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1.
Nat Commun ; 12(1): 3389, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099672

RESUMO

Bioorthogonal late-stage diversification of amino acids and peptides bears enormous potential for drug discovery and molecular imaging. Despite major accomplishments, these strategies largely rely on traditional, lengthy prefunctionalization methods, heavily involving precious transition-metal catalysis. Herein, we report on a resource-economical manganese(I)-catalyzed C-H fluorescent labeling of structurally complex peptides ensured by direct alkynylation and alkenylation manifolds. This modular strategy sets the stage for unraveling structure-activity relationships between structurally discrete fluorophores towards the rational design of BODIPY fluorogenic probes for real-time analysis of immune cell function.


Assuntos
Técnicas de Química Sintética/métodos , Corantes Fluorescentes/síntese química , Manganês/química , Peptídeos/síntese química , Compostos de Boro/química , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Carbono/química , Catálise , Membrana Celular/metabolismo , Humanos , Hidrogênio/química , Células Jurkat , Microscopia Confocal , Microscopia de Fluorescência , Imagem Molecular/métodos
2.
Immunohorizons ; 5(5): 338-348, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035081

RESUMO

Memory CD8+ T cells promote protective immunity against viruses or cancer. Our field has done a terrific job identifying how CD8+ T cell memory forms in response to Ag. However, many studies focused on systems in which inflammation recedes over time. These situations, while relevant, do not cover all situations in which CD8+ T cell memory is relevant. It is increasingly clear that CD8+ T cells with a memory phenotype form in response to infections with extensive or prolonged tissue inflammation, for example, influenza, herpes, and more recently, COVID-19. In these circumstances, inflammatory mediators expectedly affect forming memory CD8+ T cells, especially in tissues in which pathogens establish. Notwithstanding recent important discoveries, many outstanding questions on how inflammation shapes CD8+ T cell memory remain unanswered. We will discuss, in this review, what is already known and the next steps to understand how inflammation influences CD8+ T cell memory.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Inflamação/imunologia , Vírus/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/virologia , COVID-19/imunologia , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , SARS-CoV-2/imunologia
3.
BMC Infect Dis ; 21(1): 441, 2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-33985440

RESUMO

OBJECTIVES: We analysed the peripheral blood lymphocyte subsets of human immunodeficiency virus (HIV)-negative patients infected with pneumocystis pneumonia (PCP) to determine the relationships between the levels of different types of lymphocytes and the prognosis of patients. METHODS: We retrospectively reviewed HIV-negative patients with PCP diagnosed in our department. All the eligible patients underwent lymphocyte subset analysis on admission. RESULTS: A total of 88 HIV-negative PCP patients were enrolled in the study. In univariate analyses, low CD4+ T cell count, low CD8+ T cell count, and low natural killer cell (NK cell) count were associated with higher in-hospital mortality. CD8+ T cell count ≤300/µL was found to be an independent risk factor for poor prognosis in multivariate logistical regression analysis (p = 0.015, OR = 11.526, 95% CI = 1.597-83.158). Although low CD4+ T cell and NK cell counts were not independent risk factors, the mortality rates of PCP patients decreased as the CD4+ T cell and NK cell counts increased. CONCLUSION: The immune process of Pneumocystis jirovecii infection is complex but important. We propose that lymphocyte subsets could give clinicians a better understanding of patient immune status, helping with the early identification of potentially lethal infections and treatment decision making, such as adjusting the immunosuppressive regimen and choosing an appropriate patient monitoring level.


