[A MULTICENTER CLINICAL SURVEY ABOUT THE PREVALENCE OF JAPANESE CYPRESS POLLINOSIS AND THE EFFICACY OF SUBLINGUAL IMMUNOTHERAPY WITH JAPANESE CEDAR POLLEN EXTRACT DURING JAPANESE CYPRESS POLLEN DISPERSAL PERIOD].

BACKGROUND: Little is known whether sublingual immunotherapy using Japanese cedar pollen extract (cedar SLIT) is effective for not only Japanese cedar pollinosis but also Japanese cypress pollinosis. We investigated the prevalence rate of Japanese cypress pollinosis, efficacy of cedar SLIT on cypress pollinosis and patients' wish to receive cypress SLIT. METHODS: We investigated a multi-center (31 institutions), cross-sectional survey using a self-administrated questionnaire with four questions for patients received cedar SLIT aged from 5 to 69 years old. RESULTS: 2523 subjects were enrolled for analysis. 83.4% of them had pollinosis symptoms during cypress season before cedar SLIT. In such patients, 37.4% experienced lessened efficacy of cedar SLIT during cypress season. Both the prevalence of cypress pollinosis and the lessened efficacy of cedar SLIT on cypress pollinosis were significantly seen in western Japan as compared to eastern Japan. 76.1% of the subject having cypress pollinosis before SLIT wished to receive cypress SLIT if it is available. CONCLUSION: A lessened efficacy of cedar SLIT during cypress season was broadly seen in Japan, and further showed a regional difference. Together with the finding of high wish by patients, these results suggest a development of cypress SLIT is desirable.

Detection of human parechoviruses from clinical stool samples in Aichi, Japan.

Between April 1999 and March 2008, a total of 4,976 stool specimens collected from patients with suspected viral infection through infectious agent surveillance in Aichi, Japan, were tested for the presence of human parechoviruses (HPeVs). We detected HPeVs in 110 samples by either cell culture, reverse transcriptase PCR (RT-PCR), or both. Serotyping either by neutralization test or by nucleotide sequence determination and phylogenetic analysis of the VP1 region and 5' untranslated region (5'UTR) regions revealed that 63 were HPeV type 1 (HPeV-1), followed by 44 HPeV-3 strains, 2 HPeV-4 strains, and 1 HPeV-6 strain. The high nucleotide and amino acid sequence identities of the Japanese HPeV-3 isolates in 2006 to the strains previously reported from Canada and Netherlands confirmed the worldwide prevalence of HPeV-3 infection. Ninety-seven percent of the HPeV-positive patients were younger than 3 years, and 86.2% younger than 12 months. The clinical diagnoses of HPeV-positive patients were gastroenteritis, respiratory illness, febrile illness, exanthema, "hand, foot, and mouth disease," aseptic meningitis, and herpangina. Among 49 HPeV-positive patients with gastroenteritis, 35 were positive with HPeV-1 and 12 with HPeV-3, and out of 25 with respiratory illness, 11 were positive with HPeV-1 and 14 with HPeV-3. HPeV-3 seemed to be an important etiological agent of respiratory infection of children. While HPeV-1 was detected predominantly during fall and winter, the majority of the HPeV-3 cases were detected during summer and fall. A different pattern of clinical manifestations as well as seasonality suggested that there are different mechanisms of pathogenesis between HPeV-1 and HPeV-3 infections.

Suppression of histone deacetylase 3 (HDAC3) enhances apoptosis induced by paclitaxel in human maxillary cancer cells in vitro and in vivo.

Inclusion of chemotherapeutic drugs in treatment of patients with newly diagnosed head and neck cancer has improved response rates and prolonged median survival. Nevertheless, most patients with advanced head and neck cancer are destined to relapse and to develop resistance to initially used drugs such as paclitaxel. Consequently, it has been more important in cancer therapy to determine the molecular mechanisms that are related to cell-killing effects of anti-cancer agents or cancer resistance against them. Consequently, we examined whether abrogation of histone deacetylase 3 (HDAC3) expression by anti-sense oligonucleotides (ASOs) potentiates the efficacy of paclitaxel in human maxillary cancer IMC-3 cells. Here, we showed that paclitaxel-induced apoptosis was enhanced significantly by addition of ASOs for HDAC3 in cultured cells. Furthermore, paclitaxel-induced apoptosis in IMC-3 tumors transplanted in nude mice was enhanced significantly by administration of ASOs for HDAC3, thereby suppressing tumor growth. We provide new evidence that HDAC3 is a novel molecular target whose inactivation can potentiate the efficacy of anti-cancer drugs disrupting microtubules such as paclitaxel.

