Yuki Hayashida

The forkhead box M1 transcription factor as a candidate of target for anti-cancer immunotherapy

The present study attempted to identify a target antigen for immunotherapy for cholangiocarcinoma. Forkhead box M1 (FOXM1) was selected as a candidate antigen based on the data of previous cDNA microarray analysis of clinical samples of cholangiocarcinoma. The level of FOXM1 mRNA was more than 4 times higher in cancer cells in comparison to adjacent normal epithelial cells, in all of 24 samples of cholangiocarcinoma tissues. An immunohistochemical analysis also detected FOXM1 protein in the cancer cells but not in the normal cells. Twenty‐three human FOXM1‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their ability to induce HLA‐A2‐restricted T cells in HLA‐A2 transgenic mice. FOXM1362‐370 (YLVPIQFPV), FOXM1373‐382 (SLVLQPSVKV), and FOXM1640‐649 (GLMDLSTTPL) peptides primed HLA‐A2‐restricted cytotoxic T lymphocytes (CTLs) in the HLA‐A2 transgenic mice. Human CTL lines reactive to these 3 peptides could also be established from HLA‐A2‐positive healthy donors and cancer patients. Natural processing of the 3 epitopes from FOXM1 protein was confirmed by specific killing of HLA‐A2‐positive FOXM1‐transfectants by peptide‐induced CTLs. FOXM1 is expressed in various types of cancers and it is also functionally involved in oncogenic transformation and the survival of cancer cells. Therefore, FOXM1 may be a suitable target for immunotherapy against various cancers including cholangiocarcinoma.

A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T-lymphocytes reactive to tumor cells

The present study attempted to identify a useful tumor‐associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45‐like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome‐wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non‐cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti‐tumor effects of cytotoxic T‐lymphocytes (CTL) specific to CDC45L‐derived peptides induced from HLA‐A24 (A*24:02)‐positive donors. We identified three CDC45L‐derived peptides that could reproducibly induce CDC45L‐specific and HLA‐A24‐restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA‐A24. In addition, we found that CDC45L 556KFLDALISL564 was eminent in that it induced not only HLA‐A24 but also HLA‐A2 (A*02:01)‐restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L‐specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L‐derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy. (Cancer Sci 2011; 102: 697–705)

Temporal properties of retinal ganglion cell responses to local transretinal current stimuli in the frog retina.

Extracellular current stimuli have been used in both electrophysiological and clinical studies. The present study elucidates the temporal properties of the frog retinal ganglion cell response induced by local transretinal current stimuli. Two classes of spike response were recorded from the ganglion cell. One had a constant latency ranging from 1.5 to 4.5 ms after the onset of the stimulus regardless of differences in stimulus parameters. Another class had a latency that varied from trial to trial between 3.5 and 71.5 ms at the threshold even when stimulus parameters were identical. The latency became shorter and the number of spike responses increased as the charge applied via the stimulus pulse was increased by increasing the amplitude (from 50 to 200 microA) or the pulse duration (from 100 to 1000 micros). In both classes, the current stimuli with the same amount of charge induced responses of a similar latency for amplitudes between 50 and 200 microA and for pulse durations between 100 and 1000 micros.

Peptides derived from human insulin-like growth factor-II mRNA binding protein 3 can induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer cells

Insulin‐like growth factor‐II mRNA binding protein 3 (IMP‐3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP‐3 that can induce tumor‐reactive and human leukocyte antigen (HLA)‐A2 (A*02:01)‐restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP‐3‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their capacity to induce HLA‐A2‐restricted T cells in HLA‐A2.1 (HHD) transgenic mice (Tgm). We found that three IMP‐3 peptides primed HLA‐A2‐restricted CTL in the HLA‐A2.1 Tgm. Among them, human CTL lines reactive to IMP‐3 515NLSSAEVVV523 were reproducibly established from HLA‐A2‐positive healthy donors and lung cancer patients. On the other hand, IMP‐3 199RLLVPTQFV207 reproducibly induced IMP‐3‐specific and HLA‐A2‐restricted CTL from healthy donors, but did not sensitize CTL in the HLA‐A2.1 Tgm. Importantly, these two IMP‐3 peptide‐specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP‐3 and HLA‐A2. Cytotoxicity was significantly inhibited by anti‐HLA class I and anti‐HLA‐A2 monoclonal antibodies, but not by the anti‐HLA‐class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP‐3 protein was confirmed by specific killing of HLA‐A2‐positive IMP‐3‐transfectants but not the parental IMP‐negative cell line by peptide‐induced CTL. This suggests that these two IMP‐3‐derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy. (Cancer Sci 2011; 102: 71–80)

Identification of SPARC as a candidate target antigen for immunotherapy of various cancers

To establish efficient anticancer immunotherary, it is important to identify tumor‐associated antigens (TAAs) directing the immune system to attack cancer. A genome‐wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA‐A24 (A*2402)‐restricted and SPARC‐derived CTL epitopes. We previously identified H‐2Kd‐restricted and SPARC‐derived CTL epitope peptides in BALB/c mice, of which H‐2Kd‐binding peptide motif is comparable with that of HLA‐A24 binding peptides. By using these peptides, we tried to induce HLA‐A24 (A*2402)‐restricted and SPARC‐reactive human CTLs and demonstrated an antitumor immune response. The SPARC‐A24‐1143–151 (DYIGPCKYI) and SPARC‐A24‐4225–234 (MYIFPVHWQF) peptides‐reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA‐A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA‐A24 (A*2402). Furthermore, the adoptive transfer of the SPARC‐specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA‐A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.

