Quercetin reduced inflammation and increased antioxidant defense in rat adjuvant arthritis.

Novel therapies for rheumatoid arthritis also include the use of naturally occurring compounds possessing antioxidant properties. In the present work, the effects of oral administration of quercetin were investigated in a rat model of adjuvant arthritis. Arthritis was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund's adjuvant. The experimental groups were treated with an oral daily dose of 150 mg/kg b.w. of quercetin for 28 days. Results indicated that quercetin was able to ameliorate all markers of inflammation and oxidative stress measured. Quercetin lowered levels of interleukin-1ß, C-reactive protein, and monocyte chemotactic protein-1 and restored plasma antioxidant capacity. In addition, quercetin inhibited the enzymatic activity of pro-inflammatory 12/15-lipoxygenase in lung and liver and increased the expression of heme oxygenase-1 in joint and lung of arthritic rats. Finally, quercetin inhibited the 2-fold increase of NF-қB activity observed in lung, liver and joint after induction of arthritis.

ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts.

Parathyroid hormone-related peptide (PTHrP) receptors, coupled to trimeric G proteins, operate in most target cells through at least three different transduction routes: Galpha s-mediated stimulation of adenylylcyclase (AC), Galpha q-mediated activation of phospholipase Cbeta (PLC) and mitogen-activated protein kinase (MAPK) activation. In this study we investigated the relative role of different pathways in human skin fibroblast proliferation. Using chemical inhibitors and activators of signal transduction, we demonstrated that: (i) AC/cAMP and PLC/1,4,5 inositol triphosphate/diacylglycerol second-messenger systems are simultaneously activated following PTHrP binding to its receptors; (ii) the mitogenic response to PTHrP derives from a balance between two counteracting pathways--an activating route mediated by protein kinase C (PKC) and an inhibitory route mediated by protein kinase A (PKA); (iii) PTHrP mitogenic effects are largely dependent on MAPKs, whose activity can be modulated by both PKA and PKC. Our results indicate that MAPKs are common targets of both transduction routes and, at the same time, their point of divergence in mediating PTHrP dual and opposite mitogenic effects.

Skin biopsies for cell cultures from Mediterranean free-ranging cetaceans.

The aim of this study was to develop a useful method for obtaining viable tissue samples for establishing cell cultures from skin biopsies of free-ranging cetaceans. The skin biopsies were performed by two methods: dart from an air gun and dart from a crossbow. The dart tip was modified to collect tissue. The tissue was kept in tissue culture medium at ambient temperature, then processed within 24 h. Many modifications in culture technique, with respect to conventional culture methods for human fibroblasts, were made. The cultures thus obtained can be used for many purposes, including genetic and toxicological studies. In toxicology they are an alternative in vitro system for studying threatened animals such as marine mammals. In particular, fibroblasts can be used to test the vulnerability of cetaceans and pinnipeds to different environmental contaminants such as organochlorine compounds, heavy metals and polycyclic aromatic hydrocarbons.

Iron mobilization from crocidolite as enhancer of collagen content in rat lung fibroblasts.

Asbestos exposure causes pulmonary fibrosis by mechanisms that remain uncertain. There is increasing evidence that iron from asbestos is responsible for many of its effects. In this paper, we investigated the effect of iron mobilized from crocidolite asbestos on collagen content in rat lung fibroblast cultures under serum-free conditions. Crocidolite (2, 4, 6 microg/cm2 well) increased collagen content in a dose-dependent manner (+42 +/- 8, +92 +/- 10, and +129 +/- 13% vs controls). This effect was specific for collagen, since it did not alter total protein content and was inhibited by the iron chelator deferoxamine (DFO). Preincubation of crocidolite with citrate (1 mM) for 48 hr resulted in iron mobilization (51 microM) and increased collagen production (>3-fold) in treated cells. These effects occurred without the intervention of serum factors. The absence of cell damage, proliferation or lipid peroxidation leads to the supposition that iron from crocidolite per se may act as a profibrogenic agent. Although the in vivo participation of other cells and factors cannot be excluded, we conclude that iron released from crocidolite plays a role in collagen increase occurring during asbestosis.

