The application of graph theory measures in the study of functional brain networks allows for the description of their general properties and their alterations in mental illness. Among these ...measures, connectivity strength (CS) estimates the degree of functional connectivity of the whole network. Previous studies in schizophrenia patients have reported higher baseline CS values and modulation deficits in EEG spectral properties during cognitive activity. The specificity of these alterations and their relationships with pharmacological treatments remain unknown. Therefore, in the present study, we assessed functional CS on EEG-based brain networks in 79 schizophrenia and 29 bipolar patients in addition to 63 healthy controls. The subjects performed a P300 task during the EEG recordings from which the pre-stimulus and the task-related modulation CS values were computed in the global and theta bands. These values were compared between the groups and between the patients who had and had not received different treatments. The global band pre-stimulus CS was significantly higher in the schizophrenia group compared with the bipolar and control groups. Theta band CS modulation was decreased in schizophrenia and bipolar patients. Treatment with antipsychotics, lithium, benzodiazepines, and anticonvulsants did not significantly alter these CS values. The first-episode and chronic schizophrenia patients did not show significant differences in CS values. Higher global band pre-stimulus CS values were associated with worse general cognition in schizophrenia patients. These data support increased connectivity in the whole-brain network that is specific to schizophrenia and suggest a general hyper-synchronized basal state that might hamper cognition in this syndrome.
•Connectivity strength CS assesses the functional connectivity of brain networks.•Global band pre-stimulus CS values are specifically higher in schizophrenia.•Pharmacological treatment is not associated with the altered CS values.•CS values in schizophrenia are associated with worse general cognition.
NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express ...NKG2D receptors, including natural killer (NK) cells and CD8
T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D-NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated
and
the safety and cytotoxic capacity against osteosarcoma cells of CD45RA
memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR).
CD45RA
cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry.
cytotoxicity of NKG2D-CAR
CD45RA
T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the
orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rg
mice.
Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA
cells. Genetic stability was preserved in transduced cells.
, NKG2D-CAR
memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR
memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective.
Our results demonstrate NKG2D-4-1BB-CD3z CAR-redirected memory T cells target NKG2DL-expressing osteosarcoma cells
and
and could be a promising immunotherapeutic approach for patients with osteosarcoma.
.
Introduction
Approximately 10% of pediatric patients with cancer have an inherited, sometimes masked, cancer predisposition syndrome (CPS). Identifying patients with genetic susceptibility to ...malignant disease is essential for their correct diagnosis and clinical management.
Materials and Methods
Here, we present the workflow and experience of a multidisciplinary cancer predisposition unit focused on pediatric patients with cancer.
Results
Between July 2018 and July 2020, 214 patients were diagnosed with pediatric cancer in our Hospital. Of all, 49 patients were treated at the CPS unit, 48 of whom were recommended a genetic study. Mutational analysis was performed on DNA from peripheral blood samples, with approximately 45% of the patients (
n
= 22) receiving a confirmed CPS diagnosis, all of whom underwent genetic counseling. These cases represent 20% of all pediatric cancers diagnosed in the same center during this period. Most of the patients were diagnosed with hereditary retinoblastoma; however, we also identified families with Li-Fraumeni syndrome, multiple endocrine neoplasia type 2, hereditary melanoma, hereditary leiomyomatosis, and Gardner syndrome.
Conclusion
Despite its limitations regarding the type of tumors and number of patients included, this study revealed that implementing a specialized unit focused on children with cancer results in a higher diagnostic rate and better genetic counseling for patients with pediatric cancer predisposition syndromes.
Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors. ...NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3ζ signaling domain. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients' own T cells, disease progression, and death prior to return of engineered cells. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. host reactions may occur. To avoid such adverse effects, we used CD45RA
memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. CD45RA
fraction was depleted from healthy donors' non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. A total of 10
CD45RA
cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. Then, we used NKG2D-CD8TM-4-1BB-CD3ζ lentiviral vector for cell transduction (MOI = 2). NKG2D CAR T cells expanded between 10 and 13 days. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency of Medicines and Medical Devices (AEMPS) for the manufacture of investigational advanced therapy medicinal products (ATMPs). We performed four validations. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. CAR T cell final products met release criteria, except for one showing
overexpression and another with viral copy number higher than five. Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies.
The identification of the cerebral substrates of psychoses such as schizophrenia and bipolar disorder is likely hampered by its biological heterogeneity, which may contribute to the low replication ...of results in the field. In this study we aimed to replicate in a completely new sample and supplement the results of a previous study with additional data on this topic. In the aforementioned study we identified a schizophrenia cluster characterized by high mean cortical curvature and low cortical thickness, subcortical hypometabolism and progressive negative symptoms. Here, we have used magnetic resonance images from 61 schizophrenia and 28 bipolar patients, as well as 51 healthy controls and a cluster analysis to search for possible subgroups primarily characterized by cerebral structural data. Diffusion tensor imaging (fractional anisotropy, FA), cognition, clinical data and electroencephalographic (EEG) modulation during a P300 task were used to validate the possible clusters. Two clusters of patients were identified. The first cluster (29 schizophrenia and 18 bipolar patients) showed decreased cortical thickness and area values, as well as lower subcortical volumes and higher cortical curvature in some regions, as compared to the second cluster. This first cluster also showed decreased FA in frontal lobe connections and worse cognitive performance. Although this cluster also showed longer illness duration, there were first episode patients in both clusters and treatment doses and types were not different between clusters. Both clusters of patients showed decreased EEG task-related modulation. In conclusion, our data give additional support to a distinct biologically based cluster encompassing schizophrenia and bipolar disorder patients with cortical and subcortical alterations, hampered cortical connectivity and lower cognitive performance.
