RESUMO
ICAP-1 regulates ß1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4ß1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1-/- spleen T and B cells displayed upregulation of α4ß1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+ - and CD19+ -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linfócitos B , Linfócitos T CD8-Positivos , Ativação Linfocitária , Timócitos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/citologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Integrina beta1/metabolismo , Camundongos , Camundongos Knockout , Baço/citologia , Timócitos/citologia , Timo/citologiaRESUMO
T-cell development is a complex dynamic process that relies on ordered stromal signals delivered to thymus-seeding progenitors that migrate throughout different thymus microenvironments (TMEs). Particularly, Notch signaling provided by thymic epithelial cells (TECs) is crucial for T-cell fate specification and generation of mature T cells. Four canonical Notch ligands (Dll1, Dll4, Jag1 and Jag2) are expressed in the thymus, but their spatial distribution in functional TMEs is largely unknown, especially in humans, and their impact on Notch1 activation during T-lymphopoiesis remains undefined. Based on immunohistochemistry and quantitative confocal microscopy of fetal, postnatal and adult human and mouse thymus samples, we show that spatial regulation of Notch ligand expression defines discrete Notch signaling niches and dynamic species-specific TMEs. We further show that Notch ligand expression, particularly DLL4, is tightly regulated in cortical TECs during human thymus ontogeny and involution. Also, we provide the first evidence that NOTCH1 activation is induced in vivo in CD34+ progenitors and developing thymocytes at particular cortical niches of the human fetal and postnatal thymus. Collectively, our results show that human thymopoiesis involves complex spatiotemporal regulation of Notch ligand expression, which ensures the coordinated delivery of niche-specific NOTCH1 signals required for dynamic T-cell development.
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Receptor Notch1/metabolismo , Timo/crescimento & desenvolvimento , Timo/metabolismo , Adolescente , Adulto , Envelhecimento/metabolismo , Animais , Antígenos CD34/metabolismo , Criança , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Recém-Nascido , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , Proteínas Serrate-Jagged/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Timo/citologia , Timo/embriologiaRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the dysregulation of signaling pathways that control intrathymic T-cell development. Relapse rates are still significant, and prognosis is particularly bleak for relapsed patients. Therefore, development of novel therapies specifically targeting pathways controlling leukemia-initiating cell (LIC) activity is mandatory for fighting refractory T-ALL. The interleukin-7 receptor (IL-7R) is a crucial T-cell developmental pathway that is commonly expressed in T-ALL and has been implicated in leukemia progression; however, the significance of IL-7R/IL-7 signaling in T-ALL pathogenesis and its contribution to disease relapse remain unknown. To directly explore whether IL-7R targeting may be therapeutically efficient against T-ALL relapse, we focused on a known Notch1-induced T-ALL model, because a majority of T-ALL patients harbor activating mutations in NOTCH1, which is a transcriptional regulator of IL-7R expression. Using loss-of-function approaches, we show that Il7r-deficient, but not wild-type, mouse hematopoietic progenitors transduced with constitutively active Notch1 failed to generate leukemia upon transplantation into immunodeficient mice, thus providing formal evidence that IL-7R function is essential for Notch1-induced T-cell leukemogenesis. Moreover, we demonstrate that IL-7R expression is an early functional biomarker of T-ALL cells with LIC potential and report that impaired IL-7R signaling hampers engraftment and progression of patient-derived T-ALL xenografts. Notably, we show that IL-7R-dependent LIC activity and leukemia progression can be extended to human B-cell acute lymphoblastic leukemia (B-ALL). These results have important therapeutic implications, highlighting the relevance that targeting normal IL-7R signaling may have in future therapeutic interventions, particularly for preventing T-ALL (and B-ALL) relapse.