Assuntos
Subpopulações de Linfócitos/citologia , Pneumonia por Pneumocystis/imunologia , Pneumonia por Pneumocystis/mortalidade , Adulto , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Feminino , Humanos , Células Matadoras Naturais/citologia , Modelos Logísticos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Pneumonia por Pneumocystis/diagnóstico , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida
4.
Cell Prolif ; 54(7): e13056, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34021647

RESUMO

OBJECTIVES: In contrast to extensive studies on bone metastasis in advanced prostate cancer (PCa), liver metastasis has been under-researched so far. In order to decipher molecular and cellular mechanisms underpinning liver metastasis of advanced PCa, we develop a rapid and immune sufficient mouse model for liver metastasis of PCa via orthotopic injection of organoids from PbCre+ ; rb1f/f ;p53f/f mice. MATERIALS AND METHODS: PbCre+ ;rb1f/f ;p53f/f and PbCre+ ;ptenf/f ;p53f/f mice were used to generate PCa organoid cultures in vitro. Immune sufficient liver metastasis models were established via orthotopic transplantation of organoids into the prostate of C57BL/6 mice. Immunofluorescent and immunohistochemical staining were performed to characterize the lineage profile in primary tumour and organoid-derived tumour (ODT). The growth of niche-labelling reporter infected ODT can be visualized by bioluminescent imaging system. Immune cells that communicated with tumour cells in the liver metastatic niche were determined by flow cytometry. RESULTS: A PCa liver metastasis model with full penetrance is established in immune-intact mouse. This model reconstitutes the histological and lineage features of original tumours and reveals dynamic tumour-immune cell communication in liver metastatic foci. Our results suggest that a lack of CD8+ T cell and an enrichment of CD163+ M2-like macrophage as well as PD1+ CD4+ T cell contribute to an immuno-suppressive microenvironment of PCa liver metastasis. CONCLUSIONS: Our model can be served as a reliable tool for analysis of the molecular pathogenesis and tumour-immune cell crosstalk in liver metastasis of PCa, and might be used as a valuable in vivo model for therapy development.


Assuntos
Neoplasias Hepáticas/patologia , Neoplasias da Próstata/patologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Comunicação Celular , Modelos Animais de Doenças , Neoplasias Hepáticas/secundário , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/metabolismo , Microambiente Tumoral , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
5.
Nat Commun ; 12(1): 2715, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976157

RESUMO

Efficient immune responses rely on heterogeneity, which in CD8+ T cells, amongst other mechanisms, is achieved by asymmetric cell division (ACD). Here we find that ageing, known to negatively impact immune responses, impairs ACD in murine CD8+ T cells, and that this phenotype can be rescued by transient mTOR inhibition. Increased ACD rates in mitotic cells from aged mice restore the expansion and memory potential of their cellular progenies. Further characterization of the composition of CD8+ T cells reveals that virtual memory cells (TVM cells), which accumulate during ageing, have a unique proliferation and metabolic profile, and retain their ability to divide asymmetrically, which correlates with increased memory potential. The opposite is observed for naive CD8+ T cells from aged mice. Our data provide evidence on how ACD modulation contributes to long-term survival and function of T cells during ageing, offering new insights into how the immune system adapts to ageing.


Assuntos
Envelhecimento/genética , Divisão Celular Assimétrica/genética , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/genética , Serina-Treonina Quinases TOR/genética , Envelhecimento/imunologia , Animais , Divisão Celular Assimétrica/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Imunidade Inata , Interferon gama/genética , Interferon gama/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Ativação Linfocitária , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/imunologia
6.
Environ Health Prev Med ; 26(1): 50, 2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33874885

RESUMO

BACKGROUND: Asbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8+ T cells. Recently, we reported that asbestos-induced suppression of CTL induction is not due to insufficient levels of interleukin-2 (IL-2). In this study, we continue to investigate the mechanism responsible for the effect of asbestos fibers on the differentiation of CTLs and focus on interleukin-15 (IL-15) which is known to be a regulator of T lymphocyte proliferation. METHODS: For MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 µg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8+ T cells with fluorescence-labeled anti-CD3, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CD25, and anti-granzyme B antibodies using flow cytometry. To examine the effect of IL-15 on the expression level of intracellular granzyme B in proliferating and non-proliferating CD8+ lymphocytes, PBMCs were stained using carboxyfluorescein diacetate succinimidyl ester (CFSE) and then washed and used for the MLR. RESULTS: IL-15 addition partially reversed the decrease in CD3+CD8+ cell numbers and facilitated complete recovery of granzyme B+ cell percentages. IL-15 completely reversed the asbestos-induced decrease in percentage of granzyme B+ cells in both non-proliferating CFSE-positive and proliferating CFSE-negative CD8+ cells. The asbestos-induced decrease in the percentage of CD25+ and CD45RO+ cells in CD8+ lymphocytes was not reversed by IL-15. CONCLUSION: These findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.


