水上 浩明

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken beta-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

The striatum plays important motor, associative and cognitive roles in brain functions. However, the rodent dorsolateral ( the primate putamen) and dorsomedial ( the primate caudate nucleus) striatum are not anatomically separated, making it difficult to distinguish their functions. By contrast, anatomical separation exists between the caudate nucleus and putamen in primates. Here, we successfully decreased dopamine D1 receptor ( D1R) or D2R mRNA expression levels selectively in the marmoset caudate using shRNA knockdown techniques, as determined using positron emission tomography imaging with specific D1R and D2R ligands and postmortem in situ hybridization analysis. We then conducted a voxel-based correlation analysis between binding potential values of PET imaging and visual discrimination learning task performance in these genetically modified marmosets to find a critical role for the caudate D2R but no apparent role for the caudate D1R. This latter finding challenges the current understanding of the mechanisms underlying D1R activation in the caudate.

Neuronal plasticity is especially active in the young, during short windows of time termed critical periods, and loss of a critical period leads to functional limitations in the adults. The mechanism that governs the length of critical periods remains unknown. Here we show that levels of the NMDA receptor GluN2B subunit, which functions as a Ca2+ channel, declines in spinal cord synapses toward the end of the critical period for activity-dependent corticospinal synapse elimination. This period could be prolonged by blocking the decline of GluN2B, and after its termination the critical period could be reopened through upregulation of GluN2B. It is known that inhibitory neural activity increases with development in the CNS including the spinal cord. Suppression of the increasing inhibitory activity using low-dose strychnine also prolonged this critical period. During the strychnine-widened time window, Ca2+ influx through GluN2B channels returned to a level comparable to that seen during the critical period, though the level of GluN2B was slightly reduced. These findings indicate that loss of GluN2B subunits and the associated reduction in Ca2+ influx determines the end of the critical period in our in vitro CS system.

Vasopressin, a nonapeptide, signaling both as hormone in the blood and neuromodulator/neurotransmitter in the brain is considered to be causally involved in the pathological changes underlying anxiety and depression. In the present review we summarize experimental data obtained with Brattleboro rats as a model of congenital vasopressin-deficiency to test the hypothesis that central vasopressin signaling contributes to anxiety- and depression-like behavior. Male, female and lactating rats were studied. We focused on the paraventricular nucleus of the hypothalamus (PVN) and the septum, two brain areas in which vasopressin is proposed to control the endocrine and behavioral stress response, respectively. The presented data support the hypothesis that the behavioral changes seen in these rats are brought about by an altered vasopressin signaling at the brain level. Whereas vasopressin synthesized and released within the hypothalamus is primarily involved in endocrine regulation, vasopressin signaling in other brain areas may contribute to anxiety- and depression-like behavioral parameters. Further studies in this context might focus particularly on the interplay between extra-hypothalamic brain areas such as the septum and the medial amygdala.

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway.

The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR. (C) 2015 Wiley Periodicals, Inc.

Vasohibin-1 (VASH1) is a negative feedback regulator of angiogenesis, the first to be discovered, and was identified in vascular endothelial growth factor (VEGF)-stimulated vascular endothelial cells. Vasohibin-1 inhibits abnormal vascularization induced by various angiogenic factors including fibroblast growth factor and platelet-derived growth factor (PDGF), in addition to VEGF. By focusing on this characteristic of VASH1, we investigated the antitumor effects of VASH1 expression on ovarian cancer cells that produce different angiogenic factors. By using a high VEGF-producing ovarian cancer cell line, SHIN-3, and a high PDGF-producing ovarian cancer cell line, KOC-2S, the cells were transfected with either a VEGF antagonist, soluble VEGF receptor-1 (sVEGFR-1, or sFlt-1), or VASH1 genes to establish their respective cellular expression. The characteristics of these transfectants were compared with controls. We previously reported that the expression of sFlt-1 inhibited tumor vascularization and growth of high VEGF-producing ovarian cancer cells, reduced peritoneal dissemination and ascites development, and prolonged the survival time of the host. However, in the current study, the expression of sFlt-1 had no such effect on the high PDGF-producing ovarian cancer cells used here, whereas VASH1 expression inhibited tumor vascularization and growth, not only in high VEGF-producing cells, but also in high PDGF-producing cells, reduced their peritoneal dissemination and ascites, and prolonged the survival time of the host. These results suggest that VASH1 is an effective treatment for ovarian cancer cells that produce different angiogenic factors.

NLRP3 inflammasomes recognize non-microbial danger signals and induce release of proinflammatory cytokine interleukin (IL)-1 beta, leading to sterile inflammation in cardiovascular disease. Because sterile inflammation is involved in doxorubicin (Dox)-induced cardiotoxicity, we investigated the role of NLRP3 inflammasomes in Dox-induced cardiotoxicity. Cardiac dysfunction and injury were induced by low-dose Dox (15 mg/kg) administration in NLRP3-deficient (NLRP3(-/-)) mice but not in wild-type (WT) and IL-1 beta(-/-) mice, indicating that NLRP3 deficiency enhanced the susceptibility to Dox-induced cardiotoxicity independent of IL-1 beta. Although the hearts of WT and NLRP3(-/-) mice showed no significant difference in inflammatory cell infiltration, macrophages were the predominant inflammatory cells in the hearts, and cardiac IL-10 production was decreased in Dox-treated NLRP3(-/-) mice. Bone marrow transplantation experiments showed that bone marrow-derived cells contributed to the exacerbation of Dox-induced cardiotoxicity in NLRP3(-/-) mice. In vitro experiments revealed that NLRP3 deficiency decreased IL-10 production in macrophages. Furthermore, adeno-associated virus-mediated IL-10 overexpression restored the exacerbation of cardiotoxicity in the NLRP3(-/-) mice. These results demonstrated that NLRP3 regulates macrophage IL-10 production and contributes to the pathophysiology of Dox-induced cardiotoxicity, which is independent of IL-1 beta. Our findings identify a novel role of NLRP3 and provided new insights into the mechanisms underlying Dox-induced cardiotoxicity.

