Daiju Sakurai

Combination therapy of in vitro‐expanded natural killer T cells and α‐galactosylceramide‐pulsed antigen‐presenting cells in patients with recurrent head and neck carcinoma

The aim of this clinical trial was to investigate the feasibility of intra‐arterial infusion of in vitro‐expanded Vα24 natural killer T (NKT) cells combined with submucosal injection of α‐galactosylceramide (KRN7000; αGalCer)‐pulsed antigen‐presenting cells (APC). A phase I clinical study was carried out in patients with head and neck squamous cell carcinoma (HNSCC). Patients with locally recurrent HNSCC refractory to standard therapy were eligible. Eight patients received super‐selective transcatheter intra‐arterial infusion of activated Vα24 NKT cells into tumor‐feeding arteries and nasal submucosal injections of αGalCer‐pulsed APC twice with a 1‐week interval. Vα24 NKT cell‐specific immune responses, safety, and antitumor effects were evaluated. The number of Vα24 NKT cells and interferon‐γ‐producing cells in peripheral blood mononuclear cells increased in seven out of eight patients enrolled. Grade 3 toxicity with a pharyngocutaneous fistula related to local tumor reduction was observed in one patient and mild adverse events with grade 1–2 symptoms occurred in seven patients. Regarding the clinical responses, three cases exhibited a partial but significant response, four were classified as stable disease, and one patient continued to develop progressive disease. The use of the intra‐arterial infusion of activated Vα24 NKT cells and the submucosal injection of αGalCer‐pulsed APC has been shown to induce significant antitumor immunity and had beneficial clinical effects in the management of advanced HNSCC. The use of such therapeutic modalities may be helpful in the management of tumors and therefore needs to be explored in further detail. The clinical trial registration number was UMIN000000722. (Cancer Sci 2009; 100: 1092–1098)

miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC)

Background:Hypopharyngeal squamous cell carcinoma (HSCC) is an aggressive malignancy with one of the worst prognoses among all head and neck cancers. Greater understanding of the pertinent molecular oncogenic pathways could help improve diagnosis, therapy, and prevention of this disease. The aim of this study was to identify tumour-suppressive microRNAs (miRNAs), based on miRNA expression signatures from clinical HSCC specimens, and to predict their biological target genes.Methods:Expression levels of 365 human mature miRNAs from 10 HSCC clinical samples were screened using stem-loop real-time quantitative PCR. Downregulated miRNAs were used in cell proliferation assays to identify a tumour-suppressive miRNA. Genome-wide gene expression analyses were then performed to identify the target genes of the tumour-suppressive miRNA.Results:Expression analysis identified 11 upregulated and 31 downregulated miRNAs. Gain-of-function analysis of the downregulated miRNAs revealed that miR-489 inhibited cell growth in all head and neck cancer cell lines examined. The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene. Knockdown of PTPN11 resulted in the inhibition of cell proliferation in head and neck SCC cells.Conclusion:Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.

Tumor suppressive microRNA-375 regulates oncogene AEG-1 / MTDH in head and neck squamous cell carcinoma (HNSCC)

Our microRNA (miRNA) expression signatures of hypopharyngeal squamous cell carcinoma, maxillary sinus squamous cell carcinoma and esophageal squamous cell carcinoma revealed that miR-375 was significantly reduced in cancer tissues compared with normal epithelium. In this study, we focused on the functional significance of miR-375 in cancer cells and identification of miR-375-regulated novel cancer networks in head and neck squamous cell carcinoma (HNSCC). Restoration of miR-375 showed significant inhibition of cell proliferation and induction of cell apoptosis in SAS and FaDu cell lines, suggesting that miR-375 functions as a tumor suppressor. We adopted genome-wide gene expression analysis to search for miR-375-regulated molecular targets. Gene expression data and luciferase reporter assays revealed that AEG-1/MTDH was directly regulated by miR-375. Cancer cell proliferation was significantly inhibited in HNSCC cells transfected with si-AEG-1/MTDH. In addition, expression levels of AEG-1/MTDH were significantly upregulated in cancer tissues. Therefore, AEG-1/MTDH may function as an oncogene in HNSCC. The identification of novel tumor suppressive miRNA and its regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC oncogenesis.