Effect of S-carboxymethylcysteine lysine salt on mucociliary clearance in rabbits with secretory cell metaplasia.

A single intratracheal instillation of porcine pancreatic elastase (PPE, 100 U/Kg) induces in rabbits bronchial secretory cell metaplasia as well as emphysematous changes. The mucus hypersecretion and the marked reduction of ciliated cells matched by a high percentage of atypical cilia are responsible for the delayed mucociliary clearance in this model. S-Carboxymethylcysteine lysine salt (SCMC-LYS, 0.35 g/Kg b.w.), given per os daily for 10 days starting 2 days before elastase administration, significantly ameliorated the mucociliary clearance. The pharmacological treatment did not modify the degree of secretory cell metaplasia and the percentage of atypical cilia, or prevent the alveolar wall destruction. At TEM examination, the morphological aspects of secretion occurring in bronchial tree of PPE-treated animals were rarely visible in the PPE + SCMC-LYS treated group. The beneficial effect of SCMC-LYS on mucociliary clearance may be ascribed to an antisecretagogue effect of this drug through elastase inhibition and to a reduction of mucus viscosity.

Cardiac collagen changes during the development of right ventricular hypertrophy in tight-skin mice with emphysema.

The tight-skin (Tsk) mouse is a genetic model of pulmonary emphysema. In this mouse, right ventricular hypertrophy (RVH) starts to develop at approximately 8 months of age, probably as a consequence of the emphysema. The aim of the present study was to investigate cardiac collagen synthesis, content, and types both before and during the development of RVH. Collagen synthesis, assessed by the [3H]proline incorporation method, was significantly increased in the right ventricle of 3-month-old Tsk mice. This was accompanied by a marked increase in right ventricle collagen content. Collagen typing showed no difference from controls. At 8 months of age collagen synthesis had returned to control values, right ventricular collagen content was elevated but held lower values than at 3 months, and collagen typing showed a prevalence of the more compliant type III. By 16 months of age, right ventricular collagen content had returned to control values and there was a shift in collagen types due to a relative increase of the more rigid type I. At 24 months of age right ventricular collagen content was increased again and collagen type I continued to predominate. These results suggest a dynamic role for collagen both before and during the development of RVH secondary to emphysema.

Collagen breakdown products and lung collagen metabolism: an in vitro study on fibroblast cultures.

BACKGROUND: In fibrotic diseases such as pulmonary fibrosis there is evidence suggesting enhanced synthesis and degradation of lung connective tissue components, including collagen. It has therefore been hypothesised that products of collagen degradation may have a role in the promotion of collagen deposition. In support of this hypothesis, it has recently been shown that intravenous injection of lung collagen degradation products in experimental animals stimulated collagen synthesis leading to increased collagen deposition and diffuse interstitial lung disease. METHODS: Rabbit and human fibroblast cultures from lung and skin were used as an in vitro model to study the responses of these cells to rabbit collagen degradation products. The effects of an acute exposure to collagen degradation products on synthesis of collagen and noncollagenous protein have been studied in confluent cultures by [3H]-proline incorporation. The effects of collagen degradation products on fibroblast proliferation and production of genetic types of collagen have also been investigated. RESULTS: The acute exposure of rabbit lung fibroblast cultures to collagen degradation products significantly increased collagen synthesis without affecting non-collagenous protein synthesis. This effect was dose related, specific for lung fibroblasts, and species specific. Collagen degradation products altered the rate of synthesis of genetic types of collagen with a consequent decrease of type III/I+III collagen ratio (0.26 (0.04) treated with collagen degradation products; 0.45 (0.02) controls). These effects occurred without the intervention of serum factors. In addition, collagen degradation products neither affected fibroblast proliferation nor selected specific clones emphasising one type of collagen. CONCLUSIONS: These results suggest that collagen degradation products can influence lung collagen metabolism by stimulating collagen synthesis. The regulation of collagen mass by collagen degradation products may be of importance in lung collagen homeostasis in vivo.