Assuntos
Suscetibilidade a Doenças , Células-Tronco Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores de Interleucina-7/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Interleucina-7/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: GRIN-related disorders (GRD), the so-called grinpathies, is a group of rare encephalopathies caused by mutations affecting GRIN genes (mostly GRIN1, GRIN2A and GRIN2B genes), which encode for the GluN subunit of the N-methyl D-aspartate (NMDA) type ionotropic glutamate receptors. A growing number of functional studies indicate that GRIN-encoded GluN1 subunit disturbances can be dichotomically classified into gain- and loss-of-function, although intermediate complex scenarios are often present. METHODS: In this study, we aimed to delineate the structural and functional alterations of GRIN1 disease-associated variants, and their correlations with clinical symptoms in a Spanish cohort of 15 paediatric encephalopathy patients harbouring these variants. RESULTS: Patients harbouring GRIN1 disease-associated variants have been clinically deeply-phenotyped. Further, using computational and in vitro approaches, we identified different critical checkpoints affecting GluN1 biogenesis (protein stability, subunit assembly and surface trafficking) and/or NMDAR biophysical properties, and their association with GRD clinical symptoms. CONCLUSIONS: Our findings show a strong correlation between GRIN1 variants-associated structural and functional outcomes. This structural-functional stratification provides relevant insights of genotype-phenotype association, contributing to future precision medicine of GRIN1-related encephalopathies.
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Encefalopatias/patologia , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Adolescente , Animais , Encefalopatias/genética , Células COS , Criança , Pré-Escolar , Chlorocebus aethiops , Estudos de Coortes , Feminino , Células HEK293 , Humanos , Lactente , Masculino , Modelos Moleculares , Conformação Proteica , EspanhaRESUMO
Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 µg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 µg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 µg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.
Assuntos
Mycobacterium tuberculosis , Preparações Farmacêuticas , Tuberculose Resistente a Múltiplos Medicamentos , Amidoidrolases/genética , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Mutação , Pirazinamida/farmacologia , Escarro , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/ml, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.
Assuntos
Antituberculosos/farmacologia , Colorimetria/métodos , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Escarro/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Colorimetria/normas , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana/normas , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Tuberculose/microbiologia , Adulto JovemRESUMO
Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Linfócitos T/imunologia , Transcrição Gênica , Diferenciação Celular/genética , Expressão Gênica , Humanos , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timo/citologia , Timo/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Lymphoid tumor patients often exhibit resistance to standard therapies or experience relapse post-remission. Relapse is driven by Tumor Initiating Cells (TICs), a subset of tumor cells capable of regrowing the tumor and highly resistant to therapy. Growing cells in 3D gels is a method to discern tumorigenic cells because it strongly correlates with tumorigenicity. The finding that TICs, rather than differentiated tumor cells, grow in 3D gels offers a unique opportunity to unveil TIC-specific signaling pathways and therapeutic targets common to various cancer types. Here, we show that culturing lymphoid cells in 3D gels triggers reactive oxygen species (ROS) production, leading to non-tumor lymphoid cell death while enabling the survival and proliferation of a subset of lymphoma/leukemia cells, TICs or TIC-like cells. Treatment with the antioxidant N-acetylcysteine inhibits this lethality and promotes the growth of primary non-tumor lymphoid cells in 3D gels. A subset of lymphoma cells, characterized by an increased abundance of the antioxidant glutathione, escape ROS-induced lethality, a response not seen in non-tumor cells. Reducing glutathione production in lymphoma cells, either through pharmacological inhibition of glutamate cysteine ligase (GCL), the enzyme catalyzing the rate-limiting step in glutathione biosynthesis, or via knockdown of GCLC, the GCL catalytic subunit, sharply decreased cell growth in 3D gels and xenografts. Tumor cells from B-cell lymphoma/leukemia patients and λ-MYC mice, a B-cell lymphoma mouse model, overproduce glutathione. Importantly, pharmacological GCL inhibition hindered lymphoma growth in female λ-MYC mice, suggesting that this treatment holds promise as a therapeutic strategy for female lymphoma/leukemia patients.
Assuntos
Glutationa , Linfoma , Células-Tronco Neoplásicas , Espécies Reativas de Oxigênio , Animais , Humanos , Feminino , Linfoma/patologia , Linfoma/metabolismo , Linfoma/tratamento farmacológico , Glutationa/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Masculino , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Carcinogênese/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismoRESUMO
Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.