Adeno-associated virus (AAV) vector is widely used in gene transfer purposes. Not only the experimental gene transfer but also applications toward disease therapy are promising. Numerous attempts are ongoing for this purpose. One of the most prominent examples is the sustained clinical benefi t in hemophilia gene therapy targeting the liver. These successes have been brought chiefl y by the progress in vectorology, especially capsid development. At present, one of the biggest issues is the presence of neutralizing antibody (NAb) against AAV vector capsid. Challenges have been made to conquer this problem. Although there are still some hurdles for wide clinical application, use of this vector will soon be a common practice for the treatment of suitable disease conditions.

Depression is a common debilitating human disease whose etiology has defied decades of research. A critical bottleneck is the difficulty in modeling depressive episodes in animals. Here, we show that a transgenic mouse with chronic forebrain expression of a dominant negative mutant of Polg1, a mitochondrial DNA (mtDNA) polymerase, exhibits lethargic behavioral changes, which are associated with emotional, vegetative and psychomotor disturbances, and response to antidepression drug treatment. The results suggested a symptomatic similarity between the lethargic behavioral change that was recurrently and spontaneously experienced by the mutant mice and major depressive episode as defined by DSM-5. A comprehensive screen of mutant brain revealed a hotspot for mtDNA deletions and mitochondrial dysfunction in the paraventricular thalamic nucleus (PVT) with similar defects observed in postmortem brains of patients with mitochondrial disease with mood symptoms. Remarkably, the genetic inhibition of PVT synaptic output by Cre-loxP-dependent expression of tetanus toxin triggered de novo depression-like episodes. These findings identify a novel preclinical mouse model and brain area for major depressive episodes with mitochondrial dysfunction as its cellular mechanism.

Key points Direct connections between corticospinal (CS) axons and motoneurons (MNs) appear to be present only in higher primates, where they are essential for discrete movement of the digits. Their presence in adult rodents was once claimed but is now questioned. We report that MNs innervating forearm muscles in infant rats receive monosynaptic input from CS axons, but MNs innervating proximal muscles do not, which is a pattern similar to that in primates. Our experiments were carefully designed to show monosynaptic connections. This entailed selective electrical and optogenetic stimulation of CS axons and recording from MNs identified by retrograde labelling from innervated muscles. Morphological evidence was also obtained for rigorous identification of CS axons and MNs. These connections would be transient and would regress later during development. These results shed light on the development and evolution of direct CS-MN connections, which serve as the basis for dexterity in humans. AbstractRecent evidence suggests there is no direct connection between corticospinal (CS) axons and spinal motoneurons (MNs) in adult rodents. We previously showed that CS synapses are present throughout the spinal cord for a time, but are eliminated from the ventral horn during development in rodents. This raises the possibility that CS axons transiently make direct connections with MNs located in the ventral horn of the spinal cord. This was tested in the present study. Using cervical cord slices prepared from rats on postnatal days (P) 7-9, CS axons were stimulated and whole cell recordings were made from MNs retrogradely labelled with fluorescent cholera toxin B subunit (CTB) injected into selected groups of muscles. To selectively activate CS axons, electrical stimulation was carefully limited to the CS tract. In addition we employed optogenetic stimulation after injecting an adeno-associated virus vector encoding channelrhodopsin-2 (ChR2) into the sensorimotor cortex on P0. We were then able to record monosynaptic excitatory postsynaptic currents from MNs innervating forearm muscles, but not from those innervating proximal muscles. We also showed close contacts between CTB-labelled MNs and CS axons labelled through introduction of fluorescent protein-conjugated synaptophysin or the ChR2 expression system. We confirmed that some of these contacts colocalized with postsynaptic density protein 95 in their partner dendrites. It is intriguing from both phylogenetic and ontogenetic viewpoints that direct and putatively transient CS-MN connections were found only on MNs innervating the forearm muscles in infant rats, as this is analogous to the connection pattern seen in adult primates. Key points Direct connections between corticospinal (CS) axons and motoneurons (MNs) appear to be present only in higher primates, where they are essential for discrete movement of the digits. Their presence in adult rodents was once claimed but is now questioned. We report that MNs innervating forearm muscles in infant rats receive monosynaptic input from CS axons, but MNs innervating proximal muscles do not, which is a pattern similar to that in primates. Our experiments were carefully designed to show monosynaptic connections. This entailed selective electrical and optogenetic stimulation of CS axons and recording from MNs identified by retrograde labelling from innervated muscles. Morphological evidence was also obtained for rigorous identification of CS axons and MNs. These connections would be transient and would regress later during development. These results shed light on the development and evolution of direct CS-MN connections, which serve as the basis for dexterity in humans.

