RESUMO
Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3-5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.
Assuntos
Apresentação de Antígeno , Mutação da Fase de Leitura , Melanoma/imunologia , Peptídeos/genética , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , Linfócitos T/imunologia , Linhagem Celular , Códon/genética , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mudança da Fase de Leitura do Gene Ribossômico/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/imunologia , Interferon gama/farmacologia , Melanoma/patologia , Peptídeos/química , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Proteoma , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Triptofano/deficiência , Triptofano/genética , Triptofano/metabolismoRESUMO
BACKGROUND: Dengue fever is a well-studied vector-borne disease in tropical and subtropical areas of the world. Several methods for predicting the occurrence of dengue fever in Taiwan have been proposed. However, to the best of our knowledge, no study has investigated the relationship between air quality indices (AQIs) and dengue fever in Taiwan. RESULTS: This study aimed to develop a dengue fever prediction model in which meteorological factors, a vector index, and AQIs were incorporated into different machine learning algorithms. A total of 805 meteorological records from 2013 to 2015 were collected from government open-source data after preprocessing. In addition to well-known dengue-related factors, we investigated the effects of novel variables, including particulate matter with an aerodynamic diameter < 10 µm (PM10), PM2.5, and an ultraviolet index, for predicting dengue fever occurrence. The collected dataset was randomly divided into an 80% training set and a 20% test set. The experimental results showed that the random forests achieved an area under the receiver operating characteristic curve of 0.9547 for the test set, which was the best compared with the other machine learning algorithms. In addition, the temperature was the most important factor in our variable importance analysis, and it showed a positive effect on dengue fever at < 30 °C but had less of an effect at > 30 °C. The AQIs were not as important as temperature, but one was selected in the process of filtering the variables and showed a certain influence on the final results. CONCLUSIONS: Our study is the first to demonstrate that AQI negatively affects dengue fever occurrence in Taiwan. The proposed prediction model can be used as an early warning system for public health to prevent dengue fever outbreaks.
Assuntos
Dengue , Algoritmo Florestas Aleatórias , Humanos , Dengue/epidemiologia , Taiwan/epidemiologia , Temperatura , Surtos de DoençasRESUMO
Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.
Assuntos
Proteínas de Membrana/fisiologia , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ativinas/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mesoderma/crescimento & desenvolvimento , Camundongos , Modelos Biológicos , Células NIH 3T3 , RNA Mensageiro/metabolismo , XenopusRESUMO
HLA molecules presenting peptides derived from tumor-associated self-antigens (self-TAA) are attractive targets for T-cell-based immunotherapy of cancer. However, detection of such epitopes is hampered by self-tolerance and limitations in the sensitivity of mass spectrometry. Here, we used T cells from HLA-A2-negative donors as tools to detect HLA-A2-bound peptides from two leukemia-associated differentiation antigens; CD20 and the previously undescribed cancer target myeloperoxidase. A high-throughput platform for epitope discovery was designed using dendritic cells cotransfected with full-length transcripts of self-TAA and HLA-A2 to allow presentation of all naturally processed peptides from a predefined self-protein on foreign HLA. Antigen-reactive T cells were directly detected using panels of color-coded peptide-HLA multimers containing epitopes predicted by a computer algorithm. Strikingly, cytotoxic T cells were generated against 37 out of 50 peptides predicted to bind HLA-A2. Among these, 36 epitopes were previously undescribed. The allorestricted T cells were exquisitely peptide- and HLA-specific and responded strongly to HLA-A2-positive leukemic cells with endogenous expression of CD20 or myeloperoxidase. These results indicate that the repertoire of self-peptides presented on HLA class I has been underestimated and that a wealth of self-TAA can be targeted by T cells when using nontolerized T-cell repertoires.