Assuntos
Leucemia , Linfócitos T , Animais , Antígenos CD19 , Humanos , Imunoterapia Adotiva/métodos , Camundongos , RecidivaRESUMO
BACKGROUND: Bleach-sedimentation may improve microscopy for diagnosing tuberculosis by sterilising sputum and concentrating Mycobacterium tuberculosis. We studied gravity bleach-sedimentation effects on safety, sensitivity, speed and reliability of smear-microscopy. METHODS: This blinded, controlled study used sputum specimens (n = 72) from tuberculosis patients. Bleach concentrations and exposure times required to sterilise sputum (n = 31) were determined. In the light of these results, the performance of 5 gravity bleach-sedimentation techniques that sterilise sputum specimens (n = 16) were compared. The best-performing of these bleach-sedimentation techniques involved adding 1 volume of 5% bleach to 1 volume of sputum, shaking for 10-minutes, diluting in 8 volumes distilled water and sedimenting overnight before microscopy. This technique was further evaluated by comparing numbers of visible acid-fast bacilli, slide-reading speed and reliability for triplicate smears before versus after bleach-sedimentation of sputum specimens (n = 25). Triplicate smears were made to increase precision and were stained using the Ziehl-Neelsen method. RESULTS: M. tuberculosis in sputum was successfully sterilised by adding equal volumes of 15% bleach for 1-minute, 6% for 5-minutes or 3% for 20-minutes. Bleach-sedimentation significantly decreased the number of acid-fast bacilli visualised compared with conventional smears (geometric mean of acid-fast bacilli per 100 microscopy fields 166, 95%CI 68-406, versus 346, 95%CI 139-862, respectively; p = 0.02). Bleach-sedimentation diluted paucibacillary specimens less than specimens with higher concentrations of visible acid-fast bacilli (p = 0.02). Smears made from bleach-sedimented sputum were read more rapidly than conventional smears (9.6 versus 11.2 minutes, respectively, p = 0.03). Counting conventional acid-fast bacilli had high reliability (inter-observer agreement, r = 0.991) that was significantly reduced (p = 0.03) by bleach-sedimentation (to r = 0.707) because occasional strongly positive bleach-sedimented smears were misread as negative. CONCLUSIONS: Gravity bleach-sedimentation improved laboratory safety by sterilising sputum but decreased the concentration of acid-fast bacilli visible on microscopy, especially for sputum specimens containing high concentrations of M. tuberculosis. Bleach-sedimentation allowed examination of more of each specimen in the time available but decreased the inter-observer reliability with which slides were read. Thus bleach-sedimentation effects vary depending upon specimen characteristics and whether microscopy was done for a specified time, or until a specified number of microscopy fields had been read. These findings provide an explanation for the contradictory results of previous studies.
Assuntos
Técnicas Bacteriológicas/métodos , Centrifugação/métodos , Desinfecção/métodos , Mycobacterium tuberculosis/isolamento & purificação , Manejo de Espécimes/métodos , Escarro/microbiologia , Tuberculose/diagnóstico , Desinfetantes/farmacologia , Humanos , Microscopia/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Hipoclorito de Sódio/farmacologia , Fatores de Tempo , Tuberculose/microbiologiaRESUMO
Intense chemotherapy regimens of patients diagnosed with T cell acute lymphoblastic leukemia (T-ALL) have proved successful for improving patient's overall survival, especially in children. But still T-ALL treatment remains challenging, since side effects of chemotherapeutic drugs often worsen patient's quality of life, and relapse rates remain significant. Hence, the availability of experimental animal models capable of recapitulating the biology of human T-ALL is obligatory as a critical tool to explore novel promising therapies directed against specific targets that have been previously validated in in vitro assays. For this purpose, patient-derived xenografts (PDX) of primary human T-ALL are currently of great interest as preclinical models for novel therapeutic strategies toward transition into clinical trials. In this chapter, we describe the lab workflow to perform PDX assays, from the initial processing of patient T-ALL samples, genetic in vitro modifications of leukemic cells by lentiviral transduction, inoculation routes, monitoring for disease development, and mouse organ examination, to administration of several treatments.
