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We compared the amount of serum Epstein-Barr virus DNA (EBV-DNA) detected in patients with nasopharyngeal carcinoma (NPC) in a high-incidence area, represented by Taiwan, and a low-incidence area, represented by Japan, using real-time quantitative PCR. The median serum EBV-DNA value in 41 Japanese NPC cases was 5450 copies/ml, and that in in 23 Taiwanese cases was 2125 copies/ml. The median serum EBV-DNA value in all 64 NPC cases was significantly higher than in control groups. Using receiver-operating-characteristic (ROC) curves, the sensitivity and specificity of EBV-DNA quantification were determined (cut-off point, 6.87 copies/ml; sensitivity, 0.855; specificity, 0.885) and compared with those of EBV-viral-capsid-antigen (VCA) titers; the results showed that EBV-DNA was a more sensitive and specific parameter than EBV-VCA titer. Then, we analyzed 19 NPC patients in whom recurrence developed (11 Japanese and 8 Taiwanese), and 26 NPC patients in continuous remission. Although there was no significant difference in EBV-DNA values between Japanese and Taiwanese patients, the value was significantly higher in the 19 patients with recurrence than in those in remission. ROC analysis again revealed a higher diagnostic value of EBV-DNA than EBV-VCA. These results suggest EBV-DNA is a more reliable tumor marker than EBV-VCA in both high-incidence and low-incidence areas of NPC.

Background/objectives: Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor and is associated with Epstein-Barr virus (EBV) infection that exhibits type II latency. Angiogenesis is essential for tumor growth, invasion, and metastasis. Our previous studies have indicated that interleukin (IL)-8 was over-expressed in many NPC tissues and was found to be significantly correlated with angiogenesis by immunohistochemistry., Study Design: In vitro design., Methods: The influence of the EBV genome for IL-8 gene expression was studied using the EBV-genome-positive and -negative epithelial/NPC hybrid cell line NPC-KT. The EBV-positive and -negative clones were selected by polymerase chain reaction and in situ hybridization., Results: EBV-positive clones expressed abundant IL-8 mRNA compared with EBV-negative clones. This result indicated that over-expression of IL-8 depended on the presence of EBV genomes in NPC-KT cells. Two encoded genes, latent membrane protein (LMP)1 and EBV-encoded small RNAs (EBERs), expressed in NPC were transfected in EBV-negative NPC-KT cells. LMP1 transactivated the IL-8 promoter, whereas EBERs did not. Moreover, the nuclear factor (NF)-kappa B binding site in the IL-8 promoter was essential for the response to LMP1, and the activator protein (AP)-1 binding site played only a partial role., Conclusions: LMP1 induces IL-8 mainly through the activation of NF-kappa B and partly through AP-1 in NPC model cell lines, NPC-KT, and this suggests that LMP1 plays an important role in the angiogenesis of NPC.

Objectives/hypothesis: Adenoid cystic carcinoma of the head and neck (ACCHN) is characterized by late recurrence and frequent distant metastasis. Tumor attack by cytotoxic T lymphocytes and macrophages is mediated by the interaction of leukocyte function-associated antigen (LFA)-1 on lymphocytes with intercellular adhesion molecule (ICAM)-1 on the tumor surface. Thus, the reduced expression of ICAM-1 on tumor cells could contribute to their escape from host immune surveillance. To investigate the relationship between the clinical features of ACCHN and host immune surveillance, the expression of ICAM-1 and infiltration of T/natural killer (NK) cells and macrophages were immunohistochemically examined., Study Design: Retrospective analysis of immunohistochemical tumor characteristics and clinical outcome., Methods: Immunohistochemical study of ICAM-1, T/NK cells, and macrophages was performed on paraffin sections of 42 patients with ACCHN. The expression of T/NK cells and macrophages was represented by T-cell-restricted antigen (TIA)-1 and CD68 expression, respectively. The expression of these molecules and clinical features were analyzed., Results: Of 42 ACCHN cases, 15, 9, and 15 patients were classified as ICAM-1 high, TIA-1 high, and CD68 high, respectively. The TIA-1 expression scores in ICAM-1-low patients were significantly lower than those in ICAM-1-high patients (1.3 +/- 3.7 vs. 8.3 +/- 12.7, P =.0031). The CD68 expression scores in ICAM-1-low patients were also significantly lower than those in ICAM-1-high patients (9.6 +/- 9.6 vs. 21.1 +/- 17.6, P =.0047). Moreover, ICAM-1-high patients had a significantly better disease-free survival rate (P =.043)., Conclusions: Reduced expression of ICAM-1 may promote immune evasion and metastasis, resulting in poor prognosis in ACCHN.