Assuntos
Epitopos de Linfócito T/química , Antígeno HLA-A2/imunologia , Neoplasias/imunologia , Peptídeos/química , Linfócitos T Citotóxicos/citologia , Algoritmos , Apresentação de Antígeno , Antígenos CD20/metabolismo , Antígenos de Neoplasias/química , Autoantígenos/química , Citometria de Fluxo , Células HL-60 , Antígeno HLA-A2/metabolismo , Humanos , Sistema Imunitário , Imunoterapia , Espectrometria de Massas , Peroxidase/química , Ligação Proteica , RNA Mensageiro/metabolismoRESUMO
PURPOSE: Amniotic fluid embolism (AFE) is an unpredictable and unpreventable complication of maternity. The presentation may range from relatively subtle clinical events to sudden maternal cardiac arrest. However, the neglected diagnosis of non-classical form of AFE (atypical AFE) is very common. The aim of this study was to examine population-based regional data from Suzhou, China. Based on the analysis of all available case reports, we put forward an outline of atypical AFE and investigate whether any variation identified could be ascribed to methodology. METHODS: Retrospective study from January 2004 to December 2013, 53 cases was identified from the database of Center for Disease Control (CDC) in the city of Suzhou. We investigated the presentations of atypical AFE and maternal characteristics with potential factors underlying AFE. Multiple-regression analysis was used to calculate adjusted odds ratios (ORs) and 95 % confidence intervals (CIs). RESULTS: The incidence of AFE was 6.91 per 100,000 deliveries (53/766,895). Seventeen deaths occurred, a mortality rate of 32 %. Atypical AFE may as the earlier stage or mild form of AFE, there was no death case in the study with timely remedy. The atypical AFE appear is obstetric hemorrhage and/or pulmonary and renal dysfunction postpartum. Hyperfibrinolysis and coagulopathy may the early laboratory findings of atypical AFE. Atypical and classical AFE shared the same risks, such as advanced maternal age, placental abnormalities, operative deliveries, eclampsia, cervical lacerations, and induction of labor. CONCLUSION: Staying alert to premonitory symptoms of AFE is critical to turn it to a remediable disease. Patient complaints such as breathlessness, chest pain, feeling cold, distress, panic, a feeling of nausea, and vomiting should elicit close attention. The management of a suspected episode of amniotic fluid embolism is generally considered to be supportive. Hysterectomy must be performed if there is further progression of symptoms. Due to advances in acute care, mortality has decreased in recent years, highlighting the importance of early detection and treatment.
Assuntos
Embolia Amniótica/etnologia , Embolia Amniótica/etiologia , Complicações Cardiovasculares na Gravidez/diagnóstico , Adolescente , Adulto , China/epidemiologia , Parto Obstétrico/efeitos adversos , Eclampsia , Embolia Amniótica/diagnóstico , Feminino , Humanos , Incidência , Trabalho de Parto , Idade Materna , Análise Multivariada , Período Pós-Parto , Gravidez , Estudos Retrospectivos , Fatores de Risco , Adulto JovemRESUMO
Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4(+) T cells. Here, we investigated if Ii could similarly activate human CD8(+) T cells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8(+) T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naïve CD8(+) T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.
Assuntos
Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos/química , Antígenos/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endossomos/metabolismo , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Peptídeos/química , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte ProteicoRESUMO
It has been reported that long non-coding RNAs (lncRNAs) are involved in sepsis-induced liver injury, while the role of cancer susceptibility candidate 7 (CASC7) in liver injury induced by sepsis remains elusive. In our study, 62 patients and 55 healthy controls were enrolled from our hospital, from whom CASC7 and microRNA-217 (miR-217) in serum samples were detected by quantitative real-time PCR (qRT-PCR). Then the sepsis-induced liver injury mice model was established by lipopolysaccharide (LPS). The effect of CASC7 on liver injury induced by sepsis was confirmed by Hematoxylin and Eosin (HE) staining, ELISA assay, TUNEL assay, Annexin V-FITC apoptosis assay and cell counting kit-8 (CCK-8) assay, respectively. Besides, RNA pull-down, luciferase reporter gene assay, qRT-PCR, and western blot were used to evaluate the underlying mechanisms. In this study, lncRNA CASC7 was significantly increased while miR-217 was significantly decreased in patients with sepsis-induced liver injury compared with that in healthy controls. There was a negative association of CASC7 and miR-217 in serum samples from patients with sepsis-induced liver injury and healthy controls. CASC7 was upregulated in a time-dependent manner in liver tissues of LPS-treated mice. It was found that knockdown of CASC7 reduced the liver injury induced by LPS in mice. In vitro, LPS treatment enhanced cell apoptosis, while knockdown of CASC7 inhibited the role of LPS in cell apoptosis. Moreover, knockdown of CASC7 suppressed the LPS-enhanced tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß) expression. In addition, miR-217 was found to be a target of CASC7, and miR-217 mimic could reverse CASC7-promoted liver injury. Furthermore, toll-like receptor 4 (TLR4) was identified as the target of miR-217, and both CASC7 and miR-217 could downregulate the mRNA and protein level of TLR4. Additionally, TLR4 overexpression could reverse miR-217-inhibited or CASC7-promoted liver injury. Taken together, CASC7 contributes to the progression of LPS-induced liver injury via the miR-217/TLR4 axis.
