Development of a Highly Sensitive ß-Glucan Detection System Using Scanning Single-Molecule Counting Method.

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant ß-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-ß-D-glucan recognition protein (S-BGRP) and a (1→6)-ß-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-ß-D-glucan from fungi. S-BGRP and (1→6)-ß-glucanase mutant proteins reacted with ß-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived ß-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-ß-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of ß-glucan levels by the SSMC method using recombinant ß-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.

Amplification-free gene expression analysis of formalin-fixed paraffin-embedded samples using scanning single-molecule counting.

In this paper, we present the applications of our newly developed, highly sensitive fluorescent detection method referred to as scanning single-molecule counting (SSMC). We found that the target RNA added to the total RNA was detected with high sensitivity at 384 aM by combining a magnetic bead-based assay and SSMC (MB-BA + SSMC). Gene expression analysis without reverse transcription or amplification confirmed that the pattern of gene expression was identical to that of real-time polymerase chain reaction (PCR). MB-BA + SSMC was also applied to formalin-fixed paraffin-embedded (FFPE) samples. RNA fragmentation and crosslinking owing to FFPE processing slightly affected gene expression. Conversely, FFPE samples showed an increase in gene Ct values and a decrease in the number of detectable genes when analyzed using real-time PCR. Overall, our results suggested that SSMC is a powerful tool for target RNA detection and amplification-free gene expression analysis.

Scanning single-molecule counting system for Eprobe with highly simple and effective approach.

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

Relationship between globus sensation and esophageal clearance.

CONCLUSIONS: Videofluoroscopy (VF) in the prone position can diagnose a range of esophageal pathologies in all age groups. These correlate with globus sensation in younger and middle-aged patients in prone and supine positions. Abnormal esophageal clearance appears to be associated with globus sensation in young and middle-aged persons, but not in the elderly. OBJECTIVES: To assess the correlation between globus sensation and abnormal esophageal clearance by VF in relation to body position and to investigate age-related abnormal esophageal clearance, as well as to confirm the utility of VF in diagnosing globus sensation in patients. SUBJECTS AND METHODS: This was a prospective study. In 72 patients with globus sensation and 33 controls, esophageal clearance was assessed by VF in the upright, supine, and prone positions, and transfer of barium through the esophagus was classified as normal, esophageal retention, esophageal reflux, or pharyngeal reflux. The detection rate of abnormal esophageal clearance was compared among all subjects by age. RESULTS: In the upright position, VF detected no significant differences in the detection rate of abnormal esophageal clearance and reflux between the patients with globus sensation and controls; however, the detection rate of abnormal esophageal clearance was significantly higher in the prone than in the upright and supine positions (p<0.001), and in all positions was significantly higher among globus sensation patients (p<0.05). In the supine and prone positions, there were significantly higher detection rates of abnormal esophageal clearance in patients than in controls in the young group (p<0.05). Additionally in the prone position, there was a significantly higher detection rate of abnormal esophageal clearance and reflux in patients than in controls in the middle-aged group (p<0.05). In all positions, no significant differences were observed in the detection rate of abnormal esophageal clearance and reflux in the patients and controls in the elderly group.

Therapeutic effect of edaravone on inner ear barotrauma in the guinea pig.

Inner ear barotrauma (IEB) that is caused by acute pressure changes can often lead to permanent severe sensorineural hearing loss (SNHL). However, the mechanism that causes IEB is still unknown. In the current study, we assessed the involvement of reactive oxygen species (ROS) in IEB and the therapeutic effect of 3-methyl 1-phenyl-2-pyrazolin-5-one (edaravone), which is a free radical scavenger. To create the IEB model, guinea pigs were subjected to quick pressure changes that resulted in acute SNHL. The animals were then divided into two groups, an edaravone-treated IEB group and a non-treated IEB group that only received normal saline. Immunohistochemical analyses for 8-hydroxy-2-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) were performed to examine the amount of oxidative DNA damage and lipid peroxidation that occurred in guinea pig cochlea. To assess the curative efficacy of edaravone, auditory brainstem response (ABR) testing was performed to evaluate auditory function. Strong immunoreactivities against 8-OHdG and 4-HNE were observed in the inner ear tissues of the non-treated IEB group. Lesser amounts of immunoreactivity were observed in the same region of the edaravone-treated IEB group as compared to the non-treated IEB group. Furthermore, ABR measurement revealed that there was a faster improvement in the threshold shift for the edaravone-treated IEB group as compared to that of the non-treated IEB group. At the final 7-week time point, the threshold shift for the edaravone-treated IEB group was significantly smaller as compared to the non-treated IEB group. These results strongly suggest that ROS is produced in the cochlea in response to acute pressure changes and that ROS plays an important role in the pathophysiology of IEB. Furthermore, edaravone treatment had a therapeutic effect on IEB-induced acute SNHL and thus, edaravone might possibly be able to be used as a therapeutic treatment for IEB.

