Oligonucleotide-targeting periostin ameliorates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a median survival of 3-4 years after diagnosis. It is the most frequent form of a group of interstitial pneumonias of unknown etiology. Current available therapies prevent deterioration of lung function but no therapy has shown to improve survival. Periostin is a matricellular protein of the fasciclin 1 family. There is increased deposition of periostin in lung fibrotic tissues. Here we evaluated whether small interfering RNA or antisense oligonucleotide against periostin inhibits lung fibrosis by direct administration into the lung by intranasal route. Pulmonary fibrosis was induced with bleomycin and RNA therapeutics was administered during both acute and chronic phases of the disease. The levels of periostin and transforming growth factor-ß1 in airway fluid and lung tissue, the deposition of collagen in lung tissue and the lung fibrosis score were significantly reduced in mice treated with siRNA and antisense against periostin compared to control mice. These findings suggest that direct administration of siRNA or antisense oligonucleotides against periostin into the lungs is a promising alternative therapeutic approach for the management of pulmonary fibrosis.

Protein S is protective in pulmonary fibrosis.

UNLABELLED: Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. SUMMARY: Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar epithelial cells in vitro. Conclusions These observations suggest clinical relevance and a protective role of protein S in pulmonary fibrosis.

Protein S exacerbates alcoholic hepatitis by stimulating liver natural killer T cells.

BACKGROUND: Alcohol consumption is a major cause of liver injury but the mechanisms are not completely understood. Protein S (PS) is an anticoagulant glycoprotein with multiple functions. The role of PS in liver injury is unknown. OBJECTIVES: This study investigated the role of PS in acute alcoholic hepatitis. METHODS: A mouse overexpressing human PS (hPS-TG) was generated in which acute hepatitis was induced by intraperitoneal injection of ethanol. RESULTS: The levels of serum liver enzymes and liver tissue inflammatory cytokines and the degree of hepatic steatosis were significantly increased in hPS-TG mice treated with ethanol compared with ethanol-treated wild type (WT) mice. Cell expansion, activation and inhibition of apoptosis were significantly augmented in natural killer T (NKT) cells from hPS-TG mice compared with WT mice. Liver mononuclear cells from hPS-TG mice express higher levels of inflammatory cytokines than those from WT mice after stimulation with a specific stimulant of NKT cells in vitro. In a co-culture system of hepatocytes and NKT cells, the effects of PS on ethanol-mediated cell injury were suppressed by a CD1d neutralizing antibody. Alcoholic liver injury was significantly improved in mice pre-treated with PS siRNA and anti-protein S antibody compared with control mice. Patients with alcoholic hepatitis showed significantly increased plasma PS levels and enhanced liver expression of PS and CD1d compared with controls. CONCLUSIONS: The results of this study suggest that PS exacerbates acute alcoholic hepatitis by inhibiting apoptosis of activated NKT cells.

Dose-dependent differential effects of thrombin in allergic bronchial asthma.

BACKGROUND: Apart from its role in the coagulation system, thrombin plays an important role in the inflammatory response through its protease-activated receptors (PARs). However, the role of thrombin in the immune response is not clear. OBJECTIVE: To evaluate whether thrombin has a modulatory role in allergic bronchial asthma. METHODS: Bronchial asthma was induced in mice by intraperitoneal sensitization and inhalation challenge with ovalbumin. Thrombin or its inhibitors were administered by inhalation before each allergen challenge. RESULTS: Mice with low but sustained coagulation activation had reduced allergic inflammation, and allergic asthma was inhibited by low doses of thrombin but worsened by high doses. Allergic asthma was worsened by antithrombin, argatroban, hirudin, and anti-thrombomodulin antibody. Mice with a higher level of an inhibitor of both thrombin and activated protein C had worse disease. Heterozygous PAR-1 mice had less allergic inflammation, but PAR-1 agonist worsened it. Allergic bronchial inflammation was worsened in mice that received adoptive transfer of PAR-1 agonist-treated Th2 cells as compared with controls. Low levels of thrombin suppressed the maturation and secretion of cytokines in dendritic cells, but high levels enhanced this. CONCLUSIONS: The effects of thrombin on allergic asthma are dose-dependent, with detrimental effects at high doses and protective effects at low doses. These data demonstrate that thrombin modulates the outcome in allergic bronchial asthma.

Correlation of circulating dehydroepiandrosterone with activated protein C generation and carotid intima-media thickness in male patients with type 2 diabetes.