Assuntos
Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Xenoenxertos , Humanos , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cyclooxygenase 2 (COX2) has been implicated in cancer development and metastasis. We have identified several COX2-regulated inflammation-related genes in human colorectal cancer cells and shown that some of them play important roles in tumor progression. In this work, we have studied the COX2-regulated genes in the mouse colorectal cancer cell line CT26, to find that many are also regulated by COX2 over-expression. On the other hand, we generated a CT26 cell line expressing Gfp and Luciferase, to study tumor growth and metastasis in immunocompetent Balb/c mice. We then collected solid tissue, and blood samples, from healthy and tumor-bearing mice. Using the Parsortix® cell separation system and taking advantage of the fact that the tumor cells expressed Gfp, we were able to identify circulating tumor cells (CTCs) in some of the mice. We compared the mRNA expression levels of Ptgs2 and effector genes in the samples obtained from tumor-bearing or healthy mice, namely, tumor or healthy colon, Ficoll purified buffy coat, and Parsortix-isolated cells to find different patterns between healthy, tumor-bearing mice with or without CTCs. Although for genes like Il15 we did not observe any difference between healthy and tumor-bearing mice in Ficoll or Parsortix samples; others, such as Egr1, Zc3h12a, Klf4, or Nfat5, allowed distinguishing for cancer or CTC presence. Gene expression analysis in Ficoll or Parsortix processed samples, after liquid biopsy, may offer valuable diagnostic and prognostic information and thus should be further studied.
RESUMO
This paper presents the results of an experimental study on the acoustic efficiency of plastic surgical face masks. Since the very high number of disposable masks being used globally on a daily basis to face the Covid19 pandemic is posing new environmental risks, mainly connected to improper disposal, any possible improvements in the management of this waste stream is very important. In this work their potential use as sound porous absorber is discussed. Surgical face masks are mainly made of polypropylene fibers which show good acoustical properties. Their porous structure was studied through the measurement of some non-acoustic properties: bulk density, fiber diameter, porosity, flow resistivity and tortuosity. Moreover, the sound absorption performance of samples, made of scrapped face masks, with different thicknesses was evaluated using an impedance tube according to ISO 10534-2. The results obtained from the sound absorption spectra and two single indexes, Noise Reduction Coefficient and Sound Absorption Average showed a high sound absorption value over a frequency range of interest. Finally, the sound absorption spectra obtained for surgical face masks were compared with those obtained for fibrous materials currently used in building sector, suggesting that this fibrous waste could act as a possible substitute to traditional ones.
Assuntos
COVID-19 , Máscaras , Humanos , Modelos Teóricos , Porosidade , SARS-CoV-2RESUMO
The human αß T-cell receptor (TCR) is composed of a variable heterodimer (TCRαß) and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472). The role of each invariant chain in the stepwise interactions among TCR chains along the assembly is still not fully understood. Despite the high sequence homology between CD3γ and CD3δ, the clinical consequences of the corresponding immunodeficiencies (ID) in humans are very different (mild and severe, respectively), and mouse models do not recapitulate findings in human ID. To try to understand such disparities, we stably knocked down (KD) CD3D or CD3G expression in the human Jurkat T-cell line and analyzed comparatively their impact on TCRαß assembly, transport, and surface expression. The results indicated that TCR ensembles were less stable and CD3ε levels were lower when CD3γ, rather than CD3δ, was scarce. However, both defective TCR ensembles were strongly retained in the ER, lacked ζζ/CD2472, and barely reached the T-cell surface (<11% of normal controls) in any of the CD3 KD cells. This is in sharp contrast to human CD3γ ID, whose mature T cells express higher levels of surface TCR (>30% vs. normal controls). CD3 KD of human T-cell progenitors followed by mouse fetal thymus organ cultures showed high plasticity in emerging immature polyclonal T lymphocytes that allowed for the expression of significant TCR levels which may then signal for survival in CD3γ, but not in CD3δ deficiency, and explain the immunological and clinical disparities of such ID cases.
