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Hypertension (HTN) impacts almost half of adults, predisposing them to cardiovascular disease and renal damage. Salt-sensitive HTN (SSHTN) and angiotensin II (A2)-induced HTN (A2HTN) both involve immune system activation and renal innate immune cell infiltration. Subpopulations of activated [Cluster of differentiation 38 (CD38)] innate immune cells, such as macrophages and dendritic cells (DCs), play distinct roles in modulating renal function and blood pressure. It is unknown how these cells become CD38+ or which subtypes are pro-hypertensive. When bone marrow-derived monocytes (BMDMs) were grown in granulocyte-macrophage colony stimulating factor (GM-CSF) and treated with salt or A2, CD38+ macrophages and CD38+ DCs increased. The adoptive transfer of GM-CSF-primed BMDMs into mice with either SSHTN or A2HTN increased renal CD38+ macrophages and CD38+ DCs. Flow cytometry revealed increased renal M1 macrophages and type-2 conventional DCs (cDC2s), along with their CD38+ counterparts, in mice with either SSHTN or A2HTN. These results were replicable in vitro. Either salt or A2 treatment of GM-CSF-primed BMDMs significantly increased bone marrow-derived (BMD)-M1 macrophages, CD38+ BMD-M1 macrophages, BMD-cDC2s, and CD38+ BMD-cDC2s. Overall, these data suggest that GM-CSF is necessary for the salt or A2 induction of CD38+ innate immune cells, and that CD38 distinguishes pro-hypertensive immune cells. Further investigation of CD38+ M1 macrophages and CD38+ cDC2s could provide new therapeutic targets for both SSHTN and A2HTN.
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Angiotensina II , Células Dendríticas , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Imunidade Inata , Macrófagos , Animais , Angiotensina II/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Camundongos , Imunidade Inata/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Hipertensão/imunologia , Camundongos Endogâmicos C57BL , ADP-Ribosil Ciclase 1/metabolismo , Masculino , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/imunologia , Rim/imunologia , Rim/efeitos dos fármacosRESUMO
PURPOSE: PARP inhibitors (PARPi) are effective in homologous recombination repair (HRR) defective (HRD) cancers. To (re)sensitise HRR proficient (HRP) tumours to PARPi combinations with other drugs are being explored. Our aim was to determine the mechanism underpinning the sensitisation to PARPi by inhibitors of cell cycle checkpoint kinases ATR, CHK1 and WEE1. EXPERIMENTAL DESIGN: A panel of HRD and HRP cells (including matched BRCA1 or 2 mutant and corrected pairs) and ovarian cancer ascites cells were used. Rucaparib (PARPi) induced replication stress (RS) and HRR (immunofluorescence microscopy for γH2AX and RAD51 foci, respectively), cell cycle changes (flow cytometry), activation of ATR, CHK1 and WEE1 (Western Blot for pCHK1S345, pCHK1S296 and pCDK1Y15, respectively) and cytotoxicity (colony formation assay) was determined, followed by investigations of the impact on all of these parameters by inhibitors of ATR (VE-821, 1 µM), CHK1 (PF-477736, 50 nM) and WEE1 (MK-1775, 100 nM). RESULTS: Rucaparib induced RS (3 to10-fold), S-phase accumulation (2-fold) and ATR, CHK1 and WEE1 activation (up to 3-fold), and VE-821, PF-477736 and MK-1775 inhibited their targets and abrogated these rucaparib-induced cell cycle changes in HRP and HRD cells. Rucaparib activated HRR in HRP cells only and was (60-1,000x) more cytotoxic to HRD cells. VE-821, PF-477736 and MK-1775 blocked HRR and sensitised HRP but not HRD cells and primary ovarian ascites to rucaparib. CONCLUSIONS: Our data indicate that, rather than acting via abrogation of cell cycle checkpoints, ATR, CHK1 and WEE1 inhibitors cause an HRD phenotype and hence "induced synthetic lethality" with PARPi.