Vasohibin-1 (VASH1) is expressed in vascular endothelial cells stimulated by several angiogenic growth factors and displays autocrine activity to regulate angiogenesis via a negative feedback mechanism. In this study, we investigated the effect of VASH1 on ovarian cancer progression using VASH1-expressing ovarian cancer cells in vitro and in vivo. The growth ability of ovarian cancer cells engineered to express the VASH1 gene remained unchanged in vitro. However, we showed that VASH1 secretion by tumor cells inhibited the growth of human umbilical vein endothelial cells. Further, animal experiments showed that VASH1 expression inhibited tumor angiogenesis and growth. In a murine model of peritoneal dissemination of ovarian cancer cells, VASH1 inhibited peritoneal dissemination and ascites, resulting in significantly prolonged survival in mice. This indicates that VASH1 exerts an antitumor effect on ovarian cancer by inhibiting angiogenesis in the tumor environment. These findings suggest that a novel therapy based on VASH1 could be a useful therapeutic strategy for ovarian cancer.

Two-photon imaging with genetically encoded calcium indicators (GECIs) enables long-term observation of neuronal activity in vivo. However, there are very few studies of GECIs in primates. Here, we report a method for long-term imaging of a GECI, GCaMP6f, expressed from adeno-associated virus vectors in cortical neurons of the adult common marmoset (Callithrix jacchus), a small New World primate. We used a tetracycline-inducible expression system to robustly amplify neuronal GCaMP6f expression and up-and downregulate it for more than 100 days. We succeeded in monitoring spontaneous activity not only from hundreds of neurons three-dimensionally distributed in layers 2 and 3 but also from single dendrites and axons in layer 1. Furthermore, we detected selective activities from somata, dendrites, and axons in the somatosensory cortex responding to specific tactile stimuli. Our results provide a way to investigate the organization and plasticity of cortical microcircuits at subcellular resolution in non-human primates.

Recent papers have reported that oxytocin (Oxt) and the oxytocin receptor (Oxtr) may be involved in the regulation of food intake in mammals. We therefore suspected the Oxt/Oxtr system to be involved in energy homeostasis. In previous studies, we found a tendency toward obesity in Oxtr-deficient (Oxtr(-/-))mice, as well as impaired thermoregulation when these mice were exposed to cold conditions. In the present study, we observed the expression of Oxtr in the rostral medullary raphe (RMR), the brain region known to control thermogenesis in brown adipose tissue (BAT). Through immunohistochemistry, we detected neurons expressing Oxtr and c-Fos in the RMR of mice exposed to cold conditions. Up to 40% of Oxtr-positive neurons in RMR were classified as glutamatergic neurons, as shown by immunostaining using anti-VGLUT3 antibody. In addition, mice with exclusive expression of Oxtr in the RMR were generated by injecting an MV-Oxtr vector into the RMR region of Oxtr(-/-) mice. We confirmed the recovery of thermoregulatory ability in the manipulated mice during exposure to cold conditions. Moreover, mice with RMR-specific expression of Oxtr lost the typical morphological change in BAT observed in Oxtr(-/-) mice. Additionally, increased expression of the p-adrenergic receptor gene, Adrb3, was observed in BAT. These results are the first to show the critical role of RMR Oxtr expression in thermoregulation during cold conditions.

Despite the increasing commercial use of nanoparticles, little is known about their effects on placental inflammation and pregnancy complications. In this study, nanosilica (NS) particles upregulated the inflammasome component nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) and induced placental inflammation and reactive oxygen species (ROS) generation, resulting in pregnancy complications. Furthermore, NS-induced pregnancy complications were markedly improved in Nlrp3&lt sup&gt -/-&lt /sup&gt mice but not in component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-deficient (Asc&lt sup&gt -/-&lt /sup&gt ) mice, indicating the independence of NLRP3 inflammasomes. Pregnancy complications in Nlrp3&lt sup&gt -/-&lt /sup&gt and Asc&lt sup&gt -/-&lt /sup&gt mice phenotypes were dependent on the balance between interleukin (IL)-1 and IL-10. NS-induced pregnancy complications were completely prevented by either inhibition of ROS generation or forced expression of IL-10. Our findings provide important information about NS-induced placental inflammation and pregnancy complications and the novel pathophysiological roles of NLRP3 and ASC in pregnancy.

Here we investigated the transduction characteristics of adeno-associated viral vector (AAV) serotypes 1, 2, 5, 8 and 9 in the marmoset cerebral cortex. Using three constructs that each has hrGFP under ubiquitous (CMV), or neuron-specific (CaMKII and Synapsin I (SynI)) promoters, we investigated (1) the extent of viral spread, (2) cell type tropism, and (3) neuronal transduction efficiency of each serotype. AAV2 was clearly distinct from other serotypes in small spreading and neuronal tropism. We did not observe significant differences in viral spread among other serotypes. Regarding the cell tropism, AAV1, 5, 8 and 9 exhibited mostly glial expression for CMV construct. However, when the CaMKII construct was tested, cortical neurons were efficiently transduced (>similar to 70% in layer 3) by all serotypes, suggesting that glial expression obscured neuronal expression for CMV construct. For both SynI and CaMKII constructs, we observed generally high-level expression in large pyramidal cells especially in layer 5, as well as in parvalbumin-positive interneurons. The expression from the CaMKII construct was more uniformly observed in excitatory cells compared with SynI construct. Injection of the same viral preparations in mouse and macaque cortex resulted in essentially the same result with some species-specific differences. (C) 2014 The Authors. Published by Elsevier Ireland Ltd.

