The modulation of large airway smooth muscle phenotype and effects of epidermal growth factor receptor inhibition in the repeatedly allergen-challenged rat.

Allergen challenges induce airway hyperresponsiveness (AHR) and increased airway smooth muscle (ASM) mass in the sensitized rat. Whether the remodeled ASM changes its phenotype is uncertain. We examined, in sensitized Brown Norway rats, the effects of multiple ovalbumin (Ova) challenges on ASM remodeling and phenotype and the role of the epidermal growth factor receptor (EGFR) in these processes. Rats were sensitized with Ova and challenged three times at 5-day intervals with phosphate-buffered saline or Ova and pretreated with the EGFR inhibitor AG-1478 (5 mg/kg) or its vehicle dimethyl sulfoxide. Ova challenges increased ASM mass in all-sized airways and in large airway mRNA expression of smooth muscle myosin heavy chain (sm-MHC), assessed by laser capture. Myosin light chain kinase and the fast myosin isoform SM-B mRNA expressions were not affected. Ova induced AHR to methacholine, and, based on the constant-phase model, this was largely attributable to the small airways and lung derecruitment at 48 h that recovered by 1 wk. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor (HB-EGF) were increased in bronchoalveolar lavage fluid at 48 h after Ova exposure. AG-1478 inhibited AHR and prevented ASM growth. Epithelial gene expression of EGFR, HB-EGF, matrix metalloproteinase (MMP)-9, Gro-α, and transforming growth factor-ß was unaffected by Ova challenges. We conclude that EGFR drives remodeling of ASM, which results from repeated Ova challenge. Furthermore, the latter results in excessive small airway and, to a lesser degree, large airway narrowing to methacholine, and large airway gene expression of contractile protein is conserved.

Ileal smooth muscle dysfunction and remodeling in cystic fibrosis.

Patients with cystic fibrosis (CF) often suffer from gastrointestinal cramps and intestinal obstruction. The CF transmembrane conductance regulator (CFTR) channel has been shown to be expressed in vascular and airway smooth muscle (SM). We hypothesized that the absence of CFTR expression alters the gastrointestinal SM function and that these alterations may show strain-related differences in the mouse. The aim of this study was to measure the contractile properties of the ileal SM in two CF mouse models. CFTR(-/-) and CFTR(+/+) mice were studied on BALB/cJ and C57BL/6J backgrounds. Responsiveness of ileal strips to electrical field stimulation (EFS), methacholine (MCh), and isoproterenol was measured. The mass and the cell density of SM layers were measured morphometrically. Finally, the maximal velocity of shortening (Vmax) and the expression of the fast (+)insert myosin isoform were measured in the C57BL/6J ileum. Ileal hyperreactivity was observed in response to EFS and MCh in CFTR(-/-) compared with CFTR(+/+) mice in C57BL/6J background. This latter observation was not reproduced by acute inhibition of CFTR with CFTR(inh)172. BALB/cJ CFTR(-/-) mice exhibited a significant increase of SM mass with a lower density of cells compared with CFTR(+/+), whereas no difference was observed in the C57BL/6J background. In addition, in this latter strain, ileal strips from CFTR(-/-) exhibited a significant increase in Vmax compared with control and expressed a greater proportion of the fast (+)insert SM myosin isoform with respect to total myosin. BALB/cJ CFTR(-/-) ilium had a greater relaxation to isoproterenol than the CFTR(+/+) mice when precontracted with EFS, but no difference was observed in response to exogeneous MCh. In vivo, the lack of CFTR expression induces a different SM ileal phenotype in different mouse strains, supporting the importance of modifier genes in determining intestinal SM properties.

Temperature- and pH-controlled fusion between complex lipid membranes. Examples with the diacylphosphatidylcholine/fatty acid mixed liposomes.

