Prospective study of kinesthesia after ACL reconstruction.

Previous studies used a variety of methods to assess kinesthesia, thus no consensus exists regarding kinesthetic adaptation after anterior cruciate ligament (ACL) reconstruction. This study prospectively examined whether kinesthesia is adapted after ACL reconstruction, and then discussed the actual angular velocity required to properly assess kinesthesia in ACL-reconstructed patients. 31 patients were evaluated using the threshold to detect passive motion (TTDPM) test, which was applied preoperatively, and at 3, 6, and 12 months following surgery. TTDPMs were measured at 15° or 45° of knee flexion toward both extension and flexion with angular velocities of 0.1°/s or 0.2°/s. ACL-reconstructed knees showed significantly impaired TTDPMs compared to healthy knees before the operation at 15° of knee flexion toward extension and at 45° of knee flexion toward both extension and flexion at 0.2°/s (15° of knee flexion toward extension, P=0.036; 45° of knee flexion toward extension, P=0.015; 45° of knee flexion toward flexion, P=0.030). However, there were no significant differences after 3 months of follow-up. On the basis of these results, applying 0.2°/s seems appropriate to assess TTDPM for patients with an ACL reconstruction, and kinesthesia is adapted within 12 months after the operation. Sensory function and biomechanical stability are also adapted following ACL reconstruction.

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage.

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.

Purification of a low molecular weight microtubule binding protein from sea urchin eggs.

A low molecular weight microtubule binding protein(SU-MAP34) was purified from sea urchin eggs. This protein bound strongly to the microtubule formed from purified echinoderm tubulin but showed no cross-linking of microtubules. Monospecific antibody against SU-MAP34 was produced and an immunoblotting analysis showed that this protein was not a breakdown product of a protein of a higher molecular mass. Whole cell staining and confocal laser scanning microscope observation showed that SU-MAP34 localized on the filamentous structure of mitotic apparatus and this structure was identified as the microtubule with double staining using anti-SU-MAP34 and anti-tubulin. An immunoblotting experiment showed an enrichment of SU-MAP34 in a microtubule protein fraction prepared using taxol from a crude extract of the cell.

Matrix metalloproteinase digestion of aggrecan in human cartilage tumours.

Substantial experimental and clinical evidence suggests that the catabolism of extracellular matrix components is a prerequisite for invasive and metastatic behaviour of solid tumours. Chondrosarcomas are malignant cartilaginous tumours that most commonly arise in bone, and the large aggregating proteoglycan aggrecan is a major component of the extracellular matrix of these tumours. Matrix metalloproteinases (MMPs) have been implicated in tumour invasiveness. The purpose of this study was to determine whether MMPs play a role in aggrecan catabolism in cartilage tumours. In order to detect aggrecan digestion products resulting from in vivo cleavage at the MMP site, protein extracts from human articular cartilage and from various cartilage tumours were analysed by Western blot using an antibody to the FVDIPEN neoepitope generated by MMP cleavage. Examination of cartilage extracts revealed a trend of increasing aggrecan digestion at the MMP site with age. One hyaline chondrosarcoma and three osteochondromas lacked detectable aggrecan fragments with the carboxy terminal FVDIPEN neoepitope. Two osteochondromas gave weak signals. However, all chondrosarcomas with degenerating extracellular matrix or with a myxoid component exhibited strong FVDIPEN immunoreactivity. These results demonstrate that, in contrast to the benign cartilage tumour osteochondroma, human chondrosarcomas contain abundant aggrecan degradation products resulting from cleavage in vivo at the MMP site in the interglobular domain. These data support the concept that MMPs participate in the degradation of extracellular matrix in chondrosarcoma, allowing the neoplastic chondrocytes to escape local confinement, migrate, and invade neighbouring and remote tissues.

Development of an antibody-based assay for determination of baculovirus titers in 10 hours.

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.

Acute bilateral deafness with nephritis: a human temporal bone study.

Temporal bone pathology is described in a 37-year-old man who had acute, bilateral, profound sensorineural hearing loss without improvement 4 months before death. The patient had suffered from low complement nephritis, for which he had received prednisolone therapy. Autopsy revealed malignant lymphoma with non-Hodgkin's type, membranous proliferative glomerulonephritis and necrotizing vasculitis of middle and small arteries. In the temporal bone study, pathological changes were limited to the cochlear region. The vestibular structure showed no detectable pathological changes. The changes included total absence of the organ of Corti, atrophy and/or disappearance of the stria vascularis in the upper turns, collapse of Reissner's membrane in the middle turn, and new bone formation in the apical turn.