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Salt-sensitive hypertension (SSHTN) is associated with M1 macrophage polarization and inflammatory responses, leading to inflammation-associated lymphangiogenesis and functional impairment across multiple organs, including kidneys and gonads. However, it remains unclear whether promoting M2 macrophage polarization can alleviate the hypertension, inflammation, and end organ damage in mice with salt sensitive hypertension (SSHTN). Male and female mice were made hypertensive by administering nitro-L-arginine methyl ester hydrochloride (L-NAME; 0.5 mg/ml) for 2 weeks in the drinking water, followed by a 2-week interval without any treatments, and a subsequent high salt diet for 3 weeks (SSHTN). AVE0991 (AVE) was intraperitoneally administered concurrently with the high salt diet. Control mice were provided standard diet and tap water. AVE treatment significantly attenuated BP and inflammation in mice with SSHTN. Notably, AVE promoted M2 macrophage polarization, decreased pro-inflammatory immune cell populations, and improved function in renal and gonadal tissues of mice with SSHTN. Additionally, AVE decreased lymphangiogenesis in the kidneys and testes of male SSHTN mice and the ovaries of female SSHTN mice. These findings highlight the effectiveness of AVE in mitigating SSHTN-induced elevated BP, inflammation, and end organ damage by promoting M2 macrophage polarization and suppressing pro-inflammatory immune responses. Targeting macrophage polarization emerges as a promising therapeutic approach for alleviating inflammation and organ damage in SSHTN. Further studies are warranted to elucidate the precise mechanisms underlying AVE-mediated effects and to assess its clinical potential in managing SSHTN.
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Hipertensão , Inflamação , Rim , Macrófagos , Cloreto de Sódio na Dieta , Animais , Masculino , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Feminino , Hipertensão/imunologia , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/patologia , Rim/imunologia , Linfangiogênese/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos , Pressão Sanguínea/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/patologia , Modelos Animais de DoençasRESUMO
We reported that salt-sensitive hypertension (SSHTN) is associated with increased pro-inflammatory immune cells, inflammation, and inflammation-associated lymphangiogenesis in the kidneys and gonads of male and female mice. However, it is unknown whether these adverse end organ effects result from increased blood pressure (BP), elevated levels of salt, or both. We hypothesized that pharmaceutically lowering BP would not fully alleviate the renal and gonadal immune cell accumulation, inflammation, and lymphangiogenesis associated with SSHTN. SSHTN was induced in male and female C57BL6/J mice by administering nitro-L-arginine methyl ester hydrochloride (L-NAME; 0.5 mg/ml) in their drinking water for 2 weeks, followed by a 2-week washout period. Subsequently, the mice received a 3-week 4% high salt diet (SSHTN). The treatment group underwent the same SSHTN induction protocol but received hydralazine (HYD; 250 mg/L) in their drinking water during the diet phase (SSHTN+HYD). Control mice received tap water and a standard diet for 7 weeks. In addition to decreasing systolic BP, HYD treatment generally decreased pro-inflammatory immune cells and inflammation in the kidneys and gonads of SSHTN mice. Furthermore, the decrease in BP partially alleviated elevated renal and gonadal lymphatics and improved renal and gonadal function in mice with SSHTN. These data demonstrate that high systemic pressure and salt differentially act on end organ immune cells, contributing to the broader understanding of how BP and salt intake collectively shape immune responses and highlight implications for targeted therapeutic interventions.
