Engineering gamma delta T cells limits tonic signaling associated with chimeric antigen receptors

Despite the benefits of chimeric antigen receptor (CAR)–T cell therapies against lymphoid malignancies, responses in solid tumors have been more limited and off-target toxicities have been more marked. Among the possible design limitations of CAR-T cells for cancer are unwanted tonic (antigen-independent) signaling and off-target activation. Efforts to overcome these hurdles have been blunted by a lack of mechanistic understanding. Here, we showed that single-cell analysis with time course mass cytometry provided a rapid means of assessing CAR-T cell activation. We compared signal transduction in expanded T cells to that in T cells transduced to express second-generation CARs and found that cell expansion enhanced the response to stimulation. However, expansion also induced tonic signaling and reduced network plasticity, which were associated with expression of the T cell exhaustion markers PD-1 and TIM-3. Because this was most evident in pathways downstream of CD3ζ, we performed similar analyses on γδT cells that expressed chimeric costimulatory receptors (CCRs) lacking CD3ζ but containing DAP10 stimulatory domains. These CCR-γδT cells did not exhibit tonic signaling but were efficiently activated and mounted cytotoxic responses in the presence of CCR-specific stimuli or cognate leukemic cells. Single-cell signaling analysis enabled detailed characterization of CAR-T and CCR-T cell activation to better understand their functional activities. Furthermore, we demonstrated that CCR-γδT cells may offer the potential to avoid on-target, off-tumor toxicity and allo-reactivity in the context of myeloid malignancies

Characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes exposed to low frequency low energy pulsed electromagnetic fields

SummaryObjectiveThe present study describes the presence and binding parameters of the A1, A2A, A2B and A3 adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes. The effect of low frequency low energy pulsed electromagnetic fields (PEMFs) on the adenosine receptor affinity and density was studied.MethodsSaturation, competition binding experiments and Western blotting assays in the absence and in the presence of PEMFs on the adenosine receptors in bovine chondrocytes or fibroblast-like synoviocytes were performed. Thermodynamic analysis of the A2A or A3 binding was studied to investigate the forces driving drug–receptor coupling. In the adenylyl cyclase and proliferation assays the potency of typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs was evaluated.ResultsBovine chondrocytes and fibroblast-like synoviocytes expressed all adenosine receptors. PEMFs evoked an up-regulation of A2A and A3 receptors and thermodynamic parameters indicate that adenosine binding is enthalpy and entropy driven. In PEMF-treated cells the potency of typical A2A or A3 agonists on cyclic AMP assays was significantly increased when compared with the untreated cells. PEMFs potentiated the effect of A2A or A3 agonists on cell proliferation in both cell types.ConclusionsPEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities in bovine chondrocytes and fibroblast-like synoviocytes. No differences are present in adenosine affinity and in the drug–receptor interactions. Our data could be used as a trigger to future studies addressed to PEMFs and adenosine therapeutic intervention in inflammatory joint diseases

Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells

Objectives: Polycystin-1 (PC1), a signalling receptor regulating Ca2+-permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca2+ homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non-capacitative Ca2+ entry (NCCE) and Ca2+ oscillations, with downstream effects on cell proliferation. Results and discussion: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum-induced Ca2+ oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca2+ oscillations and cell proliferation are increased in PC1-mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca2+ oscillations and a molecular mechanism to explain the association between abnormal Ca2+ homeostasis and cell proliferation in autosomal dominant polycystic kidney disease

A consensus panel review of central nervous system effects of the exposure to low-intensity extremely low-frequency magnetic fields

BACKGROUND: A large number of studies explored the biological effects of extremely low-frequency (0-300 Hz) magnetic fields (ELF-MFs) on nervous system both at cellular and at system level in the intact human brain reporting several functional changes. However, the results of different studies are quite variable and the mechanisms of action of ELF-MFs are still poorly defined. The aim of this paper is to provide a comprehensive review of the effects of ELF-MFs on nervous system. METHODS: We convened a workgroup of researchers in the field to review and discuss the available data about the nervous system effects produced by the exposure to ELF-MFs. MAIN FINDINGS/DISCUSSION: We reviewed several methodological, experimental and clinical studies and discussed the findings in five sections. The first section analyses the devices used for ELF-MF exposure. The second section reviews the contribution of the computational methods and models for investigating the interaction between ELF-MFs and neuronal systems. The third section analyses the experimental data at cellular and tissue level showing the effects on cell membrane receptors and intracellular signaling and their correlation with neural stem cell proliferation and differentiation. The fourth section reviews the studies performed in the intact human brain evaluating the changes produced by ELF-MFs using neurophysiological and neuropsychological methods. The last section shows the limits and shortcomings of the available data, evidences the key challenges in the field and tracks directions for future research

Pathophysiological Role and Medicinal Chemistry of A2A Adenosine Receptor Antagonists in Alzheimer's Disease

The A(2A) adenosine receptor is a protein belonging to a family of four GPCR adenosine receptors. It is involved in the regulation of several pathophysiological conditions in both the central nervous system and periphery. In the brain, its localization at pre- and postsynaptic level in striatum, cortex, hippocampus and its effects on glutamate release, microglia and astrocyte activation account for a crucial role in neurodegenerative diseases, including Alzheimer's disease (AD). This ailment is considered the main form of dementia and is expected to exponentially increase in coming years. The pathological tracts of AD include amyloid peptide-beta extracellular accumulation and tau hyperphosphorylation, causing neuronal cell death, cognitive deficit, and memory loss. Interestingly, in vitro and in vivo studies have demonstrated that A(2A) adenosine receptor antagonists may counteract each of these clinical signs, representing an important new strategy to fight a disease for which unfortunately only symptomatic drugs are available. This review offers a brief overview of the biological effects mediated by A(2A) adenosine receptors in AD animal and human studies and reports the state of the art of A(2A) adenosine receptor antagonists currently in clinical trials. As an original approach, it focuses on the crucial role of pharmacokinetics and ability to pass the blood-brain barrier in the discovery of new agents for treating CNS disorders. Considering that A(2A) receptor antagonist istradefylline is already commercially available for Parkinson's disease treatment, if the proof of concept of these ligands in AD is confirmed and reinforced, it will be easier to offer a new hope for AD patients

