Adipose-derived mesenchymal stem cells: neuronal differentiation potential and neuroprotective action.

Le cellule mesenchimali staminali adulte derivate dal tessuto adipose (Adipose Stem Cell, ASC) rappresentano, nell\u2019ambito della terapia cellulare, un\u2019alternativa valida agli altri tipi di cellule staminali (Stem Cell, SC) poich\ue8 si possono ottenere in ampia quantit\ue0 dal tessuto adipose, possono esser facilmente coltivate in laboratorio ed espanse. Abbiamo investigato in vitro il potenziale differenziativo delle ASC in senso neuronale usando due tipi di approcci: un trattamento chimico ed un protocollo prolungato in 2 fasi, che include la formazione di sfere e il sequenziale trattamento BDNF (brain-derived neurotrophic factor) e acido retinoico (retinoic acid, RA). Dopo 30 giorni, circa il 57% di ASC mostra caratteristiche morfologiche, fenotipiche ed elettrofisiologiche suggestive di una precoce differenziazione neuronale. Infatti, le ASC assumono una forma allungata, con 2-3 processi citoplasmatici, esprimono selettivamente nestina e le molecule neuronali, fra cui il recettore di GABA-A e tirosina idrossilasi, in assenza di markers gliali. Le cellule differenziate esibiscono un potenziale di membrana negativo ( 1260 mV), correnti di potassio rettificanti ritardate e correnti TTX sensibili, elementi tipici della cellula neuronale; tuttavia non son in grado di generare un potenziale d\u2019azione. Considerando la bassa efficienza del trattamento e la incompleta differenziazione neuronale, abbiamo quindi valutato se le ASC esercitino una funzione neuro-protettiva. Usando il modello di neuroblastoma esposto a H2O2 in vitro, dimostriamo che le ASC aumentano la vitalit\ue0 cellulare (confrontate con i fibroblasti) e proteggono dall\u2019apoptosi. Un possibile meccanismo coinvolto potrebbe esser la secrezione di BDNF, come riportato per le SC mesenchimali (Mesenchymal SC, MSC) derivate da midollo osseo; infatti il medium condizionato di ASC contiene alti livelli di BDNF. Oltre a esibire neuro-protezione, fattori solubili secreti da ASC promuovono la crescita del neurite, un meccanismo aggiuntivo che pu\uf2 favorire la neurorigenerazione. Alla luce di questi dati e dell\u2019azione immunosoppressiva delle ASC recentemente da noi dimostrato (Constantin et al, 2009), le ASC possono essere una utile sorgente di MSC per il trattamento delle malattie neurodegenerative.Adult mesenchymal stem cells derived from adipose tissue (ASC) offer significant practical advantages over other types of stem cells (SC) for potential clinical applications, since they can be obtained from adult adipose tissue in large amounts, can be easily cultured and expanded with a very low risk for development of malignancies. We investigated in vitro the neuronal differentiation potential of human ASC with a chemical protocol and a prolonged two-step protocol, which included sphere formation and sequential culture in brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). After 30 days, about 57% ASC show morphological, immunocytochemical and electrophysiological evidence of initial neuronal differentiation. In fact, ASC display elongated shape with protrusion of two or three cellular processes, selectively express nestin and neuronal molecules (including GABA-A receptor and tyroxine hydroxilase) in the absence of glial phenotypic markers. Differentiated cells show negative membrane potential ( 1260 mV), delayed rectifier potassium currents and TTX-sensitive sodium currents, but they are unable to generate action potential. Considering the low efficacy and the not-fully mature neuronal differentiation, we evaluated if ASC display a neuroprotective effect. Using the H2O2-stressed neuroblastoma model in vitro, we show that ASC increase cell availability (compared to fibroblasts) and protect against apoptosis. A possible mechanism involved could be the secretion of BDNF, as reported for human BM-MSC: in this regard, we indeed find high levels of BDNF in ASCcondition medium. In addition to exert neuroprotection, soluble factors secreted by ASC promote neurite outgrowth, an additional mechanism that may favor neuroregeneration. In view of these results and their immunosuppressive action (Constantin et al, 2009), ASC may be a ready source of adult MSC to treat neurodegenerative diseases

