Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis.

The Fas ligand is predominantly expressed in activated T lymphocytes and is one of the major effector molecules of cytotoxic T lymphocytes and natural killer cells. Previously, we found excessive apoptosis of epithelial cells and infiltrating lymphocytes expressing Fas ligand mRNA in the lung tissue of bleomycin-induced pulmonary fibrosis in mice. Here we demonstrated that the administration of a soluble form of Fas antigen or anti-Fas ligand antibody prevented the development of this model and that lpr and gld mice were resistant against the induction of pneumopathy. These results suggest that the Fas-Fas ligand pathway plays an essential role in the development of pulmonary fibrosis and that preventing this pathway could have therapeutic value in lung injury and fibrosis.

The p53-Mdm2 association in epithelial cells in idiopathic pulmonary fibrosis and non-specific interstitial pneumonia.

BACKGROUND: Wild-type p53 is increased during cellular responses to various stresses. Mdm2, which is induced by p53, regulates p53 protein concentrations through the ubiquitin-proteasome pathway. AIM: To investigate whether the Mdm2 mediated ubiquitination of p53 is associated with epithelial cell apoptosis in idiopathic pulmonary fibrosis (IPF). METHODS: Immunohistochemistry and western blot analysis were carried out on lung samples obtained by lung biopsy from patients with IPF and non-specific interstitial pneumonia (NSIP). RESULTS: The expression of p53, phosphorylated p53, Mdm2, p21, and Bax was upregulated in epithelial cells from patients with IPF and NSIP compared with normal lung parenchyma. Except for p21, there was a significant increase in the expression of these factors in IPF compared with NSIP. In addition, the number of apoptotic cells and the number of p53 and Bax positive cells was increased compared with controls. p53 conjugated with Mdm2 was decreased in IPF compared with NSIP and controls. Ubiquitinated p53 was increased in both IPF and NSIP compared with controls. CONCLUSIONS: Signalling molecules associated with p53 mediated apoptosis may participate in epithelial cell apoptosis, and the attenuation of p53-Mdm2 conjugation and of p53 degradation may be involved in the epithelial cell apoptosis seen in IPF. Augmented epithelial apoptosis in IPF may lead to the poor prognosis compared with NSIP.

Apoptosis signaling pathways in lung diseases.

Evidence that apoptosis plays an important role in the pathophysiology of lung diseases has been accumulated. Apoptosis signaling is classically composed of two principle pathways. One is a direct pathway from death receptor ligation to caspase cascade activation and cell death. Death receptor ligation triggers recruitment of the precursor form of caspase-8 to a death-inducing complex, through the adaptor protein FADD, which leads to caspase-8 activation. The other pathway triggered by stimuli such as drugs, radiation, infectious agents and reactive oxygen species is initiated in mitochondria. After cytochrome c is released into the cytosol from the mitochondria, it binds to Apaf1 and ATP, which then activate caspase-9. Recently, endoplasmic reticulum has also been shown to be the organelle to execute apoptosis. Further understanding of molecular mechanisms of apoptosis and its regulation by novel drugs may lead to the development of effective strategies against lung diseases. We overview the signaling pathways of apoptosis and discuss the involvement of apoptosis in the pathophysiology of various lung diseases.

Comparison between Monte Carlo simulation and measurement with a 3D polymer gel dosimeter for dose distributions in biological samples.

In this research, we used a 135 MeV/nucleon carbon-ion beam to irradiate a biological sample composed of fresh chicken meat and bones, which was placed in front of a PAGAT gel dosimeter, and compared the measured and simulated transverse-relaxation-rate (R2) distributions in the gel dosimeter. We experimentally measured the three-dimensional R2 distribution, which records the dose induced by particles penetrating the sample, by using magnetic resonance imaging. The obtained R2 distribution reflected the heterogeneity of the biological sample. We also conducted Monte Carlo simulations using the PHITS code by reconstructing the elemental composition of the biological sample from its computed tomography images while taking into account the dependence of the gel response on the linear energy transfer. The simulation reproduced the experimental distal edge structure of the R2 distribution with an accuracy under about 2 mm, which is approximately the same as the voxel size currently used in treatment planning.