Neutrophil lysosomal dysfunctions in mutant C57 Bl/6J mice: interstrain variations in content of lysosomal elastase, cathepsin G and their inhibitors.

In this paper we report the serum antiprotease screening and the biochemical and functional characteristics of neutrophils in a variety of mouse strains with different susceptibilities for developing a protease-mediated injury. C57Bl/6J mice and their mutants tight-skin and pallid have a lower serum elastase inhibitory capacity (-30, -65 and -70% respectively) than other inbred strains (i.e. NMRI and Balb/c, which both have similar values). We demonstrate that these values are a consequence of a decreased concentration of the alpha 1-protease inhibitor for elastase [PI(E)], which is the major serum inhibitor of elastase and cathepsin G. In addition, neutrophil lysosomal dysfunctions characterized by abnormally high contents of elastase and cathepsin G, or defective lysosomal secretion are observed in tight-skin and pallid mice respectively. Another C57Bl/6J mutant with lysosomal abnormalities is the beige mouse. Negligible amounts of elastase and cathepsin G, as well as defective neutrophil degranulation, have been described previously in this strain. We found, however, discrete amounts of a latent form of neutrophil elastase that undergoes a spontaneous activation by a protease-dependent mechanism. We also report that neutrophil cathepsin G in this mouse is tightly bound to lysosomal membranes, but is released in near normal quantities during exocytosis. Cytosolic elastase and cathepsin G inhibitors, which were previously reported as being specific for the beige neutrophils, have also been detected in all the examined strains. Neutrophil functions, lysosomal enzyme content and serum antiprotease screening may represent key elements in the protease-antiprotease balance and may explain the different interstrain susceptibility to developing lesions in which an elastolytic activity has been implicated.

The pallid mouse. A model of genetic alpha 1-antitrypsin deficiency.

BACKGROUND: The current hypothesis of pulmonary emphysema is based on an alteration of the protease-antiprotease balance within the lower respiratory tract. This hypothesis derives largely from studies in emphysema patients with genetic deficiency in serum alpha 1-antitrypsin. In animals, naturally occurring deficiency in serum elastase inhibitory capacity associated with early development of emphysema has been reported in the tight-skin mouse. We describe here a mouse model of genetic deficiency of alpha 1-antitrypsin in which emphysema occurs late in life. EXPERIMENTAL DESIGN: A genetic deficiency in serum alpha 1-antitrypsin was investigated in pallid mice, a strain with spontaneous occurring emphysema. Additionally, the possible pathogenetic role of an elastase-anti-elastase imbalance in pallid mice was investigated using molecular biologic, biochemical, histologic, ultrastructural, and immunoelectron microscopic methods. RESULTS: Pallid mice have markedly low levels of serum alpha 1-antitrypsin associated with a severe deficiency in serum elastase inhibitory capacity. However, they have normal alpha 1-antitrypsin mRNA levels in the liver. At ultrastructural examination, disruption of alveolar septa is first seen at 8 months of age. At histologic examination, some patchy areas of air-space enlargement with destruction of alveolar septa are seen from 12 months of age onward. These histologic changes are paralleled by a decrease in lung elastin content. The development of the pulmonary lesions is preceded by an alveolar elastolytic burden detected by an immunogold technique. CONCLUSIONS: All these data suggest that the lung changes in pallid mice are the result of an elastolytic process due to a severe inborn deficiency of serum alpha 1-antitrypsin. This animal model reproduces important features of the human condition and may provide new insights into the pathogenesis of emphysema.

Lung collagen synthesis and deposition in tight-skin mice with genetic emphysema.

