Role of basal sweating in maintaining skin hydration in the finger: A long-standing paradox in dry skin resolved.

A long-standing paradox in dermatology is why skin dehydration in the fingers can be triggered by repeated water exposure despite the action of water to hydrate skin tissue. Potential clues might be provided by identifying a mechanism through which water is held in the skin of the fingers. We speculated that this mechanism would be impaired after repeated water exposure. Here, we investigated whether there might be glabrous skin-specific water-holding machinery and whether this machinery might be impaired in dry skin/hand eczema. We examined this by using an impression-mould technique, allowing for an accurate quantification of sweat gland/duct activity and optical coherence tomography. Unlike in hairy skin, sweat pores were rarely detected at the folds of the finger at baseline. Surprisingly, after water exposure, sweat pores at the folds opened and those at the ridges closed in healthy controls (HCs). Sweating in the dermal folds of the hands correlated with skin hydration, and decreased in dry skin/hand eczema, suggesting that its impairment may be one of the causes of dry skin. After repeated water exposure, basal sweating response at the folds was exhausted in patients with dry skin/hand eczema as well as HCs. This exhaustion was rescued by exposing individuals to high humidity. Basal sweating defects would be a target for dry skin/hand eczema. Maintaining basal sweating responses in the finger is the best preventive measures in achieving prevention of dry skin/hand eczema.

A single retinal circuit model for multiple computations.

Vision is dependent on extracting intricate features of the visual information from the outside world, and complex visual computations begin to take place as soon as at the retinal level. In multiple studies on salamander retinas, the responses of a subtype of retinal ganglion cells, i.e., fast/biphasic-OFF ganglion cells, have been shown to be able to realize multiple functions, such as the segregation of a moving object from its background, motion anticipation, and rapid encoding of the spatial features of a new visual scene. For each of these visual functions, modeling approaches using extended linear-nonlinear cascade models suggest specific preceding retinal circuitries merging onto fast/biphasic-OFF ganglion cells. However, whether multiple visual functions can be accommodated together in a certain retinal circuitry and how specific mechanisms for each visual function interact with each other have not been investigated. Here, we propose a physiologically consistent, detailed computational model of the retinal circuit based on the spatiotemporal dynamics and connections of each class of retinal neurons to implement object motion sensitivity, motion anticipation, and rapid coding in the same circuit. Simulations suggest that multiple computations can be accommodated together, thereby implying that the fast/biphasic-OFF ganglion cell has potential to output a train of spikes carrying multiple pieces of information on distinct features of the visual stimuli.

Multichannel stimulation module as a tool for animal studies on cortical neural prostheses.

Intracortical microstimulation to the visual cortex is thought to be a feasible technique for inducing localized phosphenes in patients with acquired blindness, and thereby for visual prosthesis. In order to design effective stimuli for the prosthesis, it is important to elucidate relationships between the spatio-temporal patterns of stimuli and the resulting neural responses and phosphenes through pre-clinical animal studies. However, the physiological basis of effective spatial patterns of the stimuli for the prosthesis has been little investigated in the literature, at least partly because that the previously developed multi-channel stimulation systems were designed specifically for the clinical use. In the present, a 64-channel stimulation module was developed as a scalable tool for animal experiments. The operations of the module were verified by not only dry-bench tests but also physiological animal experiments in vivo. The results demonstrated its usefulness for examining the stimulus-response relationships in a quantitative manner, and for inducing the multi-site neural excitations with a multi-electrode array. In addition, this stimulation module could be used to generate spatially patterned stimuli with up to 4,096 channels in a dynamic way, in which the stimulus patterns can be updated at a certain frame rate in accordance with the incoming visual scene. The present study demonstrated that our stimulation module is applicable to the physiological and other future studies in animals on the cortical prostheses.

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 (515) NLSSAEVVV(523) were reproducibly established from HLA-A2-positive healthy donors and lung cancer patients. On the other hand, IMP-3 (199) RLLVPTQFV(207) 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.

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 (556) KFLDALISL(564) 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.

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 D(1a) receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D(1)-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 D(1)-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 D(1)-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.

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. FOXM1(362-370) (YLVPIQFPV), FOXM1(373-382) (SLVLQPSVKV), and FOXM1(640-649) (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.

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-2K(d)-restricted and SPARC-derived CTL epitope peptides in BALB/c mice, of which H-2K(d)-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-A24-1(143-151) (DYIGPCKYI) and SPARC-A24-4(225-234) (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.

Identification of chemical compounds as an inhibitor of mitochondrial ATP synthesis, leading to an increased stress resistance and an extended lifespan in C. elegans.

It is well known that the disruption of the mitochondrial respiratory components prolongs lifespan in many species. The mitochondrial stress response can lead to an increased survival rate through the restoration of the cellular homeostasis. Therefore, developing pharmacological interventions that induce mitochondrial stress response may be desirable to delay the onset of age-related diseases and promote a healthy life. In this study, we present chemical compounds, revealed by systematic screening of chemical libraries, which inhibit mitochondrial ATP synthesis in mammalian cells. Our study demonstrates that these compounds alter the body length and promote the oxidative stress response which leads to an increased longevity in Caenorhabditis elegans. Thus, our study identifies chemical compounds that may have potential therapeutic applications through affecting the mitochondrial function.

