Persistence of TGF-beta1 induction of increased fibroblast contractility.

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.

Glucocorticoids augment fibroblast-mediated contraction of collagen gels by inhibition of endogenous PGE production.

Glucocorticoids are currently regarded as the drug of choice in the treatment of inflammatory airway and lung diseases, however, they are not routinely effective in fibrotic phases of inflammation. In the current study, glucocorticoids were investigated for their ability to affect fibroblast mediated contraction of a three dimensional collagen gel, a measure of one aspect of tissue remodeling. Dexamethasone, budesonide, hydrocortisone and fluticasone propionate were all able to significantly augment fibroblast contractility in a concentration dependent manner. Glucocorticoids also had an augmentative effect on collagen gel contraction mediated by fibroblasts from bronchi, skin and bone marrow. The increased contractility was not due to cell proliferation or to collagen degradation, since the glucocorticoids did not alter the amounts of DNA and hydroxyproline in the gels. The concentration of prostaglandin E2 (PGE2) in supernatant media was lower from glucocorticoid-treated gels compared to control gels. Consistent with this, addition of exogenous PGE2 to the culture system restored the contractile properties and indomethacin augmented contraction similar to the glucocorticoids suggesting that inhibition of prostaglandins or related eicosanoids may be the mechanism by which the increased contractility occurs. DBcAMP, forskolin and the long lasting beta2-agonist formoterol were able to reverse the effect of the glucocorticoids on fibroblast mediated collagen gel contraction suggesting that enhancers of cAMP can counteract the effect of glucocorticoids. Thus, we provide evidence that glucocorticoids have the ability to directly augment fibroblast contractility by inhibiting fibroblast endogenous PGE synthesis. The findings could be one possible mechanism to explain the poor therapeutic response to glucocorticoids on the later stages of fibrotic diseases.

8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid inhibits MAPK phosphorylation in endothelial cells induced by VEGF and bFGF.

Angiogenesis is regulated by various factors. In particular, VEGF and basic FGF are of much importance. We found that 8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid inhibited the binding of VEGF to KDR/Flk-1 (VEGF receptor-2) or Flt-1 (VEGF receptor-1) and that it inhibited the MAPK phosphorylation in HUVEC induced by VEGF or basic FGF but not by EGF. 8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid might be used as an inhibitor of VEGF and basic FGF signal transduction.

Expression of epithelial markers in nocturnal asthma.

BACKGROUND: Although the airway epithelium participates in inflammation and repair, the circadian expression of epithelial cell markers involved in these processes has not been investigated. OBJECTIVE: We sought to determine whether expression of CD51 (vitronectin and fibronectin receptor), CD54 (intercellular adhesion molecule-1), HLA-DR (activation marker), CD29 (beta1 integrin), CD49b (collagen receptor), and CD11b (complement receptor) exhibit a circadian rhythm in asthma. METHODS: Eleven patients with nocturnal asthma (NA), 9 subjects with nonnocturnal asthma (NNA), and 10 control subjects underwent bronchoscopy at 4 PM and 4 AM in a random order 1 week apart, with brushing of the proximal and distal airways. The percentage of cells staining for a particular marker was determined. RESULTS: At 4 PM, HLA-DR in the proximal airways and CD54 in the distal airways was significantly greater in control subjects as compared with asthmatic subjects (HLA-DR, control subjects: 10.0% [range, 5.0% to 21.0%]; NNA: 8.0% [range, 4.0% to 14.5%] NA: 3.5% [range, 2.0% to 6.0%], P = .01; CD54, control subjects: 17.0% [range, 8.0% to 25.0%], NNA: 8.0% [range, 5.3% to 11.5%], NA: 7.0% [range, 4.0% to 15.0%], P = .O;). At 4 AM, CD51 in the distal airways was significantly greater in patients with NA as compared with patients with NNA and control subjects (control subjects, 23.0% [range, 13.8% to 30.5%]; NNA, 32.0% [range, 13.0% to 35.0%]; NA, 40.0% [range, 23.0% to 50.0%], P = .05). Expression of CD51 in the distal airways correlated with the degree of airway obstruction (r = -0.57, P = .001). Control subjects exhibited significant circadian variation in the expression of HLA-DR in the proximal airways and CD54 in the distal airways. CONCLUSION: The increased CD51 at night in patients with NA may be related to increased airway inflammation and repair processes in response to injury. The circadian changes in CD54 and HLA-DR in control subjects require further study to determine their significance. (J Allergy Clin

Cigarette smoke extract inhibits fibroblast-mediated collagen gel contraction.

