Detrimental role of the airway mucin Muc5ac during ventilator-induced lung injury.

Acute lung injury (ALI) is associated with high morbidity and mortality in critically ill patients. At present, the functional contribution of airway mucins to ALI is unknown. We hypothesized that excessive mucus production could be detrimental during lung injury. Initial transcriptional profiling of airway mucins revealed a selective and robust induction of MUC5AC upon cyclic mechanical stretch exposure of pulmonary epithelia (Calu-3). Additional studies confirmed time- and stretch-dose-dependent induction of MUC5AC transcript or protein during cyclic mechanical stretch exposure in vitro or during ventilator-induced lung injury in vivo. Patients suffering from ALI showed a 58-fold increase in MUC5AC protein in their bronchoalveolar lavage. Studies of the MUC5AC promoter implicated nuclear factor κB in Muc5ac induction during ALI. Moreover, mice with gene-targeted deletion of Muc5ac⁻/⁻ experience attenuated lung inflammation and pulmonary edema during injurious ventilation. We observed that neutrophil trafficking into the lungs of Muc5ac⁻/⁻ mice was selectively attenuated. This implicates that endogenous Muc5ac production enhances pulmonary neutrophil trafficking during lung injury. Together, these studies reveal a detrimental role for endogenous Muc5ac production during ALI and suggest pharmacological strategies to dampen mucin production in the treatment of lung injury.

Topical tacrolimus in the management of lichen sclerosus.

The effective management of vulval lichen sclerosus (LS) currently depends upon the use of topical steroids and emollients. There are concerns with regard to the long-term toxicity of potent steroids and therefore is a need to consider effective alternatives. Immunomodulatory macrolactams offer an alternative to steroids in the management of some other inflammatory skin disorders and it would seem reasonable therefore to assess their activity in LS. This pilot study of 16 histologically confirmed cases of LS suggests that macrolactams have a positive pharmacological effect.

Underlying chronic granulomatous disease in a patient with bronchocentric granulomatosis.

We present a case of bronchocentric granulomatosis in a woman with no history of asthma who was colonised with Aspergillusfumigatus. A family history of chronic granulomatous disease prompted further testing that demonstrated severely depressed neutrophil oxidant production and gp91(phox) deficiency compatible with the X linked carrier state of chronic granulomatous disease. Only one report of the association of these two rare diseases has previously appeared in the literature. We postulate that an ineffective immune response led to the prolonged colonisation of Afumigatus resulting in a hypersensitivity reaction that was manifest clinically as bronchocentric granulomatosis.

Insulin, insulin-like growth factor-I, and platelet-derived growth factor activate extracellular signal-regulated kinase by distinct pathways in muscle cells.

We have investigated the signaling pathways initiated by insulin, insulin-like growth factor-1 (IGF-I), and platelet-derived growth factor (PDGF) leading to activation of the extracellular signal-regulated kinase (ERK) in L6 myotubes. Insulin but not IGF-I or PDGF-induced ERK activation was abrogated by Ras inhibition, either by treatment with the farnesyl transferase inhibitor FTP III, or by actin disassembly by cytochalasin D, previously shown to inhibit Ras activation. The protein kinase C (PKC) inhibitor bisindolylmaleimide abolished PDGF but not IGF-I or insulin-induced ERK activation. ERK activation by insulin, IGF-I, or PDGF was unaffected by the phosphatidylinositol 3-kinase inhibitor wortmannin but was abolished by the MEK inhibitor PD98059. In contrast, activation of the pathway involving phosphatidylinositol 3-kinase (PI3k), protein kinase B, and glycogen synthase kinase 3 (GSK3) was mediated similarly by all three receptors, through a PI 3-kinase-dependent but Ras- and actin-independent pathway. We conclude that ERK activation is mediated by distinct pathways including: (i) a cytoskeleton- and Ras-dependent, PKC-independent, pathway utilized by insulin, (ii) a PKC-dependent, cytoskeleton- and Ras-independent pathway used by PDGF, and (iii) a cytoskeleton-, Ras-, and PKC-independent pathway utilized by IGF-I.