RESUMO
The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.
Assuntos
Glutamato-Amônia Ligase/genética , Glutamina/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Glutamato-Amônia Ligase/metabolismo , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Global Health Education (GHE) focuses on training proactive global citizens to tackle health challenges in an increasingly interconnected and interdependent world. Studies show that health professionals in training have reported that GHE has improved their teamwork, responsiveness to contextual factors that impact health, and understanding of health systems; however, there is little research on the impact of GHE courses in undergraduate settings, especially in low and middle-income countries (LMICs). METHODS: Our study analyzes a multidisciplinary online global health course at Tecnologico de Monterrey, México. We conducted a cross-sectional study with pre- and post-design. Students who took the multidisciplinary course of Global Health for Leaders in the Fall of 2019 (n = 153) and Spring of 2020 (n = 348) were selected for this study. Using a five-point Likert scale (strongly agree to strongly disagree), the survey assessed seven competencies as well as questions about course expectations, takeaways, and recommendations to improve the course. We performed descriptive statistical analyses comparing the combined pre-tests (from Fall and Spring cohorts) to the combined post-tests. Fisher's exact test was used to compare the samples. RESULTS: Of the 501 pre-course surveys administered, 456 responses were completed in the pre-course and 435 in the post-course (91% overall response rate). Only 8.7% of the respondents in the pre-course survey strongly agreed that they could describe fundamental aspects of global health such as the Millennium Development Goals or Sustainable Development Goals, in contrast to a 56% of the students who strongly agreed in the post-course survey (p < 0.001). Similar differences were captured in understanding the global burden of disease, social determinants of health, the effects of globalization in health, health systems' goals and functions, and human rights. 38% felt that the course helped them develop a more empathetic perception of the suffering of others experiencing global health-related issues. CONCLUSION: In this study, we have presented our experience in teaching an online global health course for multidisciplinary undergraduates in a LMIC. The competencies reported by our students indicate that the course prepared them to confront complex global health issues.
Assuntos
Educação a Distância/estatística & dados numéricos , Saúde Global/educação , Pessoal de Saúde/educação , Estudos Interdisciplinares/estatística & dados numéricos , Estudos Transversais , MéxicoRESUMO
PURPOSE: Accurate segmentation of the hippocampus for hippocampal avoidance whole-brain radiotherapy currently requires high-resolution magnetic resonance imaging (MRI) in addition to neuroanatomic expertise for manual segmentation. Removing the need for MR images to identify the hippocampus would reduce planning complexity, the need for a treatment planning MR imaging session, potential uncertainties associated with MRI-computed tomography (CT) image registration, and cost. Three-dimensional (3D) deep convolutional network models have the potential to automate hippocampal segmentation. In this study, we investigate the accuracy and reliability of hippocampal segmentation by automated deep learning models from CT alone and compare the accuracy to experts using MRI fusion. METHODS: Retrospectively, 390 Gamma Knife patients with high-resolution CT and MR images were collected. Following the RTOG 0933 guidelines, images were rigidly fused, and a neuroanatomic expert contoured the hippocampus on the MR, then transferred the contours to CT. Using a calculated cranial centroid, the image volumes were cropped to 200 × 200 × 35 voxels, which were used to train four models, including our proposed Attention-Gated 3D ResNet (AG-3D ResNet). These models were then compared with results from a nested tenfold validation. From the predicted test set volumes, we calculated the 100% Hausdorff distance (HD). Acceptability was assessed using the RTOG 0933 protocol criteria, and contours were considered passing with HD ≤ 7 mm. RESULTS: The bilateral hippocampus passing rate across all 90 models trained in the nested cross-fold validation was 80.2% for AG-3D ResNet, which performs with a comparable pass rate (P = 0.3345) to physicians during centralized review for the RTOG 0933 Phase II clinical trial. CONCLUSIONS: Our proposed AG-3D ResNet's segmentation of the hippocampus from noncontrast CT images alone are comparable to those obtained by participating physicians from the RTOG 0933 Phase II clinical trial.