[Case report on open injuries of the larynx].

While open injury of the larynx is relatively rare, prompt and emergent control of the airway, treatment of the infected wound, and surgical repair for reconstruction of the laryngeal function are required. We report on a patient who had sustained an open injury of the larynx in whom the laryngeal function could be successfully preserved intact. The case was a 46-year-old man who had attempted suicide by cutting his throat with a kitchen knife, which resulted in complete tearing of the thyroid membrane and exposure of the upper part of the larynx. Emergent tracheotomy was performed the epiglottis was pulled up, and the thyroid cartilage was sewn to prevent occlusion of the laryngeal passage by the sagging epiglottis. In addition, we sutured together the hyoid bone and thyroid cartilage, so that the larynx could be held up and the laryngeal structure could be reconstructed satisfactorily for good deglutition. Eventually, we could close his tracheostoma, following which he regained his voice and normal swallowing function.

Further development of multiplex single nucleotide polymorphism typing method, the DigiTag2 assay.

A number of single nucleotide polymorphisms (SNPs) are considered to be candidate susceptibility or resistance genetic factors for multifactorial disease. Genome-wide searches for disease susceptibility regions followed by high-resolution mapping of primary genes require cost-effective and highly reliable technology. To accomplish successful and low-cost typing for candidate SNPs, new technologies must be developed. We previously reported a multiplex SNP typing method, designated the DigiTag assay, that has the potential to analyze nearly any SNP with high accuracy and reproducibility. However, the DigiTag assay requires multiple washing steps in manipulation and uses genotyping probes modified with biotin for each target SNP. Here we describe the next version of the assay, DigiTag2, which works with simple protocols and uses unmodified genotyping probes. We investigated the feasibility of the DigiTag2 assay by genotyping 96 target SNPs spanning a 610-kb region of human chromosome 5. The DigiTag2 assay is suitable for genotyping an intermediate number of SNPs (tens to hundreds of sites) with a high conversion rate (>90%), high accuracy, and low cost.

Pulmonary spread of laryngeal papillomatosis: radiological findings.

Laryngeal papillomatosis is a rare benign disease seen in children and young adults. The spread of laryngeal papillomas throughout the respiratory tract occurs rarely; and involvement of the distal bronchi, bronchioles, and lung parenchyma is very rare. We report a case of pulmonary spread of laryngeal papillomatosis in a 34-year-old man, focusing on the radiological evidence. Chest radiographs showed pulmonary nodules, but computed tomography scans more clearly demonstrated multiple small nodules showing cavitations and distributed centrilobularly. Pulmonary nodules and cavitations progressed gradually through the 6-year follow-up. The combination of clinical and characteristic radiological features suggests a diagnosis of pulmonary spread of laryngeal papillomatosis.

Direct assay for alpha-amylase using fluorophore-modified cyclodextrins.

A simple assay method for alpha-amylase was developed based on fluorophore-modified cyclodextrins (CDs). Four kinds of CD derivatives bearing a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety were prepared as artificial substrates for the assay method. The fluorescence intensity of all the NBD-amine-modified CDs decreased upon addition of Aspergillus oryzae alpha-amylase, indicating a reduction in hydrophobicity near the NBD-amine moiety induced by hydrolysis of the CD ring. NC4gammaCD, having a gamma-CD and an amino-tetramethylene spacer, was the most sensitive substrate for the alpha-amylase assay. The initial rate of hydrolysis of NC4gammaCD displayed a liner correlation to the concentration of the alpha-amylase. NC4gammaCD was sensitive to the alpha-amylase but was not sensitive to guest compounds that were accommodated by the native CDs.

Expression of KIT, EGFR, HER-2 and tyrosine phosphorylation in undifferentiated thyroid carcinoma: implication for a new therapeutic approach.