Identification of the H2-Kd-restricted cytotoxic T lymphocyte epitopes of a tumor-associated antigen, SPARC, which can stimulate antitumor immunity without causing autoimmune disease in mice.

We previously reported that the secreted protein acidic and rich in cystein (SPARC) was overexpressed in melanoma in humans, and the serum SPARC level was useful as a novel tumor marker for melanoma. SPARC was also reported to be overexpressed in various human cancers. In this study, we asked whether SPARC-specific cytotoxic T lymphocytes (CTL) could induce antitumor immunity to SPARC-expressing tumor in mice or not as a preclinical study of SPARC-directed anticancer immunotherapy. Because of similarities in the structural motifs of major histocompatibility complex-binding peptides between H2-Kd and HLA-A24 (A*2402), the most common human leukocyte antigen class I allele in the Japanese population, we attempted to identify the H2-Kd-restricted SPARC epitope for CTL in BALB/c mice and we found that the mouse SPARC143-151 (DYIGPCKYI) and SPARC225-234 (MYIFPVHWQF) peptides could induce peptide-reactive CTL in BALB/c mice without causing autoimmune diseases. The immunization of mice with SPARC225-234 peptide-pulsed bone marrow-derived dendritic cells (BMDC) inhibited the growth of s.c. inoculated mouse mammary cancer cell line, N2C, expressing SPARC and these mice lived longer than the mice immunized with peptide-unpulsed BMDC. In conclusion, our study indicated that SPARC peptide-based cancer immunotherapy was effective and safe at least in a mouse tumor prevention model.

Prurigo nodularis as a sweat gland/duct-related disorder: resolution associated with restoration of sweating disturbance.

Little attention has been given to the involvement of sweat glands/ducts in the pathogenesis of prurigo nodularis (PN). According to recent studies, PN is likely to develop under conditions characterized by dry skin, such as atopic dermatitis (AD), suggesting a strong impact of skin dryness on PN development. No therapeutic modalities produced complete resolution of PN without exacerbations. We previously reported that increases in skin dryness by sweating disturbance could initiate the development of AD. We investigated whether sweating responses were impaired in refractory PN lesions; and, if so, we asked whether the PN lesions could resolve by restoring sweating disturbance. Using the impression mold technique, which allows an accurate quantification of individual sweat gland/duct activity, we examined basal sweating under quiescent conditions and inducible sweating responses to thermal stimulus in PN lesions and normal-appearing skin in the same patients before and after treatment with a moisturizer or topical corticosteroids. Sweating disturbance, either basal or inducible, was most profoundly detected in the "hub" structure corresponding to the center of PN papule before the treatment. This sweating disturbance was immunohistochemically associated with the leakage of sweat into the dermis. This disturbance was restored by treatment with a moisturizer. Our limitations include a relatively small patient cohort and lack of blinding. Sweating disturbance could be one of the aggravating factors of PN development. Refractory PN with low skin hydration may resolve by restoring sweating disturbance.

Focal activation of neuronal circuits induced by microstimulation in the visual cortex.

OBJECTIVE: Microstimulation to the cortical tissue applied with penetrating electrodes delivers current that spreads concentrically around the electrode tip and is known to evoke focal visual sensations, i.e. phosphenes. However, to date, there is no direct evidence depicting the spatiotemporal properties of neuronal activity induced immediately after microstimulation and how such activity drives the subsequent local cortical circuits. APPROACH: In the present study, we imaged the spatiotemporal distribution of action potentials (APs) directly induced by microstimulation and the subsequent trans-synaptic signal propagation using a voltage-sensitive dye (VSD) and a calcium-sensitive dye (CaSD) in slice preparations of the mouse primary visual cortex. MAIN RESULTS: The directly induced APs were confined to the close vicinity of the electrode tip, and the effective distance of excitation was proportional to the square root of the current intensity. The excitation around the electrode tip in layer IV mainly propagated to layer II/III to further induce the subsequent focal activation in downstream local cortical circuits. The extent of activation in the downstream circuits was restrained by competitive interactions between excitatory and inhibitory signals. Namely, the spread of the excitation to lateral neighbor neurons along the layer II/III was confined by the delayed inhibition that also spread laterally at a faster rate. SIGNIFICANCE: These observations indicate that dynamic interactions between excitatory and inhibitory signals play a critical role in the focal activation of a cortical circuit in response to intracortical microstimulation and, therefore, in evoking a localized phosphene.

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.

Imaging of population spikes induced by repetitive stimulus pulses in mouse cerebral slices in vitro.

Effects of the repetitive current pulses of microstimulation on spatio-temporal neuronal excitations in the primary visual cortex in mouse cerebral slices in vitro were examined by utilizing the voltage-sensitive dye imaging technique. The amplitude and spatial extent of the population spike directly induced by the stimulus pulse was significantly reduced in response to successive stimulus pulses at 200 Hz. This suggested that the high-frequency microstimulation may not be efficient for inducing the neuronal spiking, at least, in vitro.