Analysis of Fas and Fas ligand expression and function in lung cancer cell lines.

The aim of this study was to investigate the expression of Fas and Fas ligand (FasL) and to determine the significance of these molecules in lung cancer cell lines. Immunoblotting, RT-PCR and flow cytometric analyses were carried out to measure the expression of Fas and FasL and to examine their interactions and effects on cell growth and apoptosis. Fas and FasL were co-expressed in most of the cell lines but to varying degrees. Apoptosis induced by the agonistic anti-Fas antibody was significantly correlated with Fas expression (P=0.0075), whereas cisplatin-induced apoptosis was not. Upregulation of Fas and FasL expression by the administration of cisplatin was found in 7 of 11 (64%) and 9 of 11 (82%) cell lines, respectively. However, cisplatin-induced apoptosis was not suppressed by antagonistic anti-FasL antibody. Thus, our data indicated that Fas and FasL were co-expressed in lung cancer cell lines, and that Fas ligation induced by agonistic anti-Fas antibody is functional and induced apoptosis that was dependent on the levels of Fas expression. In contrast, Fas-FasL interactions appeared to be non-functional. Furthermore, our results suggest that cisplatin-induced apoptosis in lung cancer cells was independent of the Fas-FasL interaction.

Respiratory failure due to vocal cord dyskinesia in olivo-ponto-cerebellar atrophy.

We present a case of OPCA with severe respiratory failure due to vocal cord dyskinesia. This vocal cord dyskinesia was found by fiberoptic bronchoscopy and confirmed by laryngeal electromyogram. Tracheal fenestration and drug therapy of haloperidol were successful for clinical improvement. To our knowledge, this is the first report of vocal cord dyskinesia in OPCA. We emphasized that OPCA could complicate vocal cord dyskinesia, causing severe respiratory failure.

Soluble form of fas and fas ligand in BAL fluid from patients with pulmonary fibrosis and bronchiolitis obliterans organizing pneumonia.

STUDY OBJECTIVES: The Fas-Fas ligand (FasL) pathway is a representative system of apoptosis-signaling receptor molecules. We previously described that this pathway may play an important role in the pathogenesis of fibrosing lung diseases. In this study, we hypothesized that soluble form of Fas (sFas) and FasL (sFasL) may also be associated with this disorder. MEASUREMENTS AND RESULTS: We measured sFas and sFasL levels in BAL fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF), interstitial pneumonia associated with collagen vascular diseases (CVD-IP), and bronchiolitis obliterans organizing pneumonia (BOOP), using enzyme-linked immunosorbent assay. BALF from all patients was obtained before prednisolone therapy. sFasL levels were relatively increased in IPF patients (p = 0.084), and significantly increased in CVD-IP patients (p < 0.05) and BOOP patients (p < 0.05), compared with control subjects. BALF sFasL levels were elevated in the IPF or CVD-IP subgroups with an indication for prednisolone therapy, compared with those without an indication for therapy. The BALF sFasL level in IPF patients was correlated with the number of total cells and lymphocytes. The BALF sFasL level in BOOP patients was relatively or significantly correlated with the number of total cells or lymphocytes, respectively. The BALF sFas level was significantly increased in BOOP patients, but not in IPF or CVD-IP patients. CONCLUSIONS: We conclude that BALF sFasL levels may be associated with the accumulation of inflammatory cells and reflect the degree of lymphocyte alveolitis in IPF. The elevation of sFasL may be associated with the deterioration of IPF and CVD-IP. The elevation of the BALF sFas level may abrogate the cytotoxicity of FasL in BOOP patients, which may be associated with better prognosis of BOOP, compared with IPF or CVD-IP.

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: IV. Isolation and identification of four novel oligosaccharide units derived from the acidic polysaccharide chain.

