A Novel Immune Evasion Mechanism of LMP-1, an EBV-Primary Oncogene, in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma is an Epstein-Barr virus (EBV)-associated tumor. Viruses that are associated with malignant transformation have evolved unique mechanisms to interfere with this interaction to evade antiviral T cell responses. EBV exploits many immune evasive strategies to successfully establish a latent infection in B cells. CD8+ T cell responses to LMP-1 are generally very low and rarely detected in healthy virus carriers. Activation of the NF-kB pathway by EBV-LMP-1 leads to an upregulation of the MHC class I antigen-processing pathway. Paradoxically, LMP-1itself induces a subdominant CD8+ T cell response and appears to have evolved to avoid immune recognition. An expression of LMP-1 in human cells enhanced the trans-presentation of CD8+ T cell epitopes; however, cis-presentation of LMP-1-derived epitopes was severely impaired. Deletion of the first transmembrane domain of LMP-1, which prevented self-aggregation, significantly enhanced the cis-presentation of T cell epitopes from this protein, whereas it lost its ability to upregulate trans-presentation. These results delineate a novel mechanism of immune evasion, which renders a virally encoded oncogene inaccessible to the conventional MHC class I pathway limiting its cis-presentation. Copyright © 2011 S. Karger AG, Basel

Retrograde mastoidectomy on demand with soft-wall reconstruction in pediatric cholesteatoma

金沢大学医薬保健研究域医学系Conclusion: Retrograde mastoidectomy with soft-wall reconstruction is an effective technique that can be used to lower the recurrence rate of cholesteatoma in the pediatric population. Objective: To evaluate surgical outcomes of retrograde mastoidectomy when using soft-wall reconstruction in pediatric cholesteatoma. Methods: A total of 25 children underwent cholesteatoma removal surgery employing soft-wall reconstruction. The cases were retrospectively reviewed. Average follow-up time was 48.7 months. In order to fully expose and extirpate the disease, the bony canal wall was removed in association with a retrograde-type mastoidectomy in all cases. The posterior ear canal defect was then reconstructed using soft tissue; i.e. temporal fascia and canal wall skin. The incidence and localization of residual and recurrent cholesteatoma, preoperative and postoperative audiogram results, pure-tone average (PTA), and airbone gap (ABG) were assessed. Results: Residual cholesteatoma was detected in 5 (20%) of 25 ears while recurrent cholesteatoma occurred in 1 (4%) of 25 ears. The mean preoperative PTA of air conduction (AC) was 39.1 dB, while the preoperative PTA of the ABGs was 28.8 dB. The mean postoperative PTA-AC and PTA-ABG were 20.9 dB and 11.7 dB, respectively. The mean hearing gain was 18.2 dB. The differences between the pre- and postoperative values were statistically significant (p < 0.05). © 2010 Informa Healthcare

Epstein-Barrウイルス転写調節因子Zタンパクおよび細胞転写調節因子c-Fosタンパクの構造と機能解析

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第987号, 学位授与年月日：平成3年3月25日,学位授与年：199

Prognostic factors for squamous cell carcinoma of the temporal bone: Extensive bone involvement or extensive soft tissue involvement?

金沢大学医薬保健研究域医学系Conclusion: Extensive bone erosion correlated with a worse prognosis of the squamous cell carcinoma (SCC) of the temporal bone but extensive soft tissue involvement did not correlate with prognosis in this study. Resectability of the tumor seems to be major prognostic factor of temporal bone SCC. Objective: Prognostic factors for SCC of the temporal bone were evaluated regarding initial clinical symptoms and radiographic imaging. Patients and methods: Clinical symptoms of the patients with primary SCC of the external auditory canal (EAC) or middle ear (ME) were reviewed based on medical records. Correlation of clinical symptoms and cancer severity staging using the modified Pittsburgh classification was analyzed, along with disease-specific survival (DSS). Results: Sixteen patients with primary SCC of the EAC (n=13) or ME (n=3) were included in the study population. DSS was not influenced by whether a hearing disturbance or otalgia was noted at the first medical examination. Extended bone involvement identified with imaging studies significantly correlated with worse prognosis (p<0.05). Prognoses of patients without extensive bone erosion were good, and extensive (≥0.5 cm) soft tissue involvement did not correlate with prognosis in this study. Prognoses of patients with insufficient bone resection or no surgery were significantly poor (p<0.01)

Malignant phosphaturic mesenchymal tumor, mixed connective tissue variant of the tongue

金沢大学医薬保健研究域医学系The majority of the oncogenic osteomalacia-associated mesenchymal tumors are considered to belong to the category of phosphaturic mesenchymal tumors, mixed connective tissue (PMTMCT) variant, of which malignant cases are very rare. Here we report a case of a recurrent malignant PMTMCT variant which arose in the tongue. The patient was treated with surgery at an initial treatment and the first recurrence. In accordance with the tumor recurrence and resection, the hypophosphatemia progressed and improved. However, hypophosphatemia did not progress after receiving radiation therapy at the second recurrence even though the recurrent tumor gradually increased its size. These results suggest clinical feature of malignant PMTMCT could be changed by radiation therapy. Thus, this report could add an insight to the nature of PMTMCT. © 2008 Elsevier Ireland Ltd. All rights reserved