Neutrophil-mediated epithelial injury during transmigration: role of elastase.

Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, beta-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases.

Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(-/-) mice.

Progressive pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the molecular basis for this susceptibility remains incompletely understood. To study this problem, we developed a model of chronic pneumonia by repeated instillation of a clinical isolate of Burkholderia cepacia (genomovar III, ET12 strain), an opportunistic gram-negative bacterium, from a case of CF into the lungs of Cftr (m1unc-/-) (Cftr(-/-)) and congenic Cftr(+/+) controls. Nine days after the last instillation, the CF transmembrane regulator knockout mice showed persistence of viable bacteria with chronic severe bronchopneumonia while wild-type mice remained healthy. The histopathological changes in the lungs of the susceptible Cftr(-/-) mice were characterized by infiltration of a mixed inflammatory-cell population into the peribronchiolar and perivascular spaces, Clara cell hyperplasia, mucus hypersecretion in airways, and exudation into alveolar airspaces by a mixed population of macrophages and neutrophils. An increased proportion of neutrophils was observed in bronchoalveolar lavage fluid from the Cftr(-/-) mice, which, despite an increased bacterial load, demonstrated minimal evidence of activation. Alveolar macrophages from Cftr(-/-) mice also demonstrated suboptimal activation. These observations suggest that the pulmonary host defenses are compromised in lungs from animals with CF, as manifested by increased susceptibility to bacterial infection and lung injury. This murine model of chronic pneumonia thus reflects, in part, the situation in human patients and may help elucidate the mechanisms leading to defective host defense in CF.

Neutrophil activation and acute lung injury.

Neutrophils are considered to be central to the pathogenesis of most forms of acute lung injury (ALI). For the sake of clarity, neutrophil involvement in ALI can be conceptualized as consisting of sequential stages, beginning with their sequestration in the pulmonary microvasculature, followed by adhesion and activation, and culminating in the production of a microbicidal or "effector" response, such as the generation of reactive oxygen species or release of proteolytic enzymes. Great strides have been made in elucidating these various stages of neutrophil involvement. Recent studies have focused on the intracellular signaling pathways that govern neutrophil activation and have elucidated complex cascades of kinases and other intracellular signaling molecules that allow for amplication of the neutrophil response, yet simultaneously confer specificity of a response. We believe that the inflammatory response in ALI may initially be adaptive, such as the pivotal role played by neutrophils in a bacterial or fungal infection. Ultimately, it is the persistence or the dysregulation of neutrophil activation that may lead to ALI. An increased understanding of how neutrophils function will facilitate the design of therapeutic strategies that retain the beneficial aspects of the inflammatory response, while avoiding unnecessary tissue damage.

Difficult asthma: consider all of the possibilities.

Asthma is a common respiratory disease that can often be managed successfully. However, there are patients that do not respond to the maximum doses of standard therapy and subsequently have a reduced quality of life. Many factors can contribute to a failure to respond to treatment, and a comprehensive approach is important when assessing and evaluating these patients. This report describes a patient referred for 'difficult to control asthma' who had multiple emergency department visits and hospitalizations. In addition to a history of wheezing, spirometry showed impaired flow and vital capacity was reduced. Further investigation showed a normal total lung capacity, and a computed tomography scan revealed main bronchus blockage by a tumour, which was confirmed by bronchoscopy. This led to a surgical resection of a mucoepidermoid carcinoma. This case highlights the need to consider all possibilities during the evaluation of patients with difficult asthma.

Deficiency of Src homology 2-containing phosphatase 1 results in abnormalities in murine neutrophil function: studies in motheaten mice.