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor ( CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3+ T-cells with stable and high-level transgene expression (similar to 95%) produced interferon. gamma upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)gamma c(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

We previously generated oxytocin (OXT)-deficient mice and oxytocin receptor (OXTR)-deficient mice. Impaired social behaviors were observed in these mice, so they may be useful as animal models for studying the regulatory mechanism of social behavior by the OXT/OXTR system in the brain. In the present review, we aimed to overview our previous works to unravel the mechanism(s) by which OXTR deficiency leads to the impairment of social behaviors; for example, abnormalities in maternal behavior and/or social memory observed in mice deficient in the OXTR will be presented. By analyzing the brain of the OXTR-modified yellow fluorescent protein knock-in mice histologically, OXTR-expressing neurons were observed conspicuously in brain regions that are related to social behaviors. We focus on the characteristics of the regions containing neurons with prominent Oxtr gene expression in the present manuscript and discuss on the mechanisms through which OXT exerts its effects on social behaviors.

Despite the increasing commercial use of nanoparticles, little is known about their effects on placental inflammation and pregnancy complications. In this study, nanosilica (NS) particles upregulated the inflammasome component nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) and induced placental inflammation and reactive oxygen species (ROS) generation, resulting in pregnancy complications. Furthermore, NS-induced pregnancy complications were markedly improved in N mice but not in component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-deficient (Asc) mice, indicating the independence of NLRP3 inflammasomes. Pregnancy complications in Nlrp3(-/-) and Asc(-/-) mice phenotypes were dependent on the balance between interleukin (IL)-1 a and IL-10. NS-induced pregnancy complications were completely prevented by either inhibition of ROS generation or forced expression of IL-10. Our findings provide important information about NS-induced placental inflammation and pregnancy complications and the novel pathophysiological roles of NLRP3 and ASC in pregnancy.

The corticospinal (CS) tract is essential for voluntary movement, but what we know about the organization and development of the CS tract remains limited. To determine the total cortical area innervating the seventh cervical spinal cord segment (C7), which controls forelimb movement, we injected a retrograde tracer (fluorescent microspheres) into C7 such that it would spread widely within the unilateral gray matter (to >80%), but not to the CS tract. Subsequent detection of the tracer showed that, in both juvenile and adult mice, neurons distributed over an unexpectedly broad portion of the rostral two-thirds of the cerebral cortex converge to C7. This even included cortical areas controlling the hindlimbs (the fourth lumbar segment, L4). With aging, cell densities greatly declined, mainly due to axon branch elimination. Whole-cell recordings from spinal cord cells upon selective optogenetic stimulation of CS axons, and labeling of axons (DsRed) and presynaptic structures (synaptophysin) through cotransfection using exo utero electroporation, showed that over-growing CS axons make synaptic connections with spinal cells in juveniles. This suggests that neuronal circuits involved in the CS tract to C7 are largely reorganized during development. By contrast, the cortical areas innervating L4 are limited to the conventional hindlimb area, and the cell distribution and density do not change during development. These findings call for an update of the traditional notion of somatotopic CS projection and imply that there are substantial developmental differences in the cortical control of forelimb and hindlimb movements, at least in rodents.

Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spines in vivo in the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)–tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopy in vivo and analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualize in vivo dendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.

The lateral habenula (LHb) regulates the activity of monoaminergic neurons in the brainstem. This area has recently attracted a surge of interest in psychiatry because studies have reported the pathological activation of the habenula in patients with major depression and in animal models. The LHb plays a significant role in the pathophysiology of depression; however, how habenular neurons are activated to cause various depression symptoms, such as reduced motivation and sleep disturbance, remain unclear. We hypothesized that dysfunctional astrocytes may cause LHb hyperactivity due to the defective uptake activity of extracellular glutamate, which induces depressivelike behaviors. We examined the activity of neurons in habenular pathways and performed behavioral and sleep analyses in mice with pharmacological and genetic inhibition of the activity of the glial glutamate transporter GLT-1 in the LHb. The habenula-specific inhibition of GLT-1 increased the neuronal firing rate and the level of c-Fos expression in the LHb. Mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype in the tail suspension and novelty-suppressed feeding tests. These animals also displayed increased susceptibility to chronic stress, displaying more frequent avoidant behavior without affecting locomotor activity in the openfield test. Intriguingly, the mice showed disinhibition of rapid eye movement sleep, which is a characteristic sleep pattern in patients with depression. These results provide evidence that disrupting glutamate clearance in habenular astrocytes increases neuronal excitability and depressive-like phenotypes in behaviors and sleep.

