Feasibility of virtual surgical simulation in the head and neck region for soft tissue reconstruction using free flap: a comparison of preoperative and postoperative volume measurement.

In the head and neck region, preoperative evaluation of the free flap volume is challenging. The current study validated preoperative three-dimensional (3D) virtual surgical simulation for soft tissue reconstruction by assessing flap volume and evaluated fat and muscle volume changes at follow-up in 13 head and neck cancer patients undergoing anterolateral craniofacial resection. Patients received 3D virtual surgical simulation, and the volume of the planned defects was estimated by surgical simulation. Following en bloc resection of the tumor, the defect in the skull base was covered using a rectus abdominis myocutaneous flap. Following surgery, computed tomography scans were acquired at day 1 and at 6 and 12 months. Virtual planned defect was on average 227 ml (range, 154-315) and was 10% smaller than the actual flap volume in patients without skin involvement of the tumor. Between day 1 and 12 months post-surgery, the volume of fat and muscle tissue in the free flap dropped by 9% and 58%, respectively. Our results indicate that 3D virtual surgical simulation provides essential information in determining the accurate volume of the required free flap for surgical defect repair and may thus help improve surgical planning and functional and esthetic outcome.

Predictive Factors Associated with Oral Intake Ability in Gastrostomy Patients Under Long-Term Care.

OBJECTIVE: To determine the physical indicators associated with oral intake status and swallowing function in gastrostomy patients under long-term care. DESIGN: Cross-sectional study. SETTING: Thirty-one hospitals that perform gastrostomy insertion, replacement and management. PARTICIPANTS: A total of 117 respondents from 31 hospitals in Japan underwent gastrostomy tube replacement and management between September 2012 and January 2014. Each participant underwent a gastrostomy at least 6 months prior to the study, and received long-term care either at home, a care facility, or a hospital. MEASUREMENTS: We conducted a questionnaire survey at Japanese hospitals and used the data obtained from 117 respondents for analysis. The survey was conducted using a questionnaire form that collected information about the following items: oral intake status, sex, age, disease history, number of days elapsed since gastrostomy, residence status, modified Rankin Scale score, consciousness, oral hygiene status, articulation and phonation, voluntary saliva swallow, Modified Water Swallow Test, and Food Test. RESULTS: Results revealed significant differences in modified Rankin Scale scores, sputum production, articulation and phonation, and voluntary saliva swallowing between patients who were orally fed and those who were not. Moreover, sputum production and voluntary saliva swallowing were strongly associated with oral intake status. Finally, sputum production, articulation and phonation, and voluntary saliva swallowing were strongly associated with swallowing function test results. CONCLUSION: Results from this study suggested that sputum production, articulation and phonation, and voluntary saliva swallowing could be used as indicators for estimating oral intake status and swallowing function in gastrostomy patients under long-term care.

Alveolar bone regeneration using absorbable poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblast growth factor.

The aim of this study was to evaluate the effects of combining a porous poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblastic growth factor (bFGF) on bone regeneration in mandibular ridges. Four full-thickness saddle-type defects (10 mm long x 5 mm deep) were symmetrically created in both edentulous mandibular alveolar ridges of 6 beagles. The dome-shaped membrane was secured to each defect site, and a gelatin sponge containing 200 microg bFGF was implanted on the left side of each defect (experimental group). Only the membranes (control group) were secured to the defect sites on the right. Three and 6 months later, 3 animals were killed. Bone regeneration was analyzed by soft X-ray photographs, micro-computed tomography (CT) images, and peripheral quantitative CT (pQCT), and then examined histologically. Soft X-ray examination revealed an increase in new bone volume in the experimental group 6 months postoperatively. pQCT showed that immature bone density was higher in the experimental group. Micro-CT images revealed well formed new bone along the original contour of the dome-shaped membrane in the experimental group. Histologically, inflammatory infiltration of tissue surrounding the membranes was slight. These results suggest that combining the poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and bFGF-gelatin sponge is promising for alveolar ridge reconstruction.

Gas6 rescues cortical neurons from amyloid beta protein-induced apoptosis.