Four novel oligosaccharide units were isolated from the acetolysis products of the acidic polysaccharide chain derived from the glycoproteins of Fusarium sp. M7-1. Their chemical structures were resolved mainly by 1H-NMR spectrometry in combination with methylation analysis and mass spectrometry. The results indicate that these oligosaccharide units originated from the side chains, GlcNAc alpha 1-->4GlcA alpha 1-->2(GlcNac alpha 1-->4)GlcA alpha 1-->2Gal, GlcNAc alpha 1-->4GlcA alpha 1-->2(GlcNAc alpha 1-->4)GlcA alpha 1-->2(GlcNac alpha 1-->4)GlcA alpha 1-->2Gal, ChN<--P--> 6Man beta 1-->4GlcA alpha 1-->2Gal, and Man beta 1-->2(ChN<--P-->6)Man beta 1-->4GlcA alpha 1-->2Gal linked together with the other units reported previously [Jikibara et al. (1992) J. Biochem. 111, 236-243] through beta 1-->6galactofuranoside linkages in the acidic polysaccharide chain.

The perforin mediated apoptotic pathway in lung injury and fibrosis.

AIMS: The perforin mediated pathway is the major pathway of cytotoxicity induced by activated T cells and natural killer cells, and may be involved in the development of pulmonary fibrosis. METHODS: Perforin and granzyme B expression were examined in idiopathic pulmonary fibrosis by means of immunohistochemistry, and perforin knockout mice were used to examine whether or not perforin mediated cytotoxicity participates in the pathophysiology of bleomycin induced pneumopathy. RESULTS: Perforin and granzyme B expression were upregulated in infiltrating lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis compared with normal lung parenchyma. Perforin and granzyme B expression were upregulated predominantly in infiltrating mononuclear cells after bleomycin instillation in wild-type mice. Although the development of bleomycin induced pneumopathy was not completely prevented, the pathological grade of inflammation and fibrosis, and the number of apoptotic cells in lung tissue, were significantly decreased in perforin knockout mice compared with wild-type mice. CONCLUSIONS: These results suggest that perforin mediated apoptosis may be associated with the pathophysiology of lung injury and fibrosis.

Morphologic study of the laryngeal taste buds in the cat.

The distribution of laryngeal taste buds (TBs) and their neutral components in the cat were investigated by immunohistochemistry and electron microscopy. The antisera used in this study were against cytokeratin, protein gene product 9.5 (PGP9.5), neuron-specific enolase (NSE), S-100 protein, calbindin D, calcitonin gene-related peptide (CGRP), and substance P (SP). Taste bud cells were specifically immunoreactive to the antibodies of human cytokeratin subtypes 8 and 18 (CAM5.2). On observation with CAM5.2, TBs were seen distributed on the laryngeal surface of the epiglottis and spread caudally along the aryepiglottic folds, reaching peak density at the laryngeal side of the arytenoid tubercle. The PGP9.5 and NSE immunoreactivities were recognized in TB cells and nerve fibers, both within the TBs and in the subepithelial connective tissue. S-100 protein immunoreactivities were not found in any of the cells in the TBs but were found exclusively in the subepithelial neural elements. The calbindin-D, CGRP, and SP immunoreactivities were confined to a part of the neural elements that was very thin. Taste pores, taste villi, neuronal varicosity, and synapselike structures were observed by scanning and transmission electron microscopic study. From these results it is considered that the TBs act as a chemical receptor.

Beaded nerve terminals in feline laryngeal mucosa.

To investigate the mechanism of airway defense reflex, beaded nerve terminals were studied by immunohistochemical techniques. In the supraglottic region the density of PGP 9.5-immunoreactive nerve fibers was the highest at the base of the glottic surface in the epiglottis, and in the glottic region it was the highest in the arytenoid region. In the subglottic region the number of positive nerve fibers was less than the number at the base of the glottic surface in the epiglottis, and when the laryngeal mucosa was processed with NaOH to dissolve the epithelium, it was possible to observe beaded nerve terminals more clearly. These beaded nerve terminals were found just beneath, in the epithelial basement membrane. Electron microscopic examination of beaded nerve terminals revealed a large quantity of secretory granules and mitochondria, suggesting that their structure is similar to that of nerve terminals. Thus these beaded nerve terminals may function as mechanoreceptors.

The fine structure of intraepithelial free nerve endings in the canine vocal cord mucosa.