Cigarette smoking, the major cause of pulmonary emphysema, is characterized by destruction of alveolar walls. Because tissue destruction represents a balance between injury and repair, we hypothesized that cigarette smoke exposure may contribute to the development of emphysema through the inhibition of tissue contraction during the repair process. To partially evaluate this hypothesis, we investigated the effects of cigarette smoke extract (CSE) on the ability of cultured fibroblasts to mediate collagen gel contraction in vitro: CSE inhibited fibroblast-mediated gel contraction in a concentration-dependent manner (P < 0.01). Production of prostaglandin E2, a known inhibitor of fibroblast contraction, was unchanged by CSE as was cell surface integrin expression. In contrast, fibronectin production by fibroblasts was inhibited (P < 0.01), and addition of exogenous fibronectin partially restored the contractile activity, thus suggesting at least one mechanism to explain inhibition of gel contraction by CSE. When CSE was treated to remove volatile components, it showed less inhibitory activity on fibroblast-mediated gel contraction. Therefore, we also examined the effects of acrolein and acetaldehyde, two volatile components of cigarette smoke. Inhibition of contraction was observed at 5 microM acrolein and at 0.5 mM acetaldehyde. In conclusion, cigarette smoke inhibited fibroblast-mediated gel contraction, and this inhibition was due, at least in part, to the volatile components of cigarette smoke and may be mediated, at least in part, by a decrease in fibroblast fibronectin production. By inhibition of repair, these smoke components may contribute to the development of pulmonary emphysema.

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture.

In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.

Mutual inhibition by TGF-beta and IL-4 in cultured human bronchial epithelial cells.

The airway epithelial cell may play a role as an effector cell, releasing various cytokines and extracellular matrix components in immune responses, inflammation, and wound repair processes, thus contributing to cytokine "networks." The cytokines transforming growth factor (TGF)-beta and interleukin (IL)-4 are though to have pivotal roles in airway diseases, with IL-4 having proinflammatory actions and TGF-beta generally regarded to mediate repair and to attenuate immune responses. In asthma, where IL-4 and TGF-beta are thought to contribute to the inflammatory process and repair, respectively, interactions between these cytokines are likely to be of importance. Therefore, we studied the potential interaction of both cytokines by measuring IL-8 and fibronectin release by cultured human bronchial epithelial cells (HBECs). IL-4 is capable of inducing IL-8 release from HBECs. This effect of IL-4 can be blocked by the concurrent presence of the cytokine TGF-beta. In contrast, TGF-beta had a modest inconsistent stimulatory effect on IL-8 release by itself and had no effect on the IL-8 release induced by tumor necrosis factor (TNF)-alpha. An antagonistic effect of IL-4 and TGF-beta was also observed on HBEC fibronectin release. TGF-beta stimulated fibronectin release, and IL-4 was able to inhibit this. This effect was not due to a redistribution of fibronectin but appeared to be due to a true reduction in synthesis. Consistent with this, IL-4 and TGF-beta effects on IL-8 and fibronectin release were paralleled by changes in mRNA levels. The ability of TGF-beta to block IL-4-induced IL-8 release is certainly not the only mechanism to inhibit IL-8 release because dexamethasone was capable of inhibiting both TNF-alpha- and IL-4-induced release of IL-8. These results indicate that TGF-beta and IL-4 can have mutually inhibitory effects. The balance determined by this reciprocal inhibition may play an important role in regulating inflammation repair and in diseases such as asthma.

Investigation of nosocomial respiratory infection due to Pseudomonas cepacia by arbitrarily primed polymerase chain reaction.

We used DNA fingerprinting by the arbitrarily primed polymerase chain reaction (AP-PCR) technique for an epidemiologic investigation of Pseudomonas cepacia nosocomial isolates obtained from patients attending our hospital. This approach was compared with conventional phenotypic typing and pulsed-field gel electrophoresis (PFGE). The patterns of gel electrophoresis of the products of AP-PCR differed significantly according to differences in the concentration of Mg2+ and in pH. AP-PCR and PFGE was identical in their resolving power, as the two methods generated four different profiles and identified the same group of strains. The AP-PCR method constitutes an easy alternative to the well-established PFGE method.

[Effect of clarithromycin on macrophage functions].

Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effect of clarithromycin (CAM) on macrophage function. We used the mouse macrophage cell line, J774.1. The following direct effects of CAM on macrophage function were evaluated: chemotaxis to CAM, chemokinetic effect of CAM, and the effect of CAM on macrophage growth. In order to examine the indirect effects of CAM on macrophage functions, we preincubated macrophages with several concentrations of CAM and then removed the CAM. Thereafter, the phagocytosis of beads, cytocidal activity against Candida albicans, and chemotaxis to lipopolysaccharide were evaluated. In addition, the indirect effects of CAM on endoxan (4 mg/ml) treated macrophage phagocytosis, cytocidal activity, and chemotaxis were evaluated. CAM (at the concentration between 0.04 and 0.2 microgram/ml) directly stimulated macrophage chemotaxis and chemokinesis. In addition, CAM dose-dependently stimulated the growth of macrophages. CAM pretreatment (for 4 hours at the concentrations between 0.04 and 0.2 microgram/ml) stimulated macrophage phagocytosis, cytocidal activity against Candida albicans, and chemotaxis to lipopolysaccharide. In addition, CAM recovered macrophage phagocytosis, cytocidal activity, and chemotaxis which were decreased after endoxan exposure. These results suggest that CAM has direct and indirect effects on macrophage functions.

Eradication of contaminating Mycobacterium chelonae from bronchofibrescopes and an automated bronchoscope disinfection machine.

The results of a follow-up study concerning the decontamination of Mycobacterium chelonae subspecies abscessus from the bronchofibrescopes and the automated bronchoscope disinfection machine are described in this paper. After modification of the methods for disinfecting the bronchofibrescopes (adding a disinfection procedure with 70% alcohol before using the automated bronchoscope disinfection machine, increasing glutaraldehyde concentration to 3%, and changing the glutaraldehyde solution once a week), and the automated bronchoscope disinfection machine (recirculating used disinfectant), M. chelonae has not been detected from either the bronchofibrescopes or the automated bronchoscope disinfection machine (examined every 6 months for 4 yr by microscopy and cultures). Moreover, no M. chelonae has been clinically detected from bronchial washings for 4 yr.

[Effect of erythromycin on macrophage functions].

Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effect of EM on macrophage function. We used the mouse macrophage cell line, J774.1. The following direct effects of EM on macrophage function were evaluated: chemotaxis to EM, chemokinetic effect by EM, and the effect of EM on macrophage growth. In order to examine the indirect effects of EM on macrophage functions, we preincubated macrophages with several concentrations of EM and then removed the EM. Thereafter, the phagocytosis of beads, cytocidal activity against Candida albicans, and chemotaxis to lipopolysaccharide were evaluated. EM (at the concentration between 0.04 and 0.2 microgram/ml) directly stimulated macrophage chemotaxis and chemokinesis. In addition, EM dose-dependently stimulated the growth of macrophages. EM pretreatment (for 4 hours at the contractions between 0.04 and 0.2 microgram/ml) stimulated macrophage phagocytosis, cytocidal activity against Candida albicans, and chemotaxis to lipopolysaccharide. These results suggest that EM has direct and indirect effects on macrophage functions.

Comparing antimicrobial activity against resistant Pseudomonas aeruginosa using an index for the absence of cross-resistance.

We devised an index to estimate the degree cross-resistance between piperacillin, ceftazidime, sulbactam/cefoperazone, amikacin, tobramycin, carumonam and imipenem against 139 separate clinical isolates of Pseudomonas aeruginosa. A negative value of the index indicated the cross-resistance whereas a positive value suggested the converse making the device an index for the absence of cross resistance (ACR). Using the ACR index, we identified pairs of antibiotics exhibiting the least degree of cross-resistance and therefore the highest potential for treating infections due to P. aeruginosa.

[Nosocomial respiratory infection caused by Xanthomonas maltophilia in immunocompromised hosts].

Between January 1988 and December 1992, 68 patients admitted to our Department of Internal Medicine with hematological malignancies or solid tumors showed colonization of the respiratory tract with Xanthomonas maltophilia (X. maltophilia). To characterize the significance of respiratory tract colonization by X. maltophilia, we retrospectively reviewed the medical records of the 68 patients colonized with this organism. Twenty-seven of these 68 patients developed pneumonia, with X. maltophilia being implicated in 10 cases. The majority of the 10 patients showed lobular infiltration on chest X-ray. Pleural effusion was observed in 2 (20%) of the 10 patients. All 68 strains of X. maltophilia were resistant to imipenem/cilastatin. Most strains (98.5%) were sensitive to latamoxef, while all strains were sensitive to minocycline. This report describes the clinical features of nosocomial X. maltophilia pneumonia in immunocompromised patients.

Myasthenia gravis associated with small-cell carcinoma of the lung.

A 49-year-old man complained of a 3-month history of progressive generalized muscle weakness. He was diagnosed as having small-cell lung carcinoma at the same time. He received an intravenous injection of edrophonium chloride with remarkable improvement of muscle strength. Electromyographic studies revealed a compound muscle action potential that decreased after repetitive stimulation. These findings were considered representative of myasthenia gravis (MG), and inconsistent with Eaton-Lambert syndrome. The appearance of MG with small-cell lung carcinoma seems to be very rare, but possible.

