Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3-specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase I trial administering repeated locoregional B7-H3 CAR T cells to children with recurrent/refractory CNS tumors and DIPG. Here, we report the results of the first three evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3 CAR T cells. Targeted mass spectrometry of CSF biospecimens revealed modulation of B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3 CAR T-cell dosing and that intracranial delivery may induce local immune activation.

Although one of the defining characteristics of Alzheimer's disease is the presence of amyloid-beta (Aβ) plaques, the early accumulation of soluble Aβ oligomers (AβOs) may disrupt synaptic function and trigger cognitive impairments long before the appearance of plaques. Furthermore, murine models aimed at understanding how AβOs alter formation and retrieval of associative memories are conducted using human Aβ species, which are more neurotoxic in the mouse brain than the native murine species. Unfortunately, there is currently a lack of attention in the literature as to what the murine version of the peptide (mAβ) does to synaptic function and how it impacts the consolidation and retrieval of associative memories. In the current study, adult mice were infused with mAβ 0, 2, 6, or 46 h after contextual-fear conditioning, and were tested 2-48 h later. Interestingly, only mAβ infusions within 2 h of training reduced freezing behavior at test, indicating that mAβ disrupted the consolidation, but not retrieval of fear memory. This consolidation deficit coincided with increased IL-1β and reduced synaptophysin mRNA levels, without disrupting other synaptic signaling-related genes here examined. Despite differences between murine and human Aβ, the deleterious functional outcomes of early-stage synaptic oligomer presence are similar. Thus, models utilizing or inducing the production of mAβ in non-transgenic animals are useful in exploring the role of dysregulated synaptic plasticity and resultant learning deficits induced by Aβ oligomers.

Clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in autism spectrum disorders (ASD), epilepsy and some inheritable metabolic disorders. Propionic acid (PPA) is a short chain fatty acid and an important intermediate of cellular metabolism. PPA is also a by-product of a subpopulation of human gut enterobacteria and is a common food preservative. We examined the behavioural, electrophysiological, neuropathological, and biochemical effects of treatment with PPA and related compounds in adult rats. Intraventricular infusions of PPA produced reversible repetitive dystonic behaviours, hyperactivity, turning behaviour, retropulsion, caudate spiking, and the progressive development of limbic kindled seizures, suggesting that this compound has central effects. Biochemical analyses of brain homogenates from PPA treated rats showed an increase in oxidative stress markers (e.g., lipid peroxidation and protein carbonylation) and glutathione S-transferase activity coupled with a decrease in glutathione and glutathione peroxidase activity. Neurohistological examinations of hippocampus and adjacent white matter (external capsule) of PPA treated rats revealed increased reactive astrogliosis (GFAP immunoreactivity) and activated microglia (CD68 immunoreactivity) suggestive of a neuroinflammatory process. This was coupled with a lack of cytotoxicity (cell counts, cleaved caspase 3' immunoreactivity), and an increase in phosphorylated CREB immunoreactivity. We propose that some types of autism may be partial forms of genetically inherited or acquired disorders involving altered PPA metabolism. Thus, intraventricular administration of PPA in rats may provide a means to model some aspects of human ASD in rats.

To test the molecular nature of the NGF receptor responsible for the ability of NGF to rescue septal cholinergic neurons following axotomy, we infused polyclonal antibodies that act as specific agonists of trkA (RTA) into the lateral ventricle of fimbria-fornix lesioned animals. Rats receiving chronic intraventricular infusions of RTA showed significantly more low affinity NGF receptor immunoreactive (p75NGFR-IR) neurons on the lesioned side than did control animals 2 weeks following unilateral fimbria-fornix lesion. RTA also initiated cholinergic sprouting. Infusions of RTA in combination with an antibody that blocks p75NGFR (REX) did not reduce the cell savings effect observed with RTA alone. However, animals infused with RTA plus REX demonstrated significantly less sprouting. These findings suggest that antibody-induced trkA activation is sufficient to mediate NGF-promoted survival of axotomized cholinergic neurons in vivo.