Assuntos
Asbestos/efeitos adversos , Linfócitos T CD8-Positivos/imunologia , Interleucina-15/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Humanos , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/metabolismo
7.
J Med Chem ; 64(9): 5802-5815, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33844923

RESUMO

Peptide stapling chemistry represents an attractive strategy to promote the clinical translation of protein epitope mimetics, but its use has not been applied to natural cytotoxic peptides (NCPs) to produce new oncolytic peptides. Based on a wasp venom peptide, a series of stapled anoplin peptides (StAnos) were prepared. The optimized stapled Ano-3/3s were shown to be protease-resistant and exerted superior tumor cell-selective cytotoxicity by rapid membrane disruption. In addition, Ano-3/3s induced tumor ablation in mice through the direct oncolytic effect and subsequent stimulation of immunogenic cell death. This synergistic oncolytic-immunotherapy effect is more remarkable on melanoma than on triple-negative breast cancer in vivo. The efficacies exerted by Ano-3/3s on melanoma were further characterized by CD8+ T cell infiltration, and the addition of anti-CD8 antibodies diminished the long-term antitumor effects. In summary, these results support stapled peptide chemistry as an advantageous method to enhance the NCP potency for oncolytic therapy.


Assuntos
Membrana Celular/metabolismo , Melanoma Experimental/terapia , Peptídeos/uso terapêutico , Venenos de Vespas/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Feminino , Interações Hidrofóbicas e Hidrofílicas , Morte Celular Imunogênica/efeitos dos fármacos , Imunoterapia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/química , Peptídeos/farmacologia , Análise de Sobrevida , Transplante Homólogo , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Venenos de Vespas/química
8.
J Cell Mol Med ; 25(10): 4870-4876, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33733611

RESUMO

Under steady-state conditions, the pool size of peripheral CD8+ T cells is maintained through turnover and survival. Beyond TCR and IL-7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8+ T cell proportion in spleens but not in thymi of mice with T cell-specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild-type (WT) and Med1-deficient CD8+ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1-deficient CD8+ T cells compared with WT counterparts. Finally, Med1-deficient CD8+ T cells exhibited a decreased expression of interleukin-7 receptor α (IL-7Rα), down-regulation of phosphorylated-STAT5 (pSTAT5) and Bim up-regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8+ T cells through IL-7Rα/STAT5 pathway-mediated cell survival.


Assuntos
Linfócitos T CD8-Positivos , Subunidade 1 do Complexo Mediador/imunologia , Receptores de Interleucina-7/imunologia , Baço/imunologia , Animais , Apoptose , Células da Medula Óssea , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Subunidade 1 do Complexo Mediador/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais , Baço/citologia
9.
Cell Prolif ; 54(5): e13028, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33738881