Objective: Oral squamous cell carcinoma (OSCC) is characterized by local invasiveness Matrix metalloproteinase-2 (MMP-2) is a key enzyme involved in local invasiveness. Type I collagen functions as a modulator of cellular function, including MMP-2 activation; additionally, it serves as a barrier against tumor invasion. This study was designed to analyze the effect of type I collagen on tumor growth and MMP-2 activation of OSCC cell lines as a function of culture conditions., Material and Methods: Two OSCC cell lines (SAS, OSC-19) were cultured under three conditions: conventional monolayer culture on plastic dishes (MP); two-dimensional culture on type I collagen gel (2D); and three-dimensional culture within a type I collagen gel (3D). MMP-2 activity was determined by means of gelatin zymography and invasive cell growth was assessed morphologically. The inhibitory effect of BB-2516 (marimastat) on SAS and OSC-19 cell growth was examined. Additionally, the effect of tissue inhibitor of MMP-1 (TIMP-1) and TIMP-2 on SAS cell growth was investigated., Results: The most intense MMP-2 activation was evident in the 3D cases in comparison with MP and 2D in both SAS and OSC-19. MMP-2 activation was correlated with cell growth. MMP-2 activation and cell growth were inhibited by BB-2516 in a dose-dependent manner. Dose-dependent inhibitory effects of TIMP-2, but not TIMP-1, against MMP-2 activation were observed in SAS., Conclusions: Three-dimensional type I collagen gel culture is a valuable method for monitoring both MMP-2 activation and invasive cell growth. Membrane type 1 matrix (MT1)-MMP supposedly plays key roles in MMP-2 activation and cell growth in 3D. Thus, this system would be useful for evaluating the inhibitory effect of molecules against MT1-MMP.

Detection thresholds are typically obtained by presenting a subject with serial dilutions of an odorant. Many factors, including the solvent used to dilute the odorant, can influence the measurement of detection thresholds. Differences have been reported in detection thresholds for phenyl ethyl alcohol (PEA) when different solvents are used. In this study we used gas chromatography (GC) to investigate further the effect of solvent on odor detection thresholds. We used a single ascending method and serial dilutions of PEA in four different solvents--liquid paraffin (LP), mineral oil (MO), propylene glycol (PG) and dipropylene glycol (DPG)--to determine the PEA thresholds for 31 adult subjects. For each solvent, we prepared eight serial log base 10 step dilutions (1-8), with corresponding liquid PEA concentrations of 6.3 x 10(1)-6.3 x 10(-6) (% v/v). We found that the threshold concentrations for PEA in LP (step 6.5) and PEA in MO (step 5.5) were significantly lower (P < 0.05) than for PEA in PG (step 4.0) and DPG (step 4.0) We then used GC to measure both the liquid and gas PEA concentrations for the dilution steps prepared with LP and PG. Although there were large threshold differences in the liquid concentrations of PEA in LP and PG, the headspace gas concentrations of PEA were the same. These results demonstrate the importance of determining the gas concentration of odorant stimuli when performing odor threshold measurements, in particular when comparing odor detection thresholds obtained using different solvents.