Tumour suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma

Background:On the basis of the microRNA (miRNA) expression signature of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-874 was significantly reduced in cancer cells. We focused on the functional significance of miR-874 in cancer cells and identification of miR-874-regulated novel cancer networks in MSSCC.Methods:We used PCR-based methods to investigate the downregulated miRNAs in clinical specimens of MSSCC. Our signature analyses identified 23 miRNAs that were significantly reduced in cancer cells, such as miR-874, miR-133a, miR-375, miR-204, and miR-1. We focused on miR-874 as the most downregulated novel miRNA in our analysis.Results:We found potential tumour suppressive functions such as inhibition of cancer cell proliferation and invasion. A molecular target search of miR-874 revealed that PPP1CA was directly regulated by miR-874. Overexpression of PPP1CA was observed in MSSCC clinical specimens. Silencing of the PPP1CA gene significantly inhibited cancer cell proliferation and invasion.Conclusion:The downregulation of miR-874 was a frequent event in MSSCC, which suggests that miR-874 functions as a tumour suppressive miRNA, directly regulating PPP1CA that has a potential role of an oncogene. The identification of novel miR-874-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

Identification of novel molecular targets regulated by tumor suppressive miR-1/miR-133a in maxillary sinus squamous cell carcinoma

Based on our microRNA (miRNA) expression signature analysis of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-1 and miR-133a were significantly reduced in tumor tissues. Quantitative real-time RT-PCR revealed that the expression levels of miR-1 and miR-133a were significantly downregulated in clinical MSSCC tumor tissues compared with normal tissues. We focused on the functional significance of miR-1 and miR-133a in cancer cells and identification of the novel cancer networks regulated by these miRNAs in MSSCC. Restoration of downregulated miRNAs (miR-1 or miR-133a) in cancer cells revealed that both miRNAs significantly inhibited cancer cell proliferation and induced cell apoptosis. Molecular target identification of these miRNAs showed that transgelin 2 (TAGLN2) and purine nucleoside phosphorylase (PNP) were regulated by miR-1 and miR-133a. Both TAGLN2 and PNP mRNA expression levels were significantly upregulated in clinical MSSCC tumor tissues. Silencing studies of target genes demonstrated that both genes inhibited cancer cell proliferation. The identification of novel miR-1/miR-133a-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

Differential effects of reduced glycoprotein VI levels on activation of murine platelets by glycoprotein VI ligands

We have investigated the effects of decreased levels of the complex between glycoprotein VI (GPVI) and the Fc receptor gamma-chain (FcRgamma) on responses to collagen and GPVI-specific ligands in murine platelets. We show that levels of GPVI-FcRgamma of the order of 50% and 20% of wild-type levels caused 2- and 5-fold shifts to the right respectively in the dose-response curve for aggregation in response to collagen, the snake toxin convulxin and the monoclonal antibody JAQ1. In addition, there is a delay in the onset of aggregation in response to collagen. In contrast, the stimulation of protein tyrosine phosphorylation by collagen (as measured after 150 s) and adhesion to a collagen-coated surface under static conditions were unaffected in platelets with 50% and 20% of wild-type levels of GPVI. In contrast, responses to a collagen-related peptide (CRP), made up of repeat glycine-proline-hydroxyproline motifs, were markedly inhibited and abolished in platelets expressing 50% and 20% of wild-type levels of GPVI respectively. We suggest that the marked effect of a reduction in GPVI levels on the CRP-induced activation of platelets is due to the multivalent nature of CRP and the fact that GPVI is its sole receptor on platelets. Thus it appears that the interaction of CRP with GPVI is determined by a combination of affinity and avidity. The observation that collagen does not behave like CRP in platelets expressing reduced levels of GPVI, even in the combined presence of blocking antibodies against integrin alpha2beta1 and GPV, suggests that collagen has a greater affinity than CRP for GPVI, and/or that other receptors are involved in its binding to platelets. The clinical significance of these results is discussed.

FcεRIγ-ITAM Is Differentially Required for Mast Cell Function In Vivo

The cross-linking of IgE-bound FcεRI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcεRIγ signaling to IgE/FcεRI-mediated mast cell responses has not yet been elucidated. In this study FcεRIγ−/− mast cells were reconstituted with either wild-type or mutant FcεRIγ in transgenic mice and transfected mast cells in vitro. We demonstrate that FcεRIγ-immunoreceptor tyrosine-based activation motif is essential for degranulation, cytokine production, and PG synthesis as well as for passive systemic anaphylaxis. Recent reports have suggested that cell surface FcεRI expression and mast cell survival are regulated by IgE in the absence of Ag, although the molecular mechanism is largely unknown. We also found that the promotion of mast cell survival by IgE without Ags is mediated by signals through the FcεRIγ-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcεRI is independent of FcεRIγ signaling. These results indicate that FcεRIγ-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.