RESUMO

OBJECTIVES: Acute lung injury (ALI) not only affects pulmonary function but also leads to intestinal dysfunction, which in turn contributes to ALI. Mesenchymal stem cell (MSC) transplantation can be a potential strategy in the treatment of ALI. However, the mechanisms of synergistic regulatory effects by MSCs on the lung and intestine in ALI need more in-depth study. MATERIALS AND METHODS: We evaluated the therapeutic effects of MSCs on the murine model of lipopolysaccharide (LPS)-induced ALI through survival rate, histopathology and bronchoalveolar lavage fluid. Metagenomic sequencing was performed to assess the gut microbiota. The levels of pulmonary and intestinal inflammation and immune response were assessed by analysing cytokine expression and flow cytometry. RESULTS: Mesenchymal stem cells significantly improved the survival rate of mice with ALI, alleviated histopathological lung damage, improved intestinal barrier integrity, and reduced the levels of inflammatory cytokines in the lung and gut. Furthermore, MSCs inhibited the inflammatory response by decreasing the infiltration of CD8+ T cells in both small-intestinal lymphocytes and Peyer's patches. The gut bacterial community diversity was significantly altered by MSC transplantation. Furthermore, depletion of intestinal bacterial communities with antibiotics resulted in more severe lung and gut damages and mortality, while MSCs significantly alleviated lung injury due to their immunosuppressive effect. CONCLUSIONS: The present research indicates that MSCs attenuate lung and gut injury partly via regulation of the immune response in the lungs and intestines and gut microbiota, providing new insights into the mechanisms underlying the therapeutic effects of MSC treatment for LPS-induced ALI.


Assuntos
Lesão Pulmonar Aguda/terapia , Linfócitos T CD8-Positivos/imunologia , Intestinos/imunologia , Pulmão/imunologia , Transplante de Células-Tronco Mesenquimais , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/mortalidade , Lesão Pulmonar Aguda/patologia , Animais , Líquido da Lavagem Broncoalveolar/química , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Microbioma Gastrointestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestinos/microbiologia , Intestinos/patologia , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Componente Principal , Taxa de Sobrevida
10.
Cell Prolif ; 54(5): e13025, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33754407

RESUMO

The peptide-based therapeutic cancer vaccines have attracted enormous attention in recent years as one of the effective treatments of tumour immunotherapy. Most of peptide-based vaccines are based on epitope peptides stimulating CD8+ T cells or CD4+ T helper cells to target tumour-associated antigens (TAAs) or tumour-specific antigens (TSAs). Some adjuvants and nanomaterials have been exploited to optimize the efficiency of immune response of the epitope peptide to improve its clinical application. At present, numerous peptide-based therapeutic cancer vaccines have been developed and achieved significant clinical benefits. Similarly, the combination of peptide-based vaccines and other therapies has demonstrated a superior efficacy in improving anti-cancer activity. We delve deeper into the choices of targets, design and screening of epitope peptides, clinical efficacy and adverse events of peptide-based vaccines, and strategies combination of peptide-based therapeutic cancer vaccines and other therapies. The review will provide a detailed overview and basis for future clinical application of peptide-based therapeutic cancer vaccines.


Assuntos
Vacinas Anticâncer/administração & dosagem , Neoplasias/terapia , Peptídeos/uso terapêutico , Adjuvantes Imunológicos/química , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Química Farmacêutica , Epitopos/imunologia , Epitopos/uso terapêutico , Eritema/etiologia , Humanos , Neoplasias/patologia , Peptídeos/efeitos adversos , Peptídeos/imunologia
11.
Cancer Med ; 10(9): 3129-3138, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33769705

RESUMO

BACKGROUND: A simple measure of immune cytolytic activity (CYT) base on mRNA expression levels of two genes, GZMA and PRF1, was recently reported. Here, we aimed to evaluate the CYT score's potential as a measure of antitumor immunity and predictor of clinical outcome in gastric cancer (GC) patients. MATERIALS AND METHODS: We evaluated the correlations between tumor-infiltrating immune cells and the CYT score in 238 GC samples from The Cancer Genome Atlas (TCGA). Next, we investigated CYT score associations with molecular subtypes, somatic mutation load, and immune checkpoint molecules in GC samples from TCGA and Asian Cancer Research Group (ACRG). Moreover, we evaluated the clinical significance of the CYT score calculated by reverse transcription (RT)-quantitative PCR (qPCR) data in 123 GC samples and the association of the CYT score with the response to anti-PD-1 therapy in 7 GC samples from Kyushu University Hospital. RESULTS: The CYT score positively correlated with the proportions of tumor-infiltrating CD8+ T cells and macrophages and negatively correlated with the proportion of regulatory T cells in GC tissues. A high CYT score was associated with common immune checkpoint molecules, a high mutation, the Epstein-Barr virus subtype, and the microsatellite instability subtype in GC. Moreover, a low CYT score was a poor prognosis factor in patients with GC. Finally, the CYT score was higher in a responder to anti-PD-1 therapy compared to nonresponders. CONCLUSION: The CYT score reflects antitumor immunity and predicts clinical outcome in GC patients.