核酸アナログ系抗ウイルス剤によるウイルス関連腫瘍治療に関する基礎研究

1)核酸アナログタイプ薬剤シドフォビルによるin vitroでの細胞障害性の検討NPC-KT細胞(EBV陽性上皮細胞)による実験を行った。シドフォビルによる細胞障害性は、1mMで0.1mMと比較して明らかに大きかった。2)リボ核酸還元酵素阻害剤(RR inhibitor)によるin vitroでの細胞障害性の検討RR inhibitorとして、hydroxyureaおよびdidoxを用いて、同じくNPC-KT細胞に対する細胞障害性を検討した。その結果、hydroxyureaでは濃度が0.125mMを超えると細胞障害性が亢進し、didoxでは0.1mM以上の濃度で細胞障害性が強く出る事が判明した。3)in vitroでの核酸アナログタイプ薬剤およびリボ核酸還元酵素阻害剤の併用による細胞障害性の検討続いて、シドフォビルおよびRR inhibitorを併用した場合、細胞障害性が増強される事が判明した。Hydroxyurea、didoxいずれの場合も、シドフォビルとの相互増強作用は相加的なものであった。さらに、PARP蛋白をウエスターン・ブロット法で同定する事により、細胞障害性の増強にアポトーシスが関与するかどうかについて検討した。シドフォビルおよびRR inhibitorを併用した場合、PARPの分解が進むことから、アポトーシスが関与する事が、判明した。4)in vivoでの核酸アナログタイプ薬剤およびリボ核酸還元酵素阻害剤の併用による細胞障害性の検討ヌードマウスにEBV陽性腫瘍を移植し、上記薬剤を併用した所、腫瘍縮小に少なくとも相加的な効果を認めた。研究課題/領域番号:16659463, 研究期間(年度):2004 – 2006出典：「核酸アナログ系抗ウイルス剤によるウイルス関連腫瘍治療に関する基礎研究」研究成果報告書　課題番号16659463（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-16659463/)を加工して作

EBウイルスリボ核酸EBERsによる上咽頭癌発癌機構の分子生物学的解析

Epstein-Barrウイルス(EBV)がコードする小RNAEBERsの発現ベクターを作成した。すなわち、EBER1、EBER2各遺伝子を各々1コピー、4コピー、10コピー直列につないでpcDNA3プラスミドに挿入した。それらを正常腎上皮細胞MDCKに形質導入し、形質転換能および増殖能を検討したところ、EBER1とEBER2を各々4コピーずつ含むプラスミドを導入した場合、軟寒天培地におけるコロニーの形成能が最も亢進した(コントロールの4倍)。EBER1とEBER2を各々10コピーずつ含むプラスミドを導入した場合のコロニー形成能は、コントロールの1.8倍、EBER1およびEBER2を各々単独で導入した場合にはコントロールと比較して、有意なコロニー形成能の亢進は認められなかった。次に、無血清培地におけるMDCK細胞の増殖能を指標としてEBERsの形質転換能を評価した。結果は、各々のコピー数のEBER1+EBER2の組み合わせのいずれにおいても有意な増殖能の変化を認めなかった。また、放射線および紫外線に対する感受性も、同様に変化を認めなかった。EBERsは核内で二本鎖RNAを形成してプロテインキナーゼR(PKR)を阻害して細胞の増殖能を促進することが示唆されている。EBERsを試験管内で合成し、MDCK細胞のリボゾームRNAをコントロールとしてPKR活性にEBERsが及ぼす影響を検討した。すなわち、PKRが燐酸化することが判明しているアミノ酸配列ILLSESRRRIRの中央のセリンと放射性同位元素γ32Pを用いてEBERsによるPKR酵素活性を評価したところ、EBERsはPKR活性に影響しないという結果を得た。上咽頭がん組織におけるEBERsの発現とEBVがん遺伝子LMP1の発現と各種臨床パラメーターとの相関を検討したところ、EBERsの発現はTstageが進行するにつれ増加する傾向にあったが統計学的に有意ではなかった。一方LMP1の発現は頸部リンパ節転移と有意に相関した。以上より、EBERs発現はMDCK細胞のanchor independentな増殖を促進するが、その現象にはPKR以外のシグナル伝達系が関与すること、EBERsは転移より増殖に関連が深いことが示唆された。EBERs is composed of EBER1 and EBER2. Transfection of EBER1+EBER2 expression vector in MDCK cell enhanced colony formation in soft agar, indicating that expression of EBERs transformed MDCK cell to potentially malignant, anchor-independent phenotype. Either EBER1 or EBER2 expression alone was not sufficient for transformation of MDCK cell to this anchor-independent character. EBERs did not protect MDCK cells from dell death in the condition of serum starvation and irradiation. EBERs are known to activates protein kinase R(PKR) which is activated by double-strand RNA. However, our in vitro kination assay revealed that EBERs did not activate PKR in MDCK cells. These results suggest that transformation of MDCK by EBERs were mediated by alternative pathway rather than PKR pathway. Expression of EBERs in nasopharyngeal carcinoma tissue tended to be correlated with advanced T stage, suggesting that EBERs promote growth and proliferation of tumor cell in nasopharyngeal carcinoma.研究課題/領域番号:13671775, 研究期間(年度):2001-2002出典：「EBウイルスリボ核酸EBERsによる上咽頭癌発癌機構の分子生物学的解析」研究成果報告書　課題番号13671775 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Cricoid ossification mimicking an impacted foreign body.