RESUMO
Cancer immunotherapy using T cell receptor-engineered T cells (TCR-Ts) represents a promising treatment option. However, technologies for pre-clinical safety assessment are incomplete or inaccessible to most laboratories. Here, TCR-T off-target reactivity was assessed in five steps: (1) Mapping target amino acids necessary for TCR-T recognition, followed by (2) a computational search for, and (3) reactivity screening against, candidate cross-reactive peptides in the human proteome. Natural processing and presentation of recognized peptides was evaluated using (4) short mRNAs, and (5) full-length proteins. TCR-Ts were screened for recognition of unintended HLA alleles, and as proxy for off-target reactivity in vivo, a syngeneic, HLA-A*02:01-transgenic mouse model was used. Validation demonstrated importance of studying recognition of full-length candidate off-targets, and that the clinically applied 1G4 TCR has a hitherto unknown reactivity to unintended HLA alleles, relevant for patient selection. This widely applicable strategy should facilitate evaluation of candidate therapeutic TCRs and inform clinical decision-making.
RESUMO
The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of T cell epitopes. However, studies on viral escape from T cell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 T cell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of T cell immunity.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Linfócitos T CD8-Positivos , COVID-19/imunologia , Epitopos de Linfócito T/genética , Epitopos Imunodominantes/genética , SARS-CoV-2/genéticaRESUMO
Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCRFLT3D/Y). TCRFLT3D/Y-redirected T cells selectively eliminated primary human AML cells harboring the FLT3D835Y mutation in vitro and in vivo. TCRFLT3D/Y cells rejected both CD34+ and CD34- AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34+ AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo.
Assuntos
Leucemia Mieloide Aguda , Proteínas Tirosina Quinases , Adulto , Humanos , Animais , Camundongos , Mutação , Proteínas Tirosina Quinases/genética , Mutação com Ganho de Função , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos de Linfócitos T/genética , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.
Assuntos
DNA Nucleotidilexotransferase , Linfócitos T , Animais , Células-Tronco Hematopoéticas , Linfócitos , Camundongos , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The basic helixloophelix protein E2-2 is known to play a role in quiescence of endothelial cells (ECs). However, it is unclear how the activity of E2-2 is controlled in the cells. In this study, we identified FAM96B as an interaction partner of E2-2. FAM96B interfered with E2-2-mediated effects on luciferase reporter activities. Furthermore, the suppression of vascular endothelial growth factor receptor 2 promoter activity by E2-2 was rescued by the expression of FAM96B in a dose-dependent manner. Interestingly, FAM96B decreased the expression of ectopic and endogenous E2-2 proteins. Mutational analysis revealed that the middle region of FAM96B is required for the limited expression of E2-2 protein. When FAM96B was expressed in ECs, the EC migration, proliferation, and tube formation were potentiated. Taken together, these findings suggest that FAM96B acts as a regulator of E2-2 through the control of its protein expression.
Assuntos
Proteínas de Transporte/metabolismo , Células Endoteliais/metabolismo , Proteínas Nucleares/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células COS , Proteínas de Transporte/genética , Movimento Celular , Proliferação de Células , Cricetinae , Sequências Hélice-Alça-Hélice , Humanos , Metaloproteínas , Camundongos , Células NIH 3T3 , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The orphan nuclear receptor TLX has been proposed to act as a repressor of cell cycle inhibitors to maintain the neural stem cells in an undifferentiated state, and prevents commitment into astrocyte lineages. However, little is known about the mechanism of TLX in neuronal lineage commitment and differentiation. A majority of adult rat hippocampus-derived progenitors (AHPs) cultured in the presence of FGF express a high level of TLX and a fraction of these cells also express the proneural gene MASH1. Upon FGF withdrawal, TLX rapidly decreased, while MASH1 was intensely expressed within 1h, decreasing gradually to disappear at 24h. Adenoviral transduction of TLX in AHP cells in the absence of FGF transiently increased cell proliferation, however, later