Metallothioneins (MTs) are ubiquitous low-molecular-weight proteins with a high affinity for heavy metal ions such as zinc, copper and cadmium. MT over-expression has been associated with resistance against anticancer drugs. In the present study we investigated 86 cases (45 cases of tumour category pT1 and 41 of category pT2) of routinely fixed and paraffin-embedded primary breast carcinomas immunohistochemically with a monoclonal antibody to an epitope of MT shared by its I and II isoforms. Immunohistochemically demonstrated MT over-expression was found in the invasive components of 7 of 32 pT1 and 17 of 28 pT2 invasive ductal carcinomas, whereas all 26 invasive lobular carcinomas gave weak or negative results. Fourteen of 17 pT2 and 2 of 7 pT1 invasive ductal carcinomas with MT over-expression developed metastases during follow-up with poor prognostic outcome. In contrast only 3 of 11 pT2 and none of the 25 pT1 cases without MT over-expression had a poor clinical course (P < 0.001). It is concluded that MT over-expression is associated with significantly poor prognosis particularly in pT2 invasive ductal breast carcinomas.

OBJECTIVE To describe our experience with ketamine sedation during infant flexible fiberoptic bronchoscopy. DESIGN Retrospective chart review. Infants were sedated with midazolam and ketamine with or without fentanyl. The sedation regimen, final procedure performed, procedure duration, and complications were recorded. Complication rates between infants 6 months of age and between infants with upper vs lower airway symptoms were compared by chi(2) test with a contingency table. RESULTS Fifty-nine procedures were performed in 55 patients aged 6.1 +/- 3.1 months (mean +/- SD). Sedation was achieved with ketamine and midazolam (n = 30) or ketamine, midazolam, and fentanyl (n = 29). Bronchoscopy with BAL was performed in 44 patients and bronchoscopy alone in 3 patients. In 11 patients, severe upper airway obstruction and/or anomalies prevented subglottic passage of the bronchoscope. One patient could not be adequately sedated. There were no major complications. Minor complications occurred in 14 patients (23.7%), most commonly mild hypoxemia (n = 9). Brief central apnea developed in three patients. Complication rates were unaffected by age or indication for bronchoscopy. CONCLUSIONS Infant flexible fiberoptic bronchoscopy can be safely and effectively performed using ketamine sedation. Complications, especially mild hypoxemia, appear more common in infants, likely due to smaller airway diameter. Regardless of the sedative(s) used, additional vigilance is required when performing bronchoscopy in this population.

SummaryBackground : Flexible fiberoptic bronchoscopy is an important diagnostic tool for pediatric pulmonologists. Because of its favorable respiratory profile, ketamine has become a popular sedative for this procedure, but may be associated with unpleasant emergence reactions in the older child. Remifentanil is a newer, ultra‐short acting opioid that has been shown to provide effective sedation and cough suppression for fiberoptic bronchoscopy when combined with intermittent propofol boluses. However, delivery of these agents as a combined, single infusion has not been described.Methods : Children ≥2 years of age undergoing fiberoptic bronchoscopy were enrolled. Remifentanil was mixed in a single syringe with undiluted propofol giving final drug concentrations of 10 mg·ml−1 of propofol and 15–20 μg·ml−1 of remifentanil. Sedation was induced with a bolus of approximately 0.1 ml·kg−1 of this mixture and maintained by titrating the drip throughout the procedure. Vital signs, sedative effectiveness, recovery patterns, and complications were prospectively recorded.Results : Fifteen patients aged 9.0 ± 5.3 years were sedated. Sedation was induced with 1.2 ± 0.4 mg·kg−1 propofol (2.4 ± 0.8 μg·kg−1 remifentanil) and maintained with 4.1 ± 1.8 mg·kg−1·h−1 propofol (0.13 ± 0.06 μg·kg−1·min−1 remifentanil). Five patients received low‐dose ketamine to augment sedation. The maximal decrease in respiratory rate was 6.1 ± 5.3 b·min−1 (27.6 ± 21%) and no patient became hypoxemic. All procedures were completed easily without significant complication. Patients recovered to baseline 13.3 ± 8.5 min following infusion discontinuation.Conclusions : A remifentanil/propofol mixture provided effective sedation and rapid recovery in pediatric patients undergoing fiberoptic bronchoscopy.