Gas6, a product of the growth-arrest-specific gene 6, protects neurons from serum deprivation-induced apoptosis. Neuronal apoptosis is also caused by amyloid beta protein (Abeta), whose accumulation in the brain is a characteristic feature of Alzheimer's disease. Abeta induces Ca(2+) influx via L-type voltage-dependent calcium channels (L-VSCCs), leading to its neurotoxicity. In the present study, we investigated effects of Gas6 on Abeta-induced cell death in primary cultures of rat cortical neurons. Abeta caused neuronal cell death in a concentration- and time-dependent manner. Gas6 significantly prevented neurons from Abeta-induced cell death. Gas6 ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Prior to cell death, Abeta increased influx of Ca(2+) into neurons through L-VSCCs. Gas6 significantly inhibited the Abeta-induced Ca(2+) influx. The inhibitor of L-VSCCs also suppressed Abeta-induced neuronal cell death. The present cortical cultures contained few non-neuronal cells, indicating that Gas6 affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that Gas6 rescues cortical neurons from Abeta-induced apoptosis. Furthermore, the present study indicates that inhibition of L-VSCC contributes to the neuroprotective effect of Gas6.

Expression of granulocyte colony-stimulating factor receptor and platelet-derived endothelial cell growth factor in oral and oropharyngeal precancerous lesions.

Epithelial hyperplasia and dysplasia have been diagnosed as precancerous lesions and have been discussed in relationship to carcinogenesis. We analyzed the immunohistochemical expression of granulocyte colony-stimulating factor receptor (G-CSFR) and platelet-derived endothelial cell growth factor (PD-ECGF) in oral and oropharynx; 33 samples of normal epithelium, 28 samples of hyperplasia, 16 samples of dysplasia and 58 samples of squamous cell carcinoma. Also, we examined mean vessel density (MVD) by using CD34 staining and proliferating cell nuclear antigen (PCNA) staining. Dysplasia and head and neck Squamous Cell Carcinoma (SCC) exhibited higher G-CSFR expression and MVD than normal or hyperplastic epithelium (p <0.01). In the PD-ECGF staining, significant differences were found between SCC and normal epithelium, hyperplasia and dysplasia (p<0.001). In dysplasia and hyperplasia, PD-ECGF expression was significantly correlated with PCNA expression (r=0.345, p=0.025), however it was not correlated with the MVD. G-CSFR expression was not correlated with either PCNA or MVD. These results suggest that G-CSFR and PD-ECGF might be concerned with different carcinogenesis pathways of the squamous cells in the oral region and that PD-ECGF may be concerned with epithelial proliferation rather than angiogenesis.

Identification of Aichi virus infection by measurement of immunoglobulin responses in an enzyme-linked immunosorbent assay.

Using inhibitory enzyme-linked immunosorbent assay, seroconversions to Aichi virus were detected in 24 (42.9%) of 56 patients with gastroenteritis in six outbreaks. Virus-specific immunoglobulin M (IgM) was detected in convalescent-phase sera from 7 of 24 patients. Of the other 17 patients, 12 developed a significant increase in both IgA and IgG levels and 5 developed a significant increase in IgG alone.

Lipopolysaccharide: neutralization by polymyxin B shuts down the signaling pathway of nuclear factor kappaB in peripheral blood mononuclear cells, even during activation.

BACKGROUND: There have been many studies on anti-lipopolysaccharide (LPS) agents and LPS-neutralizing agents; however, there have been no reports on the changes in clinical status and mediators that occur when these agents are used. Polymyxin (PMX) (treatment using a column containing polymyxin B-immobilized fiber) removed circulating endotoxin, and reduced various cytokines within 120 min, even in patients with high levels of plasma cytokines. Our purpose was examine the mechanisms of PMX treatment by which plasma cytokines are reduced by endotoxin neutralization with polymyxin B, even during therapy for sepsis and/or endotoxin shock. METHODS: We studied the interaction between nuclear factor kappaB (NF-kappaB) binding activity and tumor necrosis factor alpha (TNF-alpha) secretion in an experimental system using LPS-stimulated human peripheral blood mononuclear cells (PBMCs), after neutralization of LPS with polymyxin B. PBMCs were incubated with LPS in vitro, and TNF-alpha secretion and NF-kappaB activation were assessed. We then studied the changes in NF-kappaB activation and TNF-alpha secretion when both polymyxin B and LPS were added simultaneously and when polymyxin B was added after 30 or 120 min of incubation with LPS. RESULTS: Immediate inhibition of NF-kappaB binding activity and suppression of TNF-alpha secretion were observed after LPS neutralization with polymyxin B regardless of whether PBMCs were already producing TNF-alpha. CONCLUSIONS: These findings may indicate one of the mechanisms operating in the clinical changes that occur after circulating endotoxin removal, and are likely to have therapeutic value, even for patients with high proinflammatory cytokine levels.

