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As nanocarriers of a new generation, biomimetic nanovesicles are an emerging class of therapeutic tools whose surface is integrated or fabricated with biomaterials capable of mimicking the biological features and functions of native cells. Thanks to this, biomimetic nanovesicles, in particular, those made by plasma membrane moieties, possess greatly improved biocompatibility, high target specificity, a long retention time, and minimal undesired immune responses. For these reasons, a multitude of progenitor cells including cancer ones were employed as templates to generate biomimetic or membrane-camouflaged nanovesicles hosting different therapeutic compounds. In this contribution, different membrane-derived biomimetic vesicles (M-NVs) were generated by osmotic lysis or plasma membrane isolation approaches from normal and cancer cell lines and assayed against in vitro models of human glioblastoma. M-NVs were compared in their cellular internalization degrees of DNA and proteins, morphologically and molecularly characterized, expressing an extracellular membrane-associated marker. Then, Rose Bengal (RB), a photoactivable drug characterized by a relatively low cellular uptake, was incorporated into nascent glioblastoma-derived M-NVs and finally administered to homotypic receiving cells, showing an increased degree of internalization as well as induced cytotoxic effects, even in the absence of photodynamic direct stimulation. Similar results were also obtained assaying lyophilized M-NVs loaded with RB. In conclusion, M-NVs generated by cell membranes effectively deliver several cargoes, including therapeutic molecules, maintain functionality after lyophilization, and show significant internalization effects, making them a promising strategy for therapeutic applications against human glioblastoma cells.

Curcumin-based fluorescent probes are presented that are able to selectively bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells and specifically identify glioblastoma cells in vitro and in vivo.

Abstract We xeno-transplanted human neural precursor cells derived from induced pluripotent stem cells into the cerebellum and brainstem of mice and rats during prenatal development or the first postnatal week. The transplants survived and started to differentiate up to 1 month after birth when they were rejected by both species. Extended survival and differentiation of the same cells were obtained only when they were transplanted in NOD-SCID mice. Transplants of human neural precursor cells mixed with the same cells after partial in vitro differentiation or with a cellular extract obtained from adult rat cerebellum increased survival of the xeno-graft beyond one month. These findings are consistent with the hypothesis that the slower pace of differentiation of human neural precursors compared to that of rodents restricts induction of immune-tolerance to human antigens expressed before completion of maturation of the immune system. With further maturation the transplanted neural precursors expressed more mature antigens before the graft were rejected. Supplementation of the immature cells suspensions with more mature antigens may help to induce immune-tolerance for those antigens expressed only later by the engrafted cells.

Introduction: Spinal epidural abscess (SEA) incidence is rising. However, most series do not differentiate between SEAs associated with pyogenic infectious spondylodiscitis (PS) and SEAs limited to the epidural space.Methods: We retrospectively reviewed the records and radiological images of all patients admitted to our institutions with a diagnosis of SEA not associated with PS between January 2013 and December 2018.Results: We found three males and four females; five of the seven were intravenous drug users. All patients presented with pain: in six, it was associated with acute motor and sensory deficits, while one had only pain and paresthesias. Staphylococcus aureus was cultured from abscesses and/or from multiple blood cultures in four patients. Abscesses were localized to the cervical spine in one patient, thoracic in three, lumbar in one, and in two, the SEAs involved multiple segments. All patients but one underwent urgent open surgery. This patient had a multisegmental abscess and was successfully treated by percutaneous aspiration when pain became intractable. After abscess evacuation, the neurological deficits improved in all patients except one. The patients that were treated without spine instrumentation did not develop delayed kyphosis or instability at follow-up.Conclusion: Patients with SEAs not associated with PS are likely to present with pain and motor deficits, appear to benefit from urgent abscess evacuation, and seem to be less dependent on spine instrumentation to avoid delayed spinal deformities compared to SEA associated with PS. Finally, the lack of initial involvement of bone and intervertebral disks may suggest that at least some of the SEAs without PS originate from infection of epidural lymphatic vessels that are not present inside those structures.

Glucose transport across glioblastoma membranes plays a crucial role in maintaining the enhanced glycolysis typical of high-grade gliomas and glioblastoma. We tested the ability of two inhibitors of the glucose transporters GLUT/SLC2A superfamily, indinavir (IDV) and ritonavir (RTV), and of one inhibitor of the Na/glucose antiporter type 2 (SGLT2/SLC5A2) superfamily, phlorizin (PHZ), in decreasing glucose consumption and cell proliferation of human and murine glioblastoma cells. We found in vitro that RTV, active on at least three different GLUT/SLC2A transporters, was more effective than IDV, a specific inhibitor of GLUT4/SLC2A4, both in decreasing glucose consumption and lactate production and in inhibiting growth of U87MG and Hu197 human glioblastoma cell lines and primary cultures of human glioblastoma. PHZ was inactive on the same cells. Similar results were obtained when cells were grown in adherence or as 3D multicellular tumor spheroids. RTV treatment but not IDV treatment induced AMP-activated protein kinase (AMPKα) phosphorylation that paralleled the decrease in glycolytic activity and cell growth. IDV, but not RTV, induced an increase in GLUT1/SLC2A1 whose activity could compensate for the inhibition of GLUT4/SLC2A4 by IDV. RTV and IDV pass poorly the blood brain barrier and are unlikely to reach sufficient liquoral concentrations in vivo to inhibit glioblastoma growth as single agents. Isobologram analysis of the association of RTV or IDV and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide (TMZ) indicated synergy only with RTV on inhibition of glioblastoma cells. Finally, we tested in vivo the combination of RTV and BCNU on established GL261 tumors. This drug combination increased the overall survival and allowed a five-fold reduction in the dose of BCNU.

