Destiny of autologous bone marrow-derived stromal cells implanted in the vocal fold

OBJECTIVES: The aim of this study was to investigate the destiny of implanted autologous bone marrow-derived stromal cells (BSCs) containing mesenchymal stem cells. We previously reported the successful regeneration of an injured vocal fold through implantation of BSCs in a canine model. However, the fate of the implanted BSCs was not examined. In this study, implanted BSCs were traced in order to determine the type of tissues resulting at the injected site of the vocal fold. METHODS: After harvest of bone marrow from the femurs of green fluorescent transgenic mice, adherent cells were cultured and selectively amplified. By means of a fluorescence-activated cell sorter, it was confirmed that some cells were strongly positive for mesenchymal stem cell markers, including CD29, CD44, CD49e, and Sca-1. These cells were then injected into the injured vocal fold of a nude rat. Immunohistologic examination of the resected vocal folds was performed 8 weeks after treatment. RESULTS: The implanted cells were alive in the host tissues and showed positive expression for keratin and desmin, markers for epithelial tissue and muscle, respectively. The implanted BSCs differentiated into more than one tissue type in vivo. CONCLUSIONS: Cell-based tissue engineering using BSCs may improve the quality of the healing process in vocal fold injuries.     

Autologous replacement of the vocal fold: a new surgical approach for adduction-type spasmodic dysphonia

Many surgical approaches have been developed for the treatment of adduction-type spasmodic dysphonia (SPD). We developed and performed a new type of surgical approach (autologous replacement of the vocal fold). Our new surgical technique increases the advantages and decreases the disadvantages of previous surgical procedures in three ways: (1) It has similar effects to the previous procedures in that it prevents contraction of the thyroarytenoid muscle. (2) It decreases vocal-fold tension, as in framework surgery. (3) It reduces glottal incompetence, as does fibrinogen-glue injection, but it is more suitable because it is autologous. Furthermore, it produces increases in the mass and volume of the vocal-fold body and is also safe because the replacement tissue is autologous. The short-term results appear encouraging in preventing spastic voice while also avoiding vocal-fold atrophy. Long-term follow up will be necessary to determine the actual efficacy. However, this is clearly a possible choice as a surgical approach for treating adduction-type SPD.     

High-quality video recording and monitoring system for endoscopes

We have developed a new recording system for endoscope images with the popular mini digital video (mini DV) camera. A special adaptor screws onto the lens top, and either a flexible or rigid endoscope can be attached to the camera. Using this system, we can monitor not only the endoscopic images but also the patient's condition. Satisfactory images can be obtained even with the finest fiberscope. Nevertheless, our new system costs only USD 1,000. Therefore, the system is both cost-effective and useful for the outpatient clinic or casual setting or for house calls for the purpose of patient education.     

Autoantibodies to survivin in patients with chronic hepatitis and hepatocellular carcinoma

OBJECTIVES: Autoantibodies to tumor-associated antigens including survivin have been detected in sera from patients with hepatocellular carcinoma (HCC). However, little is known about autoantibody responses to tumor-associated antigens in patients with chronic hepatitis, which strongly predisposes to development of HCC. METHODS: We subjected sera from 57 patients with chronic hepatitis and 29 patients with HCC to an enzyme-linked immunosorbent assay (ELISA) using a full-length recombinant survivin protein. A cutoff value for positivity was determined as the mean absorbance +2SD for sera from healthy volunteers. RESULTS: In patients with chronic viral hepatitis, elevated anti-survivin antibodies were detected in 10 of 57 sera (17.5%); in HCC patients, such elevation were detected in 7 of 29 sera (24.1%). The levels of anti-survivin antibodies in HCC patients with HCV infection were significantly higher than those in the healthy control and HCC patients with HBV infection. However, there were no significant differences in the levels of anti-survivin antibodies between HCV and HCC patients with HCV infection. CONCLUSIONS: We demonstrated that elevated anti-survivin antibodies were detected for the first time in patients with chronic viral hepatitis. The results suggest that the levels of anti-survivin antibodies have no association with the progression of HCV or HBV to HCC.     

A thin carboxymethyl cellulose culture substrate for the cellulase-induced harvesting of an endothelial cell sheet

Engineered tissues constructed with two-dimensionally organized cells provide promising parts for reconstructing damaged tissues. Here we propose a new method for fabricating a 2D sheet made of an endothelial cell monolayer. First a culture substrate was prepared by treating the glass surface with an amine-terminated organosilicon derivative, followed by the covalent attachment of a thin carboxymethyl cellulose (CMC) layer. Fibronectin was immobilized onto the CMC-coated surface to promote cell adhesion. These surfaces were characterized step by step by means of contact angle measurement and X-ray photoelectron spectroscopy. Porcine aortic endothelial cells adhered to the culture substrate and consequently formed a confluent monolayer. When the substrate-cell composite was immersed in a cellulase solution, a cell sheet was spontaneously detached from the substrate due to enzymatic digestion of the CMC layer. The cell-cell connections were well preserved in the cell sheet, even after detachment from the substrate, most likely due to the fact that cellulase is harmless to mammalian cells. The cell sheet could be transferred to other culture dish with the aid of a hydrophilic membrane support, retaining the proliferation activity of the cells. The results obtained in this study demonstrate that cellulase treatment of the CMC layer is a rational and efficient method for obtaining a 2D cell sheet.     