The fusion capability of complex lipid bilayers and its pH as well as temperature sensitivity have been studied by optical and spectroscopic means. The aggregation and fusion efficiency of such lipid membranes can be optimized by controlling the phase characteristics of the individual membrane components. For a practically relevant illustration, the stoichiometric 1:2 (mol/mol) mixtures of phosphatidylcholines and fatty acids are used. Perhaps the most interesting liposomes of this kind, which are made of dipalmitoylphosphatidylcholine/elaidic acid (DPPC/ELA-COOH (1:2)), undergo a chain-melting phase transition between 42 degrees C and 48 degrees C, depending on the bulk pH value. The highest chain-melting phase transition temperatures are measured with the fully protonated fatty acids at pH < or = 5.5 and involve a change into the non-bilayer high-temperature state. Upon increasing pH, this transition reverts into an ordinary gel-to-fluid lamellar phase change and occurs at 42 degrees C, by and large. Simultaneously, the rate and the efficacy of fusion between the PC/FA and PC/FA- mixed vesicles decreases. The fusion efficacy of the PC/FA(-) mixed liposomes at pH > or = pK(FA) approximately 7.5 is practically negligible. This is largely due to the increased interbilayer repulsion and to the relatively high water-solubility of the deprotonated fatty acid molecules at high pH. While the pH-variability chiefly affects the efficacy of the intermembrane aggregation, the vesicle fusion itself is more sensitive to temperature variations. It is more likely that the temperature dependence of the intramembrane defect density is chiefly responsible for this. Optimal conditions for the fusion between DPPC/ELA-COOH (1:2) mixed vesicles are thus 3.5 < or = pH < or = 5.5 (6.3) (aggregation maximum) and T > or = 41.5 degrees C = Tm(DPPC) (defect density and fusion maximum). Under such conditions the average size of PC/FA (1:2) mixed vesicles in a 1 mM suspension increases by a factor of 10 over a period of 10 min. Interbilayer fusion can also be catalyzed by the mechanically induced local membrane defects. Freshly made liposomes thus always fuse more avidly than aged vesicles. This permits estimates of the kinetics of membrane defects annihilation based on the measured temporal dependence of the maximum fusion-rate. From such studies, a quasi-exponential decay on the time scale of 1.2 h is found for the thermolabile fusogenic DPPC/ELA-COOH liposomes.

Effects of ofloxacin on chondrogenesis in murine cartilage organoid culture.

The fluoroquinolone, ofloxacin, a widely used antimicrobial agent, has been shown to cause athropathogenic syndromes in juvenile animals. In the present study, the effect of ofloxacin on chondrogenesis in cartilage organoid cultures of limb-bud mesenchymal cells obtained from day-12 mouse embryos was investigated. Cultures treated with increasing concentrations of ofloxacin (10, 30 and 100 mug/ml medium) for 6 days showed no significant changes in overall protein content and dry weight. Collagen type II, as a specific marker for cartilage, and collagen type I, as a marker protein in the internodular loose connective tissue in this culture system, were estimated by an inhibition-ELISA after cyanogen-bromide digestion. The collagen type I content of treated cultures remained constant, but the collagen type II level decreased in a dose-dependent manner to about 40% of that of the controls. There was no detectable increase in the concentration of collagen type II in the medium suggesting that ofloxacin inhibits synthesis rather than stimulate degradation of collagen type II. Cultures treated with 100 mug ofloxacin/ml were further investigated by indirect immunofluorescence and electron microscopy. Anticollagen type II antibodies demonstrated irregularities, several defects in the cartilage nodules, and much weaker staining in the treated cultures compared with controls. Similar results were obtained with antibodies directed against the core protein of large chondroitin sulphate proteoglycan monomers. Using monoclonal antibodies specific for unsulphated, 4-sulphated and 6-sulphated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of proteoglycans, different changes in these glycosaminoglycans were observed. While unsulphated chondroitin seemed to disappear nearly completely from the cartilage matrix, the level of chondroitin 4-sulphate remained unchanged and in the case of chondroitin 6-sulphate a slight increase in staining intensity was observable in the ofloxacin-treated cultures. Ultrastructurally, there was a reduction in the number of collagenous fibrils in the cartilage matrix of treated cultures and necrotic chondroblasts could be demonstrated. The present results resemble, in some aspects, observations that have been made in vivo after ofloxacin treatment, indicating that this in vitro model may provide a suitable system for examining the mechanism of quinolone-induced athropathia.