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Pressão Sanguínea , Hipertensão , Inflamação , Rim , Camundongos Endogâmicos C57BL , Cloreto de Sódio na Dieta , Animais , Hipertensão/imunologia , Hipertensão/fisiopatologia , Hipertensão/tratamento farmacológico , Hipertensão/induzido quimicamente , Masculino , Feminino , Pressão Sanguínea/efeitos dos fármacos , Cloreto de Sódio na Dieta/efeitos adversos , Rim/imunologia , Rim/efeitos dos fármacos , Inflamação/imunologia , Linfangiogênese/efeitos dos fármacos , Anti-Hipertensivos/farmacologia , Camundongos , Hidralazina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Modelos Animais de Doenças , Gônadas/efeitos dos fármacosRESUMO
AIMS: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with complex aetiology. Despite evidence of neuromuscular junction (NMJ) denervation and 'dying-back' pathology in models of SOD1-dependent ALS, evidence in other genetic forms of ALS is limited by a lack of suitable animal models. TDP-43, a key mediator protein in ALS, is overexpressed in neurons in Thy1-hTDP-43WT mice. We therefore aimed to comprehensively analyse NMJ pathology in this model of ALS. METHODS: Expression of TDP-43 was assessed via western blotting. Immunohistochemistry techniques, alongside NMJ-morph quantification, were used to analyse motor neuron number, NMJ denervation status and terminal Schwann cell morphology. RESULTS: We present a time course of progressive, region-specific motor neuron pathology in Thy1-hTDP-43WT mice. Thy1-driven hTDP-43 expression increased steadily, correlating with developing hindlimb motor weakness and associated motor neuron loss in the spinal cord with a median survival of 21 days. Pronounced NMJ denervation was observed in hindlimb muscles, mild denervation in cranial muscles but no evidence of denervation in either forelimb or trunk muscles. NMJ pathology was restricted to motor nerve terminals, with denervation following the same time course as motor neuron loss. Terminal Schwann cells were lost from NMJs in hindlimb muscles, directly correlating with denervation status. CONCLUSIONS: Thy1-hTDP-43WT mice represent a severe model of ALS, with NMJ pathology/denervation of distal muscles and motor neuron loss, as observed in ALS patients. This model therefore provides an ideal platform to investigate mechanisms of dying-back pathology, as well as NMJ-targeting disease-modifying therapies in ALS.
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Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Camundongos , Animais , Esclerose Lateral Amiotrófica/patologia , Doenças Neurodegenerativas/patologia , Junção Neuromuscular/patologia , Neurônios Motores/patologia , Células de Schwann/metabolismo , Células de Schwann/patologia , Denervação , Proteínas de Ligação a DNA/metabolismo , Camundongos Transgênicos , Modelos Animais de DoençasRESUMO
OBJECTIVE: The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection. ANIMALS STUDIED: Three adult horses with no evidence of ophthalmic or systemic disease. PROCEDURE: Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 µg/mL, 112.5 µg/mL, 187 µg/mL, 375 µg/mL, or 750 µg/mL then stimulated to proliferate with concanavalin A (ConA). 4',6-diamidino-2-phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5-day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative-free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. RESULTS: No statistically significant comparisons in CD3+ T-lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations. CONCLUSIONS: Gentamicin had no effect on equine peripheral blood CD3+ T-lymphocyte cell viability and proliferation in concentrations ranging from "safe" to "retinotoxic" in relation to intravitreal injection volumes. Low-dose intravitreal gentamicin may not suppress the Th1- and Th17-mediated immune response.
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Doenças dos Cavalos , Uveíte , Animais , Cavalos , Gentamicinas/farmacologia , Gentamicinas/uso terapêutico , Projetos de Pesquisa , Uveíte/tratamento farmacológico , Uveíte/veterinária , Linfócitos T , Proliferação de Células , Doenças dos Cavalos/tratamento farmacológicoRESUMO
The bone cancer osteosarcoma, found mainly in adolescents, routinely forms around the growth plate/metaphysis of long bones. Bone marrow composition changes with age, shifting from a more hematopoietic to an adipocyte-rich tissue. This conversion occurs in the metaphysis during adolescence, implicating a link between bone marrow conversion and osteosarcoma initiation. To assess this, the tri-lineage differentiation potential of human bone marrow stromal cells (HBMSCs) isolated from the femoral diaphysis/metaphysis (FD) and epiphysis (FE) was characterized and compared to two osteosarcoma cell lines, Saos-2 and MG63. Compared to FE-cells, FD-cells showed an increase in tri-lineage differentiation. Additionally, differences were found between the Saos-2 cells exhibiting higher levels of osteogenic differentiation, lower adipogenic differentiation, and a more developed chondrogenic phenotype than MG63, with the Saos-2 being more comparable to FD-derived HBMSCs. The differences found between the FD and FE derived cells are consistent with the FD region containing more hematopoietic tissue compared to the FE. This may be related to the similarities between FD-derived cells and Saos-2 cells during osteogenic and chondrogenic differentiation. These studies reveal distinct differences in the tri-lineage differentiations of 'hematopoietic' and 'adipocyte rich' bone marrow, which correlate with specific characteristics of the two osteosarcoma cell lines.