Off-line leukapheresis using leukofiltration for active ulcerative colitis: a case report.

The method of leukocytapheresis for ulcerative colitis (UC) by using extracorporeal circulation (on-line system) has been reported. To perform leukocytapheresis, we have applied leukocyte elimination filters for blood transfusion to leukocytapheresis without using extracorporeal circulation (off-line leukocytapheresis system). Four hundred milliliters of peripheral blood was collected and reinfused through a leukocyte elimination filter. This procedure was repeated 5 times, and up to 2,000 ml of peripheral blood was treated. This method has been applied once a week for 5 weeks. We applied the off-line leukapheresis system to a 31-year-old male ulcerative colitis patient. As a result, the frequency of defecation and the dose of medicine were effectively decreased, and endoscopic finding was also improved. Because of the absence of complications observed with the on-line system, the off-line leukocytapheresis system that we have applied to the clinical patient is simple, safe, and useful.

Appearance of bax protein after preoperative chemoradiotherapy is a prognostic factor in maxillary cancer.

The prognosis for maxillary squamous cell carcinoma (SCC) remains poor, despite advances in combination therapy. Combined treatment with anticancer drugs and radiation therapy is aimed at inducing apoptosis. As apoptosis is regulated by several proteins, we investigated the expression of p53, Bax and Bcl-2 in maxillary SCC before treatment and after preoperative chemoradiotherapy using an immunohistochemical approach. Furthermore, apoptotic cells were visualized using an in situ apoptosis detection kit and the apoptosis index (AI) was defined as the number of positive cancer cells per 1,000 cancer cells. Expression of p53 and Bcl-2 and the Al in 23 maxillary SCCs were not associated with tumor size, lymph node metastasis, clinical stage, frequency of recurrence or 5-year survival rate either before treatment or after preoperative chemoradiotherapy. Bax expression before treatment was not correlated with any clinicopathological factors before treatment. However, no patients in the Bax-positive group (11/22 cases) after preoperative chemoradiotherapy had recurrence of maxillary SCC and all were alive after 5 years, while the 5-year survival rate was 34.1% in Bax-negative patients. These results suggest that the appearance of the Bax protein after preoperative chemoradiotherapy is a significant prognostic marker for maxillary SCC.

Expression of fas (CD95) ligand is correlated with IL-10 and granulocyte colony-stimulating factor expression in oral and oropharyngeal squamous cell carcinoma.

The Fas/Fas ligand (FasL) pathway has been shown to be an important cellular pathway mediating apoptosis. In this study we investigated the expression of Fas and FasL and the rate of spontaneous apoptosis in 58 oral and oropharyngeal squamous cell carcinoma (SCC) by using immunohistochemical techniques. There was no correlation between Fas or FasL expression and clinicopathological factors. The expression of Fas in the tumor did not affect spontaneous apoptosis of the tumor cells. However, FasL expression was associated with IL-10 and granulocyte colony-stimulating factor expression in oral and oropharyngeal SCC. These results suggested that the Fas/FasL system is connected with the expression of various factors including cytokines in tumor cells.

Discrimination between Atlantic and Pacific Subspecies of Northern Bluefin Tuna (Thunnus thynnus) by Magnetic-Capture Hybridization Using Bacterial Magnetic Particles.