Sublingual Immunotherapy with House Dust Extract for House Dust-Mite Allergic Rhinitis in Children

BACKGROUND House dust extract is used in conventional immunotherapy for house dust-mite (HDM) allergic rhinitis in Japan. However, an alternative administration route is desired. The aims of the present double blind, placebo-controlled trial were to evaluate the therapeutic efficacy and safety of sublingual immunotherapy (SLIT) with house dust extract in pediatric patients with HDM allergic rhinitis. METHODS The study population comprised 31 subjects (21 males and 10 females) aged from 7 to 15 years old. Twenty patients (the active group) received house dust extract and 11 received placebo via sublingual administration. Extract or placebo (1 ml) was administered at 10-fold dilution once weekly for 40 weeks. During the study period, the subjects recorded their daily nasal symptoms and use (dose and frequency) of other medications in a nasal allergy diary. RESULTS The symptom scores in the active group began to decrease about 24 weeks after initiation of treatment and significant differences between the active and placebo groups were observed after 30 weeks. The average scores for the last four weeks of the study were significantly lower than those for the first four weeks in the active group but not in the placebo group. The only local adverse effect was a bitter taste reported by one patient. There were no other local or systemic adverse effects associated with SLIT. CONCLUSIONS Our results suggest that SLIT with house dust extract for more than 30 weeks is safe and effective treatment for HDM allergic rhinitis in children.

Interleukin-25 and mucosal T cells in noneosinophilic and eosinophilic chronic rhinosinusitis

BACKGROUND Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous disease of uncertain pathogenesis. Memory T cells acquire additional functions during the secondary response and play important roles in chronic inflammation. OBJECTIVE To investigate characteristics of tissue memory CD4(+) T cells obtained from patients with noneosinophilic CRSwNP (NECRS) and eosinophilic CRSwNP (ECRS) by focusing on the influence of interleukin (IL)-25. METHODS Pro-allergic cytokines in tissue homogenates were measured using enzyme-linked immunosorbent assays. NP mononuclear cells and CD4(+) T cells were isolated from NPs from patients with CRSwNP. Cytokine expression and CD4(+) T-cell subpopulations were analyzed using enzyme-linked immunosorbent assay, flow cytometry, and real-time polymerase chain reaction. RESULTS The IL-25 level in NPs increased in patients with ECRS. IL-5 and IL-9 mRNA levels expressed by tissue CD4(+) T cells were significantly elevated in patients with ECRS. Most infiltrating CD4(+) T cells in ECRS and NECRS expressed CD45RO; however, regardless of the atopic status, high IL-17RB levels were detected in CD4(+) T cells from patients with ECRS. IL-17RB mRNA levels expressed by tissue CD4(+) T cells significantly correlated with the number of eosinophils in NPs. Elevation of IL-5 and IL-9 production was found in NP mononuclear cells from patients with ECRS, but not in those from patients with NECRS, by stimulation with IL-25 under T-cell receptor stimulation. CONCLUSION Interleukin-25 and a subpopulation of tissue T-helper type 2 and 9 cells that express increased IL-17RB levels could contribute to infiltration of eosinophils in NPs and could have produced the pathologic difference between NECRS and ECRS.

The Influence of Environmental Exposure to Formaldehyde in Nasal Mucosa of Medical Students during Cadaver Dissection

BACKGROUND Environmental exposure to formaldehyde is commonly associated with clinical symptoms such as mucosal irritation and olfactory disorders. However, the impact of such exposure on the development of mucosal inflammation and its outcome has not been carefully evaluated. METHODS The observational non-comparative study was planned. The study population consisted of group of 41 medical students who had signed up for a cadaver dissection course as part of their gross anatomy teaching at the school of medicine Chiba University in Japan. During such dissection course, the students are exposed to variable levels of environmental formaldehyde routinely employed for the preservation the cadavers. The subjects were evaluated by a detailed medical examination. We measured their serum IgE levels. In addition, an olfaction test and nasal mucosal sensitivity to histamine was serially determined, immediately before and after the course and 6 months after the completion of the course. RESULTS Olfactory abnormalities were observed in 13/41 (32%) subjects and increased nasal mucosal hypersensitivity to histamine was observed in 17/41 (41%) during and immediately after completion of the course. These subjects had evidence of preexisting allergic rhinitis. 6/41 (15%) other students with no prior evidence of allergic rhinitis also exhibited formaldehyde associated clinical symptoms during the dissecting course. However, the symptoms disappeared upon completion of the course in all subjects studied. CONCLUSIONS Temporary abnormalities in the olfaction test and increased nasal mucosal hypersensitivity to histamine were observed in a few students with preexisting allergic rhinitis after environmental exposure of high concentrations of formaldehyde. These effects appeared to be transient.