Neutrophils, an essential component of the innate immune system, are regulated in part by signaling pathways involving protein tyrosine phosphorylation. While protein tyrosine kinase functions in regulating neutrophil behavior have been extensively investigated, little is known about the role for specific protein tyrosine phosphatases (PTP) in modulating neutrophil signaling cascades. A key role for Src homology 2 domain-containing phosphatase 1 (SHP-1), a PTP, in neutrophil physiology is, however, implied by the overexpansion and inappropriate activation of granulocyte populations in SHP-1-deficient motheaten (me/me) and motheaten viable (me(v)/me(v)) mice. To directly investigate the importance of SHP-1 to phagocytic cell function, bone marrow neutrophils were isolated from both me/me and me(v)/me(v) mice and examined with respect to their responses to various stimuli. The results of these studies revealed that both quiescent and activated neutrophils from motheaten mice manifested enhanced tyrosine phosphorylation of cellular proteins in the 60- to 80-kDa range relative to that detected in wild-type congenic control neutrophils. MOTHEATEN: neutrophils also demonstrated increased oxidant production, surface expression of CD18, and adhesion to protein-coated plastic. Chemotaxis, however, was severely diminished in the SHP-deficient neutrophils relative to control neutrophils, which was possibly attributable to a combination of defective deadhesion and altered actin assembly. Taken together, these results indicate a significant role for SHP-1 in modulating the tyrosine phosphorylation-dependent signaling pathways that regulate neutrophil microbicidal functions.

Successful treatment of juvenile laryngeal papillomatosis-related multicystic lung disease with cidofovir: case report and review of the literature.

Cidofovir, a nucleoside analog antiviral agent, has been used with moderate success in the treatment of juvenile laryngeal papillomatosis (JLP) by direct intralesional injection. We report the first case where IV cidofovir was used successfully to treat a rare but lethal multicystic lung disease complicating JLP. A 35-year-old woman with a history of JLP requiring multiple laser ablations of laryngeal papillomata each year presented with hemoptysis and was found on CT scan to have bilateral, multiple pulmonary nodules and cysts. The results of BAL fluid analysis demonstrated no evidence of malignancy, and cultures were negative for fungi and mycobacteria. Molecular DNA typing of a biopsy specimen obtained from a laryngeal papilloma confirmed infection with human papilloma virus type 11. She received 12 months of treatment with IV cidofovir followed by 9 months of combined treatmentwith IV cidofovir and subcutaneous interferon-alpha-2A. This therapeutic regime resulted in a markedly decreased requirement for surgical removal of laryngeal papillomata, and CT scanning documented the regression of the lesions in the lung parenchyma that persisted after the discontinuation of therapy. The results of this case demonstrate that cidofovir may be used successfully to treat JLP-related lung disease and suggest that further studies are warranted.

A novel model system for characterization of phagosomal maturation, acidification, and intracellular collagen degradation in fibroblasts.

Intracellular collagen degradation by fibroblasts is an important but poorly understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival fibroblasts that express endogenous alpha(2)beta(1) integrin, the collagen receptor, would exhibit the cellular machinery required for phagosomal maturation and collagen degradation. There was a time-dependent increase of collagen bead internalization and a time-dependent decrease of bead-associated alpha(2)beta(1) integrin after initial bead binding. beta-Actin and gelsolin associated transiently with beads (0-30 min) followed by LAMP-2 (60-240 min) and cathepsin B (30-240 min). Cytochalasin D prevented phagosome formation and also prevented the sequential fusion of early endosomes with lysosomes. Collagen bead-associated pH was progressively reduced from 7.25 to 5.4, which was contemporaneous with progressive increases in degradation of bead-associated collagen (30-120 min). Concanamycin blocked acidification of phagolysosomes and collagen degradation but not phagosome maturation. Phagosomal acidification was partly dependent on elevated intracellular calcium. These studies demonstrate that the cellular machinery required for intracellular collagen degradation in fibroblasts closely resembles the vacuolar system in macrophages.

LSP1 modulates leukocyte populations in resting and inflamed peritoneum.

Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans. This study examines LSP1-deficient (Lsp1(-/-)) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen. Lsp1(-/-) mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5(-) macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased in Lsp1(-/-) mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of Lsp1(-/-) mice are similar to those of wt mice and Lsp1(-/-) mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, the Lsp1(-/-) mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells. LSP1(-) neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis. (Blood. 2000;96:1827-1835)

Neutrophil products and alterations in epithelial junctional proteins: prevention of artifactual degradation.

Recent reports of disruption of endothelial cell adherens junction proteins during neutrophil adhesion and transmigration have been challenged as being partly due to post-fixation artifactual release of neutrophil-derived proteases. In this study we examined alterations in the epithelial junctional complex during neutrophil adhesion. Using standard fixation protocols, neutrophil addition to epithelial monolayers resulted in gross disruption of apical junction protein immunofluorescence. However, the inclusion of a post fixation incubation step with formic acid resulted in epitope preservation. These observations indicate that neutrophil derived products, likely proteases, remain active despite prolonged exposure to conventional fixatives. This may result in diffuse and artifactual loss of epithelial junctional protein immunofluorescence. Formic acid prevents this loss of epitope staining and may be considered as an agent to preserve protease-sensitive endothelial or epithelial immunoreactivity.

Coagulation inhibitors in sepsis and disseminated intravascular coagulation.

Sepsis is a syndrome that is increasing in frequency and continues to be associated with an unacceptably high mortality. DIC is an important and common sequel of sepsis, is implicated in the development of multiple organ failure, and has been shown repeatedly to connote a poor prognosis. Increasing understanding of the pathogenesis of DIC has suggested several novel therapies designed to correct deficiencies in inhibitors of coagulation. To date, small randomized, controlled studies of antithrombin III concentrates in sepsis and DIC have shown a trend to increased survival, but have not achieved statistical significance. Currently, a large randomized controlled trial of antithrombin III in sepsis is being conducted. Until more data are available, important questions remain as to its proper place in the treatment of sepsis, septic shock, and DIC. Similarly, therapy with protein C and tissue factor-pathway inhibitor has been beneficial in animal models of sepsis and DIC. The results of controlled clinical trials in humans are eagerly awaited.

Role of the actin cytoskeleton in insulin action.

Insulin has diverse effects on cells, including stimulation of glucose transport, gene expression, and alterations of cell morphology. The hormone mediates these effects by activation of signaling pathways which utilize, 1) adaptor molecules such as the insulin receptor substrates (IRS), the Src and collagen homologs (Shc), and the growth factor receptor binding protein 2 (Grb2); 2) lipid kinases such as phosphatidylinositol 3-kinase (PI 3-Kinase); 3) small G proteins; and 4) serine, threonine, and tyrosine kinases. The activation of such signaling molecules by insulin is now well established, but we do not yet fully understand the mechanisms integrating these seemingly diverse pathways. Here, we discuss the involvement of the actin cytoskeleton in the propagation and regulation of insulin signals. In muscle cells in culture, insulin induces a rapid actin filament reorganization that coincides with plasma membrane ruffling and intense accumulation of pinocytotic vesicles. Initiation of these effects of insulin requires an intact actin cytoskeleton and activation of PI 3-kinase. We observed recruitment PI 3-kinase subunits and glucose transporter proteins to regions of reorganized actin. In both muscle and adipose cells, actin disassembly inhibited early insulin-induced events such as recruitment of glucose transporters to the cell surface and enhanced glucose transport. Additionally, actin disassembly inhibited more prolonged effects of insulin, including DNA synthesis and expression of immediate early genes such as c-fos. Intact actin filaments appear to be essential for mediation of early events such as association of Shc with Grb2 in response to insulin, which leads to stimulation of gene expression. Preliminary observations support a role for focal adhesion signaling complexes in insulin action. These observations suggest that the actin cytoskeleton facilitates propagation of the morphological, metabolic, and nuclear effects of insulin by regulating proper subcellular distribution of signaling molecules that participate in the insulin signaling pathway.