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Células-Tronco Mesenquimais , Osteossarcoma , Adolescente , Humanos , Osteogênese , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Células Cultivadas , Linhagem Celular , Células da Medula Óssea , Osteossarcoma/metabolismo , Células EstromaisRESUMO
Six PARP inhibitors (PARPi) are approved for cancer therapy as monotherapy agents in daily or twice-daily continuous dosing schedules to maintain the necessary continuous suppression of PARP activity. Continuous PARP inhibition is required for single-agent anticancer activity. To investigate if such intense schedules are necessary, we determined the durability of PARP inhibition up to 72 h after a 1 h pulse of 1 µM of five of the approved PARPi, rucaparib, olaparib, niraparib, talazoparib and pamiparib, in IGROV-1 and ES-2 (human ovarian cancer) cells. Rucaparib caused the most persistent inhibition of PARP activity when maintained at ≥75% at 72 h after drug withdrawal in both IGROV-1 and ES-2 cells, but inhibition was more rapidly lost with the other PARPi. PARPi are also under clinical investigation with ATR inhibitors, and thus, we evaluated the implications for scheduling with an ATR inhibitor (VE-821). Rucaparib enhanced VE-821 cytotoxicity in co-exposure, sequential and delayed (24 h drug-free) schedules in IGROV-1 and ES-2 cells. Olaparib and niraparib enhanced VE-821 cytotoxicity only in co-exposed cells and not in sequential exposures. These data have clinical implications for the scheduling of PARPi as a monotherapy and in combination with ATR inhibitors and other cytotoxic drugs.
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The contribution of immune cells in the initiation and maintenance of hypertension is undeniable. Several studies have established the association between hypertension, inflammation, and immune cells from the innate and adaptive immune systems. Here, we provide an update to our 2017 American Journal of Hypertension review on the overview of the cellular immune responses involved in hypertension. Further, we discuss the activation of immune cells and their contribution to the pathogenesis of hypertension in different in vivo models. We also highlight existing gaps in the field of hypertension that need attention. The main goal of this review is to provide a knowledge base for translational research to develop therapeutic strategies that can improve cardiovascular health in humans.
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Hipertensão , Imunidade Inata , Humanos , Hipertensão/patologia , Sistema Imunitário , InflamaçãoRESUMO
INTRODUCTION: Standardized patient (SP) methodology has been used in health professional education to help students develop communication, deeper diagnostic reasoning, and critical thinking skills. Few examples demonstrate the use of SPs to practice difficult conversations with pediatric caregivers in the pediatric dentistry literature. The objective of this educational activity was to describe the implementation of three SPs in a pediatric dentistry course for second-year dental students. METHODS: We developed three SP encounters covering interactions with caregivers of an infant with severe early childhood caries, an adolescent on the path to gender affirmation, and a child with autism and dental caries whose caregiver was resistant to fluoride- and silver-containing dental materials. We describe the case design process, rubric construction and calibration, student debriefing, and pandemic modifications. We evaluated the effectiveness of the implementation by thematic analysis of student reflections following each encounter using a qualitative descriptive framework. RESULTS: Eighty-three students completed each encounter. Qualitative analysis showed that students preferred a more realistic encounter by having a child or other distraction present. Students relied on different elements of motivational interviewing depending on the objective of each encounter and the age of the patient. Overall, the SP encounters were well received by students and faculty as an alternative or supplement to traditional student evaluation methods. DISCUSSION: We noted a number of lessons learned about implementing SP methodology in pediatric dental education. With these experiences now in place, future evaluations should measure student performance in the SP encounters against student performance during clinical care.
