A total of 68 patients were enrolled in the study. Patients were hospitalized in the second Department of Critical Care Medicine and in the fourth Department of Internal Medicine of ATTIKON University Hospital in Athens. The study was approved by the Ethics Committee of the hospital. Written informed consent was provided by patients or their relatives. All patients were older than 18 years. Exclusion criteria included neutropenia (≤500 neutrophils/μl), HIV infection or oral intake of corticosteroids at a dose equal to or higher than 1 mg/kg equivalent prednisone for at least one month.

All sequential admissions with sepsis, severe sepsis or septic shock were screened for enrolment during the period January 2006 to June 2007. Patients finally enrolled were those with septic syndrome due to VAP and those with septic syndrome caused by other types of infection, namely acute pyelonephritis, primary bacteremia, intraabdominal infection, community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP), provided that they were well-matched to patients with VAP by age, sex, underlying conditions and disease severity.

Sepsis was defined as any microbiologically documented or clinically diagnosed infection accompanied by at least two of the following: core temperature above 38°C or below 36°C; pulse rate above 90 beats/minute; respiratory rate above 20 breaths/minute or partial pressure of carbon dioxide (pCO2) below 32 mmHg; and leukocytosis (white blood cells (WBC) >12,000 cells/μl) or leukopenia (WBC <4000 cels/μl) or presence of immature forms above 10% of total WBC count 910.

Severe sepsis was defined as sepsis aggravated by the acute dysfunction of at least one organ. Acute organ dysfunction was defined as follows: acute respiratory distress syndrome, as any value of partial oxygen pressure/fraction of inspired oxygen (pO2/FiO2) less than 200 and diffuse bilateral infiltrations in chest X-ray; acute renal failure, as the production of less than 0.5 ml urine/kg/hour for at least two hours, provided that the negative fluid balance of the patient was corrected; metabolic acidosis, as any pH below 7.30 or any base deficit above 5 mEq/l and serum lactate at least more than twice the upper normal value; and acute coagulopathy, as any platelet count below 100,000 cells/μl or International Normalized Ratio above 1.5 910.

Septic shock was defined as sepsis accompanied by systolic arterial pressure lower than 90 mmHg necessitating the administration of inotropic agents 910.

Diagnosis of VAP was established if all the following criteria were met: intubation and mechanical ventilation for at least 48 hours prior to diagnosis; a new or progressive infiltrate on a chest X-ray; purulent tracheobronchial secretions; and Clinical Pulmonary Infection Score (CPIS) more than six 11121314.

Acute pyelonephritis was diagnosed in any patient presenting with all the following: fever, lumbar tenderness or radiological findings consistent with acute pyelonephritis, and pyuria defined as more than 10 WBCs/high power field or positive (+3) dipstick of urine for leukocyte esterase 15.

A diagnosis of intraabdominal infection was made in patients with temperature above 38°C or below 36°C, leukocytosis (WBC >12,000 cells/μl) and radiological findings consistent with an intraabdominal infection 15.

Primary bacteremia was defined as any positive blood culture for Gram-positive or Gram-negative microorganisms in the absence of any well-defined focus of infection, including intravascular-access devices 15.

Criteria required for the diagnosis of CAP and HAP included the presence of a new infiltrate on a chest X-ray along with two of the following: fever, leukocytosis or leukopenia, and/or purulent sputum. Pneumonia was considered as: CAP whenever the patient did not report any past hospitalization for the past 90 days or stay in a long-term care facility; or HAP when presenting more than 48 hours after hospital admission in any patient not requiring mechanical ventilation 141516.

Patients were followed up for 28 days. A complete diagnostic work-up was performed comprising history, clinical examination, blood cell counts and biochemistry, blood cultures, chest X-ray, and chest and/or abdominal computed tomography scans if considered necessary. Quantitative cultures of urine or tracheobronchial secretions (TBS) were performed and interpreted as previously described 17 depending on the patient's underlying infection. Within the first 24 hours of the advent of signs of sepsis, 15 ml of heparinized peripheral venous blood was sampled after puncture of one forearm vein under sterile conditions.

For the flow cytometric analysis, red blood cells were lysed with ammonium chloride 1 mM and WBCs were washed three times with PBS (pH 7.2; Merck, Darmstadt, Germany). WBCs were then stained with fluorocolour-conjugated monoclonal antibodies against CD3, CD4, CD8, CD(16+56), CD19 and with the protein annexin-V and propidium iodine (PI) (Immunotech, Marseille, France), and incubated for 15 minutes in the dark. Fluorocolours used were fluorescein isothiocyanate (FITC; emission 525 nm; Immunotech, Marseille, France), phycoerythrin (PE; emission 575 nm; Immunotech, Marseille, France), ECD (emission 613 nm, Immunotech, Marseille, France) and PC5 (emission 670 nm, Immunotech, Marseille, France). The following combinations were applied: anti-CD3(FITC)/CD4(PE), anti-CD3(FITC)/CD8(PE), anti-CD3(FITC)/CD(16+56)(PE), anti-CD19(FITC), annexin-V(FITC)/CD4(PE)/PI (PC5), and annexin-V(FITC)/anti-CD8(PE)/PI(PC5). Cells that stained positive for annexin-V and negative for PI were considered apoptotic.

Flow-cytometric analysis was performed on an EPICS XL/MSL flow cytometer (Beckman Coulter Co, Miami, FL, USA) with gating for mononuclears based on their characteristic forward and side scattering.