Pre-existing antibodies against adeno-associated virus (AAV), caused by natural AAV infections, interfere with recombinant AAV vector-mediated gene transfer. We studied the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 in healthy subjects (n=85) and hemophilia patients (n=59) in a Japanese population. For healthy subjects, the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 was 36.5%, 35.3%, 37.6%, 32.9%, and 36.5%, respectively, while that in hemophilia patients was 39.7%, 28.8%, 35.6%, 32.9%, and 27.4%, respectively. There was no difference in the prevalence of neutralizing antibody against each AAV serotype between the healthy subjects and the hemophilia patients. The prevalence of neutralizing antibodies against all AAV serotypes increased with age in both healthy subjects and hemophilia patients. High titers of neutralizing antibodies against AAV2 (1:224) and AAV8 (1:224) were more evident in older individuals (42 years old). Approximately 50% of all screened individuals were seronegative for neutralizing antibodies against each AAV tested, while approximately 25% of individuals were seropositive for each AAV serotype tested. The prevalence of seronegativity for all AAV serotypes was 67.0% (healthy subjects, 68.6%; hemophilia patients, 65.0%) and 18.6% (healthy subjects, 20.5%; hemophilia patients, 15.7%) in young (<42 years old) and older subjects (42 years old), respectively. The findings from this study suggested that young subjects are more likely to be eligible for gene therapy based on AAV vectors delivered via an intravascular route because of the low prevalence of antibodies to AAV capsids. J. Med. Virol. 86:1990-1997, 2014. (c) 2013 Wiley Periodicals, Inc.

Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50 x 10(11) pt/ml; CI, 4.26 x 10(11) to 6.75 x 10(11) pt/ml), vector genomes (mean, 5.75 x 10(11) vg/ml; CI, 3.05 x 10(11) to 1.09 x 10(12) vg/ml), and infectious units (mean, 1.26 x 10(9) IU/ml; CI, 6.46 x 10(8) to 2.51 x 10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.

Here we present a novel tracing technique to stain projection neurons in Golgi-like detail by double viral infection. We used retrograde lentiviral vectors and adeno-associated viral vectors (AAV) to drive "TET-ON/TET-OFF system" in neurons connecting two regions. Using this method, we successfully labeled the corticothalamic (CT) cells of the mouse somatosensory barrel field (S1BF) and motor cortex (M1) in their entirety. We also labeled contra- and ipsilaterally-projecting corticocortical (CC) cells of M1 by targeting contralateral M1 or ipsilateral Si for retrograde infection. The strength of this method is that we can observe the morphology of specific projection neuron subtypes en masse. We found that the group of CT cells extends their dendrites and intrinsic axons extensively below but not within the thalamorecipient layer in both Si BE and M1, suggesting that the primary target of this cell type is not layer 4. We also found that both ipsi- and contralateral targeting CC cells in M1 commonly exhibit widespread collateral extensions to contralateral M1 (layers 1-6), bilateral Si and S2 (layers 1, 5 and 6), perirhinal cortex (layers 1, 2/3, 5, and 6), striatum and claustrum. These findings not only strengthened the previous findings of single cell tracings but also extended them by enabling cross-area comparison of CT cells or comparison of CC cells of two different labeling.

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

Oxytocin (OXT) and OXT receptor (OXTR) have been implicated in the regulation of energy homeostasis, but the detailed mechanism is still unclear. We recently showed late-onset obesity and impaired cold-induced thermogenesis in male OXTR knockout (Oxtr(-/-)) mice. Here we demonstrate that the OXTR in the hypothalamus has important functions in thermoregulation. Male Oxtr(-/-) mice failed to maintain their body temperatures during exposure to a cold environment. Oxtr(-/-) mice also showed decreased neuronal activation in the thermoregulatory hypothalamic region during cold exposure. Normal cold-induced thermogenesis was recovered in Oxtr (/) mice by restoring OXTR to the hypothalamus with an adeno-associated virus-Oxtr vector. In addition, brown adipose tissue (BAT) in Oxtr(-/-) mice contained larger lipid droplets in both 10- and 20-week-old compared with BAT from age-matched Oxtr(-/-) control mice. In BAT, the expression level of beta 3-adrenergic receptor at normal temperature was lower in Oxtr(-/-) mice than that in control mice. In contrast, alpha 2A-adrenergic receptor expression level was higher in BAT from Oxtr(-/-) mice in both normal and cold temperatures. Because beta 3- and alpha 2A-adrenergic receptors are known to have opposite effects on the thermoregulation, the imbalance of adrenergic receptors is suspected to affect this dysfunction in the thermoregulation. Our study is the first to demonstrate that the central OXT/OXTR system plays important roles in the regulation of body temperature homeostasis.

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to alpha-ketoglutarate (alpha-KG) and acquires new activity whereby it converts alpha-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1(R132H)-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1(R132H)-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progression of the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation.

The nonapeptide oxytocin is considered beneficial to mental health due to its anxiolytic, prosocial and antistress effects, but evidence for anxiogenic actions of oxytocin in humans has recently emerged. Using region-specific manipulations of the mouse oxytocin receptor (Oxtr) gene (Oxtr), we identified the lateral septum as the brain region mediating fear-enhancing effects of Oxtr. These effects emerge after social defeat and require Oxtr specifically coupled to the extracellular signal-regulated protein kinase pathway.