The previously developed magnetic-capture hybridization technique employing bacterial magnetic particles was applied to discriminate between Atlantic and Pacific subspecies of the northern bluefin tuna (Thunnus thynnus) using specific DNA sequences. Nucleotide sequences of a 925-bp fragment (ATCO) flanking the mitochondrial ATPase and cytochrome oxidase subunit III genes in these two subspecies were compared. Two regions having single-nucleotide and three-nucleotide differences between the subspecies were adopted to design DNA probes (NR1, 21-mer; NR2, 29-mer), and two internal primer sets were designed to amplify DNA fragments containing these regions. The DNA probes were immobilized on bacterial magnetic particles via streptavidin-biotin conjugation and subjected to magnetic-capture hybridization with the digoxigenin-labeled fragments amplified using the internal primers. The luminescence intensities of DNA on bacterial magnetic particles obtained by hybridization between the probes and the complementary fragments were higher than those obtained by hybridization with noncomplementary fragments. These data suggest that this system employing DNA on bacterial magnetic particles may be useful for discrimination of these two subspecies by recognizing a single-nucleotide difference.

Application of a reverse transcription-PCR for identification and differentiation of Aichi virus, a new member of the Picornavirus family associated with gastroenteritis in humans.

Aichi viruses isolated in Vero cells from seven patients in five gastroenteritis outbreaks in Japan, five Japanese returning from Southeast Asian countries, and five local children in Pakistan with gastroenteritis were examined for differentiation based on their reactivities with a monoclonal antibody to a standard strain (A846/88) and a reverse transcription-PCR (RT-PCR) of three genomic regions. The RNA sequences were determined for 519 bases of these 17 isolates at the putative junction between the C terminus of 3C and the N terminus of 3D. The analyses revealed an approximately 90% homology between these isolates, which were then divided into two groups: group 1 (genotype A) included six isolates from four outbreaks and one isolate from a traveler and group 2 (genotype B) included one isolate from the other outbreak, four isolates from returning travelers, and all of the isolates from the Pakistani children. Based on the isolate sequences, a primer pair and a biotin-labeled probe were designed for amplification and detection of 223 bases at the 3C-3D junction of Aichi virus RNA in fecal specimens. The Aichi virus RNA was detected in 54 (55%) of 99 fecal specimens from the patients in 12 (32%) of 37 outbreaks of gastroenteritis in Japan. Of the 12 outbreaks, 11 were suspected to be due to genotype A. These results indicated that RT-PCR can be a useful tool to detect Aichi virus in stool samples and that a sequence analysis of PCR products can be employed to identify the prevalent strain in each incident.

Inhibition of cathepsin activities by vitamin B6 derivatives.

We found that pridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibited the activity of cathepsins B, K, S and L in vitro. Cathepsins activities in cultured splenocytes were suppressed by the addition of pridoxal (PL) or pridoxine (PI) in to the culture medium. A newly synthesized artificial vitamin B6 derivative, a pridoxal propionate derivative, CLIK-164, showed selective inhibition of cathepsin O/K.

Anti-metastatic efficacy and safety of MMI-166, a selective matrix metalloproteinase inhibitor.

The anti-metastatic efficacy and safety of a newly-developed matrix metalloproteinase (MMP) inhibitor were examined. MMI-166, a N-sulfonylamino acid derivative, inhibited the enzyme activity of MMP-2, 9, and 14 but not MMP-1, 3 or 7. Daily oral administration of MMI-166 resulted in potent inhibition of metastatic lung colonization of Lewis lung carcinoma injected via the tail vein and liver metastasis of C-1H human colon cancer implanted into the spleen at inhibition levels of 43% and 63%, respectively. Daily administration of MMI-166 also resulted in prolonged survival of mice given intraperitoneal implantation of Ma44 human lung cancer cells. The anti-metastatic activity of MMI-166 was as effective as that of other MMP inhibitors with broad inhibitory spectrum. MMI-166 did not affect in vitro tumor cell growth. Neither body weight losses nor hematotoxicity was observed during long-term treatment, indicating the safety of MMI-166 in mice. These results indicate that the selective MMP inhibitor MMI-166 has therapeutic potential as an anti-metastasis agent.

Phylogenic analysis of echovirus type 30 isolated from a large epidemic of aseptic meningitis in Japan during 1997-1998.