Molecular identification of enteroviruses including two new types (EV-98 and EV-107) isolated from Japanese travellers from Asian countries.

Of 58 enterovirus strains isolated from Japanese travellers returning from Asian countries, eight were non-serotypable with existing antisera. By sequencing a part of the VP1 region, six of these strains were typed as echovirus 9, enterovirus (EV)-73, EV-79 or EV-97. The nucleotide identity of the VP1 region of isolate T92-1499 to all enterovirus prototypes was <70 %. The VP1 sequence of isolate TN94-0349 was closely related to coxsackievirus (CV)-A9 (73.3 % nucleotide identity), but the virus could not be neutralized with a serum raised against the prototype CV-A9 strain. On the basis of complete molecular comparisons, T92-1499 and TN94-0349 were identified as EV-98 and EV-107, respectively, by the ICTV Picornavirus Study Group. Serum neutralization tests of Japanese individuals revealed a seroprevalence rate of 11 % for EV-73, and even lower seroprevalence rates, 1.0-3.8 %, were found for the other new enteroviruses, suggesting that prior circulation of these viruses in Japan was unlikely.

[Study on pyrogen in natural and cultivated edible mushrooms].

We examined endotoxin and pyrogen contents in several kinds of natural and cultivated edible mushrooms, as well as some cultivated vegetables. According to the Japanese Pharmacopoeia, 14th Ed., two types of endotoxin (gel-clot Limulus amebocyte lysate) test and the pyrogen test were performed using natural edible mushrooms collected in Aichi Prefecture and cultivated mushrooms and vegetables purchased at a market. The endotoxin contents of natural mushrooms were apparently higher than those of cultivated mushrooms or vegetables. The endotoxin contents in the cultivated mushrooms were slightly higher than those in the vegetables. Similar results were obtained in the pyrogen test.

Oral and oropharyngeal squamous cell carcinomas expressing CCR7 have poor prognoses.

OBJECTIVE AND METHODS: Tumor cell migration and metastasis have many similarities with leukocyte trafficking regulated by chemokines and their receptors. In this study, we investigated the expression of CC chemokine receptor 7 (CCR7) in oral and oropharyngeal squamous cell carcinomas (SCCs) using an immunohistochemical approach. RESULTS: Among 90 cases of oral and oropharyngeal SCCs, 54 cases (60%) were positive for CCR7. The CCR7-positive group had a significantly lower incidence of being disease-free and an overall lower survival rate than the CCR7-negative group (p<0.01 and p<0.01, respectively). Among the 35 patients who died of cancer, 28 patients (80%) were positive for CCR7. The expression of CCR7 was significantly associated with five clinical factors. SCCs expressing CCR7 tend to have large, lymph node metastases, progressive stages, local recurrences and cancer death. The CCR7 expression of metastatic SCCs in draining lymph nodes was detected, and the staining score of CCR7 between primary SCCs and metastatic SCCs were significantly correlated (p<0.01). CONCLUSIONS: The investigation of CCR7 expression in oral and oropharyngeal SCCs may be useful to predict patients prognoses.

Reliability of platelet-derived endothelial cell growth factor as a prognostic factor for oral and oropharyngeal carcinomas.