Cytoplasmic expression of c-erbB2 in non-small cell lung cancers

The aim of this study was to investigate the expression of c-erbB2 in non-small cell lung cancers (NSCLCs) with attention both to membranous and cytoplasmic reaction, and to try to elucidate the meaning of cytoplasmic expression of c-erbB2 in NSCLCs. Immunohistochemical c-erbB2 expression and related clinico-pathological features were examined in 312 surgically resected patient tissues of NSCLCs, including 175 cases of adenocarcinoma and 137 cases of squamous cell carcinoma. Immunostaining of inner- and ecto-domain of c-erbB2, mRNA expression and the quantitation of soluble c-erbB2 in cultured media were performed in five NSCLC cell lines. Cytoplasmic expression of c-erbB2 was observed more frequently than membranous, both in patient tissues and cell lines. Neither membranous nor cytoplasmic expression of c-erbB2 was significantly correlated with short outcome in NSCLCs. Membranous c-erbB2 was expressed by both inner and ecto-domain, while cytoplasmic c-erbB2 was expressed by either or both inner and ecto-domain. c-erbB2 mRNA was produced in most cell lines; however, the soluble form was only detectable in a cell line that only presented a membranous c-erbB2. In conclusion, cytoplasmic c-erbB2 of NSCLCs was not a full-length protein only expressed in cellular membrane, but reflected degenerated c-erbB2 fragments with less functional ability.     

Up-regulation of VGLUT2 expression in hypothalamic-neurohypophysial neurons of the rat following osmotic challenge

A second vesicular glutamate transporter (VGLUT2) has been reported to be expressed in neurosecretory neurons of the hypothalamic-neurohypophysial system. To study its role in the neurosecretory neurons, we evaluated the expression of the VGLUT2 gene in the paraventricular (PVN) and supraoptic (SON) nuclei as well as the immunoreactivity in the neurohypophysis under euhydrated and chronic hyperosmotic conditions with in situ hybridization and immunohistochemistry. The intensity of hybridization signals in the PVN, SON and thalamus of rats subjected to water deprivation for 7 days, or drinking 2% NaCl for 4 or 7 days, was compared with that of euhydrated rats (control). The overall intensity in the entire PVN or SON, but not the thalamus, was higher in osmotically stimulated rats than in controls. Within the PVN, a significantly higher intensity of signals than that of controls was found only in the dorsolateral posterior magnocellular region in 4-day salt-loaded rats and in all subregions in water-deprived or 7-day salt-loaded rats. The intensity in the SON was higher in the stimulated rats than in controls, regardless of subregions. In the neurohypophysis, VGLUT2 staining was frequently localized in vasopressin terminals of control rats and was apparently reduced in stimulated rats. These results indicate that VGLUT2 is principally expressed in magnocellular vasopressin neurons, suggesting some local effect of intrinsic glutamate on neurohypophysial hormone secretion.     

Projections to the alimentary canal from the dopaminergic neurons in the dorsal motor nucleus of the vagus of the rat

The motility of the alimentary canal is regulated not only by neurons that contain acetylcholine or adrenaline, but also by nonadrenergic noncholinergic neurons. There are many neurons containing dopamine in the dorsal motor nucleus of the vagus (DMV). We examined the projections of these dopaminergic neurons to the alimentary canal with double-labeling immunohistochemistry for tyrosine hydroxylase (TH) and the retrograde tracer cholera toxin subunit b following its injection into the subdiaphragmatic esophagus, the cardia, the pylorus, the duodenum, the jejunum, and the ascending colon. Almost all double-labeled neurons were found in the half of the DMV caudal to the area postrema. In the caudal half of the DMV, about 58% of the TH-immunoreactive neurons projected to the cardia, about 36% projected to the pylorus, and about 28% projected to the subdiaphragmatic esophagus. Only a few TH-immunoreactive neurons projected to the duodenum, the jejunum, or the ascending colon. As a whole, less than 10% of the neurons in the DMV that projected to the alimentary canal showed TH-like immunoreactivity. These results suggest that some of the dopaminergic neurons in the DMV might regulate the activities of the stomach and the subdiaphragmatic esophagus.