Gastrointestinal symptoms and altered blood phospholipid profiles have been reported in patients with autism spectrum disorders (ASD). Most of the phospholipid analyses have been conducted on the fatty acid composition of isolated phospholipid classes following hydrolysis. A paucity of information exists on how the intact phospholipid molecular species are altered in ASD. We applied ESI/MS to determine how brain and blood intact phospholipid species were altered during the induction of ASD-like behaviors in rats following intraventricular infusions with the enteric bacterial metabolite propionic acid. Animals were infused daily for 8 days, locomotor activity assessed, and animals killed during the induced behaviors. Propionic acid infusions increased locomotor activity. Lipid analysis revealed treatment altered 21 brain and 30 blood phospholipid molecular species. Notable alterations were observed in the composition of brain SM, diacyl mono and polyunsaturated PC, PI, PS, PE, and plasmalogen PC and PE molecular species. These alterations suggest that the propionic acid rat model is a useful tool to study aberrations in lipid metabolism known to affect membrane fluidity, peroxisomal function, gap junction coupling capacity, signaling, and neuroinflammation, all of which may be associated with the pathogenesis of ASD.

T cell immunotherapy is emerging as a powerful strategy to treat cancer and may improve outcomes for patients with glioblastoma (GBM). We have developed a chimeric antigen receptor (CAR) T cell immunotherapy targeting IL-13 receptor α2 (IL13Rα2) for the treatment of GBM. Here, we describe the optimization of IL13Rα2-targeted CAR T cells, including the design of a 4-1BB (CD137) co-stimulatory CAR (IL13BBζ) and a manufacturing platform using enriched central memory T cells. Utilizing orthotopic human GBM models with patient-derived tumor sphere lines in NSG mice, we found that IL13BBζ-CAR T cells improved anti-tumor activity and T cell persistence as compared to first-generation IL13ζ-CAR CD8+ T cells that had shown evidence for bioactivity in patients. Investigating the impact of corticosteroids, given their frequent use in the clinical management of GBM, we demonstrate that low-dose dexamethasone does not diminish CAR T cell anti-tumor activity in vivo. Furthermore, we found that local intracranial delivery of CAR T cells elicits superior anti-tumor efficacy as compared to intravenous administration, with intraventricular infusions exhibiting possible benefit over intracranial tumor infusions in a multifocal disease model. Overall, these findings help define parameters for the clinical translation of CAR T cell therapy for the treatment of brain tumors.

Microdialysis measurements of dopamine (DA) and DA metabolites were carried out in the putamen and substantia nigra of unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned rhesus monkeys that received intraventricular injections of vehicle or glial-derived neurotrophic factor (GDNF, 300 microg) 3 weeks prior to the microdialysis studies. Following behavioral measures in the MPTP-lesioned monkeys, they were anesthetized with isoflurane and placed in a stereotaxic apparatus. Magnetic resonance imaging (MRI)-guided sterile stereotaxic procedures were used for implantations of the microdialysis probes. Basal extracellular levels of DA and the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were found to be decreased by >95% in the right putamen of the MPTP-lesioned monkeys as compared to normal animals. In contrast, basal DA levels were not significantly decreased, and DOPAC and HVA levels were decreased by only 65% and 30%, respectively, in the MPTP-lesioned substantia nigra. Significant reductions in d-amphetamine-evoked DA release were also observed in the MPTP-lesioned substantia nigra and putamen of the monkeys as compared to normal animals. A single intraventricular administration of GDNF into one group of MPTP-lesioned monkeys elicited improvements in the parkinsonian symptoms in these animals at 2-3 weeks post-administration. In addition, d-amphetamine-evoked overflow of DA was significantly increased in the substantia nigra but not the putamen of MPTP-lesioned monkeys that had received GDNF. Moreover, post-mortem brain tissue studies showed increases in whole tissue levels of DA and DA metabolite levels primarily within the substantia nigra in MPTP-lesioned monkeys that had received GDNF. Taken together, these data support that single ventricular infusions of GDNF produce improvements in motoric behavior in MPTP-lesioned monkeys that correlate with increases in DA neuronal function that are localized to the substantia nigra and not the putamen.