金沢大学医学部附属病院耳鼻咽喉科A 54-year-old man complained of severe throat pain and showed subglottic oedema on fibre-optic endoscopy with a distinctly narrowed subglottic space on anteroposterior radiography of the neck and dense linear opacity at the level of the cricoid cartilage on lateral plain radiography. These findings suggested a foreign body just posterior to the cricopharyngeus, but a computed tomography (CT) scan demonstrated a dense calcified ridge on the posterior lamina of the cricoid cartilage but no foreign body.The patient improved symptomatically with systemic antibiotics and topical steroids, and gastrointestinal endoscopy did not detect any foreign body. This is a rare case of vertical ossification of the cricoid lamina masquerading as a foreign body

Post-operative pharyngocutaneous fistula after laryngectomy

金沢大学附属病院耳鼻咽喉科Objectives: Although organ-preserving radiotherapy or chemoradiotherapy has offered good locoregional control, many patients still experience recurrent disease requiring salvage laryngectomy. The pharyngocutaneous fistula (PCF) is a common and troublesome complication in the early post-operative period after laryngectomy. Here, we evaluated the cause of PCF after laryngectomy, with special emphasis on radiotherapy and/or chemotherapy. Patients and methods: A total of 63 consecutive patients undergoing salvage total laryngectomy for squamous cell carcinoma of the larynx at Kanazawa University Hospital from 1990 to 2005 were reviewed. Forty of the 63 had received primary total laryngectomy (PL). Ten patients underwent radiotherapy alone (SL-RT) and 13 patients underwent concurrent chemoradiotherapy (SL-CRT) followed by salvage laryngectomy. Results: Overall, 17 of the 63 patients (27.0%) developed PCF after laryngectomy. Fisher\u27s exact test showed a significant increase of PCF formation in SL-CRT (7/13, 53.8%) compared with PL (7/40, 17.5%) (p = 0.0252). There were non-significant increases of PCF formation both in SL-CRT (7/13, 53.8%) compared with SL-RT (3/10, 30.0%) (p = 0.4015), and also in SL-RT (3/10, 30.0%) compared with PL (7/40, 17.5%) (p = 0.3969). The Mann-Whitney U-test showed that the duration of PCF was significantly longer for SL-CRT PCF (121.2 ± 95.0 days) compared with those for PL (39.0 ± 55.3 days) (p = 0.0298) or SL-RT (28.0 ± 16.2 days) (p = 0.0325). However, we did not find a significant difference in the duration of PCF with respect to PL (39.0 ± 55.3 days) and SL-RT (28.0 ± 16.2 days) (p = 0.4367). Conclusions: Although radiotherapy or chemotherapy has only a limited impact on PCF formation, concurrent chemoradiotherapy significantly increases PCF formation. The addition of chemotherapy to irradiation delays PCF closure. © 2007 Elsevier Ireland Ltd. All rights reserved

Ribonucleotide reductase inhibitors enhance cidofovir-induced apoptosis in EBV-positive nasopharyngeal carcinoma xenografts

金沢大学医学部附属病院耳鼻咽喉科In nasopharyngeal carcinoma (NPC), Epstein-Barr virus (EBV) infection is mainly latent, and the tumor cells contain episomal viral DNA. We have shown that the acyclic nucleoside phosphonate analog, cidofovir [(S)-1-(3-hydroxy-2- (phosphonylmethoxypropyl)cytosine] (HPMPC), inhibits growth of NPC xenografts in nude mice by causing apoptosis. The ribonucleotide reductase (RR) inhibitors, hydroxyurea and didox (3,4-dihydroxybenzohydroxamic acid), have been demonstrated to inhibit neoplastic growth and are used as antiviral and anticancer agents. Here we show that RR inhibitors enhance the antitumor effect of cidofovir in EBV-transformed epithelial cells. MTT assays indicate that hydroxyurea and didox enhance cidofovir-induced cell toxicity in NPC-KT cells, an EBV-positive epithelial cell line derived from NPC. The effect is due to enhancement of apoptosis through the caspase cascade as shown by pronounced cleavage of poly(ADP-ribose) polymerase. Finally, hydroxyurea strikingly enhanced the cidofovir-induced growth-inhibitory effect on NPC grown in athymic mice. The results suggest that RR inhibitors should enhance the antitumor effect of acyclic nucleoside phosphonate analogs on NPC. © 2005 Wiley-Liss, Inc