Diffuse glioblastoma resembling acute hemorrhagic leukoencephalitis

We report the case of a young man with sudden onset of diplopia after an upper respiratory tract infection. Based on the first radiological findings acute hemorrhagic leukoencephalitis, a variant of acute disseminated encephalomyelitis, was suspected and treatment with high dose intravenous dexamethasone was started but it was stopped for intolerance. The patient clinically worsened, developing gait instability, ataxia and ophthalmoplegia; brain MRI performed 20 days later showed severe progression of the disease with subependymal dissemination. After brain biopsy of the right temporal lesion the histological diagnosis was glioblastoma. These findings suggest that MRI features of acute hemorrhagic leukoencephalitis may dissimulate the diagnosis of diffuse glioma/glioblastoma. This case underscores the importance of considering diffuse glioma in the differential diagnosis of atypical signs and symptoms of acute hemorrhagic leukoencephalitis and underlines the relevant role of integrating neuroradiologic findings with neuropathology

Case report: Multiple brain intravascular papillary endothelial hyperplasia: incidence, diagnostic challenges, and management approach

Multiple hemorrhagic brain lesions are mainly diagnosed based on clinico-radiological features integrated with histological data. Intravascular papillary endothelial hyperplasia (IPEH), or Masson's tumor, is a very rare entity, particularly when localized in the brain. In this study, we describe a case of multiple recurrent brain IPEHs and provide details on the diagnostic phase, therapeutic approaches, and related challenges. A 55-year-old woman presented with a relapsing neurological deficit. Brain magnetic resonance imaging (MRI) revealed a hemorrhagic right frontal-parietal lesion. When new neurological symptoms occurred, subsequent MRI scans detected more bleeding cerebral lesions. She underwent a series of single hemorrhagic lesion debulking. For any samples that underwent histopathological examination, the first results were not informative; the second and the third results revealed hemangioendothelioma (HE); and the fourth results led to the IPEH diagnosis. Interferon alpha (IFN-α) and subsequently sirolimus were prescribed. Both were well tolerated. Clinical and radiological features remained stable 43 months after starting sirolimus therapy and 132 months after the first diagnosis. To date, 45 cases of intracranial IPEH have been reported, mostly as single lesions without parenchymal location. They are usually treated by surgery and sometimes by radiotherapy upon recurrence. Our case is notable for two main reasons: because of the consecutive recurrent multifocal exclusively cerebral lesions and the therapeutic approach we used. Based on multifocal brain recurrence and good performance, we propose pharmacological therapy, including IFN-α and sirolimus, to stabilize IPEH

Response assessment of GBM during immunotherapy by delayed contrast treatment response assessment maps

IntroductionAssessing the treatment response of glioblastoma multiforme during immunotherapy (IT) is an open issue. Treatment response assessment maps (TRAMs) might help distinguish true tumor progression (TTP) and pseudoprogression (PsP) in this setting.MethodsWe recruited 16 naïve glioblastoma patients enrolled in a phase II trial consisting of the Stupp protocol (a standardized treatment for glioblastoma involving combined radiotherapy and chemotherapy with temozolomide, followed by adjuvant temozolomide) plus IT with dendritic cells. Patients were followed up till progression or death; seven underwent a second surgery for suspected progression. Clinical, immunological, and MRI data were collected from all patients and histology in case of second surgery. Patients were classified as responders (progression-free survival, PFS > 12 months), and non-responders (PFS ≤ 12), HIGH-NK (natural killer cells, i.e., immunological responders), and LOW-NK (immunological non-responders) based on immune cell counts in peripheral blood. TRAMs differentiate contrast-enhancing lesions with different washout dynamics into hypothesized tumoral (conventionally blue-colored) vs. treatment-related (red-colored).ResultsUsing receiver operating characteristic (ROC) curves, a threshold of −0.066 in VBlue/VCE (volume of the blue portion of tumoral area/volume of contrast enhancement) variation between values obtained in the MRI performed before PsP/TTP and at TTP/PSP allowed to discriminate TTP from PsP with a sensitivity of 71.4% and a specificity of 100%. Among HIGH-NK patients, at month 6 there was a significant reduction compared to baseline and month 2 in median “blue” volumes.DiscussionIn conclusion, in our pilot study TRAMs support the discrimination between tumoral and treatment-related enhancing features in immunological responders vs. non-responders, the distinction between PsP and TTP, and might provide surrogate markers of immunological response