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Cuidadores , Cárie Dentária , Adolescente , Criança , Pré-Escolar , Comunicação , Docentes , Humanos , Lactente , Odontopediatria/educaçãoRESUMO
Doping has proven to be a critical tool for enhancing the performance of organic semiconductors in devices like organic light-emitting diodes. However, the challenge in working with high-ionization-energy (IE) organic semiconductors is to find p-dopants with correspondingly high electron affinity (EA) that will improve the conductivity and charge carrier transport in a film. Here, we use an oxidant that has been recently recognized to be a very strong p-type dopant, hexacyano-1,2,3-trimethylene-cyclopropane (CN6-CP). The EA of CN6-CP has been previously estimated via cyclic voltammetry to be 5.87 eV, almost 300 meV higher than other known high-EA organic molecular oxidants. We measure the frontier orbitals of CN6-CP using ultraviolet and inverse photoemission spectroscopy techniques and confirm a high EA value of 5.88 eV in the condensed phase. The introduction of CN6-CP in a film of large-band-gap, large-IE phenyldi(pyren-1-yl)phosphine oxide (POPy2) leads to a significant shift of the Fermi level toward the highest occupied molecular orbital and a 2 orders of magnitude increase in conductivity. Using CN6-CP and n-dopant (pentamethylcyclopentadienyl)(1,3,5-trimethylbenzene)ruthenium (RuCp*Mes)2, we fabricate a POPy2-based rectifying p-i-n homojunction diode with a 2.9 V built-in potential. Blue light emission is achieved under forward bias. This effect demonstrates the dopant-enabled hole injection from the CN6-CP-doped layer and electron injection from the (RuCp*Mes)2-doped layer in the diode.
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2D polymers (2DPs) are promising as structurally well-defined, permanently porous, organic semiconductors. However, 2DPs are nearly always isolated as closed shell organic species with limited charge carriers, which leads to low bulk conductivities. Here, the bulk conductivity of two naphthalene diimide (NDI)-containing 2DP semiconductors is enhanced by controllably n-doping the NDI units using cobaltocene (CoCp2 ). Optical and transient microwave spectroscopy reveal that both as-prepared NDI-containing 2DPs are semiconducting with sub-2 eV optical bandgaps and photoexcited charge-carrier lifetimes of tens of nanoseconds. Following reduction with CoCp2 , both 2DPs largely retain their periodic structures and exhibit optical and electron-spin resonance spectroscopic features consistent with the presence of NDI-radical anions. While the native NDI-based 2DPs are electronically insulating, maximum bulk conductivities of >10-4 S cm-1 are achieved by substoichiometric levels of n-doping. Density functional theory calculations show that the strongest electronic couplings in these 2DPs exist in the out-of-plane (π-stacking) crystallographic directions, which indicates that cross-plane electronic transport through NDI stacks is primarily responsible for the observed electronic conductivity. Taken together, the controlled molecular doping is a useful approach to access structurally well-defined, paramagnetic, 2DP n-type semiconductors with measurable bulk electronic conductivities of interest for electronic or spintronic devices.
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MΦs display remarkable plasticity and the ability to activate diverse responses to a host of intracellular and external stimuli. Despite extensive characterization of M1 MΦs and a broad set of M2 MΦs, comprehensive characterization of functional phenotype and associated metabotype driving this diverse MΦ activation remains. Herein, an ex vivo model was utilized to produce 6 MΦ functional phenotypes. Isolated CD14+ PBMCs were differentiated into resting M0 MΦs, and then polarized into M1 (IFN-γ/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) MΦs. The MΦs were profiled using a bioanalyte matrix of 4 cell surface markers, â¼50 secreted proteins, â¼800 expressed myeloid genes, and â¼450 identified metabolites relative to M0 MΦs. Signal protein and expressed gene profiles grouped the MΦs into inflammatory (M1 and M2b) and wound resolution (M2a, M2c, and M2d) phenotypes; however, each had a unique metabolic profile. While both M1 and M2b MΦs shared metabotype profiles consistent with an inflammatory signature; key differences were observed in the TCA cycle, FAO, and OXPHOS. Additionally, M2a, M2c, and M2d MΦs all profiled as tissue repair MΦs; however, metabotype differences were observed in multiple pathways including hexosamine, polyamine, and fatty acid metabolism. These metabolic and other key functional distinctions suggest phagocytic and proliferative functions for M2a MΦs, and angiogenesis and ECM assembly capabilities for M2b, M2c, and M2d MΦs. By integrating metabolomics into a systems analysis of MΦ phenotypes, we provide the most comprehensive map of MΦ diversity to date, along with the global metabolic shifts that correlate to MΦ functional plasticity in these phenotypes.
