Kouichiro Tsutsumi

Detection of human papillomavirus DNA in laryngeal squamous cell carcinomas by polymerase chain reaction

The presence of human papillomavirus genomes‐16 and ‐6b in metastatic cervical lymph nodes was examined in 34 cases of laryngeal carcinomas by means of polymerase chain reaction, which had been fixed in formalin and embedded in paraffin. Human papillomavirus DNAs extracted from paraffin‐embedded tumor tissues were used for polymerase chain reaction with amplification of the E6 region of human papillomavirus genome‐16 and the El region of human papillomavirus genome‐6b. Human papillomavirus genome‐16 sequences were positively amplified in six (17.6%) metastatic tumors; ‐6b sequence was positively amplified in one (2.9%) metastatic tumor. Laryngeal carcinomas of glottic origin showed high human papillomavirus genome‐16 DNA‐positive rates (4 of 9 cases, 44.4%) compared to those of other sites. These results suggest that human papillomavirus genome‐16 infection might be closely associated with the development of some laryngeal squamous cell carcinomas of glottic origin similar to uterine cervical carcino‐genesis.

In Situ hybridization and immunohistochemical study of human papillomavirus infection in adult laryngeal papillomas

Routinely processed paraffin sections from 20 patients with adult laryngeal papillomas were examined for the presence of human papillomavirus type 11 (HPV‐11) DNA and its specific mRNA by in situ hybridization methods using 35S‐labeled RNA probes. Immunohistochemical techniques were also used to identify papillomavirus genus‐specific common antigen (pgsantigen). HPV‐11 DNA signals and/or papillomavirus genus‐specific common antigen were detected in all eight samples of multiple laryngeal papilloma. On the other hand, in 12 samples of single laryngeal papilloma, neither papillomavirus genus‐specific common antigen nor HPV‐11 DNA were detected. Four patients were positive for both HPV‐11 DNA and pgs‐antigen. In three of these four patients, HPV‐11 mRNA signals were also detected. These results provided direct evidence of the association of HPV and adult multiple laryngeal papilloma.

Caspase-9 regulates cisplatin-induced apoptosis in human head and neck squamous cell carcinoma cells

The aim of this study was to understand the molecular requirements for cisplatin-induced apoptosis in human head and neck squamous cell carcinoma (HNSCC) cell death. Cisplatin induced apoptosis in HNSCC cell lines, HSC-2, HSC-3 and HSC-4 in a dose-dependent manner. However, cisplatin did not induce the expression of Fas-ligand mRNA or upregulation of Fas protein. By caspase activation assays, cisplatin induced Caspase-3 (Casp-3), -8 and -9 activation. In all three lines tested, the use of a specific inhibitor of Casp-9 almost completely blocked cisplatin-induced apoptosis, while the use of Casp-3 and -8 inhibitors resulted in a partial blockade of cisplatin-induced apoptosis. Our results strongly suggest that Casp-9-dependent apoptosis plays an important role in cisplatin-induced HNSCC apoptosis.

Absence of Nuclear p16 From Epstein-Barr Virus-Associated Undifferentiated Nasopharyngeal Carcinomas

Objective: Epstein‐Barr virus (EBV) is detected in the majority of undifferentiated nasopharyngeal carcinomas (UNPCs, World Health Organization type III). However, the exact mechanism involved in the carcinogenesis of EBV‐associated UNPCs remains to be elucidated. An important unresolved question is: how is the normal cell cycle deregulated during EBV‐associated UNPC development? The p16CDKN2 gene encodes a nuclear protein, p16, which inhibits the D‐type cyclin/cyclin‐dependent kinase complexes that phosphorylate the retinoblastoma gene product (pRb), thus blocking G1 cell cycle progression. The objective of this study was to determine whether p16 absence is involved in the development of EBV‐associated UNPCs.

Inhibition of caspase-9 activity and Apaf-1 expression in cisplatin-resistant head and neck squamous cell carcinoma cells.