OBJECTIVE: To identify a strong prognostic biological marker for patients with oral and oropharyngeal squamous cell carcinomas. DESIGN: We evaluated the protein expressions of 26 tumor-associated factors, including cytokines and cytokine receptors (granulocyte colony-stimulating factor [G-CSF], interleukin 10 [IL-10], G-CSF receptor [G-CSFR], and IL-12 receptor); angiogenic factors (platelet-derived endothelial cell growth factor [PD-ECGF] and vessel count); cell cycle-related proteins (p27, cyclin D1, and cyclin E); apoptosis-related factors (wild-type p53, Bax, Bcl-2, apoptotic index, Fas, and Fas ligand); oncogene proteins (c-fos and c-Myc); cell-surface proteins (P-glycoprotein, multidrug resistance-associated protein, nm23, and CD40); intracellular proteins (aryl hydrocarbon receptor nuclear translocator, aryl hydrocarbon receptor, and heat shock protein 27); and DNA mismatch-repair genes (protein encoded by human mutL homologue 1 and the human mutS homologue of the chromosome 2p gene) by means of immunohistochemical analysis. SETTING: Department of Otorhinolaryngology-Head and Neck Surgery, University of Fukui, Fukui, Japan. PATIENTS: Fifty-eight patients who underwent surgical resections of oral and oropharyngeal squamous cell carcinomas. RESULTS: A low-level PD-ECGF expression, a hypovascular count, or a low-level G-CSFR expression was associated with a favorable clinical outcome using the Kaplan-Meier method. Univariate analysis showed that PD-ECGF expression (odds, 4.19; P = .02), G-CSFR expression (odds, 4.10; P = .01), and vessel count (odds, 2.80; P = .04) had significant hazard rates. When multivariate analysis was performed on 6 factors, including sex, tumor size, lymph node metastasis, PD-ECGF expression, G-CSFR expression, and vessel count, patients with a positive expression of PD-ECGF had the highest relative risk value for death due to the disease (odds, 4.94; P = .01). Also, G-CSFR was an independent prognostic indicator in the model (odds, 3.29; P = .04). No correlations between other factors and prognoses were detected. CONCLUSION: Expression of PD-ECGF was the most effective marker for making prognoses for oral and oropharyngeal squamous cell carcinomas, and G-CSFR expression was the second most effective among 26 tumor-associated factors.

Inhibition of histone deacetylase 3 stimulates apoptosis induced by heat shock under acidic conditions in human maxillary cancer.

To elucidate the molecular mechanisms for the enhancement of heat-induced apoptosis on exposure to acidic conditions, human maxillary carcinoma IMC-3 cells were heat-shocked at 44 degrees C for 30 min at either pH 7.4 or 6.7. Analyses with cDNA arrays, the reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting were performed. We found that histone deacetylase 3 (HDAC3) was specifically induced after hyperthermia at 44 degrees C for 30 min at pH 6.7. Although the cytotoxicity of heating at 44 degrees C for 30 min was enhanced by decreasing the pH from 7.4 to 6.7, it was enhanced even more by antisense RNA oligonucleotides for HDAC3. The induction of G2/M arrest after heating occurred earlier at pH 6.7 than at pH 7.4. The inhibition of HDAC3 by the antisense RNA oligonucleotides suppressed partially the induction of G2/M arrest, resulting in an enhancement of the apoptosis caused by the heating under acidic conditions. Antisense RNA oligonucleotides for HDAC3 enhanced apoptosis 48 h after hyperthermia at 43 degrees C for 30 min in vivo. Analyses of p65 activity suggested that NF-kappaB is involved in this enhancement of hyperthermia. HDAC3 may be a novel target enhancing hyperthermia and combined treatment with hyperthermia and HDAC inhibitors is a possible modality for cancer therapy.

A rapid detection method for paralytic shellfish poisoning toxins by cell bioassay.

We report here a rapid detection method for paralytic shellfish poisoning (PSP) toxins using a cultured neuroblastoma cell line, modified from the bioassay system previously established by Manger et al. [Manger, R.L., Leja, L.S., Lee, S.Y., Hungerford, J.M., Kirkpatrick, M.A., Yasumoto, T., Wekell, M.M., 2003. Detection of paralytic shellfish poison by rapid cell bioassay: antagonism of voltage-gated sodium channel active toxins in vitro. J. AOAC Int. 86 (3), 540-543]. In the present study, we made two major modifications to the previous method. The first is the use of maitotoxin, a marine toxin of ciguatera fish poisoning, which enables the incubation period to be reduced to 6 h when applied to the microplate 15 min prior to the end of the incubation. The second is the use of WST-8, a dehydrogenase detecting water-soluble tetrazolium salt for determining the target cell viability, which permits the omission of a washing step and simplifies the counting process. In addition, we attempted to reduce the required materials as much as possible. Thus, our modified method should be useful for screening the PSP-toxins from shellfish.

