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.

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.

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.

Expression of granulocyte colony-stimulating factor receptor correlates with prognosis in oral and mesopharyngeal carcinoma.

Granulocyte colony-stimulating factor receptors (G-CSFRs) have been observed on the surface of not only hematopoietic cells but also several cancer cells. The stimulation of G-CSF has been demonstrated to induce proliferation and activation of G-CSFR-positive cells. In this study, we investigated the expression of G-CSFR on the surface of tumor cells and G-CSF production in oral and mesopharyngeal squamous cell carcinoma (SCC) by an immunohistochemical approach. Of 58 oral and mesopharyngeal SCCs, 31 cases (53.4%) and 36 cases (62.1%) were positive for G-CSFR and G-CSF, respectively. There was no association between G-CSFR expression and G-CSF staining. In the group positive for G-CSFR expression, relapse was significantly more likely after primary treatment (P = 0.0069), whereas there was no association between G-CSFR expression and age, sex, tumor size, lymph node metastasis, and clinical stage. Also, the G-CSFR-positive groups had a significantly lower disease-free and overall survival rate than the G-CSFR-negative groups (P = 0.0172 and 0.0188, respectively). However, none of the clinical markers correlated significantly with G-CSF staining, nor did the status of G-CSF production influence the overall survival. The results imply that assessment of G-CSFR may prove valuable in selecting patients with oral and mesopharyngeal SCC for aggressive therapy.

Correlation of antiangiogenic and antitumor efficacy of N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA), an orally-active, selective matrix metalloproteinase inhibitor.

The antiangiogenic activity and antitumor efficacy of a newly developed matrix metalloproteinase (MMP) inhibitor were examined. 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 MMPs tested. Daily oral administration of 200 mg/kg BPHA, but not (-)BPHA in mice resulted in potent inhibition of tumor-induced angiogenesis, primary tumor growth, and liver metastasis. The growth inhibition activity of BPHA was 48% and 45% in a B16-BL6 melanoma and F2 hemangio-endothelioma model, respectively. BPHA also showed 42% inhibition of the liver metastasis of C-1H human colon carcinoma cells. These results indicate that selective MMP inhibition is correlated with antiangiogenic and antitumor efficacy and that the selective MMP inhibitor BPHA has therapeutic potential.

Purification, characterization and molecular cloning of an acidic amino acid-specific proteinase from Streptomyces fradiae ATCC 14544.

We have isolated a novel acidic amino-acid-specific proteinase from Streptomyces fradiae ATCC 14544, using benzyloxycarbonyl-L-Phe-L-Leu-L-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. A proteinase, which we propose to call SFase, was purified from the culture filtrate by salting out, repeated S-Sepharose chromatography, and affinity chromatography (CH-Sepharose-Phe-Leu-D-Glu-OMe). The purified enzyme showed a single band having an apparent molecular weight of 19,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When synthetic peptides were used as substrates, SFase showed high specificity for Z-Phe-Leu-Glu-pNA. Comparison with nitroanilides of glutamic acid and aspartic acid as substrates revealed that the reactivity was about 10-fold higher for a glutamyl bond than an aspartyl bond. SFase selectively hydrolyzed the -Glu-Ala-bond of two glutamyl bonds in the oxidized insulin B-chain within the initial reaction time until the starting material was completely digested. Diisopropylfluorophosphate and benzyloxycarbonyl-Phe-Leu-Glu chloromethylketone completely inhibited SFase, while metalloproteinase inhibitors, such as EDTA and o-phenanthrolin, did not inhibit the enzyme. The findings indicate that SFase can be classified as a serine proteinase, and is highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure and precursor of SFase, its gene was cloned from genomic DNA of the producing strain, and the nucleotide sequence was determined. Consideration of the N- and C-terminal amino-acid sequences of the mature protein of SFase indicates that it consists of 187 amino acids, which follows a prepropeptide of 170 residues. In comparison with the acidic amino-acid-specific proteinase from Streptomyces griseus (Svendsen, I., Jensen, M.R. and Breddam, K. (1991) FEBS Lett. 292, 165-167), SFase had 82% homology in the amino acid sequence. The processing site for maturation of SFase was a unique sequence (-Glu-Val-), so that the propeptide could be released by cleavage of the peptide bond between Glu and Val.

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.

Expression of platelet-derived endothelial cell growth factor in oral and oropharyngeal carcinoma.