Assuntos
Aprendizado Profundo , Hipocampo/diagnóstico por imagem , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Reprodutibilidade dos Testes , Estudos Retrospectivos , Tomografia Computadorizada por Raios XRESUMO
This study identified factors associated with adherence to a 6-month isoniazid preventive therapy (IPT) course among adolescents and children living with HIV. Forty adolescents living with HIV and 48 primary caregivers of children living with HIV completed a Likert-based survey to measure respondent opinions regarding access to care, quality of care, preferred regimens, perceived stigma, and confidence in self-efficacy. Sociodemographic data were collected and adherence measured as the average of pill counts obtained while on IPT. The rates of suboptimal adherence (< 95% adherent) were 22.5% among adolescents and 37.5% among the children of primary caregivers. Univariate logistic regression was used to model the change in the odds of suboptimal adherence. Independent factors associated with suboptimal adherence among adolescents included age, education level, the cost of coming to clinic, stigma from community members, and two variables relating to self-efficacy. Among primary caregivers, child age, concerns about stigma, and location preference for meeting a community-health worker were associated with suboptimal adherence. To determine whether these combined factors contributed different information to the prediction of suboptimal adherence, a risk score containing these predictors was constructed for each group. The risk score had an AUC of 0.87 (95% CI: 0.76, 0.99) among adolescents and an AUC of 0.76 (95% CI: 0.62, 0.90), among primary caregivers suggesting that these variables may have complementary predictive utility. The heterogeneous scope and associations of these variables in different populations suggests that interventions aiming to increase optimal adherence will need to be tailored to specific populations and multifaceted in nature. Ideally interventions should address both long-established barriers to adherence such as cost of transportation to attend clinic and more nuanced psychosocial barriers such as perceived community stigma and confidence in self-efficacy.
Assuntos
Infecções por HIV/complicações , Isoniazida/uso terapêutico , Adesão à Medicação/psicologia , Tuberculose/prevenção & controle , Adolescente , Comportamento do Adolescente/psicologia , Adulto , Fatores Etários , Cuidadores/psicologia , Cuidadores/estatística & dados numéricos , Criança , Comportamento Infantil/psicologia , Essuatíni , Feminino , Infecções por HIV/imunologia , Humanos , Masculino , Adesão à Medicação/estatística & dados numéricos , Pessoa de Meia-Idade , Autoeficácia , Estigma Social , Inquéritos e Questionários/estatística & dados numéricos , Tuberculose/imunologiaRESUMO
Graft-versus-host disease (GVHD) is the main complication after allogeneic hematopoietic stem cell transplantation. We previously unveiled a correlation between proportions of C-C motif chemokine receptor 7 (CCR7)+ T cells in the apheresis and the risk of developing GVHD. We wanted to evaluate in vivo whether apheresis with low proportion of CCR7+ cells or treatment with an anti-human CCR7 monoclonal antibody (mAb) were suitable strategies to prevent or treat acute GVHD in preclinical xenogeneic models. Therapeutic anti-CCR7 mAb was the most effective strategy in both prophylactic and therapeutic settings where antibody drastically reduced in vivo lymphoid organ infiltration of donor CCR7+ T cells, extended lifespan and solved clinical signs. The antibody neutralized in vitro migration of naïve and central memory T cells toward CCR7 ligands and depleted target CCR7+ subsets through complement activation. Both mechanisms of action spared CCR7- subsets, including effector memory and effector memory CD45RA+ T cells which may mediate graft versus leukemia effect and immunity against infections. Accordingly, the numbers of donor CCR7+ T cells in the apheresis were not associated to cytomegalovirus reactivation or the recurrence of the underlying disease. These findings provide a promising new strategy to prevent and treat acute GVHD, a condition where new specific, safety and effective treatment is needed.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Receptores CCR7 , Doença Enxerto-Hospedeiro/tratamento farmacológico , Efeito Enxerto vs Leucemia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Receptores CCR7/efeitos dos fármacos , Linfócitos TRESUMO
Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.