Erythromycin therapy for otitis media with effusion in sinobronchial syndrome.

Chronic sinusitis is frequently associated with chronic lower respiratory tract diseases, and the association is referred to as sinobronchial syndrome (SBS). This study was carried out to determine the incidence of otitis media with effusion in the patients with sinobronchial syndrome and to investigate the efficacy of low-dose and long-term erythromycin therapy for otitis media with effusion associated with sinobronchial syndrome. We have found a high incidence of otitis media with effusion in patients with sinobronchial syndrome, the morbidity rate being 54% in 50 cases studied. This ear disease seems to be the major cause of hearing disturbance in patients with sinobronchial syndrome. Sixteen patients with both sinobronchial syndrome and otitis media with effusion were given low-dose and long-term erythromycin therapy (erythromycin base, 600 mg/d for more than 4 months); of these, 13 became effusion-free and most subjects showed improvement in the symptoms of sinobronchial syndrome. The erythromycin therapy thus seems to be exceedingly effective for the treatment of sinobronchial syndrome and associated otitis media with effusion.

A histological study of the temporal bones and the nose in Wolf-Hirschhorn syndrome.

We report the histopathological findings of the temporal bones and the nasal and paranasal specimen of a 7-month-old girl diagnosed as having Wolf-Hirschhorn or 4p- syndrome (deletion of the short arm of chromosome 4). This syndrome is characterized by growth retardation, mental retardation, and multiple congenital abnormalities, including craniofacial anomalies and hearing disturbance. These temporal bones displayed malformation of the ossicles, absence of the oval windows, abnormal course of the facial nerve with incomplete bony canal, and depression of the cochlear duct and the saccule. In addition, cholesteatoma, which might be of congenital origin, was present behind the eardrum. The nasal and paranasal specimen showed bilateral complete cleft palate with normal development of paranasal sinuses, nasal septum, and conchae.

Human leukocyte antigen in childhood unilateral deafness.

Human leukocyte antigen frequencies were examined in 40 Japanese patients with severe unilateral sensorineural hearing loss found in childhood. Human leukocyte antigen-Bw54 was significantly increased in frequency. No statistically significant deviation in the Bw54 frequency was observed among several possible subgroups studied. Although this group of patients may have various causes, and underlying mechanism is still unclear, this preliminary study suggests that childhood unilateral deafness may be associated with a genetically determined factor(s).

Initial anchoring and proliferation of fibroblast L-929 cells on unstable surface of calcium phosphate ceramics.

Calcium phosphate ceramics constructed from beta-tricalcium phosphate (TCP) and hydroxyapatite (HAP) have been successfully used as implant materials. However, there is a possibility that these materials are responsible for an unwanted inflammatory response during wound healing. Since TCP is soluble in the body, the instability of this material may contribute to this inflammatory response. Using composite ceramics of TCP and HAP that possessed Ca/P molar ratios of 1.50, 1.55, 1.60, 1.64, and 1.67, the effect of surface instability on fibroblast L-929 cells was investigated. The time-dependent variation of the initial anchoring ratio, cell density, and cell viability were measured. In general, the cells were severely damaged and ruptured on the highly soluble thin surface layer of the TCP-HAP ceramics. The initial anchoring ratio for TCP-HAP ceramics was as high as that for the polystyrene dish (Lux, control). However, viability at 6 h decreased to less than 50% of the initial cell density on ceramics with a Ca/P molar ratio of 1.64 (20% TCP-80% HAP), while 85% of the cells were viable on Lux. The viability on 100% TCP, whose surface is the most highly soluble among the TCP-HAP ceramics used in this study, was reduced to 20%. Morphological observation showed that the anchored cells were ruptured when grown in culture medium on the 100% TCP. Although the high solubility of the thin surface layer on the TCP-HAP ceramics of the carrier was found to be the dominant factor in the decreasing cell viability, the initial viability was enhanced by the stabilization of the surface of the TCP-HAP ceramics by pre-incubating the scaffolds in a culture medium containing 10% fetal bovine serum for 3 d.

Visualization of a recombinant gene protein in the baculovirus expression vector system using confocal scanning laser microscopy.