Treatment of scattered glass foreign bodies in both the superficial and deep neck: a case report.

There have not been any reports about scattered glass foreign bodies in the neck, while injuries of the head and neck region as a result of traffic accidents have been frequently reported. We report the case of a 17-year-old male injured in a traffic accident, with scattered glass foreign bodies in both the superficial and deep neck. A CT scan indicated the existence of numerous glass foreign bodies in the various layers of the neck. Most of the foreign bodies were very fine or sand-like. The wounded skin was keloidal and expected to lead to remarkable facial edema of the left side. The foreign bodies and cervical keloidal lesions with small pieces of glass were carefully removed, and then reconstruction was conducted in stages using tissue expanders. The facial edema was reversed and the aesthetic reconstruction satisfied the patient.

Isolation and identification of a novel human parechovirus.

A cytopathic agent (A308/99) was isolated using Vero cells from a stool specimen of a 1-year-old patient with transient paralysis. The agent was approximately 28 nm in diameter with a distinct ultrastructure resembling the virus particle of an enterovirus. It could not be neutralized by antisera against human picornaviruses such as human enterovirus, Aichi virus or human parechovirus. The virion contained three capsid proteins with molecular masses of 38, 30.3 and 30 kDa. Determination of the complete nucleotide sequence of A308/99 revealed that the nucleotide and deduced amino acid sequences were closely related to those of human parechoviruses. When 11 regions encoding the structural and non-structural proteins were compared, A308/99 had between 75 and 97 % and 73 and 97 % nucleotide identity with human parechovirus type 1 (HPeV-1) and type 2 (HPeV-2), respectively. The most distinctive divergence was seen in VP1, which had 74.5 % and 73.1 % nucleotide identity with HPeV-1 and HPeV-2, respectively. Viruses related to A308/99 were also isolated from three patients with gastroenteritis, exanthema or respiratory illnesses. A308/99 and these other three isolates had no arginine-glycine-aspartic acid (RGD) motif, which is located near the C terminus of VP1 in HPeV-1 and HPeV-2. A seroepidemiological study revealed that the prevalence of A308/99 antibodies was low (15 %) among infants but became higher with age, reaching more than 80 % by 30 years of age. These observations indicate that A308/99 is genetically close to, but serologically and genetically distinct from, HPeV-1 and HPeV-2 and accordingly can be classified as third serotype of human parechovirus.

Isolation and characterization of a new species of kobuvirus associated with cattle.

A cytopathic agent was isolated using Vero cells from the culture medium of HeLa cells that had been used for more than 30 years in our laboratory. This agent, termed U-1 strain, was serially passed in Vero cells with distinct CPE. Particles of U-1 strain negatively stained with phosphotungstic acid exhibited a distinct surface that resembled Aichi virus. The RNA genome of U-1 strain comprises 8374 nt, with a genome organization analogous to that of picornaviruses. Possible cleavage sites of the large ORF, which encoded a leader protein prior to the capsid protein region, were assigned following amino acid alignment with Aichi virus. The virus sequence had 33 and 75 % amino acid identity with the Aichi virus VP1 and 3D regions, respectively, but no more than 23 and 36 % with those of the prototype strains of other PICORNAVIRIDAE: The dendrogram based on the P1, P2 and P3 proteins indicated that U-1 strain is genetically included in the genus Kobuvirus but is distinct from Aichi virus. Of 72 cattle sera, 43 (59.7 %) were positive for neutralizing antibody against U-1 strain at a titre of 1 : 8 or more. However, sera from 190 humans, 242 monkeys, 139 pigs, 5 horses, 22 dogs and 9 cats did not neutralize U-1 strain at a 1 : 4 dilution. RNA corresponding to U-1 strain was detected in 12 (16.7 %) of 72 faecal samples from cattle by RT-PCR. These results indicated that U-1 strain, suspected to be a contaminant from calf sera, is a new species of the genus Kobuvirus, now termed bovine kobuvirus.