AIMS: Dehydroepiandrosterone exerts a protective effect against cardiovascular diseases. However, the relationship of dehydroepiandrosterone with the anticoagulant factor activated protein C, generated by the thrombin-thrombomodulin complex on vascular endothelial cells, remains unknown. This study aimed at studying the relationship between dehydroepiandrosterone and activated protein C generation in patients with Type 2 diabetes. METHODS: Sixty-two male patients with Type 2 diabetes were enrolled in this study. Data obtained from 40 healthy male subjects were used as controls. The plasma levels of dehydroepiandrosterone, the activated protein C-protein C inhibitor complex, high-sensitivity C-reactive protein and monocyte chemoattractant protein-1 were measured by enzyme immunoassays. Carotid intima-media thickness was measured by ultrasonography. RESULTS: The plasma levels of dehydroepiandrosterone (5.15 ± 2.81 vs. 3.76 ± 2.16 ng/ml; P < 0.005) and the activated protein C-protein C inhibitor complex (1.90 ± 1.07 vs. 1.02 ± 0.51 ng/ml; P < 0.001) were significantly lower in patients with diabetes than in normal subjects. Univariate analysis showed a significant correlation of the plasma level of dehydroepiandrosterone with that of the activated protein C-protein C inhibitor complex (r = 0.48, P < 0.001), high-sensitivity C-reactive protein (r = -0.30, P < 0.05) and with the mean intima-media thickness (r = -0.28, P < 0.05) in patients with diabetes. Stepwise multiple regression analysis showed that the plasma level of dehydroepiandrosterone is significantly correlated with the plasma levels of the activated protein C-protein C inhibitor complex (F = 18.06) and high-sensitivity C-reactive protein (F = 4.94). There was no correlation between the plasma levels of dehydroepiandrosterone and monocyte chemoattractant protein-1. CONCLUSIONS: These results suggest that lower circulating levels of dehydroepiandrosterone are associated with decreased activated protein C generation and higher intima-media thickness in patients with Type 2 diabetes.

[Characteristics of hyperacuity sensitivity in normal and cyclovertical deviated subjects].

PURPOSE: To compare the sensitivity of hyperacuity of cyclovertical deviated patients with that of normal subjects. SUBJECTS AND METHODS: The sensitivity of hyperacuity was measured in 42 normal and 12 cyclovertical deviated patients, using a newly developed computerized device which randomly presents two targets opposed vertically or horizontally on a cathode ray tube(CRT) display. RESULTS: In normal subjects, lower thresholds were obtained when the targets were aligned either vertically or horizontally. These highly sensitive ranges were defined as "the neutral zone of hyperacuity". An anisotropy of the sensitivity of hyperacuity was observed. i.e., better thresholds were obtained when the offset was set away from the neutral zone, whereas worse thresholds were obtained when the offset as close to the neutral zones. In cyclovertical deviated patients, the thresholds of hyperacuity were high around the neutral zones, which may indicate dysfunction of the central nervous system. CONCLUSION: This analytical method may be useful to investigate the pathophysiology of the patients with cyclovertical deviations.

Immunohistological study of intervertebral disc herniation of lumbar spine.

In order to observe histological changes in the extruded and sequestrated intervertebral disc, we conducted pathological and immunological examinations of herniated disc materials taken at the time of discectomy. There were 49 disc materials (from 38 men and 10 women [aged 19 to 78 years; average, 36.6 years]). The herniation was classified into four types, based on the intraoperative observations: protrusion (P), subligamentous extrusion (SE), transligamentous extrusion (TE), and sequestration (S). There were 19 P type discs, 3 SE type, 10 TE type, and 17 S type. The surgical specimens were stained with hematoxylin-eosin, as well as immunohistological staining with the labelled streptavidin biotin method, using human T-cell, human B-cell, and human macrophage antibodies. Inflammatory-cell infiltration was observed at the border of the disc. These findings were present in 19 discs (70%) of the 27 discs of TE and S types (10 TE and 17 S types), but were not seen in the 22 discs of P and SE types (19 P and 3 SE types). Immunohistological staining of the area with inflammatory-cell infiltration revealed the presence of T cells and macrophages, which suggested that this cell infiltration originated from T cells and macrophages, and that the spontaneous resorption of the disc may have resulted from the phagocytic activities of these cells.

Studies on disease-modifying antirheumatic drugs. IV. Synthesis of novel thieno[2,3-b:5,4-c']dipyridine derivatives and their anti-inflammatory effect.