Inhibition of Adult Rat Retinal Ganglion Cells by D1-Type Dopamine Receptor Activation

The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D1a receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D1-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D1-type receptors, SCH-23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking I h. Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na+ current (I Na) amplitude, and tetrodotoxin, at doses that reduced I Na as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D1-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the presynaptic and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing.

Parallel inhibition of cortico-muscular synchronization and cortico-spinal excitability by theta burst TMS in humans

OBJECTIVE To investigate the after-effects of theta burst TMS (TBS) on cortico-muscular synchronization, and on cortico-spinal excitability, in humans. METHODS We studied 10 healthy subjects using a continuous paradigm of TBS (cTBS), i.e. 600 pulses in 40s. Before and after the cTBS, coherence function was computed as a measure of cortico-muscular synchronization by recording electroencephalogram (EEG) from 19 scalp sites and electromyogram (EMG) from right first dorsal interosseous (FDI) muscle during the isometric contraction. In a separate experiment, motor-evoked potentials (MEPs) in response to single TMS pulses were recorded from the FDI muscle before and after the cTBS, to measure cortico-spinal excitability. RESULTS When the cTBS was applied over the left primary motor cortex (M1), the beta-band cortico-muscular coherence for the C3 scalp site, as well as the MEP amplitude significantly decreased in 30-60 min, and then recovered to the original levels in 90-120 min. Neither sham stimulation nor cTBS applied over 2 cm posterior to M1 produced significant effects. CONCLUSIONS cTBS-over-M1 can inhibit the cortico-muscular synchronization in parallel with the decline of cortico-spinal excitability. SIGNIFICANCE Our results provide the first evidence that TBS can efficiently alter the functional cortico-muscular coupling in humans.

Ca2+ regulation by the Na+-Ca2+ exchanger in retinal horizontal cells depolarized by l-glutamate

This study is concerned with regulation of the intracellular Ca2+ concentration ([Ca2+]i) of horizontal cells isolated from cyprinid fish retinae, with the main emphasis on the role of the (Na+)-Ca2+ exchanger. An inward current was blocked by Ca2+ (4 mM) during prolonged (> 1 h) depolarization by L-glutamate (100 microM) in the whole-cell voltage-clamp configuration, suggesting the persistent activation of voltage-gated Ca2+ channels. This (Co2+)-sensitive current was absent when extracellular Na+ was replaced by Li+ to suppress (Na+)-Ca2+ exchange. Measurement of [Ca2+]i using the Fura-2 ratiometric method gave the following results. (1) L-Glutamate (100 microM) caused [Ca2+]i to increase from the resting level of 75.4+/-36.8 nM (mean +/-S.D., n = 11) to the maximum level (2.2+/-1.4 microM, n = 11) within 15 s and then to decrease to a steady level of 0.59+/-0.23 microM (n = 11). (2) Nifedipine (100 microM) lowered the L-glutamate-induced steady [Ca2+]i level, which was still higher than the resting level. (3) L-Glutamate caused [Ca2+]i to increase even after blockading the voltage-gated Ca2+ channels by nifedipine or by clamping the membrane voltage at -55 mV. (4) (Na+)-free superfusate elevated the L-glutamate-induced steady [Ca2+]i level. (5) The time course of the [Ca2+]i decrease from the L-glutamate-induced steady level to the resting level was prolonged in the (Na+)-free superfusate. These results suggest that the (Na+)-Ca2+ exchanger extrudes intracellular Ca2+ to maintain a low [Ca2+]i level by counteracting the continuous Ca2+ influx through the voltage-gated Ca2+ channels and glutamate-gated channels when horizontal cells in situ are tonically depolarized by L-glutamate released from the photoreceptors. The (Na+)-Ca2+ exchange current isolated by a voltage-clamp experiment depends exponentially on the membrane potential.

Effects of concurrent visual tasks on cortico-muscular synchronization in humans

To study the effects of external visual stimulation on motor cortex-muscle synchronization, coherence between electroencephalography (EEG) and electromyography (EMG) was measured in normal subjects under Before, Task (visual task: Ignore or Count, or arithmetic task) and After conditions. The control (Before and After) conditions required the subject to maintain first dorsal interosseous muscle contraction without visual stimulation. In the visual task, a random series of visual stimuli were displayed on a screen while the subjects maintained the muscle contraction. The subjects were asked to ignore the stimuli in the Ignore condition and to count certain stimuli in the Count condition. Also, in the arithmetic task, the subjects were asked to perform a simple subtraction. The EEG-EMG coherence found at C(3) site at 13-30 Hz (beta) was increased and sustained in magnitude during the Ignore and Count conditions, respectively. To examine the cause of the change of coherence, changes of EEG and EMG spectral power were computed for each frequency band. There was little change in the EMG spectral power in any frequency bands. While the spectral power of EEG unchanged in the beta band, it significantly increased and decreased in the range of 8-12 Hz and of 31-50 Hz, respectively, for both Ignore and Count conditions, not only at the C(3) site but at various sites as well. These results were in contrast to those obtained for the arithmetic task: the beta band EEG-EMG coherence was attenuated and the EEG spectral power at 4-7 Hz and at 31-50 Hz were significantly increased and decreased, respectively. As a conclusion, the present results are consistent with the idea that the enhanced 8-12 Hz/decreased 31-50 Hz oscillations affect strength of the beta band cortico-muscular synchronization by suppressing the visual processing.

Dissociation of Retinal Ganglion Cells Without Enzymes

We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.