Takeru Ishikawa

A newly identified MAGE‐3‐derived epitope recognized by HLA‐A24‐restricted cytotoxic T lymphocytes

Five MAGE‐3‐derived peptides carrying an HLA‐A24‐binding motif were synthesized. Binding capacity of these peptides was analyzed by an HLA‐class‐I stabilization assay. Two of the 5 peptides bound to HLA‐A*2402 molecule with high affinity, and 3 peptides with low affinity. Peripheral‐blood mononuclear cells (PBMC) depleted of CD4+T cells were stimulated with the peptides to determine whether these peptides would induce cytotoxic T lymphocytes (CTL) from PBMCs obtained from 7 healthy HLA‐A*2402+ donors. Peptide M3‐p97 (TFPDLESEF; corresponding to amino‐acid residues 97–105 of MAGE‐3), with high binding capacity to the HLA‐A*2402 molecule, elicited the peptide‐specific and HLA‐A24‐restricted CD8+CTL lines in 2 of the 7 donors, while none of the 4 other peptides induced CTL specific for the corresponding peptide in any of the donors. CTL lines induced by stimulation with peptide M3‐p97 exhibited cytolytic activities against HLA‐A*2402 transfectant cell lines (C1R‐A*2402) in the presence of peptide M3‐p97, but not in unloaded or irrelevant peptide‐pulsed C1R‐A*2402 cells. The CTL lines and a cloned CD8+CTL isolated from one of the bulk populations by limiting dilution could lyse MAGE‐3+/HLA‐A*2402+ squamous‐cell‐carcinoma(SCC) lines but neither MAGE‐3−/HLA‐A*2402+ nor MAGE‐3+/HLA‐A*2402− SCC lines, indicating that M3‐p97 can be naturally processed and presented on the tumor‐cell surface in association with HLA‐A*2402 molecules. Combined with the 4 currently reported CTL epitopes derived from MAGE‐3 and presented by HLA‐A1, HLA‐A2, HLA‐A24 or HLA‐B44, identification of this CTL epitope presented by the HLA‐A*2402 molecule will extend the application of MAGE‐3‐derived peptides for immunotherapy for cancer patients. Int. J. Cancer 81:387–394, 1999.

Single amino acid substitutions on a Japanese cedar pollen allergen (Cry j 1)-derived peptide induced alterations in human T cell responses and T cell receptor antagonism

We generated T cell clones specific to a Japanese cedar pollen allergen (Cry j 1) and investigated effects of altered T cell receptor (TCR) ligand on changes of T cell responses. One of these Cry j 1-specific T cell clones established from patients with Japanese cedar pollinosis, ST1.9, recognized an antigenic peptide Cry j 1 p335-346 in the context of HLA-DRA+DRB3*0301 molecules and secreted interleukin-4 dominantly, with a smaller amount of interferon-gamma. ST1.9 represented one of the major T cell clones specific to Cry j 1 in the donor, because a short-term cultured polyclonal T cell line specific to Cry j 1 exhibited the same character as the ST1.9. We synthesized various analog peptides derived from Cry j 1 p335-346 with single amino acid substitutions and determined key residues for interactions between TCR of ST1.9 and HLA-DR molecules. We also analyzed changes in the responses of ST1.9 to Cry j 1 p335-346-derived analog peptides. Of interest was that the substitution of 339threonine to valine resulted in a significant increase in interferon-gamma production, with no remarkable changes either in proliferative response or interleukin-4 production. Analog peptides carrying the substitutions of 339threonine to glycine or glutamine revealed TCR antagonism, without changes in their binding affinities to the DR molecule. Therefore single amino acid substitutions on an allergen peptide carrying the T cell epitope may suppress helper-T-dependent class switch pressure to IgE in B cells either by inducing increased interferon-gamma production or by inhibiting proliferative responses in helper-T cells.

