Vascular density of superficial esophageal squamous cell carcinoma determined by direct observation of resected specimen using narrow band imaging with magnifying endoscopy.

Observation of the microvasculature using narrow band imaging (NBI) with magnifying endoscopy is useful for diagnosing superficial squamous cell carcinoma. Increased vascular density is indicative of cancer, but not many studies have reported differences between cancerous and noncancerous areas based on an objective comparison. We observed specimens of endoscopic submucosal dissection (ESD) using NBI magnification, and determined the vascular density of cancerous and noncancerous areas. A total of 25 lesions of esophageal squamous cell carcinoma that were dissected en bloc by ESD between July 2013 and December 2013 were subjected to NBI magnification. We constructed a device that holds an endoscope and precisely controls the movement along the vertical axis in order to observe submerged specimens by NBI magnification. NBI image files of both cancerous (pathologically determined invasion depth, m1/2) and surrounding noncancerous areas were created and subjected to vascular density assessment by two endoscopists who were blinded to clinical information. The invasion depth was m1/2 in 20, m3/sm1 in four and sm2 in one esophageal cancer lesion. Mean vascular density was significantly increased in cancerous areas (37.6 ± 16.3 vessels/mm2) compared with noncancerous areas (17.6 ± 10.0 vessels/mm2) (P < 0.05). The correlation coefficients between vascular density determined by two endoscopists were 0.86 and 0.81 in cancerous and noncancerous areas, respectively. Receiver operating curve (ROC) analysis revealed that the area under the curve (AUC) of vascular density was 0.895 (95% CI, 0.804-0.986). For this ROC curve, sensitivity was 78.3% and specificity was 87.0% when the cutoff value of vascular density was 26 vessels/mm2. NBI magnification confirmed significant increases in vascular density in cancerous areas compared with noncancerous areas in esophageal squamous cell carcinoma. The rates of agreement between vascular density values determined by two independent operators were high.

Effect of hyaluronic acid on the excursion resistance of tendon grafts. A biomechanical study in a canine model in vitro.

The excursion resistance between the tendon and pulley is an important factor contributing to the limitation of function after surgery to the hand. The administration of hyaluronic acid (HA) in the early rehabilitation after tendon grafting may help to prevent adhesions. We evaluated changes in the excursion resistance between potential sources of flexor tendon grafts and the annular pulley in a canine model after administration of HA. The intrasynovial and extrasynovial tendons were soaked in 10 mg/ml of HA for five minutes. The excursion resistance between these tendons and the annular pulley of an intact proximal phalanx and that of the same tendons of the opposite foot without administration of HA were evaluated. The tendon of flexor digitorum profundus of the second toe without administration of HA was used as a control. The gliding resistance of canine tendons was significantly decreased after the administration of HA especially in the extrasynovial tendons. Our findings suggest that the administration of HA may improve the gliding function of a flexor tendon graft.

Experimental study of blood typing in immunosuppressant-free tracheal transplantation in dogs.

BACKGROUND: A previous report has noted that tracheal allotransplantation is possible using allografts without immunosuppressants. In the present study, we investigated whether blood typing is necessary in immunosuppressant-free tracheal allotransplantation in dogs. METHODS: In group 1, the blood types of paired dogs were mismatched (n=6, three pairs), while the blood types of paired dogs were matched in group 2 (n=6, three pairs). In paired dogs, an intrathoracic 5-ring tracheal segment was exchanged. No immunosuppressants were used. RESULTS: All animals survived for more than 8 weeks. In each group, different degrees of airway stenosis were observed in four dogs. However, no stenosis was observed in the other two dogs. Histologically, no vascular rejection was detected. Ciliated columnar epithelium covered the inner surface of the implanted graft in the long-term survival cases. CONCLUSIONS: In immunosuppressant-free tracheal allotransplantation, no hyperacute rejection occurred in any recipient dog whose blood type was mismatched with the donor. Blood typing was not the key factor to successful immunosuppressant-free tracheal allotransplantation. Immunosuppressant-free tracheal allotransplantation may be possible using mismatched blood type tracheal grafts.

Regeneration of hypogastric nerve using a polyglycolic acid (PGA)-collagen nerve conduit filled with collagen sponge proved electrophysiologically in a canine model.