Assuntos
Biomarcadores Tumorais/imunologia , Linfócitos T CD8-Positivos/imunologia , Granzimas/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Perforina/metabolismo , Neoplasias Gástricas/imunologia , Idoso , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Estudos de Coortes , Bases de Dados Genéticas , Feminino , Granzimas/genética , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Proteínas de Checkpoint Imunológico/metabolismo , Imunidade Celular , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/citologia , Masculino , Mutação/imunologia , Perforina/genética , Prognóstico , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/terapia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Resultado do Tratamento , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia
12.
Cell ; 184(7): 1804-1820.e16, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33691139

RESUMO

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.


Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos T CD8-Positivos , COVID-19 , Fragmentos Fc das Imunoglobulinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células CHO , COVID-19/imunologia , COVID-19/terapia , Chlorocebus aethiops , Cricetulus , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , SARS-CoV-2/imunologia , Células Vero , Carga Viral
13.
Methods Mol Biol ; 2294: 209-218, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33742404

RESUMO

Immune evasion hallmark has grabbed wide attention in cancer progression on the clinical level. Accordingly, innate and adaptive immune cells isolation and manipulation is essential in order to assess their activity and role in the tumor microenvironment (TME). This could open a gate toward a personalized therapy by a simple aspiration of blood sample from patients. Here, we describe the isolation of peripheral blood mononuclear cells (PBMCs) using Ficoll plus media in order to achieve the highest yield of immune cells that can be further processed and used in isolation of specific immune cells such as macrophages and cytotoxic T cells (CD8+ cells). Among the highly metastatic macrophages are the M2. This protocol describes the optimized techniques to isolate monocytes from whole blood, differentiate them into M2. This is followed by genetic and epigenetic (using synthetic nucleotides of noncoding RNAs) manipulation of these isolated immune cells in a tumor culture media, in addition to measurement of released cytokines using specific ELISA kit. In the last decade, new groups of noncoding RNAs have been emerged which are microRNAs and long noncoding RNAs. First, they were known as "junk DNA" with unknown regulatory functions. Despite the limited knowledge of these molecules, basic expression profiling is proving to be clinically relevant to cancer diagnosis, metastasis, and prognosis. Here, we describe methods used in molecular biology to assess the epigenetic expression of ncRNAs and their impact on other messenger RNA transcripts in M2 macrophages that could serve as future biomarkers in the context of tumor biology and metastasis or could open a gate in the treatment of cancer.


Assuntos
Linfócitos T CD8-Positivos/citologia , Epigenômica/métodos , Leucócitos Mononucleares/citologia , Macrófagos/citologia , Neoplasias/patologia , Cultura Primária de Células/métodos , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Meios de Cultura/química , Citocinas/genética , Citocinas/metabolismo , Humanos , Macrófagos/imunologia , Metástase Neoplásica/patologia , Neoplasias/genética , Neoplasias/imunologia , Medicina de Precisão/métodos , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
14.
Poult Sci ; 100(2): 623-630, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33518115