During 1997 to 1998, a nationwide epidemic of aseptic meningitis occurred in Japan. More than 4,500 isolates from patients with aseptic meningitis were identified as echovirus type 30. To investigate the character of these isolates, we examined the nucleotide sequences of thirty-seven geographical representatives and compared them with 50 strains isolated during the past 20 years. The phylogenic analysis used partial sequences from either the VP1 or VP4-VP2 region of the viral capsid. This analysis revealed that the isolates were divided into six genomic groups. All isolates identified during 1997-1998 belonged to only two genomic groups; these two groups are thought to be the causative viral agents involved in the recent epidemic.

[Antiangiogenic and antitumor effect of BPHA, an orally-active matrix metalloproteinase inhibitor, in in vivo murine and human tumor model].

We examined the antiangiogenic and antitumor efficacy of a newly-developed matrix metalloproteinase (MMP) inhibitor, BPHA (N-biphenyl sulfonyl-phenylalanine hydroxiamic acid). BPHA potently inhibits MMP-2, 9 and 14 but not MMP-1, 3 or 7. In contrast, (-)BPHA, an enantiomer of BPHA, was inactive against all MMP tested. Daily oral administration of BPHA in mice resulted in potent inhibition of tumor-induced angiogenesis, primary tumor growth and liver metastasis, whereas (-)BPHA did not. These results demonstrate that selective MMP inhibition is correlated with antiangiogenic and antitumor efficacy and that the selective MMP inhibitor BPHA has therapeutic potential without hematotoxic effect or loss of body weight.

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo.

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N-(L-trans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethyla mide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10(-7) M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10(-7) M in vitro.

[Bacterial infection and production of immunoglobulin in the tonsil].

Immunoglobulin (Ig) isotype switching is the process whereby B cells initially expressing IgM and/or IgD on their surface switch to other Ig heavy-chain loci. We demonstrated that TGF-beta, IL-10 and VIP in the presence of CD40 mAb, can induce isotype switching for IgA in human tonsillar B cells by the generation of switch circular DNA (S alpha/S mu), the induction of alpha germ-line transcripts and a significant amount of IgA production. IgA is the predominant immunoglobulin effector molecule of mucosal immunity that functions as the first line of specific immunologic defense against many microbial pathogens. However, IgA causes IgA nephropathy. We investigated here, whether tonsillar mononuclear cells from patients with IgA nephropathy produce HP-specific IgA and/or IgA-related cytokines by stimulation with components of HP outer membranes (OMHP). HP-specific IgA was predominately induced by tonsillar mononuclear cells of IgA nephropathy, compared to those from chronic tonsillitis. Production of IL-10 and TGF-beta was enhanced by stimulation with OMHP in tonsillar mononuclear cells from IgA nephropathy. These results suggested that local infection of HP and HP-specific IgA induction in the tonsil are associated with pathogens in IgA nephropathy.

Apoptosis-promoting gene (bax) transfer potentiates sensitivity of squamous cell carcinoma to cisplatin in vitro and in vivo.

Modulation of apoptosis may potentiate the sensitivity of tumor cells to chemotherapeutic agents, thus improving the clinical outcome of cancer treatment. Bax, an apoptosis-promoting member of the bcl-2 family, may be a key factor influencing the chemosensitivity of tumor cells, however, its involvement in cellular sensitivity to anti-cancer drugs remains uncertain in squamous cell carcinoma (SCC). To investigate the role of bax gene expression in modulating cisplatin (CDDP)-induced apoptosis in vitro, an established CDDP-resistant human head and neck SCC (IMC-3 cell line) was transfected with bax gene-bearing mammalian expression vector. Overexpression of the bax gene in CDDP-resistant IMC-3 cells elevated the CDDP susceptibility of tumor cells to a level similar to that of the parental IMC-3 cells. In an in vivo study, percutaneous transfer of apoptosis-promoting bax gene by particle-mediated (gene gun) delivery caused overexpression of Bax in SCC, which was confirmed by immunohistochemical staining, and inhibited the growth of mouse CDDP-resistant SCC. Furthermore, combination therapy with bax gene transfer and subcutaneous administration of CDDP at 3-day intervals markedly inhibited the growth of mouse SCC. Thus, overexpression of bax in SCC by a gene gun system appears to be a rational approach to improving the efficacy of chemotherapy and treatment outcome. We suggest that exogenous bax expression may have therapeutic applications for enhancing chemotherapy in SCC.