Intrastriatal transplantation of fetal human ventral mesencephalic dopaminergic neurons is an experimental therapy for patients suffering from Parkinson's disease. The success of this approach depends on several host brain parameters including neurotrophic factors and growth inhibitors that guide survival and integration of transplanted neurons. While the potential of neurotrophic factors has been extensively investigated, repression of growth inhibitors has been neglected, despite the significant effects reported in various CNS injury models. Recently, we demonstrated that infusion of neutralizing antibodies against Nogo-A into the lateral ventricles of hemi-parkinsonian rats significantly enhanced graft function. Since the Nogo-receptor 1 also interacts with other neurite growth inhibitors, we investigated whether a direct antagonization of the receptor would result in more robust effects. Therefore, rats with unilateral striatal 6-hydroxydopamine lesions were grafted with ventral mesencephalic tissue in combination with intraventricular infusions of the Nogo-receptor 1 antagonist NEP1-40. Transplanted rats receiving saline infusions served as controls. To test whether NEP1-40 treatment alone affects the remaining dopaminergic striatal fibers, rats with unilateral striatal 6-hydroxydopamine lesions were infused with NEP1-40 or saline without receiving a transplant. Motor behavior was assessed prior to the lesion as well as prior and 1, 3, and 5 weeks after the transplantations. At the end of the experimental period the number of graft-derived dopaminergic fibers growing into the host brain, the number of surviving dopaminergic neurons and graft volume were analyzed. In rats without a transplant, the density of dopaminergic fibers in the striatum was analyzed. We detected that NEP1-40 treatment significantly enhanced graft-derived dopaminergic fiber outgrowth as compared to controls while no effects were detected for graft volume and survival of grafted dopaminergic neurons. Notably, the enhanced dopaminergic fiber outgrowth was not sufficient to improve the functional recovery as compared to controls. Moreover, NEP1-40 infusions in hemi-parkinsonian rats without a transplant did not result in enhanced striatal dopaminergic fiber densities and consequently did not improve behavior. In sum, our findings demonstrate that antagonization of the Nogo-receptor 1 has the capacity to support the engraftment of transplanted mesencephalic tissue in an animal model of Parkinson's disease.

BACKGROUND Traumatic brain injury (TBI) induces edema on the uninjured side (i.e., contralateral brain tissue; CBT). We evaluated the role of AQP4 in CBT edema formation following TBI. MATERIAL AND METHODS Mild or severe TBI was induced using a controlled cortical impact model in rats, immediately followed by intraventricular siRNA infusions. The effects of AQP4 siRNA on CBT edema were assessed at up to 168 h. RESULTS Mild or severe TBI induced different patterns of CBT edema. Furthermore, following mild TBI, brain water content (BWC) was increased at 72 h thereafter and AQP4 expression was increased after 168 h, relative to non-injured rats (i.e., sham). AQP4 interference reduced AQP4 expression 48 h thereafter and BWC 72 h thereafter, relative to control siRNA. In contrast, following severe TBI, BWC was increased 1 h thereafter and AQP4 expression was transiently enhanced after 1 h, relative to sham. However, AQP4 interference reduced AQP4 expression after 1 h and BWC 24 h thereafter, relative to control siRNA. Finally, apparent diffusion coefficient (ADC) value in CBT was positively correlated with AQP4 expression level following severe, but not mild, TBI. AQP4 interference disrupted this correlation. CONCLUSIONS AQP4 interference reduces CBT edema formation, and ADC value may predict TBI severity.