The hypogastric nerve (HGN) is a sympathetic nerve in the peritoneal cavity and controls urinary and seminal functions. In this study, the regeneration of HGN was determined by using a new type of an artificial nerve conduit, polyglycolic acid (PGA)-collagen nerve conduit filled with collagen sponge in two dogs. A PGA-collagen nerve conduit (diameter=2 mm) was interposed in a 10 mm gap of the right HGN. The regeneration of the HGN was evaluated electrophysiologically 8 months after the operation. The intraluminal pressure of spermatic duct and the bladder neck were elevated 80 mmHg and 25 mmHg respectively by the stimulation across the regenerated HGN. The prostate contraction was also elicited. The responses diminished after the excision of the regenerated portion of HGN. These results proved the regeneration of HGN and this nerve conduit will be great help for patients who suffer from urinary and seminal disturbances.

Regenerative repair of the mandible using a collagen sponge containing TGF-beta1.

INTRODUCTION: Alveolar bone resorption and atrophy of the mandible are a major challenge for regeneration medicine. In the present investigation, a collagen sponge that contained TGF-beta1 was placed at a mandibular defect and the osteogenic effects of collagen-TGF-beta1, complex were evaluated. MATERIAL AND METHODS: The Pm2, Pm3, and Pm4 teeth on both sides of the mandibles of 12 adult beagle dogs (9.0-12.0 kg) were extracted. After the extraction-site wounds healed, a bone defect (10.0 x 15.0 mm-wide, 10.0 mm-deep or 10.0 x 10.0 mm-wide, 10.0 mm-deep) was created on the mandible. A collagen sponge (10.0 x 10.0 x 10.0 mm) that contained TGF-beta1 (1.0 microg, 5.0 microg, or 10.0 microg, in physiological saline) was placed at the bottom of the defect and the overlying mucous membrane was sutured with 4-0 prolene. As a control, a collagen sponge that contained physiological saline only was placed in a defect on the opposite side. Two weeks after the surgery the wounds above the bone defects on both the control and TGF-beta1-treated sides had healed completely. RESULTS: At four, six, or eight weeks post-operatively animals were killed. Soft X-ray and bone-salt measurement analyses confirmed clearly that there was greater calcified bone formation in the defects into which TGF-beta1 had been incorporated than with the control defects. The implanted collagen sponges were fully resorbed and the bone tissue had regenerated from the bottom of the defects on the TGF-beta1, side by four weeks. On the control side, no such regeneration was observed. CONCLUSIONS: These results indicate that TGF-beta1, released slowly from a collagen sponge was effective in promoting bone remodeling when applied to mandibular defects in adult dogs.

Postoperative outcome of lumbar spinal canal stenosis after fenestration: correlation with changes in intradural and extradural tube on magnetic resonance imaging.

PURPOSE: To evaluate the serial changes in clinical results and the intradural and extradural spaces on magnetic resonance imaging (MRI) after bilateral fenestration in 48 patients with lumbar spinal canal stenosis (LSCS). METHODS: A prospective interventional study was performed to study the clinical results, magnetic resonance imaging scans among patients who were followed up for more than 3 years. RESULTS: All patients showed improvement in clinical symptoms after operation, but clinical results deteriorated in 9 (19%) patients. Postoperative MRI scans showed that poor dural tube expansion, grouping of the cauda equina, and decrease in the cross-sectional area of the dural tube were factors associated with poor outcomes. The cross-sectional area of the dural tube and images of the cauda equina observed by MRI, before and after fenestration and during follow-up, reflected changes in clinical symptoms involving decompressed segments. CONCLUSION: Serial changes in the cross-sectional area of the dural tube and images of the cauda equina observed preoperatively, postoperatively, and on follow-up by MRI may be useful when evaluating patients' condition before and after operation. It is also useful for predicting outcomes.

Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene.

AIMS: This study aimed to develop a polymerase chain reaction (PCR) method to identify Lactobacillus crispatus. METHODS AND RESULTS: A primer set (CbsA2F-CbsA2R) for amplifying conserved regions of S-layer genes was designed to identify Lact. crispatus and the specificity of this set was compared with that of another primer set (Cri 16SI-Cri 16SII) which has been reported as a species-specific primer set targeting the 16S rRNA gene. Among species in the Lact. acidophilus A1-A4 groups, when KOD polymerase was used for amplification, the primer set CbsA2F-CbsA2R gave PCR products with Lact. crispatus strains only. However, when Taq polymerase was used, this primer set gave products with one Lact. amylovorus strain as well as with Lact. crispatus strains. The primer set Cri 16SI-Cri 16SII gave PCR products with Lact. crispatus strains and two Lact. acidophilus strains, regardless of whether the polymerase used was KOD or Taq. CONCLUSIONS: A PCR targeting the S-layer gene and amplified with KOD polymerase can identify Lact. crispatus accurately and rapidly. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge, this is the first paper to provide a PCR method for the specific identification of Lact. crispatus.