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Lipopolissacarídeos , Transcriptoma , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Metabolômica , FenótipoRESUMO
Synapses are particularly vulnerable in many neurodegenerative diseases and often the first to degenerate, for example in the motor neuron disease spinal muscular atrophy (SMA). Compounds that can counteract synaptic destabilisation are rare. Here, we describe an automated screening paradigm in zebrafish for small-molecule compounds that stabilize the neuromuscular synapse in vivo. We make use of a mutant for the axonal C-type lectin chondrolectin (chodl), one of the main genes dysregulated in SMA. In chodl-/- mutants, neuromuscular synapses that are formed at the first synaptic site by growing axons are not fully mature, causing axons to stall, thereby impeding further axon growth beyond that synaptic site. This makes axon length a convenient read-out for synapse stability. We screened 982 small-molecule compounds in chodl chodl-/- mutants and found four that strongly rescued motor axon length. Aberrant presynaptic neuromuscular synapse morphology was also corrected. The most-effective compound, the adenosine uptake inhibitor drug dipyridamole, also rescued axon growth defects in the UBA1-dependent zebrafish model of SMA. Hence, we describe an automated screening pipeline that can detect compounds with relevance to SMA. This versatile platform can be used for drug and genetic screens, with wider relevance to synapse formation and stabilisation.
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Avaliação Pré-Clínica de Medicamentos , Atrofia Muscular Espinal/patologia , Sinapses/patologia , Peixe-Zebra/fisiologia , Animais , Automação , Axônios/efeitos dos fármacos , Axônios/metabolismo , Dipiridamol/farmacologia , Modelos Animais de Doenças , Testes Genéticos , Atrofia Muscular Espinal/genética , Mutação/genética , Fenótipo , Terminações Pré-Sinápticas/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.
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Neoplasias Ósseas/ultraestrutura , Membrana Corioalantoide/ultraestrutura , Modelos Teóricos , Osteossarcoma/ultraestrutura , Animais , Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Membrana Corioalantoide/metabolismo , Humanos , Osteossarcoma/genética , Estudos Prospectivos , Microambiente Tumoral/genéticaRESUMO
DNA damage response (DDR) pathway prevents high level endogenous and environmental DNA damage being replicated and passed on to the next generation of cells via an orchestrated and integrated network of cell cycle checkpoint signalling and DNA repair pathways. Depending on the type of damage, and where in the cell cycle it occurs different pathways are involved, with the ATM-CHK2-p53 pathway controlling the G1 checkpoint or ATR-CHK1-Wee1 pathway controlling the S and G2/M checkpoints. Loss of G1 checkpoint control is common in cancer through TP53, ATM mutations, Rb loss or cyclin E overexpression, providing a stronger rationale for targeting the S/G2 checkpoints. This review will focus on the ATM-CHK2-p53-p21 pathway and the ATR-CHK1-WEE1 pathway and ongoing efforts to target these pathways for patient benefit.