The amplitude of the acoustic startle response is increased when elicited in the presence of brief cues that predict shock (fear-potentiated startle) and also when elicited during sustained exposure to bright light (light-enhanced startle). Although both effects are thought to reflect fear or anxiety, their neuroanatomical substrates differ. Although fear-potentiated startle is disrupted by reversible inactivation of the central nucleus of the amygdala (CeA) but not the closely related bed nucleus of the stria terminalis (BNST), light-enhanced startle is disrupted by BNST inactivation but not by CeA inactivation. Intraventricular infusions of corticotropin-releasing factor (CRF) also increase startle (CRF-enhanced startle) and this effect is mediated by CRF receptors within the BNST, with no involvement of the CeA. Together, these observations suggest that CeA- and BNST-dependent fear and anxiety may be differentially sensitive to CRF receptor blockade. We tested this by orally administering the novel, potent, and selective CRF-R1 antagonist GSK876008 to rats before CRF-enhanced, light-enhanced, or fear-potentiated startle testing. GSK876008 disrupted CRF-enhanced startle with a linear dose-response curve, and light-enhanced startle with a U-shaped dose-response curve, but did not disrupt fear-potentiated startle to a visual stimulus at any dose tested, and even augmented the response in some animals. GSK876008 also disrupted shock-related 'baseline' startle increases, which may have reflected context conditioning (shown elsewhere to also be BNST-dependent). Overall, these results suggest that short-duration CeA-dependent threat responses can be pharmacologically dissociated from longer duration BNST-dependent responses in terms of their sensitivity to CRF1 receptor antagonists.

Using a murine model of chronic ischemic cardiomyopathy caused by an old myocardial infarction (MI), we have previously found that three doses of 1 × 106 c-kit positive cardiac cells (CPCs) are more effective than a single dose of 1 × 106 cells. The goal of this study was to determine whether the beneficial effects of three doses of CPCs (1 × 106 cells each) can be fully replicated by a single combined dose of 3 × 106 CPCs. Mice underwent a 60-min coronary occlusion; after 90 days of reperfusion, they received three echo-guided intraventricular infusions at 5-week intervals: (1) vehicle × 3; (2) one combined dose of CPCs (3 × 106) and vehicle × 2; or (3) three doses of CPCs (1 × 106 each). In the combined-dose group, left ventricular ejection fraction (LVEF) improved after the 1st CPC infusion, but not after the 2nd and 3rd (vehicle) infusions. In contrast, in the multiple-dose group, LVEF increased after each CPC infusion; at the final echo, LVEF averaged 35.2 ± 0.6% (p < 0.001 vs. the vehicle group, 27.3 ± 0.2%). At the end of the study, the total cumulative change in EF from pretreatment values was numerically greater in the multiple-dose group (6.6 ± 0.6%) than in the combined-dose group (4.8 ± 0.8%), although the difference was not statistically significant (p = 0.08). Hemodynamic studies showed that several parameters of LV function in the multiple-dose group were numerically greater than in the combined-dose group (p = 0.08 for the difference in LVEF). Compared with vehicle, cardiomyocyte cross-sectional area was reduced only in the multiple-dose group (-32.7%, 182.6 ± 15.1 µm2 vs. 271.5 ± 27.2 µm2, p < 0.05, in the risk region and -28.5%, 148.5 ± 12.1 µm2 vs. 207.6 ± 20.5 µm2, p < 0.05, in the noninfarcted region). LV weight/body weight ratio and LV weight/tibia length ratios were significantly reduced in both cell treated groups vs. the vehicle group, indicating the attenuation of LV hypertrophy; however, the lung weight/body weight ratio was significantly reduced only in the multiple-dose group, suggesting decreased pulmonary congestion. Taken together, these results indicate that in mice with chronic ischemic cardiomyopathy, the beneficial effects of three doses of CPCs on LV function and hypertrophy cannot be fully replicated with a single dose, notwithstanding the fact that the total number of cells delivered with one or three doses is the same. Thus, it is the multiplicity of doses, and not the total number of cells, that accounts for the superiority of the repeated-dose paradigm. This study supports the idea that the efficacy of cell therapy in heart failure can be augmented by repeated administrations.