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•Two subgroups can be identified across schizophrenia and bipolar disorder based on neuroanatomy.•Reduced cortical thickness and increased folding characterize one of the subgroups.•That subgroup showed hampered structural connectivity and cognition.•Both groups did not differ on functional activity modulation during cognitive tasks.
Natural killer (NK) cells are lymphocytes from the innate immune system part of the first defense barrier against infected and transformed cells, representing 5%-15% of peripheral blood lymphocytes. ...The cytotoxic capacity of NK cells is controlled by a balance between inhibitory and activating NK receptors expressed on their surface, which recognize and interact with the ligands on stressed cells. The cytokines involved in NK cell activation, proliferation, survival, and cytotoxicity are signaled mainly through the Janus kinase and signal transducer and activator of transcription proteins (JAK/STAT) pathway. NK cells are also activated in response to pathogens through Toll-like receptors (TLRs) expressed on their surface. Ruxolitinib is a specific JAK1/2 inhibitor approved for treating myelofibrosis and for steroid-refractory acute and chronic graft-versus-host disease (SR-GvHD).
Purified NK cells from healthy donors were stimulated with two TOLL-like receptor ligands, LPS and CpG, in the presence of different concentrations of Ruxolitinib.
This study showed the effects of ruxolitinib on TLR4 and TLR9 ligand-activated NK cells from healthy donors. Ruxolitinib did not completely inhibit STAT3 phosphorylation and had a moderate effect on NK cell cytokine activation via the TLR pathway. Only the highest doses of ruxolitinib led to a decrease in the pro-inflammatory cytokines tumor necrosis factor α, interferon-γ, interleukin-6, and interleukin-1β. The cytotoxic capacity of stimulated NK cells versus K562, SEM, and MV-4-11 cell lines was reduced by increasing doses of ruxolitinib, but it was not completely abolished and we observed no major changes in degranulation capacity. Phenotypic changes were observed in activated NK cells in the presence of ruxolitinib. In a small cohort of pediatric patients treated with ruxolitinib for SR-GvHD, we observed no decrease in NK cell counts; however, further prospective studies with larger cohorts are necessary to confirm this finding.
In summary, our results showed that the functional capabilities and phenotype of NK cells activated through TLR4/9 agonists were not completely abolished by the inhibition of the JAK-STAT pathway by ruxolitinib.
Connexins are thought to solely mediate cell-to-cell communication by forming gap junction channels composed of two membrane-spanning hemichannels positioned end-to-end. However, many if not all ...connexin isoforms also form functional hemichannels (i.e., the precursors of complete channels) that mediate the rapid exchange of ions, second messengers and metabolites between the cell interior and the interstitial space. Electrical and molecular signaling via connexin hemichannels is now widely recognized to be important in many physiological scenarios and pathological conditions. Indeed, mutations in connexins that alter hemichannel function have been implicated in several diseases. Here, we present a comprehensive overview of how hemichannel activity is tightly regulated by membrane potential and the external calcium concentration. In addition, we discuss the genetic mutations known to alter hemichannel function and their deleterious effects, of which a better understanding is necessary to develop novel therapeutic approaches for diseases caused by hemichannel dysfunction.
This article is part of the Special Issue Section entitled ‘Current Pharmacology of Gap Junction Channels and Hemichannels’.
•Ubiquitous presence of functional hemichannels at the cell surface as a normal phase in the connexin life cycle.•Strict regulation of hemichannel activity by membrane potential and extracellular calcium.•Update of mechanisms and molecular basis of hemichannel voltage-gating and of regulation by calcium.•Hemichannel dysfunction in “connexinopathies”.
Introduction
Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, ...expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation.
Methods
In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA
-
) T cells
in vitro
and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored
in vitro
how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells’ cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and
in vivo
functionality.
Results
In vitro
, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL.
In vivo
, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and
in vivo
, they exhibited functional properties of leukemia initiating cells.
Discussion
The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.
Relapsed and refractory (R/r) disease in paediatric acute leukaemia remains the first reason for treatment failure. Advances in molecular characterisation can ameliorate the identification of genetic ...biomarkers treatment strategies for this disease, especially in high-risk patients. The purpose of this study was to analyse a cohort of R/r children diagnosed with acute lymphoblastic (ALL) or myeloid (AML) leukaemia in order to offer them a targeted treatment if available. Advanced molecular characterisation of 26 patients diagnosed with R/r disease was performed using NGS, MLPA, and RT-qPCR. The clinical relevance of the identified alterations was discussed in a multidisciplinary molecular tumour board (MTB). A total of 18 (69.2%) patients were diagnosed with B-ALL, 4 (15.4%) with T-ALL, 3 (11.5%) with AML and 1 patient (3.8%) with a mixed-phenotype acute leukaemia (MPL). Most of the patients had relapsed disease (88%) at the time of sample collection. A total of 17 patients (65.4%) were found to be carriers of a druggable molecular alteration, 8 of whom (47%) received targeted therapy, 7 (87.5%) of them in addition to hematopoietic stem cell transplantation (HSCT). Treatment response and disease control were achieved in 4 patients (50%). In conclusion, advanced molecular characterisation and MTB can improve treatment and outcome in paediatric R/r acute leukaemias.
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