[Treatment strategy for T4N1 thyroid papillary carcinoma based upon our clinical experience].

We retrospectively evaluated clinical profiles and prognoses in 152 patients with thyroid papillary carcinoma treated at Fukui Medical University between 1986 and 2000. As standard treatment, 106 (70%) underwent hemithyroidectomy to preserve the normal thyroid lobe. Subtotal thyroidectomy or total thyroidectomy was conducted on 40 cases (23%). Regional lymphnodes were extirpated in 104 (68%) with pathological N0, and radical or conservative neck dissection for 46 cases (30%) with pathological N1. Overall survival for 10 years, estimated using the Kaplan-Meier method, was 100% in both stage I and II, and 95% in stage III. Of 152 thyroid papillary cases, 22 (14%) had tumor recurrence. Of 51 in stage III, 14 (27%) had tumor recurrence. The 14 recurrent in stage III showed local extrathyroidal invasion. Note that 5 of 10 (50%) T4N1 treated with hemithyroidectomy had tumor recurrence in the residual thyroid lobe. Of 11 T4N0 cases who underwent hemithyroidectomy, none had tumor recurrence in the residual thyroid lobe. Results suggest that patients with T4N1 should be treated by total thyroidectomy and neck dissection at initial treatment. Tumor size, cervical lymphnodal metastasis, and distant metastasis may be prognostic factors for thyroid papillary carcinoma.

Expression of p27 and apoptosis in oral leukoplakia.

Recently, a close relationship between the cell cycle and apoptosis was confirmed in oral squamous cell carcinoma (SCC). To clarify the expression of cell cycle-related proteins and apoptosis in the oral premalignant state, we investigated the expression of p27, cyclin D1 and proliferating cell nuclear antigen (PCNA) using immunohistochemical staining, and apoptotic index (AI) using ApopTag in situ staining. The positive expression-score of p27 was 42.0% in leukoplakia (34.8% in hyperplasia, 55.4% in dysplasia) and 31.1% in SCC (43.3% in the early stage of SCC, 25.9% in the advanced stage of SCC). The level of p27 expression showed a peak in dysplasia and decreased in SCC. The expression of cyclin D1 was almost constant between hyperplasia and SCC. The expression of PCNA increased sequentially with disease progression. The apoptotic index (AI) in the p27-positive leukoplakia group was significantly higher than that in the p27-negative leukoplakia group (3.9 +/- 3.6 vs. 1.5 +/- 1.6, p = 0.026). From these results, we suggest that the abundance of p27 in oral leukoplakia may inhibit cell proliferation and lead premalignant tumor cells to apoptosis, and thus is concerned with prevention of tumor progression.

Gene expression analysis of human middle ear cholesteatoma using complementary DNA arrays.

OBJECTIVE: To identify genes regulated in human cholesteatoma compared with normal skin tissue using complementary DNA arrays. STUDY DESIGN: In vitro analysis. METHODS: Eight cholesteatoma and retroauricular skin samples were obtained from the same patients during surgery. Upregulated and downregulated genes were highlighted using complementary DNA arrays for screening. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining were performed to confirm the results of the complementary DNA array. RESULTS: Twelve genes were found to be induced or upregulated in cholesteatoma compared with skin samples. These included genes involved in cell proliferation and differentiation (eg, calgranulin A, calgranulin B, psoriasin, thymosin beta-10) and cell invasion (eg, cathepsin C, cathepsin D, cathepsin H). Analyses by means of reverse transcription-polymerase chain reaction showed enhanced expression of several genes including calgranulin A, calgranulin B, psoriasin, thymosin beta-10, cathepsin C, cathepsin D, and cathepsin H in cholesteatoma, supporting the findings from the gene array. In addition, it was verified by immunohistochemical analysis that the expressions of Calgranulin A, Calgranulin B, and Cathepsin D were mainly located in cholesteatoma epithelium. CONCLUSION: The observed alteration in gene expression may play a role in various mechanisms of pathogenesis in cholesteatoma.