IMMU-01. TEM-GBM: AN OPEN-LABEL, PHASE I/IIA DOSE-ESCALATION STUDY EVALUATING THE SAFETY AND EFFICACY OF GENETICALLY MODIFIED TIE-2 EXPRESSING MONOCYTES TO DELIVER IFN-A WITHIN GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract Temferon is a macrophage-based treatment relying on ex-vivo transduction of autologous HSPCs to express immune-payloads within the TME. Temferon targets the immune-modulatory molecule IFN-a, to a subset of tumor infiltrating macrophages known as Tie-2 expressing macrophages (TEMs) due to the Tie2 promoter and a post-transcriptional regulation layer represented by miRNA-126 target sequences. As of 31st May 2021, 15-patients received Temferon (D+0) with follow-up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D+13 and D+13.5, respectively. Temferon-derived differentiated cells, as determined be the number of vector copy per genome, were found within 14 days post treatment and persisted albeit at lower levels up to 18-months. Very low concentrations of IFN-a in the plasma (8.7 pg/ml-D+30) and in the CSF (1.6 pg/ml-D+30) were detected, suggesting tight regulation of transgene expression. Five-deaths occurred at D+322, +340, +402, +478 and +646 due to PD, and one at D+60 due to complications following the conditioning regimen. Eight-patients had progressive disease (range: D-11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. Four-patients underwent 2ndsurgery. Recurrent tumors had gene-marked cells and increased expression of ISGs compared to first surgery, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor-associated clones expanded in the periphery. TME characterization by scRNA and TCR-sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients, as predicted by preclinical studies

Deciphering the Labyrinthine System of the Immune Microenvironment in Recurrent Glioblastoma: Recent Original Advances and Lessons from Clinical Immunotherapeutic Approaches

The interpretation of the presence and function of immune infiltration in glioblastoma (GBM) is still debated. Over the years, GBM has been considered a cold tumor that is less infiltrated by effector cells and characterized by a high proportion of immunosuppressive innate immune cells, including GBM-associated microglia/macrophages (GAMs). In this context, the failure of checkpoint inhibitors, particularly in recurrent GBM (rGBM), caused us to look beyond the clinical results and consider the point of view of immune cells. The tumor microenvironment in rGBM can be particularly hostile, even when exposed to standard immunomodulatory therapies, and tumor-infiltrating lymphocytes (TILs), when present, are either dysfunctional or terminally exhausted. However, after checkpoint blockade therapy, it was possible to observe specific recruitment of adaptive immune cells and an efficient systemic immune response. In this review article, we attempt to address current knowledge regarding the tumor and immune microenvironment in rGBM. Furthermore, immunosuppression induced by GAMs and TIL dysfunction was revisited to account for genetic defects that can determine resistance to therapies and manipulate the immune microenvironment upon recurrence. Accordingly, we reevaluated the microenvironment of some of our rGBM patients treated with dendritic cell immunotherapy, with the goal of identifying predictive immune indicators of better treatment response

Modeling the water-energy nexus under changing energy market and climate conditions: a case study in the Italian Alps

Climate change and growing population are expected to severely affect freshwater availability by the end of 21th century. Many river basins, especially in the Mediterranean region, are likely to become more prone to periods of reduced water supply, risking considerable impacts on the society, the environment, and the economy, thus emphasizing the need to rethink the way water resources are distributed, managed, and used at the regional and river basin scale. This paradigm shift will be essential to cope with the undergoing global change, characterized by growing water demands and by increasingly uncertain hydrologic regimes. Most of the literature traditionally focused on predicting the impacts of climate change on water resources, while our understanding of the human footprint on the hydrological cycle is limited. For example, changes in the operation of the Alpine hydropower reservoirs induced by socio-economic drivers (e.g., development of renewable energy) have been already observed over the last few years and have produced relevant impacts on multiple water uses due to the altered distribution of water volumes in time and space. Modeling human decisions as well as the links between society and environmental systems becomes key to develop reliable projections on the co-evolution of the coupled human-water systems and deliver robust adaptation strategies. This work contributes a preliminary model-based analysis of the behaviour of hydropower operators under changing energy market and climate conditions. The proposed approach is developed for the San Giacomo-Cancano reservoir system located in the Lake Como catchment. The identification of the current operating policy is supported by input variable selection methods to select the most relevant hydrological and market based drivers to explain the observed release time series. The identified model is then simulated under a set of future scenarios, accounting for both climate and socio-economic change (e.g., expansion of the electric vehicle sector, load balancing from renewable energy), to eventually estimate the impacts on the multi-sector services involved (i.e., hydropower, flood protection, irrigation supply). Preliminary results show that the magnitude of the socio-economic change impacts is comparable with the one induced by climate change