The tight-skin (Tsk) mouse is a genetic model of pulmonary emphysema linked to a deficiency of serum antielastase. In this mouse occurrence of connective tissue abnormalities in various organs (systemic scleroderma) has been reported. The aim of the present work was to study lung collagen synthesis and deposition in Tsk mice. No differences in the collagen synthesis rate and morphology at the ultrastructural level were found in Tsk mice at birth. At 2 months of age, a marked increase in collagen was observed within the alveolar septa. At this time, an increased lung collagen synthesis, assessed by determining prolyl hydroxylase activity and incorporation of radiolabeled proline, was found in Tsk mice with respect to control mice. However, due to the ongoing parenchymal destruction, the values of total lung collagen at 6 and 12 months of age were only moderately but significantly increased with respect to those observed at 2 months. As a consequence, a progressive accumulation of lung collagen fibers was observed in the residual septa. The increase in collagen deposition was accompanied by a relative increase in type I collagen. Although the data in the literature would suggest a genetic cause for the lung collagen change in Tsk mice, the data presented here indicate that the change in lung collagen metabolism may be a part of a remodeling process taking place after lung destruction.

Identification of elastase in human eosinophils: immunolocalization, isolation, and partial characterization.

Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation.

An elastolytic proteinase from rabbit leukocytes: purification and partial characterization.

A proteinase with elastolytic activity was isolated from granules of rabbit bloodstream leukocytes, and purified to apparent homogeneity by a multi-step procedure consisting of ammonium sulfate precipitation, batch fractionation on DEAE-Sephadex A-50, and finally by preparative isoelectric focusing (IEF) on Sephadex G-75 Superfine. The molecule weight of the enzyme, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 28,500. This enzyme shows an isoelectric point at pH 9.0. The proteinase is active against natural elastins as well as toward Suc-(Ala)3-NA, Methoxy-Suc-(Ala)2-Pro-Val-NA, and (to a lesser extent) against Suc-(Ala)2-Pro-Leu-NA and Boc-Ala-ONp. The inhibition profile of the isolated enzyme indicates that rabbit granulocyte elastase belongs to the group of serine proteinases. Inhibition by some natural proteinase inhibitors is also observed. Unlike other mammalian elastases, it is insensitive to elastatinal.

Ultrastructural ciliary defects in children with recurrent infections of the lower respiratory tract.

One hundred fifty-four children with recurrent or chronic infections of the lower respiratory tract compatible with the diagnosis of primary ciliary dyskinesia (PCD) were evaluated for the presence of ultrastructural ciliary abnormalities. Studies were performed on multiple samples of respiratory mucosa obtained by nasal and bronchial brushing. Twenty-eight children showed ultrastructural ciliary defects compatible with the diagnosis of PCD: Twenty-four presented dynein arm deficiency (either as isolated defect or in association with microtubular abnormalities), two had ciliary aplasia, and two showed microtubular abnormalities. Eleven patients with PCD had situs viscerum inversus, bronchiectasis, and chronic sinusitis (Kartagener's syndrome); one child with Kartagener's syndrome had normal ciliary structure. The appearance of respiratory symptoms within the first month of life, the colonization by Haemophilus influenzae, and a history of recurrent rhinitis and otitis were characteristically present in children with PCD. The clinical status of those patients who reached adolescence was, in our experience, remarkably good. An early diagnosis with adequate prevention and therapy of respiratory infections may have an important role in minimizing irreversible lung damage.

Development of interstitial lung fibrosis by long-term treatment with collagen breakdown products in rabbits.

We recently demonstrated that collagen breakdown products derived from elastase digestion (CDP) can stimulate "in vivo" lung collagen synthesis. The present work deals with the morphological and biochemical characteristics of an experimental model of lung fibrosis developed in rabbit by long-term treatment with CDP. Stimulation of collagen synthesis by CDP resulted in a significant thickening of alveolar septa due to accumulation of fibroblasts and a marked deposition of collagen fibrils as revealed by light as well as electron microscopy. Biochemical analysis confirmed the increase in lung collagen deposition. Total collagen content as determined by hydroxy-proline analysis was increased in CDP-treated animals of about 56% in respect to control animals. A relative increase of type I collagen in respect to type III was also observed. An additional interesting observation was a progressive hyperplasia of type II pneumocytes. Unlike other experimental models of lung fibrosis, the collagen deposition in our condition is not preceded or associated with inflammatory or degenerative processes. This fact renders this model very suitable to study matrix-cell interactions in pulmonary fibrogenesis.