The syntheses and anti-inflammatory activities of novel thieno[2,3-b]pyridine and thieno[2,3-b:5,4-c']-dipyridine derivatives are described. These compounds were designed by modification of the quinoline template of a new type of disease-modifying antirheumatic drug (DMARD), TAK-603, and prepared by the Friedländer reaction as a key reaction. Their anti-inflammatory effects were evaluated using an adjuvant arthritis rat model. Most of the compounds which included a diethylamino moiety in the side chain had potent anti-inflammatory effect. In particular, ethyl 2-(diethylaminomethyl)-4-(3,4-dimethoxyphenyl)thieno[2,3-b:5,4-c'] dipyridine-3-carboxylate (21) exhibited more potent activity than TAK-603.

Synthesis of peptide aldehyde derivatives as selective inhibitors of human cathepsin L and their inhibitory effect on bone resorption.

Cathepsin L, a lysosomal cysteine protease, is secreted by osteoclasts and participates in bone collagen degradation. In a search for cathepsin L inhibitors as antiosteoporotic agents, a series of peptide aldehyde derivatives were prepared by two synthetic approaches, DMSO oxidation of the corresponding alcohol derivatives and DIBAL-H reduction of the corresponding N, O-dimethylhydroxylamide derivatives, and evaluated for inhibitory activity against human cathepsin L and for inhibitory effects on bone resorption. Some of the peptide aldehyde derivatives including alpha-acylamino aldehyde derivatives showed potent activities. Among these compounds, N-(1-naphthalenylsulfonyl-L-isoleucyl-L-tryptophanal (12) was selected as a candidate for further investigation. Compound 12, a potent, selective, and reversible inhibitor of human cathepsin L with an IC50 of 1.9 nM, inhibited the release of Ca2+ and hydroxyproline from bone in in vitro bone culture system and also prevented bone loss in ovariectomized mice at an oral dose of 50 mg/kg.

Treatment of fungal corneal ulcers with amphotericin B ointment.

PURPOSE: To report two patients with severe fungal corneal ulcers who were treated successfully with topical amphotericin B ointment. METHODS: Two eyes of two patients developed corneal ulcers and hypopyon after corneal foreign body removal. Aspergillus fumigatus and Fusarium solani were isolated in Patients 1 and 2, respectively. By antifungal susceptibility testing, amphotericin B was shown to have the lowest minimal inhibitory concentration. RESULT: Topical administration of amphotericin B ointment resulted in dramatic improvement in fungal corneal ulcers. CONCLUSIONS: Antifungal susceptibility tests may aid with selection of antifungal agents. Amphotericin B ointment is one of the promising therapies for keratomycosis caused by antimycotic-resistant fungi.

[Clinical applications of time-dependent perimetry (10) receptive field properties of glaucoma patients].

The receptive field center of double flash resolution (DFR) and of critical flicker fusion frequency (CFF) in a 3 degrees temporal visual field were examined in 11 normal subjects and 19 glaucoma and ocular hypertension patients. In normal subjects, the mean receptive field center of DFR was 2.17 log min of arc2 and that of CFF was 2.55 log min of arc2. In many glaucoma and ocular hypertension cases, the receptive field center of DFR (mean:2.48 log min of arc2) was significantly enlarged and came close to that of CFF (mean:2.53 log min of arc2)(p < 0.01). Although these findings were mostly seen in patients with some visual field changes, they were also encountered in 9 (70%) of the 13 cases with ocular hypertension without any visual field changes and are probably related to poor intraocular pressure control. We considered that the result was due to the dysfunction of the surrounding area of DFR that consists of interaction between sustained and transient functions of the visual pathway. This dysfunction could be closely related to the disorder of alpha-cells that occurs in the early stage of glaucoma.

The histology of lumbar intervertebral disc herniation. The significance of small blood vessels in the extruded tissue.

Six hundred surgical cases of lumbar intervertebral disc herniation were evaluated histologically for the presence of blood vessels. These patients ranged in age from 12 to 77 years. Blood vessels were observed in 57 of 101 cases of complete prolapse type of herniated disc (56.4%), 12 of 32 cases of incomplete prolapse type of herniated disc (37.5%), and 53 of 467 cases of protrusion type of intervertebral disc herniation (11.3%). The presence of blood vessels in intervertebral discs was also investigated in postmortem specimens. Blood vessels were observed in 293 of 616 intervertebral discs (T10-L5), in individuals older than 40 years of age from 100 postmortem spines. The specimen age range was 16-89 years. Most of the blood vessels seen in the extruded tissue, exposed to the epidural space in cases of complete and incomplete prolapse type of herniation, are thought to have been newly formed after herniation occurred. As invasion of the intervertebral disc by blood vessels was found to occur with the advance of age, it is possible that such blood vessels become extruded with the intervertebral disc tissue. The intervertebral disc may herniate posteriorly in three basic patterns. The first pattern is "protrusion type of herniated disc." In protrusion hernia type there is abnormal posterior bulging of the anulus fibrosus. The disc pathology is predominantly nucleus pulposus, and the peripheral layer of the anulus fibrosus remains attached to the vertebral body bony rim, however.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related phenomena in the lumbar intervertebral discs. Lipofuscin and amyloid deposition.

