Masae Yaguchi

Enhancement of immunologic tumor regression by intratumoral administration of dendritic cells in combination with cryoablative tumor pretreatment and bacillus calmette-guerin cell wall skeleton stimulation

Purpose: We developed an effective immunotherapy, which could induce antitumor immune responses against shared and unique tumor antigens expressed in autologous tumors. Experimental Design: Intratumoral administration of dendritic cells is one of the individualized immunotherapies; however, the antitumor activity is relatively weak. In this study, we attempted to enhance the antitumor efficacy of the i.t. dendritic cell administration by combining dendritic cells stimulated with Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) additionally with cryoablative pretreatment of tumors and analyzed the therapeutic mechanisms. Results: These two modifications (cryoablation of tumors and BCG-CWS stimulation of dendritic cells) significantly increases the antitumor effect on both the treated tumor and the untreated tumor, which was distant at the opposite side, in a bilateral s.c. murine CT26 colon cancer model. Further analysis of the augmented antitumor effects revealed that the cryoablative pretreatment enhances the uptake of tumor antigens by the introduced dendritic cells, resulting in the induction of tumor-specific CD8+ T cells responsible for the in vivo tumor regression of both treated and remote untreated tumors. This novel combination i.t. dendritic cell immunotherapy was effective against well-established large tumors. The antitumor efficacy was further enhanced by depletion of CD4+CD25+FoxP3+ regulatory T cells. Conclusions: This novel dendritic cell immunotherapy with i.t. administration of BCG-CWS–treated dendritic cells following tumor cryoablation could be used for the therapy of cancer patients with multiple metastases.

Functional recovery after spinal cord injury in mice through activation of microglia and dendritic cells after IL-12 administration.

We have previously reported that the transplantation of dendritic cells (DCs) brings about functional recovery after spinal cord injury in mice through the activation of endogenous microglia/macrophages and neural stem/progenitor cells. In this study, the effect of interleukin‐12 (IL‐12), which is secreted from DCs, was evaluated for the treatment of spinal cord injury in mice. Administration of IL‐12 into the injured site significantly increased the number of activated microglia/macrophages and DCs as well as the expression of brain‐derived neurotrophic factor surrounding the lesion site. Immunohistochemical analyses showed that de novo neurogenesis and remyelination were induced by IL‐12 treatment. Furthermore, an open field test using Basso‐Beattie‐Brenham scoring revealed a significant improvement of locomotor function in mice treated with IL‐12. These results suggest that IL‐12 administration into the injured spinal cord results in a functional recovery through the activation of microglia/macrophages and DCs.

Characterization of the Properties of Seven Promoters in the Motor Cortex of Rats and Monkeys After Lentiviral Vector-Mediated Gene Transfer

Lentiviral vectors deliver transgenes efficiently to a wide range of neuronal cell types in the mammalian central nervous system. To drive gene expression, internal promoters are essential; however, the in vivo properties of promoters, such as their cell type specificity and gene expression activity, are not well known, especially in the nonhuman primate brain. Here, the properties of five ubiquitous promoters (murine stem cell virus [MSCV], cytomegalovirus [CMV], CMV early enhancer/chicken β-actin [CAG], human elongation factor-1α [EF-1α], and Rous sarcoma virus [RSV]) and two cell type-specific promoters (rat synapsin I and mouse α-calcium/calmodulin-dependent protein kinase II [CaMKIIα]) in rat and monkey motor cortices in vivo were characterized. Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors expressing enhanced green fluorescent protein (EGFP) under the control of the various promoters were prepared and injected into rat and monkey motor cortices. Immunohistochemical analysis revealed that all of the VSV-G-pseudotyped lentiviral vectors had strong endogenous neuronal tropisms in rat and monkey brains. Among the seven promoters, the CMV promoter showed modest expression in glial cells (9.4%) of the rat brain, whereas the five ubiquitous promoters (MSCV, CMV, CAG, EF-1α, and RSV) showed expression in glial cells (7.0-14.7%) in the monkey brain. Cell type-specific synapsin I and CaMKIIα promoters showed excitatory neuron-specific expression in the monkey brain (synapsin I, 99.7%; CaMKIIα, 100.0%), but their specificities for excitatory neurons were significantly lower in the rat brain (synapsin I, 94.6%; CaMKIIα, 93.7%). These findings could be useful in basic and clinical neuroscience research for the design of vectors that efficiently deliver and express transgenes into rat and monkey brains.