We present a very rare case of mucosal melanoma with osteocartilaginous differentiation (MMOD). This is the first report of MMOD originating in the sinonasal cavity. Preoperative diagnosis of this tumor from specimens resected for biopsy alone is very difficult, because it has 2 histopathologic components: a primary melanoma site and the part of the melanoma with osteocartilaginous differentiation. The immunohistochemical technique is useful in histopathologic diagnosis of this tumor. Malignant melanoma usually shows reactivity with MelanA (melanoma tumor antigen) and S-100 proteins. We diagnosed this case as MMOD because both regions in this tumor were stained with MelanA and S-100 proteins. Magnetic resonance imaging provides the best clinical ability to detect malignant melanoma in the sinonasal region. In this case, the melanotic melanoma site in the posterior ethmoid sinus appeared hyperintense to gray matter on T1-weighted image magnetic resonance imaging. Thus, it would have been possible to diagnose this case as MMOD before operation, even though we could not obtain a biopsy specimen from the posterior ethmoid sinus lesion.

We have shown that the EBV oncoprotein, latent membrane protein 1 (LMP1), induces a constellation of tumor-invasiveness factors. Fibroblast growth factor (FGF)-2 is angiogenic as well mitogenic. Although FGF-2 does not contain a hydrophobic signal sequence for secretion, FGF-2 is released extracellularly. However, the mechanism by which FGF-2 is released is unclear. Here we show first that LMP1 induces in epithelial cells the expression of FGF-2 mRNA and protein through both LMP1 COOH-terminal activation domains, CTAR 1 and CTAR 2, which can activate nuclear factor (NF)-kappaB signaling and also the p38 mitogen-activated protein kinase pathway. Coexpression of IkappaBalpha (S32A/S36A), which cannot be phosphorylated and prevents NF-kappaB activation, with LMP1 inhibited induction of FGF-2 by LMP1, which suggests that LMP1 induces FGF-2 via NF-kappaB signaling. Moreover, unlike phorbol 12-myristate 13-acetate LMP1 also induced the release of the M(r) 18,000 isoform of FGF-2 protein. Transfection of Ad-AH cells with LMP1 deletion mutants lacking either CTAR 1 or CTAR 2 also induced the release of the protein. Secretion was confirmed in 293 cells, which do not contain detectable endogenous FGF-2 protein, cotransfected with FGF-2 and LMP1. Finally, Na(+)/K(+)-ATPase participates in FGF-2 release, independently of the classical endoplasmic reticulum/Golgi pathway. In this study, the release of M(r) 18,000 FGF-2 protein was partially suppressed by ouabain, which inhibits the activity of Na(+)/K(+)-ATPase alpha1 subunit, but not by Brefeldin A, which inhibits the endoplasmic reticulum/Golgi-dependent secretory pathway. In contrast, the release of M(r) 18,000 FGF-2 protein was almost completely inhibited by IkappaBalpha (S32A/S36A). These results suggest that FGF-2 release is independently mediated by NF-kappaB signaling, not simply a consequence of induction itself. Thus, NF-kappaB signaling is involved in induction of expression and release of FGF-2 by LMP1.

Olfactory neurons are unique in the mammalian nervous system because of their capacity to regenerate in adult animals. It has been shown that olfactory receptor cells located in the olfactory epithelium are replaced on a continuous basis and in response to injury throughout the life span of most species. NGF, which is one of the neurotrophic factors, is present in many areas of the central and peripheral nervous system. It has been shown that NGF in the olfactory bulb plays a role in the survival of cholinergic neurons in the horizontal limb of the diagonal band (HDB). Recent studies of NGF in the olfactory bulb suggest that it is involved in the development, maintenance, and regeneration of olfactory receptor cells. In this study, we review reports examining the relationship between NGF in the olfactory bulb and neuronal regeneration and development in the mammalian olfactory systems. Low- and high-affinity NGF receptor immunoreactivity is markedly expressed during regeneration and at different stages of development in the mouse olfactory system. This level of immunoreactivity is no longer present after completion of regeneration and at maturation. Other findings indicate that NGF injected into the olfactory bulb is transported retrogradely to the olfactory epithelium. It has also been shown that continuous anti-NGF antibody injection into the olfactory bulb causes degeneration and olfactory dysfunction. Administration of NGF directory into nasal cavity results in an increase in the expression of olfactory marker protein within the olfactory epithelium in axotomized rats. These findings suggested that the presence of NGF in the olfactory bulb plays an essential role in regeneration, maintenance, and development in the olfactory system of mammals., (Copyright 2002 Wiley-Liss, Inc.)