Stromal cell-derived factor-1 and its receptor CXCR4 are essential regulators of the neurogenesis that occurs in the adult hippocampal dentate gyrus. However, the effects of CXCR7, a new atypical receptor of stromal cell-derived factor-1, on hippocampal neurogenesis after a stroke remain largely unknown. Our study is the first to investigate the effect of a CXCR7-neutralizing antibody on neurogenesis in the dentate gyrus and the associated recovery of cognitive function of rats in the chronic stage of cerebral ischemia. The rats were randomly divided into sham, sham + anti-CXCR7, ischemia and ischemia + anti-CXCR7 groups. Endothelin-1 was injected in the ipsilateral motor cortex and striatum to induce focal cerebral ischemia. Sham group rats were injected with saline instead of endothelin-1 via intracranial injection. Both sham and ischemic rats were treated with intraventricular infusions of CXCR7-neutralizing antibodies for 6 days 1 week after surgery. Immunofluorescence staining with doublecortin, a marker for neuronal precursors, was performed to assess the neurogenesis in the dentate gyrus. We found that anti-CXCR7 antibody infusion enhanced the proliferation and dendritic development of doublecortin-labeled cells in the dentate gyrus in both ischemic and sham-operated rats. Spatial learning and memory functions were assessed by Morris water maze tests 30-32 days after ischemia. CXCR7-neutralizing antibody treatment significantly reduced the escape latency of the spatial navigation trial and increased the time spent in the target quadrant of spatial probe trial in animals that received ischemic insult, but not in sham operated rats. These results suggest that CXCR7-neutralizing antibody enhances the neurogenesis in the dentate gyrus and improves the cognitive function after cerebral ischemia in rats. All animal experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of China Medical University (CMU16089R) on December 8, 2016.

Autophagy alterations have been observed in a variety of neurological disorders, however, very few studies have focused on autophagy alterations in epilepsy. The ketogenic diet (KD) likely ameliorates neuronal loss in several seizure models. However, whether this neuroprotective function occurs via starvation-induced autophagy and its prevalence in chronic kindled seizures remains unknown. The aim of this study was to determine the role of autophagy following seizure under KD, and the potential mechanism involved. Pentylenetetrazol (PTZ)-kindled rats, which were fed a Normal diet (ND) or KD, were pretreated with intraventricular infusions of saline, autophagy inducer rapamycin (RAP), or inhibitor 3-methyladenine (3-MA). KD alleviated seizure severity, decreased the number of Fluoro-jade B (FJB)-positive cells in the hippocampus of kindled rats. These effects were abolished by 3-MA pretreatment. RAP pretreatment did not affect seizure severity, but decreased the number of FJB-positive cells in ND group. KD decreased the percentage of damaged mitochondria in kindled group. Hippocampal Beclin-1 was increased by KD in vehicle group. The autophagy proteins Atg5, Beclin-1 and the ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3-I in kindled KD-fed rats were higher, and the autophagy substrate P62 was lower than those in the kindled ND-fed rats, indicating an increase in autophagy following KD. Pretreatment with RAP increased the level of LC3-II/LC3-I, and pretreatment with 3-MA increased the level of P62 in KD-fed rats. To further clarify the mechanism of autophagy protection, the levels of key mitochondria related molecules were examed. The results showed that mitochondrial cytochrome c was up-regulated, cytosolic cytochrome c and the downstream cleaved caspase-3 was down-regulated in KD-fed rats, indicating a decrease in mitochondrial apoptosis. Taken together, our results indicated that KD activates autophagic pathways and reduces brain injury during PTZ-kindled seizures. The neuroprotective effect of KD is likely exerted via a reduction of mitochondrial cytochrome c release.