Slowing of sensory conduction of the median nerve and carpal tunnel syndrome in Japanese and American industrial workers.

As part of a continuing study of the causes of carpal tunnel syndrome (CTS) in industry, we measured sensory conduction of the median nerve in 101 Japanese furniture factory workers. We used the maximum latency difference (MLD) with a critical value of > or = 0.40 msec to indicate abnormal slowing of nerve conduction. The prevalence of slowing in the Japanese workers was 17.8%, while the prevalence of probable CTS (based on symptoms only) was 2.5%, and the prevalence of definite CTS (probable CTS confirmed by slowing) was 2.0%. The most important factor predicting the MLD was the body mass index. The MLD was the most important factor predicting probable CTS. The prevalence of slowing in the Japanese workers was not significantly different from the prevalence of slowing in 316 American workers from four industries (22.0%), but the prevalences of probable CTS and definite CTS were much lower in the Japanese. The meaning of these findings is discussed.

[Epidemiological study on nosocomial infection of Pseudomonas cepacia by plasmid analyses].

In order to clarify the usefulness of plasmid analyses in epidemiological study, plasmid DNA profiles, restriction fragment patterns of chromosomal DNA analysed by pulsed-field gel electrophoresis (PFGE), and patterns of extracellular products were performed in twenty-four nosocomial strains of Pseudomonas cepacia isolated at the Kagawa Medical School Hospital. Fifteen strains were obtained from 15 patients admitted in A ward, three strains were obtained from nebulizer devices used in A ward, 5 strains were obtained from 5 patients admitted in B ward, and 1 strain was obtained from a patient admitted in C ward. Three different patterns which differed from ward to ward were clearly distinguished by PFGE patterns and patterns of extracellular products. On the other hand, plasmid DNA profiles were different in some strains obtained from B ward. These results suggested that plasmid analyses combined with other typing methods are useful in more precise epidemiological analysis on nosocomial infection of P. cepacia.

Nosocomial outbreak of Pseudomonas cepacia respiratory infection in immunocompromised patients associated with contaminated nebulizer devices.

From May 1990 to August 1991, 36 patients admitted to the Department of Internal Medicine in a medical school hospital with hematological malignancies or solid tumors, developed respiratory tract colonization with Pseudomonas cepacia. Sixteen (44.4%) of these patients developed pneumonia, and four (11.1%) died of respiratory failure due to P. cepacia pneumonia. Extensive survey of the hospital environment as well as equipment showed that nebulizer devices used by the patients for inhalation were contaminated with P. cepacia. Phenotypic characteristics, (production of hemolysin and extracellular enzymes [lipase, lecithinase and protease]), the Analytical Profile Index 20 NE pattern, and the pattern of DNA fingerprinting by pulse-field gel electrophoresis in clinically isolated strains and strains derived from nebulizer devices were compared. The strains of P. cepacia obtained from patients in the Department of Internal Medicine were indistinguishable from each other and also from those isolated from nebulizer devices, but were different from those isolated from patients in other departments at the same time. These results demonstrated that the outbreak of P. cepacia respiratory colonization in immunocompromised patients was a nosocomial acquisition, and probably occurred by transmission through contaminated nebulizer devices.

Experimental pneumonia produced by clinical isolates of Pseudomonas cepacia in mice.

An experimental Pseudomonas cepacia lung infection was induced in ddY mice pretreated with cyclophosphamide. A single dose of 250 mg of cyclophosphamide per kg resulted in leukopenia which lasted for four days. At the lowest PMN levels, the mice were exposed to various doses of bacteria by either intratracheal inoculation or aerosol inhalation. Experimental pneumonia was established by intratracheal inoculation of 1 x 10(7) - 2 x 10(8) colony-forming units of P. cepacia. The duration of survival time of the mice and the number of viable bacteria in their lungs were determined. A dramatic rise in the viable counts of P. cepacia was found within 24 hours after intratracheal inoculation of 1 x 10(8) colony-forming units of P. cepacia. It was impossible to establish P. cepacia pneumonia by aerosol inhalation, since the bacteria were immediately cleared from the lung. Mice which had not been treated with cyclophosphamide remained healthy and did not show any lung lesions. Thus, neutrophils appear to play an important role in the early defense mechanisms of the lung against P. cepacia. This animal model could be of use in evaluating additional therapies for the infection, and the pathologic determinants of infection caused by P. cepacia.