Epidemiologic studies of nosocomial bacterial pneumonia in patients requiring mechanical ventilation have been limited because of the poor reliability of diagnosis procedures in this setting. To determine prognostic and descriptive factors of ventilator-associated (V-A) pneumonia, we prospectively studied 567 patients who had been receiving mechanical ventilation for more than 3 days in our unit. Fiberoptic bronchoscopy using a protected specimen brush (PSB) was performed on each patient suspected of having pneumonia because of the presence of a new pulmonary infiltrate and purulent tracheal secretions. The diagnosis of V-A pneumonia was retained only if PSB specimens yielded greater than 10(3) cfu/ml of at least one microorganism, unless this result was established to be a false positive result on follow-up. V-A pneumonia developed in 49 patients for a total of 52 episodes (9%). The actuarial risk of V-A pneumonia was 6.5% at 10 days, 19% at 20 days, and 28% at 30 days of ventilation. Patients with pneumonia were significantly older (65 versus 57 yr of age, p less than 0.01) and more frequently had severe underlying illnesses (24 versus 10%, p less than 0.01) than did patients without pneumonia. A total of 84 microorganisms (51 gram-negative and 33 gram-positive) were isolated in significant concentrations from PSB specimens. Pseudomonas aeruginosa and Staphylococcus aureus were involved in 31 and 33% of these pneumonias, respectively. Forty percent of all specimens yielded a polymicrobial flora with more than one potential pathogen.(ABSTRACT TRUNCATED AT 250 WORDS)

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To determine the usefulness of samples obtained by bronchoscopy using a protected specimen brush and evaluated by quantitative culture techniques in establishing the diagnosis of nosocomial pneumonia in patients requiring mechanical ventilation, we prospectively studied 147 ventilated patients suspected of having nosocomial pneumonia because of the presence of a new pulmonary infiltrate and purulent tracheal secretions. Positive cultures of protected brush specimens (greater than 10(3) cfu/ml) were found in only 45 patients (31%). Subsequent follow-up confirmed the diagnosis of pneumonia in 34 of 45 patients, and in only 4 of 45 patients was a positive culture firmly established to be a false positive result. No patient with less than 10(3) cfu/ml was subsequently shown to have had pneumonia, and the diagnosis was definitely excluded in 72 of 102 patients by the absence of pneumonia at autopsy or recovery without antibiotic therapy. In contrast, when 16 clinical variables were evaluated by stepwise logistic regression analysis, no combination could be identified that was useful in distinguishing patients with bacterial pneumonia. Furthermore, when the actual costs of evaluation and therapy of our patients were compared with the projected costs entailed in treating all patients suspected of having pneumonia with antibiotics, evaluation using the protected specimen brush and quantitative cultures was less expensive after only 6 days of treatment. These results suggest that the appearance of pulmonary infiltrates and purulent tracheal secretions does not result from bacterial pneumonia in a majority of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

To identify risk factors for and prognostic indicators of the nosocomial acquisition of multiresistant Acinetobacter baumannii in an intensive care unit, we prospectively studied 40 patients: 13 who were infected with this organism and 27 who were colonized. Isolates were identified by pulsed-field gel electrophoresis; the infected/colonized patients were compared with 348 noninfected, noncolonized patients by logistic regression analysis and with matched historical controls in a cohort study. The severity of illness (evaluated by the APACHE II score; P < .05) and previous infection (P < .001) were retained as independent risk factors for acquiring A. baumannii. Logistic regression analysis selected a high APACHE II score (P < .01) and the acquisition of A. baumannii (P < .01) as factors independently associated with death. The acquisition of A. baumannii was associated not only with high mortality but also with a length of stay on the intensive care unit in excess of that due to the underlying disease alone; specifically, the attributable mortality was 25%, with a risk ratio for death of 2.0 (95% confidence interval, 1.11-3.62), and the duration of stay for infected/colonized patients was 10.3 days longer than that for controls (P < .001).

The enteropathogenic bacterium Yersinia enterocolitica counteracts host defense mechanisms by interfering with eukaryotic signal transduction pathways. In this study, we investigated the mechanism by which Y. enterocolitica prevents macrophage tumor necrosis factor-␣ (TNF␣) production. Murine J774A.1 macrophages responded to Y. enterocolitica infection by rapid activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH 2 -terminal kinase (JNK). However, after initial activation, the virulent Y. enterocolitica strain harboring the Y. enterocolitica virulence plasmid caused a substantial decrease in ERK1/2 and p38 tyrosine phosphorylation. Simultaneously, the virulent Y. enterocolitica strain gradually suppressed phosphorylation of the transcription factors Elk-1, activating transcription factor 2 (ATF2), and c-Jun, indicating time-dependent inhibition of ERK1/2, p38, and JNK kinase activities, respectively. Analysis of different Y. enterocolitica mutants revealed that (i) MAPK inactivation parallels the inhibition of TNF␣ release, (ii) the suppressor effect on TNF␣ production, which originates from the lack of TNF␣ mRNA, is distinct from the ability of Y. enterocolitica to resist phagocytosis and to prevent the oxidative burst, (iii) the tyrosine phosphatase YopH, encoded by the Y. enterocolitica virulence plasmid, is not involved in the decrease of ERK1/2 and p38 tyrosine phosphorylation or in the cytokine suppressive effect. Altogether, these results indicate that Y. enterocolitica possesses one or more virulence proteins that suppress TNF␣ production by inhibiting ERK1/2, p38, and JNK kinase activities.