resulted in neuronal differentiation by inducing MASH1, Neurogenin1, DCX, and MAP2ab. Furthermore, TLX directly targets and activates the MASH1 promoter through interaction with Sp1, recruiting co-activators whereas dismissing the co-repressor HDAC4. Conversely, silencing of TLX in AHPs decreased beta-III tubulin and DCX expression and promoted glial differentiation. Our results thus suggest that TLX not only acts as a repressor of cell cycle and glial differentiation but also activates neuronal lineage commitment in AHPs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipocampo/crescimento & desenvolvimento , Neurogênese/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ativação Transcricional , Adenoviridae , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Linhagem da Célula/genética , Células Cultivadas , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Histona Desacetilases/análise , Humanos , Proteínas Associadas aos Microtúbulos/análise , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neuropeptídeos/análise , Regiões Promotoras Genéticas , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Fator de Transcrição Sp1/análise , Fator de Transcrição Sp1/metabolismo , Tubulina (Proteína)/análiseRESUMO
OBJECTIVE: To evaluate the effects of low-molecular-weight heparin (LMWH) combined with low-dose aspirin (LDA) in pregnant women with a history of pregnancy-related hypertensive disorders. METHODS: In the current retrospective stratified cohort study, 33 women with previous hypertensive disorders of pregnancy treated with LMWH and LDA were compared with 37 control women who did not undergo LMWH or LDA treatment. Rates of pre-eclampsia recurrence, placental abruption, and other adverse outcomes for the fetuses and pregnant women were compared in the two groups. RESULTS: The pre-eclampsia recurrence rates were 12/33 (36.4%) in the LMWH + LDA group and 28/37 (75.7%) in the control group (P < 0.01). In stratified cohort analysis, pregnant women with a history of early-onset pre-eclampsia, a body mass index of at least 24 (calculated as weight in kilograms divided by the square of height in meters), and aged less than 35 years benefited from LMWH + LDA treatment. In women with chronic hypertension or a history of placental abruption there was no protective effect. There were no significant differences in other adverse outcomes such as placental abruption and small size for gestational age in fetuses or pregnant women in the two groups. CONCLUSION: Administration of LMWH + LDA only lowered the risk of pre-eclampsia recurrence in subgroups of pregnant women with a history of pregnancy-associated hypertensive disorders.
Assuntos
Aspirina/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Pré-Eclâmpsia/tratamento farmacológico , Descolamento Prematuro da Placenta/epidemiologia , Adulto , Anticoagulantes/uso terapêutico , Estudos de Coortes , Feminino , Idade Gestacional , Humanos , Gravidez , Complicações na Gravidez/epidemiologia , Estudos RetrospectivosRESUMO
OBJECTIVE: The aim of this study was to investigate the potential role of ADAMTS13 analysis in the early recognition and management of thrombotic thrombocytopenic purpura (TTP) in pregnant women. METHODS: Five cases of TTP were evaluated retrospectively. Clinical and laboratory findings, von Willebrand factor (vWF)-cleaving metalloprotease (ADAMTS13) activity and maternal and neonatal outcome were recorded and analysed. RESULTS: Five cases were all nulliparous. ADAMTS13 assay was performed and the enzyme activity was less than 5% of the normal controls in three cases. Gene mutation in the 9th exon resulting in amino acid exchange 349ArgâCys in ADAMTS13 was identified in one patient. After treatment including transfusion of fresh-frozen plasma (n = 5), packed red blood cells (n = 5), platelet transfusions (n = 2) and/or continued renal replacement therapy (CRRT) (n = 1) and plasma exchange (n = 2), three patients were alive, one died on postpartum day 6 in hospital without plasma exchange and one of familial TTP died three months after discharge. CONCLUSION: With increasing awareness, extra-attention must be paid to patients with thrombotic microangiopathy and to measurement of ADAMTS13 activity for early diagnosis. Although severe ADAMTS13 deficiency may be helpful for TTP, it may not be sensitive enough to identify all TTP patients. Therefore, despite ADAMTS13 result positive or negative, prompt aggressive management should include early termination of pregnancy, plasma transfusion and/or plasma exchange.