RESUMO

The immunomodulatory effect of Acanthopanax senticosus polysaccharide (ASPS) on immunosuppressed chickens induced by cyclophosphamide (Cy) was observed in this study. Four hundred 7-day-old chickens were randomly divided into 4 groups: vaccinated control group (VC group), Cy-challenged control group (Cy group), Cy-challenged + low-dose ASPS group (ASPSL + Cy group), and Cy-challenged + high-dose ASPS group (ASPSH + Cy group). All groups except the VC group were injected with Cy at a dose of 80 mg/kg/day of BW for 3 successive days to induce immunosuppression. At the age of 10 d, the ASPSL + Cy group and ASPSH + Cy group were intramuscularly injected with 0.2 mL of ASPS at the dose of 100 and 200 mg/mL/day, respectively, once a day for 3 successive days. The Cy group was injected with saline solution in the same way as the 2 ASPS groups. At the age of 14 d, the chickens were vaccinated with Newcastle disease (ND) vaccine in all groups. On day 7, 14, 21, and 28 after the vaccination, BW, lymphocyte proliferation, the serum antibody titers of the ND vaccine, the proportion of CD4+ and CD8+ T lymphocytes, and the concentrations of interferon gamma and IL-2 were determined. The results showed that chickens were injected with Cy at a dose of 80 mg/kg of BW for 3 d displayed lower immune responses than the control group, indicating that the immunosuppressive model was successfully established. At most time points, both high and low doses of ASPS could significantly promote lymphocyte proliferation; enhance BW, antibody titers, and the proportion of CD4+ and CD8+ T lymphocytes; and raised the concentrations of interferon gamma and IL-2 in Cy-treated chickens compared with those in the Cy control group (P < 0.05). These results indicated that ASPS could resist immunosuppression induced by Cy and may be a new-type immune adjuvant to improve vaccination in normal and immunosuppressed chickens.


Assuntos
Galinhas/imunologia , Eleutherococcus/imunologia , Imunossupressão/veterinária , Doença de Newcastle , Vacinas Virais , Adjuvantes Imunológicos , Animais , Anticorpos/sangue , Contagem de Linfócito CD4/métodos , Contagem de Linfócito CD4/veterinária , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Ciclofosfamida/administração & dosagem , Imunossupressores/administração & dosagem , Interferon gama/sangue , Interleucina-2/sangue , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/imunologia , Polissacarídeos/imunologia , Distribuição Aleatória
15.
J Virol ; 95(9)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33568510

RESUMO

In this placebo-controlled phase II randomized clinical trial, 103 human immunodeficiency virus type 1 (HIV-1)-infected patients under cART (combined antiretroviral treatment) were randomized 2:1 to receive either 3 doses of DNA GTU-MultiHIV B (coding for Rev, Nef, Tat, Gag, and gp160) at week 0 (W0), W4, and W12, followed by 2 doses of LIPO-5 vaccine containing long peptides from Gag, Pol, and Nef at W20 and W24, or placebo. Analytical treatment interruption (ATI) was performed between W36 to W48. At W28, vaccinees experienced an increase in functional CD4+ T-cell responses (P < 0.001 for each cytokine compared to W0) measured, predominantly against Gag and Pol/Env, and an increase in HIV-specific CD8+ T cells producing interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-α) (P = 0.001 and 0.013, respectively), predominantly against Pol/Env and Nef. However, analysis of T-cell subsets by mass cytometry in a subpopulation showed an increase in the W28/W0 ratio for memory CD8+ T cells coexpressing exhaustion and senescence markers such as PD-1/TIGIT (P = 0.004) and CD27/CD57 (P = 0.044) in vaccinees compared to the placebo group. During ATI, all patients experienced viral rebound, with the maximum observed HIV RNA level at W42 (median, 4.63 log10 copies [cp]/ml; interquartile range [IQR], 4.00 to 5.09), without any difference between arms. No patient resumed cART for CD4 cell count drop. Globally, the vaccine strategy was safe. However, a secondary HIV transmission during ATI was observed. These data show that the prime-boost combination of DNA and LIPO-5 vaccines elicited broad and polyfunctional T cells. The contrast between the quality of immune responses and the lack of potent viral control underscores the need for combined immunomodulatory strategies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01492985.)IMPORTANCE In this placebo-controlled phase II randomized clinical trial, we evaluated the safety and immunogenicity of a therapeutic prime-boost vaccine strategy using a recombinant DNA vaccine (GTU-MultiHIV B clade) followed by a boost vaccination with a lipopeptide vaccine (HIV-LIPO-5) in HIV-infected patients on combined antiretroviral therapy. We show here that this prime-boost strategy is well tolerated, consistently with previous studies in HIV-1-infected individuals and healthy volunteers who received each vaccine component individually. Compared to the placebo group, vaccinees elicited strong and polyfunctional HIV-specific CD4+ and CD8+ T-cell responses. However, these immune responses presented some qualitative defects and were not able to control viremia following antiretroviral treatment interruption, as no difference in HIV viral rebound was observed in the vaccine and placebo groups. Several lessons were learned from these results, pointing out the urgent need to combine vaccine strategies with other immune-based interventions.