In 69 lumbar intervertebral discs from 69 autopsies, lipofuscin was seen in discs from individuals older than 50 years of age and amyloids in the discs of individuals older than 40 years of age. In 261 intervertebral disc tissue specimens collected at the time of surgery for disc herniation, lipofuscin was seen in the discs of individuals older than 20 years of age and amyloids in the discs of individuals older than 15 years of age. It is speculated that amyloids and lipofuscin are a sign of aging in the intervertebral disc as in other tissues. Furthermore, amyloids and lipofuscin were seen in the operated discs of young individuals than in the autopsy cases.

Effects of color CRT display on pupil size in color-blind subjects.

The effects of color cathode ray tube (CRT) display on the pupil size in color-blind subjects were studied experimentally. Red, magenta, green, cyan, yellow, and white were presented on the CRT. Five protan subjects, five deutans, and five normal subjects were tested using infrared pupillography. In the protan group, the pupils were significantly less sensitive to red (wavelength: 600 nm) targets compared to the other colors. In the deutans and the control group, there were non-significant changes in pupil size in response to the colors. Dominant wavelengths above 600 nm in color CRTs should be avoided in routine work presentation because of less sensitivity in protanopias.

In vitro retinal and erythrocyte polyol pathway regulation by hormones and an aldose reductase inhibitor.

The effects of a high-glucose medium, insulin, and an aldose reductase inhibitor (ONO-2235) on sorbitol accumulation were compared in the human erythrocyte and the rabbit retina, while the effects of epinephrine on in vitro sorbitol accumulation were investigated in the human and rabbit retina. In both erythrocytes and the retina, linear increments of sorbitol accumulation were observed in a dose-dependent manner with 5 to 50 mM glucose. These increments were markedly inhibited by 100 microM ONO-2235 but not by insulin (400 microU/ml). In the presence of 5 mM glucose, a dose-dependent increase of the sorbitol content of the rabbit retina was seen following epinephrine stimulation (0.4-4.0 microM and this was markedly reduced by 100 microM ONO-2235. Moreover, both 50 mM glucose and 4.0 microM epinephrine increased the sorbitol content of the retina from a diabetic patient, and the glucose-induced increment in sorbitol was significantly reduced by 100 microM ONO-2235. Our data suggested that aldose reductase inhibitors might be useful for the treatment of diabetic retinopathy, since the polyol pathway appears to be an important factor in its pathogenesis, and that catecholamines might have some role in the activation of the retinal polyol pathway.

Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses.

To study the relationships between the changes due to aging in lumbar intervertebral discs and the development of protrusion or prolapse, we carried out histological studies on operative specimens of thirty-one discs, of which twenty-two had been protruded and nine, prolapsed. The specimens were obtained during twenty-nine operations for herniation of a lumbar intervertebral disc in patients who were sixty years old or older. Changes in the anulus fibrosus were more extensive in the nine prolapsed discs than in the twenty-two protruded discs. Of the nine prolapsed discs, myxomatous degeneration, fibrosis, and swollen anular fibers were found in all nine, and cysts were seen in five. Of the twenty-two protruded discs, only five showed myxomatous degeneration; ten, fibrosis; one, a cyst; and sixteen, swollen fibers. For comparison, we also studied specimens that had been obtained at operation from twenty-one other patients, twenty to fifty-nine years old, who had a prolapsed disc. The anulus showed myxomatous degeneration in all twenty-one specimens, cysts in eight, and fibrosis in ten. In addition, we examined 368 autopsy specimens from people who had been between twenty-five and eighty-five years old at the time of death. In many of the subjects who had died in the sixth decade of life or later, we found that the orientation of the inner fiber bundles of the anulus fibrosus was reversed, so that they bulged inward. The reversal appeared to be the result of myxomatous degeneration of the middle fibers of the anulus, atrophy of the nucleus, and narrowing of the disc space. These histological findings suggest explanations for the predominance of protrusions of the nucleus pulposus in patients who are less than sixty years old and of prolapse of the anulus fibrosus in the few patients who are more than sixty years old who have herniation of an intervertebral disc.