IL-4 and IL-13 are related cytokines which induce both pro-and anti-in¯ammatory e ects depending on the cell type they act upon and the nature of the receptors expressed. The type I receptor complex is composed of the IL-4Ra and gc and only binds IL-4, whereas, in the type II receptor, IL-4Ra dimerizes with IL-13Ra1 upon either IL-4 or IL-13 binding. Another ligand binding chain potentially implicated in the IL-4/IL-13 receptor has been described, the IL-13Ra2, but the regulation of its expression and its role in IL-4/IL-13 transduction is poorly understood. In this study we report that IL-4 and IL-13 upregulate IL-13Ra2 at both the mRNA and protein levels in the keratinocyte cell line HaCaT. In these cells, IL-4 or IL-13 were shown to activate the Janus Kinases JAK1 and JAK2, the transcription factor STAT6, and the ERK and p38 mitogen-activated protein kinases. We show that IL-4 or IL-13-induced IL-13Ra2 mRNA expression was inhibited by the ERK inhibitor U0126, the JAK inhibitor AG490 and, to a lesser extent, the p38 MAPK inhibitor SB203580. Moreover, expression of a constitutive active mutant of STAT6 alone did not modify IL-13Ra2 mRNA expression, but potentiated the e ects of IL-4 or IL-13 on IL-13Ra2 expression. The constitutive active mutants of MEK1 or MKK6 increased the level of expression of IL-13Ra2 mRNA even in absence of stimulation. Our ®ndings demonstrate, for the ®rst time, that IL-4 and IL-13 can induce IL-13Ra2 expression in keratinocytes, and that the ERK and p38 MAPK together with JAK2 and STAT6 play a critical role in this process. Oncogene (2001) 20, 6660 ± 6668.

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Reactive oxygen species modulate major cellular functions by mechanisms which are still poorly understood. Recently, H,O, has been reported to stimulate the activity of the mitogen-activated protein kinases (MAPKs) ERK and JNK, and the expression of the proto-oncogenes c-fos and c-jun. As their expression is enhanced by H,O, in astrocytes, we studied whether these MAPKs were stimulated by H,O, in primary cultured astrocytes. The result was positive, a maximum of stimulation being reached with 200 pM H,O, (0.3 pmol H,O,/cell) for both ERK and JNK. ERK was previously reported to stimulate cytosolic phospholipase A, phosphorylation and activity. H,O, stimulated the release of arachidonic acid in astrocytes, as already reported in other cell types. We found also that cPLA, phosphorylation was increased by H,O,. Moreover, the stimulation by H,O, of ERK and JNK was decreased by phospholipase A, activity inhibitors. When astrocytes were incubated first with eicosatetraynoic acid, a structural analogue competing in arachidonic acid metabolism, the stimulation of JNK by H,O, was also inhibited, suggesting the involvement of arachidonic acid metabolites. Cyclooxygenase or cytochrome P450 nionooxygenase inhibitors failed in decreasing the MAPK stimulation by HZ02, whereas lipoxygenase inhibitors completely abolished that of JNK. Mitogenicity has been reported to be stimulated by H,O, in other cell types. Although ERK was strongly and durably stimulated by 200 pM H,O, in astrocytes, at the same extent as by mitogenic growth factors, basal thymidine incorporation rate was decreased by more than 80% after 12-15 h. Moreover, the stimulation of thymidine incorporation induced by basic fibroblast growth factor was transiently abolished by H,O,. Furthermore, H,O, likely induced the expression of CLIOO/PACl/MKP-l, a dual specificity phosphatase which has been implicated in ERK and JNK inactivation in the nucleus. Finally, the prior treatment of astrocytes with MK886, a 5-lipoxygenaseactivating protein inhibitor, prevented JNK from stimulation, but did not prevent thymidine incorporation from inhibition, both induced by H,O,. These results strongly suggest an involvement of arachidonic acid and/or its metabolites in the stimulation of both ERK and JNK following the oxidative stress evoked by H,O,, which induced a cell cycle arrest probably independent of the stimulation of JNK.