Adaptation of glioblastoma to caloric restriction induces compensatory changes in tumor metabolism that are incompletely known. Here we show that in human glioblastoma cells maintained in exhausted medium, SHC adaptor protein 3 (SHC3) increases due to down-regulation of SHC3 protein degradation. This effect is reversed by glucose addition and is not present in normal astrocytes. Increased SHC3 levels are associated to increased glucose uptake mediated by changes in membrane trafficking of glucose transporters of the solute carrier 2A superfamily (GLUT/SLC2A). We found that the effects on vesicle trafficking are mediated by SHC3 interactions with adaptor protein complex 1 and 2 (AP), BMP-2-inducible protein kinase and a fraction of poly ADP-ribose polymerase 1 (PARP1) associated to vesicles containing GLUT/SLC2As. In glioblastoma cells, PARP1 inhibitor veliparib mimics glucose starvation in enhancing glucose uptake. Furthermore, cytosol extracted from glioblastoma cells inhibits PARP1 enzymatic activity in vitro while immunodepletion of SHC3 from the cytosol significantly relieves this inhibition. The identification of a new pathway controlling glucose uptake in high grade gliomas represents an opportunity for repositioning existing drugs and designing new ones.

The lack of direct neurophysiological recordings from the thalamus and the cortex hampers our understanding of vegetative state/unresponsive wakefulness syndrome and minimally conscious state in humans. We obtained microelectrode recordings from the thalami and the homolateral parietal cortex of two vegetative state/unresponsive wakefulness syndrome and one minimally conscious state patients during surgery for implantation of electrodes in both thalami for chronic deep brain stimulation. We found that activity of the thalamo-cortical networks differed among the two conditions. There were half the number of active neurons in the thalami of patients in vegetative state/unresponsive wakefulness syndrome than in minimally conscious state. Coupling of thalamic neuron discharge with EEG phases also differed in the two conditions and thalamo-cortical cross-frequency coupling was limited to the minimally conscious state patient. When consciousness is physiologically or pharmacologically reversibly suspended there is a significant increase in bursting activity of the thalamic neurons. By contrast, in the thalami of our patients in both conditions fewer than 17% of the recorded neurons showed bursting activity. This indicates that these conditions differ from physiological suspension of consciousness and that increased thalamic inhibition is not prominent. Our findings, albeit obtained in a limited number of patients, unveil the neurophysiology of these conditions at single unit resolution and might be relevant for inspiring novel therapeutic options.

Recent evidence suggests that adult bone marrow stem cells reduce tissue damage and promote repair following CNS ischemic injury. Since granulocyte-colony stimulating factor (G-CSF) mobilizes hematopoietic stem cells to the circulating compartment, here we tested whether administration of this drug modifies the outcome of a permanent occlusion of the middle cerebral artery in adult mice. To elucidate the behavior and fate of blood-borne cells in the ischemic brain, we produced chimeric animals, in which hematopoietic derivatives are genetically tagged. G-CSF administration enhances the proliferation of microglia in the uninjured CNS but has no effect on the amount of hematopoietic cells that infiltrate the ischemic tissue and on the size of the lesion. The blood-borne elements acquire different mesodermal identities but fail to adopt neural phenotypes, even though they occasionally fuse with Purkinje neurons. These results indicate that G-CSF treatment does not exert a significant beneficial effect on the ischemic injury.

CNS repair by cell transplantation requires new neurons to integrate into complex recipient networks. We assessed how the migratory route of transplanted granule neurons and the developmental stage of the host rat cerebellum influence engraftment. In both embryonic and postnatal hosts, granule cells can enter the cerebellar cortex and achieve correct placement along their natural migratory pathway. Donor neurons can also reach the internal granular layer from the white matter and integrate following an unusual developmental pattern. Although the frequency of correct positioning declines in parallel with cortical development, in mature recipients correct homing is more frequent through the unusual path. Following depletion of granule cell precursors in the host, more granule neurons engraft, but their ability for achieving correct placement is unchanged. Therefore, while the cerebellar environment remains receptive for granule cells even after the end of development, their full integration is partially hindered by the mature cortical architecture.

The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.