Expression of the recombinant protein beta-galactosidase in the Spodoptera frugiperda Sf-9 insect cell line infected by the Autographa californica nuclear polyhedrosis virus expressing beta-galactosidase (AcNPV-betagal) was visualized using confocal scanning laser microscopy with fluorescent staining of both the recombinant protein and the cell nucleus. The average size of the insect cells and the intracellular DNA concentration both increased markedly, respectively reading 3.8- and 2.3-fold the values before infection. The average beta-galactosidase activity began to increase at 20-24 h post infection and finally reached 1.9 x 10(4) units/ml. As the post infection time increased, the stained nucleus images expanded and spread broadly. Beta-galactosidase was first identified by fluorescent staining at 12 h post-infection, filled the cell at 27 h, began to be released at 36 h, and finally spread out of the cell. The locations of the nucleus and expressed beta-galactosidase were identified from computerized tomograms and 3-dimensional images.

Sudden deafness with severe loss of cochlear neurons.

We report our postmortem findings in the temporal bones of a patient who, at the age of 13, after having had a cold, experienced a sudden and profound hearing loss in her right ear. She died of nasopharyngeal carcinoma at 41. The patient's right temporal bone showed a complete loss of cochlear neurons, but the organ of Corti was well-preserved. These findings indicate a primary degeneration of the cochlear nerve. The authors speculated that the possible cause of this sudden deafness was severe neuronitis of the cochlear nerve.

Morphological changes of vestibular ganglion cells in human fetuses and in pediatric patients.

The temporal bone histopathology of human vestibular ganglion cells of fetuses and pediatric patients was studied. In the first study, we traced the morphological changes in vestibular ganglion cells in human fetuses ranging from 13 weeks to 39 weeks of gestational age by using 13 temporal bone serial sections. Vestibular ganglion cells had reached histological maturity by the 24th week of gestation and the volume of vestibular ganglion cell cytoplasm increased until the 39th week of gestation. In the second study, the temporal bone serial sections of seven neonates, eight infants and five children were investigated to reveal pathological changes in vestibular ganglion cells. Morphological changes in vestibular ganglion cells in human fetuses were revealed. Vestibular ganglion cells were changed pathologically by intracranial disease and variety etiology affecting the inner ear, because these are located in the internal auditory canal between the brain and labyrinth.

Prognostic factors for persistent middle ear effusion after acute otitis media in children.

The present study was carried out to determine the clinical course of acute otitis media (AOM) in children and to analyze the risk factors that lead to persistent effusion. One hundred and twenty children aged 9 months to 10 years diagnosed as having AOM were included in this study. Sixty-two children (52%) recovered from AOM without middle ear effusions within a month. In 26 cases (22%), middle ear effusions resolved after 1 to 3 months, while 32 children (26%) had persistent effusions for more than 3 months after the onset of acute inflammation. Statistical analysis between the group showing quick recovery and that with persistent effusion was carried out in relation to various clinical factors at the onset. The significant risk factors were: younger age, bilateral AOM, presence of otorrhea, tympanogram type B or C2 on an AOM ear. Moreover, the most significant prognostic factor was a tympanogram type B or C2 on an opposite ear at the acute onset. On the other hand, factors such as sex, fever, history of otitis media, season at onset or complications did not correlate with the duration of persistent effusion. From these results we conclude that careful examination should be performed not only on the ear with AOM but also on the opposite ear in order to predict the course of otitis media.

Cholesteatoma debris as an activator of human monocytes. Potentiation of the production of tumor necrosis factor.

Tumor necrosis factor (TNF) is a cytokine which stimulates osteoclastic bone resorption and inhibits collagen synthesis in vitro. In this study the effect of human cholesteatoma debris and its constituents on the production of TNF-alpha by human monocytes in vitro was studied. Cultured human peripheral monocytes secreted TNF into the culture medium when exposed to cholesteatoma debris in a dose-dependent manner. The TNF production, however, was partially inhibited by the treatment of the debris with polymyxin B which inhibits biological activities of lipopolysaccharide (LPS). When individual constituents of cholesteatoma debris, i.e. keratin, cholesterol, lauric acid and LPS, were added to the cultured monocytes at concentrations equivalent to those in the debris, significant production of TNF was observed only with the keratin and LPS. These data suggest that cholesteatoma debris is a potent activator of the TNF production of human monocytes in vitro, and that LPS and keratin are responsible for the production.

Activation of peritoneal macrophages with human cholesteatoma debris and alpha-keratin.