Lymph node metastasis is the most important prognostic factor of endometrial cancer. However, effective therapy has not been established against lymph node metastasis. In this study, we explored the efficacy of gene therapy targeting lymph node metastasis of endometrial cancer by suppressing the action of vascular endothelial growth factor (VEGF)-C through soluble VEGF receptor-3 (sVEGFR-3) expression. For this purpose, we first conducted a model experiment by introducing sVEGFR-3 cDNA into an endometrial cancer cell line HEC1A and established HEC1A/sVEGFR-3 cell line with high sVEGFR-3 expression. The conditioned medium of HEC1A/sVEGFR-3 cells inhibited lymphatic endothelial cell growth in vitro, and sVEGFR-3 expression in HEC1A cells suppressed in vivo lymph node and lung metastases without inhibiting the growth of a subcutaneously inoculated tumor. To validate the therapeutic efficacy, adeno-associated virus vectors encoding sVEGFR-3 were injected into the skeletal muscle of mice with lymph node metastasis. Lymph node and lung metastases of HEC1A cells were completely suppressed by the muscle-mediated expression of sVEG-FR-3 using adeno-associated virus vectors. These results suggest the possibility of gene therapy against lymph node and lung metastases of endometrial cancer by using muscle-mediated expression of sVEGFR-3.

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19(+) tumor lesions, and their ability to provide antitumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-gamma in response to CD19, and lysed both Raji and Daudi CD19(+) human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2(-/-)gamma c(-/-) immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma. (C) 2013 Elsevier Inc. All rights reserved.

Introduction: Factor VIII (FVIII) treatment for hemophilia A has difficulties in correcting bleeding diathesis in the presence of inhibitors. Materials and Methods: An adeno-associated virus type 8 (AAV8) vector containing the factor VII (FVII) gene or the activated factor VII (FVIIa) gene was used to investigate the therapeutic effect of FVII or FVIIa overexpression in FVIII-deficient mice with inhibitors. Results: Following repeated human FVIII injection, FVIII-deficient mice developed anti-human FVIII antibodies that cross-reacted with mouse FVIII. High transgene expression of murine FVII or murine FVIIa was achieved using the AAV8 vector and resulted in increased blood FVII activity greater than 800% of normal murine FVII levels in vector-injected FVIII-deficient mice. Thromboelastography analysis showed significant improvements in clotting time, clot formation time, a angle, and mean clot firmness in AAV8 vector-injected FVIII-deficient mice with inhibitors. Overexpression of FVIIa ameliorated the bleeding phenotype of FVIII-deficient mice with inhibitors and significantly increased the survival rate after tail clipping. In addition, overexpression of FVII increased the survival rate of FVIII-deficient mice with inhibitors after tail clipping though it was not as efficient as FVIIa overexpression. Conclusions: These data suggest that FVII overexpression is an alternative strategy for the treatment of hemophilia A with inhibitors. (C) 2013 Elsevier Ltd. All rights reserved.

We previously reported on a monoclonal antibody (mAb) that targeted amyloid beta (A beta) protein. Repeated injection of that mAb reduced the accumulation of A beta protein in the brain of human A beta transgenic mice (Tg2576). In the present study, cDNA encoding the heavy and light chains of this mAb were subcloned into an adeno-associated virus type 1 (AAV) vector with a 2A/furin adapter. A single intramuscular injection of 3.0x10(10) viral genome of these AAV vectors into C57BL/6 mice generated serum anti-A beta Ab levels up to 0.3 mg/ml. Anti-A beta Ab levels in excess of 0.1 mg/ml were maintained for up to 64 weeks. The effect of AAV administration on A beta levels in vivo was examined. A significant decrease in A beta levels in the brain of Tg2576 mice treated at 5 months (prophylactic) or 10 months (therapeutic) of age was observed. These results support the use of AAV vector encoding anti-A beta Ab for the prevention and treatment of Alzheimer's disease.

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 +/- 2.10-10.1 +/- 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 +/- 2.59-12.68 +/- 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 +/- 1.06-9.0 +/- 2.37%) in the presence of NAbs (14-56x dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.

Distinct anatomical regions of the neocortex subserve different sensory modalities and neuronal integration functions, but mechanisms for these regional specializations remain elusive. Involvement of epigenetic mechanisms for such specialization through the spatiotemporal regulation of gene expression is an intriguing possibility. Here we examined whether epigenetic mechanisms might play a role in the selective gene expression in the association areas (AAs) and the primary visual cortex (V1) in macaque neocortex. By analyzing the two types of area-selective gene promoters that we previously identified, we found a striking difference of DNA methylation between these promoters, i.e., hypermethylation in AA-selective gene promoters and hypomethylation in V1-selective ones. Methylation levels of promoters of each area-selective gene showed no areal difference, but a specific methyl-binding protein (MBD4) was enriched in the AAs, in correspondence with expression patterns of AA-selective genes. MBD4 expression was mainly observed in neurons. MBD4 specifically bound to and activated the AA-selective genes both in vitro and in vivo. Our results demonstrate that methylation in the promoters and specific methyl-binding proteins play an important role in the area-selective gene expression profiles in the primate neocortex. © 2013 the authors.

This study examined the role of the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO) in cervical cancer progression and the possible use of this enzyme for cervical cancer therapy. We analyzed IDO protein expression in 9 cervical cancer cell lines (SKG-I, -II, -IIIa, -IIIb, SiHa, CaSki, BOKU, HCS-2 and ME-180) stimulated with interferon-(gamma). IDO expression was observed in all cell lines except for SKG-IIIb. We transfected the human cervical cancer cell line CaSki that constitutively expresses IDO with a short hairpin RNA vector targeting IDO, and established an IDO-downregulated cell line to determine whether inhibition of IDO mediates cervical cancer progression. IDO downregulation suppressed tumor growth in vivo, without influencing cancer cell growth in vitro. Moreover, IDO downregulation enhanced the sensitivity of cervical cancer cells to natural killer (NK) cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. These findings indicate that downregulation of IDO controls cervical cancer progression by activating NK cells, suggesting IDO as a potential therapy for cervical cancer.