Histopathologic study on tumor infiltration into the intervertebral disc.

This study was performed in order to understand the reasons why the metastasis of malignancy into the disc is rare in contrast to the commonly seen vertebral metastasis. By histopathologic studies of 14 vertebral columns with vertebral metastases of various malignant tumors and 616 disc specimens, the authors found three pathways for an intravertebral tumor to invade into the adjacent disc: 1) direct infiltration from the rim of the vertebral body not covered by the cartilagenous plate, 2) infiltration from the side of the vertebral body close to the vertebral corner, through the subspace beneath the longitudinal ligament, and 3) hematogenous invasion via small vessels in the subspace beneath the longitudinal ligament. The authors concluded that the cartilagenous plate between the vertebral body and the disc as well as high intradiscal pressure would be acting as a barrier against tumor invasion from the vertebral body into the disc, and suggests that gradual increase of capillaries in the disc with age may enhance hematogenous invasion into the disc in rare occasions.

Retinal embolism from left atrial myxoma.

Retinal artery embolism that had arisen as a complication of a left atrial myxoma was diagnosed in a young man who had suddenly developed a visual disorder in his left eye. A tumor in the left atrium was surgically excised. Pathologically, retinal embolism secondary to atrial myxoma is rare. To our knowledge, only 15 cases have been documented so far. The present case was only the second case of an isolated retinal embolism that had developed without systemic involvement, and the first case of retinal artery branch embolism that had resulted from a left atrial myxoma.

[Clinical application of time dependent perimetry (9). The relations between stimulus area and double flash resolution, and critical flicker frequency].

Double flash resolution (DFR) and critical flicker fusion frequency (CFF) at the center of the visual field and the 5 degrees superior visual field were measured in normal subjects as a function of the stimulus area. Six stimulus sizes, ranging from a visual angle of 5' to 3 degrees 36' were used. The following results were obtained. First, the CFF increased as the stimulus area became larger at both the center and the 5 degrees superior visual field. Secondly, at the center of the visual field, the DFR initially increased as the stimulus area became larger and then decreased. Thirdly, at the 5 degrees superior visual field, this inhibitory phenomenon was also encountered with great individual variations and was not so evident as at the center of the visual field. Fourthly, the increasing rate of sensitivity was found to be greater at the 5 degrees superior visual field than at the center of the visual field in both CFF and DFR. From these results, we speculated that the CFF was detected by the transient system, which exhibited a weaker or no surround inhibition, and that the DFR in the center of the visual field was detected by the sustained system, which exhibited great surround inhibition. The DFR at the 5 degrees superior visual field could be detected by both transient and sustained systems.

False-negative lumbar discograms. Correlation of discographic and histological findings in postmortem and surgical specimens.

To investigate the causes of false-negative discograms, 181 lower thoracic and lumbar intervertebral discs that had been removed as part of en bloc specimens during thirty autopsies were studied first by discography and then histologically. Comparison of the results of the two methods showed that if fissures and cysts were present in a degenerated anulus fibrosus, but did not establish continuity between the nuclear cavity and the site of a herniation, the discogram was false-negative. Under these circumstances, the inner fiber bundles of the anulus fibrosus were intact and their orientation was often reversed, so that they bulged inward. This finding suggested that a protrusion or a prolapse of tissue from just the anulus fibrosus might have been developing. Ten of the fifty-seven discs that had such changes in the orientation of the fibers had a histologically proved protrusion or prolapse of the anulus fibrosus. However, the discograms showed protrusion in only six of the ten discs and demonstrated a false-negative result in the other four. The cases of seventy-seven patients in whom discography had been performed and a herniation had been subsequently confirmed at operation were also studied. Fifty-nine of the patients had a protrusion and eighteen had a prolapse of the disc. The discograms were falsely interpreted as negative in 32 per cent (nineteen) of the fifty-nine patients who had a protrusion and in 56 per cent (ten) of the eighteen who had a prolapse. Histologically, the prolapses were interpreted as protrusions of a portion of the anulus fibrosus. It was concluded that false-negative discograms are more frequent when a protrusion or a prolapse involves the anulus fibrosus rather than the nucleus pulposus, and that a negative discogram does not exclude the possibility of extensive degeneration of the anulus fibrosus.