The effect of human cholesteatoma debris on mouse peritoneal macrophages was studied in vivo. The number of macrophages and lymphocytes increased 5 days after injection of the debris into the peritoneal cavity. A similar increase in peritoneal cells was observed when an urea-extracted fraction of the cholesteatoma debris or alpha-keratin, a major component of the debris, was injected. Both cholesteatoma debris- and alpha-keratin-elicited macrophages exhibited a greater response of luminol-dependent chemiluminescence upon exposure to zymosan, suggesting that the elicited macrophages were activated. In contrast, other constituents of the debris, such as cholesterol or fatty acid--with the exception of lipopolysaccharide (LPS)--failed to elicit or activate peritoneal macrophages at the similar doses detected in the debris. The chemiluminescent response of macrophages obtained by injecting LPS was, however, much lower than that of alpha-keratin-induced macrophages. These results indicate that cholesteatoma debris is capable of eliciting and activating macrophages, and that alpha-keratin is responsible for the activation.

Histopathological study of otitis media with effusion.

Thirteen temporal bones with otitis media with effusion (OME) taken from ten patients were studied. The effusions in the tympanum were histologically classifed into serous and mucoid types. In the serous effusion, neutrophils were dominantly distributed and the mucosa and submucosa showed inflammatory signs. Few epithelial cells and lymphocytes were observed in the effusion of the mucoid type. Eosinophils were rarely seen in the effusions of both types. The presence of inflammatory process was a common finding in OME, particularly with serous effusion. A case, with an immunologic disorder suspected, showed an extensive proliferation of mucosa and unusually thick liquid in the middle ear. This histological feature of the case did not resemble those of other ears with mucoid type of effusion.

Erythromycin inhibition of lipopolysaccharide-stimulated tumor necrosis factor alpha production by human monocytes in vitro.

The mechanism of clinical effectiveness of low-dose and long-term erythromycin (EM) treatment for diffuse panbronchiolitis, sinobronchial syndrome, and associated otitis media with effusion was investigated by studying the effects of EM on tumor necrosis factor alpha (TNF-alpha) production by cultured human monocytes stimulated with lipopolysaccharide. At concentrations of 0.1 microgram/mL or more, EM inhibited TNF-alpha release from human monocytes stimulated by lipopolysaccharide in a dose-dependent manner. Of the other macrolides tested, roxithromycin, an EM derivative, also showed significant inhibition of TNF-alpha production, whereas josamycin failed to inhibit TNF-alpha release from monocytes. Nonmacrolidic drugs such as minocycline hydrochloride, ofloxacin, or penicillin G had no significant effect on TNF-alpha production. These results suggest that the clinical improvement of chronic respiratory diseases by EM may depend on the suppression of production of inflammatory cytokines such as TNF-alpha.

Experimental mumps labyrinthitis in monkeys (Macaca irus)--immunohistochemical and ultrastructural studies.

Three monkeys (Macaca irus) were inoculated with mumps virus into unilateral cochleas and their inner ear were examined by immunofluorescent microscopy and transmission electronmicroscopy. The temporal bones were removed after survival period of 14 days when serological tests disclosed elevation of anti-mumps antibody titers. Immunofluorescent microscopy revealed that the viral antigen was positive in the stria vascularis. The ultrastructural study revealed that the pathologic changes in the cochleas were marked in the organ of Corti and stria vascularis. The outer hair cells were more susceptible to the infection than the inner hair cells. In the stria vascularis, both marginal and intermediate cells were affected. It was possible to find some of marginal cells in the basal turn shedding a large number of mature virions into the endolymph. These pathologic changes observed in the cochleas of the monkeys were similar to those previously revealed in the guinea pig cochleas and thus were considered as the specific features of acute mumps labyrinthitis.

Cochlear neuronal loss are determined by use of a neurofilament protein antibody in cases of bilateral profound deafness.

The temporal bones of two patients with profound bilateral deafness from infancy were studied immunohistochemically, using a neurofilament protein antibody to detect the cochlear neuronal elements. One patient exhibited Mondini dysplasia of the inner ear, with the organ of Corti almost completely deteriorated. The other patient is the first reported case involving complete aplasia of the organ of Corti in all turns. In both cases, the immunohistochemical staining clearly revealed a severe reduction in the number of afferent neurons, such as dendrites, spiral ganglion cells and cochlear axons. The number of efferent spiral bundles in the osseous spiral lamina and intraganglionic portion also decreased in parallel with the reduction in the number of cochlear afferent neurons. Our results are inconsistent with previously reported cases of presbycusis and acquired deafness induced by the measles virus, in which efferent neurons were preserved while afferent neurons degenerated. The loss of both the efferent and afferent neurons might be characteristics of congenital deafness.