Role of dynamic MRI in the evaluation of head and neck cancers treated with radiation therapy

PURPOSE To study the usefulness of dynamic magnetic resonance imaging in the evaluation of head and neck cancers treated with radiation therapy. METHODS AND MATERIALS Seventy-six patients (58 males and 18 females; ages 20-82) with head and neck cancers (10 nasopharyngeal carcinomas, 22 mesopharyngeal carcinomas, 10 hypopharyngeal carcinomas, 16 oral cavity carcinomas, 11 lingual carcinomas, and 7 laryngeal carcinomas) were treated by radiation therapy combined with concomitant low-dose cisplatinum. Magnetic resonance imaging (MRI) was performed before and 2 weeks after the irradiation in all cases. After bolus administration of gadopentetate dimeglumine (Gd-DTPA) (0.1 mmol kg), images were obtained every 30 s (repetition time 200 ms, echo time 16 ms) using a 1.5 or 0.5-T superconductive unit. Biopsy or surgery was performed after radiation therapy and the histologic findings were correlated with the MRI findings (T1, T2, dynamic, and enhanced T1). RESULTS Complete remission, partial response, and no response were obtained in 18, 36, and 7 cases, respectively. Dynamic MRI correctly diagnosed 17 of the 18 complete remission cases, 33 of the 36 partial response cases, and all of the 7 no-response cases. The accuracy of dynamic MRI, T1-weighted image, T2-weighted image, and Gd-enhanced T1-weighted image was 94.4%, 68%, 82%, and 86%, respectively. CONCLUSION Dynamic MRI proved to be useful in the evaluation of the radiation therapy of head and neck cancers.

Histamine application to the nasal mucosa induces release of calcitonin gene-related peptide and substance P from peripheral terminals of trigeminal ganglion: A morphological study in the guinea pig

Short-term effects of application of histamine to the nasal mucosa on trigeminal ganglion neurons containing calcitonin gene-related peptide (CGRP) and substance P (SP) were examined in guinea pig. Immunoreactivities to CGRP and SP in these neurons were decreased 30 min after the histamine application. The decreases were most marked at 1-3 h after application, after which the immunoreactivities began to increase, reaching the base line by 6 h after the application. The immunoreactivities to CGRP and SP in the nerve endings of nasal mucosa were not decreased. The expression of mRNAs for both peptides in the soma of trigeminal neurons was unchanged. The histamine application to the nasal mucosa may cause release of CGRP and SP from terminals of peripheral processes of trigeminal ganglion neurons, and enhance axonal transport of these peptides, but does not affect their biosynthesis in the soma of trigeminal ganglion neurons.

Immunoregulatory Effect of Substance P in Human Eosinophil Migratory Function

Substance P (SP) is a tachykinin involved in the regulation of inflammatory processes. To investigate a modulatory role of the neuropeptide SP in allergic inflammation, we studied its priming effect on human eosinophil chemotaxis and kinetic responses towards platelet activating factor (PAF) and recombinant human interleukin 5 (rhIL-5). Blood was obtained from normal subjects and eosinophils were separated by Percoll discontinuous density gradient centrifugation. High purification was obtained by negative selection procedure (CD16-beads) and the experiments were performed in a 48-well microchemotaxis Boyden chamber. In the present study we demonstrate a potent synergistic effect of 1OOnM dose of SP on the migratory function of human eosinophils stimulated by PAF and rhIL- 5. This synergism was chemotaxis specific and was abolished by NK-1 receptor antagonist (FK888). The results suggest that neurogenic stimuli may play a significant role in eosinophil infiltration via its priming effect on the cell.

Omalizumab is more effective than suplatast tosilate in the treatment of Japanese cedar pollen-induced seasonal allergic rhinitis

Background Seasonal allergic rhinitis (SAR) induced by Japanese cedar pollens is a major problem in Japan. Omalizumab, a humanized monoclonal anti‐IgE antibody, improves symptoms associated with SAR, but a comparative study with an anti‐allergy drug has not yet been conducted.