The pandemic within the pandemic: the surge of neuropsychological disorders in Italian children during the COVID-19 era

Background: Quarantine and isolation measures during COVID-19 pandemic may have caused additional stress and challenged the mental health of the youth. Aim of the study is to investigate the COVID-19 pandemic impact on neuropsychological disorders (NPD) of Italian children and adolescents to provide general pediatric recommendations. Material and methods: A retrospective multicenter observational study was planned by the Italian Pediatric Society (SIP) to explore the impact of COVID-19 on the access of children to pediatric Emergency Departments (pED) for the evaluation of neuropsychological symptoms, collecting the classification codes of diagnoses between March 1, 2019 and March 2, 2021. The period study was split into two sub-periods: a pre COVID-19 period (from March 1 2019 to March 1, 2020) and a COVID-19 period (from March 2, 2020 to March 2, 2021). As additional information, data on NPD hospitalizations in any pediatric department of the involved centers were recorded. Results: During the study period, a total of 533,318 children were admitted to the pED involved in the study. Despite a 48.2% decline of pED admissions, there was a significant increase (83.1%) in patient admissions for NPD. The most frequent NPD conditions which increased during the COVID-19 pandemic were suicidal ideation (+ 147%), depression (+ 115%), eating disorder (+ 78.4%), and psychosis (+ 17.2%). During the pandemic period, a 39.5% increase in NPD hospitalizations was observed as well. The NPD disorders that mostly required hospitalizations were suicidal ideation (+ 134%), depression (+ 41.4%), eating disorder (+ 31.4%), and drug abuse (+ 26.7%). COVID-19 pandemic had a major impact on children's health, mainly on their NPD development. Neuropsychological assessment should be required at the primary level, in the pediatrician's office, to facilitate early capture of the sign of impairment and provide an adequate treatment. Conclusion: SIP underlines the psychological consequences of COVID 19 pandemic on the youngest and recommends an early identification of NPD in the pediatric population to avoid other serious consequences for children's physical and mental health

Odontostomatologic management of patients receiving oral anticoagulant therapy: a retrospective multicentric study

Introduction: Today, we frequently find patients taking oral anticoagulant therapy (OAT), a prophylaxis against the occurrence of thromboembolic events. An oral surgeon needs to know how to better manage such patients, in order to avoid hemorrhagic and thromboembolic complications. Materials and methods: A group of 193 patients (119 men aged between 46 and 82 and 74 women aged between 54 and 76) undergoing OAT for more than 5 years were managed with a standardized management protocol and a 2-months follow-up. The aim of the present study was to apply a protocol, which could provide a safe intra- and postoperative management of patients on OAT. Results: Among the 193 patients, only 2 had postoperative complications. Conclusions: We think that the protocol used in the present study can be used for complete safety in the treatment of this type of patients. Keywords: Oral Anticoagulant Therapy (OAT), Tranexamic Acid, Oral Surger

Study on psychoeducation enhancing results of adherence in patients with schizophrenia (SPERA-S): study protocol for a randomized controlled trial.

BACKGROUND: Poor adherence to pharmacotherapy negatively affects the course and the outcome of schizophreniaspectrum psychoses, enhancing the risk of relapse. Falloon and coworkers developed a Psychoeducation Program aimed at improving communication and problem-solving abilities in patients and their families. This study set out to evaluate changes in adherence to pharmacotherapy in patients diagnosed with schizophrenia-spectrum psychoses, by comparing one group exposed to the Falloon Psychoeducation Program (FPP) with another group exposed to family supportive therapy with generic information on the disorders. METHODS: 340 patients diagnosed with schizophrenia and related disorders according to standardized criteria from 10 participating units distributed throughout the Italian National Health System (NHS), will be enrolled with 1:1 allocation by the method of blocks of randomized permutations. Patients will be reassessed at 6, 12 and 18 months after start of treatment (duration: 6 months).The primary objective is to evaluate changes in adherence to pharmacotherapy after psychoeducation. Adherence will be assessed at three-month intervals by measuring blood levels of the primary prescribed drug using high pressure liquid chromatography, and via the Medication Adherence Questionnaire and a modified version of the Adherence Interview. Secondary objectives are changes in the frequency of relapse and readmission, as the main indicator of the course of the disorder.Enrolled patients will be allocated to the FPP (yes/no) randomly, 1:1, in a procedure controlled by the coordinating unit; codes will be masked until the conclusion of the protocol (or the occurrence of a severe negative event). The raters will be blind to treatment allocation and will be tested for blinding after treatment completion. Intention-to-treat will be applied in considering the primary and secondary outcomes. Multiple imputations will be applied to integrate the missing data. The study started recruitment in February 2013; the total duration of the study is 27 months. DISCUSSION: If the psychoeducation program proves effective in improving adherence to pharmacotherapy and in reducing relapse and readmissions, its application could be proposed as a standard adjunctive psychosocial treatment within the Italian NHS