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Pontos de Checagem do Ciclo Celular , Dano ao DNA , Reparo do DNA , Neoplasias/enzimologia , Proteínas Quinases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
PARP inhibition results in the accumulation of DNA SSBs, causing replication stress (RS) and lesions that can only be resolved by homologous recombination repair (HRR). Defects in HRR, e.g., due to BRCA2 mutation, confer profound sensitivity to PARP inhibitor (PARPi) cytotoxicity. In response to RS, CHK1 is activated to signal to S and G2/M cell cycle checkpoints and also to HRR. To determine the relative contribution of these two functions of CHK1 to survival following PARPi exposure, we investigated the effects of rucaparib (a PARPi) and PF-477736 (a CHK1 inhibitor) alone and in combination in cells with mutated and corrected BRCA2. The BRCA2 mutated V-C8 cells were 1000× more sensitive to rucaparib cytotoxicity than their matched BRCA2 corrected V-C8.B2 cells, but no more sensitive to PF-477736 despite having seven-fold higher levels of RS. PF-477736 caused a five-fold enhancement of rucaparib cytotoxicity in the V-C8.B2 cells, but no enhancement in the V-C8 cells. This differential sensitivity was not due to a difference in PARP1 or CHK1 expression or activity. PF-477736 increased rucaparib-induced RS (γH2AX foci) and completely inhibited RAD51 focus formation, indicating a profound suppression of HRR. Our data suggested that inhibition of HRR was the main mechanism of sensitisation to rucaparib, compounded with an inhibition of cell cycle checkpoints by PF-477736.
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Autism spectrum disorder (ASD) is genetically heterogeneous with convergent symptomatology, suggesting common dysregulated pathways. In this study, we analyzed brain transcriptional changes in five mouse models of Pitt-Hopkins syndrome (PTHS), a syndromic form of ASD caused by mutations in the TCF4 gene, but not the TCF7L2 gene. Analyses of differentially expressed genes (DEGs) highlighted oligodendrocyte (OL) dysregulation, which we confirmed in two additional mouse models of syndromic ASD (Ptenm3m4/m3m4 and Mecp2tm1.1Bird). The PTHS mouse models showed cell-autonomous reductions in OL numbers and myelination, functionally confirming OL transcriptional signatures. We also integrated PTHS mouse model DEGs with human idiopathic ASD postmortem brain RNA-sequencing data and found significant enrichment of overlapping DEGs and common myelination-associated pathways. Notably, DEGs from syndromic ASD mouse models and reduced deconvoluted OL numbers distinguished human idiopathic ASD cases from controls across three postmortem brain data sets. These results implicate disruptions in OL biology as a cellular mechanism in ASD pathology.
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Transtorno do Espectro Autista/genética , Impressões Digitais de DNA , Hiperventilação/genética , Deficiência Intelectual/genética , Bainha de Mielina/genética , Transcriptoma/genética , Envelhecimento , Animais , Contagem de Células , Fácies , Regulação da Expressão Gênica , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Knockout , Oligodendroglia/metabolismo , PTEN Fosfo-Hidrolase/genética , Cultura Primária de Células , Transdução de Sinais/genética , Fator de Transcrição 4/genéticaRESUMO
BACKGROUND: Previous data suggests that anti-OX40 mAb can elicit anti-tumor effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. Human trials with anti-OX40 antibodies have shown that these entities are well tolerated but to date have delivered disappointing clinical responses, indicating that the rules for the optimal use of anti-human OX40 (hOX40) antibodies is not yet fully understood. Changes to timing and dosages may lead to improved outcomes; however, here we focus on addressing the role of agonism versus depleting activity in determining therapeutic outcomes. We investigated a novel panel of anti-hOX40 mAb to understand how these reagents and mechanisms may be optimized for therapeutic benefit. METHODS: This study examines the binding activity and in vitro activity of a panel of anti-hOX40 antibodies. They were further evaluated in several in vivo models to address how isotype and epitope determine mechanism of action and efficacy of anti-hOX40 mAb. RESULTS: Binding analysis revealed the antibodies to be high affinity, with epitopes spanning all four cysteine-rich domains of the OX40 extracellular domain. In vivo analysis showed that their activities relate directly to two key properties: (1) isotype-with mIgG1 mAb evoking receptor agonism and CD8+ T-cell expansion and mIgG2a mAb evoking deletion of Treg and (2) epitope-with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumor models by engaging these different mechanisms. CONCLUSION: These findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T-cell expansion or Treg depletion might be preferred according to the composition of different tumors. As many of the current clinical trials using OX40 antibodies are now using combination therapies, this understanding of how to manipulate therapeutic activity will be vital in directing new combinations that are more likely to improve efficacy and clinical outcomes.