Hydrocephalus, bronchiectasis, and ciliary aplasia.

A girl presented in the neonatal period with hydrocephalus, bronchiectasis, and ciliary aplasia. A common defect both in respiratory tract cilia and in ventricular ependyma cilia may explain the association of the two diseases.

In vivo stimulation of lung collagen synthesis by collagen derived peptides.

In the present work we examined the "in vivo" effects of collagen breakdown products derived from elastase digestion on lung collagen synthesis in rabbits. It was found that i.v. injection of collagen peptides greatly enhances the collagen synthesis rate while does not affect the synthesis of non collagenous proteins. The increase of incorporation of 3H-proline in lung collagen parallels that of prolyl hydroxylase activity. The collagen synthesis, expressed as fractional rate (% day), amounted to 15% day in treated animals, resulting in a significant increase with respect to controls (11.7% day). The observations reported provide evidence that collagen breakdown products stimulate lung collagen synthesis and may play a role in collagen homeostasis.

Serum antielastase deficiency in tight-skin mice with genetic emphysema.

The tight-skin (Tsk) mouse is a model of genetically determined emphysema. The cause for the development of the lung lesion is unknown. In the present study we investigated the lung morphometry and the serum elastase inhibitory capacity (EIC) of Tsk mice. Mean interalveolar distance was significantly greater (+60%) in Tsk mice than in C57 Bl/6J, NMRI, and Balb/c mice, which have similar values. Serum of Tsk mice against mouse leukocyte elastase (MLE) has significantly lower EIC values than that of NMRI, Balb/c (-64%), and C57 Bl/6J (-50%) mice. Similar results were obtained when porcine pancreatic elastase (PPE) was used. Against human leukocyte elastase (HLE), however, there was no difference among the strains, all of which had high EIC values. Preincubation of mouse (C57 Bl/6J) serum with chloramine-T (CT) resulted in an almost complete inhibition of EIC against MLE and PPE but only in a 20% inhibition against HLE using a synthetic substrate. Using elastin Congo Red as substrate, CT inhibited EIC against MLE and PPE by approximately 70% but did not affect the EIC against HLE. These results indicate that (1) the Tsk mouse can be considered a model of severe inborn deficiency of serum antielastase activity which is associated with emphysema; and (2) MLE and PPE can be considered interchangeable in studies of serum EIC in the mouse. On the other hand, the differences between MLE and HLE preclude the use of HLE for EIC determination in this species.

Immunoelectron-microscopic demonstration of elastase in emphysematous lungs of tight-skin mice.

The tight-skin (Tsk) mouse has recently been proposed as a genetic model of emphysema. A morphometric study has shown that emphysema develops quickly, between 15 days and 1 month after birth. Previous biochemical and ultrastructural investigations of the lungs of 1- and 2-month-old Tsk mice revealed the presence of an ongoing elastolytic process. The goal of the present study was to investigate the role of mouse leukocyte elastase (MLE) in the development of emphysema in 1-month-old Tsk mice. Using electron microscopy and an immunogold labeling technique with rabbit anti-MLE IgG, MLE was localized within the lung neutrophils of control and Tsk mice. MLE was also found associated with elastin in the alveolar septa of Tsk but not of control mice. Little or no labeling was associated with other components (collagen, pneumocytes, and endothelium) of alveolar septa of Tsk mice. Lung elastin of control mice, or of control mice rendered emphysematous with porcine pancreatic elastase, showed negligible gold particle density when incubated with gold-conjugated rabbit IgG. Thus, under the present experimental conditions, an aspecific labeling of elastin is unlikely. This study indicates that MLE may be one of the factors responsible for the rapid development of emphysema in Tsk mice.