The KIT, epidermal growth factor receptor (EGFR) and HER-2 oncoproteins have tyrosine kinase activity and are molecular targets in human cancer therapy. To clarify the significance of KIT, EGFR, and HER-2 in undifferentiated thyroid carcinoma (UTC), the expression of these receptors and tyrosine phosphorylation was examined immunohistochemically in resected cases of UTC and papillary thyroid carcinoma (PTC). KIT, EGFR, and HER-2 were also examined at the protein and mRNA levels in five UTC cell lines. KIT expression (1+), EGFR overexpression (2+/3+), HER-2 expression (1+), and tyrosine phosphorylation were detected immunohistochemically in 40%, 70%, 10%, and 50% of the 10 UTC. In 20 PTC, KIT, EGFR, and HER-2 were not detected, but tyrosine phosphorylation was detected in 25% of cases. In the five UTC cell lines, KIT expression (1+), EGFR overexpression (3+), HER-2 expression (1+), and tyrosine phosphorylation were detected immunocytochemically in 60%, 100%, 20%, and 40%, respectively. Western blot analysis did not detect KIT expression, but did detect EGFR and HER-2 expression in all five cell lines. Real-time polymerase chain reaction detected KIT mRNA in two of the cell lines (40%), EGFR in five (100%), and HER-2 in three (60%). The present findings suggest that EGFR overexpression was involved in the proliferation and development of UTC and was frequently accompanied by tyrosine phosphorylation. Expression of KIT and HER-2 appeared to be weak but significant, suggesting a possible role in the development of UTC. Molecular therapies targeting KIT, EGFR, HER-2, and/or tyrosine phosphorylation might be indicated for UTC.

DigiTag assay for multiplex single nucleotide polymorphism typing with high success rate.

As a consequence of Human Genome Project and single nucleotide polymorphism (SNP) discovery projects, several millions of SNPs, which include possible susceptibility SNPs for multifactorial diseases, have been revealed. Accordingly, there has been a strong drive to perform the investigation with all candidate SNPs for a certain disease without decreasing the number of analyzed SNPs. We developed DigiTag assay, which uses well-designed oligonucleotides called DNA coded numbers (DCNs) in multiplex SNP genotype analysis. During the analysis, the information of a genotype is converted to one of the DCNs in a one to one manner using oligonucleotide ligation assay (encoding). After the encoding reaction, only the DCNs regions and not the SNP specific regions are amplified using the universal primers and then SNP genotype is read out using DNA capillary arrays. DigiTag assay was found to be successful in SNP genotyping, giving a high success rate (24 of 27 SNPs) for randomly chosen SNPs. Moreover, this assay has the potential to analyze almost all kinds of the target SNPs by applying mismatch-induced probes and redesigned primer pairs at a low-cost.

[Identification of sentinel lymph node in neck-node-negative oral and pharyngeal carcinoma study of patients, it's feasibility, and problems].

We assessd the feasibility and problems associated with sentinel lymph node (SLN) study in 13 cases of oral and pharyngeal squamous cell carcinoma (SCC) that were neck-node-negative clinically. The primary sites were the tongue (n = 10), other sites in the oral cavity (n = 2), and the mesopharynx (n = 1). The day before surgery, tracer was injected into the submucosa around the tumor, and scintigraphic images were acquired 2 hours later. The SLN was identified intraoperatively with a handheld gamma probe, and neck dissection, including the SLNs, was performed. Radioactivity within the nodes was confirmed with a well type scintillation counter, and all resected lymph nodes were histologically examined for metastasis. The SLN was identified in every case. There were regional lymphnode metastases in 4 cases, and metastasis to the SLNs was present in all of 4 cases. Thus, the SLN concept was valid for head and neck SCC, sentinel node navigation surgery (SNNS) was thought to be applied in stage NO SCC of the head and neck. If SNNS is performed, about 70% of patients do not require neck dissection. SNNS is feasible and cost-effective in these cases. We used two different tracers: phytate and tin colloid, and found that phytate was more useful. To avoid the effects of the shine-through phenomenon, it was thought that some directions of lymphoscintigram should be taken. For intraoperative identification of the SLNs, care should be taken to the angle of gamma probe. SLN study leads to clarify each patient's lymphoid flow mapping, and it is also useful to determine the dissection area of selective neck dissection.

Feasibility and cost-effectiveness of sentinel lymph node radiolocalization in stage N0 head and neck cancer.