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.

The purpose of this study was to explore the possibility of targeted molecular therapy with anti-epidermal growth factor receptor (anti-EGFR) antibody (cetuximab) for the treatment of mucinous ovarian carcinoma. We analyzed EGFR protein expression and KRAS gene mutations in 5 mucinous ovarian carcinoma cell lines RMUG-L, RMUG-S, MN-1, OMC-1 and MCAS and evaluated the in vitro and in vivo effects of cetuximab on each. EGER expression was observed in all cell lines except for MN-1 cells, and a KRAS gene mutation at codon 12 was detected only in the MCAS cell line. Cetuximab inhibited RMUG-L and OMC-1 cell growth in vitro and completely blocked RMUG-L tumor growth in vivo. On the other hand, cetuximab did not affect MCAS cell growth in vitro and only partially reduced the MCAS tumor growth in vivo. These results suggest the possibility of targeted molecular therapy with cetuximab for mucinous ovarian carcinoma cells lacking a KRAS gene mutation.

Varying degrees of metabolic abnormalities mediated by chronic inflammation are implicated in the chronic glomerular injuries associated with obesity. Interleukin (IL)-10, a pleiotropic cytokine, exerts anti-inflammatory effects in numerous biological settings. In the present study, we explored the biological benefits of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against the pathological renal characteristics observed in Zucker fatty rats (ZFRs). We injected an AAV vector, encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male ZFRs at 5 weeks of age. Subsequently, the renal pathophysiological changes were analyzed. Persistent IL-10 expression significantly reduced the urinary protein excretion of ZFRs compared with GFP expression (47.1 +/- 11.6 mg per mg.creatinine versus 88.8 +/- 30.0 mg per mg.creatinine, P<0.01). The serum levels of IL-10 negatively correlated with the urinary protein in AAV-treated rats (r = -0.78, P<0.01). Renal hypertrophy, increased widths in the glomerular basement membrane, and the lack of uniformity and regularity of the foot process of the visceral glomerular epithelial cells of ZFRs were significantly blunted by IL-10 expression. IL-10 also abrogated the downregulation of glomerular nephrin observed in ZFRs treated with the GFP vector. Our findings provide insights into the potential benefit of the anti-inflammatory effects of IL-10 on the overall management of glomerulopathy induced by the metabolic disorders associated with obesity. Gene Therapy (2012) 19, 476-482; doi:10.1038/gt.2011.183; published online 24 November 2011

This study examined the role of the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO) in ovarian cancer progression, and the possible application of this enzyme as a target for ovarian cancer therapy. We transfected a short hairpin RNA vector targeting IDO into the human ovarian cancer cell line SKOV-3, that constitutively expresses IDO and established an IDO downregulated cell line (SKOV-3/shIDO) to determine whether inhibition of IDO mediates the progression of ovarian cancer. IDO downregulation suppressed tumor growth and peritoneal dissemination in vivo, without influencing cancer cell growth. Moreover, IDO downregulation enhanced the sensitivity of cancer cells to natural killer (NK) cells in vitro, and promoted NK cell accumulation in the tumor stroma in vivo. These findings indicate that downregulation of IDO controls ovarian cancer progression by activating NK cells, suggesting IDO targeting as a potential therapy for ovarian cancer.

Phenylketonuria (PKU) is a common genetic disorder arising from a deficiency of phenylalanine hydroxylase. If left untreated, the accumulation of phenylalanine leads to brain damage and neuropsychological dysfunction. One of the abnormalities found in hyperphenylalaninemic patients and a mouse model of PKU is an aminergic deficit in the brain. We previously showed correction of hyperphenylalaninemia and concomitant behavioral recovery in PKU mice after liver-targeted gene transfer with a viral vector. Here, we addressed whether such a functional recovery was substantiated by an improved amine metabolism in the brain. After gene transfer, brain dopamine, norepinephrine, and serotonin levels in the PKU mice were significantly elevated to normal or near-normal levels, along with systemic improvement of phenylalanine catabolism. The results of biochemical analyses validated the efficacy of PKU gene therapy in the central nervous system. NeuroReport 23:30-34 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Controlling lymph node metastasis is currently a key issue in cancer therapy. Lymph node metastasis is one of the most important prognostic factors in various types of cancers, including endometrial cancer. Vascular endothelial growth factor-C (VEGF-C) plays a crucial role in lymphangiogenesis, and is implicated to play an important role in lymph node metastasis. To evaluate the role of VEGF-C in lymph node metastasis, we developed an animal model by using an endometrial cancer cell line, HEC1A. This cell line is not invasive by nature and secretes moderate amounts of VEGF-C; intrauterine injection of HEC1A cells into Balb/c nude mice resulted in uterine cancer with lymph node metastasis after 8 weeks. To analyze the contribution of VEGF-C to lymph node metastasis, its corresponding gene was stably introduced into HEC1A cells (HEC1A/VEGF-C), which then produced more than 10 times the amount of VEGF-C. The number of lymph node metastases was significantly higher in HEC1A/VEGF-C cells than in HEC1A cells (3.2 vs 1.1 nodes/animal, respectively). Augmented lymphangiogenesis was observed within tumors when HEC1A/VEGF-C cells were inoculated. These results indicate that VEGF-C plays a critical role in lymph node metastasis, in addition to serving as a platform to test the efficacy of various therapeutic modalities against lymph node metastasis. (Cancer Sci 2011; 102: 22722277)