Transplantation of dendritic cells promotes functional recovery from spinal cord injury in common marmoset

We previously reported that implantation of dendritic cells (DCs) into the injured site activates neural stem/progenitor cells (NSPCs) and promotes functional recovery after spinal cord injury (SCI) in mice. Working toward clinical application of DC therapy for SCI, we analyzed whether DCs promote functional recovery after SCI in a non-human primate, the common marmoset (CM). CMs are usually born as dizygotic twins. They are thus natural bone marrow and peripheral blood chimeras due to sharing of the placental circulation between dizygotic twins, leading to functional immune tolerance. In this study, to identify adequate CM donor-and-host pairs, mixed leukocyte reaction (MLR) assays were performed. Then, CM-DCs were generated from the bone marrow of the twin selected to be donor and transplanted into the injured site of the spinal cord of the other twin selected to be host, 7 days after injury. Histological analyses revealed fewer areas of demyelination around the injured site in DC-treated CMs than in controls. Immunohistochemical analysis showed that more motor neurons and corticospinal tracts were preserved after SCI in DC-treated CMs. Motor functions were evaluated using three different behavior tests and earlier functional recovery was observed in DC-treated CMs. These results suggest DC therapy to possibly be beneficial in primates with SCI and that this treatment has potential for clinical application.

Isolation and characterization of dendritic cells from common marmosets for preclinical cell therapy studies

Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC‐associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)‐DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen‐presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM‐derived CD11c+ cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)‐12, and to stimulate Xenogenic T cells. Moreover, BM‐derived DCs produced the neurotrophic factor NT‐3, which is also found in murine splenic DCs. These results suggest that BM‐derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.

A simple behavioral test for locomotor function after brain injury in mice

To establish a simple and reliable test for assessing locomotor function in mice with brain injury, we developed a new method, the rotarod slip test, in which the number of slips of the paralytic hind limb from a rotarod is counted. Brain injuries of different severity were created in adult C57BL/6 mice, by inflicting 1-point, 2-point and 4-point cryo-injuries. These mice were subjected to the rotarod slip test, the accelerating rotarod test and the elevated body swing test (EBST). Histological analyses were performed to assess the severity of the brain damage. Significant and consistent correlations between test scores and severity were observed for the rotarod slip test and the EBST. Only the rotarod slip test detected the mild hindlimb paresis in the acute and sub-acute phase after injury. Our results suggest that the rotarod slip test is the most sensitive and reliable method for assessing locomotor function after brain damage in mice.

Cofilin1 Controls Transcolumnar Plasticity in Dendritic Spines in Adult Barrel Cortex

During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.

Avian sarcoma leukosis virus receptor-envelope system for simultaneous dissection of multiple neural circuits in mammalian brain

Significance Genetic dissection of multiple neural pathways remains challenging because of the limited number of genetic methods that can be used simultaneously. To overcome this limitation, we used modified avian sarcoma and leukosis virus envelopes and receptors to develop highly orthogonal genetic tools that can achieve expression of different genes in different target cells. From in vitro and in vivo screens, we identified tools that can specifically transfer genes of interest into mammalian neurons via engineered receptors, with minimal unintended interactions. Using this approach, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different cortical regions. Thus, our approach provides independent, simultaneous, and specific genetic tools for manipulating intermingled neural pathways in vivo. Pathway-specific gene delivery is requisite for understanding complex neuronal systems in which neurons that project to different target regions are locally intermingled. However, conventional genetic tools cannot achieve simultaneous, independent gene delivery into multiple target cells with high efficiency and low cross-reactivity. In this study, we systematically screened all receptor–envelope pairs resulting from the combination of four avian sarcoma leukosis virus (ASLV) envelopes (EnvA, EnvB, EnvC, and EnvE) and five engineered avian-derived receptors (TVA950, TVBS3, TVC, TVBT, and DR-46TVB) in vitro. Four of the 20 pairs exhibited both high infection rates (TVA–EnvA, 99.6%; TVBS3–EnvB, 97.7%; TVC–EnvC, 98.2%; and DR-46TVB–EnvE, 98.8%) and low cross-reactivity (<2.5%). Next, we tested these four receptor–envelope pairs in vivo in a pathway-specific gene-transfer method. Neurons projecting into a limited somatosensory area were labeled with each receptor by retrograde gene transfer. Three of the four pairs exhibited selective transduction into thalamocortical neurons expressing the paired receptor (>98%), with no observed cross-reaction. Finally, by expressing three receptor types in a single animal, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different somatosensory areas. Thus, we identified three orthogonal pairs from the list of ASLV subgroups and established a new vector system that provides a simultaneous, independent, and highly specific genetic tool for transferring genes into multiple target cells in vivo. Our approach is broadly applicable to pathway-specific labeling and functional analysis of diverse neuronal systems.

A new glass-coated tungsten optrode enclosing multiple optic fibers for fluorescence measurement, optogenetic photo-stimulation, and single-unit recording in deep brain regions

increasing the local electrical field of the observed region by increasing their resistance higher than around. The Ca imaging result indicated that the neurons not only on the electrode but ones around it responds. We found that the stimulating voltage where first Ca spikes occur has threshold. Also, we analyzed the Ca response by the raster plot of spikes, and confirmed specific Ca propagation between neurons. The trigger of the response is the Ca spike of on-electrode neurons. In conclusion, this device is the best tool to observe the change of the plasticity of neuronal synaptic connection by controlling stimulus timing of multiple electrode lines. Research fund: Strategic Research Foundation Grant-aided Project for Private Universities from Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT), 2008–2012 (S0801008).