Assuntos
Vacinas contra a AIDS , Antirretrovirais/uso terapêutico , Infecções por HIV/terapia , Vacinas de DNA , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Adulto , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , Humanos , Imunização Secundária , Masculino , Pessoa de Meia-Idade , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia
16.
Mol Immunol ; 133: 53-62, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33631555

RESUMO

T cells can be subdivided into a number of different subsets that are defined by their distinct functions. While the specialization of different T cell subsets is partly achieved by the expression of specific genes, the overall transcriptional profiles of all T cells appear very similar. Alternative mRNA splicing is a mechanism that facilitates greater transcript/protein diversity from a limited number of genes, which may contribute to the functional specialization of distinct T cell subsets. In this study we employ a combination of short-read and long-read sequencing technologies to compare alternative mRNA splicing between the CD4 and CD8 T cell lineages. While long-read technology was effective at assembling full-length alternatively spliced transcripts, the low sequencing depth did not facilitate accurate quantitation. On the other hand, short-read technology was ineffective at assembling full-length transcripts but was highly accurate for quantifying expression. We show that integrating long-read and short-read data together achieves a more complete view of transcriptomic diversity. We found that while the overall usage of transcript isoforms was very similar between the CD4 and CD8 lineages, there were numerous alternative spliced mRNA isoforms that were preferentially used by one lineage over the other. These alternative spliced isoforms included ones with different exon usage, exon exclusion or intron inclusion, all of which are expected to significantly alter the protein sequence.


Assuntos
Processamento Alternativo/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , RNA Mensageiro/genética , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genética , Transcriptoma/genética
17.
Cancer Med ; 10(5): 1805-1814, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33560598

RESUMO

BACKGROUND: Disparities in colon cancer (CC) outcomes may be due to a more aggressive phenotype in African American patients in the setting of a decreased tumor immunity, though the precise mechanism for this result has not been well elucidated. To explore the molecular factors underlying CC disparities, we compared the immunogenomic signatures of CC from African American and European American patients. METHODS: We identified all CC patients from the publicly available Cancer Genome Atlas for whom race and survival data are available. Immunophenotype signatures were established for African American and European American patients. Comparisons were made regarding survival and a multivariable linear regression model was created to determine the association of immune cellular components with race. Differential gene expression was also assessed. RESULTS: Of the 254 patients identified, 58 (23%) were African American and 196 (77%) were European American. African American patients had a decreased progression free survival (p = 0.04). Tumors from African American patients displayed a reduced fraction of macrophages and CD8+ T cells and an increased fraction of B cells compared with tumors from European Americans. Differences persisted when controlling for sex, age, and disease stage. Immunostimulatory and immunoinhibitory gene profiles including major histocompatibility complex expression differed by race. CONCLUSIONS: Differences in the tumor immune microenvironment of African American as compared to European American CC specimens may play a role in the survival differences between the groups. These differences may provide targeted therapeutic opportunities.