Platelet-derived endothelial cell growth factor (PD-ECGF) was isolated as an endothelial cell mitogen from platelets. In this study, we investigated the expression of PD-ECGF and counted microvessels in 58 oral and oropharyngeal squamous cell carcinoma (SCC) specimens by an immunohistochemical technique to examine their prognostic significance and performed tumor in vitro sensitivity to 5-fluorouracil (5-FU) and cisplatin as determined by a bioluminescence assay of the ATP values of tumor cells after continuous exposure. The percentage of PD-ECGF-positive tumor cells (PD-ECGF score) was correlated with the frequency of the recurrence of disease (P=0.0043) but not with sex, tumor size, metastasis, or clinical stage. Overall survival of the high PD-ECGF expression group (>40% PD-ECGF score) was shorter than the low expression (<40%) group (P=0.0365). Vessel count was correlated with lymph node metastasis and clinical stage. The survival of patients with hypervascularity (more than the median of intratumor vessel counts, >82) was shorter than that of those with hypovascularity (vessel count <81, P=0.0446). However, there was no association between PD-ECGF expression and vessel count. Cox proportional multivariate analysis showed that PD-ECGF expression was the most significant independent prognostic indicator for overall survival. The susceptibility to 5-FU cytotoxicity in the extremely high PD-ECGF expression groups (>70% of PD-ECGF score) was significantly higher than that in the low group, whereas there was no difference in their sensitivity to cisplatin. These results showed that carcinoma cells with high PD-ECGF expression were sensitive to 5-FU in spite of poor prognosis. These data provide further information when deciding on adjuvant therapy for oral and oropharyngeal SCCs.

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.

Isolation and characterization of human reg protein produced in Saccharomyces cerevisiae.

reg was originally identified as a gene expressed during the regeneration of insulin-producing pancreatic beta-cells of the rat. We built an expression vector containing human reg cDNA to drive Saccharomyces cerevisiae to synthesize the reg protein, and purified it from the culture medium. The 144-amino acid sequence of the recombinant protein was consistent with that deduced from the cDNA and genomic DNA sequence except that the signal sequence of 22 amino acids was eliminated, and the amino-terminal residue of the protein was pyroglutamic acid. The secondary structure of the reg protein was predicted by determination of the intramolecular cystine linkage and of alpha-helix and beta-sheet contents.

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.

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.

Homology modeling of gelatinase catalytic domains and docking simulations of novel sulfonamide inhibitors.

Three-dimensional models for the catalytic domain of gelatinases (MMP-9 and -2) have been constructed based on the X-ray crystal structure of MMP-3. Conformations of the loop segment which forms the bottom half of the S1' subsite but shows conformational diversity among the crystal structures of other MMPs have been explored by simulated annealing of each gelatinase model complexed with two highly potent "probe" inhibitors. Representative catalytic domain models have been selected for each gelatinase from the set of generated conformations based on shape complementarity of the loop to the probe inhibitors. The single model selected for MMP-9 was utilized to explain the structure-activity relationship of our novel sulfonamide inhibitors. Molecular dynamics (MD) simulations of the complex models revealed important features of the binding mechanism of our inhibitors: (i) the ligand carboxylate group coordinating to the catalytic zinc ion and hydrogen bonding to the Glu219 side chain, (ii) one of the sulfonyl oxygens forming hydrogen bonds with the main chain NHs (Leu181 and Ala182), (iii) the sulfonyl substituent making extensive hydrophobic contact with the S1' subsite. The gauche conformation exclusively adopted by the sulfonamide C-N-S-C torsion plays an important role in achieving the third binding feature by properly directing the substituent into the S1' subsite. Improvement of the inhibitory activity according to straight elongation of the sulfonyl substituent was attributed to an increase of the hydrophobic contact between the substituent and the S1' subsite. Structural modifications which alter the straight shape of the substituent lead to deterioration of the activity. On the other hand, the two candidate models selected for MMP-2 differ in the bottom shape of the S1' subsite: one with a channel-like subsite and the other with a pocket-like subsite resembling that of the MMP-9 model. The bottom shape was experimentally probed by chemical synthesis of inhibitors having elongated sulfonyl substituents whose terminal alkyl groups were shown by MD simulations to protrude from the S1' subsite bottom into the solvent. Gelatinase assays of these inhibitors showed that elongation of the substituent significantly reduces activity against MMP-9 while retaining activity against MMP-2, consequently increasing the selectivity between MMP-2 and -9. The results confirm that MMP-9 has a pocket-like S1' subsite with a floorboard and MMP-2 has a channel-like S1' subsite.

Highly selective and orally active inhibitors of type IV collagenase (MMP-9 and MMP-2): N-sulfonylamino acid derivatives.

Various N-sulfonylamino acid derivatives were synthesized and evaluated for their in vitro and in vivo activities to inhibit type IV collagenase (MMP-9 and MMP-2). When the amino acid residue and the sulfonamide moiety were modified, their inhibitory activities were greatly affected by the structure of the sulfonamide moiety. A series of aryl sulfonamide derivatives containing biaryl, tetrazole, amide, and triple bond were found to be potent and highly selective inhibitors of MMP-9 and MMP-2. In addition, these compounds were orally active in animal models of tumor growth and metastasis. These results revealed the potential of the N-sulfonylamino acid derivatives as a new type of candidate drug for the treatment of cancer.