Therapeutic Approaches in Adult Primary Spinal Cord Astrocytoma: A Systematic Review

The issue: Gliomas are primary tumors arising from supporting cells of the central nervous system (CNS), usually in the brain. The 2021 World Health Organization (WHO) classifies gliomas as adult-type diffuse gliomas or circumscribed astrocytic gliomas depending on their histology and molecular features. Spinal astrocytic gliomas are very rare, and nowadays no standard of therapy is available. Treatment options are limited: surgery is often not radical, and adjuvant therapies include mostly radiotherapy (RT) or systemic chemotherapy (CHT). There is lack of knowledge about the efficacy and safety of therapies and their multidisciplinary approaches. The aim of the review: A systematic review of the literature from January 2000 to June 2021 was performed, including both clinical trials and observational studies on histological adult primary spinal cord astrocytomas (SCA), with a minimum follow-up of 6 months and reporting the overall survival, progression-free survival or clinical neurological outcome after any therapeutic approach (surgery, RT or CHT). What are the main findings? A total of 1197 citations were identified by the Medline search and additional records; based on our inclusion criteria, 18 studies were included with a total of 285 adult patients. We documented the lack of any clinical trial. What are the conclusions? The available literature data are limited to series/retrospective studies, including heterogeneous patients, i.e., astrocytoma as well as ependymoma or pediatric/adult age, with scanty data on the outcomes of interest. No clinical trials have been run. Due to the rarity of this disease, multicentric clinical trials with molecular investigations are mandatory to better manage such a rare disease

ABCC3 Expressed by CD56dim CD16+ NK Cells Predicts Response in Glioblastoma Patients Treated with Combined Chemotherapy and Dendritic Cell Immunotherapy

Recently, we found that temozolomide (TMZ) can upregulate the expression of the multidrug-resistance protein ABCC3 in NK cells from both glioma-bearing mice and glioblastoma patients treated with dendritic cell immunotherapy combined with TMZ, allowing NK cells to escape apoptosis and favoring their role as antitumor effector cells. Here, we demonstrate that CD56dim NK cells expressing CD16+ are predominant in patients surviving more than 12 months after surgery without disease progression. CD56dim CD16+ NK cells co-expressed high levels of ABCC3 and IFN-γ. Notably, not only basal but also TMZ-induced ABCC3 expression was related to a strong, long-term NK cell response and a better prognosis of patients. The identification of the single nucleotide polymorphism (SNP) rs35467079 with the deletion of a cytosine (−897DelC) in the promoter region of the ABCC3 gene resulted associated with a better patient outcome. ABCC3 expression in patients carrying DelC compared to patients with reference haplotype was higher and modulated by TMZ. The transcription factor NRF2, involved in ABCC3 induction, was phosphorylated in CD56dim CD16+ NK cells expressing ABCC3 under TMZ treatment. Thus, ABCC3 protein and the SNP −897DelC can play a predictive role in patients affected by GBM, and possibly other cancers, treated with dendritic cell immunotherapy combined with chemotherapy

Neuronal differentiation potential of human adipose-derived mesenchymal stem cells

reserved9noPRIN 2005Adult mesenchymal stem cells derived from adipose tissue (A-MSC) have the capacity to differentiate in vitro into mesenchymal as well as endodermal and ectodermal cell lineages. We investigated the neuronal differentiation potential of human A-MSC with a protocol which included sphere formation followed by culture in brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). After 30 days, about 57% of A-MSC showed morphological, immunocytochemical and electrophysiological evidence of initial neuronal differentiation. In fact, A-MSC displayed elongated shape with protrusion of two or three cellular processes, selectively expressed nestin and neuronal molecules (including GABA receptor and tyroxine hydroxilase), but not glial phenotypic markers. Differentiated cells showed negative membrane potential (–60 mV), delayed rectifier potassium currents and TTX-sensitive sodium currents. Such changes were stable for at least 7 days after the removal of differentiation medium. In view of these results and the easy accessibility of adipose tissue, A-MSC may be a ready source of adult MSC with neuronal differentiation potential, an useful tool to treat neurodegenerative diseases.mixedAnghileri E; Marconi S; Pignatelli A; Cifelli P; Galié M; Sbarbati A; Krampera M; Belluzzi O; Bonetti BAnghileri, E; Marconi, S; Pignatelli, Angela; Cifelli, Pierangelo; Galié, M; Sbarbati, A; Krampera, M; Belluzzi, Ottorino; Bonetti, B