OBJECTIVES: To determine the feasibility of sentinal lymph node (SN) radiolocalization and to assess the cost-effectiveness of the SN navigation surgery strategy in patients with stage N0 squamous cell carcinoma (SCC) of the head and neck. Patients Eleven consecutive patients with stage N0 SCC of the head and neck. METHODS: Head and neck lymphoscintigraphy was performed 2 hours after the injection of technetium Tc 99m tin colloid or phytate. A handheld gamma probe was used to detect the SN before and directly after making a skin incision. Nodes were evaluated histopathologically for micrometastasis. To determine the expected cost savings, a decision tree sensitivity analysis was designed based on the 2 competing strategies: ipsilateral neck dissection vs SN navigation surgery. The costs referred to billed costs based on the Japanese national insurance reimbursement system. RESULTS: The sensitivity of SN navigation surgery in our series was 100% (11/11) on a patient-by-patient basis and 94% (17/18) on a node-by-node basis. Micrometastasis was found in 36% (4/11). Assuming the micrometastasis prevalence, sensitivity, and specificity of navigation surgery for detecting SN to be 30%, 90%, and 100%, respectively, the decision tree sensitivity analysis showed that introduction of SN navigation surgery in place of ipsilateral neck dissection would yield cost savings of $1218 (US) per stage N0 patient in Japan and avoid 7 surgical deaths per 1000 patients who are supposed to undergo neck dissection in the neck dissection strategy. Break-even point analysis for the SN navigation surgery strategy showed that the threshold value required more than 41 patients for the savings to begin to accrue. CONCLUSION: Our results indicate that SN navigation surgery using radiolocalization is feasible and cost-effective, based on decision tree sensitivity analysis, in patients with stage N0 SCC of the head and neck.

Feasibility of sentinel lymph node radiolocalization in neck node-negative oral squamous cell carcinoma patients.

We examined the reliability of the use of lymphoscintigraphy (LS) and a hand-held gamma probe (GP) to identify the sentinel lymph node (SLN), and sequentially determined the feasibility of SLN radiolocalization in clinical neck node-negative oral squamous cell carcinoma (SCC) patients. A radiolabel with the unfiltered (99m)Tc tin colloid was injected submucosally around the primary site followed by LS at 2-hour intervals. Preoperative localization was performed with GP. After en bloc removal of the regional lymphatics, histopathologic results for the nodes were compared with the SLN radiolocalization. The LS and GP counts were well correlated, and there was concordance between SLN and neck node status in 7 of 8 cases (87.5%). We thus considered that our concept in this study was valid in determining the necessity for neck dissection for those node-negative oral SCC patients.

Concurrent chemoradiotherapy with low-dose cisplatin plus 5-fluorouracil for the treatment of patients with unresectable head and neck cancer.

OBJECTIVE: The purpose of this study was to determine the efficacy of concurrent chemoradiotherapy using conventional radiotherapy combined with low-dose daily 5-fluorouracil (5FU) and cisplatin (CDDP) for the locally unresectable head and neck cancer patients. PATIENTS AND METHODS: From September 1996 through December 2000, we carried out a phase II study of concurrent chemoradiotherapy with low-dose CDDP plus 5FU for the treatment of patients with unresectable squamous cell carcinoma of the head and neck. Chemoradiotherapy consisted of irradiation with 1.6-2.0 Gy/day for 5 days per week up to a total dose 68 Gy and CDDP 3 mg/m2 by intravenous infusion over 1 h plus 5FU 150 mg/m2 by intravenous infusion over 24 h per day for 5 days per week. RESULTS: Ninety percent of the patients had stage IV disease, including 65% of patients with T4 disease. Thirty-three patients (83%) received the full treatment as planned; 39 (98%) received full-dose radiotherapy and 33 (83%) full-dose chemotherapy. Of the 40 patients evaluable for response, 20 (50%) achieved complete response (CR) and 12 (30%) partial response with an overall response rate of 80%. Among the 20 CR patients, 15 underwent endoscopic blind biopsies and 4 had positive lesions. The most frequently observed toxicity was mucositis. Ten patients developed grade III mucositis, and 3 patients required enteral nutritional support through a feeding tube. Grade III leukopenia, anemia and thrombocytopenia were observed in 28, 25 and 20% of the patients, respectively. The median duration of follow-up at the time of analysis was 18 months. The median survival time was 23 months. The responders survived longer (34 months) than the nonresponders (4 months; p < 0.05). CONCLUSION: This regimen is safe and efficacious in the treatment of patients with advanced unresectable head and neck cancer.