Dyspnea, the subjective symptom feeling of breathlessness, is a common symptom in terminally ill patients with cystic fibrosis (CF). The palliation of the dyspnea is a reasonable goal to improve patient comfort as the progression of the disease worsens. We report the successful use of inhaled fentanyl for 3 days in a 17-year-old female with terminal CF lung disease, as measured by improved oxygenation and a reduction in the modified Borg score, and the subjective feeling of less air hunger reported by parents and patient.

Respiratory syncytial virus (RSV) infection is associated with epithelial cell death and vigorous inflammation. In mouse models, and in immunosuppressed patients, CD8+T cells are necessary for RSV clearance.In vitro,RSV has been shown to induce expression of several proteins on the respiratory epithelial cell, including RSV proteins, ICAM-1, and MHC class I, that can potentially interact with CD8+T cells in initiating apoptosis of the target cell. One mechanism of T-cell-directed cell death is the interaction of FasL on the CD8+T lymphocytes and Fas expressed on the target cell. In order to determine the ability of RSV to induce Fas on the respiratory epithelium, we studied the RSV infection of a human respiratory epithelial cell line (A549)in vitro.Fas mRNA and protein levels are increased two-to-fourfold following RSV infection, and transcriptional upregulation of Fas was demonstrated using promoter/reporter gene constructs. RSV infection directly resulted in cellular apoptosis, and the frequency of apoptotic cells was further increased by cross-linking with antibodies to Fas. These data demonstrate that RSV infection induces cellular apoptosis and suggest that interactions of surface Fas with T cells may further augment this processin vivo.

In an earlier study, we showed that a recombinant adenovirus vector with deletions in the E1 and E3 regions of the viral genome (AV1LacZ4) induces expression of interleukin (IL)-8 in A549 cells (a human respiratory cell line). IL-8 can be induced through several pathways, including activation by IL-1. We tested the hypothesis that the induction of IL-8 by the AV1LacZ4 adenovirus is accomplished by means of the IL-1/IL-8 activation pathway, which could be blocked by IL-1 receptor antagonist (IRAP). Viral infections of A549 cells were performed at a multiplicity of infection (MOI) of 50 in the presence and absence of IRAP (50 ng/ml). A549 cells were also stimulated with tumor necrosis factor (TNF)-alpha (100 ng/ml), a known stimulant of IL-8, in the presence and absence of IRAP. IL-8 expression was evaluated by Northern blot analysis and enzyme-linked immunosorbent assay. Levels of IL-8 protein and messenger RNA (mRNA) were greater in the infected cells than in the uninfected ones at 24, 48, and 96 h (P < 0.01). Virus-infected cells treated with IRAP expressed 75% less IL-8 mRNA and protein (P < 0.01) than did untreated cells, whereas IRAP pretreatment of TNF-alpha-stimulated cells did not affect IL-8 production. IL-1 production by the virus-infected cells was detectable by concentration of the supernatants and reverse transcription-polymerase chain reaction. We conclude that IL-8 is produced by virus vector-infected cells, partly through IL-1 activation that can be downregulated by IRAP.

The purpose of the studies described here was to test the hypothesis that overexpression of the human interleukin-1 receptor antagonist (IL-1ra) in the distal airway epithelia of mice would result in amelioration of the inflammatory effects of IL-1alpha. The coding region of the human IL-1ra gene was placed under transcriptional control of the 5' flanking region of the human SP-C gene. Transgenic mice were generated by pronuclear injection of the transgene and identified by Southern blot analysis of genomic DNA. RNA expression of the transgene was confirmed by Northern blot analysis. In order to determine whether expression of the transgene conferred protection against inflammatory stimuli, control and transgenic mice were treated with IL-1alpha by intratracheal instillation. Six hours after treatment, bronchoalveolar lavage was performed, which revealed a statistically significant decrease in the degree of neutrophilia in the transgenic mice as compared with control mice. Furthermore, there was a significant reduction in the whole-lung myeloperoxidase concentration. Reverse transcription-polymerase chain reaction analysis of whole-lung RNA revealed a significant reduction in the messenger RNA/beta-actin ratio of macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-2 in the transgenic animals as compared with controls. The results of these studies indicate that distal airway epithelial cell expression of human IL-1ra results in partial protection from IL-1alpha-induced airway inflammation and injury.