Although mesenchymal stem cells (MSCs) play pivotal supportive roles in hematopoiesis, how they interact with hematopoietic stem cells (HSCs) is not well understood. We investigated the interaction between HSCs and surrogate MSCs (C3H10T1/2 stromal cells), focusing on the molecular events induced by cell contact of these bipartite populations. C3H10T1/2 is a mesenchymal stromal cell line that can be induced to differentiate into preadipocytes (A54) and my oblasts (M1601). The stromal cell derivatives were cocultured with murine HSCs (Lineage-Sca1+), and gene expression profiles in stromal cells and HSCs were compared before and after the coculture. HSCs gave rise to cobblestone areas only on A54 cells, with nine fold more progenitors than on M1601 or undifferentiated C3H10T1/2 cells. Microarray-based screening and a quantitative reverse transcriptase directed-polymerase chain reaction showed that the levels of Notch ligands (Jagged1 and Deltalike 3) were increased in A54 cells upon interaction with HSCs. On the other hand, the expression of Notch1 and Hes1 was unregulated in the HSCs cocultured with A54 cells. A Tran swell assay revealed that the reciprocal up regulation was dependent on cell-to-cell contact. The result suggested that in the hematopoietic niche, HSCs help MSCs to produce Notch ligands, and in turn, MSCs help HSCs to express Notch receptor. Such a reciprocal up regulation would reinforce the downstream signaling to determine the fate of hematopoietic cell lineage. Clarification of the initiating events on cell contact should lead to the identification of specific molecular targets to facilitate HSC engraftment in transplantation therapy. © Journal of Stem Cells and Regenerative Medicine. All rights reserved.

The superficial layer of the superior colliculus (sSC) receives visual inputs via two different pathways: from the retina and the primary visual cortex. However, the functional significance of each input for the operation of the sSC circuit remains to be identified. As a first step toward understanding the functional role of each of these inputs, we developed an optogenetic method to specifically suppress the synaptic transmission in the retino-tectal pathway. We introduced enhanced halorhodopsin (eNpHR), a yellow light-sensitive, membrane-targeting chloride pump, into mouse retinal ganglion cells (RGCs) by intravitreously injecting an adeno-associated virus serotype-2 vector carrying the CMV-eNpHR-EYFP construct. Several weeks after the injection, whole-cell recordings made from sSC neurons in slice preparations revealed that yellow laser illumination of the eNpHR-expressing retino-tectal axons, putatively synapsing onto the recorded cells, effectively inhibited EPSCs evoked by electrical stimulation of the optic nerve layer. We also showed that sSC spike activities elicited by visual stimulation were significantly reduced by laser illumination of the sSC in anesthetized mice. These results indicate that photo-activation of eNpHR expressed in RGC axons enables selective blockade of retino-tectal synaptic transmission. The method established here can most likely be applied to a variety of brain regions for studying the function of individual inputs to these regions.

Background Classical phenylketonuria (PKU) arises from a deficiency of phenylalanine hydroxylase (PAH) that catalyses phenylalanine oxidation in the liver. Lack of PAH activity causes massive hyperphenylalaninemia and consequently severe brain damage. Preclinical studies showed that conventional adeno-associated virus (AAV) vectors could correct hyperphenylalaninemia in a mouse model of PKU, although limitations such as very large dose requirement and relative inefficiency in female animals were recognized. Method An AAV8-pseudotyped vector was constructed with a self-complementary AAV (scAAV) genome for efficient liver transduction and expression. Following vector injection to PKU mice, blood Phe was periodically measured by an enzymatic fluorometric assay. In vivo Phe oxidation was evaluated by a non-invasive breath test using [1-(13)C] Phe. Vector copy number in the host tissues was determined by quantitative polymerase chain reaction. Results A single injection of 1 x 10(11)-1 x 10(12) particles of the scAAV8 vector resulted in a reduction of blood Phe to normal or near-normal levels for more than 1 year in both genders. The treated animals showed normal level of in vivo Phe oxidation. The presence of > 1 copy of vector DNA per diploid genome in the liver was associated with normal blood Phe in the AAV-treated PKU mice. Conclusions Complete phenotypic correction of PKU mice was achieved by the scAAV8 vector for the longest duration reported to date. The vector overcame the female-specific disadvantage in AAV-mediated liver transduction; thus, it offers a promising platform of long-lasting gene therapy for PKU. Copyright (C) 2011 John Wiley & Sons, Ltd.

Gene therapy is considered as one of the innovative treatment modalities for diabetic retinopathy (DR). Since genuine animal models of DR are limited, only a few studies have reported the efficacy of gene therapy. For preclinical study of DR, spontaneously diabetic Torii (SDT) rat is a valuable model. Fortunately, we could evaluate the efficacy of adeno-associated virus (AAV)-mediated gene therapy in SDT rats and proved that sFlt-1 expression prevented DR progression. Because of a limited number of large-animal models of DR, it is uncertain whether gene therapy experiments using dogs or monkeys allow reliable conclusions. On the other hand, owing to the recent progress in AAV-mediated gene therapy for retinal diseases in monkeys and humans, gene therapy for DR using AAV vectors may become a reality in the near future. © Mizukami et al.