Histamine acts directly on calcitonin gene-related peptide- and substance P-containing trigeminal ganglion neurons as assessed by calcium influx and immunocytochemistry

Primary cultures of rat trigeminal ganglion cells were exposed to histamine, and the intracellular free-calcium concentration, [Ca2+]i, was measured by the calcium-sensitive dye fura-2. Histamine increased the [Ca2+]i of the neurons. Pretreatment of the cells with histamine H1-receptor blocker, or removal of extracellular calcium, abolished the response, however, the response was not altered by pretreatment with H2-blocker. Immunocytochemical analysis showed that these cultured cells that responded to histamine identically showed substance P- or calcitonin gene-related peptide-like immunoreactivity.

Thermal and Photochemical Control of Molecular Orientation of Azo‐Functionalized Polymer Liquid Crystals and Application for Photo‐Rewritable Paper

A photo-responsive multi-bilayered film consisting of azobenzene polymer liquid crystals (PA6Az1) and poly(vinyl alcohol) (PVA) has been prepared on a glass substrate by alternate spin coating of the polymer solutions. The reflectivity of the multi-bilayered film disappears by annealing at 80 °C. The disappearance of the reflection by the annealing is related to the thermal out-of-plane molecular orientation of PA6Az1 even in the multi-bilayered film, which leads to a very small difference in refractive indices between PA6Az1 and PVA. The reflectance of the multi-bilayered film is increased again by UV irradiation because of the transformation from the out-of-plane orientation to an in-plane random orientation. In this way, on-off switching of the reflection is achieved by combination of the thermally spontaneous out-of-plane molecular orientation and following photoisomerization of PA6Az1 comprising the multi-bilayered film.

Adoptive Immunotherapy for Head and Neck Cancer with Killer Cells Induced by Stimulation with Autologous or Allogeneic Tumour Cells and Recombinant Interleukin-2

Peripheral blood lymphocytes drawn by leukapheresis using Haemonetics V50 were mixed and cultured with autologous or allogeneic tumour cell line to activate killer cells by tumour antigenic stimulation, and further with recombinant interleukin-2 (rIL-2). Killer cells were intra-arterially infused, as a primary therapy, in 5 patients with maxillary and one with lingual cancer (squamous cell carcinoma). Effects on reduction of primary tumour size were significantly high without any severe side effects. The effects were interpreted mainly by direct day-by-day observation of the site, findings of CT and histology. Histological findings of the tissue obtained by surgical operation performed after adoptive immunotherapy were remarkable changes, such as infiltration of lymphoid cells around the cancer nets, degeneration of cancer cells, infiltration of scavenger macrophages (giant cells) and so on. The results suggested that adoptive immunotherapy by the killer cells can be a powerful treatment to bring the cancer under control, in with combination of other therapies.

Human eotaxin induces eosinophil-derived neurotoxin release from normal human eosinophils

Background: Eosinophil granule proteins deposition at the site of allergic inflammation contributes to the late-phase reaction of hypersensitivity diseases. In the present communication, we describe the effect of human eotaxin on normal human eosinophil exocytosis measured as degranulation of eosinophil-derived neurotoxin (EDN). Methods: Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Purified eosinophils were stimulated with various concentrations of eotaxin and the amount of EDN released was analysed by radioimmunoassay. Flow cytometry was used to examine the surface expression of adhesion molecules on eosinophils. Results: Eotaxin significantly induced EDN release in a dose-dependent manner. The potency of eotaxin in this effect was equal to that of RANTES, and comparable to that of platelet-activating factor. Eotaxin-induced EDN release was blocked by cytochalasin B in a dose-dependent manner. The surface expression of CD11a, CD11b, CD18 and VLA-4 adhesion molecules on normal human eosinophils were not modulated by eotaxin stimulation. Conclusions: These results indicate that eotaxin may play an important role not only as a selective chemotaxin for the cell type but also as a secretagogue. Furthermore, they demonstrate a degranulation mechanism(s) involving cytoskeletal changes which is probably independent of the quantitative expression of adhesion molecules.