Evaluation of peripheral nerve regeneration across an 80-mm gap using a polyglycolic acid (PGA)--collagen nerve conduit filled with laminin-soaked collagen sponge in dogs.

We evaluated peripheral nerve regeneration using a novel artificial nerve conduit. The conduit was made of a polyglycolic acid(PGA) - collagen tube filled with laminin- soaked collagen sponge. We implanted this nerve conduit across an 80mm gap in the peroneal nerve of dogs. Histological observation 12 months after implantation showed numerous unmyelinated and myelinated nerve fibershad regenerated beyond the gap. Neurofilaments were widely observed immunohistochemically in the regenerated nerve segments. These findings indicated that newly regenerated axons had extended across the gap and connected into the distal nerve segments. Compound muscle action potentials(CMAPs) and somatosensory evoked potentials (SEPs) were recorded in all dogs. At 12 months, the CMAPs indicated complete recovery, while the SEPs showed incomplete but substantial recovery. Walking patterns had returned to near-normal 12 months after implantation. Use of this nerve conduit can lead to peripheral nerve elongation and favorable functional recovery across a wider nerve gap.

Inhibition of the adherence of Escherichia coli strains to basement membrane by Lactobacillus crispatus expressing an S-layer.

AIMS: This study aimed to evaluate the efficiency with which Lactobacillus crispatus JCM 5810 inhibited the adhesion of enteric pathogens to a synthetic basement membrane and to elucidate the mechanism underlying the inhibition. METHODS AND RESULTS: Lactobacillus crispatus JCM 5810 inhibited the adhesion of three diarrhoeagenic Escherichia coli strains to a reconstituted basement membrane preparation called Matrigel, used as a model of a damaged intestinal tissue site. Inhibition was also observed with the use of immobilized laminin, a major component of Matrigel, but diminished after the removal of S-layer protein (CbsA) from JCM 5810 cells. The isolated CbsA inhibited the adhesion of E. coli to both Matrigel and immobilized laminin. Lactobacillus crispatus JCM 5810 and CbsA seem to inhibit pathogenic E. coli from adhering to basement membrane via competition with laminin molecules for binding sites. CONCLUSIONS: These results suggested that not only Lact. crispatus JCM 5810 cells but CbsA alone might prevent pathogens from colonizing damaged intestinal tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to show the applied aspect of Lactobacillus S-layer protein.

Regeneration of canine peroneal nerve with the use of a polyglycolic acid-collagen tube filled with laminin-soaked collagen sponge: a comparative study of collagen sponge and collagen fibers as filling materials for nerve conduits.

A novel artificial nerve conduit was developed and its efficiency was evaluated on the basis of promotion of peripheral nerve regeneration across an 80-mm gap in dogs. The nerve conduit was made of a polyglycolic acid-collagen tube filled with laminin-soaked collagen sponge. Conduits filled with either sponge- or fiber-form collagen were implanted into an 80-mm gap of the peroneal nerve (five dogs for each form). Twelve months postoperatively nerve regeneration was superior in the sponge group both morphometrically (percentage of neural tissue: fiber: 39.7 +/- 5.2, sponge: 43.0 +/- 4.5, n=3) and electrophysiologically (fiber: CMAP 1.06 +/- 0.077, SEP 1.32 +/- 0.127 sponge: CMAP 1.04 +/- 0.106, SEP 1.24 +/- 0.197, n=5), although these differences were not statistically significant. The observed regeneration was complementary to successful results reported previously in the same model, in which collagen fibers exclusively were used. The results indicate a possible superiority of collagen sponge over collagen fibers as filling materials. In addition, the mass-producibility, superior scaffolding potential, and capacity for gradual release of soluble factors of the sponge provide make it an attractive alternative to fine fibers, which are both technologically difficult and costly to produce. This newly developed nerve conduit has the potential to enhance peripheral nerve regeneration across longer gaps commonly encountered in clinical settings.

Tracheal allotransplantation in beagle dogs without immunosuppressants.