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.

Matrix metalloproteinases (MMPs) play an important role in degradation of extracellular matrix (ECM), which is an essential step in the cascade of metastasis. Various types of MMPs are expressed and activated in head and neck squamous cell carcinoma (HNSCC) as well as other human cancers. MMP-2 is a prominent predictor of poor prognosis. Membrane type 1-MMP (MT1-MMP) was originally identified as an activator of MMP-2. In addition to the original role, recent studies show other important functions of MT1-MMP such as degradation of type I collagen and cleavage of CD44. Tissue inhibitor of MMP-2 (TIMP-2) was identified as an inhibitor of MMP-2 and MT1-MMP. However, TIMP-2 was reported to be essential for cell-mediated activation of MMP-2, and thus the contribution of TIMP-2 to tumor invasion has remained controversial. Some studies also suggested a role of TIMP-2 as a predictor of poor prognosis. Thus, inhibition of MMP activation by TIMPs is not a suitable strategy for suppressing invasion and metastasis. Instead of TIMP-2, various MMP inhibitors (MMPI) such as BB-2516 have been investigated with regard to suppression of tumor progression and improvement of prognosis in patients with advanced cancers, which resulted in no clinical efficacy. MMPs are especially important in the early stage of cancer progression, and thus strategies for future MMPI trials should be reconsidered.

Objective: A rapid increase of penicillin resistance in Streptococcus pneumoniae has recently been reported in most areas of the world. Penicillin-resistant S. pneumoniae and other resistant bacteria are the principal causes of recurrent acute otitis media (AOM). Penicillin-resistant S. pneumoniae was examined so that we could investigate the bacteriologic and clinical interpretations of nasopharyngeal flora from healthy children., Methods: We obtained nasopharyngeal swab specimens from healthy children attending a day care center and from children attending a public health examination in Kanazawa, Japan. We also obtained clinical specimens from children with AOM who visited the Kanazawa University Hospital and 4 other hospitals in Ishikawa Prefecture in Japan., Results: The chief bacteria from the children were S. pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Penicillin-resistant S. pneumoniae was identified in 75% of children attending a day care center. On the other hand, S. pneumoniae was identified in 37% of children who were not attending day care. Of the children with AOM, penicillin-resistant S. pneumoniae was identified in 77%., Conclusion: Our results suggest that there is a strong relationship between day care attendance and nasopharyngeal carriage of S. pneumoniae. The carriage rate of penicillin-resistant S. pneumoniae in healthy children in day care centers was very high and similar to the carriage rate of young patients with AOM in Japan.

A thrombosed varicose vein arising in the neck is quite rare. A 68-year-old Japanese male was referred to our hospital because of a mass in his left upper neck. The mass did not change in size in response to strain resulting from Valsalva's manouvre. Magnetic resonance imaging (MRI) showed iso-signal intensity of the mass on T1-weighted images and a target-like signal arrangement (concentric hyper-, hypo-, hyper-signal intensity from outside to inside) on T2-weighted images. Surgical excision revealed that the tumour arose from the intramuscular small vein in the sternocleidomastoid muscle. The pathological examination revealed the mass to be a thrombosed varicose vein with capillarization in the dilated vein wall. The de-oxygenation and degradation of haemoglobin were thought to be responsible for these characteristic MRI findings. The concentric signal distribution on MRI strongly suggested this pathology.