For the isolation of monocytes, blood was layered over Ficoll Hypaque (Biochrom, Berlin, Germany) and centrifuged. Isolated peripheral blood mononuclear cells (PBMCs) were washed three times with PBS (pH 7.2) and incubated with RPMI 1640 media enriched with 10% fetal bovine serum (FBS) and 2 mM glutamine, 100 U/ml penicillin G and 0.1 mg/ml streptomycin (Sigma Co, St Louis, MO, USA) in 75 cm³ flasks. After one hour of incubation at 37°C in 5% CO2, non-adherent cells were removed. Adherent monocytes were thoroughly washed with Hank's solution (Biochrom, Berlin, Germany), harvested with a 0.25% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA) solution (Biochrom, Berlin, Germany). Their purity was more than 95% as defined after staining with anti-CD14 and analysis by a flow cytometer.

Isolated monocytes were counted in a Neubauer plate by trypan blue exclusion of dead cells, distributed in two wells of a 12-well plate and cultured with RPMI 1640 media supplemented with 10% FBS and 2 mM glutamine with or without the addition of 10 ng/ml of purified endotoxin (lipopolysaccharide (LPS)) derived from Escherichia coli O155:H5 (Sigma Co, St Louis, MO, USA). After incubation for 24 hours at 37°C in a 5% CO2 atmosphere, supernatants were collected and stored at -70°C until assayed for cytokines.

Estimation of TNFα and IL-6 in supernatants was performed by an ELISA (Diaclone, Paris, France). Lowest detection limits were 15.75 pg/ml for TNFα and 6.25 pg/ml for IL-6. Concentrations were adjusted as pg/10⁴ live cells.

In an attempt to explain our findings, PBMCs of healthy volunteers were exposed to isolates of TBS from patients with VAP and to blood isolates of patients with bloodstream infections enrolled in this study. Current theories attribute pathogenesis of VAP to the aspiration of microbes colonizing the oropharynx in the lower respiratory tract. According to the theories, bacteria replicate gradually and when their growth surpasses a certain threshold then VAP develops 1819. In an attempt to reproduce the above sequence of events in vitro, PBMCs were isolated from five healthy volunteers as described above. They were distributed in wells of a 12-well plate in RPMI 1640 media supplemented with 10% FBS and 2 mM glutamine, 100 U/ml penicillin G and 0.1 mg/ml streptomycin (Sigma Co, St Louis, MO, USA). These PBMCs were stimulated by four different isolates: one of Acinetobacter baumannii and another of Pseudomonas aeruginosa isolated at a count of 1 × 10⁶ cfu/ml or more from TBS of two different patients with VAP; and one of A. baumannii and another of P. aeruginosa isolated from blood of two different patients with bacteremia. All isolates were grown for 12 hours in Mueller-Hinton broth (Oxoid Ltd, London, UK) in a shaking-water bath at 37°C. Then a log-phase inoculum of 5 × 10⁷ cfu/ml was prepared in Mueller-Hinton broth using the 0.5 standard of the McFarland climax. Appropriate amounts of that inoculum were used for cell stimulation in four different patterns, as follows.

Pattern A was non-stimulated PBMCs incubated for 4.75 hours in growth medium at 37°C in 5% CO2.

Pattern B was sequential stimulation in three steps mimicking pathogenesis of VAP. In the first step, PBMCs were exposed for 15 minutes at 37°C in 5% CO2 in 1 × 10³ cfu/ml of each of the VAP pathogens. Then the plate was centrifuged, supernatants were discarded and the cell pellet was dissolved in 2.4 ml of growth medium. In the second step, the same procedure as in the first step was repeated after two hours. In the third step, after two hours of incubation at 37°C in a 5% CO2 atmosphere, PBMCs were stimulated with 1 × 10⁶ cfu/ml of each of the two pathogens for 30 minutes. These inocula were selected for stimulation in an attempt to generate conditions of bacterial growth similar to those existing in patients with VAP. Then, the plate was centrifuged.

Pattern C was an abrupt stimulation with VAP pathogens. The first two steps of pattern B were performed but instead of stimulation with 1 × 10³ cfu/ml inoculum, Mueller-Hinton broth was added in the plates. The third step was repeated as in pattern B.

Pattern D was an abrupt stimulation with pathogens causing bacteremia mimicking the pathogenesis of bacteremia. After incubation for 4.15 hours at 37°C in 5% CO₂ PBMCs were exposed for 30 minutes to 1 × 10⁶ cfu/ml of each of the two pathogens causing bacteremia. Then the plate was centrifuged.

For all the above patterns, after centrifugation of the plate and removal of supernatants, adherent cells were harvested with a 0.25% trypsin/0.02% EDTA solution (Biochrom, Berlin, Germany). Flow cytometric analysis of apoptosis was performed after staining collected cells with annexin-V(FITC)/anti-CD4(PE)/PI(PC5) and annexin-V(FITC)/anti-CD14(PE)/PI(PC5). To exclude debris or red blood cells, collected cells were also stained with anti-CD45 (ECD); their purity was more than 95%.

Septic patients were divided in two groups, those with VAP and those suffering from other infections. Results were expressed as means (standard deviation) for parametric variables and as medians (interquartile range) for non-parametric variables. Comparisons of baseline quantitative characteristics between groups were performed by the Student's t-test and of baseline qualitative characteristics by the chi-squared test. Comparisons of non-parametric quantitative characteristics between groups were performed by the Mann-Whitney U test.

Both groups of patients were additionally divided in two subgroups each, depending on the positive response of monocytes to LPS-stimulation with or without TNFα production. A more than five-fold increase of TNFα production following stimulation was considered a positive response. Survival of two subgroups was estimated by Kaplan-Meier analysis; comparisons were performed by the log-rank test.

Apoptosis of each pattern of stimulation of PBMCs was expressed by means (standard error); comparisons were performed by analysis of variance after Bonferroni correction. Any value of P below 0.05 was considered significant.