukin (IL)-6, IL-8 and G-CSF were estimated by ELISA. CD34/CD45 cells were determined after incubation with anti-CD45 FITC and anti-CD34 PE monocloncal antibodies and flow cytometric analysis. Samples from eight healthy volunteers served as controls. RESULTS: Median of CD34/CD45 absolute count of controls was 1.0/mul. Respective values of the total study population were 123.4, 112.4, 121.5 and 120.9/mul on days 1, 3, 5 and 7 (p < 0.0001 compared to controls). Positive correlations were found between the absolute CD34/CD45 count and the absolute monocyte count on days 1, 5 and 7. Survival was prolonged among patients with less than 310/microl CD34/CD45 cells on day 1 compared to those with more than 310/microl of CD34/CD45 cells (p: 0.022). Hazard ratio for death due to sepsis was 5.47 (p: 0.039) for CD34/CD45 cells more than 310/microl. Median IL-6 on day 1 was 56.78 and 233.85 pg/ml respectively for patients with less than 310/microl and more than 310/microl CD34/CD45 cells (p: 0.021). CONCLUSION: Stem cells are increased in peripheral blood over all days of follow-up compared to healthy volunteers. Patients with counts on day 1 less than 310/microl are accompanied by increased survival compared to patients with more than 310/microl.