Activation of matrix metalloproteinase-2 (MMP-2) is a common event in head and neck squamous cell carcinoma. An OSC-19 cell line, derived from human oral squamous cell carcinoma and known to metastasize to cervical lymph nodes, was implanted into the lingual margin of mice. The effect of marimastat (BB-2516), a broad MMP inhibitor, on the suppression of regional cervical lymph node metastasis was evaluated with an orthotopic implantation nude mice model. Marimastat was given immediately after OSC-19 implantation and continuously administered by an osmotic pump. The mice were divided into three groups by marimastat dose; Group A; 0 mg/kg/day, Group B; 30 mg/kg/day, and Group C; 150 mg/kg/day. Twenty-one days after implantation, primary oral tumors and cervical lymph nodes were resected. Cervical lymph node status was microscopically examined. Activation of MMP-2 in primary oral tumor was examined by gelatin zymography. Both cervical lymph node metastasis and activation of MMP-2 were significantly suppressed in Group C (P < 0.05). Moreover, the Group C mice had a significantly better survival than group A (P = 0.0026). There was a significant difference between Group A and Group C in terms of proliferation of tumor cells by proliferating cell nuclear antigen immunostaining (P = 0.0120). These results suggest a positive role for marimastat in the inhibition of MMP-2 activation and prevention of cervical lymph node metastasis in oral squamous cell carcinoma (OSCC). Improvement of survival in patients with OSCC could be expected using adjuvant therapy with marimastat.

Systemic or topical application of glucocorticoid is the treatment of choice for olfactory disturbance. Recently, Na(+)/K(+) ATPase and glucocorticoid receptor immunoreactivity in the olfactory mucosa was reported. To elucidate a glucocorticoid action on Na(+)/K(+) ATPase production, an animal model was produced by an intra-nasal application of 5% ZnSO(4) solution to Wistar rats. Dexamethasone was injected i.p. (0.01 mg/100 g) for 14 days after the insult. Histologically, the regeneration process was completed on day 14 in both dexamethasone- and saline-injected control rats. We used a quantitative polymerase chain reaction (PCR) method to evaluate mRNA production of Na(+)/K(+) ATPase and glucocorticoid receptor. In dexamethasone-injected rats, up-regulation of glucocorticoid receptor mRNA (95% more than control rats, P = 0.00068, unpaired t-test) and of Na(+)/K(+) ATPase mRNA expression (76% more than control rats, P = 0.0042) was observed on day 14. The increased Na(+)/K(+) ATPase expression in the regenerated olfactory mucosa is thought to be beneficial for an active uptake of K(+), which is released during excitation, around olfactory neurons and for the transepithelial absorption of Na(+) from olfactory mucus. Dexamethasone may thus contribute to the recovery of function after the morphological regeneration in part, at least, through its receptor by regulation of the ionic concentration in the olfactory mucosal microenvironment.

Malignant myoepitheliomas of the salivary gland are very rare tumors which may either arise de novo or develop in a pre-existing pleomorphic adenoma. We report a case of malignant myoepithelioma of the minor salivary gland that progressed from benign pleomorphic adenoma. The original pleomorphic adenoma was resected in 1979 (the '79 tumor). The first recurrent tumor was operated in 1995 (the '95 tumor). The '95 tumor was diagnosed as pleomorphic adenoma. Although the myoepithelial tumor component was more prevalent in the '95 tumor, histological features of the first recurrent tumor were similar to the '79 tumor. The second recurrent tumor showed more aggressive clinical features (the '98 tumor). It also showed massive growth of myoepithelial tumor cells with bone invasion, which led to the diagnosis of the '98 tumor as malignant myoepithelioma. With adequate surgical margin, the patient has been free from tumor recurrence for 20 months.

Hydrazinoethanols 1 were reacted with aldehydes 2 and 6 and glutaraldehyde (14) in aqueous solution to give dioxatetraazaperhydroanthracenes 3, 7, 12, and 13 and -perylenes 15 in yields of 19-88 and 42-72%, respectively. Compounds 3, 7, 12, and 15 were deduced by (13)C-NMR spectra to have two C(2) symmetry axes, while compound 12 was shown to have a symmetry axis by X-ray crystallography. The most favorable stereoisomers were consistent with predictions obtained by the semiempirical molecular orbital method AM1. The structure of compound 15 was confirmed by X-ray crystallography.