Age-related changes in the spatiotemporal responses to electrical stimulation in the visual cortex of rats with progressive vision loss.

The Royal College of Surgeons (RCS) rat gradually loses vision due to retinal degeneration. Previous physiological studies have depicted the progressive loss of optical responses in the visual pathway, including the primary visual cortex (V1), over the course of retinal degeneration in the RCS rat. However, little is known about how the excitability of the V1 circuit changes during over the course of the gradual loss of visual signal input from the retina. We elucidated the properties of responses to electrical stimulations directly applied to V1 at different stages of vision input loss in the RCS rat in reference to those of the Long-Evans (LE) rat, using in vivo voltage-sensitive dye imaging. The V1 neuronal network of the RCS rat exhibited an excitatory response comparable to the LE rat. The excitatory response was maintained even long after total loss of the visual signal input from the retina. However, the response time-course suggested that the suppressive response was somewhat debilitated in the RCS rat. This is the first experiment demonstrating the long-term effect of retinal degeneration on cortical activities. Our findings provide the physiological fundamentals to enhance the preclinical research of cortical prostheses with the use of the RCS rat.

Retinal Circuit Emulator With Spatiotemporal Spike Outputs at Millisecond Resolution in Response to Visual Events.

To gain insights on how visual information of the real world is filtered, compressed, and encoded by the vertebrate retinas, emulating in silico the spatiotemporal patterns of the graded and action potentials of neuronal responses to natural visual scenes on biological time scale is a feasible approach. As a basic platform for such an emulation, we here developed a compact hardware system comprising an analog silicon retina and a field-programmable gate array module. With utilizing the Izhikevich formalism, a retinal circuit model that emulates spiking of ganglion cells was implemented in this system. The emulated spike timing had the resolution of about 2 ms relative to the stimulus onset and was little affected by timings of the synchronous frame sampling in the silicon retina. Thus, the emulator can mimic the event-driven spike outputs of biological retinas. The system was useful for simultaneously visualizing neural images of both the graded potentials and the spikes in response to real live visual scenes. Since our emulator system is reconfigurable, it provides a flexible platform for investigating visual functions of retinal circuits under natural visual environment.

Effects of visual stimulation on cortico-spinal coherence during isometric hand contraction in humans.

The effects of visual stimuli on cortico-spinal synchronization were investigated by measuring the coherence between electroencephalogram (EEG) and electromyogram (EMG) during isometric contraction of the first dorsal interosseous muscle of the right hand. Because a spinal motoneuron and the corresponding muscle fibers form a motor unit with one-to-one correspondence of their action potentials, the EMG indirectly measures the activity of the corresponding spinal neuronal group. The tasks were isometric contraction (Control condition); and isometric contraction with concurrent ignoring of visual stimuli (VS condition). By comparing the Control and VS conditions, the following results were obtained. The coherence increased significantly in magnitude, but was unchanged in frequency range (beta band) and scalp location; the EEG and EMG spectral power in the beta band were unchanged in amplitude; and the alpha and gamma bands of EEG spectral power were significantly increased and decreased, respectively. These findings suggest that the cortico-muscular coherence reflects the cognitive effort needed to maintain isometric muscle contraction. When visual stimuli need to be ignored, the cognitive effort and cortico-spinal coherence are enhanced.

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.

The interdependence and independence of amacrine cell dendrites: patch-clamp recordings and simulation studies on cultured GABAergic amacrine cells.

Previously we reported that cultured rat GABAergic amacrine cells can evoke subthreshold graded depolarization and action potentials. Both types of electrical signals are thought to contribute to neurotransmitter release from their dendrites, because Ca(2+) channels in amacrine cells can be activated at a subthreshold level (around -50 mV). The aim of the present study is to describe the spatiotemporal pattern of the spread of these electrical signals in an amacrine cell, using a computer simulation study. The simulation is based on physiological data, obtained by dual whole-cell patch-clamp recordings on the soma and the dendrites of cultured rat GABAergic amacrine cells. We determined passive and active properties of amacrine cells from the physiological recordings. Then, using the NEURON simulator, we conducted computer simulations on a reconstructed model of amacrine cells. We show that graded potentials and action potentials spread through amacrine cells with distinct patterns, and discuss the electrical interrelationship among the dendrites of an amacrine cell. Subthreshold graded potentials applied to a distal dendrite were sufficiently localized, so that each dendrite could behave independently (dendritic independence). However, at a suprathreshold level, once action potentials were triggered, they propagated into every dendrite, exciting the entire cell (dendritic interdependence). We also showed that GABAergic inhibitory inputs on the dendrites suppress the dendritic interdependence of amacrine cells. These results suggest that an inhibitory amacrine cell can mediate both local and wide-field lateral inhibition, regulated by the spatiotemporal pattern of excitatory and inhibitory synaptic inputs on its dendrites.