To determine the role of surfactant protein-A (SP-A) in host defense, the murine SP-A locus was targeted by homologous recombination to produce mice lacking SP-A. SP-A-/- and wild-type mice were infected with mucoid Pseudomonas aeruginosa by intratracheal instillation. Pulmonary bacterial loads were greater in SP-A-/- than in wild-type mice, with increased numbers of mucoid P. aeruginosa in lung homogenates at 6 and 24 h after infection. Pulmonary infiltration with polymorphonuclear leukocytes (PMN) was similar in both groups; however, an earlier influx of PMN into the lung occurred in the SP-A-/- mice. The number of bacteria phagocytosed by alveolar macrophages was decreased in the SP-A-/- mice at 1 h after infection. Superoxide-radical generation by PMN was similar for the SP-A-/- and wild-type mice, but nitrite levels were increased in SP-A-/- mice. Concentrations of tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2 (proinflammatory cytokines) were greater in bronchoalveolar lavage fluid at 2 h after infection in SP-A-/- mice. SP-A plays an important role in the pathogenesis of mucoid P. aeruginosa infection in the lung in vivo by enhancing macrophage phagocytosis and clearance of bacteria, and by modifying the inflammatory response.

A significant number of pulmonary exacerbations in patients with cystic fibrosis (CF) and asthma are associated with respiratory virus infections. The molecular mediators of this process are beginning to be understood. Viral infection of respiratory epithelial cultures in vitro leads to the production of intercellular adhesion molecule-1 (ICAM-1) (a ligand for inflammatory cell adhesion and activation) and a number of proinflammatory cytokines. Human gene therapy vectors derived from human adenoviruses (AV) are currently under evaluation for CF transmembrane regulator (CFTR) gene delivery to the airway epithelium of CF patients. However, studies in animal models using these AV vectors demonstrate pulmonary inflammation following AV exposure. Using an in vitro model, we examined the hypothesis that exposure of respiratory epithelial cells to AV vectors results in upregulation of ICAM-1 gene expression. Infections were performed using a replication-deficient, first-generation AV vector. A549 cells (a human pulmonary adenocarcinoma cell line) were exposed to AV at multiplicity of infection of 50-150 plaque-forming units/cell (resulting in > 90% of cells expressing the reporter gene by 48 hr following exposure). Measurements of ICAM-1 expression were made at time intervals following virus exposure using enzyme immunoassay, flow cytometry, and Northern blot analysis. Cell-bound ICAM-1 was significantly increased 96 hr following vector exposure, two to four times control, p < 0.001). The AV-exposed A549 cells also supported increased levels of adhesion of activated neutrophils 96 hr following AV exposure (four times control, p < 0.001) that was blocked by antibody to CD18. AV exposure of A549 monolayers increases expression of biologically active ICAM-1. Strategies to minimize host cellular proinflammatory responses to the replication-deficient AV vectors may improve their safety for gene therapy.

Eosinophilic infiltration and damage to airway epithelium are characteristic features of asthma. To assess possible interactions between eosinophils and airway epithelium, Percoll-purified human peripheral blood eosinophils were evaluated for their ability to adhere to respiratory epithelial cell (REC) cultures. REC (an immortalized cell line, A549, and primary bronchial epithelial cells) were grown in 96-well tissue culture plates, treated with proinflammatory cytokines (TNF-alpha or IL-1 beta), and eosinophil adhesion to these tissues was determined. Cytokine treatment of the REC cultures significantly increased expression of intercellular adhesion molecule-1 (ICAM-1) (P < 0.01). Eosinophils demonstrated a variable baseline adhesion to untreated REC which was then significantly increased following activation with phorbol myristate acetate (PMA) (P < 0.01). Furthermore, treatment of REC monolayers with TNF-alpha or IL-1 beta significantly increased adhesion of PMA-stimulated eosinophils (P < 0.01). To delineate the adhesion proteins involved in the cell-cell interactions, assays were performed in the presence of specific blocking monoclonal antibodies to eosinophil CD18, CD11a, or CD11b, and REC ICAM-1 molecules. Blocking antibodies to ICAM-1 had no significant effect on levels of eosinophil adhesion. In contrast, antibodies to CD18, CD11a, and CD11b significantly decreased (P < 0.01) eosinophil adhesion, thus demonstrating pivotal roles for the CD11/CD18 (beta 2) integrins, but not necessarily for ICAM-1, in interactions between the REC and eosinophils. These data demonstrate that TNF-alpha and IL-1 beta increase eosinophil adhesion to human respiratory epithelial cell cultures by induction of ligands recognized by eosinophil beta 2 integrins.

The interrelated systems which control proteolysis may go into disequilibrium during pregnancy so that a renin-like enzyme in the plasma produces effects such as the early clinical manifestations of pre-eclampsia; the overactivity of the reninlike enzyme may in turn lead to undue activity of other physiological systems including those of the coagulation and complement systems.