We have previously reported that cisplatin induces caspase-9 (Casp9) activation in head and neck squamous cell carcinoma cells in vitro (HNSCCs). Our purpose here was to examine whether HNSCCs selected for resistance to cisplatin fail to exhibit Casp9 activation in response to cisplatin. The cisplatin-resistant HNSCCs (HSC-2CR) were selected from cisplatin-sensitive HNSCCs (HSC-2) for growth in the presence of cisplatin. Following cisplatin treatment, protelyzed Casp9 subunits were detected in HSC-2, but not detected in HSC-2CR. Using a direct enzymatic assay measuring cleavage of the synthetic peptide substrate (LEHD-AFC), Casp9 activity in cisplatin-treated HSC-2CR was less than that in cisplatin-treated HSC-2. Apoptotic protease-activating factor 1 (Apaf-1) has been shown to participate as an adaptor molecule in Casp9 activation. In the presence of cytochrome c (Cyt c) released from mitochondria, Apaf-1 binds to Casp9 and causes its activation. HSC-2 expressed 2-fold higher levels of Apaf-1 compared with HSC-2CR. On the other hand, following cisplatin treatment, the same degree of increase in cytoplasmic Cyt c was detected in both HSC-2 and HSC-2CR. These results suggest that in a certain type of HNSCCs, the inhibition of Casp9 activity and Apaf-1 expression may represent a mechanism of acquired cisplatin resistance.

Objective tinnitus caused by an aberrant internal carotid artery

We report the case of a 29 year-old woman who complained of pulsatile tinnitus and impaired hearing. On otoscopic examination, her right tympanic membrane was observed to be in contact with a mass in the middle ear cavity, with the formation of a meniscus at the point of contact. Using a high-sensitivity microphone inserted into the external auditory canal, we recorded pulsatile tinnitus that was synchronous with the electrocardiogram. Magnetic resonance angiography revealed that the middle ear mass was an aberrant internal carotid artery coursing through the hypotympanum.

Crosslinking of the CD69 molecule enhances S100A9 production in activated neutrophils.

Expression of CD69 on neutrophils and generation of anti‐CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti‐CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint‐composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte‐macrophage colony stimulating factor (GM‐CSF)‐activated neutrophils was crosslinked by anti‐CD69 monoclonal antibodies, and then intracellular proteins were detected using 2‐dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up‐regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS‐27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS‐27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL‐1β production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti‐CD69 autoantibodies possibly affect joint‐composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.

Identification of β‐Tubulin Isoform V as an Autoantigen in Allergic Rhinitis by a Proteomic Approach

Autoantibodies to IgE and p2‐adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2‐dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, β‐tubulin isoform V (β‐tubV), using a recombinant protein and then measured prevalence of the anti‐β‐tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti‐β‐tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and β‐tubV is one of the major autoantigens in allergic rhinitis.

A case of carcinoid tumor of the middle ear.

We report here a case of a carcinoid tumor observed in the middle ear (ME), which was initially diagnosed as ME adenoma. The patient was a 64-year-old woman who was first seen in our hospital in March 2001 for a 7-month hearing loss. On otoscopic examination, a whitish mass could be observed through the intact tympanic membrane. High-resolution computed tomography demonstrated a tumor-like lesion in the ME with no evidence of bone destruction. A myringotomy and biopsy were performed and an initial diagnosis of ME adenoma was made. Light microscopy showed fragments of cellular tissue in which both glandular (adenomatous) and trabecular (carcinoid) growth patterns could be identified, but neuroendocrine differentiation was not detected by immunohistochemistry (negative staining for chromogranin A and synaptophysin). On the basis of this diagnosis, the patient underwent a tympanomastoidectomy in June 2001, in which the presumed ME adenoma was completely excised and the diagnosis was modified to ME carcinoid tumor. Immunohistochemical examinations at that time showed positive staining of the tumor cells for chromogranin A and synaptophysin. This case suggests the difficulties in distinguishing ME carcinoid tumors from ME adenomas. The patient is without recurrence of her disease to date.

A case of an inverted tooth in the nasal cavity

We report a case of an inverted tooth in the nasal cavity. The patient was a 27-year-old man who attended our hospital in May 1998, complaining of left cheek-pain. There was nothing remarkable in his medical or family history. Fiberscopic (intranasal) and radiological examinations revealed a white foreign body in the left nasal cavity, within 2 cm of the left nostril. This foreign body was diagnosed as an inverted tooth. It was removed under general anesthesia and found to be 17 mm in length. Although the tooth showed a single root, it possessed two cusps and we deduced it to be a molar.