Innervation of the epithelium on the undersurface of the canine vocal cords was investigated by transmission electron microscopy. In the subepithelial lamina propria, nerve bundles containing unmyelinated fibers were observed. The nerve bundles, encircled by basal lamina, were enclosed by a thin connective tissue layer and by flattened fibroblast-like cells. With nerve bundles approaching the epithelium, the axons divided repeatedly and entered the epithelial layer. In the epithelial cells, nerve axons formed knob-like swellings that contained a small number of large granular vesicles and a large number of small agranular vesicles. A sensory function responsive to irritant chemical stimuli and to mechanical stimuli is presumed for these vesicle-containing nerve processes.

Substance P immunoreactive nerve fibers of the canine laryngeal mucosa.

Substance P (SP) immunoreactive nerve endings in the laryngeal mucosa were studied by PAP immunohistochemistry with light and electron microscopy. SP immunoreactive sensory endings were observed in the epithelium as intra-epithelial free nerve endings and taste bud-like structures. A small number of autonomic SP immunoreactive nerve fibers were observed running parallel to arterioles which were over 30 micron in diameter and terminated in glandular cells. Contrary to findings by silver impregnation, intraepithelial free nerve endings were more frequently observed on the lower surface of the vocal cord. The taste bud-like structures were classified into two different types: simple terminations and reticular terminations, according to the mode of the SP immunoreactive nerve fiber. Immature or degenerated taste bud-like structures in the larynx were assumed to be mechanical receptors because these receptors lacked outer taste pores and taste hairs.

Sensory nerve endings in the mucosa of the epiglottis--morphologic investigations with silver impregnation, immunohistochemistry, and electron microscopy.

This study was conducted in order to investigate the structure of sensory nerve endings of the human epiglottis and substance P immunoreactive nerve fibers of the canine epiglottis in relationship to physiologic functions of the larynx. The human epiglottis was observed by light microscopy (silver impregnation) and electron microscopy, and the canine epiglottis was studied by peroxidase-anti-peroxidase (PAP) immunohistochemistry. The results are summarized as follows: (1) In the membranes of the epiglottis, we observed free endings of simple or complex tree shape, corpuscle endings with glomerular patterns, and taste-bud-like structures, and (2) electron microscopic studies revealed varicosity of the terminal axon with processes that contained small, clear and large, dense cored vesicles. Substance P was observed in these structures, and it was suggested that substance P was related to perception in the larynx.

Intracordal injection increases glottic closing force in recurrent laryngeal nerve paralysis.

Glottic closing pressure during swallowing was measured in the cat with a catheter pressure transducer to study the effectiveness of intracordal injection in increasing glottic pressure in unilateral recurrent laryngeal nerve paralysis. Swallows were elicited by pouring water into the pharynx while the animal was under light anesthesia with ketamine. Peak pressure of the glottic closure for the control group during deglutition was 68.0 +/- 10.5 mm Hg (mean +/- standard deviation). Peak pressure decreased to 22.0 +/- 3.6 mm Hg just after sectioning of the unilateral recurrent laryngeal nerve, and rose to 39.8 +/- 8.3 mm Hg by silicon injection into the paralyzed vocal fold. In a study of chronic cases 1 month or more after unilateral recurrent laryngeal nerve section, peak pressure was 49.1 +/- 23.4 mm Hg, and varied widely from 21 to 92 mm Hg because of differences in the position of the paralyzed vocal fold and the degree of compensation by the unaffected vocal fold. In the group that had the paralyzed vocal fold fixed in the median position, peak pressure was almost the same as that of the control group. When the paralyzed vocal fold was fixed in either the paramedian or lateral position, peak pressure was 33.3 +/- 7.0 mm Hg. This value was significantly elevated to 45.8 +/- 10.4 mm Hg by injection of silicon, though it remained lower than that of the control. These results suggest that the decrease in glottic closing force during swallowing as a result of unilateral recurrent laryngeal nerve lesion is compensated for by the unaffected vocal fold to some degree and is improved by intracordal injection.

Laryngeal reflex mechanism during deglutition--observation of subglottal pressure and afferent discharge.