Assuntos
Afro-Americanos/genética , Neoplasias do Colo/etnologia , Neoplasias do Colo/imunologia , Grupo com Ancestrais do Continente Europeu/genética , Microambiente Tumoral/imunologia , Afro-Americanos/estatística & dados numéricos , Fatores Etários , Idoso , Linfócitos B/citologia , Linfócitos T CD8-Positivos/citologia , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Grupo com Ancestrais do Continente Europeu/estatística & dados numéricos , Feminino , Expressão Gênica , Genes MHC Classe I , Genes MHC da Classe II , Humanos , Imunidade/genética , Imunidade Celular , Imunofenotipagem , Modelos Lineares , Macrófagos/citologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Intervalo Livre de Progressão , Fatores Sexuais
18.
Cell Res ; 31(3): 272-290, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33473155

RESUMO

How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8+ resident-memory (TRM) and CD4+ T-helper-17 (TH17) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4+ T-cells with T-helper-1 characteristics (TH1-like) and CD8+ T-cells expressing exhaustion markers (TEX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.


Assuntos
Imunidade Adaptativa , Lavagem Broncoalveolar , COVID-19/diagnóstico , COVID-19/imunologia , Imunidade Inata , Análise de Célula Única , Líquido da Lavagem Broncoalveolar , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Comunicação Celular , Perfilação da Expressão Gênica , Humanos , Pulmão/virologia , Macrófagos Alveolares/citologia , Monócitos/citologia , Neutrófilos/citologia , Fenótipo , Análise de Componente Principal , RNA-Seq , Células Th17/citologia
19.
Nat Commun ; 12(1): 99, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397934

RESUMO

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.


Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Timo/citologia , Timo/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade/metabolismo , Ativação Linfocitária/genética , Camundongos Endogâmicos C57BL , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
20.
BMC Infect Dis ; 21(1): 57, 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33435865

RESUMO

BACKGROUND: In December 2019, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, Hubei, China. Moreover, it has become a global pandemic. This is of great value in describing the clinical symptoms of COVID-19 patients in detail and looking for markers which are significant to predict the prognosis of COVID-19 patients. METHODS: In this multicenter, retrospective study, 476 patients with COVID-19 were enrolled from a consecutive series. After screening, a total of 395 patients were included in this study. All-cause death was the primary endpoint. All patients were followed up from admission till discharge or death. RESULTS: The main symptoms observed in the study included fever on admission, cough, fatigue, and shortness of breath. The most common comorbidities were hypertension and diabetes mellitus. Patients with lower CD4+T cell level were older and more often male compared to those with higher CD4+T cell level. Reduced CD8+T cell level was an indicator of the severity of COVID-19. Both decreased CD4+T [HR:13.659; 95%CI: 3.235-57.671] and CD8+T [HR: 10.883; 95%CI: 3.277-36.145] cell levels were associated with in-hospital death in COVID-19 patients, but only the decrease of CD4+T cell level was an independent predictor of in-hospital death in COVID-19 patients. CONCLUSIONS: Reductions in lymphocytes and lymphocyte subsets were common in COVID-19 patients, especially in severe cases of COVID-19. It was the CD8+T cell level, not the CD4+T cell level, that reflected the severity of the patient's disease. Only reduced CD4+T cell level was independently associated with increased in-hospital death in COVID-19 patients. TRIAL REGISTRATION: Prognostic Factors of Patients With COVID-19, NCT04292964 . Registered 03 March 2020. Retrospectively registered.


Assuntos
Linfócitos T CD4-Positivos/citologia , COVID-19/sangue , SARS-CoV-2/imunologia , Adulto , Idoso , Linfócitos T CD8-Positivos/citologia , COVID-19/diagnóstico , COVID-19/mortalidade , COVID-19/terapia , Comorbidade , Feminino , Seguimentos , Hospitalização , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Pandemias , Alta do Paciente , Prognóstico , Estudos Retrospectivos , SARS-CoV-2/genética
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