Assuntos
Proteínas ADAM/análise , Complicações Hematológicas na Gravidez/diagnóstico , Púrpura Trombocitopênica Trombótica/diagnóstico , Proteínas ADAM/genética , Proteína ADAMTS13 , Adulto , Autoanticorpos/metabolismo , Transfusão de Componentes Sanguíneos , Evolução Fatal , Feminino , Humanos , Mutação , Troca Plasmática , Gravidez , Complicações Hematológicas na Gravidez/terapia , Púrpura Trombocitopênica Trombótica/terapia , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Epitopos/imunologia , Neoplasias/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Eletroporação/métodos , Epitopos/genética , Humanos , Imunoterapia/métodos , Neoplasias/terapia , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/imunologia , Transfecção/métodosRESUMO
The efficacy of Fluorouracil (FU) in the treatment of colorectal cancer (CRC) is greatly limited by drug resistance. Autophagy has been implicated in chemoresistance, but the role of selective autophagic degradation in regulating chemoresistance remains unknown. In this study, we revealed a critical role of ABHD5 in charging CRC sensitivity to FU via regulating autophagic uracil yield. We demonstrated that ABHD5 localizes to lysosome and interacts with PDIA5 to prevent PDIA5 from interacting with RNASET2 and inactivating RNASET2. ABHD5 deficiency releases PDIA5 to directly interact with RNASET2 and leave RNASET2 in an inactivate state, which impairs RNASET2-mediated autophagic uracil yield and promotes CRC cells to uptake FU as an exogenous uracil, thus increasing their sensitivity to FU. Our findings for the first time reveal a novel role of ABHD5 in regulating lysosome function, highlighting the significance of ABHD5 as a compelling biomarker predicting the sensitivity of CRCs to FU-based chemotherapy.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Autofagia , Neoplasias Colorretais/terapia , Fluoruracila/farmacologia , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinogênese/patologia , Quimioterapia Adjuvante/métodos , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Conjuntos de Dados como Assunto , Progressão da Doença , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/uso terapêutico , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Estimativa de Kaplan-Meier , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Ribonucleases/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Uracila/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herein, we show that both exogenously transfected and endogenously activated p53 repress promoter activity and expression of PDGFRB. p53 binds the proximal promoter containing the CCAAT motif as examined by EMSA and chromatin immunoprecipitation. However, gradual induction of p53 in tet-onSAOS2 cells resulted in a transient increase of the PDGFRB-promoter activity and its expression. As binding of p53 to the promoter increased, previously bound p73, DeltaNp73, c-Myc, HDAC1 and HDAC4 were dismissed from the repressed promoter, and p300 was recruited. The transient increase of the promoter activity was therefore induced by the release of the p73, Myc and HDACs, previously shown to act as repressors to this promoter. Along with further increase of p53, p300 was replaced by HDAC1 and HDAC4, resulting in decreased PDGFRB expression. For the repression, acetylation of the C-terminal lysines of p53 is important, and both acetyl-K373p53 and methyl-K370p53 became bound to the promoter. The acetyl-K373p53 was accumulated in the nucleus and colocalized with promyelocytic leukemia protein. Mitomycin treatment of MEF induced similar epigenetic modification of p53 and its binding to the promoter chromatin. Addition of a PDGFR tyrosine-kinase inhibitor to p53-inducing tet-onSAOS2 increased the number of apoptotic cells. These results suggest that p53 represses the PDGFRB promoter, facilitating the p53-induced apoptosis, whereas tumor cells with p53 mutation or a high level of DeltaNp73 or Myc could become refractory to the regulation.
Assuntos
Regiões Promotoras Genéticas , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteína Supressora de Tumor p53/fisiologia , Apoptose , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/fisiologia , Histona Desacetilase 1 , Histona Desacetilases/fisiologia , Humanos , Cinética , Metilação , Mitomicina/farmacologia , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Repressoras/fisiologia , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19- K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467-80. ©2018 AACR.
Assuntos
Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Eletroporação , Expressão Gênica , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Receptores KIR/antagonistas & inibidoresRESUMO
The developing central nervous system is known to be highly vulnerable to X-irradiation. Although glial cells are involved in various brain functions, knowledge on the effects of X-irradiation on glial cells is limited. Therefore, the purpose of the present study was to evaluate the effects of prenatal X-irradiation on glial cells. Pregnant Wistar rats were exposed to X-irradiation at a dose of 1.0 Gy on day 15 of gestation. Their offspring were examined at 7 weeks of age. The forebrain weight of X-irradiated rats was significantly lower than that of the age-matched controls. Histological quantification with stereology of the somatosensory cortex (SC) revealed no significant difference in the numerical density of glial cells between the X-irradiated and control rats. However, the glial cells in the X-irradiated animals had significantly larger nuclear size. We had previously reported that a similar X-irradiation paradigm resulted in no significant change in the numerical density of neurons in the SC. According to the results of the present study, there were no significant differences in the glial cell-to-neuron ratios between the X-irradiated and control animals. Taken together, it is speculated that prenatal X-irradiation has an equal effects on the numerical densities of glial cells and neurons.