BACKGROUND: The antigenicity of tracheal grafts is still unclear. We investigated the possibility of performing tracheal allotransplantation without immunosuppressants. METHODS: Intrathoracic five-ring tracheal replacements were performed in beagle dogs without immunosuppressants (n = 18). The dogs were divided into 9 pairs, and grafts were exchanged within the pairs. In group 1 (n = 6), the paired dogs were blood relatives, whereas in group 2 (n = 12), the paired dogs were not related. Full-thickness skin transplantation was also performed in both groups. RESULTS: In group 1, 5 animals survived uneventfully for more than 3 months. No stenosis was observed in any of the dogs. In group 2, the grafts were incorporated by the host trachea in 2 dogs. Four animals died of airway obstruction within 3 months. Moderate or slight airway stenosis was observed in 6 dogs. Rejection was confirmed by histologic examination. In both groups, all of the skin allografts were destroyed within 2 weeks. CONCLUSIONS: After tracheal allotransplantation, long-term survival was achieved, especially in recipient dogs that were blood relatives of donors. We conclude that it is possible to perform tracheal allotransplantation using histocompatible matched grafts without immunosuppressants.

Sagittal splitting laminoplasty for spinal canal enlargement for ossification of the spinal ligaments (OPLL and OLF).

Patients have been followed up radiologically after sagittal splitting laminoplasty (SSL) for ossification of the posterior longitudinal ligament (OPLL) and ligamentum flavum. The enlarged spinal canal was maintained satisfactorily in all the cases, and the posterior spinal elements were also almost completely restored. In most cases, the range of the vertebral motion was good with more than 50% of the preoperative status. Postoperative instability of the vertebral column was not identified in any case. Mild cervical kyphosis was noted in 6% of cervical OPLL patients. There were no patients with neurological deterioration. Using SSL, reconstruction and restoration of the posterior spinal elements and retention of the enlarged spinal canal were accomplished safely and adequately.

Inhibition of adhesion of food-borne pathogens to Caco-2 cells by Lactobacillus strains.

AIMS: To select adhesive strains among strains of Lactobacillus and to apply them to inhibit adhesion of food-borne pathogens. METHODS AND RESULTS: Twelve Lactobacillus strains (10 from intestine) were examined for adhesion using Caco-2 cell cultures. The two most adhesive strains, Lactobacillus crispatus JCM 8779 and Lact. reuteri JCM 1081, were used to test antiadhesion activity against enterotoxigenic Escherichia coli, Salmonella typhimurium and Enterococcus faecalis strains. Adhesion of the pathogens was inhibited by both Lactobacillus strains. Adhesion of Ent. faecalis was especially strongly inhibited by JCM 8779. Although antimicrobial activity was not detected in the culture supernatant fluid by agar well diffusion assay, the supernatant fluid obtained from the harvested JCM 8779 cell suspension showed bactericidal activity against Ent. faecalis. CONCLUSION: The strong antiadhesion activity of JCM 8779 against Ent. faecalis appears to be due to the combined effect of both bactericidal activity and competition for attachment site. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that Lact. crispatus produces a bactericidal substance.

[Identification of Vibrio vulnificus from patients with otitis media and septicemia by PCR method].

Sucrose-negative colonies were isolated on TCBS agar plate from otorrhea specimens from otitis media patient in the Adult Diseases Examination Center, Hirosaki Medical Association. The isolate was identified as Vibrio vulnificus by nested PCR method which amplify V. vulnificus-specific sequences directed against 23S rRNA genes. The PCR method was applied to identify 6 strains originally isolated from septicemia patients in Kurashiki Central Hospital and formerly identified as V. vulnificus by phenotypic characteristics. When examined by the API20E system, the above PCR confirmed-V. vulnificus isolates were correctly identified as V. vulnificus with % ID 99.8, though this gave 3 different profiles. Cytotoxin-hemolysin gene was detected in otorrhea strain as well as septicemia strains by PCR method. The above results suggest that PCR method targeted cytotoxin-hemolysin gene is suitable for rapid and accurate identification of the isolate, because the result is obtained in less than 4 h. To our knowledge this is the first report on the V. vulnificus infection in Aomori Prefecture and the isolation from otorrhea.

Analysis of sputum taken from wheezy and asthmatic infants and children, with special reference to respiratory infections.