IL-10 expression is associated with the expression of platelet-derived endothelial cell growth factor and prognosis in oral and oropharyngeal carcinoma.

We investigated the expression of IL-10 in oral and oropharyngeal squamous cell carcinoma (SCC) specimens by an immunohistochemical technique. Of 58 SCC, 13 (22%) and 35 (60%) cases showed intense and moderate positive staining of IL-10, respectively. There was no association between the staining of IL-10 and clinicopathological features. However, the patients with intense staining of IL-10 had a significantly lower overall survival rate than those with moderate or negative staining of IL-10 (P = 0.019). In addition, the patients with intense staining of IL-10 had the highest score of platelet-derived endothelial cell growth factor (PD-ECGF), which is established as a poor prognostic indicator (P = 0.0105). These results suggested that IL-10 contributes to the clinical outcome of oral and oropharyngeal SCC.

Expression of hMSH2 correlates with in vitro chemosensitivity to CDDP cytotoxicity in oral and oropharyngeal carcinoma.

We investigated the expression of hMSH2, a human mutS homologue from chromosome 2p, in oral and oropharyngeal squamous cell carcinoma (SCC) by an immunohistochemical technique and performed tumor in vitro chemosensitivity testing. In 58 oral and oropharyngeal SCC, the hMSH2 positive score was inversely associated with tumor size, but not with other clinical parameters. Among five anticancer drugs (cisplatin (CDDP), 5-FU, peplomycin, mitomycin C and doxorubicin), only for CDDP was sensitivity to cytotoxicity correlated with the hMSH2 positive score. The susceptibility of hMSH2-positive tumors to CDDP killing was significantly higher than that of hMSH2-negative tumors. Immunohistochemical results regarding hMSH2 are promising in the evaluation of the sensitivity of cancer cells to CDDP cytotoxicity and enable one to select patients for adjuvant chemotherapy for oral and oropharyngeal SCC.

Expression of multidrug resistance-associated protein (MRP) in head and neck squamous cell carcinoma.

We investigated the expression of multidrug resistance-associated protein (MRP) in 115 cases of head and neck squamous cell carcinoma (H&NSCC) by immunohistochemistry and examined the relationship between MRP expression and clinical factors. Thirty-four (30%) of 115 cases of H&NSCC had expression of MRP. The clinical stage was inversely associated with the expression of MRP (P = 0.0090), but not with age, sex, tumor size, metastasis, recurrence, death from disease or overall survival rate for 5 years. In vitro chemosensitivity to five chemotherapeutic agents (cis-diamminedichloroplatinum, 5-fluorouracil, peplomycin, mitomycin C and Adriamycin) was tested by ATP assay and no correlation between the sensitivity of tumor cells to the cytotoxicity of any drug and MRP expression was found. These results suggest that the resistance to anticancer drugs is not dependent only on the expression of MRP in H&NSCC.

Decreased expression of Bax is correlated with poor prognosis in oral and oropharyngeal carcinoma.

We investigated the expression of apoptosis-related factors, p53, Bax, Bcl-2, and spontaneous apoptosis in 57 cases of oral and oropharyngeal squamous cell carcinoma (SCC) by immunochemical staining and ApopTag kit. Positive expression of Bax was inversely associated with advanced tumor stage (P = 0.0225), lymph node metastasis (P = 0.0225), clinical stage (P = 0.0083) and poor prognosis (P = 0.0478). Positive expression of p53 was related to poor prognosis (P = 0.0445) and was associated with negative expression of Bax (P = 0.0439). The apoptosis index did not correlate with clinical outcome. These results suggest that abnormality of Bax expression plays an important role in tumor progression in oral and oropharyngeal 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.

Coupling between Cbz-Arg-OH and Leu-X catalyzed by trypsin and papain.

Trypsin [EC 3.4.21.4] and papain [EC 3.4.22.2] catalyzed the coupling between Cbz-Arg-OH and Leu-X (X=-NH2, -OEt, -OBut, or -NH) in solution. The optimum pH for synthesis by trypsin and by papain was 6.5-7.0 and 5.5, respectively. Papain possessed reactivity several times higher than that of trypsin. To shift the reaction toward synthesis, the concentration of amine components must be at least several times higher than that of the carboxyl component (Cbz-Arg-OH). The effect of the concentration of Leu-X depended upon the nature of X; higher concentrations were required in the following order: -NH2 > -OEt, -OBut > -NH. High concentrations of organic cosolvents were also significant for promoting the synthesis.