In this investigation, particular attention was paid to elucidate the laryngeal reflex mechanism of protective closure and the sensory function of the larynx during deglutition. For this purpose, three different experimental procedures were adopted: (1) subglottal pressure of felines was measured during deglutition using a pressure transducer; (2) subglottal pressure of human beings was measured during deglutition using a pressure transducer; and (3) afferent discharges from superior and recurrent laryngeal nerves of felines were recorded. The following conclusions appear justified. (1) Feline and human subglottal pressure during deglutition showed the following pattern. The pressure rises with onset of deglutition, temporarily drops during laryngeal elevation, rises again during the downward movement of the larynx, and drops again at the end of the glutition. This pattern was not affected by the resection of the unilateral recurrent laryngeal nerve. (2) The superior laryngeal nerve is involved in the sensory function of the pharynx, larynx, and trachea. At least two types of afferent discharges from superficial and internal sensory nerves are suspected. Afferent discharges from the recurrent laryngeal nerves in the larynx and trachea are not as distinct as those of the superior laryngeal nerve, and this seems to correspond with various changes in the thorax. During deglutition, afferent discharges were recorded from superior to recurrent laryngeal nerves.

Glottic closure during swallowing in the recurrent laryngeal nerve-paralyzed cat.

Glottic closing pressure and time were quantitatively analyzed during deglutition and in reflex glottic closure elicited by superior laryngeal nerve stimulation by means of a catheter pressure transducer in the cat. Duration and peak pressure of glottic closure during deglutition were 322.6 +/- 32.2 msec (mean +/- SE) and 57.5 +/- 6.0 mmHg, respectively, whereas peak pressure of the reflex glottic closure was 21.7 +/- 6.1 mmHg in control animals. When the recurrent laryngeal nerve was denervated unilaterally, decrease in peak glottic closing pressure on swallowing was only about 36%, whereas the peak pressure of reflex glottic closure was markedly diminished to 4.5 +/- 4.6%. When bilateral recurrent laryngeal nerves denervated, decrease in peak pressure during deglutition showed no greater significance than it did after unilateral denervation. Inferior constrictors myotomy in addition to bilateral recurrent laryngeal nerve denervation reduced peak pressure to nearly zero. These results indicate that on swallowing, the inferior constrictors cooperate with the intrinsic laryngeal adductors, thus playing a very important role in reinforcing glottic closure, a function that is unlikely during reflex glottic closure.

Experimental study on expanded polytetrafluoroethylene (E-PTFE) for portal vein reconstruction with combined operation of the digestive tract.

For evaluating the resistance of Expanded Polytetrafluoroethylene (E-PTFE) to infection, an E-PTFE vascular graft was exposed to intestinal contents and transplanted into the canine portal vein. In group A, E-PTFE tubular graft was inserted into the portal vein defect. In group B, choledochojejunostomy was also performed combined with the E-PTFE graft interposition. In an early stage infiltration of leukocytes was observed around the graft removed from the dog. However, exploration of the periprosthetic adventitial attachment revealed no infiltration of leukocytes in both groups more than three months after operation. The results showed no morphological difference in the formation of neointima and no statistical discrepancy of patency rates (X2-test; 0.5 less than p less than 0.75). These results demonstrated superiority of the E-PTFE vascular substitute in terms of being antithrombogenic and refractory to infection.

The effect of a macrolide antibiotic against experimental sinusitis.

The effect of a macrolide antibiotic on experimentally induced sinusitis in rabbits was evaluated. Compared with the control group, the effect on sinus mucosal pathology was significantly superior in the drug-treated groups. Inflammation models as used in the present study will contribute to the establishment of a suitable pharmacotherapy for sinusitis.

A study of experimental sinusitis in rabbits.

Sinusitis was experimentally induced in rabbits by injecting one ml of casitone semi-liquid agar containing Staphylococcus aureus 209P in the order of 10(7) per ml into rabbit's bilateral paranasal sinuses (which had been sensitized with a 2.5% concentration of a purified egg white albumin solution) through a percutaneous route with a sterilized syringe. Two weeks after the last injection the rabbits were sacrificed and dissected. Light microscopic (LM) and scanning electron microscopic (SEM) examination of pathomorphological changes in this experimental model revealed that chronic inflammation developed in the mucous membranes of the paranasal sinuses at a high incidence.