The extracellular matrix in cartilage organoid culture: biochemical, immunomorphological and electron microscopic studies.

Limb bud mesenchymal cells obtained from day-12 mouse embryos were grown at high density on a membrane filter (pore size 0.2 micron) at the medium/air interphase. Chondrogenesis in this so-called cartilage organoid culture was monitored quantitatively by immunological estimation of type I and type II collagen and qualitatively by indirect immunofluorescence and electron microscopy in the course of a 36 days culture period. Three stages of cartilage development could be substantiated: 1. Formation of cartilage between days 2 and 7; 2. maturation of cartilage between days 9 and 13; 3. degeneration of cartilage beginning at day 20. Differentiation in cell aggregates and a loose mesenchymal tissue occurred during the first two days of the culture period. Type II collagen synthesis started in cell aggregates two days after plating and after 6 days in culture distinct cartilage nodules had developed which were embedded in loose connective tissue that contained type I collagen. During this period the type II collagen content increased progressively from 2.3 micrograms (day 3) to nearly 40 micrograms (day 7) per mg dry weight, whereas the type I collagen level increased more linearly from 2.7 to 21.3 micrograms/mg dry weight. The second period was characterized by enlargement and fusion of cartilage nodules and a diminished increase in type II collagen content from 45 to 60 micrograms/mg dry weight. Enlargement and fusion occurred by matrix production as well as by transformation of perichondrial cells into chondroblasts. Type I collagen synthesis enhanced from 29 to 54 micrograms/mg. Hypertrophic chondrocytes could be demonstrated ultrastructurally. At the third stage a nearly continuous layer of cartilage on the membrane filter covered by noncartilagenous tissue had developed. To some extent chondrocytes lost their matrix capsule and changed into fibroblast-like cells accompanied by a switch of collagen synthesis from type II to type I collagen. Quantitative studies yielded a constant level of about 60 micrograms/mg type II collagen and a further increase in type I collagen from 77 to 116 micrograms/mg dry weight. This study reveals an in vitro model of a prolonged, but almost identical image of chondrogenesis in vivo prior to endochondral mineralization which may be useful for investigations on cartilage differentiation, maturation and degeneration.

Kidney-derived urinary antigens assayed with monoclonal antibodies for the detection of renal damage.

For the quantitation of kidney-derived Urinary Antigens (UA) monoclonal antibodies specific for antigens localized in cells of defined subunits of the nephron were applied in sandwich ELISA. Antigen excretion was measured in the urine of healthy individuals, patients suffering from various diseases, kidney transplant recipients, and healthy volunteers receiving therapeutic doses of antibiotic drugs. In healthy individuals, in patients with diseases primarily affecting the glomerulus, and in inactive phases of chronic diseases antigen excretion was low. Toxic drug effects enhanced antigenuria. Excretion of some or all of the antigens always indicated tubular alterations. The tests thus provide information on location and extent of acute primary tubular damage.

Urinary kidney-derived antigens determined by tests built on monoclonal antibodies: new markers for kidney damage.

We have developed a series of sandwich ELISA for the quantitation of kidney derived urinary antigens (UA) utilizing monoclonal antibodies specific for antigens localized in cells of defined subunits of the nephron of human kidney. Antigens derived from the distal and proximal parts of the tubular system as well as antigens localized over its entire length can be detected and quantified in urine samples. Antigen excretion was measured in the urine of healthy individuals, patients with various diseases with and without kidney involvement, kidney transplant recipients, and healthy volunteers after receiving antibiotics. Low antigen excretion values were found in healthy individuals, patients with diseases primarily affecting the glomerulus, and inactive phases of chronic diseases. Toxic side effects of drugs were reflected by slightly (antibiotic drugs) or strongly (cytostatic drugs) enhanced antigenuria. Excretion of some or all of the antigens was always indicative of massive alteration of tubular structures, such as in acute phases of tubulointerstitial disease or during rejection episodes in kidney transplant recipients. The results obtained indicate that it is possible to obtain information on the location and extent of acute primary processes in the kidney with these tests.