Children who are destined to develop asthma are considered to be susceptible to a variety of respiratory pathogens. To elucidate respiratory inflammation among these children, we measured the levels of eosinophil cationic protein (ECP) and tryptase in sputum taken from three different groups of wheezy infants and young children: those with a first wheeze (n = 15); those with recurrent wheeze (n = 27); and those with recurrent wheeze with respiratory distress, namely asthma (n = 56). The numbers of eosinophils or metachromatic cells determined by microscopic analysis of sputum samples were also evaluated in combination with the ECP and tryptase levels. Although neither sputum ECP nor tryptase was a clear discriminative marker that differentiated the three different types of wheezy disease, ECP levels in sputum from the asthma group were significantly higher (2,269.2 +/- 6,216.8 ng/g) than those in the recurrent wheezy group (440.3 +/- 1,199.8 ng/g) or in the first-wheeze group (209.0 +/- 172.9 ng/g). A similar trend was observed with tryptase levels in sputum, but there were no significant differences among the three groups. Sputum taken from asthmatic children showed a marked accumulation of eosinophils. However, an accumulation of eosinophils in sputum (even in the presence of an elevated level of sputum ECP) was not identified in the asthmatic infants < 1 year of age. An accumlation of eosinophils in sputum was not evident until children became > 1 year old and thereafter the eosinophils rapidly increased in number until the children reached 5 years of age. It was noteworthy that sputa positive for pathogenic bacteria, taken from the 1- and 2-year-old asthmatic infants, had a tendency to show high levels of ECP but a reduced number of eosinophils. Along with the wheezy episodes induced by viral infection, primarily and occasionally in combination with secondary bacterial infection, eosinophil activation and infiltration may develop. These predestined immune reactions to various pathogens might be associated with triggering the onset of asthma.

Artificial trachea and long term follow-up in carinal reconstruction in dogs.

We have already reported successful carinal reconstruction of the trachea with an observation period of 1 - 2 years. In this study, we evaluate the long-term safety and efficacy of the reconstruction after 5-years of follow-up. The Y-shaped Marlex mesh tube was reinforced with a polypropylene spiral and coated with atelocollagen made from porcine skin. The prosthesis was 60 mm long with an outer diameter of 18 mm. Replacement of the tracheobronchial bifurcation was preformed through a right thoracotomy in a beagle dog. Bronchoscopical examination and sampling of the tracheal epithelium was performed periodically to check the function of cilia. The implanted prothesis was promptly infiltrated by the surrounding connective tissue and completely incorporated by the host trachea and bronchus. Bronchoscopically, sufficient epithelization was confirmed from the upper to the lower site of anastomosis. After 5 years neither stenosis nor dehiscence was observed. In spite of there being mesh-exposure at the luminal surface, the dog had no clinical symptoms until sacrifice for pathological examination. The bent frequency of the cilia was maintained within the normal range, indicating functional recovery of the regenerating airway. Our tracheal prosthesis is promising for clinical repair of the tracheobronchial bifurcation.

Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus.

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

The evaluation of corticosteroid therapy in conjunction with plasma exchange in the treatment of renal cholesterol embolic disease. A report of 5 cases.

In this report, we describe 5 patients with cholesterol atheroembolic renal failure. In 3 of the 5 patients, combined therapy with corticosteroids and plasma exchange was performed. These 3 patients survived, with 2 showing an improvement in renal function. The 2 remaining patients died of multifactorial causes. The literature on therapy for cholesterol atheroembolic renal failure is reviewed and the efficacy of combined therapy by use of corticosteroids and plasma exchange is evaluated.

Usefulness of a highly sensitive urinary and serum IL-6 assay in patients with diabetic nephropathy.

BACKGROUND/AIM: Interleukin-6 (IL-6) promotes the growth of renal mesangial cells. IL-6 may play a major role in such mesangial proliferation, but there has been little research on IL-6 in relation to diabetic nephropathy because of the difficulty in measuring urinary and serum IL-6 levels. Using a newly developed, highly sensitive IL-6 assay, we studied the relationship between serum and urinary IL-6 and diabetic nephropathy. METHODS: We investigated 72 patients with type 2 diabetes. Urinary and serum IL-6 concentrations were measured using a chemiluminescent enzyme immunoassay with a detection limit of 0.11 pg/ml. RESULTS: There was a significant increase of the serum IL-6 level as diabetic nephropathy progressed, with the level being 1.4 +/- 0.3 pg/ml in patients with normal albuminuria, rising to 2.4 +/- 0.6 pg/ml in patients with microalbuminuria and then to 4.4 +/- 0.8 pg/ml in those having proteinuria. The serum IL-6 level was also significantly correlated with fibrinogen and aortic pulse wave velocity. The urinary IL-6 level was also significantly increased in diabetic patients as nephropathy progressed. Both serum and urinary IL-6 levels were high in the group with nephropathy, but there was no correlation between the two. CONCLUSION: The urinary IL-6 level seems to be a good indicator of diabetic nephropathy, and atherosclerotic changes were related to the serum IL-6 level. The serum IL-6 may, therefore, be useful in the evaluation of atherosclerosis including nephropathy.